The Effect And Mechanism Of Everolimus Induce Fibroblast Apoptosis And Reduce Postoperative Knee Fibrosis By Activating Fibroblasts Autophagy

Objective:To investigate the inhibitory effect of Everolimus(EVE)on postoperative knee fibrosis and its possible mechanism.Methods:Local fibrotic tissues from patients undergoing revision surgery for artificial knee joints were histologically stained and cell identification.Then the rabbit knee fibrosis model was established.After anesthesia,the medial skin of the knee joint was incised,the medial femoral condyle was exposed.After removed the bone cortex of about 1.0×1.0cm,normal saline and 30ng/ml EVE respectively were performed with local treatment.4 weeks later,specimens were taken to make paraffin sections.Immunofluorescence staining of paraffin sections(IF-P)confirmed that fibroblasts are the main components of fibrotic tissue.The inhibitory effect of EVE on postoperative fibrosis of rabbit knee joint was observed by HE staining,and the effect of EVE on collagen deposition in postoperative fibrosis tissue of rabbit knee model was observed by Masson and Sirius red staining.Subsequently,fibroblasts derived from local fibrotic tissue of human were cultured in vitro,and the cells were treated with different concentrations of EVE.CCK-8 and flow cytometry were used to detect the effects of EVE on fibroblast viability and apoptosis.LC3 immunofluorescence(IF)and transmission electron microscopy(TEM)were used to detect the effect of EVE on fibroblast autophagy;Western blot(WB)was used to detect experession of EVE on apoptosis-related proteins(Bcl-2,Bax,cleaved-PARP),autophagy-related proteins(LC3,Atg5 and Beclin-1)and PI3K/AKT/mTOR signal-related proteins.In order to explore the potential relationship between EVE-activated fibroblast autophagy and EVE-induced fibroblast apoptosis,the effect of inhibiting autophagy on EVE-induced apoptosis of fibroblasts were observed after pretreatment with autophagy inhibitor(3-MA).Results:The histological staining of human knee fibrotic tissue showed that there were dense fibrosis formation and abundant collagen tissue deposition in the local area.IF-P results confirmed that the fibrotic tissue contained a large number of fibroblasts.Animal experiments showed that:IF-P confirmed that the main constituent cells of rabbit knee fibrosis tissue are fibroblasts,which are consistent with the main constituent cells of human knee fibrosis tissue after surgery.The results of HE staining confirmed that the degree of fibrosis in the knee operation area which treated by 3 0ng/ml EVE was significantly less than that which treated by normal saline.The results of Masson staining and Sirius Red staining showed that the collagen content of the knee operation area which treated by 30ng/ml EVE was significantly lower than that which treated by normal saline.In vitro:the results of CCK-8 showed that EVE can inhibit the activity of fibroblasts.Flow cytometry showed that EVE can increase the apoptosis rate of fibroblasts.WB showed that EVE can up-regulate the expression of apoptotic proteins Bax and cleaved-PARP,while down-regulate the expression of anti-apoptotic protein Bcl-2.TEM results showed that the number of autophagosomes in the fibroblasts of the EVE treatment group was significantly increased compared with the control group.IF results showed that after treating with EVE,the green fluorescence around the nucleus was obviously gathered,and it gradually increased in the manner of concentration-dependment.WB showed that EVE can up-regulate the expression of autophagy-related proteins(LC3,Atg5 and Beclin-1).After combined with the autophagy inhibitor(3-MA),IF results showed that the green fluorescence accumulated around the nucleus after EVE treatment decreased.And WB results showed that the expression of autophagy-related proteins(LC3,Atg5 and Beclin-1)also decreased.At the same time,flow cytometry confirmed that the apoptosis rate of EVE up-regulated decreased,the high expression of apoptotic proteins Bax and cleaved-PARP decreased,and the expression of anti-apoptotic protein Bcl-2 increased.Treatment of fibroblasts with EVE can down-regulate the expression levels of p-PI3K,p-AKT,and p-mTOR,while 3-MA can partially reverse the expression levels of p-PI3K,p-AKT,and p-mTOR down-regulated by EVE.Conclusion:Fibroblast hyperproliferation is one of the factors of postoperative knee fibrosis and EVE can reduce postoperative knee fibrosis.EVE can inhibit PI3K/AKT/mTOR signaling pathway to activate fibroblast autophagy and induce apoptosis,which may be the molecular mechanism of EVE inhibiting postoperative knee fibrosis.