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丹酚酸B对非酒精性脂肪性肝病细胞模型氧化应激的影响
Effect of salvianolic acid B on oxidative stress in a cell model of nonalcoholic fatty liver disease
文章发布日期:2018年09月10日  来源:  作者:陈小青,任俞霏,孔维宗,等  点击次数:558次  下载次数:39次

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【摘要】:目的通过体外油酸(OA)诱导HepG2细胞建立非酒精性脂肪性肝病(NAFLD)细胞模型,给予丹酚酸B(Sal B)干预NAFLD细胞,探讨Sal B对NAFLD细胞氧化应激的影响及其机制。方法将HepG2细胞分为对照组、模型组(OA)及治疗组(OA+丹酚酸B),CCK8法检测细胞标准生长曲线,筛选OA最佳的药物干预浓度及干预时间;油红O染色观察细胞内脂质蓄积情况;检测ALT、AST、TG、TC含量;荧光显微镜检测细胞内活性氧(ROS)的含量;TBA法检测细胞内丙二醛(MDA)含量;RT- qPCR和Western Blot 分别检测SOD2及SIRT3 的mRNA及蛋白的表达。运用SIRT3过表达质粒上调SIRT3后给予Sal B干预,通过Western Blot筛选转染试剂和质粒的最合适转染比例,并检测SIRT3与SOD2的相对表达量变化。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t法。结果1 mmol/L 24 h为OA最佳造模浓度及时间。细胞培养上清液中ALT、AST模型组较对照组升高,干预组较模型组降低,3组间比较差异均有统计学意义(F值分别为1240.075、471.989,P值均<0.05)。细胞内TG、TC模型组较对照组升高,干预组较模型组降低,3组间比较差异均有统计学意义(F值分别为97.530、39.824,P值均<0.01)。模型组MDA较对照组升高,干预组较模型组明显降低,3组间比较差异有统计学意义(F=336.67,P<0.01)。3组间细胞SIRT3 mRNA及SOD2 mRNA表达量比较差异均有统计学意义(F值分别为119.35、32.005,P值均<0.01),模型组较正常对照组显著降低,干预组较模型组明显升高(P值均<0.05)。3组细胞SIRT3及SOD2蛋白表达量比较差异均有统计学意义(F值分别67.093、70.314,P值均<0.01),模型组较对照组降低,治疗组较模型组升高(P值均<0.01)。Western Blot检测转染和(或)加药后SIRT3、SOD2 蛋白的表达,4组细胞SIRT3、SOD2蛋白表达量不同,差异均有统计学意义(F值分别为287.2、179.8,P值均<0.01),空白质粒+Sal B组以及质粒组较空白质粒组升高,质粒+Sal B组较质粒组升高(P值均<0.05)。结论Sal B可减轻NAFLD细胞脂质蓄积状态和氧化应激反应,对 NAFLD细胞发挥保护作用。
【Abstract】:ObjectiveTo establish a cell model of nonalcoholic fatty liver disease (NAFLD) by inducing HepG2 cells with oleic acid (OA) in vitro, and to investigate the effect of salvianolic acid B (Sal B) on oxidative stress in NAFLD cells and its possible mechanisms via the intervention of NAFLD cells with Sal B. MethodsHepG2 cells were divided into control group, model group (treated with OA), and intervention group (treated with OA+Sal B). The CCK8 method was used to plot the standard cell growth curve, and the optimal drug intervention concentration and intervention time were determined for OA. Oil red O staining was used to observe intracellular lipid accumulation. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), and total cholesterol (TC) were measured. A fluorescence microscope was used to measure the content of reactive oxygen species (ROS) in cells. The TBA method was used to measure the content of malondialdehyde (MDA) in cells. RT-qPCR and Western blot were used to measure the mRNA and protein expression of SOD2 and SIRT3. After SIRT3 was upregualted with SIRT3 overexpression plasmid, Sal B intervention was performed. Western blot was used to screen out the optimal ratio of transfection reagent to plasmid, and the change in relative expression of SIRT3 and SOD2 was measured. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsThe optimal concentration of OA for modeling was 1 mmol/L. The model group had higher levels of ALT and AST in cell culture supernatant than the control group, and the intervention group had lower levels of ALT and AST than the model group; there were significant differences between the three groups (F=1240.075 and 471.989, both P<0.05). The model group had higher levels of TG and TC in cells than the control group, and the intervention group had lower levels of TG and TC than the model group; there were significant differences between the three groups (F=97.530 and 39.824, both P<0.01). The model group had a higher MDA level than the control group, and the intervention group had a significantly lower MDA level than the model group; there was a significant difference between the three groups (F=336.67, P<0.01). There were significant differences in the mRNA expression of SIRT3 and SOD2 between the three groups (F=119.35 and 32.005, both P<0.01); the model group had significantly lower mRNA expression of SIRT3 and SOD2 than the control group (P<0.05), and the intervention group had significantly higher mRNA expression of SIRT3 and SOD2 than the model group (P<0.05). There were significant differences in the protein expression of SIRT3 and SOD2 between the three groups (F=67.093 and 70.314, both P<0.01); the model group had significantly lower protein expression of SIRT3 and SOD2 than the control group (P<0.01), and the intervention group had significantly higher protein expression of SIRT3 and SOD2 than the model group (P<0.01). The Western blot showed significant differences in the protein expression of SIRT3 and SOD2 between the empty plasmid+Sal B group, plasmid group, empty plasmid group, and plasmid+Sal B group (F=287.2 and 179.8, both P<0.01); the empty plasmid+Sal B group and plasmid group had significantly higher protein expression of SIRT3 and SOD2 than the empty plasmid group (P<0.05), and the plasmid+Sal B group had significantly higher protein expression of SIRT3 and SOD2 than the plasmid group (P<0.05). ConclusionSal B can reduce lipid accumulation and oxidative stress response in NAFLD cells and thus exert a protective effect on NAFLD cells.
【关键字】:脂肪肝; 丹酚酸B; 氧化性应激
【Key words】:fatty liver; salvianolic acid B; oxidative stress
【引证本文】:CHEN XQ, REN YF, KONG WZ, et al. Effect of salvianolic acid B on oxidative stress in a cell model of nonalcoholic fatty liver disease[J]. J Clin Hepatol, 2018, 34(10): 2175-2181. (in Chinese)
陈小青, 任俞霏, 孔维宗, 等. 丹酚酸B对非酒精性脂肪性肝病细胞模型氧化应激的影响[J]. 临床肝胆病杂志, 2018, 34(10): 2175-2181.

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