摘要目的：探讨氢气（H2）对高糖诱导大鼠雪旺细胞多聚二磷酸腺苷核糖聚合酶-1（PARP-1）依赖性细胞死亡（PARthanatos）的抑制作用，并探讨其机制。方法将原代培养的大鼠雪旺细胞按随机数字表法分为空白对照组（C组）、H2对照组（H2组）、高渗对照组（M组）、高糖处理组（HG组）、H2治疗组（HG+H2组）5组。H2组和HG+H2组用饱和富氢培养基（含H20.6mmol/L）培养，3个对照组用低糖DMEM培养基（总含糖量5.6mmol/L）培养。HG组和HG+H2组加入44.4mmol/L葡萄糖（培养基总含糖量50mmol/L）培养，C组和H2组加等量生理盐水，M组加等量甘露醇。各组相应处理48h后计算乳酸脱氢酶（LDH）释放率以反映细胞毒性，用酶联免疫吸附试验（ELISA）测定亚硝酸基阴离子（ONOO-）和8-羟基脱氧鸟苷（8-OHdG）含量以反映氧化应激损伤和DNA损伤，用蛋白质免疫印迹试验（WesternBlot）检测多聚二磷酸腺苷核糖（PAR）的蛋白表达，用免疫荧光法检测凋亡诱导因子（AIF）的核转位。结果与C组相比，HG组和HG+H2组细胞毒性明显增加〔LDH释放率：（61.40±2.89）%、（42.80±2.32）%比（9.92±0.38）%，均P＜0.01〕，细胞内ONOO-和8-OHdG含量明显增加〔ONOO-（ng/L）：853.58±51.00、553.11±38.66比113.56±14.22，8-OHdG（ng/L）：1177.37±60.97、732.06±54.29比419.67±28.77，均P＜0.01〕，PAR蛋白表达明显增加（积分A值：0.603±0.028、0.441±0.010比0.324±0.021，均P＜0.01）；而HG+H2组细胞毒性、ONOO-和8-OHdG含量、PAR表达均较HG组明显降低（均P＜0.01）。H2组及M组各项指标与C组比较差异均无统计学意义。免疫荧光显示，C组、H2组和M组AIF均表达在细胞质中；HG组AIF则在整个细胞中表达，且细胞核中表达尤为明显；HG+H2组细胞核中有少量AIF表达。说明高糖可以刺激AIF的核转移，而H2可以抑制这一过程。结论高糖处理大鼠雪旺细胞可以使其DNA损伤增加，从而加强其PARthanatos；而H2不仅可以减轻受损细胞的DNA损伤，亦可以抑制这一特殊的死亡过程，降低细胞毒性，具有细胞保护效应。

abstractsObjective To investigate the protective effects and underlying molecular mechanisms of hydrogen (H2) on high glucose-induced poly (ADP-ribose) polymerase-1 (PARP-1) dependent cell death (PARthanatos) in primary rat Schwann cells. Methods Cultured primary rat Schwann cells were randomly divided into five groups: blank control group (C group), H2 control group (H2 group), high osmotic control group (M group), high glucose treatment group (HG group), and H2 treatment group (HG+H2 group). The cells in H2 group and HG+H2 group were cultured with saturated hydrogen-rich medium containing 0.6 mmol/L of H2, and those in three control groups were cultured with low sugar DMEM medium containing 5.6 mmol/L of sugar, and the cells in HG and HG+H2 groups were given 44.4 mmol/L of glucose in addition (the medium containing 50 mmol/L of glucose), the cells in C group and H2 group were given the same volume of normal saline, and the cells in M group were given the same volume of mannitol. Cytotoxicity was evaluated using lactate dehydrogenase (LDH) release rate assays after treatment for 48 hours in each group. The contents of peroxynitrite (ONOO-) and 8-hydroxy-2-deoxyguanosine (8-OHdG) reflecting oxidative stress injury and DNA damage were detected by enzyme linked immunosorbent assay (ELISA). Poly (ADP-ribose) (PAR) protein expression was analyzed by Western Blot, and immunofluorescence staining was used to determine the nuclear translocation of the apoptosis-inducing factor (AIF). Results The cytotoxicity in HG and HG+H2 groups was significantly increased as compared with that of C group [LDH release rate: (61.40±2.89)%, (42.80±2.32)% vs. (9.92±0.38)%, both P < 0.01], the levels of ONOO- and 8-OHdG were markedly elevated [ONOO- (ng/L): 853.58±51.00, 553.11±38.66 vs. 113.56±14.22; 8-OHdG (ng/L): 1 177.37±60.97, 732.06±54.29 vs. 419.67±28.77, all P < 0.01], and the PAR protein expression was up-regulated (A value: 0.603±0.028, 0.441±0.010 vs. 0.324±0.021, both P < 0.01). The cytotoxicity, the levels of ONOO- and 8-OHdG, and PAR expression in HG+H2 group were significantly lower than those of the HG group (all P < 0.01). There were no significant differences in above parameters between H2 group as well as M group and C group. It was shown by immunofluorescence that AIF was expressed in the cytoplasm in C group, H2 group and M group, AIF was expressed in the whole cell in HG group, and the expression in the nucleus was particularly increased. A small amount of AIF expression was found in the nucleus of HG+H2 group, which indicated that high glucose could promote the AIF nuclear translocation, and that hydrogen-rich medium could prevent the process of translocation. Conclusions High glucose levels could enhance DNA damage that enhance PARthanatos in primary rat Schwann cells. However, H2 can not only reduce DNA damage of injured cells, but also inhibit the special death process, reduce the cell toxicity, all of which have protective effects.