低浓度亚硝酸钠预处理对高浓度亚硝酸钠损伤PC12细胞的保护作用

Protective effect of low concentration sodium nitrite preconditioning on PC12 cells lesioned by high concentration sodium nitrite

目的探讨低浓度亚硝酸钠(NaNO2)预处理对高浓度亚硝酸钠损伤PC12细胞的保护作用。方法 NaNO20.14 mmol·L-1处理PC12细胞24 h,然后用NaNO245 mmol·L-1再处理2 h制作预处理模型,噻唑蓝(MTT)法检测细胞的存活率,流式细胞术和Hoechst 33258/PI双染检测细胞凋亡,比色法检测超氧化物歧化酶(SOD)、过氧化氢酶(CAT)及谷胱甘肽过氧化物酶(GSH-Px)活性和丙二醛(MDA)含量,Western印迹法检测缺氧诱导因子-1α(HIF-1α)和凋亡相关蛋白表达。结果与NaNO245 mmol·L-1处理组相比,NaNO20.14 mmol·L-1预处理+NaNO245 mmol·L-1组的PC12细胞存活率增加、凋亡减少(P<0.05);细胞SOD、CAT活性和GSH-Px含量明显增加,MDA含量明显下降,促凋亡相关蛋白Bax,胱天蛋白酶9,胱天蛋白酶3表达明显下降,凋亡抑制蛋白Bcl-2和HIF-1α表达明显升高(P<0.05);加入一氧化氮特异性清除剂c-PTIO可以逆转这种现象(P<0.05)。结论低浓度NaNO2预处理增加PC12细胞抗氧化能力,拮抗高浓度NaNO2诱导的细胞凋亡,机制与NaNO2还原为一氧化氮和增加HIF-1α表达有关。

OBJECTIVE To investigate the protective effect and mechanisms of low concentration sodium nitrite（NaNO2） preconditioning on high concentration NaNO2 induced damage in PC12 cells.METHODS A model of low concentration NaNO2 preconditioning was established after PC12 cells were pretreated with NaNO2 0.14 mmol·L-1 for 24 h,then exposed to NaNO2 45 mmol·L-1 for 2 h.The cell viability was measured by MTT method,the apoptosis was evaluated by flow cytometry and Hoechst 33258/PI staining,catalase（CAT）,superoxide dismutase（SOD） glutathione peroxidase（GSH-Px） activities and malondialdehyde（MDA） levels were determined by colorimetric,hypoxia-inducible factor 1α（HIF-1α） and apoptosis-associated protein expression were detected by Western blotting.RESULTS Compared with NaNO2 45 mmol·L-1 alone group,the cell viability was significantly elevated by NaNO2 0.14 mmol·L-1 pretreament while the apoptosis rate decreased（P0.05）.The activity of CAT,SOD and GSH-Px in NaNO2 0.14 mmol·L-1 pretreated group was significantly increased,the MDA level decreased,the expression of Bax,caspase-9 and caspase-3 decreased,HIF-1α and Bcl-2 increased（P0.05）.Nitric oxide specific scavenger c-PTIO could reverse these changes（P0.05）.CONCLUSION Low concentration NaNO2 can increase the anti-oxidant activity and expression of HIF-1α,and resist the apoptosis induced by high concentration NaNO2.Its mechanism might be related to the reduction of NaNO2 to NO and the increased expression of HIF-1α.