Abstract Background Intervertebral disc degeneration (IDD) is a chronic degenerative disorder marked by nucleus pulposus cells (NPCs) senescence and extracellular matrix (ECM) degradation, which is a key pathological factor leading to low back pain. Current clinical treatments are mainly limited to symptomatic relief without effectively reversing the degenerative process. Results We revealed that lysyl oxidase-like protein 2 (LOXL2) is involved in the molecular mechanism of NPCs senescence through the regulation of the Notch signaling pathway, and developed a novel therapeutic strategy. We propose an injectable, degradable and ROS-responsive hydrogel delivery system (LNP-LOXL2@Gel), which mimics the biomechanical properties of natural intervertebral disc tissues and can be used as a sustained-release vehicle for LNP-LOXL2. Its ROS scavenging ability complements the intracellular anti-aging effect of LOXL2, blocking the cascade of “oxidative stress-cellular senescence-ECM degradation”. In the rat IDD model, LNP-LOXL2@Gel not only reduced nucleus pulposus senescence, but also inhibited ECM catabolism by decreasing the expression of ROS in the microenvironment, thus partially restoring the physiological function of the intervertebral disc. Conclusions LOXL2 expression is down-regulated in degenerated discs, and its overexpression inhibits the Notch pathway and delays NPCs senescence. The LNP-LOXL2@Gel hydrogel system, which can effectively alleviate IDD by synergistically integrating the antiaging effect of LOXL2 and the ROS scavenging ability of PVA-tsPBA, provides a new targeting therapeutic strategy for the clinical treatment of IDD. Graphical Abstract [42]graphic file with name 12951_2025_3718_Figa_HTML.jpg Supplementary Information The online version contains supplementary material available at 10.1186/s12951-025-03718-y. Keywords: Intervertebral disc degeneration, Senescence, Lysyl oxidase-like protein 2, Extracellular matrix, Notch signaling pathway Introduction Low back pain (LBP) has become an urgent public health problem with the aging of the global population and the increasing prevalence of age-related diseases [[43]1, [44]2]. Intervertebral disc degeneration (IDD) is the main cause of LBP, which can lead to a series of conditions such as disc herniation, spinal stenosis, and spinal instability, seriously affecting the quality of life of patients [[45]3]. It is generally thought that a number of factors, such as aging, mechanical compression, and heredity, contribute to the development of IDD [[46]4–[47]6]. Currently, the treatment of IDD relies mainly on medications and physical therapy, but it can only temporarily relieve symptoms and cannot block the progression of the disease [[48]7, [49]8]. Although surgery can be used as a last resort, it will damage the anatomical structure of the intervertebral disc (IVD), affecting functional reconstruction, and may lead to complications such as degeneration of neighboring segments [[50]9]. Therefore, the search for novel intervention targets is of great significance for preventing and treating IDD, and delaying or even reversing its progression. Cellular senescence, defined by irreversible cell cycle arrest, is a key factor in the pathogenesis of age-related dysfunction and chronic degenerative diseases [[51]10, [52]11]. Studies have shown that senescent nucleus pulposus cells (NPCs) show an accumulation trend during the course of IDD [[53]12–[54]14]. Senescence of NPCs is closely related to molecular mechanisms such as abnormal release of inflammatory factors, elevated levels of oxidative stress, and mitochondrial dysfunction [[55]15–[56]17]. Under physiological conditions, reactive oxygen species (ROS) are mainly concentrated in mitochondria and maintain a dynamic balance [[57]18]. However, in degenerated IVD, ROS accumulate in a closed microenvironment, leading to mitochondrial dysfunction and cellular senescence [[58]19]. In addition, depolarized mitochondria further produce more ROS, thus creating a vicious cycle that leads to sustained oxidative stress and mitochondrial damage, thereby accelerated NPCs senescence [[59]19]. The human LOXL2 gene is located on chromosome 8p21.3 region and encodes a protein consisting of 774 amino acids [[60]20]. LOXL2 has been strongly implicated in the regulation of cellular senescence [[61]21–[62]23]. Down-regulation of LOXL2 expression induced a senescent phenotype in hepatocellular carcinoma cells, accompanied by upregulated expression of senescence markers p21 and p16 [[63]24]. In addition, adenovirus-mediated delivery of the LOXL2 gene or systemic overexpression of LOXL2 in transgenic mice prevented senescence-associated osteoarthritis of the knee [[64]25]. However, studies of LOXL2 in the IDD are relatively limited. Here we found that LOXL2 was significantly downregulated in degenerated nucleus pulposus (NP) samples. We further experimentally verified that LOXL2 was down-regulated in degenerated NP tissues and negatively correlated with the grade of IDD, whereas the expression of p21 and p16 was up-regulated with the progression of degeneration. This suggests that LOXL2 may affect disc degeneration by regulating NPCs senescence. Gene therapy is gradually demonstrating its incomparable advantages and great potential [[65]26]. However, the problems of nucleic acid degradation, short duration of action, and low efficiency of targeted delivery seriously limit the therapeutic effect [[66]26]. Lipid nanoparticles (LNPs), as efficient and biocompatible delivery vehicles, have been widely used for nucleic acid delivery [[67]27], but challenges remain in the treatment of degenerative disc disease. Directly injected LNP drug solutions are prone to leakage from the injury site, which not only leads to decreased drug bioavailability but also to systemic toxicity [[68]28]. Since NP tissues are highly hydrated gel-like structures, hydrogels are ideal carriers due to their similar mechanical properties [[69]29]. What’s more, the responsive hydrogel system is able to sense the degenerative IVD microenvironment and dynamically release therapeutic drugs, showing great potential for clinical application and providing a new solution for the treatment of IDD [[70]30, [71]31]. In this study, we developed an injectable, ROS-responsive hydrogel system (LNP-LOXL2@Gel), which can continuously release LNP loaded with LOXL2 (LNP-LOXL2) to attenuate the senescence phenotype of NPCs and inhibit the process of IDD. We first demonstrated that LOXL2 attenuates mitochondrial damage and cellular senescence in response to intracellular oxidative damage. Mechanistically, LOXL2 binds to the NOTCH1 promoter to suppress Notch signaling and inhibit NPCs senescence. Then, we loaded LNP-LOXL2 into PVA-tsPBA hydrogel to make a multifunctional hydrogel system. The sensitivity of its boronate ester bond to ROS enabled the system to realize the precise controlled release of LNP-LOXL2 according to the elevated ROS levels in the microenvironment of the degenerated IVD. In a rat model of IDD, the combination of PVA-tsPBA hydrogel and LNP-LOXL2 showed synergistic therapeutic effects, alleviating the NPCs senescence and ECM degradation. Materials and methods Clinical specimen Human NP tissue samples were obtained from patients who underwent lumbar discectomy in the Second Affiliated Hospital of Nanchang University. All patients were examined by MRI before surgery and grouped according to the Pfirrmann grading system for imaging evaluation [[72]32]. Detailed information is listed in Table S2. The study protocol was reviewed and approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University. Cell transfection LOXL2 overexpression plasmid pcDNA-LOXL2 and empty vector pcDNA were provided by Focus Bioscience (Shanghai, China). The plasmids were transfected into 293 T cells using Lipofectamine 2000 (Invitrogen, USA). The viral supernatant was collected 48 h after transfection. The collected lentiviral supernatants were co-cultured with NPCs, with the fresh medium supplanted 24 h after infection. Reactive oxygen species (ROS) detection Dihydroethidium (DHE, Beyotime, China) and MitoSOX (AbMole BioScience, USA) were used as fluorescent probes for cytoplasmic and mitochondrial ROS, respectively. The fluorescence intensity was visualized by fluorescence microscopy after completing the staining of the samples according to the instruction procedure. Preparation and characterization of hydrogels PVA-tsPBA hydrogels were prepared as described previously [[73]31]. To prepare LNP-LOXL2@Gel for in vivo experiments, 10 µL LOXL2 plasmid-LNP complex (containing 2 µg of LOXL2 plasmid) was introduced into the same volume of tsPBA solution. Subsequently, 25 µl of the mixture for was combined with 25 µl of PVA to form 50 µl of PVA-LNP-LOXL2@Gel, with a final plasmid concentration of 40 µg/ml [[74]33]. FT-IR spectroscopy (Thermo Scientific Nicolet iS10, USA) was used to analyze in order to examine the functional groups in the PBA-tsPBA based hydrogels. The HAAKE MARS40 Rheometer (Thermo Fisher Scientific, USA) was used to evaluate the rheological properties and self-healing capabilities of hydrogels. The energy storage modulus, loss modulus and critical strain of hydrogel samples were tested as described previously [[75]34]. In addition, cutting and reconnection experiments were performed to investigate the fusion mechanism and to assess the self-healing ability of the hydrogels. The ultrastructure of the hydrogel was then observed using SEM (Sigma 360, Zeiss). Morphological observations of LNP’s encapsulated in hydrogels were performed by scanning electron microscopy (SEM, Zeiss Ultra 55, Germany) and laser confocal microscopy (Olympus, Japan). Confocal scanning was performed to detect the Cy3 signal of the LNPs and the images were reconstructed in three dimensions. Animal models and treatment Male Sprague-Dawley rats (2 months old) were used to construct the IDD model as described previously [[76]35]. After the experimental animals were induced anesthesia by a mixture of isoflurane and oxygen, the caudal (C7/8) intervertebral disc was localized by palpation. After the surgical area was cleaned with povidone-iodine, a 21G puncture needle was inserted perpendicular to the caudal vertebrae, slowly penetrating the annulus fibrosus to the NP region, rotated 360° and maintained for 30 s to induce degeneration. Then, 5 µl of material (or PBS) was injected into the NP tissue through a 22-gauge needle. The rats were routinely housed for 4 weeks, during which time they were observed daily for activity status. The rats were divided into Sham, PBS (degeneration + PBS), Gel (degeneration + hydrogel), LNP-LOXL2 (degeneration + LNP-LOXL2) and LNP-LOXL2@Gel group (degeneration + LNP-LOXL2@Gel), and the corresponding interventions were carried out after successful puncture. Intervertebral disc imaging evaluation At postoperative weeks 4 and 8, disc height index (DHI) was determined by radiography, while Pfirrmann grading was performed by MRI T2-weighted imaging, which was used to systematically evaluate the pathological progression of IDD. Histological analysis and immunohistochemistry After radiological examination was completed, rat intervertebral disc tissue samples were collected, fixed in 4% paraformaldehyde, dehydrated in gradient ethanol, paraffin-embedded and prepared for sectioning. Hematoxylin-eosin (HE) staining and Safranin O-Fast Green staining were used for histologic evaluation. The expression levels of LOXL2, COL2A1 and p16 proteins were also detected by immunohistochemical staining. Statistical analysis Experimental data are presented as mean ± standard deviation. Differences