Effects Of ASIC1a-Mediated Aupothagy On Apoptosis Of Rat Articular Chondrocytes

Objective: Rheumatoid arthritis(RA)is a chronic,systemic autoimmune disease that eventually leads to joint damage and loss of function.In our previous study,we found that acid-sensitive ion channel 1a(ASIC1a)was significantly overexpressed in the rat model of adjuvant arthritis(AA)for the first time,and played an important role in the pathogenesis of cartilage and bone tissue destruction in AA rats.Further study found that ASIC1 a may be involved in the pathological process of RA through the mediated autophagy and apoptosis of articular chondrocytes.As two forms of programmed death,autophagy and apoptosis are closely related and complicated.The main purpose of this study was to investigate the effect of ASIC1 a mediated autophagy on apoptosis of rat articular chondrocytes,and to explore the role of autophagy in the pathogenesis of RA.Methods: AA rat model was established in vivo,and the inflammation was induced at0,15,25 and 35 days,respectively.The dynamic changes of autophagy and apoptosis were observed.Using amiloride to block ASIC1 a in AA rats,the correlation between ASIC1 a and autophagy was observed,and also the correlation between ASIC1 a with apoptosis.In vivo and in vitro,autophagy specific inhibitor 3-methyladenine(3-MA)and autophagy inducer Rapa were used to block or induce autophagy,and the effects of ASIC1 a mediated autophagy on apoptosis of articular chondrocytes in rats were observed.Body mass index,arthritis score and the expression level of inflammatory factors in serum of AA rats were used to determine whether the establishment of AA rat model was successful or not.The pathological changes of anklebone in AA rats were detected by HE staining.Immunohistochemstry(HIC)detection LC3 and type II collagen(Co II)expression.Tunel assay was used to detect the apoptosis of AA rat chondrocytes.Tunel and immunofluorescence were used to detect the co-expression of autophagy and apoptosis.Western blot was used to detect the expression of target proteins(Co II,Agg,LC3,Beclin1,cleaved Caspase-3,cleaved Caspase-9,cleaved Caspase-PARP).The morphological changes of chondrocyte apoptosis were observed by Hoechst33342 staining.The apoptosis rate of chondrocytes was observed by flow cytometry.Results: On the 14 th day after inflammation in AA rats and compared with the normal group,body weight increased slowly,joints became inflamed and stiff,disordered arrangement of chondrocytes,and activities were limited.The level of inflammatory factors in serum increased detected by ELISA.In the ankle joint section,synovial tissue hyperplasia,massive inflammatory cell infiltration,disordered chondrocytes,and loss of Co II were observed,indicating the success of the model establishment.Autophagy and apoptosis were expressed dynamically in different stages of adjuvant-induced inflammation.After blocked ASIC1 a in vivo,both autophagy and apoptosis of chondrocytes were inhibited,suggesting that both autophagy and apoptosis of chondrocytes occurred simultaneously in the development process of AA rats,possibly mediated by ASIC1 a.When the autophagy inhibitor 3-MA was used to treat AA rats,the joint inflammation and damage were intensified.However,after the treatment with the autophagy inducer Rapa in AA rats,joint inflammation and damage were alleviated.Moreover,apoptosis of articular chondrocytes was up-regulated after autophagy was inhibited,while apoptosis of articular chondrocytes was down-regulated after autophagy was induced.In vitro experiments further confirmed that autophagy can inhibit apoptosis of articular chondrocytes.These experimental results suggest that autophagy may protect articular chondrocytes by inhibiting apoptosis during the occurrence and development of adjuvant arthritis,and reveal that ASIC1 a mediated autophagy may be an important target for the treatment of RA.Conclusions: The results showed that autophagy and apoptosis of articular chondrocytes were expressed simultaneously in the pathological process of RA,and were closely related to ASIC1 a.Autophagy had a protective effect on articular chondrocytes by inhibiting apoptosis and joint inflammation.For the first time,We confirmed the dynamic changes of autophagy and apoptosis of articular chondrocytes in AA rats,and the important role of autophagy in regulating chondrocytes inflammation.These results suggest that autophagy may be a potential target for RA therapy.