BMSC-Derived Exosomes Alleviate Intervertebral Disc Degeneration by Modulating AKT/mTOR-Mediated Autophagy of Nucleus Pulposus Cells

Abstract

The pathogenesis of intervertebral disc degeneration (IDD) is still unclear. It has been shown that the pathological process of IDD is most closely related to inflammation of nucleus pulposus cells (NPCs), in which inflammatory factors play an important role. Exosomes are the main paracrine mediators and are microvesicles with biological functions similar to those of the cells from which they are derived. Studies have shown that bone mesenchymal stem cells (BMSCs) can inhibit apoptosis of NPCs by sending exosomes as anti-inflammatory and antioxidant, which has been proved to be effective on IDD. However, the specific mechanism of inhibiting apoptosis of NPCs is still unclear. In our study, BMSC-derived exosomes (BMSC-Exo) were isolated from the BMSC culture medium, and their antiapoptotic effects were evaluated in cells and rat models to explore the possible mechanisms. We observed that BMSC-Exo promotes autophagy in NPCs and inhibits the release of inflammatory factors such as IL-1β and TNF-α in LPS-treated NPCs and inhibits apoptosis in NPCs. Further studies showed that BMSC-Exo inhibited the Akt-mTOR pathway. Intramuscular injection of BMSC-Exo alleviates disc degeneration in rat IDD models. In conclusion, our results suggest that BMSC-Exo can reduce NPC apoptosis and alleviate IDD by promoting autophagy by inhibiting the Akt-mTOR pathway. Our study confers a promising therapeutic strategy for IDD.

Figure 1

Morphology and characterization of BMSCs, NPCs, and BMSC-Exos: (a) P0-generation BMSC morphology (scale bar: 200 μm); (b) Oil Red O staining (scale bar: 100 μm); (c) Alizarin Red staining (scale bar: 100 μm); (d) Alcian Blue staining (scale bar: 50 μm), n = 3; (e) flow cytometric analysis of BMSC surface markers (CD29, CD90, CD34, and CD45); (f) NPC morphology (scale bar: 200 μm); (g) toluidine blue staining of NPCs (scale bar: 100 μm); (h) type II collagen immunochemical staining of NPCs (scale bar: 100 μm); (i) transmission electron microscopy (TEM) image of BMSC-Exo (scale bar: 200 nm); (j) NanoSight tracking analysis (NTA) for the diameters of BMSC-Exo; (k) Western blot analysis of CD9, CD63, and TSG101, n = 3; (l) analysis of cellular exosome uptake by NPCs. n = 3. Scale bar: 25 μm.

Figure 2

BMSC-Exo inhibits NPC apoptosis by promoting autophagy: (a) the integrated optical density (IOD) of NPCs treated with LPS gradient concentration; (b) IOD of NPCs treated with BMSC-Exo gradient concentration; (c) the mRNA expression of NPCs after incubation with LPS, BMSC-Exo, or 3-MA; (d–g) protein levels of autophagy and apoptosis-related protein (Col2a1, Baxter, LC3, Beclin-1, and Cleaved Caspase-3) in NPCs after being treated with LPS, BMSC-Exo, or 3-MA. n = 6. Data are presented as mean ± SD. Statistical analysis was performed with Student's t-test and one-way ANOVA followed by Tukey's multiple comparisons test. ^∗P < 0.05; ^∗∗P < 0.01.

Figure 3

Western blot detects the relative expression changes of p-Akt, Akt, p-mTOR, mTOR, and LC3 in each group. n = 6. ^∗P < 0.05; ^∗∗P < 0.01.

Figure 4

Results of the experiments in live animals. (a) MRI findings after injection of exosomes and IGF-1 into the caudal disc of a rat. (b) Histological analysis of HE and S/O staining discs. Immunohistochemical staining of type II collagen showed the presence of type II collagen in the exosome group, but little in the control and exosome+IGF-1 groups. Scale bar: 100 μm. (c) Thompson scores of three groups. The score of the exosome group is lower than that of the other groups. (d) Histological score showed significant imaging improvement at week 8 of the therapy with exosome, but higher in the control and exosome+IGF-1 groups; (e) Immunohistochemical staining of type II collagen area (%). ^∗∗P < 0.01.

Figure 5

MSC-derived exosomes mitigate apoptosis against LPS-induced apoptosis by promoting nucleus pulposus cell autophagy, which is achieved through the Akt-mTOR signaling pathway.