between groups were analyzed using independent samples t-tests or one-way ANOVA, with p-values < 0.05 considered statistically significant. All statistical analyses were performed using GraphPad Prism 10.1.2. Additional methods and materials are included in the Supplementary Information. Results Expression pattern of senescence-related genes in degenerated NP tissues We systematically analyzed senescence-related genes (SRGs) in NP tissues. As shown in Figs. [77]1A and 830 candidate SRGs were first obtained by integrating the [78]GSE70362 dataset with the CellAge database. Differential expression analysis identified a total of 90 SRGs with significant expression changes, of which 47 were downregulated and 43 were upregulated. These differentially expressed SRGs were visualized by heatmap (Fig. [79]1B). KEGG pathway enrichment analysis showed that differentially expressed genes (DEGs) were enriched in cellular senescence, ROS, and p53 signaling pathways (Fig. [80]1C). GO enrichment analysis showed that in biological processes, DEGs were mainly enriched in response to oxidative stress, cell growth regulation, and response to ROS; in cellular components, these genes were primarily enriched in spindle, PML body and autophagosome (Fig. [81]1D). PPI network showed that the differentially expressed SRGs closely interacted with each other at the protein level (Fig. [82]1E). Fig. 1. [83]Fig. 1 [84]Open in a new tab Expression pattern of senescence-related genes in degenerated NP tissues. (A) Workflow of SRGs analysis in NP tissues; (B) Heatmap of differentially expressed SRGs; (C) KEGG pathway analysis; (D) GO analysis; (E) Protein molecule interaction network LOXL2 is the key SRG in intervertebral disc degeneration To further identify key SRGs, we filtered the above 90 differentially expressed SRGs by three machine learning algorithms (Fig. [85]2A). Eight feature genes were obtained by the LASSO algorithm (Fig. [86]2B), and 10 feature genes were obtained by the SVM-REF algorithm and RF algorithm respectively (Fig. [87]2C, D). The genes obtained by the three algorithms were taken to intersect to obtain the key gene LOXL2, and the Venn diagram was plotted (Fig. [88]2E). The ROC curves demonstrated the superior ability of LOXL2 to diagnose IDD versus control samples (Fig. [89]2F). And LOXL2 expression was negatively correlated with disc degeneration grade (Fig. [90]2G). To validate LOXL2 expression, independent datasets [91]GSE70362, [92]GSE56081, [93]GSE167199, and [94]GSE42611 were downloaded. The box plot demonstrated that the expression level of LOXL2 was significantly reduced in degenerated NP tissues (Fig. [95]2H). Similarly, the expression level of LOXL2 was reduced in TNF, IL-1β and serum starvation-treated rat NPCs (Fig. [96]2I). Fig. 2. [97]Fig. 2 [98]Open in a new tab Three machine learning algorithms screened the key senescence gene LOXL2. (A) Flowchart of key gene screening and analysis; (B) LASSO Cox regression analysis; (C) SVM-REF analysis; (D) Random forest analysis; (E) Venn plot of the intersection taken by three machine learning algorithms. (F) ROC curve of LOXL2; (G) Correlation between LOXL2 expression and disc grade; (H) Differential expression of LOXL2 in internal and external datasets; (I) Differential expression of Loxl2 in rat NPCs. *p < 0.05, **p < 0.01 Subsequently, GSEA was performed to explore the biological functions and potential pathways associated with LOXL2. the ECM and ECM constituents were positively correlated with LOXL2. Ribonucleoprotein complex biogenesis, ribosome biosynthesis, and ribosomes in organelles were negatively associated with LOXL2 (Figure S1A). We also noted that pathways were associated with LOXL2, such as “cytokine-cytokine receptor signaling,” “extracellular matrix-receptor interactions,” and “adhesion spots” (Figure S1B). GeneMANIA shows LOXL2-interacting proteins and associated functions (Figure S1C). Non-coding RNA (ncRNA) plays an important role in the regulation of mRNA expression. To explore the upstream of LOXL2, we predicted 21 miRNAs that may target LOXL2 and further identified 40 lncRNAs that can interact with these miRNAs. Based on these regulatory relationships, we constructed a ceRNA network (Figure S1D). To verify the reliability of this network, we utilized independent databases to validate the expression levels of ncRNAs. The results showed that five miRNA expressions were up-regulated (Figure S1E) and four lncRNA expressions were down-regulated (Figure S1F) in degenerating NP tissues. LOXL2 expression is downregulated in degenerated NP tissue Based on the Pfirrmann grading system, NP tissues with different degrees of degeneration showed characteristic signal intensity changes on T2-weighted MRI (Fig. [99]3A). This result indicated a progressive decrease in the water content of the NP tissues with the progression of IDD. IHC results showed that the expression level of p21 and p16 was significantly up-regulated in the degeneration group compared to the control group, while the expression of LOXL2 tended to be down-regulated (Fig. [100]3B-E). The qPCR results were consistent with this (Fig. [101]3I, J). Further correlation analysis showed that p21 and p16 mRNA expression was positively correlated with degeneration grade, while LOXL2 expression was negatively correlated. (Fig. [102]3F-H). Western blot assay results were highly consistent with the changes in mRNA expression, and the ECM markers ACAN and COL2A1 were downregulated in degenerated NP tissues (Fig. [103]3K, L). Fig. 3. [104]Fig. 3 [105]Open in a new tab LOXL2 expression is downregulated in degenerated intervertebral discs. (A) Representative MRI images of intervertebral discs with different grades of degeneration; (B) IHC staining of LOXL2, p21, and p16 in NP tissues; (C-E) quantitative analyses of immunohistochemistry; (F-H) linear