虫草多糖对肿瘤坏死因子α诱导肝细胞凋亡的影响

Effect of Cordyceps polysaccharides on hepatocyte apoptosis induced by tumor necrosis factor-α

目的探讨虫草多糖对肝细胞凋亡的影响和作用机制。方法以人正常肝细胞L02作为研究对象,将不经任何药物处理的人正常肝细胞L02作为正常组;利用不同浓度的TNFα(5、10、20、40 ng/ml)进行干预24 h后筛选肝细胞凋亡模型的造模条件并作为模型组;根据实验设计,使用3种不同浓度的虫草多糖(50、100、200μg/ml)预作用12 h后给予或者不给予筛选后的TNFα模型浓度24 h作为实验组,收取样本进行检测,采用CCK8法检测细胞增殖活力;采用Annexin V/PI双染法检测肝细胞凋亡数;采用RT-PCR法检测细胞凋亡(Bax、caspase3、caspase8、caspase9、死亡受体Fas)的mRNA表达;采用蛋白免疫印迹法检测cleaved-caspase3、cleaved-caspase8蛋白表达。计量资料多组间比较采用单因素方差分析或Kruskal-Wallis H检验,进一步两两比较采用LSD-t检验或Dunnett-t检验。结果CCK8法检测结果显示,40 ng/ml的TNFα诱导L02肝细胞24 h后,L02肝细胞增殖较正常组显著降低(73.54%±14.19%vs 100.00%±23.61%,P<0.01),3种不同浓度的虫草多糖在对肝细胞无明显细胞毒性的前提下,较模型组(93.02%±7.21%),细胞增殖均显著升高(P值均<0.01),分别为108.10%±9.05%、114.30%±8.79%、117.70%±9.66%。Annexin V/PI双染法检测结果显示,与正常组细胞凋亡数量比较,模型组细胞凋亡数量显著升高(7.71%±1.20%vs 11.57%±1.41%,P<0.05),3种不同浓度的虫草多糖较模型组(18.91%±0.80%)均明显降低细胞凋亡数量,分别为15.16%±0.16%、13.28%±1.57%、16.91%±0.21%(P值均<0.05)。RT-PCR法和蛋白免疫印迹检测结果显示,40 ng/ml TNFα造模后细胞凋亡相关的Bax、死亡受体Fas的mRNA表达较正常组均增强,差异均有统计学意义(P值均<0.05),并且激活形式的cleaved-caspase3、cleaved-caspase8的蛋白表达显著增强(P值均<0.05),与模型组比较,50μg/ml的虫草多糖可显著降低Bax、caspase3、caspase9的mRNA表达和cleaved-caspase8蛋白表达,100μg/ml的虫草多糖可显著降低Bax、caspase3、caspase8、Fas、caspase9 mRNA和cleaved-caspase8蛋白表达,200μg/ml的虫草多糖可显著降低caspase3、caspase8、Fas、caspase9 mRNA和cleaved-caspase3、cleaved-caspase8蛋白表达,3种虫草多糖浓度作用具有一定量效趋势。结论虫草多糖可以有效抑制TNFα诱导的L02正常肝细胞凋亡。

Objective To investigate the effect of Cordyceps polysaccharides on hepatocyte apoptosis and its mechanism of action.MethodsNormal human L02 hepatocytes without any drug treatment was established as normal group.The L02 hepatocytes were treated with different concentrations of tumor necrosis factor-α(TNF-α)(5,10,20,and 40 ng/ml)for 24 hours to screen out the modeling conditions for the model of hepatocyte apoptosis,which were established as model group.According to the experimental design,L02 hepatocytes pretreated with three different concentrations of Cordyceps polysaccharides(50,100,and 200μg/ml)for 12 hours,with or without TNF-αtreatment for 24 hours at the selected concentration,were established as experimental group.Related samples were collected for analysis.CCK8 assay was used to measure cell proliferation;Annexin V/PI double staining was used to measure the number of apoptotic hepatocytes;RT-PCR was used to measure the mRNA expression of cell apoptosis genes(Bax,caspase-3,caspase-8,caspase-9,and death receptor Fas);Western blotting was used to measure the protein expression of cleaved-caspase-3 and cleaved-caspase-8.A one-way analysis of variance or the Kruskal-Wallis H test was used for comparison of continuous data between multiple groups,and the least significant difference t-test or the Dunnett-t test was used for further comparison between two groups.Results CCK8 assay showed that compared with the normal group,the L02 hepatocytes induced by 40 ng/ml TNF-αfor 24 hours had a significant reduction in proliferation(73.54%±14.19%vs 100.00%±23.61%,P<0.01),and compared with the model group under the premise that Cordyceps polysaccharides had no obvious cytotoxicity to hepatocytes at the three concentrations,the 50,100,and 200μg/ml experimental groups had a significant increase in cell proliferation(108.10%±9.05%/114.30%±8.79%/117.70%±9.66%vs 93.02%±7.21%,all P<0.01).Annexin V/PI double staining showed that compared with the normal group,the model group had a significant increase in the number of apoptotic cells(11.57%±1.41%vs 7.71%±1.20%,P<0.05),and compared with the model group,the 50,100,and 200μg/ml experimental groups had a significant reduction in the number of apoptotic cells(15.16%±0.16%/13.28%±1.57%/16.91%±0.21%vs 18.91%±0.80%,all P<0.05).RT-PCR and Western blotting showed that compared with the normal group,the L02 hepatocytes induced by 40 ng/ml TNF-αhad significant increases in the mRNA expression of the apoptosis-related genes Bax,caspase-9,and death receptor Fas(all P<0.05),as well as significant increases in the protein expression of the activated forms of cleaved-caspase-3 and cleaved-caspase-8(P<0.05).Compared with the model group,the 50μg/ml experimental group had significant reductions in the mRNA expression of Bax,caspase-3,and caspase-9 and the protein expression of cleaved-caspase-8;the 100μg/ml experimental group had significant reductions in the mRNA expression of Bax,caspase-3,caspase-8,Fas,and caspase-9 and the protein expression of cleaved-caspase-8;the 200μg/ml experimental group had significant reductions in the mRNA expression of caspase-3,caspase-8,Fas,and caspase-9 and the protein expression of cleaved-caspase-3 and cleaved-caspase-8.The three concentrations of Cordyceps polysaccharides had a certain dose-effect trend.ConclusionCordyceps polysaccharides can effectively inhibit the apoptosis of normal L02 hepatocytes induced by TNF-α.