Abstract An ultrasound-responsive silk fibroin (SF)-graphene oxide (GO)-based hydrogel (SF/GO-gel) was created in this study to facilitate the sustained delivery of troxerutin (Trox) in order to alleviate intervertebral disc degeneration (IDD). The SF/GO-gel@Trox system exhibited exceptional biocompatibility, mechanical robustness, and controlled drug release. In vivo, X-ray and MRI demonstrated that SF/GO-gel@Trox substantially preserved disc height index and hydration in comparison to IDD. Histology also confirmed the preservation of extracellular matrix expression. In a mechanistic manner, SF/GO-gel@Trox activated tyrosine-protein kinase receptor (Tie2), which subsequently promoted Casitas B-lineage Lymphoma (CBL)-mediated K48-linked ubiquitination and degradation of epidermal growth factor receptor (EGFR). Consequently, NF-κB-driven inflammation and senescence were suppressed. The protective effects of SF/GO-gel@Trox were demonstrated by transcriptomics and functional assays to be underpinned by Tie2/PI3K-Akt signaling, whereas degeneration was exacerbated by Tie2 knockdown. It is important to note that SF/GO-gel@Trox stabilized phosphorylated Tie2 (Y992), which in turn improved the interaction between CBL and EGFR, thereby accelerating the turnover of EGFR. In a rat IDD model, Tie2 overexpression was able to mitigate disc structural damage through hydrogel delivery, whereas concurrent EGFR expression reversed these benefits. The SF/GO-gel platform facilitated the localized, ultrasound-triggered release of Trox, providing a novel approach to IDD therapy that targets the Tie2/EGFR axis. These results emphasize the potential of SF/GO-gel@Trox as a multifunctional system that can effectively combat IDD by utilizing coordinated anti-inflammatory, pro-anabolic, and anti-catabolic mechanisms. Keywords: Intervertebral disc degeneration, Ultrasound-responsive drug delivery system, Graphene oxide, Silk fibroin, Hydrogel, Troxerutin Graphical abstract A: Synthesis of composite hydrogel. B: Schematic diagram of animal models and molecular mechanism diagram of this study. C: The effects of composite hydrogel. Image 1 [37]Open in a new tab 1. Introduction Intervertebral disc degeneration (IDD) is a common degenerative spinal disorder characterized by the structural degradation and functional decline of the intervertebral disc (IVD). It is a primary contributor to chronic low back pain and radiculopathy [[38]1]. The pathophysiology of IDD involves a multitude of mechanisms, such as age-associated decreases in IVD hydration and proteoglycan content, persistent mechanical stress, genetic predisposition, inflammatory processes, oxidative stress, and impaired nutrient supply to the IVD [[39][2], [40][3], [41][4], [42][5], [43][6]]. As the illness advances, individuals may endure chronic pain, restricted movement, and potential disability, markedly diminishing their quality of life [[44]7,[45]8]. Furthermore, IDD impose a substantial socioeconomic burden, characterized by increased healthcare costs, reduced labor productivity, and diminished work capacity, thereby constituting a critical global public health challenge [[46]9]. Consequently, clarifying its fundamental causes and enhancing preventative and treatment options are of considerable clinical and societal significance. Troxerutin (Trox), a trihydroxyethylated derivative of rutin derived from rutaceae plants, exhibits considerable therapeutic potential in the prevention and treatment of various disorders due to its multifaceted pharmacological actions that target multiple pathways [[47]10,[48]11]. This compound primarily exerts its free radical scavenging effects through modulation of the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) antioxidant pathway, while concurrently inhibiting the activation of inflammatory signaling pathways, such as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Consequently, it exhibits notable antioxidative and anti-inflammatory properties [[49][12], [50][13], [51][14], [52][15]]. Trox markedly diminishes vascular permeability and improves venous tone in the vascular system, hence reducing symptoms of chronic venous insufficiency and varicose veins, such as lower limb edema and pain [[53]16]. In the context of metabolic disorders, it mitigates oxidative stress and suppresses inflammatory responses, thereby demonstrating protective effects against diabetes and its associated complications, such as nephropathy, retinopathy, and neuropathy [[54]17,[55]18]. Trox not only mitigates blood-brain barrier disruption and slows neuronal death in neuroprotection but also demonstrates therapeutic potential in cerebral ischemia-reperfusion injury and neurodegenerative disorders, including Alzheimer's disease [[56]19,[57]20]. Recent research indicates that Trox can modulate the expression of proteins involved in autophagy and apoptosis, thereby exerting protective effects in liver fibrosis and specific tumor models [[58]15,[59]21]. Owing to its diverse pharmacological properties, favorable safety profile, and systemic protective effects, Trox has emerged as a crucial medication in vascular protection, antioxidant therapy, and the management of chronic diseases, offering a novel therapeutic alternative for age-related conditions. Graphene oxide (GO), a significant derivative of graphene, features a distinctive two-dimensional nanosheet architecture with several surface functional groups (e.g., hydroxyl, carboxyl, and epoxy groups) [[60]22]. These characteristics confer upon GO remarkable hydrophilicity, an elevated specific surface area, and superior biocompatibility, rendering it particularly advantageous for biomedical applications [[61]23]. The π-conjugated architecture and surface functional groups of GO provide optimal binding sites for drug loading, thereby enhancing drug-loading capacity through physical adsorption and enabling chemical modification for functionalization [[62]24]. The integration of GO into hydrogel systems markedly improves their overall efficacy. GO nanosheets establish many connections with hydrogel networks, enhancing mechanical strength and extending sustained drug release [[63]25,[64]26]. An optimal GO content can improve the compressive properties of hydrogels to levels akin to those of native nucleus pulposus (NP) tissue, while also markedly extending the duration of drug release Moreover, GO exhibits free radical-scavenging capabilities, thereby mitigating oxidative stress at implantation sites and promoting a favorable microenvironment for tissue regeneration [[65]27]. GO exhibits remarkable adsorption capacity for aromatic small-molecule pharmaceuticals, including the anticancer medicine doxorubicin, much exceeding that of traditional nanocarriers [[66]28,[67]29]. Ultrasound, defined by sound waves with frequencies beyond the range of human hearing, induces numerous physical and chemical effects as it propagates through biological tissues Ultrasound-responsive drug delivery systems represent an advanced therapeutic approach that leverages the physical properties of ultrasound to achieve spatiotemporally controlled drug release, thereby ensuring precise and effective treatment outcomes [[68]30,[69]31]. These systems typically consist of two primary components: (1) drug carriers, such as nanoparticles, liposomes, or microspheres, which are designed to encapsulate and transport therapeutic agents to specific target sites, and (2) external ultrasound devices that are calibrated to emit precise frequencies and intensities, thereby facilitating the controlled release of the drug at the lesion site. The primary benefits encompass improved local drug concentration, less off-target toxicity, and lowered systemic adverse effects [[70]32]. This study presents the development of an ultrasound-responsive silk fibroin (SF)/GO hydrogel system designed for sustained drug release, capitalizing on the synergistic benefits of GO and ultrasound-mediated drug delivery. The methodology integrates GO within traditional hydrogel matrices, preserving the hydrogel's intrinsic three-dimensional porous structure and biocompatibility, while augmenting drug-loading capacity and enabling controlled release kinetics. This novel composite drug delivery system is projected to provide an effective localized therapeutic strategy for IDD, promoting targeted drug delivery and prolonged release. Additionally, we examined Trox-mediated targeting of tyrosine-protein kinase receptor (Tie2) to modulate NP cell proliferation and apoptosis both in vitro and in vivo, and assessed the therapeutic efficacy of SF/GO-gel@Trox in the repair of IVD in a rat IDD model. 2. Methodology and materials 2.1. Construction of composite hydrogel 2.1.1. Materials SF was extracted in-house from domesticated silkworm cocoons with a concentration of 6 wt% GO power (purity ≥98 %) was purchased from Suzhou Carbon Feng Graphene Technology Co., Ltd., China. Type XIV from Streptomyces griseus was purchased from Sigma-Aldrich Chemical Co., Ltd., China. Deionized water was prepared in-house for laboratory use. All chemicals were used as received without further purification. 2.1.2. Preparation of SF protein solution The cocoons were initially subjected to a degumming process, which involved segmenting them into smaller pieces and subsequently boiling them in a 0.02 M sodium carbonate (Na[2]CO[3]) solution for a duration of 30 min. This was followed by multiple rinses with deionized water until a neutral pH was achieved. The degummed silk fibers were then dissolved in a 9.3 M lithium bromide (LiBr) solution at a temperature of 60 °C for 1 h to ensure complete dissolution. The solution was transferred to dialysis tubing (MWCO 12,000) and dialyzed against deionized water at 4 °C for 3 days, with water changes every 6 h. Following dialysis, the solution underwent centrifugation to eliminate impurities. Subsequently, the SF solution was concentrated using dialysis membranes with a molecular weight cut-off (MWCO) of 3500 in a 20 % polyethylene glycol (PEG) solution, resulting in a high-concentration SF solution (6 wt%) for subsequent applications.2.1.3 Preparation of GO dispersion. An appropriate amount of GO powder was dissolved in deionized water to prepare dispersions with concentrations of 6 mg/mL and 3 mg/mL. The dispersions were sonicated using an ultrasonic cell disruptor at 300 W for 30 min to ensure complete dispersion. 