regression analyses between the mRNA levels of LOXL2, p16, and p21 and the disc grades; (I, J) qPCR analyses of LOXL2, p16, and p21 mRNA levels; (K, L) WB analysis of LOXL2, p16 and p21 protein levels in human intervertebral discs; (M, N) WB analysis of LOXL2, p16 and p21 protein levels in rat intervertebral discs; (O) HE, SO, IHC, IF staining of rat intervertebral discs. *p < 0.05, **p < 0.01, *** p < 0.001 We constructed a rat IDD model for further validation. The qPCR results showed that the expression of p21 and p16 mRNA were up-regulated (Figure S2A), whereas the expression of Loxl2 mRNA was down-regulated (Figure S2B), and this trend was further verified in Western blot (Fig. [106]3M, N). The down-regulated of LOXL2 was verified by histologic staining (HE and SO), IHC and IF (Fig. [107]3O). IHC results showed that p21 and p16 were up-regulated in degenerating rat NP tissues (Figure S2C). Overexpression of LOXL2 inhibits the senescence process of NPCs NPCs were isolated from rat normal NP tissues, and then LOXL2 overexpressing cell clones were constructed by lentiviral vectors to explore the function of LOXL2 (Fig. [108]4A). Under light microscopy NPCs were elongated, spindle-shaped or irregularly polygonal (Figure S3A). Immunofluorescence staining showed abundant expression and uniform distribution of ACAN and COL2A1 in NPCs (Figure S3B, C). To determine the optimal concentration of TBHP for inducing senescence in NPCs, we set a concentration gradient of 0, 25, 50, 75, 100, 150, and 200µM. CCK8 showed that when the concentration hit 50µM, NPC cell viability dramatically declined, and decreased in a dose-dependent manner as the concentration increased (75–200µM) (Figure S3D). Therefore, we chose 50µM TBHP as the treatment concentration for the subsequent experiments. Fig. 4. [109]Fig. 4 [110]Open in a new tab Overexpression of LOXL2 inhibits the senescence process of nucleus pulposus cells. (A) Schematic flowdiagram of the experimental design; (B) WB detection of protein expression of LOXL2, ACAN, COL2A1, p21, p16 afterTBHP treatment; (C) Immunofluorescence of LOXL2; (D) Crystal violet staining and quantitative analysis of cellularclones; (E) EdU staining and quantitative analysis; (F) SA-β-gal staining and quantitative analysis ; (G) DHE stainingand quantitative analysis of ROS levels; (H, I) Protein expression of ECM-related genes, SASP, and senescencerepresentativegenes in NP cells; (J) Heatmap of qPCR demonstrating ECM-related gene, SASP, and senescencerepresentativegene expression; (K) MitoSOX/MitoTracker staining to detect the level of ROS in the mitochondria; (L)JC-1 fluorescent dye to detect the mitochondrial membrane potential. *p < 0.05, **p < 0.01, ***p < 0.001. The qPCR results showed that TBHP treatment up-regulated the mRNA expression levels of p21 and p16, while causing a down-regulation of Loxl2, Acan and Col2a1 (Figure S3E). Western blot showed a trend consistent with the above (Fig. [111]4B, S3F). Immunofluorescence staining showed that the fluorescence intensity of p16 and p21 was enhanced in the cells of the TBHP-treated group (Figure S3G), while the LOXL2 was weakened (Figure S3H). To clarify the molecular features of TBHP-induced senescence in NPCs, we performed mRNA sequencing. PCA results showed that the TBHP-treated and control NPCs exhibited grouping aggregation (Figure S3I), indicating significant differences in the transcriptome of the two groups. Further analysis revealed that both the oxidative stress-related senescence gene set (Figure S3J) and the DNA damage-induced senescence gene set (Figure S3K) showed differential expression patterns. In summary, we confirmed that TBHP effectively induced NPCs into senescence and down-regulated LOXL2 expression in senescent NPCs. We constructed cells with stable overexpression of LOXL2 by infecting NPCs with lentiviral vector (Lv-LOXL2). According to the qPCR results, the Lv-LOXL2 group had higher levels of LOXL2 mRNA expression (Figure S3L), while LOXL2 protein expression also showed a significant increase (Figure S3M, N). Moreover, immunofluorescence showed positive staining of LOXL2 in both cytoplasm and nucleus (Fig. [112]4C). Crystalline violet and EdU staining showed that TBHP treatment inhibited the proliferation of NPCs, and LOXL2 overexpression partially reversed this effect (Fig. [113]4D, E). In addition, SA-β-gal staining showed that overexpression of LOXL2 reduced the number of SA-β-gal positive cells upon TBHP treatment (Fig. [114]4F). DHE staining showed that ROS levels were reduced in the Lv-LOXL2 group compared with the Lv-NC group (Fig. [115]4G). Moreover, overexpression of LOXL2 reversed TBHP-induced p21 and p16 expression (Fig. [116]4H, I). Notably, TBHP treatment also upregulated the expression of SASPs (Adamts5, Mmp13, Il-1β, Il-6), and overexpression of LOXL2 was able to partially reverse the effects of TBHP (Fig. [117]4J). Mitochondrial dysfunction is one of the key features of cellular senescence. The results showed that mitochondrial ROS levels were elevated in NPCs after treatment with TBHP, and LOXL2 overexpression inhibited TBHP-induced mitochondrial oxidative stress (Fig. [118]4K). To further evaluate the mitochondrial membrane potential changes, we used JC-1 fluorescent probe for detection. The results showed that the red/green fluorescence ratio of JC-1 in NPCs was reduced after TBHP treatment. Notably, LOXL2 overexpression significantly ameliorated this pathological change and maintained the stability of mitochondrial membrane potential (Fig. [119]4L). Sequencing of mRNA from NPCs after overexpression of LOXL2 To elucidate the molecular mechanism of LOXL2 action in NPCs, we performed mRNA sequencing. As seen in the heat map (Fig. [120]5A) and volcano plot (Fig. [121]5B), a total of 601 DEGs were detected compared with the control group, of which 