Effects Of ASIC1a-mediated Autophagy On Senescence Of Rat Articular Chondrocytes Under Extracellular Acidification

ObjectiveAcid-sensing ion channels（ASICs）are a class of ligand-gated ion channels that are members of the DEG/ENa C superfamily and can be activated by decreases in extracellular p H or increases in H⁺concentration.Studies have shown that ASIC1a plays an important role in the development of several tissues acidifying diseases such as inflammation,ischemia,and hypoxia.A common feature in these pathological responses is local tissue acidification,and the activated acid-sensitive ion channel ASIC1a mediates the inward flow of extracellular Ca²⁺and Na⁺plasma and activates various pathophysiological effects.Our group has shown that ASIC1a is strongly expressed on rat articular chondrocytes and is involved in the process of tissue acidification-induced articular chondrocyte damage.Recent studies have shown that the progression of osteoarthritis is accompanied by a decrease in tissue microenvironment p H,cartilage damage,local bone erosion,and chondrocyte senescence.It has also been shown that there is excessive activation of autophagy on articular chondrocytes in osteoarthritis and that it plays an important role in the destruction of articular chondrocytes,but the exact mechanism is not known.Therefore,in this study,SPF-grade rat primary chondrocytes were used to study the effect of ASIC1a-induced autophagy on the senescence of rat articular chondrocytes and its mechanism by using extracellular acidification stimulation to simulate the synovial fluid environment in patients with osteoarthritis.MethodsIn this study,rat articular chondrocytes cultured in vitro were isolated and extracted by type II collagenase digestion.1.Different time of extracellular acidification:the primary chondrocytes were acidified under the condition of extracellular acidification p H6.0 at different time points（0、12、24、36、48h）,and the senescence-related proteins p16、p21、p53 and autophagy-related proteins Beclin-1 and LC3B-Ⅱ;were detected by Western blot method to observe the aging phenomenon of chondrocytes by detecting the expression ofβ-galactosidase.2.Chondrocytes were divided into four groups:Control group,p H6.0 group,Pc Tx1 group,and p H6.0+Pc Tx1 group.The expression of p16、p21、p53、Beclin-1 and LC3B-Ⅱprotein was detected by Western blot;the expression ofγ-H2A.X and Lamin B1 was detected by immunofluorescence;the expression of mitochondrial membrane potential was detected by Rhodamine123 staining;reactive oxygen species（ROS）was detected by fluorescence probe DCFH-DA.The number of autophagosomes was detected by the transmission electron microscope.3.The effects of different concentrations of CQ on the viability of chondrocytes were detected by adding autophagy blocker chloroquine.After selecting the appropriate concentration of CQ,chondrocytes were divided into four groups:Control group,p H6.0group,CQ group,and p H6.0+CQ group.The expression of senescence-related proteins p16、p21、p53 and autophagy-related protein Beclin-1,LC3B-Ⅱwere detected by,Western blot under the condition of p H6.0.The senescence of chondrocytes was observed by detecting the expression ofβ-galactosidase in treated cells;the expression ofγ-H2A.X and Lamin B1 in chondrocytes was detected by immunofluorescence;the expression of mitochondrial membrane potential was detected by Rhodamine123 staining;reactive oxygen species（ROS）was detected by fluorescence probe DCFH-DA.Results1.Effect of extracellular acidification on senescence of rat articular chondrocytes cultured in vitro.The results of Western blot showed that the expression of aging-related proteins p16、p21、p53 gradually increased,while the autophagy-related proteins Beclin-1 and LC3B-Ⅱincreased.Therefore,we speculated that acidification induced cell senescence and autophagy increased gradually.By detecting the expression ofβ-galactosidase,it was found that the phenomenon of cell senescence was more obvious after acidification for48 h under the condition of p H6.0.On this basis,the treatment time of extracellular acidification p H6.0,for 48 h was selected as the in vitro model of articular chondrocyte senescence.2.Effect of ASIC1a on chondrocyte senescence induced by extracellular acidification.Western blot detection showed that the expression of aging-related proteins p16,p21,p53 and autophagy-related proteins Beclin-1 and LC3B-Ⅱin the acidification group were significantly higher than those in the control group,and TEM detection showed that there were more autophagy bodies in the cytoplasm of acidified chondrocytes.However,the expression of aging-related proteins p16、p21、p53and autophagy-related proteins Beclin-1 and LC3B-Ⅱin the acidified group was significantly lower than that in the acidified group,and the number of autophagosomes in TEM was less than that in the acidified group.The expression of ASIC1a specific antagonist Pc Tx1 was significantly decreased in the acidified group.In Rhodamine123 staining and reactive oxygen species detection,it was found that the mitochondrial membrane potential of the acidified group was lower than that of the control group,and that of reactive oxygen species was higher than that of the control group.The mitochondrial membrane potential of the p H6.0+Pc Tx1 group was higher than that of the p H6.0 acidified group,and that of reactive oxygen species was lower than that of the acidified group.In immunofluorescence,the fluorescence expression ofγ-H2A.X in the p H6.0 group was higher than that in the control group,the expression in the p H6.0+Pc Tx1 group was lower than that in p H6.0 group,the fluorescence expression of Lamin B1 in p H6.0 group was lower than that in the control group,and the expression in p H6.0+Pc Tx1 group was significantly higher than that in p H6.0 group.3.Effect of autophagy in extracellular acidification of ASIC1a-mediated chondrocyte senescence.Western blot detection showed that the expression of chondrocyte aging-related proteins p16、p21、p53 and autophagy-related protein Beclin-1,LC3B-Ⅱin the CQ+p H6.0 group was lower than that in p H6.0 group,the positive rate ofβ-galactosidase in the CQ+p H6.0 group was lower than that in p H6.0 group,and the fluorescence expression ofγ-H2A.X and Lamin B1 in CQ+p H6.0 was significantly lower than that in p H6.0.Conclusion1.Extracellular acidification stimulation can induce the senescence of rat articular chondrocytes cultured in vitro.with the increase of acidity,the activity of chondrocytes decreases and the level of autophagy increases gradually.2.Inhibition of ASIC1a expression can inhibit the senescence of rat chondrocytes induced by extracellular acidification.3.Blocking autophagy can inhibit the senescence of rat articular chondrocytes induced by acid,and its mechanism may be achieved by blocking ASIC1a,to inhibit autophagy,and then inhibit the senescence of chondrocytes.