Aging-associated cellular phenotypes ameliorated by OKS in aging NPCs

To restore youthful epigenetics in senescent NPCs, we constructed a plasmid that expressed pluripotency-associated genes (OKS) to promote cellular reprogramming, activate pluripotency genes, and remodel the epigenome (Fig. 1a). First, we successfully constructed plasmid vectors that could simultaneously overexpress OKS, as verified by cutting the plasmid with a restriction endonuclease and analyzing the resulting fragments (Fig. S1a, b). The NPCs’ replicative senescence model (passage 6, P6) was used to investigate whether partial overexpression of OKS could promote regeneration of senescent NPCs. As shown in and Fig. S1c, d, the OKS were overexpressed in senescent NPCs after OKS plasmid transfection. The expression of p16INK4a (P16), p21CIP1 (P21), P53, Atf3 and Gadd45b, age-related stress response genes in the p53 tumor suppressor pathway, were downregulated in senescent NPCs after treatment of OKS plasmid (Fig. 1b–f). The number of foci for histone γ-H2A·X, a marker of nuclear DNA double-strand breaks associated with senescent30, was significantly reduced by partial expression of OKS compared with the aging NPCs (Fig. 1g). Increased histone H4K20 trimethylation (H4K20me3) and downregulated H3K9 trimethylation (H3K9me3) are two of the epigenetic marks associated with aging.30,31,32,33 As shown in Fig. 1h and Fig. S2, the expression of H4K20me3 was decreased and H3K9me3 was upregulated after OKS plasmid transfection compared with the aging NPCs. Lamins (mainly laminA/C) are components of the nuclear lamina, maintaining proper architecture of the nuclear envelope, and nuclear envelope abnormalities are associated with senescence.7,34,35 Importantly, over expression of OKS in senescent NPCs significantly decreased the blebbing in the nuclear envelope compared to aging cells, indicating OKS could retain nuclear envelope architecture (Fig. 1i). Furthermore, senescence associated β-galactosidase (SA-β-Gal) activity decreased in senescent NPCs after induction of OKS (Fig. 1j, l). Together, these results suggested that OKS plasmid ameliorated age-associated hallmarks in senescent NPCs.

Fig. 1

Partial expression of OKS ameliorated cellular phenotypes associated with aging and restored normal function of NPCs. a A schematic diagram illustrating the experimental design. The replicative senescence model of NPCs (passage 6, P6) was used to explore the physical function of OKS plasmids. RT-qPCR analysis of expression level of P16 (b), P21 (c), P53 (d), Atf3 (e) and Gadd45b (f) in Young group (passage 2 NPCs), Aging group (passage 6 NPCs) and Aging+OKS group (passage 6 NPCs treated with OKS plasmids). (n = 3/group). g Representative confocal immunofluorescence micrographs showing the expression of γ-H2A·X foci (green) in Young, Aging and Aging+OKS groups. Scale bar, 20 μm. h Representative confocal immunofluorescence micrographs showing the expression of H4K20me3 (green) in Young, Aging and Aging+OKS groups. Scale bar, 20 μm. i Representative immunofluorescence micrographs showing the expression Lamin A/C (red) of nuclear abnormality in Young, Aging and Aging+OKS groups. White arrows indicate blebbing in the nuclear envelope in cells. Scale bar, 50 μm. Representative images of β-Gal staining (j) and quantification (l) in Young, Aging and Aging+OKS groups. (n = 7-8/group). Scale bar, 100 μm. Representative immunofluorescence micrographs of EdU (k) and quantification (m) in Young, Aging and Aging+OKS groups. (n = 5/group). Scale bar, 100 μm. RT-qPCR analysis of senescence-associated anabolism factors Col2 (n), Acan (o), and catabolism factors Mmp13 (p), Adamts5 (q) in Young, Aging and Aging+OKS groups. (n = 4/group). RT-qPCR analysis of senescence-associated inflammatory factors Il-6 (r), Il-1β (s) in Young, Aging and Aging+OKS groups. (n = 3-4/group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1, NS means none sense, one-way ANOVA with Tukey’s multiple comparisons. Data are presented as the mean ± SD

