Estrogen Antagonizes ASIC1a-induced Chondrocyte Mitochondrial Stress In Rheumatoid Arthritis

Rheumatoid arthritis（RA）is a chronic autoimmune disease with unknown etiology,which is related to gender differences.The peak incidence of RA in women is reached after menopause,with temporary relief of symptoms after ovulation,suggesting that changes in estrogen levels play a key role in the development of RA.The damage of articular cartilage caused by chondrocyte death is the main cause of RA joint dysfunction.Tissue acidification is an important factor leading to cell death.Acid-sensitive ion channel1a（ASIC1a）is an important ion channel for the corresponding extracellular acidification signal,which mediates the influx of calcium ions and leads to chondrocyte and tissue damage.Previous research results show that estrogen induces ASIC1a protein degradation through autophagy lysosome pathway,which protects chondrocytes and articular cartilage.Calcium overload induces mitochondrial stress mediating cell death and tissue damage in pathological conditions,but its potential function in RA is unclear.Objectives:This study focuses on elucidating the important role of mitochondrial stress in cartilage injury caused by ASIC1a and the regulation of ASIC1a function by estrogen,aiming to provide new insights into the pathogenesis and therapeutic strategies of RA.Methods:1.Effect of ASIC1a activation on mitochondrial stress.Chondrocytes were stimulated with p H 6.0 acid for different times（0 h,45 min,1.5h,3 h,6 h,12 h）.The m RNA levels of mitochondrial stress protein（HSP10,LONP1,Clp P）were detected by real-time quantification-polymerase chain reaction（RT-q PCR）,and mitochondrial stress protein（HSP10 and LONP1）fluorescence levels were detected by laser confocal.Mitochondrial membrane potential was detected by JC-1,and fluorescent probes targeting mitochondrial reactive oxygen species（mt ROS）were used to detect change in mitochondrial mt ROS level.Finally,mitochondrial morphological changes were observed by transmission electron microscopy.The cells were pretreated with ASIC1a specific inhibitor Pc Tx-1 and then acid stimulated for 6 h.The mitochondrial stress protein fluorescence level was detected by immunofluorescence,the mitochondrial membrane potential and mt ROS were detected by laser confocal,the mitochondrial morphological changes were observed by transmission electron microscopy.2.Effect of ASIC1a-mediated calcium ion influx on mitochondrial stress.Chondrocytes were stimulated with p H 6.0 acid for different times（0 h,45 min,1.5h,3 h,6 h,12 h）.Fura-3/AM probes labeled intracellular Ca²⁺was detected by laser confocal microscopy.The cells were divided into control group,p H 6.0 group,and extracellular calcium chelator（EGTA）+p H 6.0 group.The m RNA and fluorescence levels of mitochondrial stress proteins HSP10,LONP1,and Clp P were detected by RT-q PCR and immunofluorescence,mitochondrial membrane potential and mt ROS were detected by laser confocal.3.Effect of estrogen on ASIC1a function.Rat articular chondrocytes were treated with estrogen at different times（0 h,12 h,24h,36 h,48 h,72 h）.Detection of ASIC1a protein expression by western blot.Rat articular chondrocytes were pretreated with estrogen or Pc Tx-1 and stimulated with p H 6.0.ASIC1a fluorescence levels were detected by immunofluorescence,and intracellular Ca²⁺was labeled by Fura-3/AM probe and detected by laser confocal microscopy.4.Effect of estrogen on ASIC1a-mediated mitochondrial stressRat articular chondrocytes were treated with estrogen and Pc Tx-1 respectively.The m RNA levels of mitochondrial stress proteins HSP10,LONP1 and Clp P were detected by RT-q PCR.Rat articular chondrocytes were treated with estrogen for 48 h and stimulated with p H 6.0 for 6 h.The fluorescence levels of mitochondrial stress proteins HSP10,LONP1,and Clp P were detected by immunofluorescence.The mitochondrial membrane potential and mt ROS levels were detected by laser confocal microscopy.The effect of estrogen on the morphological changes of mitochondria was observed by transmission electron microscopy.5.Effects of estrogen on articular cartilage in AA ratsFemale SD rats were divided into the following groups after ovarian removal surgery:normal group,AA model group,estrogen treatment group（low dose:0.1 mg/kg;medium dose:0.2 mg/kg;high dose:0.3 mg/kg）,and positive drug tretinoin（TA）group.The sham-operated group had only surgery without ovarian removal.Histopathological lesions of the joints were observed by hematoxylin-eosin staining（HE）,toluidine blue was used to stain the joints to observe cartilage damage.The expression of mitochondrial stress proteins（HSP10,LONP1,Clp P）was detected by immunohistochemistry.Results:1.Effect of ASIC1a activation on mitochondrial stress.Compared with normal rat articular chondrocytes,acid stimulation led to an increase in mitochondrial stress protein m RNA and fluorescence levels,a decrease in mitochondrial membrane potential,an increase in mt ROS release,and mitochondrial structural damage such as swelling,cristae breakage disruption,and membrane disruption in mitochondria.The optimal time of acid stimulation was determined by the above experiments as 6 h.Compared with the p H 6.0 group,inhibition of ASIC1a could significantly reduce mitochondrial stress protein m RNA and fluorescence levels,elevate mitochondrial membrane potential,decrease mt ROS release,and improve mitochondrial structural damage.2.Effect of ASIC1a-mediated calcium ion influx on mitochondrial stress.The result showed that ASIC1a activation increased intracellular calcium ion concentration in a time-dependent manner.Compared with the p H 6.0 group,treatment with EGTA could significantly decrease m RNA and fluorescence levels of mitochondrial stress proteins HSP10,LONP1,and Clp P,increase membrane potential of mitochondria,decrease mt ROS release.3.Effect of estrogen on ASIC1a function.The result showed that estrogen decreased ASIC1a protein levels in a time-dependent manner,and the optimal action time of estrogen was 48 h.Chondrocytes treatment with estrogen and Pc Tx-1 could significantly reduce ASIC1a fluorescence and Ca²⁺levels compared with the p H 6.0 group.4.Effect of estrogen on ASIC1a-mediated mitochondrial stressCompared with the acid-stimulated group alone,estrogen pretreatment for 48 h could significantly reduce the m RNA and fluorescence levels of mitochondrial stress proteins HSP10,LONP1,and Clp P,restore the mitochondrial membrane potential,and reduce the release of mt ROS.Moreover,estrogen significantly improved a series of structural damage of mitochondria caused by acidosis.5.Effects of estrogen on articular cartilage in AA ratsCompared with AA rats in model group,estrogen treatment could significantly reduce synovial infiltration and cartilage damage in ovariectomized AA rats in a dose-dependent manner.Compared with the normal group rats,the expression of mitochondrial stress proteins HSP10,LONP1,and Clp P in the cartilage tissue of AA rats in the model group increased significantly.The use of estrogen treatment reduced the expression levels of HSP10,LONP1,and Clp P.Conclusions:1.ASIC1a activation-mediated calcium ion influx induces mitochondrial stress in rat chondrocytes.2.Estrogen antagonizes mitochondrial stress by decreasing ASIC1a activity and protects articular cartilage in AA rats.