Abstract Background Obesity-induced osteoporosis is a prevalent complication among obese individuals. Conventional anti-osteoporosis medications often lack therapeutic specificity and may exacerbate lipid metabolism disorders. Consequently, identifying suitable pharmacological interventions for obesity-induced osteoporosis, elucidating its underlying biological mechanisms, and developing nanodrug delivery systems with enhanced biocompatibility and targeted delivery remain significant challenges. Methods This study reveals that the pathogenesis of obesity-induced osteoporosis is primarily driven by excessive mitophagy. Notably, Exendin-4 (Ex-4) has been shown to ameliorate mitophagy and mitigate obesity-induced osteoporosis. The nanocomposite DSPE-PEG-ALN (DPA)@Neutrophil membrane (NM)@Cu-TCPP(Zn)/Ex-4 (CTZE), characterized by high biocompatibility and reactive oxygen species (ROS) responsiveness, effectively targets bone tissue, reduces ROS levels, and regulates the release of Cu^2+, Zn^2+, Ex-4, and Alendronate (ALN). This composite interferes with B-cell lymphoma-2 (BCL2)- Beclin-1 (BECN1) binding via the tet methylcytosine dioxygenase 2 (TET2)/PTEN-induced putative kinase protein 1 (PINK1)/Parkin (E3 ubiquitin-protein ligase parkin) pathway, thereby promoting osteoblast differentiation and mineralization. The safety and efficacy of this nano-delivery platform were validated in a mouse model of obesity-induced osteoporosis. Conclusions In summary, our study illustrates that excessive mitophagy plays a crucial role in obesity-induced osteoporosis. Furthermore, DPA@NM@CTZE exhibits significant potential for the precise treatment of obesity-induced osteoporosis, mitigating the side effects of Ex-4, and enhancing the bone microenvironment. Keywords: Obesity-induced osteoporosis, Mitophagy, Exendin- 4, Cu-TCPP(Zn), Alendronate Graphical abstract [45]Image 1 [46]Open in a new tab 1. Introduction With the advancement of the economy and the transformation of dietary patterns, the prevalence of obesity has been rising annually. A high-fat diet (HFD) is a significant factor contributing to obesity. It is projected that by 2024, the global obesity rate will surpass 20 %, and by 2030, the adult obesity rate in China is anticipated to reach 65.3 % [[47]1,[48]2]. Obesity can lead to excessive accumulation of saturated fatty acids in the blood, impaired osteoblast function, and imbalance of bone metabolism. Therefore, compared with normal patients, obese patients have a higher risk of developing osteoporosis [[49]3] (see [50]Scheme 1). Schematic 1. [51]Schematic 1 [52]Open in a new tab DPA@NM@CTZE can regulate excessive mitochondrial autophagy in osteoblasts, and enhance osteoblast mineralization and differentiation. Traditional pharmacological treatments for osteoporosis encompass agents that bone minerality-promoting drugs, such as calcium and vitamin D; bone formation stimulators, including parathyroid hormone analogs like teriparatide; and bone resorption inhibitors, such as bisphosphonates and calcitonin. These medications effectively promote osteoblast proliferation and inhibit osteoclast activity. However, their clinical application is constrained by limited bioavailability and the potential for accumulation in hepatic and renal tissues. Furthermore, increasing the dosage of these drugs can exacerbate the risk of metabolic disturbances [[53]4]. Obesity-induced osteoporosis refers to osteoporosis resulting from lipid metabolism disorders, characterized by a diminished uptake and response of osteoblasts to pharmacological treatments in obese patients [[54]5,[55]6]. Consequently, clinicians are focusing on drugs to adjust lipid disorders and treat obesity-related osteoporosis. Mitochondria serve as the primary organelles involved in intracellular lipid metabolism, facilitating energy production and the elimination of excess reactive oxygen (ROS). Damage to mitochondrial function can lead to the onset of oxidative stress and metabolic disorders [[56]7]. Mitophagy, is a critical biological mechanism that preserves mitochondrial integrity and homeostasis [[57]8]. This process involves the degradation of impaired mitochondrial components via lysosomal pathways, particularly under pathological conditions such as inflammation, exposure to toxins, and nutrient deprivation, thereby ensuring the maintenance of normal lipid metabolism [[58]9]. Recent research has demonstrated a significant association between osteoporosis and mitophagy [[59]10]. In bone marrow mesenchymal stem cells (BMSCs) derived from ovariectomy (OVX) mice, there is a noted reduction in mitophagy [[60]11]. This reduction is accompanied by an inhibition of osteoblast differentiation and an increase in osteoclast activity, ultimately leading to the development of osteoporosis [[61]12,[62]13]. Osteoblast dysfunction induced by type 2 diabetes mellitus (T2DM) can be ameliorated, and bone tissue health can be restored by enhancing mitochondrial quality and the level of mitophagy [[63]14,[64]15]. While research on mitophagy and osteoporosis is currently a prominent area of investigation, the majority of studies have concentrated on the association between mitophagy and diabetic osteoporosis as well as OVX-induced osteoporosis [[65]16,[66]17]. In contrast, there is a paucity of research examining the relationship between mitophagy and obesity-induced osteoporosis. Moreover, current studies do not clarify the mechanisms behind obesity-induced osteoporosis nor do they explore treatments that improve the bone microenvironment. Therefore, elucidating the relationship between mitophagy and obesity-induced osteoporosis, along with identifying methods to improve the bone microenvironment, represents a promising avenue for clinical intervention. Exendin-4 (Ex-4) is an analog of glucagon-like peptide-1 (GLP-1) that targets the GLP-1 receptor, enhances islet cell function, and lowers blood glucose levels [[67]18]. In comparison to GLP-1, it exhibits a prolonged half-life, extended pharmacokinetic profile, and increased duration of action. Furthermore, when compared to other anti-osteoporosis medications and metabolic agents, the activation of the GLP-1 receptor is characterized by its ability to restore bone homeostasis, decrease cellular insulin resistance (IR), and facilitate the bidirectional regulation of osteoblasts and osteoclasts [[68]19]. Following Ex-4 treatment in OVX mice, there is a significant improvement in bone turnover markers. Specifically, serum levels of alkaline phosphatase (ALP), osteocalcin (OCN), and the N-terminal propeptide of procollagen type Ⅰ (PⅠNP) are elevated, contributing to enhanced bone strength and the prevention of osteoporosis progression [[69]20]. Therefore, Ex-4 has the potential to be a significant candidate for the amelioration of obesity-induced osteoporosis. Despite the widespread use of Ex-4, precise dosage control remains challenging, easily leading to gastrointestinal discomfort and an increased risk of liver and kidney failure. Consequently, the development of a nanodrug delivery platform designed to facilitate the targeted delivery of Ex-4 to the abnormal bone microenvironment, while ensuring controlled release, represents a promising strategy to enhance both the efficacy and safety of this therapeutic agent. We developed a biosimulated nanoplatform designed to target high ROS environments within bone tissue for the delivery of Ex-4. Neutrophil membranes (NM) were isolated from mouse bone marrow and subsequently modified with alendronate (ALN) to encapsulate nanoparticles [[70]21]. These NM-based bionic drug delivery nanocarriers exhibit several advantageous properties, including an extended half-life, high delivery efficiency, favorable biocompatibility, and controlled release characteristics [[71]22,[72]23]. ALN has been extensively utilized to enhance the targeting capabilities of nanoparticles in osteoporotic tissues [[73]24,[74]25]. The Copper (Cu) - Tetrakis (4-carboxyphenyl) porphyrin (TCPP)/Zinc (Zn) (CTZ)structure is a metal-organic framework characterized by a Cu^2+ metal core, Zn^2+ modification, and serving as the ligand [[75]13]. CTZ exhibits several advantageous properties, including a large surface area, high porosity, and facile drug delivery capabilities. Research indicates that both Cu^2+ and Zn^2+ ions facilitate the proliferation and differentiation of osteoblasts, with Zn^2+ being a critical element for cellular metabolism. This element can respond to acidic environments, enhance the degradation of endogenous inflammatory substances such as ROS, and improve the bone microenvironment. Consequently, this bio-simulated nanoplatform demonstrates excellent biocompatibility, bone-targeting capabilities, and the potential to enhance osteoblast activity. In conclusion, this study elucidates the potential interrelationship between obesity-induced osteoporosis, mitophagy, and Ex-4. We have developed a biosimulated nanoplatform (DPA@NM@CTZE) designed for the precise delivery of Ex-4, characterized by excellent biocompatibility, bone-targeting capabilities, and responsiveness to ROS. Our research aims to address the following questions: (1) Is mitophagy dysfunction a pivotal factor in the pathogenesis of obesity-induced osteoporosis? (2) Can Ex-4 ameliorate obesity-induced osteoporosis by enhancing mitophagy? (3) Does the DPA@NM@CTZE formulation exhibit superior therapeutic efficacy compared to Ex-4 alone, and what are the underlying biological mechanisms? (4) Can DPA@NM@CTZE safely and effectively mitigate obesity-induced osteoporosis. We believe this study can advance research on using Ex-4 to treat obesity-induced osteoporosis and offer a new approach for precise treatment using biosimulated nanomaterials combined with drugs. 2. Experimental section 2.1. Materials Items and chemicals utilized in this experiment: MC3T3-E1 subclone 24 (EallBio, China), Alpha modification of Eagle's medium (α-MEM, EallBio, China), Fetal bovine serum (FBS, CELL-BOX, China), Trypsin (NCM, China), A mixture of protease and phosphatase inhibitors (NCM, China), Phosphate-buffered saline (PBS, EallBio, China), Bovine Serum Albumin (BSA, Solarbio, China), Cell Counting Kit-8 (CCK-8, NCM, China), Bicin-choninic Acid (BCA, NCM, China), Enhanced chemiluminescent reagent (NCM, China), RIPA assay buffer (NCM, China), Blotting reagent (NCM, China), Color page gel rapid preparation kit (Epizyme, USA), Multicolor prestained protein ladder (Epizyme, USA), Mitotracker Red CMXRos (Beyotime, China), Mitochondrial membrane potential (ΔΨm) assay kit with JC-1 (Beyotime, China), ROS kit (Beyotime, China), 4′,6-Diamidino-2-phenylindole (DAPI, Beyotime, China), Hoechst (Beyotime, China), 4 % Paraformaldehyde (PFA, Solarbio, China), sodium citrate buffer (Solarbio, China), formalin-EDTA decalcifying solution (Solarbio, China), Hematoxylin-eosin stain kit (H&E, Solarbio, China), Modified Masson's trichrome stain kit (Masson, Solarbio, China), Triton X-100 (Servicebio, China), Enhanced endogenous peroxidase blocking buffer (Beyotime, China), Diaminobenzidine (DAB, Zsbio, China), Tris buffered saline with Tween-20 (TBST, Solarbio, China), TRIzol (Sigma, USA), ALN (Sigma, USA), Ex-4 (Selleck, USA), Dexamethasone (MCE, USA), β-Glycerophosphate disodium salt pentahydrate (MCE, USA) and Vitamin C (MCE, USA), Palmitic acid (Pa, Kunchuang Technology, China), Bafilomycin A1 (BafA1, MCE, USA). 