Experimental Study Of The Effect Of MiR-155 On The Regulation Of Rheb/mTOR/Atg1 Signaling Pathway In A Rat Model Of Intervertebral Disc Degeneration

Objective: To investigate the use of stem cells in combination with known treatments for intervertebral disc degeneration(IDD).However,cancer and immunogenicity problems have limited the use of stem cells in therapy.Mesenchymal stem cells(MSCs)deliver information via paracrine,and the mechanism of action of exosomes as a material for intercellular communication is also of great interest,as our team found significant differences in miR-155 expression in exosomes and activated autophagy by regulating the Rheb / mTOR / Atg1 pathway.But it has not been tested in animals.The purpose of this study was to validate the regulatory and therapeutic role of miRNA-155 in exosomes in rat intervertebral discs by regulating the Rheb / mTOR / Atg1 pathway,and to address the problems in animal models following drug administration.Methods:(1)A rat model of intervertebral disc degeneration was established: Rats intervertebral discs were pricked with fibrillation and fed for 4 weeks,and a sham operation group was set up and validated by HE staining after 4 weeks.(2)Experimental grouping and intervention methods: miRNA-155 was administered in gelatin methacrylate(GelMA)hydrogel and injected into the degenerated intervertebral discs of rats.The groups were divided into normal group,model group,GelMA hydrogel group and GelMA+miRNA155-agomir group according to different intervention methods.(3)Detection indexes: Histopathological changes in each grouping were observed by HE staining.q RT-PCR was used to determine the expression levels of miR-155 in the myeloid cells of each group.m RNA expression changes of autophagy-related proteins Beclin1 and LC3,and Rheb,mTOR and ATG1 in the signalling pathway were detected,and Western-blot technique was used to The final flow cytometric assay can be used to assess the extent of apoptosis in myeloid cells.Results:(1)Compared with their normal counterparts,the expression levels of miR-155 were significantly lower in the model group(P<0.001),the expression levels of autophagy marker Beclin1 were higher in the model group(P<0.05),the expression levels of autophagy marker LC3 and Rheb were significantly higher(P<0.01),the expression levels of mTOR were significantly higher(P<0.01),and the expression levels of ATG1 were similarly higher in the model group(P<0.05).ATG1 expression levels were also increased(P<0.05).The apoptosis rate of nucleus pulposus was significantly higher in the model group than in the normal group(P<0.001).HE staining showed that there was a large amount of connective tissue in the model group compared to the normal group,and the fibrous ring of the tissue had obvious necrosis,and the line between the nucleus pulposus and the fibrous ring was not clear.(2)There was no significant difference in the expression levels of miR-155,Beclin1,LC3,Rheb and ATG1 in the GelMA hydrogel group compared to the model group,while flow cytometry results showed that the apoptosis rate of myeloid nuclei in the GelMA hydrogel group was significantly reduced compared to that in the model group(P<0.01).However,the GelMA hydrogel group showed no significant inflammatory cell infiltration.(3)Compared with the model group,GelMA+miRNA155-agomir group showed a significant increase in miR-155expression(P<0.01),a significant increase in Beclin1 and LC3 expression(P<0.001),and a significant decrease in Rheb expression(P<0.001),a significant decrease in mTOR expression,and a significant increase in ATG1 expression.Significantly increased,both of which were significant(P<0.001).Medullary cells in the GelMA+miRNA155-agomir group had significantly lower apoptosis rates by flow cytometry compared to the model group(P<0.001),while apoptosis rates were significantly lower in the GelMA+miRNA155-agomir group compared to the GelMA hydrogel group(P<0.001).On HE staining,the GelMA+miRNA155-agomir group showed slight abnormalities in the overall disc structure compared to the model group,with visible nucleus pulposus and fibrous ring structure and no significant fibrous ring necrosis and no significant inflammatory cell infiltration overall.Conclusion:(1)Exogenous injection of miR-155 into the intervertebral disc can enhance the autophagy degree of nucleus pulposus cells.(2)miR-155 can regulate myeloid autophagy in rats by regulating the Rheb/mTOR/Atg1 signaling pathway.Elevated miR-155 expression can promote myeloid autophagy and reduce the rate of myeloid apoptosis,thus improving the effect of IDD.