The Role Of MiR155 In BMSC Exosomes Mediating Autophagy Through Rheb/mTOR Pathway In Intervertebral Disc Degeneration

Objective The treatment of intervertebral disc degeneration based on stem cells has been studied extensively.However,the mechanism of the interaction between bone marrow mesenchymal stem cells(BMSC)and nucleus pulposus cells(NPC)remains unclear.As an important paracrine mechanism in cell-to-cell communication,exosomes have received increased attention.The present study aimed to explore the protective effect of BMSC-derived exosomes on NPC apoptosis and intervertebral disc degeneration(IDD),and to explore the regulatory effect of mi RNAs(mi RNAs)in BMSC exosomes on NPC apoptosis and its related mechanisms.Methods Rat NPC and BMSC were isolated and cultured in vitro,and an Oxygen glucose deprivation(OGD)model was established to simulate the pathological state of intervertebral disc degeneration in vivo,and contactless co-culture was performed.The m RNA and mi R-155 expression changes of Beclin1,LC3,Rheb,m TOR and ATG1 in NPC were detected by q RT-PCR.The contents of Beclin1,LC3,Rheb,m TOR and ATG1 fusion proteins in NPC were detected by immunofluorescence staining.The above-mentioned related proteins were detected again by immunoblotting test,and the degree of autophagy and the degree of NPC apoptosis were evaluated by the content of Bcl-2 and Bax in nucleus pulposus cells and related changes.Results(1)Compared with the blank control group,the expression of mi R-155 in the degeneration group was significantly decreased(P<0.05),while the expression levels of Rheb and m TOR were significantly increased(P<0.05).ATG1 in the nucleus pulposus cells of the degeneration group The expression of NPC was significantly lower than that of the blank control group(P<0.01),and the expression levels of autophagy marker proteins Beclin1 and LC3 were also decreased(P<0.05).Increased expression of the death marker Bax.(2)Compared with the degeneration group,the expression level of mi R-155 in the co-culture group was significantly increased(P<0.001),the expression of Rheb and m TOR was significantly decreased(P<0.01),and the expression level of ATG1 was significantly increased(P<0.001).The expression of autophagy markers Beclin1 and LC3 in the co-culture group was significantly higher than that in the degeneration group(P<0.01),the level of autophagy in NPC was significantly increased,the expression of Bcl-2 was increased,and the expression of Bax was decreased.(3)Compared with the co-culture group,the expression level of mi R-155 in the autophagy inhibition group and the reduced mi R-155 expression group decreased(P<0.001),the expression of Rheb and m TOR increased(P<0.01),and the expression of ATG1 was increased(P<0.01).The levels were significantly decreased(P<0.001),the expression levels of Beclin1 and LC3 were significantly decreased(P<0.01),the level of NPC autophagy decreased,the expression of Bcl-2 decreased,and the expression of Bax increased.Conclusion(1)Under the condition of glucose and oxygen deprivation,the degree of autophagy of NPC decreased and the level of apoptosis was enhanced,which proved that NPC entered a degenerate state after modeling by glucose and oxygen deprivation.(2)After non-contact co-culture of BMSCs with NPCs under the condition of glucose and oxygen deprivation,the degree of autophagy and anti-apoptosis of NPCs increased,which reversed the degree of NPC degeneration.BMSCs have a certain therapeutic effect on intervertebral disc degeneration.(3)During the non-contact co-culture of BMSCs and NPCs,mi R-155 was an important substance carried by BMSC exosomes;mi R-155 in exosomes enhanced the autophagy intensity of rat nucleus pulposus cells and made nucleus pulposus cells anti-apoptotic The ability of apoptosis was enhanced,thereby improving the degree of degeneration of nucleus pulposus cells;reducing the expression of mi R-155 in BMSCs or adding an autophagy inhibitor to the co-culture system reversed the above process.(4)mi R-155 in exosomes can regulate Rheb,which is a negative regulator of autophagy,thereby affecting the Rheb/m TOR/ATG1 signaling pathway to achieve the purpose of regulating the autophagy level of NPC and improve the effect of IDD.