432 were up-regulated and 169 were down-regulated. The PCA results showed that LOXL2 overexpression led to alterations of the transcriptome features of the NPCs (Fig. [122]5C). GO analysis showed an enrichment of extracellular matrix-regulated, cell cycle-related biological processes (Fig. [123]5D). In terms of cellular components, pathways related to ECM were enriched. In addition, molecular functions showed enrichment in growth factors and cell adhesion binding pathways (Fig. [124]5D). Based on the results of GO analysis, we further screened for DEGs associated with key biological processes such as cell cycle, ECM remodeling and cell division (Fig. [125]5E). In addition, KEGG pathway enrichment results indicated that the DEGs were mainly involved in important biological pathways such as cytokine-cytokine receptor interaction pathway, Notch signaling pathway, and IL-17-mediated signal transduction pathway (Fig. [126]5F). GSEA analysis showed that a variety of signaling pathways related to cell proliferation and senescence were altered after LOXL2 overexpression, and the top ten pathways that were most up- and down-regulated are shown in Fig. [127]5G. LOXL2 overexpression promoted the expression of cell cycle-related genes (Fig. [128]5H), while inhibiting the activity of Notch signaling pathway (Fig. [129]5I), suggesting that Notch signaling may be be involved in the LOXL2-mediated senescence inhibition process in NPCs. Fig. 5. [130]Fig. 5 [131]Open in a new tab Sequencing of mRNA from nucleus pulposus cells after overexpression of LOXL2. (A, B) Heatmaps and volcano plots demonstrating the overall changes in the transcriptome; (C) PCA analysis; (D, E) GO enrichment analysis; (F) KEGG pathway analysis; (G) Top 10 pathways activated and repressed after overexpression of LOXL2; (H) GSEA analysis of the cell cycle; (I) GSEA analysis of the Notch pathway Overexpression of LOXL2 inhibits the Notch pathway Previous research has shown that the Notch signaling pathway plays a role in regulating cellular senescence [[132]36, [133]37]. Notably, the components of the Notch signaling pathway (including Notch1, Jag1 and Psen1) showed up-regulation in LOXL2 overexpressing NPCs (Fig. [134]6A). In addition, cell cycle-related genes also showed differential expression changes (Fig. [135]6A). In degenerated NP tissues, we confirmed that the expression of key effector molecules of Notch signaling, NOTCH1, HES1, and HEY1, was significantly upregulated (Fig. [136]6B, S4A). The qPCR results showed a 1.5-2.2-fold increase in NOTCH1, HES1, and HEY1 mRNA expression (Figure S4B), and Western blot further demonstrated that their expression was also elevated (Fig. [137]6C, D). Immunofluorescence demonstrated that TBHP activated the Notch signaling in NPCs (Fig. [138]6E). Fig. 6. [139]Fig. 6 [140]Open in a new tab Overexpression of LOXL2 inhibits the Notch pathway. (A) Heatmap of cell cycle, Notch-related pathway genes expression; (B) Immunohistochemical staining of NOTCH1, HES1, and HEY1 in human NP tissues; (C, D) WB detection of NOTCH1, HES1, and HEY1 in human NP tissues; (E) Immunofluorescence of TBHP-treated NPCs; (F) mRNA levels of Notch1, Acan, Col2a1, p21, and p16; (G, H) Protein level expression of Notch1, Acan, Col2a1, p21, and p16; (I, K, M) Effects of DAPT on cell proliferation, ROS accumulation, and senescence phenotypes in TBHP-treated NPCs; (J, L, N) Quantitative analyses of EdU staining, DHE staining, and SA-β-gal staining; (O,P) DAPT inhibits TBHP-induced impairment of mitochondrial function; (Q,R) WB detection of protein levels of NOTCH1, HES1, and HEY1; (S) CHIP-qPCR validation of the binding relationship between LOXL2 and NOTCH1 promoters. *p < 0.05, **p < 0.01, *** p < 0.001 In view of the potential role of the Notch signaling in cellular senescence, we intervened with the γ-secretase inhibitor DAPT, a NOTCH1-specific activation inhibitor. The results showed that DAPT treatment not only inhibited the Notch signaling pathway, but also ameliorated the catabolic abnormalities of ECM and partially suppressed the expression of p21 and p16 (Fig. [141]6F-H). EdU staining showed that TBHP treatment reduced the proliferation of NPCs, while DAPT increased the proliferation of NPCs when compared to DMSO-treated controls (Fig. [142]6F-H). In addition, ROS levels were significantly lower in DAPT-treated NPCs compared with DMSO-treated controls (Fig. [143]6K, L), and the proportion of senescent cells in the DAPT group was lower than that in the DMSO-treated group (Fig. [144]6M, N). MitoSOX staining showed that DAPT treatment inhibited the TBHP-induced elevation of mitochondrial ROS (Fig. [145]6O). Moreover, DAPT intervention also maintained the stability of membrane potential (Fig. [146]6P). The above findings indicated that DAPT effectively delayed the senescence process of NPCs by inhibiting the Notch signaling pathway. To further clarify the regulatory relationship between LOXL2 and Notch signaling pathway, we analyzed the expression changes of key molecules of Notch signaling after overexpression of LOXL2. The results showed that the overexpression of LOXL2 significantly down-regulated the expression levels of NOTCH1, HES1 and HEY1 (Figure S4C, Fig. [147]6Q, R). CHIP- qPCR results showed that LOXL2 could specifically bind to the promoter region of NOTCH1 (Fig. [148]6S). The above results suggest that LOXL2 may inhibit the transcription of NOTCH1 by directly acting on its promoter region. Characterization of LNP-LOXL2@Gel Naked nucleic acids are susceptible to enzymatic degradation in vivo, and encapsulation of LNPs protects the nucleic acids [[149]27]. However, direct injection of LNPs may result in leakage, leading to random diffusion of the drug. We developed a PVA-tsPBA hydrogel-based delivery system (LNP-LOXL2@Gel), for the efficient delivery of LNP-LOXL2. The hydrogel