2.1.3. Fabrication of SF/GO-gel 5 mL of SF solution was mixed with 5 mL of GO dispersion at appropriate concentrations to obtain mixed solutions with SF:GO ratios of 20:1 and 10:1. SF/GO-5 %-gel and SF/GO-10 %-gel were prepared by ultrasonic treatment of SF solution using a cell pulverizer. Ultrasound was administered over four cycles at an amplitude of 15 %, with each cycle comprising 15 s of sonication followed by a 5-s pause. Subsequently, the mixture was promptly transferred to an oven set at 37 °C to facilitate gelation. The SF/GO-gel loaded with Trox (denoted as SF/GO-gel@Trox) was synthesized based on the optimal ratio of SF/GO-gel. Add 50 mg of Trox to the GO dispersion and stir magnetically at 4 °C for 30 min to ensure uniformity. Then add 5 mL of SF solution and mix evenly. Treated with the same cell pulverizer process. 2.2. Characterization of mixed solution and SF/GO-gels Rheological measurements of the mixed solution were conducted utilizing a rheometer (HAAKE MARS60, Thermo Fisher Scientific, USA) at a controlled temperature of 37 °C, employing a parallel plate configuration with a diameter of 20 mm. The storage modulus (G′) and loss modulus (G″) were assessed across an angular frequency spectrum ranging from 0.1 to 100 rad/s. In a comparable manner, the hydrogel was subjected to characterization under identical conditions to determine G′ and G″ as functions of angular frequency.2.3 Characterization of SF/GO-gels. 2.2.1. Morphological analysis The hydrogel samples were frozen at −20 °C overnight and then freeze-dried for two days. The samples were mounted on aluminum stubs with conductive adhesive and sputter-coated with Au. The microstructure of the scaffold was observed using a field-emission scanning electron microscope (SEM; Sigma 300, Zeiss, Germany). 2.2.2. Compositional analysis The Fourier-transform infrared (FT-IR) spectra of the scaffold were acquired in attenuated total reflectance (ATR) mode utilizing a Nicolet iS20 spectrometer (Thermo Scientific, USA), covering a spectral range from 4000 to 400 cm^−1. X-ray diffraction (XRD) analysis was conducted using a SmartLab diffractometer (Rigaku, Japan) with Cu Kα radiation (λ = 1.54 Å) at an operational voltage of 40 kV and a current of 30 mA, with the 2θ angle spanning from 3° to 50°. 2.3.3 Swelling ratio. The freeze-dried scaffolds were immersed in phosphate-buffered saline (PBS, pH 7.4) at 37 °C for 1, 2, 3, 6, and 12 h. After removal, excess surface liquid was gently blotted with filter paper, and the weight (W[1]) was recorded. The swelling ratio (SR%) was calculated as: SR (%) = [(W[1] - W[0])/W[0]] × 100 %. 2.2.3. Degradation rate The freeze-dried scaffolds (initial weight W[0]) were incubated in PBS containing 2 U/mL protease (Type XIV from Streptomyces griseus, 3.5 U/mg) at 37 °C. The enzyme solution was refreshed every 2 days to maintain activity. At specified time points (3, 7, 14, and 28 days), the scaffolds were rinsed with deionized water, freeze-dried, and weighed (W[2]). The degradation rate (DR%) was calculated as: DR (%) = [(W[0] − W[2])/W[0]] × 100 %. 2.2.4. Mechanical properties Uniaxial compression tests were performed using a universal testing machine (INSTRON 5944, USA) to evaluate the mechanical properties of the scaffolds. Cylindrical hydrogel samples were compressed vertically at a speed of 3 mm/min. 2.2.5. Viral release rate A virus titer kit (OBiO) was utilized to quantify the viral release from the hydrogel into PBS under agitation conditions. At each designated time point, a consistent volume of 500 μl was extracted to evaluate the viral concentration, with an equal volume of fresh PBS subsequently added to maintain the system. Specifically, 500 μl of crosslinked composite hydrogel was mixed with 500 μl of PBS, and the PBS was collected and replaced every five days over a 30-day period. 2.3.5 In vitro release of Trox from SF/GO-gel@Trox. The quantification of Trox release from SF/GO-gel@Trox under ultrasonic conditions was conducted utilizing ELISA. Initial experiments were performed to establish the ultrasonic parameters, which included a frequency of 1 MHz, a temperature of 25 °C, a pH of 7.4, a duty cycle of 20 %, a power of 2 W/cm^2, and an irradiation duration of 3 min. Two experimental groups, each containing equivalent amounts of Trox, were prepared: one group was subjected to ultrasonic treatment, while the other served as a control without ultrasonic exposure. The release was seen at 1, 3, 5, 8, 10, 15, 20, 25, and 30 days. Subsequent to treatment, the samples underwent centrifugation at 3000 g for 5 min at ambient temperature. The supernatant was gathered and preserved at −80 °C until analysis. The average release rate is calculated based on the cumulative release data. The cumulative release Rn is determined using the formula: [MATH: Rn=Ve∑k=1nCkWtotal×100% :MATH] , where Ve is the volume of each sample used for analysis, Ck is the Trox concentration in the release solution at the k-th sampling, and W[total] is the total amount of Trox loaded into the hydrogel. To obtain the average release rate, the difference in cumulative release ΔR between consecutive time points is divided by the corresponding time interval Δt, giving Average Release Rate = ΔR/Δt. The concentration of Trox was quantified utilizing an ELISA kit (Nanjing Jiancheng Bioengineering Institute, A142-1-1), with all assessments conducted in triplicate. 2.2.6. Safety assessment 2.2.6.1. CCK8 and cell live/dead assay The composite hydrogel was immersed in full medium for 48 h to prepare the leaching solution. In accordance with the manufacturer's guidelines, the CCK8 (C0037, Beyotime, China) and Calcein/PI Cell Viability/Cytotoxicity Assay Kit (C2015S, Beyotime, China) were employed to assess the impact of the leaching solution on cellular proliferation at 1, 3, and 6 days. 2.2.6.2. Biocompatibility and organ toxicity in vivo study The biocompatibility of SF/GO-gel@Trox was determined by their subcutaneous implantation in rats. To evaluate the biocompatibility and potential immunogenicity, liver and kidney function were analyzed. The major organs (heart, liver, spleen, lung, and kidney) were sectioned and analyzed by hematoxylin and eosin (HE) staining for biocompatibility evaluation. 2.2.6.3. In vitro hemolysis assay of SF/GO-gel@Trox An in vitro hemolysis study was performed to evaluate the safety of SF/GO-gel@Trox. Initially, 1 mL of human blood was procured in an EDTA anticoagulant tube and subsequently diluted with 2 mL of saline. The diluted blood sample was subjected to centrifugation at 2000 rpm for a duration of 5 min, following which the supernatant was carefully removed. The erythrocytes were then resuspended in saline solution and underwent three to four washing cycles until the supernatant was visually clear. Finally, the erythrocytes were resuspended in 2 mL of saline solution. The hemolysis assay comprised three groups: 5 mL saline, 5 mL PBS, 5 mL Triton, and 5 mL SF/GO-gel@Trox, each with three duplicates. All tubes were pre-warmed to 37 °C for 30 min prior to the test. Subsequently, 0.1 mL of diluted red blood cells was introduced into each tube, stirred gently, and incubated at 37 °C for 60 min. Following centrifugation at 1200 g for 5 min, the absorbance of the supernatant was assessed at 545 nm utilizing a spectrophotometer. 2.3. Collection of clinical samples and ethical review The Ethics Committee and Institutional Animal Care and Use Committee of Tongji University Affiliated East Hospital authorized this study. All experiments adhered to the updated Declaration of Helsinki and institutional protocols for animal research. Samples were collected from nine patients undergoing IVD removal and spinal fusion surgery for lumbar spinal stenosis (clinical details provided in [71]Table S1). The severity of IDD was evaluated preoperatively utilizing the Pfirrmann grading method on sagittal T2-weighted MRI. 2.4. Cellular isolation and cultivation Two-month-old SD rats were sedated with 2 % sodium pentobarbital at a dosage of 50 mg/kg and subsequently killed. In sterile conditions, the annulus fibrosus (AF) was dissected, and NP tissue was removed with ophthalmic forceps. After a 4-h enzymatic digestion using 0.1 % collagenase II, NP cells were isolated through centrifugation. The cells were subsequently cultured in F12-DMEM complete medium, supplemented with 10 % fetal bovine serum (A5256701, Gibco) and 1 % penicillin/streptomycin (15070063, Gibco). The media was replaced every three days, and cells from passages 2 to 3 were utilized for research. In vitro investigations involved treating NP cells with interleukin-1β (IL-1β) at a concentration of 10 ng/mL (RIL1BI, Thermo Fisher), Trox at 10 μM (HY-N0139, MedChemExpress), or HY-143404 at 10 μM (MedChemExpress). The transfection section provides a comprehensive account of lentiviral transfection. In cycloheximide (CHX) (66-81-9, Sigma-Aldrich) chase tests, transfected (lentivirus) cells were administered 50 μg/mL CHX for different durations prior to Western blot (Wb) examination. 2.5. Wb Cells were lysed using RIPA buffer (P0013, Beyotime, China) supplemented with PMSF (P0100, Solarbio, China). Protein concentrations were determined using a BCA assay kit (P0009, Beyotime, China), and the proteins were separated by SDS-PAGE. Post-electrophoresis, proteins were transferred onto PVDF membranes and blocked with 5 % skim milk for 1 h at room temperature. Subsequently, the membranes were incubated with primary antibodies (refer to [72]Table S2) overnight at 4 °C. After washing with TBST (PS103, Enzyme, China), HRP-conjugated secondary antibodies (anti-rabbit or anti-mouse) were applied for 1 h at room temperature. Protein bands were visualized using an ECL detection system. 