2.2. Extraction of NM Euthanized C57BL/6J mice were immersed in 75 % ethanol for 20 min. Subsequently, the femur was isolated, and the distal portion was excised. Bone marrow was extracted by flushing the medullary cavity with 10 % FBS in α-MEM, and the resulting fluid was collected and prepared as a single-cell suspension. Reagents were added according to the guidelines provided for the Neutrophil Isolation Kit (Solarbio, China) [[76]26]. Following the protocol, a High-speed floor centrifuge (Thermo Fisher Scientific, USA) was employed with the parameters (1,000g, 30 min). The neutrophil layer was carefully aspirated and transferred, washed with 10 mL of PBS, and centrifuged using an Eppendorf centrifuge (Germany) at 250g for 10 min. This washing step was repeated 3 times to obtain clean NM. Subsequently, NM was prepared using a hypotonic swelling method. Neutrophils were placed in a hypotonic lysis buffer composed of Tris-HCl (30 mM, pH 7.5), D-mannitol (225 mM), sucrose (75 mM), and a protease inhibitor, and stored in a refrigerator at 4 °C overnight. Purified NM was obtained through grinding and centrifugation, and re-suspended in PBS for subsequent use [[77]23]. The BCA kit was employed to quantitatively measure the key membrane protein content on the NM surface, using bovine serum albumin as a standard [[78]27,[79]28]. 2.3. Preparation and characterization of DPA@NM@CTZE Following established research methodologies, two-dimensional (2D) metal-organic framework (MOF) nanomaterials were synthesized. The preparation process for 2D CTZ involved the synthesis of Zn(NO[3])[2]·6H[2]O (267 mg, Aladdin, USA), pyrazine (24 mg, Aladdin, USA), and polyvinylpyrrolidone (PVP, 600 mg, Aladdin, USA) in N,N-dimethylformamide (DMF, Rhawn, USA). These components were subsequently dissolved in 360 mL of ethanol (v/v = 3/1). Once completely dissolved, 120 mL of the solution was transferred into a test tube, and meso-TCPP (120 mg, Aladdin, USA) was introduced. The solution was heated in an oil bath (80 °C, 2 h), and Cu(NO[3])[2]·3H[2]O (144 mg, Aladdin, USA) was added for another 12 h. The mixture was then centrifuged, rinsed with ethanol, and freeze-dried to produce CTZ powder. The CTZ nanosheet solution, with a concentration of 4 mg/mL, underwent ultrasonic probe treatment at a power of 300 W, with 5 s on and 5 s off intervals, maintained at room temperature for 4 h. Subsequently, the solution was subjected to centrifugation and washing processes, resulting in the collection of CTZ nanosheets with reduced diameters. CTZE preparation process: CTZE was thoroughly combined with 2 mL of Ex-4 (1 mg/mL) and stirred at ambient temperature for a duration of 24 h. Subsequently, CTZE was isolated by centrifugation at 12,000 rpm for 10 min. DPA preparation process: In 10 mL of ultrapure water, ALN (16 mg) and DSPE-PEG-NHS (50 mg, Ponsure, China) were dissolved and stirred at 37 °C over a 24 h period. Subsequently, the solution underwent dialysis for an additional 24 h, followed by freeze-drying to yield DPA. NM@CTZE preparation process: NM (1 mg/mL) and CTZE (100 μg/mL) were combined in a 10:1 ratio, and dissolution was facilitated using an ultrasonic probe pre-cooled to 4 °C, operating at 100 W for 5 min. Subsequently, the mixture was extruded approximately 11 times through a 0.4 μm polycarbonate membrane (Sigma, USA) to yield mature NM@Cu-TCPP (Zn)/Ex-4 [[80]29]. DPA@NM@CTZE preparation process: NM@Cu-TCPP (Zn)/Ex-4 (1 mg/mL) and DPA (1 mg/mL) were incubated in a PBS shaker at 37 °C with a pH of 7.4 for 30 min to synthesize DPA@NM@CTZE. Characterization: The presence of key membrane proteins, including Integrin β1, β2, and CXC chemokine receptor 2 (CXCR2) on NM, was confirmed through Western blot (WB) analysis, thereby validating the successful extraction of NM. The 2D and 3D morphologies of CTZ and DPA@NM@CTZE were examined using scanning electron microscopy (SEM, ZEISS, Germany) and transmission electron microscopy (TEM, FEI, USA). Energy dispersive X-ray spectroscopy (EDS) provided a visual representation of the spatial distribution of elements within CTZ, supporting the hypothesis that Cu and Zn are predominantly located in the core, while P derived from membrane phospholipids, is distributed in the outer core-shell structure. X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific, USA) was employed to ascertain the valence states of surface elements in CTZ materials. The microstructure and composition of CTZ were characterized using X-ray diffraction (XRD, Malvern, Netherlands). Fourier transform infrared spectroscopy (FTIR, Thermo Fisher Scientific, USA) was utilized to assess the key molecular structure features of DPA, as well as to evaluate its synthesis efficiency and chemical stability. Dynamic light scattering (DLS, Malvern, UK) was applied to measure the hydrodynamic diameter and Zeta potential variations of the aforementioned materials, assessing the dispersion and stability of the nanoparticles in solution. The absorption properties of various concentrations of Ex-4 were analyzed using Ultraviolet–Visible Spectroscopy (UV–Vis, Shimadzu, Japan), and the status of Ex-4 loaded with CTZ was examined. 2.4. The ROS response of DPA@NM@CTZE was assessed DPA@NM@CTZE was introduced into a PBS solution containing varying concentrations of hydrogen peroxide (H[2]O[2]) at 0, 0.1, and 1 mM. This mixture was then placed in a dialysis bag and subjected to shaking incubation at 37 °C with a speed of 200 rpm. Samples of the released liquid were collected at specified time intervals (5, 10, 15, 20, and 25 h). To keep the volume of the dialysis bag steady, an equal amount of PBS was added. The concentration of Ex-4 in the released samples was quantified using high-performance liquid chromatography (HPLC, Agilent Technologies, USA) [[81]24]. 2.5. Cell culture MC3T3-E1 cells were grown in α-MEM with 10 % FBS and 0.01 % penicillin-streptomycin-amphotericin B. For lentivirus (LV) and small interfering RNA (siRNA) transfection, cells were cultured in α-MEM without antibiotics. Cells were maintained in a tri-gas incubator (Thermo Fisher Scientific, USA), and the culture medium was routinely refreshed. Cells were passaged using trypsin when they reached 70–80 % confluency. 2.6. Animal culture All C57BL/6J mice utilized in this study were procured from the Animal Center of the First Hospital of Jilin University. The animal experiments were evaluated, approved, and overseen by the Animal Ethics Committee of the First Hospital of Jilin University (Issue No. 20230908 (0655)). The mice were housed in controlled environments with a temperature range of 20–25 °C and relative humidity levels between 40 and 70 %. In the first animal experiment, 15 male C57BL/6J mice (eight weeks) underwent a one-week acclimatization period before being randomly assigned to three groups: the Sham group (n = 5), which received a maintenance diet; the HFD group (n = 5), which was fed an HFD; and the HFD + Ex-4 group (n = 5), which received an HFD supplemented with Ex-4. The latter two groups were given an HFD for eight weeks, then received intra-peritoneal Ex-4 injections for ten weeks before euthanasia. In the second animal experiment, we acquired male C57BL/6J mice (n = 60, eight weeks). From these, 25 mice were randomly selected for euthanasia, and their bilateral femurs were harvested for NM extraction. After a one-week period, The other 35 mice were randomly distributed among the respective groups: the Sham group (fed a maintenance diet, n = 5), the HFD group (HFD, n = 5), the HFD + Ex-4 group (HFD with Ex-4, n = 5), the HFD + CTZ group (HFD with CTZ, n = 5), the HFD + DPA@NM@CTZE group (HFD + DPA@NM@CTZE, n = 5), the HFD + DPA@NM@CTZE + LV-NC group (HFD + DPA@NM@CTZE + LV-NC, n = 5) and the HFD + DPA@NM@CTZE + LV-shTet2 (tet methylcytosine dioxygenase 2) group (HFD + DPA@NM@CTZE + LV-shTet2, n = 5). LV-shNC/LV-shTet2 (25 μl) was administered into the medullary cavity via syringe every two weeks after eight weeks of an HFD, targeting the epiphyseal region of the distal femur. Drugs and nanoparticles were administered via the tail vein for ten weeks before euthanasia. Throughout this period, measurements of body weight, fasting blood glucose levels (FBG, mmol/L), and fasting insulin levels (FINS, ng/mL) were conducted. Upon completion of the experiment, the mice were euthanized, and both femurs were collected for micro-computed tomography (micro-CT, Aartselaar, Belgium) and histological analyses. Additionally, blood samples were obtained for subsequent experiments, and the heart, liver, spleen, and kidneys were collected for further analyses. To assess the bone targeting of the bionic nanoplatform, either PBS or ^Cy 5DPA@NM@CTZE was injected into the HFD group of mice, and major organs were collected for in vivo fluorescence imaging 30 min later. 2.7. CCK-8 assay A total of 2500 cells were introduced into each well. After 12 h of adhesion, Pa and Ex-4 were added. Following incubation, 10 μL of CCK-8 working solution was added to each well and incubated for 0.5–1 h to avoid bubbles. Finally, absorbance at 450 nm was measured using a Multi-functional enzyme label instrument (BMG LABtech, Germany), and the data were analyzed. 