A decline in self-renewal capacity is one of the main characteristics of NPCs senescence. By EdU assay, the percent of EdU+ cells in the Aging+OKS group significantly increased compared with the Aging group, indicating that overexpression of OKS could reverse the impaired proliferation capacity of aging NPCs (Fig. 1k, m). In addition, senescent NPCs may suffer from synthetic and catabolic disequilibrium. The partial overexpression of OKS in senescent NPCs promoted function recovery in senescent NPCs by boosting the expression of anabolism factor Col2 and Acan, and downregulating the expression of catabolism factors Mmp13 and Adamts5 (Fig. 1n–q). The western blotting (WB) experiments further confirmed the downregulation of the expression of ADAMTs5 and MMP13 proteins after OKS treatment (Fig. S3a, b). By analyzing the immunofluorescence signals of ACAN and MMP13, we also observed an increase in ACAN expression and a decreased expression of MMP13 in the Aging+OKS group (Fig. S3c, d). These results further confirmed the ability of OKS to enhance function recovery in senescent NPCs. Senescent cells induce the formation of a complex, multicomponent SASP by secreting a range of cytokines and inflammatory factors. In the local microenvironment, the SASP alters the biological behavior of adjacent cells.4,5,7 Therefore, it is also necessary to find out whether OKS can reduce inflammation. As shown in Fig. 1r, s and Fig. S4a, b, partial expression of OKS could reduce the expression of IL-6, TNF-α and IL-1β compared with the Aging group. Collectively, these results showed that partial expression of OKS in senescent NPCs alleviated age-associated hallmarks, restored proliferation capacity and metabolic equilibrium, and decreased inflammation, indicating OKS could remodel the senescent process and promote a youthful state of NPCs.

Cavin2-modified exosomes enhanced the uptake of OKS by aging NPCs

Low toxicity and high transfection efficiency are two of the most important factors for successful gene therapy.23 Exosomes represent a cutting-edge platform for targeted gene delivery, offering unique advantages such as stability, low immunogenicity, and efficient cell penetration.29,36 Therefore, we used BMSCs-derived exosomes as carriers to deliver OKS plasmid into senescent NPCs (Fig. 2a). BMSCs were obtained from the femur and tibial bone marrow of rats, and displayed spindle-like morphology (Fig. S5a). BMSCs are able to differentiate to osteocyte and adipocyte (Fig. S5b). The results of flow cytometry showed that BMSCs were positive for CD73, CD90 and CD105, and negative for HLA-DR, CD19 and CD11b (Fig. S5c). The above results suggested that BMSCs were successfully extracted. And then, the exosomes (C-Exo) were isolated from the BMSCs. Transmission electron microscopy (TEM) images showed that the majority of the particles exhibited a cup- or round-shaped morphology and the diameter of the exosomes was approximately 121 nm (Fig. S6a, b). As shown in Fig. 2b, we found that less exosomes taken up by the aging NPCs than the young ones. To guarantee that OKS can efficiently remodel the epigenetics of aging cells, it is crucial for enhancing the uptake efficiency of C-Exo in aging NPCs. The exosomes primarily gain access to NPCs through caveolae/lipid raft-dependent endocytosis.37 Our experimental results also demonstrated that C-Exo entered into NPCs through caveolae/lipid raft-dependent endocytosis (Fig. 2c, d and Fig. S7a). Cavin2 plays a crucial role in the formation of plasma membrane curvature and the endocytosis of extracellular cargoes by recruiting polymerase I and transcript release factor (PTRF) and binding to caveolin-1.38 The expression of Cavin2 decreased in aging NPCs (Fig. 2e), which may be the cause of the decline in phagocytic ability within the aging NPCs. So, to improve phagocytic ability, introduction of exogenous Cavin2 may provide a potential solution. Then, we produced exosomes with Cavin2 surface modification (M-Exo) through a three step strategy: (1) constructed Cavin2-Lamp2b overexpression plasmid (Fig. S5a, b), (2) transfected the plasmids into BMSCs, (3) isolated M-Exo from the culture medium of M-BMSCs. The morphology and size of the C-Exo and M-Exo were similar (Fig. S6). The results of WB analysis revealed robust expression of Cavin2 in M-Exo (Fig. S8c and Fig. 2f). To evaluate the uptake effects of M-Exo on aging NPCs, Paul Karl Horan 26 dye (PKH26) was used to label exosomes. Compared with the Aging + C-Exo group, the uptake of M-Exo increased in aging NPCs, suggesting that modified Cavin2 on exosomes enhanced the uptake of aging NPCs (Fig. 2h). For young NPCs, whether the exosomes were modified with Cavin2 or not, there was no difference in their ability to exosomes taken-up. The results of flow cytometry were consistence with the laser confocal detection (Fig. 2g and Fig. S7b). Lysosome escape is indeed crucial for nanoparticles to exert their cellular effects effectively. The fluorescence of PKH26 was completely overlapped with lysosomes after 1 h, partially out of alignment with lysosomal fluorescence after 2 h, and only a small portion of PKH26 fluorescence was overlapped with lysosomes after 9 h of incubation, indicating the exosomes effectively escaped from the lysosomes (Fig. 2i and Fig. S9). Taken together, these results indicated that the Cavin2 modified exosomes can be engulfed into senescent NPCs and escape from the lysosomes to function.

