The Role And Mechanism Of Lamin B1 In The Senescence Of Osteoarthritis Articular Chondrocytes Mediated By Acid-sensitive Ion Channel 1a

Objective Osteoarthritis（OA）is a common and debilitating chronic joint disease,which is characterized by degeneration of articular cartilage and the aging of chondrocytes.Acidsensitive ion channel 1a（ASIC1a）is a proton-activated cationic channel abundant in chondrocytes,which senses and regulates joint cavity p H.Our previous study demonstrated that ASIC1 a was involved in acid-induced rat articular chondrocyte senescence,but the mechanistic basis remained unclear.In this study,we explored the mechanism of ASIC1 a in chondrocyte senescence and OA.The results showed that senescence-related-β-galactosidase,senescence-related markers（p53 and p21）and the autophagy-related protein Beclin-1 were found to be increased,but Lamin B1 was found to be reduced with acid（p H 6.0）treatment.These effects were inhibited by ASIC1 aspecific blocker psalmotoxin-1 or ASIC1a-short hairpin RNA respectively in chondrocytes.Moreover,Silencing of Lamin B1 enhanced ASIC1a-mediated chondrocyte senescence,this effect was reversed by overexpression of Lamin B1,indicating that Lamin B1 was involved in ASIC1a-mediated chondrocyte senescence.Further,blockade of ASIC1 a inhibits acid-induced autophagosomes and Beclin-1 protein expression,suggesting that ASIC1 a is involved in acid-induced chondrocyte autophagy.Blocking autophagy with chloroquine inhibited Beclin-1 and increased Lamin B1 in acidinduced chondrocyte senescence.We further demonstrated that ASIC1a-mediated reduction of Lamin B1 expression was caused by autophagy pathway-dependent protein degradation.Finally,blocking ASIC1 a protected cartilage tissue,restored Lamin B1 levels and inhibited chondrocyte senescence in a rat OA model.In summary,these results indicate that blockade of ASIC1 a inhibits osteoarthritis chondrocyte senescence by promoting autophagy pathway-dependent Lamin B1 protein degradation.In this regard,we will use cultured rat primary chondrocyte to construct an in vitro model,and perform repetitive verification of some important results on Human cartilage C28/I2 cell lines,and construct an in vivo OA rat model.The following studies are carried out:Methods1.The effect of silencing ASIC1 a on the senescence of rat articular chondrocyte induced by extracellular acidification: The treatment was divided into NC control group,p H6.0+NC group,ASIC1 a silent group,p H 6.0+ASIC1a silent group,and β-galactosidase staining to detect articular chondrocyte senescence;Western blot method to detect ASIC1 a,p21,p53,Lamin B1 and Beclin-1 protein expression;Cellular immunofluorescence to detect Lamin B1 expression in rat primary chondrocyte;RTq PCR to detect ASIC1 a gene expression.2.The effect of blocking protein synthesis pathway,autophagy lysosome pathway and proteasome pathway on the senescence of chondrocyte induced by extracellular acidification: Treatment was divided into normal group,p H 6.0 group,tool drug group,and p H 6.0+ tool drug group.Western blot method was used to detect the expression of Beclin-1,p21,p53 and Lamin B1;RT-q PCR was used to detect the expression of Lamin B1 gene;cell immunofluorescence was used to detect the expression of Lamin B1 and γ-H2 A.X in primary rat chondrocyte;Transmission electron microscope was used to observe the ultrastructure of autophagosomes;β-galactosidase staining was used to detect the senescence of articular chondrocyte.3.The effect of silent/overexpression Lamin B1 on the senescence of chondrocytes induced by extracellular acidification: Treatment was divided into NC control group,p H 6.0 group,silence/overexpression Lamin B1 group,p H 6.0+silence/overexpression Lamin B1 group,β-galactosidase staining was used to detect the senescence of articular chondrocyte;Western blot method was used to detect the expression of p21,p53,Lamin B1 and Beclin-1 protein;cell immunofluorescence was used to detect the expression of Lamin B1 and γ-H2 A.X in chondrocytes;RT-q PCR to detect the expression of Lamin B1 gene.4.Blocking the effect of ASIC1 a on the aging of OA rats and its protective effect on OA:Male SD rats were 6-8 weeks,about 200-220 g,randomly divided into normal group,model group and treatment groups.There were 8 rats in each of the normal group and the model group,and 8 rats in each of the treatment groups: Pc Tx1 group and hyaluronic acid（HA,1%）group.HE staining,Safranin O-fast green and toluidine blue staining were used to observe the pathological changes of the knee joint;immunohistochemistry was used to detect the expression of Lamin B1,p21 and p53 proteins in the knee cartilage;electronic imaging of articular cartilage observation and Pelletier score.Results1.Silencing ASIC1 a inhibits chondrocyte senescence induced by extracellular acidification: Compared with the p H 6.0 group,the ASIC1 a silence group significantly reduced the positive rate of senescent cells after acidification induction.And it could increase the expression of Lamin B1 protein,inhibited the expression of senescencerelated proteins p53 and p21 and autophagy-related protein Beclin-1.2.The silencing of Lamin B1 promoted ASIC1a-mediated chondrocyte senescence: β-galactosidase staining showed that compared with the normal group,the positive rate of senescent cells was significantly increased in the Lamin B1 silenced group,and the expression of senescence-related proteins p53 and p21 obviously increased.3.ASIC1a-mediated Lamin B1 protein degradation by autophagy: In p H 6.0 group adding CQ（20 μM）could reverse the decrease in Lamin B1 protein expression induced by extracellular acidification,and inhibited senescence-related proteins p53 and p21 and autophagy-related protein Beclin-1.4.Blockade of ASIC1 a protected chondrocyte on the rat OA model: Cartilage-specific staining showed that blocking ASIC1 a with Pc Tx1 significantly reduced the destruction of cartilage structure in OA rats.Blockade of ASIC1 a significantly restored Lamin B1,whereas downregulated the expression of p21 and p53 in OA cartilage.ConclusionIn summary,the micro-environment of extracellular acidification activated ASIC1 a,degraded Lamin B1 protein through the autophagy pathway,destroy the structure and function of the nucleus,and accelerated the process of osteoarthritis.It provides a new mechanism for osteoarthritis treatment and cartilage chondrocyte senescence.