2.8. Protein extraction and WB A 100 μL RIPA buffer was introduced to each well and putted on ice for 20 min to promote cell lysis. Following complete lysis, proteins were collected using a cell scraper and transferred. The sample was then subjected to sonication using a pre-cooled ultrasonic cell disruptor operating at 40 W, with 5 s pulses and 5 s intervals, for a total duration of 5 min. Subsequently, the lysate was centrifuged at 14,000 rpm for 15 min at 4 °C. An aliquot of 80 μL of the supernatant was carefully extracted, ensuring minimal disturbance of the pellet. The remaining 20 μL of the protein solution was reserved for subsequent quantitative analysis using the BCA protein assay. Based on the quantitative results, an appropriate volume of 1 × SDS-PAGE loading buffer was added, followed by the 5 × SDS-PAGE loading buffer. The mixture was heated to ensure complete protein denaturation. The extracted proteins were stored at −80 °C for future analysis. The proteins were separated using SDS-PAGE at an appropriate concentration and subsequently transferred to a PVDF membrane (Sigma, USA) in an ice bath. Following the completion of the membrane transfer, the protein was blocked with a 5 % Blotting reagent, and then incubated with the primary antibody on a shaking platform at 4 °C overnight. WB using antibodies including: TET2 (Abcam, USA), COL1A1 (Collagen I, Abcam, UK), RUNX2 (Runt-related transcription factor 2, CST, USA), PINK1 (PTEN-induced putative kinase protein 1, Bioss, China), Parkin (E3 ubiquitin-protein ligase parkin, Servicebio, China), LC3B (Light Chain 3, Abmart, China), ATG4B (Autophagy Related 4B Cysteine Peptidase, Proteintech, China), BCL2 (B-cell lymphoma-2, Abmart, China), BECN1 (Beclin-1, Abmart, China), ACTB (Beyotime, China), Integrin β1 (MCE, USA), Integrin β2 (Bio-Techne, USA), CXCR2 (Proteintech, China), HRP-labeled Goat Anti-Rabbit IgG (H + L) (Beyotime, China), HRP-labeled Goat Anti-Mouse IgG (H + L) (Beyotime, China), KEAP1 (Kelch Like ECH Associated Protein 1, Proteintech, China),NRF2 (Nuclear factor erythroid 2-related factor 2, Proteintech, China). 2.9. ROS assay Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probes permeate freely through cellular membranes and emit green fluorescence upon reaction with ROS. Prior to detection, DCFH-DA was diluted to a concentration of 1 μmol/L in α-MEM. The cells were gently washed twice with α-MEM, after which 1 mL of the DCFH-DA working solution was added to each well and fluorescence was observed and captured using a Nikon laser confocal microscope (Nikon, Japan). 2.10. Mitochondrial morphology The MitoTracker Red CMXRos storage solution (200 mM, 1 μL) was diluted in 1 mL of α-MEM to obtain a working solution with a concentration of 200 nM. The pre-treated cells were gently washed twice with α-MEM, after which 1 mL of the MitoTracker Red CMXRos working solution was added. The cells were then incubated at 37 °C for 30 min. Following incubation, the cells were again gently washed twice with α-MEM. A Nikon laser confocal microscope (Nikon, Japan) was subsequently employed for observation and imaging. 2.11. ΔΨm The kit was utilized to detect changes in the ΔΨm of MC3T3-E1 cells under various conditions using JC-1 dye (a mitochondrial membrane potential indicator). In healthy mitochondria, JC-1 accumulates in the mitochondrial matrix and forms red aggregates. However, upon mitochondrial damage, JC-1 remains in its green monomeric form due to its inability to aggregate. The pre-treated cells were washed twice with α-MEM, followed by the addition of 1 mL of JC-1 staining solution to each well. The cells were then incubated at 37 °C for 30 min. After incubation, the JC-1 staining solution was gently washed off twice, and 3 mL of α-MEM was added. Observations and imaging were conducted using a Nikon laser confocal microscope (Nikon, Japan). 2.12. Lysosome and mitochondrial colocalization The cells were prepared as a suspension and counted to achieve a concentration of 5 × 10^4 cells per well, which were then distributed onto a glass substrate Petri dish (Nest, USA). Various treatment conditions were applied for a duration of 48 h. Post-treatment, the cells were gently rinsed twice with PBS, followed by fixation using Beyotime (China) for 10 min. Subsequently, 1 mL of pre-cooled methanol was added, and the samples were placed in a −20 °C refrigerator for 10 min. After this step, the samples were washed 3 times with pre-cooled PBS, each wash lasting 5 min. Triton X-100 (1 mL) was then added for 10 min, and the samples were incubated with an Immunostaining blocking solution (Beyotime, China). Following washing with PBS, the Cytochrome c oxidase IV (COX IV, CST, USA) -specific primary antibody was applied and incubated overnight. After 24 h, the procedure was repeated, and the Lysosomal associated membrane protein 1 (LAMP1, Abcam, UK) primary antibody was added and incubated overnight. Subsequently, Alexa Fluor®488 Anti-Rabbit antibody (Abcam, UK) was applied to LAMP1, and Alexa Fluor®647 Anti-Mouse antibody (CST, USA) was used. In cases where MC3T3-E1 cells were transfected with LV-shPink1 or LV-shTet2, Alexa Fluor®555 Anti-Mouse antibody (CST, USA) was employed to prevent fluorescence overlap. The samples were incubated at room temperature for 1 h, followed by the addition of Antifade Mounting Medium with DAPI (1 mL, 5 min, Beyotime, China). The samples were rinsed twice with PBS and subsequently analyzed and photographed using a Nikon laser confocal microscope (Nikon, Japan). 2.13. TEM The pretreated cells were digested with trypsin. Following centrifugation, 2 mL of glutaraldehyde fixative (Biosharp, China) was added to precipitate the cells along the tube wall, and the samples were fixed for 24 h. This was followed by treatment with Osmium (VIII) oxide (Sigma, USA). After fixation, the samples were dehydrated using a graded series of alcohols for 20 min and then soaked in acetone for an additional 20 min. The samples were embedded in Spurr resin (HEAD, AT) and sectioned using a Leica EM UC7 ultra-thin microtome (Leica, Germany). The sections were stained with uranyl acetate dihydrate (Chemical Book, China) and lead citrate (Chemical Book, China) for 5 min. Finally, the samples were analyzed and imaged using TEM. 2.14. ALP staining The pre-treated cells were prepared as a cell suspension, counted, and distributed onto a 12-well plate (1.5 × 10^4 cells/well). After 12 h, an osteogenic induction solution consisting of 10 % FBS and dexamethasone (10 nmol/L) was introduced. Differentiation was induced using β-glycerophosphate disodium salt pentahydrate (10 mmol/L) and vitamin C (50 μg/mL). At one week, the culture medium was supplemented with or without Pa, Ex-4, DPA@NM@CTZ, or DPA@NM@CTZE. Following treatment, the cells were incubated with the BCIP/NBT ALP color development kit (Beyotime, China) for 30 min in the absence of light. The color reaction was terminated using double-distilled water (ddH[2]O), and images were captured. 2.15. Alizarin red S (ARS) staining The pre-treated cells were prepared as a cell suspension, counted, and distributed into a 12-well plate (1.5 × 10^4 cells/well). Following cell adhesion, an osteogenic induction solution was introduced to promote mineralization. The medium either included or excluded Pa, Ex-4, CTZ, or DPA@NM@CTZE, with the treatment period lasting four weeks. Post-treatment, the cells were gently rinsed three times with PBS, followed by the addition of 1 mL of 4 % PFA to each well for fixation at room temperature for 30 min. Subsequently, the cells were gently rinsed three times with PBS, and 1 mL of ARS Solution (OriCell, USA) working solution was added to each well for 10 min. The wells were then washed 3 times with PBS before imaging. 2.16. Proteomic analysis (label-free) Shanghai Genechem Company conducted the proteomic analysis. In summary, MC3T3-E1 cells were cultured in 10 % FBS α-MEM, with or without the addition of Pa, for a duration of 48 h. Subsequently, samples (n = 3) were submitted to Genechem for label-free protein quantification. The experimental procedure was as follows: The α-MEM medium was removed, and SDT Lysis Buffer was added. The samples were then subjected to sonication and a boiling water bath. Following this, centrifugation was performed at 14,000 g for 15 min using an Eppendorf centrifuge (Eppendorf, Germany) to separate the supernatant. Bicin-choninic Acid was then added for quantification. Subsequent analyses included SDS-PAGE gel electrophoresis, FASP enzymatic digestion, and mass spectrometry (Thermo Fisher Scientific, USA). Data acquisition and protein qualitative and quantitative analyses were conducted using MaxQuant software (version 1.6.17.0). The STRING database (version 11.5) was employed to create a network of protein-protein interactions (PPI). setting a minimum interaction confidence threshold of 0.7. Visual analysis was conducted using Cytoscape software (version 3.9.1) to elucidate both direct and indirect interactions among target proteins. Significantly differentially expressed proteins were identified using screening thresholds of a false discovery rate (FDR) corrected by the Benjamini-Hochberg method of less than 0.05 and |fold change (FC)| > 1.2. A volcano plot was generated with the ggplot2 package in R (version 3.3.1) to illustrate the distribution of protein expression differences between samples. The functional annotation of differentially expressed proteins was performed utilizing the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A systematic analysis was conducted on the Biological Process (BP), Molecular Function (MF), Cellular Component (CC), and signaling pathways associated with these proteins, with a significance threshold set at p < 0.05. To comprehensively assess the biological significance of the proteome, gene set enrichment analysis (GSEA, version 4.2.3) was employed for the functional annotation of the corresponding genes of the entire genome's proteins. The MSigDB databases (C5: GO and C2: KEGG v7.4) gene sets were utilized. Significance is determined by a |Normalized Enrichment Score (NES)| greater than 1 and an FDR q-value less than 0.25. 2.17. The Pink1and Tet2 LV transfection LV-NC and LV-shPink1 were procured from Genechem (Shanghai, China) to downregulate the expression of Pink1 (Serial number: 58770-2) and Tet2 (Serial number: 135756-1). The sequence for the former was GCTGCAAATGTGCTGCACTTA and GCGGTAATTGACTACAGCAAA, while the sequence for the latter was GGAGCTATTTGCTGAAGAATA and GCTACAGTTTCTGCCCATTCT. LV particles were utilized at a multiplicity of infection (MOI) of 100, as determined by preliminary testing. After 72 h post-transfection into MC3T3-E1 cells, transfection efficiency was assessed and quantified using an Inverted fluorescence microscope (Olympus, Japan). Subsequently, the cells were subjected to selection with puromycin (8 μg/mL) to establish stable transfected cell lines. The transfection efficiency of the virus was further evaluated through WB and Quantitative Real-time PCR (RT-qPCR) analyses. 2.18. Bcl2 knockdown The CALNP RNAi transfection reagents and siRNA targeting Bcl2 were procured from D-Nano Therapeutics (China). Following a 72 h transfection of osteoblasts with LV-shTet2 and subsequent selection using puromycin, si-Bcl2 was introduced into the cells utilizing the CALNP RNAi transfection reagent. The transfection efficiency was confirmed through WB and RT-qPCR analyses. The sequences of the Bcl2 siRNA oligonucleotides were as follows: si-Bcl2-2 sense strand, 5′-GGAUGACUGAGUACCUGAA-3′, and antisense strand, 5′-UUCAGGUACUCAGUCAUCC-3'. The sense sequence of the negative control was 5′-UUCUCCGAACGUGUCACGU-3′, and the antisense sequence was 5′-ACGUGACACGUUCGGAGAA-3'. 