Fig. 2

Cavin2-modified exosomes could be uptaken by aging NPCs via Caveolae-dependent endocytosis. a Schematic graph of the preparation for engineering exosomes. b Representative confocal fluorescence micrographs of Young and Aging NPCs, co-cultured with C-Exo. The exosomes were labeled with PKH26 (red) and the cytoskeleton was labeled with FITC (green). Scale bar, 20 μm. c NPCs were cultured under different inhibition conditions. Representative confocal fluorescence micrographs showed the internalization of PKH26-labeled (red) exosomes in these groups. Chlorpromazine: clathrin-mediated endocytosis inhibitor; Wortmannin: inhibitor of the phosphoinositide 3-kinase; Dynasore: dynamin inhibitor; Filipin: caveolae-dependent endocytosis inhibitor. Scale bar, 20 μm. d Fluorescence intensity of PKH26-labeled C-Exo taken up by NPCs under different inhibition conditions by flow cytometry (n = 3/group). e WB analysis of Cavin2 in Young and Aging NPCs. f WB analysis of CD9, CD63, CD81 and Cavin2 in C-Exo and M-Exo. g Fluorescence intensity of PKH26-labeled C-Exo or M-Exo taken up by Young or Aging NPCs by flow cytometry (n = 3/group). h Representative confocal fluorescence micrographs of PKH26-labeled C-Exo or M-Exo (red) taken up by Young or Aging NPCs. The cytoskeleton was labeled with FITC (green). Scale bar, 20 μm. i Representative confocal fluorescence micrographs of lysosomes (green) and exosomes (red) in NPCs co-cultured with exosomes for 1 h, 2 h and 9 h. Scale bar, 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1, one-way ANOVA with Tukey’s multiple comparisons. Data are presented as the mean ± SD

OKS@M-Exo reduced the aging characteristics of senescent NPCs

We next assessed whether OKS@M-Exo treatment was able to restore youthful epigenetics of aging NPCs (Fig. 3a). First, we observed and analyzed the exosomes before and after electroporation through TEM and nanoparticle tracking analysis (NTA), and found that electroporation didn’t affect the morphology and size of the exosomes (Fig. S10a, b). To confirm OKS plasmids loaded into M-Exo via electroporation, we extracted OKS plasmids from OKS@M-Exo. Agarose Gel Electrophoresis was used to test the cut OKS plasmids by the restriction endonuclease. As shown in Fig. S10c, two fragments of OKS plasmid cut by restriction endonuclease were observed, indicating successful transfection of OKS plasmids into OKS@M-Exo. Moreover, the efficiency of loading OKS plasmids into exosomes by electroporation was approximately 35% (Fig. S10d).