2.6. Lentiviral transfection Lentiviral vectors were engineered and assembled by GeneChem (Shanghai, China) in accordance with established techniques. The coding sequences of Casitas B-lineage lymphoma (CBL), Tie2, and epidermal growth factor receptor (EGFR) were inserted into the PGMLV-SC5-ZsGreen1-Puro vector, incorporating Myc, His, or Flag tags. HA-tagged ubiquitin (Ub) and its variants (18712, 121151, 121152, 22902, 17607, 17606, 17605, 17604) were acquired from Addgene. Point mutations in Tie2 were generated utilizing the QuikChange Mutagenesis Kit (Agilent Technologies) and confirmed through DNA sequencing. Lentiviral shRNAs targeting CBL, Tie2, and EGFR (shEGFR: 5′-AGTAACAGGCTCACCCAAC-3′; shCBL: 5′-GACAAGAAGATGGTGGAGAAG-3′; shTie2: GE Dharmacon V2LHS_93160 and V2LHS_232742) were transfected employing Lipo3000 (Invitrogen). NP cells at 40–60 % confluency were subjected to lentiviral transduction with a multiplicity of infection (MOI) of 20. The media was replenished every other day after 12 h until full confluency was achieved for following trials. 2.7. Immunoprecipitation (IP) For the co-immunoprecipitation (Co-IP) procedure, cellular lysates were prepared using an immunoprecipitation buffer (P0013, Beyotime) supplemented with PMSF and phosphatase inhibitors. The lysates underwent pre-clearance with protein A/G agarose beads, followed by incubation with primary antibodies overnight at 4 °C. Subsequently, protein A/G beads were introduced for an additional 4-h incubation. The immune complexes were then subjected to three washes prior to being boiled in loading buffer for subsequent Wb analysis.2.9 Immunofluorescence (IF) After 48 h of treatment, the cells and paraffin sections were fixed in 4 % paraformaldehyde for 15 min at 4 °C. They were then permeabilized using 0.1 % Triton X-100 and subsequently blocked with 5 % goat serum (P0096/C0265, Beyotime).Primary antibodies were incubated overnight at 4 °C, then followed by FITC- or Cy3-conjugated secondary antibodies (Abclonal, AS011/AS007) for 1 h in the absence of light. Nuclei were stained with DAPI (Beyotime, C1006) for a duration of 5 min. Images were obtained with a Leica DMI 6000B microscope and subsequently analyzed with ImageJ. 2.8. In vivo experiments One-hundred male SD rats, aged two months, were maintained in a controlled environment with a 12-h light/dark cycle at 22 °C, sourced from SLAC Laboratory Animal Co., Shanghai, China. An IDD model was created in the caudal spine utilizing a needle puncture technique. Subsequently, the rats were anesthetized, and 2 μl of hydrogel were injected into the IVD using a 31-gauge needle, which was maintained in position for 5 min to prevent leakage. Two rounds of experiments were conducted: Round 1: Control (no treatment), IDD (no injection), SF/GO-gel, and SF/GO-gel@Trox (ultrasound: 1 MHz, 2 W/cm^2, 20 % duty cycle, 3 min/session, 2–3 sessions/week). Round 2: Control, IDD, SF/GO-gel@His-Tie2, and SF/GO-gel@His-Tie2+Flag-EGFR. Rats were euthanized at specified intervals. 2.9. Radiological assessment Anteroposterior X-rays of the caudal spine were acquired at 0, 4, and 8 weeks. Imaging data were evaluated utilizing a PACS system. Included measurements: 1) Direct IVD and vertebral height; 2) Disc height index (DHI) = (IVD height/adjacent vertebral length) × 100 %; 3) Percentage DHI = (post-injection DHI/pre-injection DHI) × 100 %. An MRI (Philips-Achieva 3.0T, Netherlands) was conducted for IDD assessment. 2.10. Histopathological examination IVD tissues were preserved in 10 % formalin for 48 h, subjected to decalcification in 10 % EDTA (Sigma) for 6 months, and subsequently embedded in paraffin. Coronal slices (5 μm) were stained with HE or Safranin O/Fast Green (SO) and photographed using an Olympus FV-1000 microscope. 2.11. β-Galactosidase staining Cellular senescence was evaluated utilizing a β-galactosidase staining kit (C0602, Beyotime). Cells were fixed using 0.2 % glutaraldehyde for 15 min at 37 °C, thereafter washed with PBS, and treated with X-gal (pH 6.0) at 37 °C for 16–24 h. Senescent cells (blue) were enumerated using a microscope. 2.12. Immunohistochemistry (IHC) NP tissues were fixed in 4 % paraformaldehyde, subsequently embedded in paraffin, and sectioned to a thickness of 5 μm. Antigen retrieval was performed at 60 °C using a citrate buffer, after which endogenous peroxidase activity was quenched with 3 % hydrogen peroxide. Sections were incubated with primary antibodies overnight at 4 °C, subsequently treated with HRP-conjugated secondary antibodies and subjected to DAB staining. Images were obtained with a Leica microscope and subsequently analyzed with ImageJ. 2.13. EdU assay NP cells (2 × 10^4/well) were grown in 12-well plates and subjected to EdU (BeyoClick™ EdU-488, C0071S, Beyotime, China) treatment for 2 h. Cells were fixed, permeabilized, and stained using Hoechst 33342. EdU-positive cells (red) and nuclei (blue) were seen by fluorescence microscopy. 2.14. Statistical examination All trials were conducted a minimum of three times. Data were analyzed utilizing SPSS 25.0 (Chicago, IL) employing one-way ANOVA and Tukey's post-hoc test. A p-value of less than 0.05 was deemed statistically significant. 3. Results 3.1. Material characterization The SF and GO were uniformly combined in varying ratios, resulting in the rapid formation of hydrogels at 37 °C following crosslinking facilitated by a cell disruptor. The procedural workflow is depicted in [73]Fig. 1A. [74]Fig. 1B illustrates the outcomes for SF, SF/GO-5 %, and SF/GO-10 % post-treatment with the cell disruptor, as they were introduced into a culture dish via an 18G needle at 37 °C. This demonstrates that all solutions exhibited excellent injectability and underwent swift gelation upon injection. The freeze-dried gel network structures were analyzed by SEM, and the results are shown in [75]Fig. 1C. The freeze-dried SF, SF/GO-5 %, and SF/GO-10 % hydrogels all exhibited uniform porous microstructures with pore sizes around 100 μm. Notably, the pore size distribution in pure SF was relatively irregular. In contrast, the incorporation of GO led to a more uniform distribution of pore sizes. Uniform and larger pores facilitate the exchange of nutrients, oxygen, and carbon dioxide, thereby enhancing the proliferation and differentiation of NP cells. AFM analysis of the sonicated SF/GO-5 % solution revealed numerous fibrous SF structures attached to the surface and periphery of GO, suggesting the potential for hydrogel network formation ([76]Fig. 1D). Further FTIR and XRD analyses were performed to examine the chemical composition changes in SF, SF/GO-5 %, and SF/GO-10 % hydrogels ([77]Fig. 1E–G). In the XRD patterns, SF/GO-5 % and SF/GO-10 % exhibited characteristic peaks of GO near 10° and 42°, confirming the successful incorporation of GO into SF. In the FTIR spectra of typical SF, the characteristic vibration bands of amide I (C=O stretching) and amide II (secondary NH bending) are within the range of 1700-1450 cm^−1. In the amide I band, random coils and α-helix structures are typically observed at 1640-1660 cm^−1, while β-sheet structures are usually located at 1620–1637 cm^−1 [[78]33,[79]34]. In the FTIR spectra of [80]Fig. 1F–G, the C=O peak of the directly freeze-dried SF sample appeared at 1640.2 cm^−1, while sonicated SF, SF/GO-5 %, and SF/GO-10 % exhibited blue shifts in the C=O peaks, appearing at 1618.5, 1620.4, and 1620.4 cm^−1, respectively, indicating a transition to β-sheet secondary structures, which is the primary reason for the gelation of SF, SF/GO-5 %, and SF/GO-10 %. Additionally, compared to pure SF, SF/GO-5 % and SF/GO-10 % exhibited enhanced broad peaks in the 3200–3500 cm^−1 range and a new epoxy C=O peak near 1726 cm^−1, further confirming the successful incorporation of GO into the SF network. Fig. 1. [81]Fig. 1 [82]Open in a new tab Hydrogel preparation and characterization. A: Hydrogel preparation process. B: SF, SF/GO-5 %, and SF/GO-10 % solutions were injected onto culture dishes using a needle at 37 °C after cell disruptor treatment, demonstrating good injectability for all solutions with rapid gelation post-injection. C: SEM analysis was employed to examine the freeze-dried gel network structure. D: AFM analysis of SF/GO-5 % solution revealed numerous fibrous SF proteins attached to and surrounding GO surfaces, suggesting potential for hydrogel network formation. E–G: Further chemical composition analysis of SF, SF/GO-5 %, and SF/GO-10 % hydrogels was conducted using FTIR and XRD. SF, silk fibroin; GO, graphene oxide; Trox, Troxerutin; AFM, atomic force microscopy; FTIR, Fourier transform infrared spectroscopy; XRD, X-ray diffraction; SEM, scanning electron microscopy. On days 1, 3, and 6, there were negligible numbers of inactivated cells across all groups, and no significant differences were detected in the live/dead cell ratio among the groups ([83]Fig. 2A). To further assess potential visceral toxicity, comprehensive histological evaluations were conducted on tissue sections from the heart, liver, spleen, lungs, and kidneys ([84]Fig. 2B). The results of the CCK-8 assay corroborated these observations ([85]Fig. 2C). Regarding biotoxicity, the hemolysis rate of SF/GO-gel@Trox was less than 1 %, which was lower than that of Triton ([86]Fig. 2D). Additionally, liver and kidney functions remained stable, with no significant deviations observed in their respective functional markers, as illustrated in [87]Fig. 2E–F. In addition, when investigating the regulation of GO doping concentration on the compressive strength of SF-based hydrogels, we found that the compressive strengths of SF, SF/GO-5 %, and SF/GO-10 % hydrogels reached 2.074 ± 0.104 kPa, 4.051 ± 0.203 kPa, and 2.785 ± 0.139 kPa, respectively ([88]Fig. 2G). Notably, when the GO doping amount increased to 5 %, the mechanical strength of the composite hydrogel increased by 95.3 % compared to the pure SF matrix, which is attributed to the uniform dispersion of GO nanosheets in the SF three-dimensional network and their excellent mechanical enhancement effect. When the concentration of GO was further elevated to 10 %, a 19.6 % reduction in compressive strength was observed. This decline can likely be attributed to agglomeration resulting from strong van der Waals forces and π-π interactions between nanosheets, as well as the disruption of the SF network continuity due to the increased GO content. These findings