2.19. RT-qPCR RNA was extracted from cells utilizing the TRIzol reagent. The quality of the RNA samples was assessed using an ultraviolet spectrophotometer (Thermo Fisher Scientific, USA). Subsequently, equal amounts of RNA, dNTP (Monad, China), qPCR Mix (Monad, China), ddH2O, and cDNA were combined in a PCR tube and subjected to PCR amplification using a qPCR instrument (Thermo Fisher Scientific, USA). Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) served as the reference gene. The primer sequences were as follows: Tet2: 5′-CTGCTGTTTGGGTCTGAAGGAAGG-3' (forward) and 5′-GTTCtgctGGtctgtgggagaatg-3' (reverse); Bcl2: 5′-GGTGGGGTCATGTGTGTGG-3' (forward) and 5′-CGGTTCAGGTACTCAGTCATCC-3' (reverse); Pink1: 5′-CACACTGTTCCTCGTTATGAAGA-3' (forward) and 5′-CTTGAGATCCCGATGGGCAAT-3' (reverse); Gapdh: 5′-GGTTGTCTCCTGCGACTTCA-3' (forward) and 5′-TGGTCCAGGGTTTCTTACTCC-3' (reverse). Following data acquisition, normalization was performed using the positive control, and the quantification of relative mRNA expression levels was done through the 2^−ΔΔCt method. 2.20. Immunohistochemistry (IHC) Following the dissection of the mice, femur samples were fixed in 4 % PFA for 24 h. Subsequently, they were decalcified using a Formalin-EDTA Decalcifying Solution for two weeks, dehydrated with a Leica instrument (Germany), embedded in paraffin, and sectioned longitudinally at a thickness of 5 μm. Prior to experimentation, the sections were incubated at 65 °C for 24 h, dewaxed in xylene the following day, rehydrated, and antigen retrieval, followed by natural cooling to room temperature. Enhanced endogenous peroxidase blocking buffer was then applied for 10 min to eliminate endogenous peroxidase activity. Subsequently, the samples were treated with an immunostaining blocking solution for 60 min, followed by incubation with anti-OCN (Abcam, UK), anti-RUNX2 (Proteintech, China), and anti-LC3B (Proteintech, China) antibodies overnight. On the following day, after equilibration to room temperature, the sections were incubated with a Horseradish peroxidase-conjugated secondary antibody (Proteintech, China) and stained using a DAB chromogenic kit (Zsbio, China) for 5 min. The sections were counterstained with hematoxylin for 30 s, mounted, and subsequently imaged using the Olympus VS200 full slide scanning system (Olympus, Japan). 2.21. H&E and masson staining The pre-treated sections were subjected to roasting at 60 °C for a duration of 3 h, followed by immersion in xylene for dewaxing and subsequent hydration using a gradient of alcohol concentrations. In accordance with the instruction manual, H&E staining was employed to assess the morphology of bone trabeculae and quantify the number of adipocytes within the bone marrow matrix. Additionally, Masson staining was utilized to evaluate the arrangement and distribution of bone trabeculae within the bone tissue. 2.22. Three-point bending test The fresh mouse femurs were horizontally positioned on a universal testing machine scaffold (Shimadzu, Japan) and subjected to compression under specified parameters (0.5 N, 1 mm/min) until fracture occurred. Data acquisition was conducted using Trapezium X software, and a load-displacement curve was generated, incorporating parameters such as ultimate load and stiffness. 2.23. Micro-CT imaging and microstructural parameters The mouse femur was preserved in 10 % formalin and subsequently sent to Pingsen Scientific (Jiangsu, China) for analysis. The femur was positioned between an X-ray source and a complementary metal-oxide semiconductor (CMOS) detector and subjected to micro-CT scanning (n = 5). During the scanning process, the femur underwent a 360° rotation along its longitudinal axis within the scanning field, with 1000 projections acquired. The CMOS detector captured the images, which were then transmitted to a computer. The image processing software Avatar (version 2.0.12.5) was utilized to reconstruct the images using the Feldkamp-Davis-Kress (FDK) algorithm, with a reconstruction pixel size of 0.008 × 0.008 × 0.009 mm and a reconstruction matrix of 2000 × 2000 × 1101. The scanning region for the three-dimensional histomorphological images was defined as 5 mm proximal to the growth plate of the distal femur, focusing on the trabecular bone region at 1 mm. The bone histomorphometric analysis was conducted 1 mm proximal to the distal growth plate, targeting the trabecular bone region at 0.5 mm. The parameters analyzed included bone volume fraction (BV/TV, %), trabecular thickness (Tb. Th, μm), bone surface area per unit volume (BS/BV, %), trabecular separation (Tb. Sp, μm), trabecular number (Tb. N, 1/mm), and bone mineral density (BMD, g/cm^3). 2.24. Calcein fluorescence double labeling experiment Calcein (Solarbio, China) and Sodium citrate buffer (Solarbio, China) were thoroughly mixed and administered via intraperitoneal injection into the mice two weeks prior to euthanasia. This procedure was repeated one week before euthanasia. Following euthanasia, the femurs of the mice were harvested and fixed. After 1 day, the samples underwent dehydration using a tissue dehydrator (Leica, Germany), were embedded in methyl methacrylate, and sectioned along the distal coronal plane of the femur using a Hard tissue microslicer (Leica, Germany) to produce slices with a thickness of 50 μm. The deposition of calcein on the trabecular bone surface was subsequently examined using Fluorescence microscopy (Olympus, Japan). 2.25. Enzyme-linked immunosorbent assay (ELISA) Prior to blood collection, the mice underwent a 12 h fasting period, followed by anesthesia. Blood samples were then collected via the retro-orbital sinus. Subsequently, fasting plasma glucose (FPG) levels were determined using a Blood glucose meter (OMRON, Japan). The blood samples were then subjected to centrifugation (3000 rpm, 15 min). Total cholesterol (TC, mmol/L) was measured according to the instructions provided with the assay kit. Additionally, the concentrations of triglycerides (TG, mmol/L), low-density lipoprotein cholesterol (LDL-C, mmol/L), and high-density lipoprotein cholesterol (HDL-C, mmol/L) were quantified using kits (Solarbio, China). FINS were also assessed using Solarbio kits. PINP and Cross-Linked C-telopeptide of Type Ⅰ Collagen (CTX-Ⅰ) assessed using YaJi Biological kits. 2.26. Statistical analysis A minimum of three independent replicates was conducted for each data group, with the mean and standard deviation (SD) subsequently calculated. Statistical analysis of the data was performed using GraphPad Prism (version 9.5). Student's t-test was used for comparisons when dealing with normally distributed data with sample sizes of n ≤ 2.For multiple groups with normally distributed data (n ≥ 3), one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was employed. The Kruskal-Wallis test was utilized for comparing data that did not follow a normal distribution. A p-value of less than 0.05 was considered indicative of a statistically significant difference. 3. Results and discussion 3.1. Ex-4 can enhance obesity-induced osteoporosis The bone-lipid balance refers to the dynamic equilibrium between adipose tissue and skeletal tissue within the bones, with the accumulation of fat in bone tissue being closely associated with metabolic disorders [[82]30]. Obesity has been identified as a significant predictor of osteoporosis [[83]31]. To investigate the potential of Ex-4 in ameliorating obesity-induced osteoporosis, we developed a model of obesity-related osteoporosis and assessed the effects of both obesity and Ex-4 on bone tissue. This evaluation was conducted through a comprehensive analysis ([84]Fig. 1A). Fig. 1. [85]Fig. 1 [86]Open in a new tab In a model of obesity-induced osteoporosis, Ex-4 demonstrated a significant therapeutic effect in ameliorating osteoporosis. (A) Schematic representation of the obesity-induced osteoporosis mouse model. (B) Micro-CT images of the distal femur in mice following HFD and Ex-4 treatment. The measurement scale is 1 mm. (C-D) The histological images stained with H&E and Masson. Scale bar: 100 μm. (E) Bone morphometric parameters: bone mineral density (BMD), bone volume fraction (BV/TV), bone surface area to bone volume ratio (BS/BV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). (F) IHC staining of OCN and RUNX2, with black arrows indicating positively stained osteoblasts. The red arrow points to the fat droplets. Scale bar: 400 μm. The data are presented as Mean ± standard deviation (SD). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 5. Indices related to lipid metabolism, such as TC, TG, LDL-C, and those related to glucose metabolism, including FBG and FINS, were significantly elevated, indicating a higher level of IR. In comparison to the HFD group, mice in the HFD + Ex-4 group exhibited a significant reduction in body weight, as well as notable improvements in blood glucose and lipid levels, with a concomitant alleviation of IR ([87]Fig. S1A, B and C). We found that HFD group significantly reduced PⅠNP and increased CTX-Ⅰ levels, but Ex-4 only effectively countered PⅠNP, primarily impacting bone formation ([88]Fig. S1E and F). Micro-CT results demonstrated that bone trabeculae were continuous and uniformly distributed in the Sham group. In contrast, the HFD group showed a significant reduction in bone mass and trabecular continuity. Ex-4 administration raised BMD to 0.5 mg/cm^3 and TB. N to 3.8 mm^−1, significantly lowering fracture risk ([89]Fig. 1B). Further analysis of bone morphological parameters revealed that, compared to the Sham group, BV/TV, BS/BV and Tb. Th were significantly decreased, while Tb. Sp was increased in the HFD group, resulting in severe bone loss. Treatment with Ex-4 ameliorated these conditions ([90]Fig. 1E). The results suggest that a model of obesity-induced osteoporosis was successfully developed, with obesity predominantly impacting the trabecular architecture of bone within the medullary cavity. The findings indicated a significant reduction in the bending strength and load-bearing capacity of bone tissue in the HFD group. Notably, under identical pressure conditions, the deformation of bone tissue in the HFD group was markedly more pronounced than in the other groups. Conversely, the bone tissue of mice in the HFD + Ex-4 group exhibited significant improvements, demonstrating enhanced resistance to deformation and a reduced risk of fracture ([91]Fig. S1D). H&E staining, along with Masson staining, revealed that in the HFD group, there was a pronounced fracture of the trabecular bone, accompanied by significant thinning of the trabeculae. Additionally, the bone marrow cavities were notably enlarged, with an increased presence of lipid droplets, indicative of a precarious porous bone tissue structure ([92]Fig. 1C and D). The normal mineralization function of bone tissue is intrinsically linked to its strength. Calcein labeling was employed to assess the mineralization capacity of bone tissue. In the HFD group, there was a reduction in the calcein-labeled area and a decline in the mineralization rate, suggesting a diminished mineralization capacity of osteoblasts. Conversely, in the HFD + Ex-4 group, the mineralization capacity of osteoblasts was restored, as evidenced by a significant increase in calcein-labeled areas and an enhanced mineralization rate ([93]Fig. S1H and I). The findings revealed that in the HFD group, there was a marked reduction in osteoblast staining intensity, a decreased number of positive osteoblasts on the trabecular surface, and significantly diminished expressions of OCN and RUNX2. Conversely, in the HFD + Ex-4 group, both the intensity of osteoblast staining and the expression of osteoblast markers were significantly enhanced ([94]Fig. 1F, [95]Fig. S1G). These results suggest that obesity can substantially impair bone tissue, leading to functional deficits in osteoblasts. Furthermore, Ex-4 appears to ameliorate obesity-induced osteoporosis and improve lipid metabolism within bone tissue. However, the underlying mechanisms remain unclear, necessitating further exploration through in vitro studies to elucidate the specific BP involved. 