Fig. 3

OKS@M-Exo mitigated the aging characteristics of aging NPCs. a A schematic diagram illustrating the experimental design. OKS plasmid transfected into the exosomes via electroporation. Different indicators, associated with aging and NPCs’ function, were detected. b RT-qPCR analysis of stress response genes in the P53 pathway including P21, Gadd45b, Atf3, P16, P53 in aging NPCs treated with Control, OKS, M-Exo and OKS@M-Exo groups. (n = 4/group). c, g Representative confocal immunofluorescence micrographs showing the expression of γ-H2A·X foci (green) (c) and quantification (g) in aging NPCs treated with Control, OKS, M-Exo and OKS@M-Exo groups (n = 17–32/group). Scale bar, 20 μm. d Representative confocal immunofluorescence micrographs showing the expression of H4K20me3 (green) in aging NPCs treated with Control, OKS, M-Exo and OKS@M-Exo groups. Scale bar, 20 μm. Representative immunofluorescence micrographs of EdU (green) (e) and quantification (f) in aging NPCs treated with Control, OKS, M-Exo and OKS@M-Exo groups (n = 5/group). Scale bar, 100 μm. h The culture medium concentration of IL-6 in aging NPCs treated with Control, OKS, M-Exo and OKS@M-Exo groups were determined by ELISA assay (n = 3/group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1, NS means none sense, one-way ANOVA with Tukey’s multiple comparisons. Data are presented as the mean ± SD

The aging NPCs were used as Control group and treated with OKS plasmid (OKS group), M-Exo (M-Exo group) and OKS@M-Exo (OKS@M-Exo group) (Fig. 3). OKS@M-Exo has no effect on the proliferation of normal NPCs, suggesting that it’s a safe way of administration (Fig. S11a, b). As shown in and Fig. S12, the expressions of Oct4, Sox2, Klf4 proteins in aging NPCs increased after OKS@M-Exo treatment. Meanwhile, we performed a dual-luciferase reporter assay to evaluate the transcriptional activity of plasmids delivered into NPCs via exosomes. The results showed that both OKS plasmids (delivered with transfection reagent) and OKS@M-Exo treatment increased luciferase activity (Fig. S13). Compared with Control, OKS and M-Exo groups, the mRNA expression of P21, Gadd45b, Atf3, P16 and P53 was downregulated after OKS@M-Exo treatment (Fig. 3b). The number of foci for histone γ-H2A·X and the fluorescence intensity of H4K20me3 were significantly reduced by OKS@M-Exo treatment in aging NPCs compared with Control, OKS and M-Exo groups (Fig. 3c, d, g). And the expression of H3K9me3 was significantly increased in the OKS@M-Exo group compared with the other three groups (Fig. S14). These results suggested that OKS@M-Exo could ameliorate age-associated hallmarks. Meanwhile, OKS@M-Exo promoted the expression of ACAN and Col2, reduced the expression of MMP13 and ADAMTs5, and balanced anabolic and catabolic metabolism balance, thus restoring the normal structure of ECM (Figs. S15, S16). The proliferation of aging NPCs was rejuvenated after OKS@M-Exo treatment (Fig. 3e, f). Furthermore, the expression of inflammation-related factors, such as TNF-α, IL-1β and IL-6, were inhibited in aging NPCs after exposure to OKS@M-Exo (Fig. S17a, b and Fig. 3h). Altogether, these results showed that loading OKS into the exosomes could mitigate the aging characteristics of NPCs, and restore the metabolic balance and proliferative capacity of aging NPCs.

OKS@M-Exo ameliorated IVDD and relieved LBP in vivo

We next assessed whether OKS@M-Exo treatment was able to restore youthful epigenetics of aging NPCs and mitigate IVDD. To investigate the therapeutic effects of OKS@M-Exo in vivo, a rat model of IVDD was created by inducing needle puncture in rat coccygeal discs. The needle puncture can simulate the pathological process of IVDD39,40,41, and also lead to signs of IVD aging.42 Imaging and histological tests were performed on clinical patients’ specimens and constructed rat IVDD models (Fig. 4a). The NP specimens were obtained from patients with mild and severe degeneration, demonstrated by magnetic resonance imaging (MRI) images (Fig. 4b), and detected the aging-related indicators including P21 and P16. As shown in Fig. 4c, the expression of P21 and P16 increased in severe degenerated IVD. The animal model was used to detect the degeneration and aging indicators of the rats’ caudal vertebra. After 2 weeks of puncture, the coccyx of the rats experienced IVDD, which verified by MRI, Micro-CT, hematoxylin-eosin (H&E) and Safranin O-Fast Green Staining (Safranin O) (Fig. S18a–c). Meanwhile, the expression of P21 increased, suggesting that the pathological process of IVDD could lead to senescence of NPCs in IVD (Fig. S18d). Due to IVDD and aging appeared 2 weeks after acupuncture of the coccygeal disc in rats, we performed drug administration 2 weeks after puncture to explore the therapeutic effect of OKS@M-Exo.

