RhoA激酶的硫巯基化修饰在硫化氢抗大鼠神经细胞缺氧性损伤中的作用

Role of S-sulfhydration of RhoA kinase in neuroprotection of hydrogen sulfide against hypoxic injury

目的研究RhoA激酶2(ROCK_(2))的硫巯基化修饰在硫化氢(H_(2)S)抗大鼠神经细胞缺氧性损伤中的作用。方法原代培养大鼠海马神经细胞,给予硫氢化钠(NaHS)和硫巯基化抑制剂二硫苏糖醇DTT预处理,构建细胞缺氧复氧模型。检测细胞活力、乳酸脱氢酶(LDH)和神经特异性烯醇化酶(NSE)活性以及细胞凋亡。纯化神经细胞中硫巯基化蛋白,检测ROCK_(2)和硫巯基化修饰ROCK_(2)(SSH-ROCK_(2))蛋白表达以及活性。结果100、200μmol·L^(-1)NaHS明显提高缺氧复氧神经细胞的活力、降低上清中LDH和NSE活性、减少细胞凋亡,提示NaHS的神经保护作用;但是DTT可减弱NaHS对缺氧复氧神经细胞的保护作用。此外,100、200μmol·L^(-1)NaHS可减少海马神经细胞中ROCK_(2)蛋白表达,并通过增加ROCK_(2)蛋白硫巯基化修饰而降低ROCK_(2)蛋白活性。结论H2S对大鼠海马神经元H/R损伤有明显的保护作用,其机制与硫巯基化修饰致ROCK_(2)活性降低及ROCK_(2)蛋白表达减少有关。

Aim To investigate the role of S-sulfhydration of RhoA kinase 2 in the neuroprotection of hydrogen sulfide(H_(2)S)against hypoxic injury.Methods Rat hippocampal neurons were primarily cultured and treated with exogenous H_(2)S donor NaHS(50,100,200μmol·L^(-1))and S-sulfhydration inhibitor DTT(50μmol·L^(-1) )during 4 hours of hypoxia and 12 hours of reoxygenation.Cell viability,the lactate dehydrogenase(LDH)activity and neuron-specific enolase(NSE)activity released from injured neuron to culture supernatant,and the proportion of apoptotic cells were measured to assess the neuroprotection of H2S,and the role of S-sulfhydration in the neuroprotection of H2S was preliminarily explored.In addition,the S-sulfhydrated proteins in neurons were isolated and purified by modified biotin-switch assay.And then,the RhoA kinase 2(ROCK_(2))expression and activity,and S-sulfhydrated ROCK_(2) were detected to further confirm the role H2S on the S-sulfhydrated ROCK_(2) by Western blot and assay kits,respectively.Results The decrease of cell viability,and the increase of LDH and NSE released from injured neuron to culture supernatant and cell apoptosis after hypoxia/reoxygenation(H/R)were significantly inhibited by 100 and 200μmol·L^(-1)NaHS.Compared with the effect of 200μmol·L^(-1) NaHS,the neuroprotection of 200μmol·L^(-1) NaHS could be inhibited by co-application with DTT.Furthermore,100 and 200μmol·L^(-1) NaHS could reduce the expression of ROCK_(2) protein and restrain ROCK_(2) activity via promoting the S-sulfhydryl modification of ROCK_(2) protein in hippocampal neurons.Conclusions H2S exerts protective effect on H/R injury of rat hippocampal neurons via down-regulation of ROCK_(2) expression and inhibition of ROCK_(2) activity by S-sulfhydration modification.