Study On The Mechanism Of MSC-Exos Regulating The Autophagy Of Vascular Endothelial Cells Mediated By MTOR Signal Pathway In Rat SANFH Model

Objective: The aim of this study was to investigate the correlation between PI3K/Akt/mTOR signaling and autophagy regulatory genes Beclin1 and MAP1LC3 in SANFH,and to investigate the impact of MSC-Exos on VEC autophagy and the ability of angiogenesis in SANFH on the regulation of VEC autophagy and angiogenesis in SANFH.Methods: Ten healthy rats were selected at 15 days of age,and three generations of intra-femoral microcirculatory VEC were extracted and cultured and then grouped.The first group was the control group,which was normal cultured VEC without any treatment;the second group added methylprednisolone(MPS group)to achieve a final concentration of 1 μmol/L;the third group added methylprednisolone with 200 μL MSC-Exos(MPS+MSC-Exos group).Extract VECs cultured in each group and detect their cell viability using CCK-8 Kit at 12,24,48,and 72 hours after administration;Transwell measured Cell migration;Immunofluorescence staining(GFP)was used to detect the expression levels of GFP-Beclin1,GFP-LC3,GFP PI3 KI,GFP-mTOR,and GFP-Akt proteins in VEC.The expression levels of Beclin1,MAP1LC3,PI3 KI,mTOR and Akt in vascular endothelial cells were measured by Western blotting,and the expression of6-keto-PGF1a(VEC injury marker)in VEC was detected by ELISA.Finally,statistical analysis was applied to calculate whether there was a statistical difference between the control and experimental groups at each time point.Results:(1)The protein expressions of Beclin1 and MAP1LC3 in MPS group were lower than those in the control group,while the protein expressions of PI3 K,Akt and mTOR were higher than those in the control group.MPS+MSC-Exos group was between the two groups;(2)At different time points,the cell activity of the control group was almost unchanged.The cell activity of the MPS group was significantly lower than that of the control group at each time point,and the MPS+MSC-Exos group was between the two groups;(3)6-keto-PGF1 αThere was no significant change in the content of MPS in the control group,and the MPS group showed an overall upward trend over time.The MPS+MSC-Exos group was higher than the control group,but lower than the hormone group;(4)At different time points,the cell migration level of the control group was almost unchanged.The cell migration level of the MPS group was significantly lower than that of the control group,and the MPS+MSC-Exos group was between the two groups.Conclusion:(1)The decrease of physiological autophagy of VEC under hormonal action can lead to the occurrence of SANFH;(2)Hormones inhibit VEC physiological autophagy by enhancing PI3K/Akt/mTOR signaling pathway,thereby damaging VEC and leading to the development of SANFH;(3)MSC-exos may enhance VEC physiological autophagy by inhibiting the PI3K/Akt/mTOR signaling pathway,and may delay or repair avascular necrosis of femoral head cells by repairing VEC damage,ultimately achieving the therapeutic effect of SANFH.