Fig. 4

Radiological results of animal experiments indicated OKS@M-Exo could alleviate IVDD. a A schematic diagram illustrating the experimental design. Clinical patients conducted imaging examinations, and took the patients’ NP tissue specimens for histological testing during the operation. Imaging and histological tests were performed 2 weeks after the puncture of the rat tail spine. b Representative T2-weighted MRI scans of human lumbar spine (the red arrow indicated the IVD). c Representative immunohistochemical (IHC) analysis of human IVD sections to assess the expression of P16 and P21. Scale bar, 50 μm. d The schematic graph of IVDD modeling and the treatments. Different coccygeal IVD of each rat tail was divided into five groups for puncture and injection with various treatment groups. Radiological tests were performed at weeks 4 and 8 after treatment. Sham group: normal IVD (n = 5), PBS group: punctured IVD treated with PBS (n = 5), OKS group: punctured IVD treated with OKS plasmids (n = 5), M-Exo group: punctured IVD treated with M-Exo (n = 5), OKS@M-Exo group: punctured IVD treated with OKS@M-Exo (n = 5). e Representative Micro-CT images of coccygeal IVD in rats at weeks 4 and 8 after treatment. f The quantification of DHI changes in Sham, PBS, OKS, M-Exo and OKS@M-Exo groups at weeks 4 and 8 after treatment. (n = 5/group). g Representative T2-weighted MRI sagittal and cross-sectional images of coccygeal IVD in rats. h The quantification of gray values of coccygeal IVD in Sham, PBS, OKS, M-Exo, OKS@M-Exo groups at weeks 4 and 8 after treatment (n = 5/group). i The Pfirrmann classification of coccygeal IVD at weeks 4 and 8 after treatment to evaluate the degenerated level of IVD. (n = 5/group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1, NS means none sense, one-way ANOVA with Tukey’s multiple comparisons. Data are presented as the mean ± SD

To verify that OKS@M-Exo can be taken up by NPCs, we locally injected either unlabeled or PKH26-labeled OKS@M-Exo (PKH26-Exo) into the IVD. As shown in Fig. S19, two days after the injection, OKS@M-Exo were mainly endocytosed by NPCs. Additionally, we tested the local expression levels of the three transcription factors. The results showed that the expression of Oct4, Klf4 and Sox2 in the OKS@M-Exo group was significantly higher than in the other four groups. (Fig. S20). Micro-CT and MRI were performed to measure the degree of IVDD at weeks 4 and 8 after injection of drugs (Fig. 4d). Disc height index (DHI), which refers to the variation of disc height, can reflect the degree of IVDD. A lower DHI indicates a greater degree of disc degeneration. Compared with PBS, OKS and M-Exo groups, the DHI in the OKS@M-Exo group was significantly improved and similar to that of the Sham group at weeks 4 and 8 after treatment (Fig. 4e, f). Subsequently, MRI scans were obtained to evaluate alterations in disc water content. This was analyzed using the grayscale values at the center of the discs, where higher grayscale values signify lower water content. There was a high T2-weighted signal intensity in sham group, and a low T2-weighted signal intensity in PBS group. As shown in Fig. 4g, the T2-weighted signal intensity of the OKS@M-Exo group was the highest among the PBS, OKS and M-Exo groups, similar to the Sham group. The OKS@M-Exo group had the lowest grayscale compared with PBS, OKS and M-Exo groups, suggesting that the water content of the OKS@M-Exo group was significantly restored (Fig. 4h). Pfirrmann grade, a semi-quantitative visual classification system, is currently recognized as a classification method for diagnosing the degree of IVDD.43 The Pfirrman MRI score of OKS@M-Exo group also remarkably decreased compared with PBS, OKS and M-Exo groups (Fig. 4i). These results suggested that OKS@M-Exo OKS@M-Exo had a better therapeutic effect on IVDD.