corroborate the nonlinear relationship between GO content and mechanical properties. Further rheological analysis revealed the material's phase transition characteristics. All rheological properties were measured at 37 °C ([89]Fig. 2H–I). In the solution state, G′ was generally lower than the loss modulus G″, confirming that the system exhibited typical fluid characteristics (tanδ = G''/G'>1). After treatment with the cell disruptor and standing at 37 °C, the system rapidly underwent sol-gel transition, forming a three-dimensional network structure with significant elastic-dominated characteristics (G'>G″). Particularly noteworthy is that the G′ value of the SF/GO-5 %-gel composite system increased compared to SF-gel, indicating that the introduction of GO enhanced the network strength. Similarly, further increase of GO significantly reduced G′, which is unfavorable for network formation. [90]Fig. 2J illustrates the swelling ratio variation of SF-based hydrogels over time. All hydrogels exhibited rapid swelling responses upon immersion in PBS solution. Notably, the GO-incorporated sample groups showed slightly enhanced swelling ratios, likely attributable to the structural optimization of SF's three-dimensional crosslinked network by GO introduction, which improved water diffusion and storage. [91]Fig. 2K presents quantitative analysis of the degradation rates of SF-based hydrogels in simulated in vitro conditions. All hydrogels demonstrated increasing degradation rates over time, reaching 66.25 ± 5.29 %, 56.51 ± 3.25 %, and 51.26 ± 4.28 % for SF, SF/GO-5 %, and SF/GO-10 % respectively after 28 days. This trend indicates that the degradation rate of SF hydrogels varies with GO content, demonstrating the tunability of SF hydrogel degradation rates according to specific requirements. Fig. 2. [92]Fig. 2 [93]Open in a new tab Functional characterization of the hydrogels. A: Cell compatibility of the prepared hydrogels indicated by live/dead staining on days 1, 3, and 6. B: Visceral toxicity analysis in vivo, showing HE-stained sections of major organs (heart, liver, spleen, lungs, and kidneys). C: Effect of the prepared hydrogels on NP cell proliferation measured by CCK8 assay. D: Hemolytic activity of the prepared hydrogels in rat red blood cells. E–F: Liver function tests (ALT and AST) and kidney function tests (UA and BUN). G: Stress-strain curves of the prepared hydrogels. H–I: Phase transition characteristics of the prepared hydrogels analyzed by rheological measurements, with all rheological properties measured at 37 °C. J: Swelling ratio of the prepared hydrogels. K: Degradation rate of the prepared hydrogels. All experiments were repeated three times. All data are expressed as the mean ± SD. One-way ANOVA and Tukey's multiple comparisons test were used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ALT, alanine aminotransferase; AST, aspartate transaminase; UA, uric acid; BUN, blood urea nitrogen; HE, hematoxylin and eosin; SF, silk fibroin; GO, graphene oxide; Trox, Troxerutin. The three-dimensional network architecture of the SF/GO hydrogel provides a substantial specific surface area, thereby significantly enhancing its drug loading capacity. This unique structure facilitates uniform distribution of the medication and enables controlled and sustained drug release through the modulation of the hydrogel's porosity and crosslinking density. For example, GO/chitosan composite hydrogel membranes fabricated by electrostatic self-assembly technique have superior sustained-release capabilities [[94]35]. SF/GO hydrogels demonstrate advantageous properties for biocompatibility and safety. Studies demonstrate that nano-SF/GO can be internalized by cells without exhibiting considerable cytotoxicity, rendering it highly advantageous for biological applications [[95]36]. Furthermore, the in vivo durability and biocompatibility of GO hydrogels can be improved through chemical modification or by integrating them with other biocompatible materials. Further, we investigated whether ultrasound intervention affects the release of Trox from SF/GO-gel@Trox ([96]Fig. 3A). SF/GO-5 %-gel exhibits a sustained drug release profile, maintaining gradual release kinetics throughout the 24-h observation period and demonstrating prolonged release capability extending up to one month ([97]Fig. 3B–C). Ultrasound promotes the release of Trox from SF/GO-gel@Trox, and within a certain range, the amount of Trox released increases with higher ultrasound amplitude, increased duty cycle, and prolonged duration ([98]Fig. 3D–G). Correspondingly, the weight loss of hydrogels was more rapid under ultrasound intervention ([99]Fig. 3H). The ultrasound-responsive medication delivery system integrated with SF/GO-gel@Trox exemplifies an advanced biomedical technology with extensive applications. The advantages encompass spatiotemporal controllability, non-invasiveness, and profound tissue penetration [[100]37]. Modifiable ultrasonic parameters (e.g., frequency, intensity, duration) provide on-demand medication release. SF/GO's remarkable mechanical strength, biocompatibility, colloidal stability, extensive surface area, and elevated adsorption capacity render it optimal for drug delivery [[101]38]. In this system, SF/GO-gel functions as a drug carrier, with ultrasound inducing targeted drug release through cavitation effects (increasing drug penetration via microbubble dynamics) [[102]39]. The surface functional groups of SF/GO-gel, including carboxyl and hydroxyl groups, enhance drug loading and stability through mechanisms such as hydrogen bonding and electrostatic interactions. Ultrasound-responsive SF/GO-gel holds promise for applications in oncological therapies, including tumor-targeted chemotherapy, as well as anti-inflammatory treatments. The surface can be modified with targeting moieties, such as antibodies and peptides, to improve lesion-specific delivery. This advanced approach, characterized by increased degrees of GO oxidation and hydrogel crosslinking densities, offers potential innovative solutions for regenerative medicine, antibacterial dressings, and other applications. Fig. 3. [103]Fig. 3 [104]Open in a new tab The drug release profiles of SF/GO-gel@Trox under ultrasound conditions are presented. A: Ultrasound intervention schematic diagram of Trox release from SF/GO-gel@Trox. B: Drug release curves of SF, SF/GO-5 %, and SF/GO-10 % hydrogels. C: 24-h drug release profiles of SF, SF/GO-5 %, and SF/GO-10 % hydrogels. D: Comparison of drug release between ultrasound-treated and non-ultrasound-treated SF/GO-5 %-gel@Trox. E–G: Drug release curves of SF/GO-5 %-gel@Trox under different ultrasound parameters. H: Weight loss curves of SF/GO-5 %-gel@Trox with and without ultrasound treatment. All experiments were repeated three times. All data are expressed as the mean ± SD. One-way ANOVA and Tukey's multiple comparisons test were used for statistical analysis. SF, silk fibroin; GO, graphene oxide; Trox, Troxerutin. In our study, we have demonstrated that ultrasound can significantly modulate the binding energy between Trox and GO, as evidenced by the dynamic docked molecules in [105]Fig. S1A–B. Specifically, the binding energy decreased from −872.52 kJ/mol (without ultrasound) to −727.39 kJ/mol (with ultrasound), indicating a weaker interaction between Trox and GO under ultrasound treatment. This reduction in binding energy is primarily attributed to the decrease in both Lennard-Jones (LJ) and Coulombic interactions, as shown in our calculations. The Interactions Graphical Method (IGM) visualization further supports these findings, illustrating that more Trox binds to GO in the no ultrasound state, and extensive van der Waals interactions form between them. This suggests that ultrasound treatment can effectively disrupt the non-covalent interactions, facilitating the release of Trox from the GO matrix. In addition, we employed specific ultrasound parameters (1 MHz, 2 W/cm^2, 20 % duty cycle, 3 min/session, 2–3 sessions/week) to induce drug release from SF/GO-gel implanted in IVD. The experimental results demonstrated variations in drug release efficiency among hydrogels with different GO concentrations under these ultrasound parameters, with the SF/GO-5 %-gel showing the most significant drug release effect. However, this outcome was not attributed to the highest heat generation during ultrasound application to the SF/GO-5 %-gel leading to optimal drug release. Specifically, although cavitation effects occur during ultrasound treatment, the temperature variations among hydrogels with different GO concentrations showed no significant differences under our experimental ultrasound settings in [106]Fig. S1C–D. While the ultrasound frequency (1 MHz) and power density (2 W/cm^2) were not high, the pulsed mode (20 % duty cycle) enabled intermittent energy delivery, allowing timely heat dissipation and preventing excessive temperature rise that could cause structural alterations in GO or thermal degradation of drugs. Furthermore, the short duration of each ultrasound session (3 min/session) and limited treatment frequency (2–3 sessions/week) under these mild ultrasound conditions effectively controlled temperature elevation, thereby maintaining the structural stability of the GO hydrogels. Therefore, ultrasound primarily accelerated drug release by modifying the binding energy between GO and the drug molecules. The mechanical shear forces generated by ultrasound cavitation weakened chemical bonds or van der Waals forces on the GO surface, thereby promoting drug release. These changes in binding energy were independent of GO concentration but closely related to the inherent characteristics of ultrasound itself. Therefore, the observed differences in drug release efficiency - particularly the optimal performance of the SF/GO-5 %-gel under these ultrasound parameters are more likely attributable to ultrasound-induced modifications of the binding energy between GO and drugs rather than thermal effects. This finding holds significant implications for optimizing drug release strategies in GO hydrogel-based IVD implantation therapies and provides theoretical foundations for subsequent clinical applications. 