system was able to efficiently maintain the bioactivity of LOXL2, and at the same time could respond to the high ROS levels in the microenvironment of the degenerated disc to achieve sustained release of LNP-LOXL2. Figure [150]7A shows a schematic of the preparation process of LNP-LOXL2@Gel. LNP-LOXL2 was first dispersed in a 6% tsPBA solution and subsequently mixed with an equal volume of 6% PVA solution to form a stable LNP-LOXL2-loaded hydrogel system via a rapid cross-linking reaction. FT-IR analysis confirmed that the spectral peaks of the hydrogels included those of PVA and tsPBA (Figure S6A), consistent with previous studies [[151]38]. Fig. 7. [152]Fig. 7 [153]Open in a new tab Characterization of LNP-LOXL2@Gel. (A) Schematic of the synthesis of ROS-responsive LNP-LOXL2@Gel; (B) Self-repairing behavior of LNP-LOXL2@Gel; (C) Injectability of LNP-LOXL2@Gel; (D) Compression and tensile properties of the hydrogel demonstrated; (E) Energy storage modulus (G’) and loss modulus (G”) of LNP-LOXL2@Gel; (F) G’ and G“ measured by strain amplitude scanning at a fixed frequency (1 Hz); (G) Variation of G’ and G” of LNP-LOXL2@Gel with temperature; (H) Dynamic shear rheological properties of LNP-LOXL2@Gel; (I) SEM microscopy images showing the adhesion of LNP-LOXL2 on the surface of the PVA-tsPBA hydrogel; (J) LNP- LOXL2@Gel in 3D and 2D images of LNP-LOXL2 distribution; (K) In vivo bioluminescence imaging after injection of LNP-LOXL2 and LNP-LOXL2@Gel Ideal disc repair hydrogels should have mechanical properties comparable to those of natural disc tissues, including suitable cushioning capacity, flexibility, and elasticity. In this study, the self-repairing ability and mechanical properties of LNP-LOXL2@Gel hydrogel were systematically evaluated. The results showed that the severed hydrogel sections could be fused autonomously within 10 min to restore the complete structure (Fig. [154]7B). Injectability experiments confirmed that the hydrogel was able to pass through a syringe needle smoothly and maintain structural stability in an aqueous environment (Fig. [155]7C). In addition, the hydrogel exhibited good compression and tensile properties (Fig. [156]7D). Next, we evaluated the ROS responsiveness of LNP-LOXL2@Gel. We used 50 nM TBHP treatment to simulate an oxidative stress microenvironment, and the hydrogel exhibited significant ROS-responsive properties: under TBHP conditions, the degradation rate reached 80% within 5 days, whereas the PBS group still retained > 20% of the hydrogel on day 15 (Figure S6B). PicoGreen quantitative assay showed that pcDNA-LOXL2 exhibited a significant burst release on day 3, and then reached a release plateau on day 7 (Figure S6C), further confirming the ROS responsiveness of the material. We further evaluated the biocompatibility of LNP-LOXL2@Gel in vitro. The results of live/dead cell staining and CCK-8 assay confirmed that neither the Gel group nor the LNP-LOXL2@Gel group significantly affected cell proliferation (Figure S6D, E). We conducted a systematic study of the rheological properties of LNP-LOXL2@Gel. Frequency scans showed that the storage modulus (G’) and loss modulus (G’) remained almost constant when the frequency was increased from 0.01 to 100 Hz, revealing the stability of the hydrogel in the natural range (Fig. [157]7E). The strain scan of the hydrogel showed that the G’ and G’’ of the hydrogel remained almost constant until the strain reached about 63% (Fig. [158]7F). Temperature scanning tests showed that the G’ of the samples increased very slowly with increasing temperature, indicating that the dynamic regulation of the hydrogel stiffness was slightly affected by temperature (Fig. [159]7G). The hydrogel’s self-healing capabilities were shown by successive alternating strain scans. When a high dynamic strain (100%) was applied, G’ was lower than that of the hydrogel, indicating collapse of the hydrogel network. Once a lower strain (1%) was applied, the hydrogel structure was immediately restored (Fig. [160]7H). Scanning electron microscopy (SEM) observations confirmed the 3D network of the hydrogel, as well as the successful encapsulation and homogeneous distribution of LNP-LOXL2 nanoparticles in the hydrogel porous structure (Fig. [161]7I). In addition, micrographs exhibited 3D and 2D uniform distribution of fluorescently labeled LNP-LOXL2 in the hydrogels (Fig. [162]7J). LNP-LOXL2@Gel remained in the intervertebral disc for up to 14 days compared to direct injection of LNP-LOXL2, indicating better retention of LOXL2 with the hydrogel delivery system (Fig. [163]7K). LNP-LOXL2@Gel inhibited disc degeneration At 4/8 weeks postoperatively, the disc condition of each group was assessed by radiographic imaging and histology (Fig. [164]8A), and the effect of tissue repair was evaluated using the Disc Height Index (DHI) (Fig. [165]8B). The analysis of the X-ray imaging revealed a decrease in the DHI in the PBS and Gel groups compared to the control group. The LNP-LOXL2 and LNP-LOXL2@Gel groups both partially maintained disc height, and the LNP-LOXL2@Gel group showed a stronger treatment effect. (Fig. [166]8C, E). The MRI findings further confirmed that the PBS and Gel groups exhibited typical degenerative features, including signal attenuation of the vertebral T2-weighted image and a decrease in the height of the spinal interspace; in contrast, the LNP-LOXL2@Gel group showed significant improvement in disc signal intensity and intervertebral space height (Fig. [167]8D). Quantitative assessment of the degree of intervertebral disc degeneration using the Pfirrmann scoring system revealed that the scores of the PBS and Gel groups were higher than those of the normal control group, whereas the scores of the LNP-LOXL2@Gel group were lower than those of these two model groups (Fig. [168]8F). Fig. 8. [169]Fig. 8 [170]Open