CAJ | 학술논문

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(1×104/ml,200μl/孔)或6孔板(1× 106/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.
목적 평개경기대고당유도대서설왕세포양화응격손상적영향급기여다취ADP핵당취합매-1(PARP-1)의뢰성세포사망(parthanatos)적관계.방법 원대대서설왕세포배양우RSM배양기,제비단세포현액,분별접충우96공판(1×104/ml,200μl/공)혹6공판(1× 106/ml,2 ml/공).채용수궤수자표법,장기분위5조(n=30):대조조(C조)、경기조(H2조)、고당조(HG조)、고당+경기조(HG+H2조)、고삼대조조(M조).C조、HG조화M조용정상배양기배양;H2조화HG+H2조용포화부경배양기배양.기중HG조화HG+H2조분별가입포도당50 mmol/L;C조화H2조가입등용량생리염수;M조가입감로순44.4 mmol/L.분별부육48 h후,응용CCK-8법측정세포활력;DCFH-DA실험검측세포내활성양(ROS)수평;ELISA법검측8-간탈양조감(8-OHdG)적농도;Western blot법검측PARP-1、렬해PARP-1(cleaved-PARP-1)、다취ADP핵당(PAR)、조망유도인자(AIF)총단백급핵단백표체수평.계산PARP-1활성(cleaved-PARP-1/PARP-1)화AIF핵전위정황(AIF핵단백/AIF총단백).결과 여C조화H2조비교,HG조화HG+H2조세포활력강저,PARP-1활성、ROS、8-OhdG、PAR표체수평승고,AIF핵전위증다(P＜0.05);여HG조비교,HG+H2조세포활력승고,PARP-1활성、ROS、8-OhdG、PAR표체수평강저,AIF핵전위감소(P＜0.05);C조여M조각지표비교차이무통계학의의(P＞0.05).결론 경기가감경고당유도대서설왕세포양화응격손상,기궤제여억제parthanatos유관.
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 (1×104 cells/ml,200 μl/well) or in 6-well plate (1 × 106 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.