金雀根通过阻断TRPM7抑制Erastin诱导的软骨细胞铁死亡

Caragana sinica root inhibits Erastin-induced chondrocyte ferroptosis by blocking TRPM7

目的:探讨金雀根(Caragana sinica root,CSR)对Erastin诱导的软骨细胞铁死亡模型中铁死亡的影响及可能的机制。方法:培养C28/I2软骨细胞系,构建Erastin诱导铁死亡的细胞模型。采用MTT法检测细胞活力;Calcein/PI染色观察细胞活性;通过试剂盒检测细胞乳酸脱氢酶(LDH)及总谷胱甘肽(GSH)水平;荧光探针BODIPY 581/591 C11标记检测活性氧(ROS)水平;Rh123和JC-1染色观察线粒体膜电位(ΔΨm)的变化;Western blot检测铁死亡相关蛋白(ACSL4、GPX4)和TRPM7蛋白的表达。结果:Erastin处理可降低软骨细胞活力,增加细胞毒性,诱发氧化应激,破坏ΔΨm,并上调ACSL4蛋白表达,同时下调GPX4蛋白表达,诱导软骨细胞铁死亡。相反,金雀根可以使细胞活力恢复,降低氧化应激反应,从而抑制软骨细胞铁死亡。此外,金雀根能降低Erastin引起的TRPM7蛋白表达水平的升高。结论:Erastin诱发了C28/I2软骨细胞脂质过氧化反应,引起线粒体损伤及铁死亡;金雀根可能通过阻断TRPM7抑制软骨细胞铁死亡,从而发挥保护软骨细胞的作用。

AIM:To investigate the effect of Caragana sinica root(CSR)on ferroptosis in the Erastininduced chondrocyte ferroptosis model and possible mechanisms.METHODS:The C28/I2 chondrocyte cell line was cultured to construct a cell model of Erastin-induced ferroptosis.Cell viability was detected by MTT assay;cell death was observed by Calcein/PI staining;cell lactate dehydrogenase(LDH)and total glutathione(GSH)levels were detected by the kit;reactive oxygen species(ROS)levels were detected by fluorescent probe BODIPY 581/591 C11 labeling;mitochondrial membrane potential(ΔΨm)changes were observed by Rh123 and JC-1 staining;Western blot was used to detect the expression of ferroptosis-related proteins(ACSL4,GPX4)and TRPM7 proteins.RESULTS:Erastin treatment decreased chondrocyte viability,increased cytotoxicity,induced oxidative stress,disruptedΔΨm,and up-regulated ACSL4 protein expression,while down-regulating GPX4 protein expression and inducing chondrocyte ferroptosis.In contrast,CSR restored cell viability and reduced oxidative stress,thereby inhibiting chondrocyte ferroptosis.In addition,CSR reduced the Erastin-induced increase in TRPM7 protein expression level.CONCLUSION:Erastin induced lipid peroxidation in C28/I2 chondrocytes,causing mitochondrial damage and ferroptosis;CSR may inhibit chondrocyte ferroptosis by blocking TRPM7,thus exerting a protective effect on chondrocytes.