黄芪甲苷联合丹参酚酸B对肝细胞过氧化损伤的保护作用及其机制研究
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摘要
目的:探讨黄芪甲苷联合丹参酚酸B对HHL-5肝细胞过氧化损伤的保护作用机理。方法:采用1640完全培养基培养HHL-5细胞株,3次传代后分为正常组、过氧化损伤模型组、黄芪甲苷组、丹参酚酸B组、黄芪甲苷联合丹参酚酸B组。采用过氧化氢诱导细胞过氧化损伤,丹参酚酸B和黄芪甲苷给药浓度均是10~(-5)mol/L。qRT-PCR法检测细胞TLR4、NF-κB mRNA的表达水平,细胞爬片免疫组化法检测TLR4、NF-κB蛋白的表达,ELISA法检测细胞上清液中TNFα和SOD的含量。结果:与正常组比较,模型组TLR4、NF-κB mRNA和蛋白的表达水平显著增加,细胞上清液中TNFα的含量明显升高,SOD的含量明显降低(P<0.001)。与模型组比较,丹参酚酸B和黄芪甲苷干预组TLR4、NF-κB mRNA和蛋白的表达水平显著下降,细胞上清液中TNFα的含量明显降低,SOD的含量明显升高(P<0.01)。与黄芪甲苷或丹参酚酸B组比较,黄芪甲苷联合丹参酚酸B干预组TLR4、NF-κB mRNA和蛋白的表达水平显著下降,细胞上清液中TNFα的含量明显降低,SOD的含量明显升高(P<0.05)。结论:丹参酚酸B和黄芪甲苷能保护肝细胞的过氧化损伤且二者具有协同作用,该保护作用与抑制TLR4-NF-κB-TNFα炎症损伤通路相关。
Objective:To investigate the protective effects and mechanism of Astragalus Ⅳ combined with salvianolic acid B on peroxidation injury of HHL-5 liver cells.Methods:HHL-5 cells were cultured in 1640 complete medium.After 3 passages,they were divided into normal group,peroxidative injury model group,Astragalus Ⅳ group,salvianolic acid B group,Astragalus Ⅳ combined with salvianolic acid B group.Hydrogen peroxide was used to the induce cell peroxidation injury.The concentration of Astragalus Ⅳ and salvianolic acid B were 10-5 mol/L.The expression levels of TLR4 and NF-κB mRNA were detected by qRT-PCR method.The expression levels of TLR4 and NF-κB protein were detected by immunohistochemistry method.The contents of TNF-α and SOD in cell supernatant were detected by ELISA method.Results:Compared with the normal group,the expression levels of TLR4,NF-κB mRNA and protein increased significantly.The content of TNF-α increased while SOD decreased significantly in supernatant in the model group(P<0.001).Compared with the model group,the expression levels of TLR4,NF-κB mRNA and protein decreased significantly and the content of TNF-α decreased while SOD increased significantly in Astragalus IV group and salvianolic acid B group(P<0.01).Compared with Astragalus IV group or salvianolic acid B group,the expression levels of TLR4,NF-κB,mRNA and protein decreased significantly and the content of TNF-α decreased while SOD increased significantly in Astragaloside IV combined with salvianolic acid B group(P<0.05).Conclusion:Astragaloside IV and salvianolic acid B can protect hepatocytes from peroxidative injury and two drugs have synergistic effects.The protective effects of Astragaloside IV and salvianolic acid B were partly by inhibiting TLR4-NF-κB-TNFα pathway.
Objective:To investigate the protective effects and mechanism of Astragalus Ⅳ combined with salvianolic acid B on peroxidation injury of HHL-5 liver cells.Methods:HHL-5 cells were cultured in 1640 complete medium.After 3 passages,they were divided into normal group,peroxidative injury model group,Astragalus Ⅳ group,salvianolic acid B group,Astragalus Ⅳ combined with salvianolic acid B group.Hydrogen peroxide was used to the induce cell peroxidation injury.The concentration of Astragalus Ⅳ and salvianolic acid B were 10-5 mol/L.The expression levels of TLR4 and NF-κB mRNA were detected by qRT-PCR method.The expression levels of TLR4 and NF-κB protein were detected by immunohistochemistry method.The contents of TNF-α and SOD in cell supernatant were detected by ELISA method.Results:Compared with the normal group,the expression levels of TLR4,NF-κB mRNA and protein increased significantly.The content of TNF-α increased while SOD decreased significantly in supernatant in the model group(P<0.001).Compared with the model group,the expression levels of TLR4,NF-κB mRNA and protein decreased significantly and the content of TNF-α decreased while SOD increased significantly in Astragalus IV group and salvianolic acid B group(P<0.01).Compared with Astragalus IV group or salvianolic acid B group,the expression levels of TLR4,NF-κB,mRNA and protein decreased significantly and the content of TNF-α decreased while SOD increased significantly in Astragaloside IV combined with salvianolic acid B group(P<0.05).Conclusion:Astragaloside IV and salvianolic acid B can protect hepatocytes from peroxidative injury and two drugs have synergistic effects.The protective effects of Astragaloside IV and salvianolic acid B were partly by inhibiting TLR4-NF-κB-TNFα pathway.
引文
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[2] Kgatle MM,Spearman CW,Kalla AA,et al.DNA Oncogenic Virus-Induced Oxidative Stress,Genomic Damage,and Aberrant Epigenetic Alterations[J].Oxid Med Cell Longev,2017,2017:3179421.
[3] Tu T,Bühler S,Bartenschlager R.Chronic viral hepatitis and its association with liver cancer[J].Biol Chem,2017,398(8):817-837.
[4] 黄建生.复方丹参滴丸加黄芪对乙肝肝硬化肝纤维化指标的影响[J].湖南中医杂志,2006,22(3):17-18.
[5] 霍璞,曹永年.黄芪、丹参注射液治疗慢性乙肝血瘀证40例[J].基层医学论坛,2004,8(1):95.
[6] Ahn CB,Je JY,Kim YS,et al.Induction of Nrf2-mediated phase II detoxifying/antioxidant enzymes in vitro by chitosan-caffeic acid against hydrogen peroxide-induced hepatotoxicity through JNK/ERK pathway[J].Mol Cell Biochem,2017,424(1-2):79-86.
[7] Alam P,Parvez MK,Arbab AH,et al.Quantitative analysis of rutin,quercetin,naringenin,and gallic acid by validated RP- and NP-HPTLC methods for quality control of anti-HBV active extract of Guiera senegalensis[J].Pharm Biol,2017,55(1):1317-1323.
[8] Ivanov AV,Valuev-Elliston VT,Tyurina DA,et al.Oxidative stress,a trigger of hepatitis C and B virus-induced liver carcinogenesis[J].Oncotarget,2017,8(3):3895-3932.
[9] Cai J,Han T,Nie C,et al.Biomarkers of oxidation stress,inflammation,necrosis and apoptosis are associated with hepatitis B-related acute-on-chronic liver failure[J].Clin Res Hepatol Gastroenterol,2016,40(1):41-50.
[10] Lee JH,Won JH,Choi JM,et al.Protective effect of ellagic acid on concanavalin A-induced hepatitis via toll-like receptor and mitogen-activated protein kinase/nuclear factor κB signaling pathways[J].J Agric Food Chem,2014,62(41):10110-10117.
[11] Zhou GY,Yi YX,Jin LX,et al.The protective effect of juglanin on fructose-induced hepatitis by inhibiting inflammation and apoptosis through TLR4 and JAK2/STAT3 signaling pathways in fructose-fed rats[J].Biomed Pharmacother,2016,81:318-328.