3.2. Ex-4 enhances osteoblast mineralization and differentiation in high lipid conditions To demonstrate the sustained osteogenic efficacy of Ex-4 in vitro, we employed an osteoblast model under a high lipid environment using Pa. An inhibition rate of 20–25 % was noted with a Pa concentration of 0.2 mmol/L for 48 h ([96]Fig. 2A). WB analysis further confirmed that under these conditions, the expression levels of osteoblast-related proteins COL1A1 and RUNX2 were significantly decreased. However, the addition of Ex-4 led to a marked increase in the expression of osteoblast-related proteins, indicating that Ex-4 retains a significant capacity to enhance osteoblast function in vitro ([97]Fig. 2B). Fig. 2. [98]Fig. 2 [99]Open in a new tab Investigation into the Alterations in Gene Expression Profiles of Osteoblasts in a High-Fat Environment. (A) CCK-8 assay was utilized to determine the optimal treatment concentration and duration. (B) Western blot (WB) analysis was conducted to assess the impact of Ex-4 on the protein expression levels in osteoblasts across different experimental groups. (C-F) ARS and ALP assays were employed to evaluate and quantify the osteogenic mineralization and differentiation potential of osteoblasts in both Pa and Ex-4 environments. (G) A protein-protein interaction (PPI) network. (H) A volcano plot was created to show the differences in protein expression (fold change >1.2) between the control and Pa-treated groups. (I) Enrichment analyses of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed, with the red box indicating factors potentially implicated in Pa-induced damage to osteoblasts. (J-K) Gene Set Enrichment Analysis (GSEA) for GO and KEGG pathways was conducted, with the red box highlighting the biological processes potentially involving the protein of interest. Values are represented as Mean ± SD. Statistical significance was denoted as follows: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3. The health of bone tissue is intrinsically linked to the mineralization capacity of osteoblasts, which serves as a direct indicator of bone formation potential. The findings demonstrated that, in comparison to the Control group, Pa significantly diminished the mineralization capacity of osteoblasts, as evidenced by a reduction in the number of mineralized nodules and a decrease in staining intensity. However, these negative effects were ameliorated in the Pa + Ex-4 group ([100]Fig. 2C and E). ALP is a key marker of early osteogenic differentiation, with elevated ALP secretion signifying an enhanced differentiation capacity of osteoblasts [[101]32]. In the Control group, ALP staining was intense. Pa notably decreased ALP secretion, whereas Ex-4 significantly enhanced ALP staining and boosted osteoblast differentiation ([102]Fig. 2D and F). We treated osteoblasts with Pa for 48 h and conducted a proteomic analysis. The protein interaction network was constructed using the STRING database, and Cytoscape software was employed to identify the top 10 key genes exhibiting the highest degree of connectivity: Rps27a, Rps14, Pno1, Rps29, Hspa5, Hspa1b, Rpl10, Dimt1, Dnttip2, and Stat1. These genes predominantly participate in ribosomal function (Rps27a, Rps14, Rps29, Rpl10, Dimt1) and stress response mechanisms (Hspa5, Hspa1b). Furthermore, the genes ranked 11 to 20 also garnered our attention due to their associations with critical BP, including cell cycle regulation (Cdkn1b, Tk1), autophagy and lysosomal degradation (Sqstm1, Atp6v0c), and bone development (Col6a1) ([103]Fig. 2G). These findings indicate that lipid toxicity may influence osteogenic differentiation through various mechanisms, including the disruption of protein synthesis, stress responses, and cellular homeostasis. Subsequently, we identifying 339 proteins that were upregulated and 245 proteins that were downregulated (FC > 1.2, p < 0.05) ([104]Fig. 2H). To further substantiate these findings, we performed an enrichment analysis on these differentially expressed proteins. The KEGG pathway enrichment analysis revealed significant enrichment in pathways related to IR, cholesterol metabolism, and the cell cycle. GO analysis demonstrated that BP were primarily associated with cholesterol metabolic processes, fatty acid beta-oxidation, skeletal system development, and oxidation-reduction processes. CC analysis indicated that lysosomes and the mitochondrial matrix were compromised in a high-fat environment. MF analysis showed enrichment in alterations of key enzymes involved in lipid metabolism, such as Acyl-CoA hydrolase and Palmitoyl-CoA hydrolase ([105]Fig. 2I). Additionally, GSEA corroborated these results, showing significant enrichment of fatty acid metabolism and oxidation disruption in both GO and KEGG analyses ([106]Fig. 2J and K). The mitochondrial matrix is crucial for sustaining the transmembrane potential and removing ROS. Damage to it can disrupt mitophagy, hinder oxidative phosphorylation and glycolysis, and block Adenosine triphosphate (ATP) production [[107]33,[108]34]. Lysosomes act as regulators in response to environmental changes and cellular damage, with their primary functions encompassing monitoring, modification, repair, degradation, and replacement [[109]35,[110]36]. Based on the aforementioned proteomic sequencing results, we hypothesize that Pa may disrupt the osteoblast metabolic pathway by impairing the interaction between mitochondria and lysosomes, ultimately leading to osteoblast damage. 3.3. Ex-4 enhances bone formation in high lipid conditions and is linked to mitophagy Mitochondria are essential organelles involved in cellular energy metabolism, responsible for energy production and the clearance of ROS [[111]37]. Mitophagy is crucial for maintaining mitochondrial homeostasis, stabilizing cellular energy metabolism, and facilitating normal fatty acid and glucose metabolism [[112]38]. We hypothesize that Pa may impede the lipid metabolism of osteoblasts, induce mitochondrial damage, promote the overproduction of ROS, and adversely affect the processes of mineralization and differentiation in bone [[113]39]. To validate our hypothesis, we conducted ROS detection and confirmed that Pa induces a substantial increase in ROS production, leading to elevated oxidative stress levels in osteoblasts. Furthermore, Ex-4 was found to effectively clear ROS and mitigate cellular damage ([114]Fig. 3A and F). The KEAP1/NRF2 pathway, influenced by ROS, is crucial for cellular regulation. In Pa conditions, KEAP1 levels rise while NRF2 levels drop. Adding Ex-4 reverses this effect, suggesting that the KEAP1/NRF2 axis helps manage cellular functions and lower oxidative stress in such environments ([115]Fig. S2A and B). Fig. 3. [116]Fig. 3 [117]Open in a new tab The enhancement of Ex-4 on osteoblasts within a Pa environment is associated with mitophagy. (A) and (F) depict intracellular reactive oxygen species (ROS) levels using Dichlorodihydrofluorescein diacetate (DCFH-DA) (green) staining, with quantitative analysis provided. Scale bar: 100 μm. (B) It illustrates the alterations in mitochondrial morphology across different environments, as marked by MitoTracker. Scale bar: 20 μm. (C) and (G) Current alterations in mitochondrial membrane potential (ΔΨm) are identified using the JC-1 fluorescent probe. Scale bar: 30 μm. (D) and (H) It assesses the colocalization levels of mitochondria and lysosomes in various environments, with COX IV (red), LAMP1 (green), and DAPI (blue) marking mitochondria, lysosomes, and nuclei, respectively. Scale bar: 20 μm. (E) It employs transmission electron microscopy (TEM) to observe the formation of mitochondria and autophagosomes in osteoblasts under different conditions. Scale bar: 2 μm. (I-J) It utilizes WB analysis to examine autophagy-related proteins ATG4B and LC3B. Results are expressed as Mean ± SD, with significance denoted by ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, n = 3. In the Control group, mitochondria exhibited smooth filaments and a well-defined structure. However, following treatment with Pa, significant mitochondrial damage was observed, characterized by fragmented and indistinct structures. Notably, Ex-4 demonstrated the ability to restore damaged mitochondria and reduce mitochondrial fragmentation induced by Pa ([118]Fig. 3B). The maintenance of ΔΨm at normal levels is crucial for preserving mitochondrial function. Our study demonstrated that treatment with Pa significantly decreased ΔΨm, whereas Ex-4 treatment was able to restore ΔΨm and sustain mitochondrial homeostasis ([119]Fig. 3C and G). In the Pa + Ex-4 group, the colocalization of lysosomes and mitochondria also significantly decreased ([120]Fig. 3D and H). Under normal conditions, the mitochondria exhibited an oval or round morphology, uniform distribution, distinct inner membrane folds, and an intact cristae structure. However, following treatment with Pa, the mitochondria displayed swelling, loss of wrinkles, and a blurred or absent cristae structure. Additionally, some mitochondria exhibited disrupted outer membrane continuity and content leakage, resulting in pronounced vacuolation. A significant number of autophagosomes were observed, containing digestive organelles. These findings suggest that the mitochondria in osteoblasts have experienced severe damage, compromising their structural and functional integrity ([121]Fig. 3E). Based on the results, we predicted that impaired mitophagy is crucial in Pa-induced cell damage. WB detection showed a significant increase in the key mitophagy proteins, ATG4B and LC3B, in the Pa group, suggesting that mitophagy is a significant mediator in this context ([122]Fig. 3I and J). We treated the cells with the lysosomal inhibitor BafA1 and found that LC3B accumulated further in osteoblasts in a high-lipid environment. This suggests that the increased autophagy induced by Pa may not be due to impaired autophagosome clearance ([123]Fig. S2C and D). In summary, PA has been shown to elevate ROS levels, disrupt mitophagy homeostasis, interfere with normal cellular metabolism, and inhibit lipid metabolic processes. Conversely, Ex-4 has demonstrated the ability to ameliorate IR, reduce excessive ROS, mitigate mitochondrial damage, and facilitate the restoration of normal cellular metabolism. Currently, despite Ex-4's status as a first-line pharmacological agent for weight management, its use in isolation lacks specificity and is associated with an increased risk of gastrointestinal discomfort, as well as hepatic and renal impairment. Therefore, it is imperative to formulate strategies that facilitate the precise and safe targeting of bone tissue while concurrently reducing the occurrence of adverse side effects. 