Experimental Study On The Role Of Autophagy And Apoptosis Regulated By 3-MA In SANFH

Objective: Steroid-induced avascular necrosis of the femoral head(SANFH)has always been a challenging disease.Heavy use of glucocorticoids will cause ischemia,hypoxia and oxidative stress of the femoral head.finally,it can induce the abnormal expression of genes related to autophagy and apoptosis signal pathway,leading to osteonecrosis.3-Methyladenine has a significant inhibitory effect on the production of autophagosomes and can effectively inhibit the process of autophagy,so it is one of the most commonly used autophagy inhibitors.In this study,the effect of 3-MA on SANFH was studied in vivo.By detecting the changes of m RNA and protein expression of Beclin-1 and LC3 in autophagy signal pathway and Caspase-3 and Caspase-9 in apoptosis signal pathway,to analyze the role of autophagy and apoptosis induced by glucocorticoid in SANFH,and to evaluate whether 3-MA can provide a new direction for the prevention and treatment of SANFH.Methods: We selected 60 SD rats and randomly divided them into three groups to establish SANFH animal model.Groups A,B and C received intramuscular injection of normal saline,methylprednisolone,methylprednisolone and 3murma A respectively as the control group,providing data basis for the other two groups.The ischemic necrosis of bone tissue cells in each group was observed by light microscope and transmission electron microscope,and the pathological changes of bone tissue cells in each group were observed by Hoechst staining and HE staining.The m RNA and protein expressions of Caspase-3,Caspase-9,Beclin-1,LC3 in bone tissue cells of each group were detected by q RT-PCR test and Western blotting test,and the correlation analysis was carried out.Results:(1)According to the observation of light microscope and transmission electron microscope,as well as the results of Hoechst staining and HE staining,the results showed that the tissue of the femoral head of the control group was normal and uniform,the articular surface of the bone was smooth,and there were no obvious pathological changes of bone cells.On the other hand,the action of methylprednisolone can lead to irregular arrangement of osteocytes,sparse trabeculae,disordered structure,partial fracture,a large number of osteocytes in the femoral head tissue,an increase in the number of empty bone lacunae,a significant increase in the number of autophagy bodies in cells,and gradually aggravated with time.3-MA intervention can significantly alleviate the above pathological manifestations.(2)The results of q RT-PCR and WB showed that the m RNA expression and protein content of related genes Beclin-1,LC3,Caspase-3 and Caspase-9 in bone tissue cells of rats injected with methylprednisolone were significantly increased,but the expression levels of related m RNA and protein in osteoblasts were significantly decreased after the intervention of methylprednisolone plus 3-MA,and the pathological changes of osteonecrosis of the femoral head were slighter and progressed more slowly.Conclusion:(1)The excessive application of glucocorticoid will lead to the loss of the original rules of the tissue and cell row of the femoral head,the disorder of trabecular structure and the phenomenon of fracture.With the progress of the experiment,after the action of methylprednisolone,the number of hollow bone lacunae and autophagosomes increased greatly,a large number of osteocytes necrosed,and finally the macroscopic joint collapse of the femoral head appeared.(2)Glucocorticoid-induced SANFH is closely related to autophagy and apoptosis.abnormal m RNA and protein expressions of Beclin-1,LC3,Caspase-3 and Caspase-9,which are related to autophagy and apoptosis,were observed in group B and C.with the progress of the experiment,the expression of related genes in group B and C increased significantly,the number of autophagy corpuscles increased and cell necrosis.(3)3-MA,an inhibitor of autophagy,can inhibit the process of autophagy and affect the course of SANFH.Compared with group B,the m RNA and protein expression of Beclin-1,LC3,Caspase-3 and Caspase-9 in group C were significantly lower,the number of autophagosomes and the degree of cell necrosis were lower in group C.It is speculated that3-MA may treat or delay the occurrence and development of SANFH by regulating the occurrence and development of autophagy.This is likely to become a new target in the treatment of SANFH and provide new inspiration and ideas for the prevention and treatment of SANFH.