| Rheumatoid arthritis(RA)is an autoimmune disease characterized by persistent inflammatory reactions in the joint sites that eventually lead to irreversible destruction of cartilage and bone tissue.A growing body of evidence shows that there are significant gender differences in the incidence and prognosis of RA.Epidemiological data show that the prevalence of RA in women increases significantly after menopause,which may be caused by the lack of the protective effect of estradiol.The main pathological feature of RA is the acidification of tissue fluid in the articular cavity.A large number of studies have shown that extracellular acidification can induce autophagy of articular chondrocytes.Acid-sensing ion channels(ASICs)are cationic channels that can be activated by extracellular H~+,and their activation is closely related to acid-induced chondrocyte injury.Our research group previously demonstrated that ASIC1a was highly expressed in the articular cartilage of rats with Adjuvant arthritis(AA)model and played a role in articular cartilage injury,and the protective effect of ASIC1a on articular chondrocytes of AA rats was achieved by inhibiting autophagy of chondrocytes.Recent studies have found that estradiol can significantly increase the activity of acidification induced chondrocytes In vitro and inhibit the expression of ASIC1a.However,the protective effect of estradiol on articular cartilage tissue of AA rats and the specific effect of estradiol on ASIC1a-mediated autophagy of articular chondrocytes and its mechanism remain unclear.In order to study the protective effect of estradiol on the articular cartilage of AA rats and its specific mechanism,this subject conducts the following experimental research:1.Estradiol protects articular cartilage in AA rats(1)In vivo experiment:ovary extraction(OVX)was used to remove rat ovaries to eliminate the interference of estradiol in the experiment;Freund’s complete adjuvant(CFA)induced AA rat adjuvant arthritis model,and then given different concentrations Estradiol E2(0.1,0.2,0.3 mg/kg)and the positive drug triamcinolone acetonide(TA)were used to treat AA rats.The paw swelling instrument was used to detect the secondary paw swelling degree,the hematoxylin-eosin staining(HE staining)method was used to observe the histopathological changes of the ankle joint of AA rats,and the cartilage damage was observed by toluidine blue staining.Score it;Enzyme linked immunosorbent assay(ELISA)detects the concentration of estradiol and related inflammatory factors(IL-1β,TNF-α)in rat serum.The results show that the treatment of estradiol can reduce the swelling of the paws of AA rats after ovarian removal,reduce the infiltration of inflammatory substances and cartilage destruction in the joint tissues,and reduce the inflammatory factors IL-1βand TNF-αin the serum of AA rats.Concentration,protect joint cartilage.(2)In vitro experiment:Taking extracellular acidified rat articular chondrocytes as an experimental model,giving different concentrations of estradiol(0,1.25×10~5,2.5×10~5,5×10~5,1.0×10~6,2.0×10~6n M)to stimulate chondrocytes,using MTT experiment to explore females The effect of glycol on the survival rate of rat articular chondrocytes induced by acid.The results show that:estradiol can increase the viability of chondrocytes under acid stimulation.2.Estradiol reduces the level of ASIC1a and autophagy in rat articular cartilage(1)In vivo experiment:immunohistochemical method was used to detect the expression of ASIC1a and autophagy-related proteins(Beclin1,LC3,p62)in cartilage tissue of AA rats after estradiol and TA treatment,and IPP6.0 was used to quantify them;Western blot to detect the expression of ASIC1a and autophagy-related proteins(Beclin1,LC3,p62)in rat articular cartilage tissue.The results showed that the treatment of estradiol can reduce the expression of ASIC1a and the level of autophagy-related proteins in the articular cartilage tissue of AA rats(the expression of Beclin1 and LC3 decreased,and the expression of p62 increased).(2)In vitro experiment:The rat articular chondrocytes were stimulated in an acidic environment with p H 6.0,and different concentrations of estradiol(0,1.25×10~5,2.5×10~5,5×10~5,1.0×10~6,2.0×10~6n M)were given at different time points(0,12,24,48h)treatment of chondrocytes,western blot to detect the expression of ASIC1a and autophagy-related proteins(Beclin1,LC3,p62);immunofluorescence to detect the expression of ASIC1a.The results showed that:estradiol can reduce the expression of ASIC1a and autophagy-related protein levels in acid-induced rat articular chondrocytes(decreased expressions of Beclin1 and LC3,and increased expression of p62).3.Estradiol inhibits ASIC1a-mediated autophagy in rat articular chondrocytes through the receptor GPER1(1)Use estradiol receptor ERαand GPER1 specific inhibitors MPP(20 mmol/L),G15(15μmol/L)and small interfering RNA(si RNA)to inhibit and silence ERαand GPER1,and western blot to detect ERαand GPER1 As well as ASIC1a protein expression;the results show that compared with inhibiting ERα,inhibiting GPER1 can significantly reverse the effect of estradiol-induced ASIC1a expression reduction,which proves that estradiol down-regulates ASIC1a in rat articular chondrocytes induced by acid through the GPER1 receptor.expression.(2)The effective concentration of GPER1 receptor agonist G1 was selected by Western blot.The results showed that:when the G1 concentration was 100 nmol/L,the activation of GPER1 and the inhibition of ASIC1a and autophagy began to show significant differences;giving G1(100 nmol/L)And specific inhibitor G15(15μmol/L)to treat chondrocytes,western blot to detect autophagy-related proteins(Beclin1,LC3,p62)and the protein expression of ASIC1a;q RT-PCR to detect autophagy-related genes(Beclin1,LC3,Atg5)and ASIC1a gene expression,immunofluorescence detection of ASIC1a and autophagy-related protein(Beclin1,LC3,p62)expression.The results showed that activation of GPER1 can reduce the expression of ASIC1a in chondrocytes and the level of autophagy(decreased expression of Beclin1 and LC3,and increased expression of p62).(3)Western blot was used to observe the role of PI3K-AKT-m TOR,an important signaling pathway downstream of ASIC1a and GPER1 in extracellular acidified rat articular chondrocytes.By blocking the phosphorylation of AKT signaling pathway related proteins AKT,m TOR,and S6K1,to analyze the effect of PI3K-AKT-m TOR signaling pathway inhibition on ASIC1a expression and autophagy levels in chondrocytes down-regulated by GPER1;q RT-PCR detects ASIC1a and Expression of autophagy-related genes(Beclin1,LC3,Atg5).The results show that GPER1 agonist G1 activates the PI3K-AKT-m TOR signaling pathway,and pathway inhibitors can significantly reduce the down-regulated expression of ASIC1a and autophagy by G1.(4)Small interfering RNA(si RNA)and a specific inhibitor Pc TX-1 were used to silence and inhibit ASIC1a,respectively,to explore the effect of ASIC1a inhibition on acid-induced autophagy in rat articular chondrocytes;Western blot was used to detect Beclin1,LC3 and p62 Protein expression,q RT-PCR to detect the expression of related genes(Beclin1,LC3,Atg5).The results show that inhibition of ASIC1a can reduce the autophagy level of extracellular acidified rat articular chondrocytes.Conclusion:1.Estradiol can protect articular cartilage tissues and cells from autophagy injury in AA rats.2.The protective effect of estradiol is achieved through its receptor GPER1 inhibiting ASIC1a and then reducing autophagy.The underlying mechanism is related to the PI3K-AKT-m TOR signaling pathway. |