3.4. Construction and characterization of DPA@NM@CTZE We developed a novel nanoplatform for biosimulation, designated as DPA@NM@CTZE. The synthesis process is illustrated in Graphical abstract. To construct the nanoparticle shell, we combined NM and ALN. NM offers several advantageous properties, including high biocompatibility, targeted delivery, intelligent controlled release, and enhanced stability [[124]40]. These attributes facilitate stability in the bloodstream and enable the nanoparticles to evade immune detection [[125]41,[126]42]. ALN is a primary osteoporosis treatment known for its strong bone-targeting ability, as documented in numerous studies [[127]43,[128]44]. The alteration of NM by ALN can modulate the osteoblast-osteoclast equilibrium, facilitating a reduction in osteoclast activity and degradation of the bone tissue surface, thereby inhibiting bone resorption [[129]45]. According to previous research methods, the 2D CTZ structure was synthesized and subsequently combined with Ex-4 to form CTZE [[130]46]. Concurrently, DPA@NM was amalgamated with CTZE, resulting in the production of DPA@NM@CTZE via the extrusion method. The 2D CTZ structure was synthesized and combined with Ex-4 to create CTZE. At the same time, DPA@NM was amalgamated with CTZE, resulting in the formation of DPA@NM@CTZE through the extrusion method [[131]23,[132]47]. The analysis of SEM and TEM images reveals that the 2D CTZ nanosheets exhibit a characteristic irregular sheet-like morphology, aligning with the structural features commonly observed in porphyrin ligand-based MOF materials [[133]48]. The nanosheet exhibits a size of approximately 100 nm, suggesting that it possesses the characteristics typical of 2D crystals ([134]Fig. 4A, B and C). EDS analysis revealed that C and N originated from the TCPP organism, while the presence of Cu and Zn confirmed the incorporation of metal nodes and the formation of metal-ligand coordination bonds within the MOF ([135]Fig. 4D, [136]Fig. S3A). These findings substantiate that the 2D layered structure of the CTZ MOF possesses a substantial specific surface area and numerous coordination sites, thus showing considerable promise for use in drug delivery. Subsequently, XPS helped in determining the chemical states on the surface of different elements within the CTZ material. The analysis identified the presence of C 1s, N 1s, O 1s, Cu 2p, and Zn 2p. Furthermore, two distinct spin-orbit splitting peaks corresponding to Zn 2p[3/2] and Zn 2p[1/2] were detected. The position and intensity of these peaks suggest that Zn ions are uniformly incorporated into the porphyrin ring structure, thereby maintaining a stable chemical state ([137]Fig. 4E, [138]Fig. S3B). XRD pattern exhibits six distinct characteristic diffraction peaks at 5.8°, 7.5°, 12.5°, 19.8°, 22.5°, and 30.9°. This diffraction pattern aligns with the typical structure of a porphyrin-based MOF, suggesting that CTZ forms a 3D network structure characterized by regular porosity ([139]Fig. 4F) [[140]46]; Pronounced diffraction peaks at low angles (5.8° and 7.5°) indicate the presence of large aperture structures, while peaks at 30.9° are indicative of smaller atomic arrangement characteristics. The occurrence of these specific crystal faces corroborates the bimetallic cooperative structure, with Cu serving as the primary metal center and Zn as a secondary metal within the porphyrin ring. Fig. 4. [141]Fig. 4 [142]Open in a new tab The characterization of DPA@NM@CTZE. (A) Scanning electron microscopy (SEM) image of CTZ. Scale bar: 100 μm. (B-C) Transmission electron microscopy (TEM) analysis of CTZ and NM@CTZE. Scale bar: 50 nm, 100 nm, 50 nm. (D) Energy-dispersive X-ray spectroscopy (EDS) analysis of CTZ. Scale bar: 2.5 μm. (E) X-ray photoelectron spectroscopy (XPS) employed to investigate the surface chemical states of various elements in CTZ. (F) X-ray diffraction (XRD) utilized to examine the characteristic diffraction peaks of CTZ. (G) Fourier Transform Infrared Spectroscopy (FTIR) was employed to examine the molecular structure of DPA. (H) The effectiveness of NM@CTZE synthesis was evaluated through Zeta potential analysis. (I) The trend in Zeta potential variation following CTZ loading in Ex-4 was investigated. (J) The expression levels of key membrane proteins, including Integrin β1, Integrin β2, and CXCR2, on the surface of NM were analyzed using WB. (K) The ROS responsiveness of DPA@NM@CTZE was assessed. Data are presented as Mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3. FTIR spectroscopy provides critical insights into the molecular structure of DPA. The spectral region spanning 2850–2920 cm^−1 is indicative of different stretching vibrations of the CH[2] groups within the phospholipid's double fatty acid chains, as well as the stretching vibrations of the carbonyl (C=O) groups, which manifest as pronounced peaks near 1740 cm^−1. Additionally, the expanded spectral region highlights the O–H stretching vibration at approximately 3300 cm^−1. Further analysis confirmed the successful binding of the phosphonic acid group of ALN to the terminal DPA, substantiating the molecular interaction ([143]Fig. 4G). Upon coating the NM, DLS analysis indicated an increase in the particle size of the CTZ nanosheet from 125.7 nm to 145.7 nm ([144]Fig. 4H). The Zeta potential analysis revealed a distinctive pattern of interaction between CTZ and Ex-4, with CTZ exhibiting a pronounced negative potential of −14.5 mV. Upon adsorption of Ex-4 onto the CTZ surface to form the CTZE complex, the potential value increased to −6.4 mV, indicating a significant reduction in the degree of negative charge ([145]Fig. 4I, [146]Fig. S3C). UV–Vis analysis revealed that varying concentrations of Ex-4 exhibited distinct absorption peaks at 280 nm, demonstrating a positive correlation with Ex-4 concentration ([147]Fig. S3E). Similarly, CTZE displayed comparable absorption peaks at 280 nm, confirming the successful encapsulation of Ex-4 ([148]Fig. 4J). Furthermore, BCA analysis revealed a strong linear correlation between Ex-4 concentration and absorbance, indicating a drug loading efficiency of approximately 19.1 % ([149]Fig. S3F). We propose that the efficient binding of CTZE can be attributed to: 1) the positive charge region of Ex-4 generating effective electrostatic attraction with the CTZ surface; and 2) the aromatic porphyrin ring system of CTZ facilitating π-π stacking interactions with the aromatic amino acid residues in Ex-4. This non-covalent interaction synergistically complements electrostatic adsorption, thereby enhancing the binding stability between the two entities. The BCA curve showed a strong linear correlation (R^2 = 0.9975) between NM (specify what NM stands for) concentration and bovine serum albumin at 562 nm absorbance, confirming the successful extraction and enrichment of NM ([150]Fig. S3D). CXCR2, Integrin β1, and Integrin β2 are specific proteins localized on the surface membranes of neutrophil cells. The successful expression of these surface receptors and adhesion molecules in such cells serves as an indicator of the efficacy of the neutrophil cell membrane extraction technique. These proteins enhance the targeted delivery of coated nanoparticles to osteoblasts with elevated ROS levels, enabling precise drug release, minimizing systemic drug exposure, and maximizing the homing effect of the nanoparticles [[151]28,[152]49]. Integrin β1, Integrin β2 and CXCR2, were identified through WB analysis, with results indicating a significant concentration of these proteins in the NM group. This finding establishes a solid experimental basis for validating the efficacy of NM in future research endeavors ([153]Fig. 4K). The aforementioned results demonstrate that Ex-4 effectively enhances osteogenic differentiation and mitigates ROS. Clinically, Ex-4 is administered via subcutaneous injection; however, it lacks specific tissue targeting post-absorption, leading to localized concentrations. Excessive accumulation in the kidneys and liver can result in hepatorenal toxicity. Given the controlled release and superior ROS-responsive properties of NM, we evaluated the efficacy of DPA@NM@CTZE in an osteoblast environment, induced by PA with elevated ROS levels, through in vitro drug release assays. HPLC analysis revealed minimal release of Ex-4 in a normal PBS solution. However, in PBS solutions with H[2]O[2] concentrations of 1 mM and 10 mM, Ex-4 release increased in a concentration-dependent manner, with 23.6 % and 43.8 % of Ex-4 released at 24 h, respectively ([154]Fig. 4L). The detection results demonstrate that DPA@NM@CTZE exhibits exceptional responsiveness to ROS, thereby preventing drug leakage in normal tissue environments. Furthermore, it facilitates personalized and precise drug release tailored to the varying cellular environments associated with the elevated ROS levels linked to obesity-induced osteoporosis. 