三种天然植物水溶物对H_(2)O_(2)损伤草鱼原代肝细胞的保护作用

Protective Effects of Three Natural Plants Water Soluble Matter on Primary Hepatocyte of Grass Carp(Ctenopharyngodon idellus)Damaged by Hydrogen Peroxide

为了探讨单一天然植物水溶物和复合植物水溶物对H_(2)O_(2)损伤草鱼原代肝细胞的修复作用,以诃子(terminalia chebularetz,TCR)、天然植物复合物1(RAT)、天然植物复合物2(JLT)水溶物冻干粉为试验材料,维生素C(VC)作为对照,以草鱼(Ctenopharyngodon idellus)离体培养的原代肝细胞为试验对象,利用H_(2)O_(2)建立损伤模型后分别添加含有0.1 mg/mL的TCR、RAT和JLT的细胞培养液培养24 h后检测细胞状态。结果表明:RAT、TCR和VC组与H_(2)O_(2)组相比细胞活力显著提高(P<0.05)。与H_(2)O_(2)组相比,RAT、TCR和VC组过氧化氢酶(CAT)活性均极显著上升(P<0.01),天然植物水溶物组和VC组谷胱甘肽(GSH)含量极显著升高(P<0.01),丙二醛(MDA)含量显著下降(P<0.05),超氧化物歧化酶(SOD)活性极显著下降(P<0.01),RAT组和JLT组细胞凋亡率显著降低(P<0.05),天然植物水溶物组和VC组活性氧(ROS)水平均极显著降低(P<0.01),天然植物水溶物组线粒体膜电位(MMP)均有不同程度的升高。电镜结果显示H_(2)O_(2)组细胞线粒体肿胀、嵴消失,天然植物水溶物组细胞嵴消失情况减缓,自噬小体和自噬溶酶体数量略有增加。Western Blot结果显示,与H_(2)O_(2)组相比,天然植物水溶物组Bax蛋白的表达量均有显著或极显著降低(P<0.05或P<0.01),凋亡蛋白活性被抑制。转录组结果显示,天然植物水溶物能减缓细胞损伤是多个通路共同作用的结果。综上所述,天然植物水溶物对草鱼原代肝细胞的氧化应激损伤具有缓解作用,减少细胞损伤程度,对肝细胞的保护与损伤修复作用具有一定效果。

In order to investigate the repair effect of single natural plant water soluble matter and compound plant water soluble matter on primary hepatocytes of grass carp injured by hydrogen peroxide.Terminalia chebula Retz(TCR),RAT and JLT were used as test materials,VC was used as the contrast material,and the primary hepatocytes of grass carp cultured in vitro were used as the test object,after the injury model was established by hydrogen peroxide,the cell culture medium containing 0.1 mg/mL TCR,RAT and JLT was added for 24 hours,respectively.The results showed that cell viability of RAT,TCR and VC groups was significantly higher than that of H_(2)O_(2) group(P<0.05).Compared with H_(2)O_(2) group,CAT enzyme activity increased significantly in RAT,TCR and VC groups(P<0.01),GSH content increased significantly in all natural plant water soluble matter groups and VC group(P<0.01),MDA content and SOD activity decreased significantly(P<0.05).ROS level decreased significantly in all natural plant water extract groups and VC group(P<0.01),MMP increased significantly in JLT group and TCR group.The results of electron microscope showed that mitochondria swelled and cristae disappeared in H_(2)O_(2) group,the disappearance of cristae slowed down in natural plant water soluble matter group,and the number of autophagy bodies and autophagy lysosomes increased slightly.Western blot showed that compared with H_(2)O_(2) group,the expression of Bax protein in all natural plant water-soluble groups decreased significantly(P<0.01),and the activity of apoptotic protein was inhibited.Transcriptome results show that natural plant water-soluble substances could slow down cell damage,which was the result of the joint action of multiple pathways.In conclusion,natural plant water soluble matter could alleviate the oxidative stress injury of grass carp primary hepatocytes,reduce the degree of cell injury,and have a certain effect on the protection of liver.