3.2. In animal studies of SF/GO-gel@Trox The advancement of IDD was assessed by X-ray ([107]Fig. 4A and [108]Fig. S2A–C), utilizing DHI% as a principal indicator. No significant variations in DHI% were identified among groups at baseline (0 weeks) (p > 0.05). At weeks 4 and 8, all groups except the control demonstrated a decrease in DHI%, with the SF/GO-gel@Trox group exhibiting a considerably lesser reduction compared to the IDD and SF/GO-gel groups (p < 0.05), so confirming that SF/GO-gel@Trox effectively preserved IVD height. MRI, the benchmark for IDD evaluation, precisely indicated alterations in IVD hydration. In accordance with X-ray findings, the SF/GO-gel@Trox group exhibited significantly smaller decrease in MRI signal intensity at weeks 4 and 8 compared to the IDD and SF/GO-gel groups ([109]Fig. 4B and [110]Fig. S2D–F) (p < 0.05). Fig. 4. [111]Fig. 4 [112]Open in a new tab Effects of SF/GO-gel@Trox on histological scores and ECM corresponding to rat coccygeal IVDs after the intervention. A: Representative X ray radiographs of the rat tail at 0, 4 and 8 weeks after the intervention. B: Representative MRI scans were obtained at 0, 4 and 8 weeks after the intervention. C: Macroscopic IVD images, fitting picture, and the relative area of NP at 8 weeks after the intervention. D: Representative HE staining and SO staining images of rat coccygeal IVDs corresponding to different treatment groups (coronal position) at 4 weeks after the intervention. E: Representative HE staining and SO staining images of rat coccygeal IVDs corresponding to different treatment groups (coronal position) at 8 weeks after the intervention. F–G: IF staining of COL2 and ACAN at 4 weeks after the intervention. H–I: IF staining of COL2 and ACAN at 8 weeks after the intervention. All experiments were repeated six times. All data are expressed as the mean ± SD. One-way ANOVA and Tukey's multiple comparisons test were used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ECM, extracellular matrix; IVD, intervertebral disc; NP, nucleus pulposus; HE, hematoxylin and eosin. SO, safranin-o/fast green; COL2, Collagen type II; ACAN, aggrecan; IF, immunofluorescence; SF, silk fibroin; GO, graphene oxide; Trox, Troxerutin. The macroscopic morphological features of IVD demonstrated a significant correlation with the severity of IDD and the Pfirrmann grading system. The control group and the group treated with SF/GO-gel@Trox preserved normal IVD architecture, characterized by adequate hydration and well-defined NP-AF boundaries. In contrast, the IDD group and the SF/GO-gel group displayed disrupted IVD structures, including NP contraction and increased fibrous tissue proliferation. Quantitative analysis indicated that, in addition to the control group, the SF/GO-gel@Trox group exhibited considerably greater NP relative areas compared to the other two groups ([113]Fig. 4C) (p < 0.05). Histological research revealed preserved NP structures in control HE stains, whereas the IDD and SF/GO-gel groups demonstrated structural degradation ([114]Fig. 4D–E and [115]Fig. S3A–B). The SF/GO-gel@Trox group maintained partial NP tissue for up to 4–8 weeks, albeit with compromised AF alignment. SO staining revealed significant proteoglycan depletion in the IDD and SF/GO-gel groups, accompanied by a modest decrease in the SF/GO-gel@Trox group. At weeks 4 and 8, histological scores were significantly higher in the intervention groups compared to the control group (p < 0.05). Nonetheless, the SF/GO-gel@Trox group demonstrated significantly lower scores than both the IDD and SF/GO-gel groups (p < 0.05).IF staining at weeks 4 and 8 revealed that, excluding the control, the SF/GO-gel@Trox group exhibited elevated collagen type II (COL2) and aggrecan (ACAN) expression compared to the other intervention groups ([116]Fig. 4F–I and Figures S3C-D) (p < 0.05). We acknowledge that one of the limitations of this study that providing in vivo treatment data comparing SF/GO-gel@Trox with SF-gel@Trox (without GO) would provide definitive evidence of GO's critical contribution to enhancing IDD treatment efficacy. However, our previous comparative studies of SF, SF/GO-5 %, and SF/GO-10 % hydrogels demonstrated that the SF/GO-5 % formulation exhibited the most favorable combination of mechanical properties, biocompatibility, and controlled drug release characteristics. Specifically, the SF/GO-5 % hydrogel showed superior sustained release performance with a more gradual initial burst release profile compared to both pure SF and SF/GO-10 % hydrogels, making it particularly suitable for long-term therapeutic applications. Therefore, all subsequent in vivo and in vitro experiments in this study were conducted using the SF/GO-5 % hydrogel as the carrier platform, which represents the fundamental research workflow and rationale of our investigation. In addition, we conducted supplementary comparative studies at the cellular level to evaluate the protective effects of SF/GO-5 %-gel@Trox versus SF-gel@Trox on NP cells following IL-1β modeling. This will be elaborated in detail in the following section. Meanwhile, we plan to conduct additional in vivo experiments to further validate these findings in subsequent research. 3.3. Functional Validation of SF/GO-gel@Trox This research rigorously assessed the therapeutic impacts of SF/GO-gel@Trox on NP cells and clarified its fundamental molecular mechanisms. To examine its protective function, we developed a cellular damage model with 10 ng/mL IL-1β activation. The findings indicated that SF/GO-gel@Trox markedly mitigated IL-1β-induced cytotoxicity: the EdU assay showed an increase in the positive cell rate in the IL-1β group following SF/GO-gel@Trox treatment ([117]Fig. 5A). Additional validation via β-galactosidase staining corroborated that SF/GO-gel@Trox pretreatment significantly mitigated IL-1β-induced cellular senescence ([118]Fig. S4A). Consistent IF findings demonstrated that SF/GO-gel@Trox administration enhanced COL2 expression while diminishing senescence markers such as a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), Cyclin-dependent kinase inhibitor 2A (p16), and Cyclin-dependent kinase inhibitor 1A (p21), showing its protective benefits against IL-1β-induced degenerative and senescent phenotypes ([119]Fig. 5B–E). In addition, we conducted supplementary comparative studies at the cellular level to evaluate the protective effects of SF/GO-5 %-gel@Trox versus SF-gel@Trox on NP cells following IL-1β modeling ([120]Fig. S5A–B). Our current in vitro experiments using IL-1β-treated NP cells confirm that SF-gel@Trox effectively alleviates disc degeneration phenotypes, as evidenced by increased COL2 expression and decreased levels of degenerative/senescence markers (ADAMTS4, p16, and p21) ([121]Fig. S5A–B). Importantly, the therapeutic effects were further enhanced in the SF/GO-gel@Trox group, with more pronounced improvements in extracellular matrix (ECM) preservation and cellular senescence markers. These cellular-level findings complement our material characterization data and support our selection of SF/GO-5 % as the optimal delivery vehicle. Fig. 5. [122]Fig. 5 [123]Open in a new tab Effects of SF/GO-gel@Trox on NP cell proliferation, senescence and apoptosis. A: Under IL-1β stimulation, EdU assay demonstrated SF/GO-gel@Trox pretreatment significantly affected NP cell proliferation rates with EdU-positive cell quantification using ImageJ software; B–E: IF staining imaging revealed degeneration- and senescence-related marker patterns in NP cells post SF/GO-gel@Trox treatment under inflammatory conditions; F–I: Transcriptome sequencing yielded expression heatmaps with GO annotation and KEGG pathway results; J–M: combined PI3K inhibitor HY-143404 treatment maintained SF/GO-gel@Trox's regulatory effects on degeneration/senescence markers confirmed by IF. All experiments were repeated three times. All data are expressed as the mean ± SD. One-way ANOVA and Tukey's multiple comparisons test were used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. IL-1β, interleukin-1β; NP, nucleus pulposus; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; PI3K, Phosphoinositide 3-Kinase; IF, immunofluorescence; SF, silk fibroin; GO, graphene oxide; Trox, Troxerutin. We conducted transcriptome sequencing analysis to elucidate the mechanistic underpinnings. Gene ontology functional annotation (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis indicated that the phosphoinositide 3-kinase (PI3K)/protein kinase B(Akt) signaling pathway likely mediates the regulatory effects of SF/GO-gel@Trox ([124]Fig. 5F–I). This discovery was corroborated by inhibitor experiments: treatment with a PI3K inhibitor HY-143404 partially abolished the protective effects of SF/GO-gel@Trox against NP cells degeneration and senescence ([125]Fig. 5J–M and [126]Fig. S4B). These findings collectively indicate that SF/GO-gel@Trox mostly postpones NP cells degeneration and senescence via the stimulation of the PI3K/Akt signaling pathway. Trox, a naturally occurring flavonoid, exhibits significant efficacy in antioxidant, anti-inflammatory, and vasculoprotective functions, alongside considerable promise in functional control [[127]40,[128]41]. Its antioxidant properties play a crucial role in neutralizing free radicals and mitigating cellular damage induced by oxidative stress, which holds significant implications for the prevention and treatment of cardiovascular diseases, neurological disorders, and certain types of cancer. The anti-inflammatory effects are achieved through the suppression of pro-inflammatory mediators, including tumor necrosis factor-α (TNF-α) and interleukins (ILs), thereby alleviating symptoms associated with chronic inflammatory disorders such as arthritis and inflammatory bowel disease [[129]42,[130]43]. Furthermore, Trox regulates essential cellular signaling pathways (e.g., PI3K/Akt) to control cell proliferation, differentiation, and apoptosis, therefore preserving normal cellular and tissue functions for disease prevention and therapy [[131]41]. These regulatory systems highlight its therapeutic significance in addressing diabetic complications, hepatic