3.5. DPA@NM@CTZE enhances osteogenic differentiation by suppressing the PINK1/Parkin pathway through ROS reduction DPA@NM enhances the targeted delivery of CTZE to bone tissue exhibiting elevated levels of oxidative stress. In response to ROS stimulation, the material undergoes decomposition, resulting in the release of Cu^2+ and Zn^2+. Due to the antioxidant properties of Cu^2+ and its involvement in the formation of coenzymes essential for sustaining normal cellular metabolism, a deficiency in this ion has been identified as a potential contributing factor to the development of osteoporosis [[155]50]. Zn^2+ exhibit notable anti-inflammatory properties and contribute to the reduction of ROS. Furthermore, as a vital element for bone development, Zn^2+ is vital in facilitating the mineralization and differentiation of osteoblasts [[156]51,[157]52]. To investigate the scavenging effect of DPA@NM@CTZE on ROS, ROS levels were assessed as follows: In comparison to the Pa group, DPA@NM@CTZ demonstrated a significant ROS clearance capability. Notably, the ROS levels in the DPA@NM@CTZE group were comparable to those in the Control group ([158]Fig. 5A and F). These findings indicate that DPA@NM@CTZE effectively mitigates ROS and protects cells from elevated oxidative stress. Additionally, mitophagy analysis revealed that treatment with DPA@NM@CTZE resulted in more elongated and evenly distributed mitochondria, with clearly defined cell boundaries ([159]Fig. 5B). Fig. 5. [160]Fig. 5 [161]Open in a new tab DPA@NM@CTZE reduces excessive mitophagy, enhancing osteoblast function under high lipid conditions. (A) and (F) show changes in intracellular ROS levels post-treatment with various materials. Scale bar: 100 μm. (B) Displays mitochondrial morphological changes in treated osteoblasts. Scale bar: 20 μm. (C) and (G) JC-1 detection of the improvement in ΔΨm by DPA@NM@CTZE. Scale bar: 30 μm. (D) and (H) The effects of different materials on osteoblasts in a Pa environment were analyzed by assessing the degree of co-localization of mitochondria and lysosomes. Scale bar: 20 μm. (E) The effects of different material components on mitochondria and autophagosomes were observed at the microscopic level using TEM. Scale bar: 2 μm. (I-J) WB was used to detect the changes in PINK1/Parkin autophagy pathway, the autophagy-related proteins and osteoblast proteins. Data are presented as Mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3. Both DPA@NM@CTZ and DPA@NM@CTZE were shown to enhance the formation of mineralized nodules and the secretion of ALP in osteoblasts under Pa conditions. Notably, the latter formulation demonstrated superior efficacy compared to a simple mixture of Ex-4 and DPA@NM@CTZ, indicating an improved therapeutic effect ([162]Fig. S4A and B). The ΔΨm results indicate that DPA@NM@CTZE effectively preserved ΔΨm potential energy after reducing excess ROS ([163]Fig. 5C and G). Mitochondrial and lysosome colocalization results were similar, with DPA@NM@CTZE showing a smaller colocalization area than the Ex-4 + DPA@NM@CTZ group, preventing mitophagy dysfunction ([164]Fig. 5D and H). TEM analysis revealed a substantial production of autophagosomes in the Pa group, which encapsulated mitochondria undergoing digestion. In contrast, the DPA@NM@CTZE group exhibited a reduced presence of lysosomes, alongside a uniform distribution of mitochondria, well-defined outlines, and distinct mitochondrial cristae structures. These findings indicate that the application of nanoparticles facilitated the restoration of mitochondria to a healthy state ([165]Fig. 5E). PINK1/Parkin signaling pathway represents a well-established mechanism of mitophagy, playing a critical role in the removal of damaged mitochondria and the maintenance of mitochondrial homeostasis [[166]53]. The results from WB analyses indicated that mitophagy in osteoblasts was significantly upregulated following Pa treatment, as evidenced by the marked increase in the levels of PINK1 and Parkin. The pathophysiology of osteoblasts in this context differs from that observed in diabetic osteoporosis, primarily due to the induction of mitophagy in a high-fat environment. Excessive mitophagy has been shown to impair the differentiation and mineralization capabilities of osteoblasts, diminish ATP production, and elevate ATG4B and LC3B. Notably, this detrimental condition was ameliorated by the administration of DPA@NM@CTZ and DPA@NM@CTZE ([167]Fig. 5I and J). Based on these findings, we propose that the PINK1/Parkin signaling pathway plays a critical role in the pathology of osteoblasts subjected to pa-induced damage. To investigate whether Pa mediates excessive mitophagy injury via the PINK1/Parkin pathway, consequently impairing osteoblast function, we transfected MC3T3-E1 cells with LV-shPink1 to suppress Pink1 expression. The efficiency of transfection was confirmed through inverted microscopy, RT-qPCR, and WB analysis ([168]Figs. S5A, B, C and D). Additionally, ROS production was assessed, revealing a significant reduction in ROS levels following transfection with LV-shPink1 ([169]Fig. 6A and F). To assess the potential detrimental effects of inhibiting Pink1 expression on osteoblast mineralization and differentiation, we conducted ARS and ALP assays. The findings indicated that transfection with LV-shPink1 did not adversely affect osteoblasts. Moreover, it synergistically interacted with DPA@NM@CTZE to enhance the mineralization and differentiation capacities of osteoblasts impaired by Pa exposure ([170]Fig. S5E and F). Mitochondrial morphology staining corroborated these results, demonstrating that inhibition of PINK1 protein led to a reduction in mitochondrial fragmentation, with clearer boundaries and elongated shapes ([171]Fig. 6B). In summary, LV-shPink1 does not exert toxic effects on mitochondrial morphology and significantly enhances osteoblast function. Fig. 6. [172]Fig. 6 [173]Open in a new tab DPA@NM@CTZE enhances osteoblast function by inhibiting the PINK1/Parkin signaling pathway. (A) and (F) Show the impact of LV-shPink1 transfection on ROS levels in osteoblast. scale bar: 100 μm. (B) Illustrates the effects of LV-shPink1 and DPA@NM@CTZE on mitochondrial morphology. scale bar: 20 μm. (C) and (G) Indicate that inhibiting the PINK1/Parkin pathway increases ΔΨm scale bar: 30 μm. (D) and (H) To prevent self-fluorescence post-LV-shPink1 transfection, mitochondria, lysosomes, and the nucleus were labeled with COX IV (red), LAMP1 (yellow), and DAPI (blue), respectively, to observe mitochondria-lysosome colocalization. Scale bar: 20 μm. (E) To examine mitochondrial and autophagosome changes post-transfection. Scale indicator: 2 μm. (I-J) Impact of LV-shPink1 transfection on mitophagy and osteogenic proteins. Data as Mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3. ΔΨm are essential for PINK1 transport. Following intervention with JC-1, it was observed that the reduction in ΔΨm induced by Pa was restored through LV-shPink1 intervention. Furthermore, LV-shPink1 demonstrated a synergistic effect with DPA@NM@CTZE, enhancing the amelioration effect ([174]Fig. 6C and G). Subsequent experiments involving mitochondrial and lysosome co-localization revealed that LV-shPink1 contributed to a reduction in the co-localization area, the number of lysosomes, and the fluorescence intensity of LAMP1. These findings suggest that Pa-induced excessive mitophagy was effectively corrected ([175]Fig. 6D and H). TEM analysis demonstrated that a reduction in PINK1 protein expression led to the normalization of mitochondrial structure, a decrease in the number of autophagosomes, and a uniform distribution of mitochondria ([176]Fig. 6E). Furthermore, transfection with LV-shPink1 did not adversely affect MC3T3-E1 cells. When used in conjunction with DPA@NM@CTZE, it enhanced the resilience of osteoblasts in a high-lipid environment. WB was employed to elucidate the critical role of the PINK1/Parkin signaling pathway. Inhibition of PINK1 protein expression resulted in a reduction of Parkin activation, a decrease in the expression levels of mitophagy-related proteins ATG4B and LC3B, and an upregulation of osteogenic proteins COL1A1 and RUNX2 ([177]Fig. 6I and J). The findings suggest that DPA@NM@CTZE is highly responsive to ROS. It slows Ex-4 release and generates Cu^2+ and Zn^2+, which inhibit the PINK1/Parkin pathway by reducing ROS levels, thereby shielding osteoblasts from environmental damage. 3.6. DPA@NM@CTZE modulates the PINK1/Parkin pathway by influencing TET2, which boosts BCL2 expression and decreases autophagy vesicle formation Based on preliminary proteomic findings, the process of bone differentiation is inhibited, with excessive mitophagy following Pa treatment identified as the primary inducing factor. Research has demonstrated that TET2 can induce mitophagy in osteoclasts triggered by ovarian resection, thereby promoting osteoclast differentiation and exacerbating osteoporosis [[178]54]. TET2 is a critical demethylase that significantly influences the regulation of cell differentiation and gene expression. BCL2 serves as a pivotal anti-apoptotic factor, modulating the mitochondrial clearance process. The interaction between BCL2 and BECN1 attenuates autophagosome production, thereby preserving mitophagy homeostasis. We proposed that DPA@NM@CTZE may influence the TET2/PINK1/Parkin signaling pathway, thereby enhancing BCL2 binding to BECN1 and mitigating excessive mitophagy. To investigate the potential regulatory roles of TET2 and BCL2 in osteoblast differentiation within a model of obesity-induced osteoporosis in mice, we initially performed WB analyses. The findings indicated that in the Pa group, there was an upregulation of TET2 and BECN1 expression, whereas BCL2 expression was downregulated. Treatment with Ex-4 or DPA@NM@CTZE resulted in a reduction of TET2 expression and an increase in BCL2 expression ([179]Fig. 7A and B). Fig. 7. [180]Fig. 7 [181]Open in a new tab TET2 enhances mitophagy in DPA@NM@CTZE. (A-B) WB analysis assessed changes in TET2 and autophagosome proteins BCL2 and BECN1, with data shown as Mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3. We explored TET2's role in osteoblast differentiation under high lipid conditions and BCL2's impact on mitophagy. LV-shTet2 was used to suppress Tet2 in osteoblasts, and si-Bcl2 was introduced into LV-shTet2 knocked-down MC3T3-E1 cells. Transfection efficiency was confirmed using an inverted fluorescence microscope, RT-qPCR, and WB ([182]Figs. S6A, B, C, D, E, F and G). Osteogenic ARS and ALP staining were conducted to assess the impact of LV-shTet2 and LV-shTet2 + si-Bcl2 transfection on osteoblasts. The findings indicated no significant damage from LV-NC, LV-shTet2, or LV-shTet2 + si-Bcl2 transfection. DPA@NM@CTZE + LV-shTet2 notably enhanced bone mineralization and differentiation ([183]Fig. S6H and I). Additionally, LV-shTet2 transfection significantly reduced ROS expression in the Pa environment to levels similar to the Control group, but ROS levels increased after Bcl2 knockdown ([184]Fig. 