Histological analysis of the IVD in rats were conducted at weeks 4 and 8 after drug administration. The IVD in Sham and OKS@M-Exo groups showed a normal microstructure, in which the NP was intact and had a clear boundary with the AF. In PBS group, the NP was absent, and the IVD was disorganized. To the contrary, the IVD microstructure of OKS and M-Exo groups were not clear, but there was still some collagen in the disc tissue at weeks 4 and 8 after treatment (Fig. 5a and Fig. S21a). The histological score of OKS@M-Exo group was significantly lower than that of PBS, OKS, M-Exo groups, similar to that of Shame group at weeks 4 and 8 after treatment (Fig. S21b, c). The IHC results showed that Sham group and OKS@M-Exo groups had a stronger expression of ACAN and lower expression of MMP13, which were conducive to maintaining the normal structure and biomechanical properties of ECM. While the expression level of ACAN and MMP13 in OKS and M-Exo groups were not significantly different from those in PBS group (Fig. 5b, c). The results of Fig. 3 showed that OKS@M-Exo could ameliorate cellular phenotypes associated with aging. Therefore, we used IHC to detect the expression of aging-related indicators, and the results showed that OKS@M-Exo could downregulate the expression of P21 in the degenerative IVD tissue compared to PBS, OKS and M-Exo groups (Fig. 5d). Above all, these results suggested that OKS@M-Exo can maintain the normal structure and metabolic balance of IVD and reduce the expression of age-related factors, then play a role in alleviating IVDD.

Fig. 5

Histological results of animal experiments. a Representative H&E staining images of IVD in Sham, PBS, OKS, M-Exo and OKS@M-Exo groups at weeks 4 and 8 after treatment. Scale bar, 500 μm. Representative IHC analysis of rat IVD sections to assess ACAN (b), MMP13 (c), P21 (d) in Sham, PBS, OKS, M-Exo and OKS@M-Exo groups at weeks 4 and 8 after treatment. Scale bar, 500 μm

We next examined the ability of OKS@M-Exo to relieve LBP in vivo. An animal model of anterior lumbar puncture in rat was constructed to assess the pain-associated behavioral changes (Fig. 6a). The rats were divided into three groups: Sham group, OKS@M-Exo group and PBS group. The results of MRI showed that OKS@M-Exo maintained the normal structure of IVD, indicating a better therapeutic effect on IVDD (Fig. 6b). Pressure algometry thresholds (PATs) was used to assess pain sensitivity and threshold levels in response to applied pressure (Fig. 6c). The baseline PATs of the Sham group (749.5 ± 27.6), OKS@M-Exo group (710.7 ± 29.1) and PBS group (767.4 ± 39.3) was similar at day 0 (without surgery). The PATs of the OKS@M-Exo group and the PBS group were significantly lower than that of the Sham group after weeks 1 and 2 post-surgery (without treatment), indicating successful induction of LBP. After the OKS@M-Exo treatment, we found that the PATs gradually increased and reached 744.8 ± 40.1 at weeks 6. Meanwhile, the PATs of the PBS treatment were tended to be stable with 513.2 ± 28.1 at weeks 6. The above results indicated that OKS@M-Exo could relieve back pain in rats (Fig. 6c). The increase of PATs in Sham group may be associated with the rise in animals’ body weight. The Von Frey and Hargreaves tests were conducted, which indicate radiation-induced leg pain. There was no significant difference between the Sham group, OKS@M-Exo group and PBS group (Fig. 6d, e). This lack of difference may be attributed to the fact that the anterior puncture did not rupture the posterior margin of the AF to form a disc herniation and did not stimulate the nerve roots to cause leg pain. During IVDD, nerve endings expand from the outer AF to the inner AF and NP regions of the disc, accompanied by increased levels of neurotrophic factors, such as nerve growth factor (NGF). These factors can lead to nerve sensitization.6 As shown in Fig. 6f, compared with the PBS group, the expression of NGF in IVD of OKS@M-Exo group decreased, indicating that nerve ingrowth reduced after OKS@M-Exo treatment and thereby alleviating LBP. Taken together, these results suggested that OKS@M-Exo can effectively mitigate IVDD and alleviate LBP.

