Effect Of Rapamycin-mTOR Pathway-induced Autophagy On Inhibition Of Chondrocyte Apoptosis

BACKGROUND & OBJECTIVE: Osteoarthritis(OA)is a chronic pain-disabled disease associated with apoptosis of articular chondrocytes and degradation of extracellular matrix(ECM).In recent years,it has been found that the loss of autophagy in chondrocytes plays a key role in the progression of OA.Autophagy is an adaptive response in which cells degrade intracellular components under stress conditions to maintain essential cellular functions.Rapamycin(RAPA)is a novel macrolide immunosuppressant that inhibits mTORC1 in the mTOR signaling pathway and induces autophagy initiation.Studies have shown that autophagy has a protective effect on OA chondrocytes.At present,the effect of autophagy on chondrocyte apoptosis has rarely been reported.In this study,the method of in vitro culture of chondrocytes was used to investigate the possible injury mechanism of dexamethasone(DEX)-induced chondrocyte apoptosis,and the effect of autophagy on DEX-induced chondrocyte apoptosis was studied by RAPA activation of chondrocyte autophagy.In order to provide a new basis for the prevention and treatment of OA.METHODS: Normal and OA chondrocytes were isolated and cultured from 5patients with femoral neck fracture who underwent hip arthroplasty and 7 patients with advanced OA who underwent total knee arthroplasty.The second-generation chondrocytes successfully cultured were divided into the following 6 groups: normal chondrocytes plus BSA were added to a final concentration of 100 ug/ml for 12 h;normal cartilage DEX cells were supplemented with DEX-BSA to a final concentration of 100 ug/ml for 12 h;normal The cartilage RAPA group was added with RAPA to a final concentration of 1mmol/L for 2 hours,then DEX-BSA was added to a final concentration of 100ug/ml for 12h;OA chondrocytes plus BSA to a final concentration of 100ug/ml for 12h;OA cartilage DEX group cells were treatedwith DEX-BSA to a final concentration of 100 ug/ml for 12 h;the OA cartilage RAPA group was added with RAPA to a final concentration of 1 mmol/L for 2 hours,and then DEX-BSA was added to a final concentration of 100 ug/ml for 12 h.The cell viability was detected by MTT assay,the number of autophagosomes in chondrocytes was detected by LC3 autophagy,and the expression of total LC3 B,LC3B-?II,ULK1 and Beclin1 in autophagy was detected by Western-Blot.Cellular ULK1 was detected by quantitative RT-PCR.mRNA expression of Beclin1,LC3,TNF-α and IL-1 was detected by ELISA.The concentrations of IL-1 and TNF-α were detected by ELISA.The apoptosis of chondrocytes was detected by flow cytometry and TUNEL.RESULTS: MTT assay showed that the survival rate of DEX-BSA group was lower than that of BSA group(P<0.001).Compared with the DEX-BSA group in normal or OA chondrocytes,the survival rate of the RAPA+DEX-BSA group was increased(P<0.001).Fluorescence spectrophotometric analysis of autophagosomes in LC3 autophagy showed that in the OA chondrocytes,the DEX-BSA group was lower than the BSA group(P<0.05).The RAPA+DEX-BSA group was elevated in the normal(P<0.05)and OA chondrocytes(P<0.001)compared with the DEX-BSA group.Western-Blot test showed that in normal or OA cartilage,compared with the DEX-BSA group,RAPA DEX-BSA total LC3B(P<0.001),LC3B-II(P<0.001),and ULK1(P<0.001)protein expression were all elevated.The Beclin1 protein expression in the RAPA+DEX-BSA group was higher than that in the DEX-BSA group in normal chondrocytes(P<0.001)and OA chondrocytes(P<0.01).Compared with the BSA group,the expression of ULK1(P<0.01),Beclin1(P<0.001),total LC3B(P<0.001),and LC3B-II(P<0.001)protein in the DEX-BSA group decreased in OA chondrocytes.In normal chondrocytes,the expression of ULK1(P<0.001),Beclin1(P<0.001),total LC3B(P<0.01),and LC3B-II(P<0.001)in the DEX-BSA group was lower than that in the BSA group.Real-time PCR showed that the gene expression of Beclin1(P<0.001),LC3(P<0.01)and ULK1(P<0.001)decreased in the DEX-BSA group compared with the BSA group in normal chondrocytes.Compared with the BSA group,the gene expression of TNF-α and IL-1 in the DEX-BSA group was increased(P<0.01),whether in normal or OA chondrocytes.In normal chondrocytes,LC3(P<0.05),Beclin1(P<0.001),and ULK1(P<0.001)genes were elevated in the RAPA+DEX-BSA group compared with the DEX-BSA group,but TNF-α(P<0.01)and IL-1(P<0.05)gene expression decreased.In OA chondrocytes,compared with DEX-BSA group,the expression of ULK1(P<0.01)and LC3(P<0.05)in RAPA+DEX-BSA group was higher,but there was no difference in Beclin1 gene expression(P=0.09),The expression of both TNF-α and IL-1 decreased(P<0.01).ELISA showed that the TNF-α and IL-1 protein concentrations in the DEX-BSA group were higher in normal chondrocytes(P<0.001)and OA chondrocytes(P<0.01)compared with the BSA group.Regardless of normal or OA chondrocytes,TNF-α(P< 0.001)and IL-1(P<0.01)decreased in the RAPA + DEX-BSA group compared with DEX-BSA group.Flow cytometry showed that in the normal cartilage or OA chondrocytes,the apoptosis rate was increased in the DEX-BSA group compared with the BSA group(P<0.001).The apoptosis rate of the RAPA+DEX-BSA group was lower than that of the DEX-BSA group in normal cartilage(P<0.01)and OA chondrocytes(P<0.001).TUNEL assay showed that in OA chondrocytes,the formation of apoptotic bodies in the DEX-BSA group was increased and the fluorescence spectrophotometry was increased compared with the BSA group(P<0.05).In normal chondrocytes,compared with the BSA group,the formation of apoptotic bodies in the DEX-BSA group increased,but the fluorescence spectrophotometry did not increase significantly(P=0.19).Regardless of normal or OA chondrocytes,the apoptotic bodies of the RAPA+DEX-BSA group were lower than the DEX-BSA group.CONCLUSIONS: DEX can reduce the survival rate of chondrocytes and increase apoptosis,leading to destruction of chondrocytes and involvement in the pathogenesis of OA.The mechanism may be related to the increase of TNF-α and IL-1 expression and the reduction of autophagy.RAPA can activate autophagy in chondrocytes,reduce the expression of TNF-α and IL-1 in chondrocytes and inhibit the apoptotic pathway to enhance cell viability and reduce or alleviate the damage of DEX on chondrocytes.It has cartilage protection against DEX-induced apoptosis.