8A, [185]Fig. S6J). The morphology of mitochondria improved following the transfection of either LV-shTet2 or LV-shTet2 + si-Bcl2 into MC3T3-E1 cells. A similar trend was observed for ΔΨm; however, this trend was halted upon the reduction of Bcl2 expression ([186]Fig. 8B). We hypothesize that this may be due to the increased production of autophagosomes, which disrupts the normal mitochondrial structure, leading to damage in the mitochondrial matrix and an inability to maintain normal ΔΨm ([187]Fig. 8C, [188]Fig. S6K). Results from mitochondrial and lysosome colocalization studies indicated that the use of LV-shTet2 and DPA@NM@CTZE significantly reduced both the colocalization area and the production and loss of lysosomes ([189]Fig. 8D, [190]Fig. S6L). Fig. 8. [191]Fig. 8 [192]Open in a new tab TET2 plays a crucial role in DPA@NM@CTZE's inhibition of autophagosome formation. (A) Assess the impact of reduced Tet2 and Bcl2 expression with DPA@NM@CTZE on intracellular ROS levels. Scale bar: 100 μm. (B) Examine how LV-shTet2 and si-Bcl2 transfection affects mitochondrial morphology Scale bar: 20 μm. (C) Evaluate the influence of transfected cells, with or without LV-shTet2 and si-Bcl2, on osteoblasts' cell ΔΨm. Scale bar: 30 μm. (D) Colocalization images show mitochondria (red) and lysosomes (yellow) in LV-shTet2 and si-Bcl2 transfected cells. Scale: 20 μm. (E) TEM analysis reveals the impact of reduced Tet2 and Bcl2 on mitochondria and autophagosomes in osteoblasts. Scale: 2 μm. (F–G) WB assess the effects of decreased Tet2 and Bcl2 on mitophagy and osteoblast protein expression. Data: Mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3. TEM analysis revealed that mitochondria in the Pa + DPA@NM@CTZE + LV-shTet2 group exhibited a uniform distribution, with well-defined mitochondrial structures and prominent ridges and folds ([193]Fig. 8E). Subsequent WB validation demonstrated that a reduction in TET2 expression was associated with decreased levels of PINK1 and Parkin, as well as a reduction in autophagy-related proteins ATG4B and LC3B. Conversely, there was an observed increase in the expression of osteogenic proteins Col1A1 and RUNX2. The combined effect of reduced TET2 expression and DPA@NM@CTZE treatments resulted in increased BCL2 expression. However, the beneficial effects of the nanoparticles were diminished when BCL2 was knocked down ([194]Fig. 8F and G). We think that this is attributable to the absence of BCL2-mediated inhibition of autophagosome production. In conclusion, TET2 serves as a crucial regulatory protein in the modulation of Pa-induced excessive mitophagy. The compound DPA@NM@CTZE has been shown to inhibit the TET2/PINK1/Parkin signaling pathway, thereby reducing the formation of BCL2-BECN1 dependent autophagosomes and mitigating excessive mitophagy. Consequently, this leads to enhanced mineralization and differentiation capabilities of osteoblasts. These findings elucidate the biological mechanisms underlying the enhanced effects of Ex-4 and highlight the potential of DPA@NM@CTZE as an innovative multifunctional nanoplatform that not only increases efficacy but also enhances the safety profile of Ex-4. 3.7. DPA@NM@CTZE can lower TET2 levels and enhance osteoporosis caused by obesity We created an obesity-induced osteoporosis mouse model. We injected 25 μl of LV-shTet2 and LV-NC into the bone marrow of the mice's distal femoral epiphysis every two weeks ([195]Fig. 9A). Fig. 9. [196]Fig. 9 [197]Open in a new tab DPA@NM@CTZE can enhance metabolic function and facilitate the repair of osteoporosis induced by obesity. (A) It illustrates a flowchart detailing the experimental approach for addressing obesity-induced osteoporosis. (B) and (E) It provides representative micro-CT images following caudal intravenous administration of DPA@NM@CTZE. The following indices were evaluated: BMD, BV/TV, BS/BV, Tb.N, Tb.Th, and Tb.Sp. Scale bar: 1 mm. (C-D) It depicts histomorphological changes in the distal femur bone of mice, analyzed via H&E and Masson staining, post-treatment with DPA@NM@CTZE and LV-shTet2. Scale bar: 400 μm. (F-G) Quantitative analysis of H&E and Masson staining. Data are presented as Mean ± SD. Statistical significance is indicated as follows: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 5. The drug was administered through the tail vein, and after a ten weeks treatment period, the group receiving transfection with HFD + DPA@NM@CTZE in conjunction with LV-shTet2 exhibited a significant reduction in body weight, enhanced lipid and glucose metabolism indices, and a mitigation of IR compared to the HFD group ([198]Fig. S7A, B and C). Subsequently, we conducted a re-evaluation of PⅠNP and CTX-Ⅰ levels, discovering that the combination of DPA@NM@CTZE with LV-shTet2 resulted in a significant increase in PⅠNP levels and a decrease in CTX-Ⅰ levels in HFD mice. Notably, the primary effect of this intervention appears to be enhanced osteogenesis ([199]Fig. S7D and E). Micro-CT analysis indicated significant bone mass loss in the HFD group. Treatment with Ex-4 or DPA@NM@CTZE alone improved bone mass and trabecular continuity, but combining DPA@NM@CTZE and LV-shTet2 achieved better results. Quantitative analysis of bone parameters demonstrated that the DPA@NM@CTZE and LV-shTet2 treatments significantly enhanced BMD (0.54 mg/cm^3), BV/TV (16.9 %), and BS/BV (58 %), as well as Tb. N (4.8 mm^−1) and Tb. Th (74 mm^−1). Additionally, these treatments reduced Tb. Sp (218 μm) and decreased fracture risk ([200]Fig. 9B and E). We investigated the impact of DPA@NM@CTZE and LV-shTet2 on mouse organs using H&E staining on liver, kidney, spleen, and heart tissues. The results showed no significant organ damage or accumulation of the treatment ([201]Fig. S7F). PBS and DPA@NM@CTZE were given to mice with obesity-induced osteoporosis. Imaging showed that the nanoparticles mainly gathered in the osteoporotic areas of the long bones, with low fluorescence in the liver and little accumulation in other organs ([202]Fig. S7G). The study shows that DPA@NM@CTZE is efficiently eliminated from mouse organs. The addition of NM and ALN improved bone targeting and ROS responsiveness, reducing potential harm from accumulation. Subsequently, the results from three-point bending tests indicated that, in addition to the enhancement effects of Ex-4, DPA@NM@CTZ also contributed to the improvement of bone mechanical function in these mice. Furthermore, the knockdown of Tet2 expression led to an additional enhancement in both the bending strength and load-bearing capacity of the bone tissue ([203]Fig. S7H). H&E and Masson staining techniques were employed to further investigate the bone microstructure. The findings indicated that DPA@NM@CTZE significantly enhances trabecular bone continuity. Moreover, the administration of LV-shTet2 in conjunction with DPA@NM@CTZE further ameliorates trabecular damage and decreases trabecular separation ([204]Fig. 9C and D). IHC analysis revealed a significant upregulation of LC3B, in the Pa group, while the expression levels of osteogenesis-associated proteins OCN and RUNX2 decreased. These alterations were reversed by treatments with Ex-4, DPA@NM@CTZ, and DPA@NM@CTZE. Notably, the DPA@NM@CTZE + LV-shTet2 group exhibited the most pronounced improvement ([205]Fig. 10A and B). Calcein labeling of mineralization experiments corroborated this trend of improvement ([206]Fig. 10C and D). Fig. 10. [207]Fig. 10 [208]Open in a new tab The impact of Tet2 knockdown on DPA@NM@CTZE expression. (A-B) It shows IHC images of LC3B, OCN, and RUNX2. Scale bar: 400 μm. (C-D) It displays calcein double-standard images and analysis. Scale bar: 100 μm. Results are expressed as Mean ± SD, with significance denoted by ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, n = 5. In the HDF + CTZ group, significant changes in morphological indicators such as BMD and TB. N were observed. Both Cu^2+ and Zn^2+ effectively enhanced osteogenesis and boosted osteoblast activity and mineralization, consistent with previous studies [[209]55]. The combination of Ex-4 and ALN significantly increased the osteogenic proteins RUNX2 and OCN and improved PⅠNP in mice with obesity-induced osteoporosis. Notably, unlike the first animal experiment, the osteoclast activity marker CTX-I significantly decreased, likely due to the effects of ALN, Zn^2+, and Cu^2+. ALN is known to inhibit osteoclast activity, Cu^2+ can improve dental osteoporosis, and Zn^2+ reduces ROS and enhances the bone microenvironment [[210]56,[211]57].Thus, the DPA@NM@CTZE material optimally utilizes each component to combat obesity-induced osteoporosis. 4. Conclusion In conclusion, this study created the DPA@NM@CTZE biosimulation nanoplatform to address obesity-related osteoporosis, identifying that the condition primarily results from lipid metabolism disorders and overactive mitochondrial autophagy in osteoblasts. DPA@NM@CTZE can mitigate high ROS environments by releasing ALN, Cu^2+, Zn^2+, and Ex-4. Additionally, inhibiting the TET2/PINK1/Parkin pathway enhances BCL2-BECN1 binding, countering the effects of high-fat environments. This pioneering research explored the biological mechanism of obesity-induced osteoporosis and successfully developed a safe and precise biological simulation nanoplatform for treatment. It has significant clinical potential in transforming the formulation of Ex-4 drugs and providing more effective treatment options for patients with obesity-induced osteoporosis. CRediT authorship contribution statement Qifan Yang: Writing – original draft, Methodology, Data curation, Conceptualization. Jing Liu: Methodology, Investigation, Conceptualization. Yanwei Liu: Data curation. Shun Liu: Data curation. Xiaokang Wei: Software, Methodology. Yilin Yang: Validation, Methodology. Weijie Zhang: Validation, Methodology. Shuqi Zhang: Validation, Methodology. Maosheng Zhang: Validation, Methodology. Bin Liu: Writing – review & editing. Xinyu Wang: Writing – review & editing, Supervision. Dong Zhu: Writing – review & editing, Writing – original draft, Validation, Supervision, Funding acquisition, Conceptualization. Ethics approval and consent to participate The Animal Ethics Committee of the First Hospital of Jilin University approved all animal experiments in this project (Issue No. 20230908 (0655)). Consent for publication All authors consented to publish the article. Funding This work was supported by the National Key R&D Program of China (2022YFC2405805), the National Natural Science Foundation of China (Grant No. 12072129), and the Doctor of Excellence Program (DEP) at The First Hospital of Jilin University (JDYY-DEP-2024048). Declaration of competing interest The authors report no conflicts of interest. Acknowledgements