illnesses, and several metabolic disorders. Recent study reveals more pharmacological advantages, including neuroprotective properties that could improve cognitive performance and mitigate dementia [[132]44]. Trox demonstrates a favorable safety profile and minimal adverse effects, suggesting considerable potential as a natural supplement in public health initiatives. Future research should aim to elucidate its molecular targets, evaluate its clinical efficacy and safety, and provide a scientific foundation for the development of novel therapeutic strategies. In conclusion, Trox, as a multifunctional natural compound, shows substantial promise for health improvement and disease prevention, warranting further investigation and application.3.4 Functional Validation of Tie2. This study thoroughly clarifies the essential function of Tie2 in IDD and its molecular mechanisms. Transcriptome study of NP cells first demonstrated a substantial increase of Tie2 expression subsequent to SF/GO-gel@Trox therapy ([133]Fig. 6A). To assess its clinical significance, we obtained NP tissues from nine patients, with three exhibiting mild, moderate, and severe degeneration, respectively, as per the Pfirrmann grading system. Wb and IHC investigations revealed a significant reduction in Tie2 protein expression in Pfirrmann grade III and V NP tissues compared to grade II (P < 0.001), indicating a negative connection with Pfirrmann grades ([134]Fig. 6B–E). In vitro studies have demonstrated that stimulation with IL-1β (10 ng/mL for three days) significantly decreases the levels of both Tie2 and phosphorylated Tie2 (p-Tie2 Y992) in NP cells (refer to [135]Fig. 6M–R and Figure S6H-M). Fig. 6. [136]Fig. 6 [137]Open in a new tab Tie2 modulates degeneration and senescence in NP cells. A: Differential gene expression analysis by transcriptome sequencing in NP cells following SF/GO-gel@Trox intervention. B–C: Wb analysis revealed the expression levels of Tie2 protein in human NP tissues with differing degeneration degrees (n = 3). D–E: IHC staining revealed the expression levels of Tie2 protein in human NP tissues with differing degeneration degrees (n = 3). F: NP cells treated with SF/GO-gel@Trox were exposed to CHX for specified durations. Cell lysates were then subjected to IB to determine relative Tie2 protein levels normalized to β-actin. G: Wb analysis of total Tie2 protein and its phosphorylated form (p-Tie2 Y992) in NP cells following SF/GO-gel@Trox intervention. H: NP cells transfected with His-Tie2 Y992A mutant were treated with CHX for indicated time periods. IB of cell lysates was performed to assess Tie2 protein stability relative to β-actin. I: Wb analysis of degeneration markers (COL2 and ADAMTS4) and senescence markers (P21 and P16) protein expression levels in NP cells at 48 h post-transfection with shTie2 following SF/GO-gel@Trox treatment. J: NP cells were transduced with the for 72 h. Degeneration and senescence-associated proteins COL2, ADAMTS4, p21, and p16 were evaluated using Wb analysis. Band density was assessed by semiquantitative analysis (n = 3). K: NP cells were treated with IL-1β (10 ng/mL, 72 h) followed by Tie2 overexpression. Cell proliferation rates were assessed by EdU assay. L: Molecular docking pattern of Trox and Tie2. Trox appears rose red and Tie2 protein appears light blue. M–R: Representative IF staining images showing NP cells treated with IL-1β and labeled with anti-Tie2, phosphorylated Tie2, degeneration markers antibodies (COL2 and ADAMTS4), and senescence markers (p16 and p21) antibodies. All experiments were repeated three times. All data are expressed as the mean ± SD. One-way ANOVA and Tukey's multiple comparisons test were used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. NP, nucleus pulposus; IHC, immunohistochemistry; Wb, western blot; IB, immunoblotting; CHX, cycloheximide; COL2, collagen type II; ADAMTS4, a disintegrin and metalloproteinase with thrombospondin motifs 4; p16, cyclin-dependent kinase inhibitor 2A; p21, cyclin-dependent kinase inhibitor 1A; Tie2, tyrosine-protein kinase receptor; IL-1β, interleukin-1β; EdU, 5-ethynyl-2‘-deoxyuridine; IF, immunofluorescence; SF, silk fibroin; GO, graphene oxide; Trox, Troxerutin. Subsequent experiments demonstrated that Trox not only extended the half-life of Tie2 protein but also elevated its total protein and phosphorylation levels at the Y992 location ([138]Fig. 6F–G and [139]Fig. S6A–B). Mutation at the Tie2 Y992 location, resulting in diminished phosphorylation, markedly reduced protein stability and expedited disintegration ([140]Fig. 6H and [141]Fig. S6C). Notably, the administration of SF/GO-gel@Trox significantly upregulated COL2 expression while concurrently reducing senescence markers, including ADAMTS4, P16, and p21. These protective effects were substantially abrogated following Tie2 knockdown, as illustrated in [142]Fig. 6I, [143]Figure S6D, and Figures S7A-D.We successfully created a Tie2-knockout NP cell line by lentiviral transfection. Wb and IF analyses demonstrated that the deletion of Tie2 not only exacerbated degenerative features but also upregulated the expression of senescence markers (refer to [144]Fig. 6J, [145]Figure S6E, and Figures S7E-H).In contrast, Tie2 overexpression markedly mitigated the cytotoxicity of IL-1β (10 ng/mL): EdU-positive rates rose ([146]Fig. 6K and [147]Fig. S6F), with β-galactosidase staining validating its anti-senescence actions against IL-1β induction ([148]Fig. S6G). Furthermore, molecular docking also suggests that Trox can bind to Tie2 ([149]Fig. 6L). Mechanistic investigations revealed that the PI3K/Akt signaling pathway mediates the protective effects of Tie2. Transcriptome sequencing and GO/KEGG analysis demonstrated substantial enrichment of this pathway ([150]Fig. S8A–E). Experiments indicated that the PI3K inhibitor HY-143404 largely negated the advantageous effects of Tie2 overexpression on cellular degeneration and senescence ([151]Fig. S8F–I), thereby showing that Tie2 predominantly sustains NP cells homeostasis via the activation of the PI3K/Akt pathway. Tie2 is a receptor primarily localized in vascular endothelial cells, where it plays a critical role in angiogenesis, maintaining vascular stability, and regulating inflammatory processes [[152]45]. Through its interaction with angiopoietin (Ang) family members, Tie2 regulates endothelial cell viability, motility, and vascular integrity [[153]46]. Under healthy settings, the Ang-1/Tie2 signaling pathway enhances endothelial cell survival and vascular stability by activating the PI3K/Akt pathway [[154]47], while simultaneously inhibiting the nuclear translocation of forkhead box protein O1 (FOXO1) and NF-κB activity to diminish inflammatory cytokine expression [[155]48,[156]49]. The functional state of Tie2 is closely associated with its phosphorylation status [[157]50]. Tie2 phosphorylation is modulated by angiopoietin family ligands; Ang1 binding promotes phosphorylation and activates downstream pathways such as PI3K-Akt to sustain vascular integrity, while Ang2 competitively obstructs Tie2 phosphorylation in the presence of Ang1, resulting in vascular destabilization [[158]51,[159]52]. Although direct evidence demonstrating that Trox influences Tie2 phosphorylation levels is lacking, its well-documented antioxidant and anti-inflammatory effects on the vascular endothelium may indirectly impact Tie2 functionality. Trox promotes the PI3K/Akt pathway to modulate hypoxia-inducible factor 1-α (HIF-1α) expression, thus alleviating oxidative stress and inflammation-effects that may synergize with Tie2 signaling, as Tie2 activation also diminishes inflammation through the PI3K-Akt pathway [[160]53]. Furthermore, Trox exhibits endothelial protective properties across various disease contexts, potentially through the regulation of vascular homeostasis via the Tie2 pathway. Trox may maintain Tie2 phosphorylation through two mechanisms: increasing Ang-1/Tie2 binding affinity and suppressing the activity of protein phosphatases [[161]54]. Tie2 autophosphorylation at pivotal residues (Tyr992, Tyr1100) may function as a molecular switch that activates downstream PI3K/Akt and MAPK pathways [[162]55], enhancing endothelial survival and vascular stability, while also inducing conformational alterations that inhibit recognition by E3 ubiquitin ligases (e.g., c-CBL), consequently diminishing receptor ubiquitination and degradation [[163]56]. The phosphorylation state of Tie2 has a direct impact on its ubiquitination, with dephosphorylation triggering internalization and degradation, thereby impairing it signaling capacity. Our research demonstrates that reduced phosphorylation of Tie2 leads to increased polyubiquitination and subsequent proteasomal degradation. However, treatment with Trox effectively mitigates this effect, thereby maintaining the stability of the Tie2 protein. These findings provide novel insights into the vasculoprotective mechanisms of Trox and suggest that regulating the balance between Tie2 phosphorylation and ubiquitination may represent a promising therapeutic strategy for addressing vascular degenerative diseases.3.5 Trox Modulates EGFR via Tie2. Transcriptomic analysis of NP cells subjected to shTie2 treatment demonstrated a notable increase of EGFR expression ([164]Fig. 7A). To ascertain its clinical significance, we obtained NP tissues from nine patients, with three exhibiting mild, moderate, and severe degeneration, respectively, as per the Pfirrmann grading system. The findings from Wb analysis and IHC demonstrated a significant upregulation of EGFR protein expression in Pfirrmann grade III and V NP tissues relative to grade II, as illustrated in [165]Fig. 7B–E (p < 0.001). This suggests a positive correlation between EGFR expression and increasing Pfirrmann grades. Furthermore, EGFR protein levels elevated subsequent to shTie2 transfection ([166]Fig. 7F and [167]Fig. S9A). Fig. 7. [168]Fig. 7 [169]Open in a new tab Mechanism of Tie2 regulation of EGFR and functional verification of EGFR. A: Differential gene analysis of transcriptome sequencing in NP cells after shTie2 transfection intervention; B–E: Wb and IHC analysis showed