17β-estradiol Mediates Mitophagy In ATDC5 Chondrocytes Via GPR30/PI3K/Akt Signaling Pathway

Objective:G protein-coupled estrogen receptor-30（GPR30）is an estrogen membrane receptor that is present in a variety of cell lines and mediates rapid cell signaling.PI3K/Akt is an important signaling pathway involved in the physiological regulation of various cells and is involved in autophagy.Mitophagy is present in a variety of cells and usually protects cells.Articular cartilage damage is the main pathological feature of osteoarthritis（OA）.Autophagy in chondrocytes is closely related to the pathogenesis of OA.The expression of GPR30 was detected by mouse ATDC5 chondrocyte culture in vitro,and the effect of estrogen on the expression of GPR30 was observed.The expressions of LC-3,TOM20,Hsp60,Akt,mTOR,P38 and JNK in the control group and the experimental group were analyzed,and the viability and proliferation of ATDC5chondrocytes were observed.Treating ATDC5 chondrocytes with GPR30 blocker or PI3K blocker to study the effect of 17β-estradiol on autophagy mechanism.Although estrogen has been shown to be useful in the treatment of osteoporosis,and involving in bone tissue metabolism,its mechanism of action remains unclear.By studying in vitro mouse chondrocyte culture,to investigate the mechanism of17β-estradiol mediated GPR30 and PI3K/Akt signaling pathways in inhibiting mitophagy in ATDC5 chondrocytes.Provide a theoretical basis for protecting and improving chondrocyte function and preventing OA.Methods:The mouse cartilage primary cell line ATDC5 was cultured for 24 hours by serum-free.Treating ATDC5 chondrocytes with different concentrations of 17β-estradiol or GPR30 inhibitor（G15）,or PI3K inhibitor（LY294002）for 24 h by group.Using western blot and immunofluorescence（IF）staining to detect the expression of GPR30 in ATDC5 chondrocytes.Observing the proliferation and viability of ATDC5 chondrocytes by MTT assay.The viability of autophagy-tagged protein activity and signaling key proteins of LC-3,TOM20,Hsp60,Akt,mTOR,P38 and JNK were observed by protein preparation and Western blot.Using SPSS 15.0 software to analyze data and assess cell viability and proliferation.Each group of data was expressed by（x±s）.The homogeneity of the data was verified by Student’s t test or one-way analysis of variance（ANOVA）followed by LSD test.P<0.05 was considered statistically significant.Results:GPR30 is expressed detected in cells,and 10^-7mol/L concentration of17β-estradiol can improve the expression level of GPR30.With 10^-8mol/L and 10^-7mol/L of 17β-estradiol can reduce autophagosomes and improve the activity of TOM20 protein.The 10^-7mol/L concentrations of 17β-estradiol can improve viability and proliferation of cells in vitro,and the activity of autophagy-tagged protein Hsp60,but the activity of LC-3 was decreased.Treating cells with 10^-7mol/L concentration of 17β-estradiol,the phosphorylation levels of Akt protein and mTOR were increased,but has no effect on the activity of P38 and JNK.Conclusion:17β-estradiol promotes the expression of GPR30 protein in ATDC5chondrocytes,activates the intracellular PI3K/Akt signaling pathway,and inhibits mitophagy in ATDC cells.