氢气对高糖诱导大鼠雪旺细胞氧化应激损伤的影响：与parthanatos的关系

Effects of hydrogen on oxidative stress injury induced by high glucose in rat Schwann cells： the relationship with parthanatos

目的 评价氢气对高糖诱导大鼠雪旺细胞氧化应激损伤的影响及其与多聚ADP核糖聚合酶-1（PARP-1）依赖性细胞死亡（parthanatos）的关系.方法 原代大鼠雪旺细胞培养于RSM培养基,制备单细胞悬液,分别接种于96孔板（1×10^4/ml,200μl/孔）或6孔板（1× 10^6/ml,2 ml/孔）.采用随机数字表法,将其分为5组（n=30）：对照组（C组）、氢气组（H2组）、高糖组（HG组）、高糖＋氢气组（HG＋H2组）、高渗对照组（M组）.C组、HG组和M组用正常培养基培养;H2组和HG＋H2组用饱和富氢培养基培养.其中HG组和HG＋H2组分别加入葡萄糖50 mmol/L;C组和H2组加入等容量生理盐水;M组加入甘露醇44.4 mmol/L.分别孵育48 h后,应用CCK-8法测定细胞活力;DCFH-DA实验检测细胞内活性氧（ROS）水平;ELISA法检测8-羟脱氧鸟苷（8-OHdG）的浓度;Western blot法检测PARP-1、裂解PARP-1（cleaved-PARP-1）、多聚ADP核糖（PAR）、凋亡诱导因子（AIF）总蛋白及核蛋白表达水平.计算PARP-1活性（cleaved-PARP-1/PARP-1）和AIF核转位情况（AIF核蛋白/AIF总蛋白）.结果 与C组和H2组比较,HG组和HG＋H2组细胞活力降低,PARP-1活性、ROS、8-OhdG、PAR表达水平升高,AIF核转位增多（P＜0.05）;与HG组比较,HG＋H2组细胞活力升高,PARP-1活性、ROS、8-OhdG、PAR表达水平降低,AIF核转位减少（P＜0.05）;C组与M组各指标比较差异无统计学意义（P＞0.05）.结论 氢气可减轻高糖诱导大鼠雪旺细胞氧化应激损伤,其机制与抑制parthanatos有关.

Objective To evaluate the effects of hydrogen on oxidative stress injury induced by high glucose in Schwann cells and its relationship with PARP-1-dependent cell death （parthanatos）.Methods Primary rat Schwann cells were cultured in 96-well plate （1×10^4 cells/ml,200 μl/well） or in 6-well plate （1 × 10^6 cells/ml,2 ml/well） with RSM culture medium and were randomly divided into 5 groups （n=30 each）：control group （group C）,hydrogen group （group H2）,high glucose group （group HG）,high glucose plus hydrogen group （group HG＋H2） and high osmotic control group （group M）.The cells were cultured in the common culture medium in C,HG and M groups.The cells were cultured in hydrogen-rich culture medium in H2 and HG ＋ H2 groups.In HG and HG ＋ H2 groups,50 mmol/L of glucose was added to the culture medium.In C and H2 groups,the equal volume of normal saline was added to the culture medium.In M group,mannitol 44.4 mmol/L was added to the culture medium.The cells were then incubated for 48 h.After 48 h of incubation,the cell viability was measured using CCK-8 assay,intracellular reactive oxygen species （ROS） level was detected by flow cytometry,the concentration of 8-hydroxy-2-deoxy Guanosine （8-OHdG） was determined by ELISA,and the expression of poly （ADP-ribose）-polymerase-1 （PARP-1）,cleaved-PARP-1,poly （ADP-ribose） （PAR）,and apoptosis-inducing factor （AIF） in the total protein and nucleus was measured by Western blot.PARP-1 activity （cleaved-PARP-1/PARP-1） and AIF nuclear translocation were recorded.Results Compared with C and H2 groups,the cell viability was significantly decreased,and PARP-1 activity,expression of ROS,8-OhdG and PAR,and AIF nuclear translocation were increased in HG and HG ＋ H2 groups.Compared with HG group,the cell viability was significantly increased,and PARP-1 activity,expression of ROS,8-OhdG and PAR,and AIF nuclear translocation were decreased in HG＋H2 group.There was no significant difference in each parameter between M and C groups.Conclusion Hydrogen can reduce oxidative stress injury induced by high glucose in Schwann cells,and the mechanism is related to inhibition of parthanatos.