in a new tab LNP-LOXL2@Gel inhibited disc degeneration. (A) Schematic diagram showing the process of the in vivo experiment; (B) Disc height index (DHI) calculation method; (C) Representative images of X-ray and MRI at postoperative weeks 4 and 8; (D, E) DHI, Pfirrmann scores of different experimental groups (n = 6). (F) HE and SO staining at 4 and 8 weeks postoperatively. (G) Histologic grade changes at 4 and 8 weeks postoperatively. *p < 0.05, ** p < 0.01, *** p < 0.001 After confirming the differences in the degree of disc degeneration by imaging assessment, we performed histological staining of the disc tissue. The results revealed that the histological structure of the IVD in the control group remained intact, and the demarcation between the NP and the annulus fibrosus was clearly distinguishable; in comparison, both the PBS group and the gel group exhibited obvious degenerative features, including reduced intervertebral space height, disturbed nucleus pulposus structure with fissure formation, inward subsidence of the annulus fibrosus, and a blurred NP-AF boundary. In contrast, the histological structure of the discs in the LNP-LOXL2@Gel group remained relatively intact, and the degree of degeneration was significantly reduced, and its morphological characteristics were close to those of the control group, and the histologic scores were reduced (Fig. [171]8G, H). Together, these results suggest that LNP-LOXL2@Gel can ameliorate the pathologic process of IDD. Finally, we evaluated the in vivo safety of Gel, LNP-LOXL2 and LNP-LOXL2@Gel. At postoperative week 8, we collected blood and major organs from rats. Blood count analysis showed no significant changes in leukocyte, erythrocyte, and platelet counts compared to controls (Figure S5A). In addition, when assessing serum biochemical metabolism, we observed no significant changes in markers associated with liver (ALT, AST, TBIL) and kidney (CRE, UA, UREA) function (Figure S5B). Histopathologic analysis of major organs by H&E staining showed no significant damage in the PBS, Gel, LNP-LOXL2, and LNP-LOXL2@Gel groups compared with controls (Figure S5C). These results confirmed the in vivo safety of Gel, LNP-LOXL2 and LNP-LOXL2@Gel. IHC of different groups of intervertebral discs On the basis of systematic assessment of the morphological characteristics of rat intervertebral disc tissues in each group, this study further examined the expression of key regulatory proteins, such as LOXL2, COL2A1 and p16. The IHC results showed that the expression of LOXL2 in the LNP-LOXL2@Gel group was significantly higher than that in the other puncture groups at 4 and 8 weeks (Fig. [172]9A), and in addition, the signal intensity of COL2A1, an important indicator of ECM content, showed similarity to LOXL2 (Fig. [173]9B). In addition, the expression of p16 was significantly lower in the LNP-LOXL2@Gel group than in the other puncture groups (Fig. [174]9C). These differences were further illustrated by semi-quantitative staining analysis (Fig. [175]9D-F). The above results showed that LOXL2 overexpression not only downregulated the levels of aging-related markers, but also inhibited the degradation of ECM in vivo. Fig. 9. [176]Fig. 9 [177]Open in a new tab Immunohistochemical images of different groups of intervertebral discs. (A, D) Immunohistochemical staining and quantification of LOXL2 protein-positive cells; (B, E) immunohistochemical staining and quantification of COL2A1-positive area; (C, F) immunohistochemical staining and quantification of p16-positive cells Discussion As an age-dependent degenerative disease, the incidence of IDD has increased with the acceleration of global population aging [[178]1, [179]2]. With the aging and external environmental factors, the function of NPCs gradually declines and cell senescence occurs, which plays a key role in the pathological progression of IDD [[180]39]. Senescence of NPCs leads to a significant decrease in their metabolic activity and matrix synthesis capacity, which impairs the structural stability and biomechanical properties of IVD [[181]39]. Therefore, delaying the aging of NPCs may become a key strategy for the treatment of IDD. In this study, we demonstrated that LOXL2 was down-regulated in degenerated NP tissues and TBHP-induced NPCs, whereas the senescence markers p21 and p16 were up-regulated with the progression of degeneration. Overexpression of LOXL2 significantly up-regulated the expression of ACAN and COL2A1 in NPCs, attenuated mitochondrial damage, and effectively suppressed the cell senescence. Mechanistically, LOXL2 binds to the NOTCH1 promoter to inhibit Notch signaling and suppresses NPCs senescence. Based on the above findings, we present a strategy for treating IDD using an injectable, ROS-responsive and LNP-LOXL2 loaded hydrogel (LNP-LOXL2@Gel). LOXL2 is a copper-dependent amine oxidase that plays different roles in normal development and disease processes [[182]40]. LOXL2 gene deletion leads to increased perinatal lethality and abnormal skeletal development [[183]41–[184]43]. LOXL2 knockdown not only inhibits chondrocyte development, but is also accompanied by upregulation of SNAIL expression and downregulation of SOX9 expression [[185]44]. It was found that LOXL2 was mainly localized in the nucleus [[186]45]. Similarly, immunofluorescence results of NPCs showed that LOXL2 expression was higher in the nucleus than in the cytoplasm. Notably, LOXL2 is closely associated with cellular senescence. During the process of senescence, fibroblasts and epithelial cells show elevated levels of LOXL2 expression [[187]21, [188]22]. In addition, adenovirus-mediated delivery of the LOXL2 or systemic overexpression of LOXL2 in transgenic mice prevented progressive and senescence-associated osteoarthritis of the knee joints [[189]25]. In this study, we demonstrated