Dorsal Root Ganglion Maintains Stemness of Bone Marrow Mesenchymal Stem Cells by Enhancing Autophagy through the AMPK/mTOR Pathway in a Coculture System

Abstract

Our previous studies found that sensory nerve tracts implanted in tissue-engineered bone (TEB) could result in better osteogenesis. To explore the mechanism of the sensory nerve promoting osteogenesis in TEB in vitro, a transwell coculture experiment was designed between dorsal root ganglion (DRG) cells and bone marrow mesenchymal stem cells (BMSCs). BMSC proliferation was determined by CCK8 assay, and osteo-, chondro-, and adipogenic differentiation were assessed by alizarin red, alcian blue, and oil red staining. We found that the proliferation and multipotent differentiation of BMSCs were all enhanced in the coculture group compared to the BMSCs group. Crystal violet staining showed that the clone-forming ability of BMSCs in the coculture group was also enhanced and mRNA levels of Sox2, Nanog, and Oct4 were significantly upregulated in the coculture group. Moreover, the autophagy level of BMSCs, regulating their stemness, was promoted in the coculture group, mediated by the AMPK/mTOR pathway. In addition, AMPK inhibitor compound C could significantly downregulate the protein expression of LC3 and the mRNA level of stemness genes in the coculture group. Finally, we found that the NK1 receptor antagonist, aprepitant, could partly block this effect, which indicated that substance P played an important role in the effect. Together, we conclude that DRG could maintain the stemness of BMSCs by enhancing autophagy through the AMPK/mTOR pathway in a transwell coculture system, which may help explain the better osteogenesis after implantation of the sensory nerve into TEB.

GPR30 Alleviates Pressure Overload-Induced Myocardial Hypertrophy in Ovariectomized Mice by Regulating Autophagy

“…F-actin of H9c2 cells were stained with FITC-phalloidin and examined by fluorescence microscopy. Consistent with the results in vivo, AngII could induce cardiomyocyte hypertrophy and increase the expression of hypertrophic genes ( Figure S1 and Table S1 ) [ 31 , 32 , 33 ], and G1 attenuated H9c2 cells hypertrophy and downregulated the expression of hypertrophy genes ( Figure S1 and Figure 6 A,B). It should be noted that G1 did not change the cell area of H9c2 without AngII since there was no significant difference between the G1 and CON groups in cell area or hypertrophic gene expression ( Figure S1 and Figure 6 A,B).…”

Modern Trends for Peripheral Nerve Repair and Regeneration: Beyond the Hollow Nerve Guidance Conduit

“…Xu et al (2011) developed an vitro study in 2011 which demonstrated that a co-culture of BMSCs and DRGs explants stimulated neurite growth and neuronal cell survival through the up-regulation of wide list of secretory proteins, including bFGF, NGF, CNTF, and BDNF. Also, by means of using a co-culture system of DRGs and BMSCs in a trans-well, it was found that the presence of DRGs helped keep the stemness of BMSCs using the AMPK/mTOR signaling (Zhang et al, 2018).…”

Exploring the effect of the “quaternary regulation” theory of “peripheral nerve-angiogenesis-osteoclast-osteogenesis” on osteoporosis based on neuropeptides

“…Fu et al (60) used SP to intervene in BMSCs and found that SP promoted the expression of Bcl-2 and increased the ratio of Bcl-2 to Bax, confirming that SP inhibited the apoptosis of BMSCs through NK-1 receptors. Zhang et al (61) found in the co-culture experiments of dorsal root ganglion (DRG) cells and BMSCs that DRG enhanced the autophagy level of BSMCs through the AMPK/mTOR signaling pathway, thereby maintaining the differentiation activity, and this process was related to the release of substance P. Therefore, SP can stimulate the proliferation, differentiation, and mineralization of pre-osteoblasts and improve the activity of late osteoblasts, but the specific mechanism needs to be further studied (62).…”