Fig. 6

OKS@M-Exo effectively reduced the occurrence of LBP in the rat model of anterior lumbar puncture. a The schematic graph illustrated that OKS@M-Exo could alleviate LBP. The rats were divided into three groups, underwent anterior lumbar disc puncture (L4/5) and injection of different drugs to detect pain behavior. Sham group: normal IVD (n = 6), PBS group: punctured IVD treated with PBS (n = 6), OKS@M-Exo group: punctured IVD treated with OKS@M-Exo (n = 5). b Representative T2-weighted MRI sagittal images of lumbar IVD in rats (the red arrow indicated the IVD). c Temporal changes of PATs for the lumbar L4/5 regions in Sham, OKS@M-Exo and PBS groups. d Graph comparing the mechanical threshold (Von Frey test) in Sham, OKS@M-Exo and PBS groups. e Graph comparing the thermal withdrawal latency (Hargreaves test) in Sham, OKS@M-Exo and PBS groups. f Left: representative fluorescence images NGF (red) immunostaining at IVD. Yellow boxes indicate the area shown at ×30 magnification in images below. Right: quantification of NGF+ cells in Sham, OKS@M-Exo and PBS groups (n = 5 or 6/group). Scale bar, 500 μm (above), scale bar, 50 μm (below). *P < 0.05, **P < 0.01, ****P < 0.000 1, one-way ANOVA with Tukey’s multiple comparisons. Data are presented as the mean ± SD

RNA-seq confirmed OKS@M-Exo alleviated the progression of IVDD

To get insight into the molecular pathways of the protective effect of OKS@M-Exo on IVDD, we conducted transcriptomics RNA-seq analysis of IVD treated with or without OKS@M-Exo at 1 month after treatment (Fig. 7a). Differentially expressed genes (DEGs) were identified based on significant expression level differences (log2 FC > 1 and FDR < 0.05) between OKS@M-Exo and PBS groups. Transcriptome sequencing revealed distinct expression profiles in OKS@M-Exo group compared to PBS group, with 357 DEGs identified—215 upregulated and 142 downregulated genes (Fig. 7b and Fig. S22). Notably, genes related to ECM catabolic metabolism, such as Mmp3, Mmp13, Adamts5 and senescence related genes, including Tp53, Cdkn1a and Cdkn2a, were significantly downregulated in OKS@M-Exo group. In contrast, ECM anabolic metabolism genes, Col2a1 and Acan, were significantly upregulated in OKS@M-Exo group compared with PBS group. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed significant enrichment of DEGs in OKS@M-Exo group within pathways such as “cell cycle”, “cell mitosis”, “p53 signaling pathway”, “cellular senescence” and “ECM-receptor interaction” (Fig. 7c). The “cell cycle” and “cell mitosis” are closely related to cell proliferation capacity. In addition, the “p53 signaling pathway” and “cellular senescence” are intricately linked to the aging process. The pathway “ECM-receptor interaction” is strongly correlated with the balance of ECM metabolism. Meanwhile, Gene Ontology (GO) enrichment analysis was used to classify and enrich for gene ontology functions of DEGs between OKS@M-Exo and PBS groups. Cellular component (CC) represents the location and organization of proteins in cells. Biological process (BP) denotes the biological activities in which genes and proteins are involved. Molecular function (MF) describes the activities and properties of protein molecules in biochemical reactions. The results of CC enrichment indicated that DEGs were associated with spindle components, encompassing spindle, spindle pore, spindle midzone, and microtubule, all of which played a role in cell mitosis (Fig. 7d). BP enrichment suggested that DEGs were mostly involved in cell proliferation processes, including cell division, chromosome segregation, mitotic spindle organization and cell cycle (Fig. 7e). Additionally, MF enrichment revealed that the DEGs were predominantly related to mitosis, including microtubule binding, ATP binding, and DNA replication origin binding (Fig. 7f). In summary, the KEGG and GO analysis results revealed that OKS@M-Exo could restore the proliferation ability and maintain the metabolic balance by reshaping the epigenetic information of aging NPCs.

Fig. 7

The RNA-seq analysis was performed between the OKS@M-Exo and PBS groups. a A schematic diagram illustrating the experimental design. The rats were divided into two groups, underwent coccygeal puncture (Co 5/6, Co 6/7) and injection of different drugs to perform RNA-Seq. PBS group: punctured IVD treated with PBS (n = 3), OKS@M-Exo group: punctured IVD treated with OKS@M-Exo (n = 3). b Volcano plot of differentially expressed genes (DEGs) at weeks 4 after treatment. c The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results for the DEGs between OKS@M-Exo and PBS groups. Top twenty pathways were showed. The DEGs were filtered by Gene Ontology (GO) analysis in CC (d), BP (e) and MF (f). Top ten classifications of DEGs were showed in the graphic