Effect of CaR regulating TRPC6 mediated autophagy on superoxide dismutase(SOD)and NADPH oxidase 2(NOX2)in chondrocytes
Objective To observe the effect of calcium-sensing receptor(CaR)regulating TRPC6-medi-ated autophagy on superoxide dismutase(SOD)and NADPH oxidase 2(NOX2)in chondrocytes.Methods The articular cartilage of 3-5 day old SD rats was digested with collagenase and subcultured.The third-gen-eration cultured cells were inoculated into 96-well plates and induced with interleukin-1β(10 ng/mL).The chondrocytes were divided into control group,CaR(10 μg/L)group.U73122(5 μg/L)group and CaR(10μg/L)+U73122(5 μg/L)group.After 24 hours of co-culture,the levels of SOD and NOX2 in the superna-tant of each group were detected by enzyme-linked immunosorbent assay.Real-time quantitative polymerase chain reaction was used to detect the expression of SOD,NOX2 and Beclin1 mRNA in each group,and West-ern-blot was used to detect the expression of SOD,NOX2 and Beclin-1 protein.Results The primary articular chondrocytes were triangular or irregular or polygonal under the microscope,and the distribution was sparse.After 24 hours of culture,adherent growth was observed.The third generation of chondrocytes showed blue-stained nuclei,occasionally purple-red heterochromatic granules in the intercellular substance and cytoplasm.The nucleus was stained blue,and the cytoplasmic region was brownish yellow.The level of SOD in the super-natant of chondrocytes,the relative expression of SOD,Beclin-1 mRNA and protein,and the relative expres-sion of NOX2 mRNA in the control group were significantly lower than those in the CaR(10 μg/L)group.The level of NOX2 and the relative expression of NOX2 protein were significantly higher than those in the CaR(10 μg/L)group,and the differences were statistically significant(P＜0.05).The levels of SOD,the relative expression of SOD,Beclin-1 mRNA and protein in the supernatant of chondrocytes in the U73122(5μg/L)group and CaR(10 μg/L)+U73122(5 μg/L)group were significantly lower than those in the control group,and the levels of NOX2,the relative expression of NOX2 mRNA and protein were significantly higher than those in the control group,the differences were statistically significant(P＜0.05).Conclusion CaR re-duces the level of oxidative stress in chondrocytes through TRPC6-mediated autophagy,which may be an im-portant mechanism to delay the occurrence and development of osteoarthritis.
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维普