that LOXL2 is closely associated with NPCs senescence. In degenerated NP tissues, the expression levels of LOXL2 were significantly down-regulated and tended to decrease with increasing degeneration. In addition, LOXL2 inhibited TBHP-induced mitochondrial damage and NPC cellular senescence. To further elucidate the molecular mechanisms by which LOXL2 regulates senescence in NPCs, we performed transcriptome sequencing. KEGG analyses showed that cytokine-cytokine receptor interactions, Notch, and IL-17 signaling pathways were enriched when LOXL2 was overexpressed. GSEA analysis suggested that LOXL2 overexpression caused down-regulation of this pathway, suggesting that Notch signaling pathway may be involved in the inhibitory effect of LOXL2 on the senescence of NPCs. Several studies have revealed the differential expression profile of Notch receptors and their important regulatory roles in different aging models. Replicative aging is associated with upregulation of NOTCH1 in normal human prostate cells and esophageal keratinocytes [[190]46, [191]47]. Notch signaling can accelerate senescence by inducing p21 expression through enhanced p53 transcriptional activity [[192]36, [193]48]. In our study, the Notch signaling pathway was activated in TBHP-induced senescent NPCs, as evidenced by the up-regulation of NOTCH1 and its downstream effectors HES1 and HEY1, which in turn promoted cellular senescence. DAPT significantly inhibited these effects. Although LOXL2 supplementation is effective in rescuing senescence in NPCs, nucleic acid degradation, loss of therapeutic efficacy over time, and insufficient delivery to target tissues limit its therapeutic translation. To overcome these bottlenecks, delivery strategies based on LNPs provide an ideal solution [[194]49]. With their natural multifunctional properties and low immunogenicity, LNP-mediated gene delivery systems can overcome the technical bottlenecks of traditional gene therapy, such as poor stability, susceptibility to degradation, and difficulty in targeted delivery [[195]50].Particularly noteworthy is the endogenous phospholipid bilayer structure of LNPs, which enhances its compatibility with the internal environment of the organism and promotes benign gene-vector-organism interactions [[196]51]. These properties make LNPs an ideal nanotherapeutic platform for the treatment of IDD. Effective delivery of LNP-LOXL2 is essential for the satisfactory outcome in the treatment of IDD. Direct injection carries the risk of drug leakage through the needle hole, which may reduce drug efficacy and lead to leakage-related complications. Hydrogels and hydrogel microspheres based on natural biopolymers have been demonstrated to have applications in the protection and delivery of therapeutic drugs for IDD [[197]29, [198]52]. Among many hydrogel materials, PVA-tsPBA hydrogel has attracted much attention due to its unique performance advantages [[199]31]. It is not only widely used for delivering drugs and bioactive factors, but its ROS-responsive properties also give it a unique advantage in the treatment of IDD [[200]53–[201]55]. PVA-tsPBA hydrogels are able to scavenge ROS from the tissue microenvironment, which is usually characterized by elevated ROS levels in IDD [[202]56]. This property allows PVA-tsPBA hydrogel to achieve targeted drug release in the microenvironment of IDD. We loaded LNP-LOXL2 into PVA-tsPBA hydrogels. In contrast to traditional hydrogels limited to either mechanical repair or unimodal therapy, our system integrates the dual advantages of mechanical support and pathological microenvironmental modulation [[203]57, [204]58]. In vivo experiments showed that LNP-LOXL2@Gel exhibits superior therapeutic efficacy compared to LNP-LOXL2 alone. This enhancement was mainly attributed to the targeted delivery and sustained release properties of the ROS-responsive hydrogel. More importantly, our system not only reversed the senescence of NPCs, but also inhibited the catabolism of extracellular matrix by reducing the expression of ROS levels in the microenvironment. The extracellular ROS scavenging ability of PVA-tsPBA hydrogel and the intracellular antiageing effect of LOXL2 formed a perfect synergistic effect, blocking the the vicious cycle of “oxidative stress-cellular senescence”. Our LNP-LOXL2@Gel hydrogel system realizes the cascade regulation of NP cellular senescence and extracellular ROS. Besides the significant findings of this study, several limitations remain. First, while our study highlights LOXL2’s anti-senescence effects, its role in intervertebral disc inflammation remains unclear. Although results revealed LOXL2’s association with IL-1β and IL-6, further experiments are needed to determine whether LOXL2 directly regulates inflammatory pathways. Second, fundamental differences exist between rat and human models regarding cell populations, spinal anatomy, and biomechanical forces. Consequently, these inevitable differences must be considered when interpreting results from preclinical animal models. Conclusion In this work, we showed that the expression of LOXL2 was down-regulated in degenerated IVD and that the degree of degeneration was negatively connected with the expression level. Overexpression of LOXL2 up-regulated ACAN and COL2A1, and delayed TBHP-induced senescence of NPCs by inhibiting the Notch signaling pathway. Based on this, we constructed the LNP-LOXL2@Gel hydrogel system, which integrates the anti-aging effect of LOXL2 with the ROS scavenging ability of PVA-tsPBA to synergistically block the vicious cycle of “oxidative stress-cellular senescence”. In vivo experiments demonstrated that the LNP-LOXL2@Gel hydrogel system alleviated disc degeneration, providing a new strategy for IDD treatment. Supplementary Information [205]Supplementary Material 1^ (1.5MB, pdf) Acknowledgements