Mechanism Of Vascular Endothelial Cell Autophagy In Rat SANFH Model

Objective:The pathogenesis of steroid-induced femoral head ischemic necrosis has not been determined yet.Studies have shown that bone cell autophagy and apoptosis,gene polymorphism and vascular endothelial cell injury are all involved in the pathogenesis of SANFH^[1-3].However,the relationship between SANFH and vascular endothelial cell autophagy has not been fully elucidated.This study aims to explore the role of VEC autophagy in SANFH,to explore the relationship between SANFH and VEC autophagy in femoral head by studying autophagy related genes Beclin1,MAP1LC3 and PI3K/Akt/mTOR signaling pathways,and to study the possibility of mTOR-si RNA repairing VEC injury.Methods:The rat model of SANFH was established,and the rats were divided into 3 groups and injected with normal saline respectively.Methylprednisolone;The number of autophagosomes in the three groups was observed by transmission electron microscopy at 12,24,48 and 72h,respectively.The m RNA expressions of Beclin1,MAP1LC3,PI3K,Akt and mTOR were detected by RT-PCR.At 1w,2w,3w and 4w,the protein expression of the above factors was detected by immunofluorescence and Western blot,and the expression of6-Keto-PGF1α,a marker of vascular endothelial cell injury,was detected by ELISA.Finally,statistical methods were used for statistical analysis to determine whether there were statistical differences between the control group and the experimental group at different time points.Results:（1）Vacant bone lacunae in the control group were rare in HE staining and more in the hormone group,more in the mTOR-si group and less in the hormone group.（2）After 4 weeks of modeling,autophagosomes were observed by electron microscopy in the control group,but not in the hormone group and the mTOR-si group,but the mitochondrial structure of vascular endothelial cells in the mTOR-si group was still discernible.（3）The content of 6-Keto-PGF1αin the control group had no significant change,while that in the hormone group increased as time went on.mTOR-si group was higher than the control group,but lower than the hormone group.（4）The m RNA and protein expressions of Beclin1 and MAP1LC3 in the control group were higher than those in the hormone group,while the m RNA and protein expressions of PI3K,Akt and mTOR were lower than those in the mTOR-si group.Conclusion:（1）Abnormal VEC autophagy induced by hormones is associated with the pathogenesis of SANFH;（2）With the prolongation of hormone time,the expression of autophagy related genes Beclin1 and MAP1LC3 in VEC of femoral head gradually decreased,and the expression of PI3K/Akt/mTOR signaling pathway related factors gradually increased;（3）Inhibit the protective effect of physiological autophagy on VEC,resulting in damage of VEC,and then SANFH;（4）PI3K/Akt/mTOR signaling pathway can be inhibited by mTOR-si RNA,enhance physiological autophagy,and play a certain role in the repair of VEC injury and SANFH remission.