the expression levels of EGFR protein in human NP tissues with different degeneration degrees. F: Wb analysis of EGFR protein levels in NP cells after shTie2 transfection treatment. G: After treating NP cells with SF/GO-gel@Trox intervention with CHX for the specified durations, cell lysates were subjected to IB to determine the relative levels of EGFR compared to β-actin. H: After treating NP cells transfected with shTie2 with CHX for the specified durations, cell lysates were subjected to IB to determine the relative levels of EGFR compared to β-actin. I: His-Tie2 was transfected into NP cells for 72 h, followed by MG132 treatment for 6 h. J: After shEGFR treatment in NP cells, wb was used to analyze the protein expression levels of degeneration markers (COL2 and ADAMTS4) and senescence markers (p21 and p16). K: Wb analysis of total NF-κB protein and its phosphorylated protein levels in NP cells after co-transfection with shTie2 and/or shEGFR. L: NP cells were transfected with His-Tie2 for 72 h. Cell lysates were IP with EGFR antibody, followed by IB with Ub antibody. All input lysates were subjected to EGFR and β-actin detection. M: NP cells were transfected with shCBL for 72 h. Cell lysates were IP with EGFR antibody, followed by IB with Ub antibody. All input lysates were subjected to EGFR and β-actin detection. N: After treating NP cells transfected with shCBL with CHX for the specified durations, cell lysates were subjected to IB to determine the relative levels of EGFR compared to β-actin. O: Myc-CBL was transfected into NP cells for 72 h, followed by MG132 treatment for 6 h. P: NP cells were transfected with Flag-EGFR and specified Ub mutants along with Myc-CBL. Cell lysates were subjected to IP and IB with the specified antibodies. Q: NP cells were transfected with Myc-CBL and HA-Ub-K48 along with Flag-EGFR or its mutants, then treated with MG132 (20 μM). Cell lysates were subjected to IP and IB with the specified antibodies. All experiments were repeated three times. All data are expressed as the mean ± SD. One-way ANOVA and Tukey's multiple comparisons test were used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Tie2, tyrosine-protein kinase receptor; EGFR, epidermal growth factor receptor; NP, nucleus pulposus; Wb, western blot; IHC, immunohistochemistry; CHX, cycloheximide; IB, immunoblotting; COL2, collagen II; ADAMTS4, a disintegrin and metalloproteinase with thrombospondin motifs 4; p16, cyclin-dependent kinase inhibitor 2A; p21, cyclin-dependent kinase inhibitor 1A; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; IP, immunoprecipitation; Ub, ubiquitin; CBL, Casitas B-lineage lymphoma; MG132, carbobenzoxy-Leu-Leu-leucinal; SF, silk fibroin; GO, graphene oxide; Trox, Troxerutin. Subsequent analysis indicated that SF/GO-gel@Trox facilitates EGFR degradation by reducing its protein half-life ([170]Fig. 7G and [171]Fig. S9B), while shTie2 transfection significantly inhibited EGFR protein degradation ([172]Fig. 7H and [173]Fig. S9C). Significantly, pretreatment with the proteasome inhibitor MG132 mitigated the expedited EGFR degradation caused by Tie2 overexpression ([174]Fig. 7I). We effectively established an EGFR-knockout NP cell line through lentiviral transfection. Wb analysis confirmed that the downregulation of EGFR alleviated cellular degenerative characteristics and reduced the expression of senescence markers, as illustrated in [175]Fig. 7J and [176]Fig. S9D. To clarify the regulation mechanism of EGFR, we conducted Wb comparisons across three cell groups: control, Tie2-knockdown, and Tie2/EGFR double-knockdown. Experimental data indicated that Tie2 knockdown markedly elevated phosphorylation levels of critical NF-κB pathway proteins, whereas simultaneous EGFR deletion further inhibited system activation ([177]Fig. 7K and [178]Fig. S9E). EGFR is a pivotal receptor tyrosine kinase that is integral to cell proliferation, differentiation, survival, and migration. Upon ligand binding, EGFR experiences autophosphorylation, activating downstream signaling pathways such as rat sarcoma viral oncogene homolog (Ras)-rapidly accelerated fibrosarcoma kinase (Raf)-mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase (ERK) and PI3K-Akt-mammalian target of rapamycin (mTOR), therefore regulating cellular growth and survival [[179]57,[180]58]. Abnormal EGFR activation is significantly linked to numerous illnesses, including malignant cancers. Recent research has increasingly concentrated on the involvement of EGFR in IDD [[181]59]. EGFR is potentially implicated in the pathogenesis of IDD through its modulation of inflammatory responses, ECM degradation, and the activation of various cellular signaling pathways. In degenerated IVD tissues, there is a significant upregulation of EGFR expression, and its activation may accelerate the progression of IDD through multiple mechanisms. Primarily, excessive activation of EGFR signaling leads to abnormal proliferation of NP cells and exacerbates inflammatory processes, resulting in increased production of matrix-degrading enzymes, such as matrix metalloproteinases (MMPs) and ADAMTs. These enzymes facilitate the degradation of COL2 and proteoglycans within the ECM. Secondly, EGFR can suppress NP cells autophagy and enhance apoptosis through downstream pathways like mitogen-activated protein kinase (MAPK)/ERK, hence compromising IVD cellular homeostasis [[182]60]. Moreover, mechanical stress may induce aberrant EGFR phosphorylation in IVD cells, potentially enhancing inflammatory signals (e.g., NF-κB pathway activation) via positive feedback mechanisms, so fostering a pro-degenerative milieu. As previously demonstrated, shTie2 transfection significantly impeded the degradation of the EGFR protein. Our subsequent objective was to determine the effect of Tie2 on the ubiquitination of EGFR. The overexpression of Tie2 was found to enhance the ubiquitination levels of endogenous EGFR in NP cells ([183]Fig. 7L). Further screening identified CBL as the primary E3 ubiquitin ligase associated with EGFR.CBL knockdown significantly reduced EGFR ubiquitination levels ([184]Fig. 7M). CHX chase studies demonstrated that CBL absence markedly impeded EGFR degradation ([185]Fig. 7N and [186]Fig. S9F). In NP cells transfected with Myc-CBL WT and subjected to 20 μM MG132 treatment for 6 h before collection, Wb analysis confirmed that CBL regulates EGFR protein levels via proteasomal ubiquitin-mediated degradation ([187]Fig. 7O). These data combined indicate that CBL functions as an EGFR-interacting protein that regulates EGFR protein homeostasis. Ubiquitination can transpire via seven specific lysine residues (K6, K11, K27, K29, K33, K48, and K63) on Ub. We co-transfected Myc-CBL and Flag-EGFR into NP cells using a panel of Ub mutants that retained just single lysine residues, alongside either wild-type Ub or specific mutants. This study illustrates that CBL selectively enhances K48-linked ubiquitination ([188]Fig. 7P). A comprehensive analysis was performed on seven previously identified potential EGFR ubiquitination sites, as cataloged in PhosphoSitePlus. To elucidate the specific catalytic sites of CBL on EGFR, we engineered a series of EGFR mutants incorporating Lys-to-Arg substitutions. The co-transfection of each EGFR mutant with HA-Ub-K48 and Myc-CBL into NP cells revealed that only two mutations, K489R and K538R, exhibited diminished CBL-mediated ubiquitination ([189]Fig. 7Q). Current literature demonstrates that the phosphorylation of CBL family members is crucial for substrate recognition, indicating that the phosphorylation state modulates their enzymatic activity. Our findings indicate that the mutation at the Y992 site in Tie2 resulted in a reduction of Tie2 phosphorylation, as well as a decrease in both the total CBL protein and its phosphorylated forms (refer to [190]Fig. S10A). Consequently, the half-life of the EGFR protein was extended, and the degradation rate diminished under these circumstances ([191]Fig. S10B). The overexpression of the kinase-dead Tie2 L914F mutant resulted in a reduction of CBL phosphorylation levels ([192]Fig. S10C). Moreover, the endogenous binding of EGFR to CBL was diminished subsequent to the knockdown of Tie2 in NP cells ([193]Fig. S10D–E). Treatment with the Tie2 activator SF/GO-gel@Trox significantly improved CBL-EGFR interaction, consistent with our earlier finding that SF/GO-gel@Trox expedites EGFR degradation ([194]Fig. S10F–G). The Y992 mutation in Tie2, however, obstructed CBL-EGFR binding ([195]Fig. S10H–I). Notably, in the investigation of SF/GO-gel@Trox's influence on the modulation of CBL-EGFR interaction through Tie2 regulation, it was observed that the Y992 mutation in Tie2 inhibited the CBL-EGFR interaction under identical SF/GO-gel@Trox treatment conditions ([196]Fig. S10J–K).A complicated regulatory interaction exists between Tie2 and EGFR, wherein Tie2 activation markedly suppresses EGFR activity and facilitates its destruction. Activated Tie2 may recruit the protein tyrosine phosphatase PTPN2 to dephosphorylate EGFR at essential tyrosine residues (e.g., Y1045), so directly blocking it signaling [[197]61,[198]62]. Simultaneously, the activation of Tie2 enhances the activity of CBL-family E3 ubiquitin ligases, leading to increased K48-linked polyubiquitination of EGFR. This process accelerates the proteasomal degradation of EGFR, thereby reducing its protein levels and attenuating prolonged signaling. Moreover, Tie2 and EGFR can create heterodimers on the cell membrane [[199]63,[200]64], potentially modifying EGFR conformation and impeding ligand-induced activation, so further inhibiting it signaling. In our study, we have demonstrated that the Tie2/EGFR axis plays a crucial role in regulating IDD by modulating the balance between anabolic and catabolic processes in NP cells. Specifically, we found that activation of Tie2 leads to the degradation of EGFR, thereby suppressing downstream signaling pathways that drive inflammation and cellular senescence. This finding is consistent with previous studies showing that EGFR signaling is involved in the pathogenesis of IDD through the activation of inflammatory pathways and the promotion of matrix-degrading enzymes [[201]65,[202]66]. The NF-κB pathway is a well-known driver of inflammation and cellular senescence, both of which are critical factors in IDD [[203]67]. Our results indicate that activation of Tie2 can suppress NF-κB signaling, thereby reducing the production of pro-inflammatory cytokines and the expression of senescence markers. This suppression is likely mediated through the degradation of EGFR, as EGFR signaling has been shown to activate NF-κB via multiple mechanisms, including the phosphorylation of IκB and the subsequent nuclear translocation of NF-κB subunits [[204]68,[205]69]. By reducing EGFR levels, Tie2 activation effectively attenuates NF-κB-driven inflammation and senescence, contributing to the preservation of disc health. With the aging and degeneration of the IVD, the expression of the NP progenitor cell marker Tie2 is downregulated, leading to a gradual decline in its regenerative potential [[206]70]. The PI3K-Akt pathway is another key signaling pathway involved in cell survival, proliferation, and anabolic processes. Previous studies and our study demonstrate that Tie2 activation enhances PI3K-Akt signaling, promoting the cells survival and function [[207]71,[208]72]. Theoretically, the activation of PI3K-Akt signaling by Tie2 not only supports cell survival but also promotes the expression of anabolic markers such as COL2 and ACAN, which are essential for maintaining the structural integrity of the IVD. The interactions between the Tie2/EGFR axis and other signaling pathways highlight the complexity of IDD pathogenesis. Our study suggests that Tie2 activation can modulate multiple pathways simultaneously, providing a coordinated response to maintain disc health. By suppressing EGFR signaling, Tie2 reduces inflammation and senescence driven by NF-κB, while simultaneously promoting anabolic processes through the PI3K-Akt pathway. This dual action underscores the potential of Tie2 as a therapeutic target for IDD, as it can address both the catabolic and anabolic aspects of the disease. 3.4. Verification of mechanism in vivo As shown in [209]Fig. 8A, at 4 weeks post-implantation, substantial luminescence from fluorescent-labeled viruses persisted in the hydrogel, thereby confirming the hydrogel's sustained viral release capability, with complete disappearance observed by the 8th week. [210]Fig. S11A illustrates the hydrogel's smooth microstructure, characterized by uniform pores measuring 10 μm, within which 100 nm viral particles are embedded. [211]Fig. S11B demonstrates a sustained release of viral particles in vitro over a period of approximately four weeks, thereby confirming the hydrogel's capability for controlled release through its stable degradation. Fig. 8. [212]Fig. 8 [213]Open in a new tab Effects of SF/GO-gel@virus on radiographic evaluations corresponding to rat coccygeal IVDs after the intervention. A: Representative bioluminescent imaging in the ROI in overlay images at 0, 4, and 8 weeks. B: Macroscopic IVD images, fitting picture, and the relative area of NP at 8 weeks after the intervention. C: Representative X ray radiographs of the rat tail at 0, 4 and 8 weeks after the intervention. D: Representative MRI scans were obtained at 0, 4 and 8 weeks after the intervention. All experiments were repeated six times. All data are expressed as the mean ± SD. One-way ANOVA and Tukey's multiple comparisons test were used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. IVD, intervertebral disc; NP, nucleus pulposus; MRI, magnetic resonance imaging; SF, silk fibroin; GO, graphene oxide; ROI, Region of interest. In IVD morphological assessment ([214]Fig. 8B), macroscopic morphological parameters were shown to be strongly correlated with IDD advancement and Pfirrmann grading. The control group and the SF/GO-gel@His-Tie2 group preserved normal IVD architecture, demonstrating adequately hydrated tissue with distinct delineation between the NP and AF. In contrast, the IDD group and the SF/GO-gel@His-Tie2 + Flag-EGFR group demonstrated pronounced tissue disorganization, as evidenced by a reduction in NP volume and an increase in fibrous tissue proliferation. Quantitative research indicated that the SF/GO-gel@His-Tie2 group exhibited a considerably larger relative NP area compared to the other intervention groups. The radiographic assessment revealed the varying impacts of therapies on the progression of IDD ([215]Fig. 8C–D). X-ray measurements indicated that the SF/GO-gel@His-Tie2 group demonstrated markedly smaller reductions in DHI% at both 4 and 8 weeks in comparison to the IDD group and the SF/GO-gel@His-Tie2 + Flag-EGFR group. Consistent MRI data indicated a significant reduction in signal intensity attenuation within the SF/GO-gel@His-Tie2 group, collectively affirming the protective role of Tie2 and highlighting the potential pro-degenerative effects associated with EGFR. Histopathological analyses corroborated these observations ([216]Fig. 9A–B and [217]Fig. S12A–B). HE staining revealed superior preservation of NP tissue architecture in the SF/GO-gel@His-Tie2 group, but the SF/GO-gel@His-Tie2 + Flag-EGFR group exhibited significant structural disruption. SO labeling similarly demonstrated a reduced loss of proteoglycans in the SF/GO-gel@His-Tie2 group. IF experiments conclusively demonstrated that the SF/GO-gel@His-Tie2 therapy greatly preserved COL2 and ACAN levels ([218]Fig. 9C–F and [219]Fig. S12C–D). Fig. 9. [220]Fig. 9 [221]Open in a new tab Effects of SF/GO-gel@virus on radiographic evaluations corresponding to rat coccygeal IVDs after the intervention. A: Representative HE staining and SO staining images of rat coccygeal IVDs corresponding to different treatment groups (coronal position) at 4 weeks after the intervention. B: Representative HE staining and SO staining images of rat coccygeal IVDs corresponding to different treatment groups (coronal position) at 8 weeks after the intervention. C–D: IF staining images of COL2 and ACAN at 4 weeks after the intervention. E–F: IF staining images of COL2 and ACAN at 8 weeks after the intervention. All experiments were repeated six times. All data are expressed as the mean ± SD. One-way ANOVA and Tukey's multiple comparisons test were used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. IVD, intervertebral disc; HE, hematoxylin and eosin; SO, safranin O-fast green; IF, immunofluorescence; COL2, collagen type II; ACAN, aggrecan; SF, silk fibroin; GO, graphene oxide. IDD is a progressive disorder characterized by structural and biochemical alterations in the IVD, exhibiting distinct stage-dependent pathological features: early stages are dominated by inflammation and oxidative stress, while advanced stages manifest primarily as ECM degradation and cellular senescence [[222]73,[223]74]. Given these pathological characteristics, developing stage-specific therapeutic strategies is crucial. Ultrasound-triggered drug delivery technology has emerged as an ideal approach for IDD treatment due to its unique advantages [[224][75], [225][76], [226][77], [227][78], [228][79]]: (1) Precise targeted delivery: ultrasound can be accurately focused on degenerated discs to achieve localized drug release through enhanced tissue permeability, minimizing systemic side effects; (2) Stage-regulated drug release: by adjusting ultrasound parameters (frequency, intensity, and duration), anti-inflammatory drugs can be released during inflammatory phases (high-frequency short-duration), while matrix-repairing and anti-senescence agents can be delivered in advanced degeneration (low-frequency long-duration); (3) Enhanced drug penetration: ultrasound cavitation creates transient pores in disc matrix, significantly improving bioavailability of macromolecular drugs; (4) Controlled release kinetics: enabling sustained drug delivery to manage chronic disease progression. The SF/GO-gel @Trox developed in this study demonstrates exceptional biocompatibility, mechanical robustness, and controlled drug release properties. In vivo experiments confirmed that ultrasound-triggered Trox release effectively maintained disc height and hydration (validated by X-ray and MRI), with histological analysis showing preserved ECM expression. This technology offers novel perspectives for managing IDD and other degenerative spinal disorders through precise drug delivery control, enhanced bioavailability, and stage-specific therapeutic effects. 4. Conclusion This study demonstrated that the ultrasound-mediated SF/GO-gel@Trox system effectively alleviates degeneration and senescence in NP cells. This innovative drug delivery platform exhibits enhanced biocompatibility, mechanical properties, swelling behavior, and sustained drug release capabilities. Furthermore, our research revealed that Trox-activated Tie2 influences K48-linked ubiquitination of EGFR through CBL, while the knockdown of Tie2 exacerbates characteristics of NP degeneration and senescence. In the context of CBL-mediated ubiquitination and degradation of EGFR, we identified that both Tie2 autophosphorylation and Tie2-induced CBL phosphorylation are critical for facilitating the interaction between CBL and EGFR. These findings provide novel insights into the regulatory mechanisms governing EGFR and CBL, suggesting that reduced levels of EGFR protein may alleviate the characteristics associated with NP degeneration and senescence. Ethics statement. The study was reviewed and approved by Ethics Committee of Shanghai East Hospital (TJBB05022101). CRediT authorship contribution statement Youfeng Guo: Writing – original draft, Methodology, Formal analysis, Data curation, Conceptualization. Xiao Liu: Funding acquisition, Data curation, Conceptualization. Chao Wang: Methodology, Investigation, Data curation. Shuguang Wang: Methodology, Investigation, Funding acquisition, Formal analysis. Yufeng Huang: Investigation, Funding acquisition. Shuo Ding: Funding acquisition. Zeyu Wang: Funding acquisition. Feng Wang: Writing – review & editing, Writing – original draft, Funding acquisition, Conceptualization. Consent for publication All patients gave their consent for publication. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments