大黄素恢复自噬减轻脂多糖诱导的急性肝损伤

Emodin reactivated autophagy and alleviated D-galactosamine/lipopolysaccharide-induced acute liver injury

目的探讨大黄素在D-氨基半乳糖胺(D-galactosamine,D-GalN)/脂多糖(lipopolysaccharide,LPS)诱导的急性肝损伤中的保护作用及其机制。方法将40只BALB/c雄性小鼠按随机数字法分为5组(n=8),对照组、大黄素组、D-GalN/LPS组、大黄素+D-GalN/LPS组和自噬抑制剂3-MA+大黄素+D-GalN/LPS组。经小鼠腹腔注射D-GalN(700 mg/kg)/LPS(10μg/kg)诱导急性肝损伤模型。3-MA(15 mg/kg)和(或)大黄素(20 mg/kg)腹腔注射于模型建立前30 min,6 h后在麻醉下处死小鼠并采集血、肝组织标本。采用比色定量法检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平、肝组织髓过氧化物酶(MPO)活性,采用ELISA法检测血清中肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)的水平,HE染色观察肝组织病理学改变,Western blot检测肝组织自噬蛋白LC3-Ⅱ、Beclin1的表达量,并分析实验动物生存率。定量资料比较采用单因素方差分析,采用SNK-q检验进行两两比较,方差不齐时采用Games-Howell检验。结果与对照组相比,D-GalN/LPS组AST、ALT、TNF-α、IL-6水平和MPO活性[(2476.80±263.14)U/L、(271.71±47.15)U/L、(537.92±89.35)pg/mL、(169.74±25.52)pg/mL、(1.37±0.22)U/mg]显著升高(P<0.05)。与D-GalN/LPS组相比,大黄素+D-GalN/LPS组AST、ALT、TNF-α、IL-6水平和MPO活性[(1248.01±380.70)U/L、(142.59±34.63)U/L、(288.91±67.21)pg/mL、(61.83±13.64)pg/mL、(0.80±0.21)U/mg]显著降低(P<0.05)。与D-GalN/LPS组相比,大黄素+D-GalN/LPS组可明显减轻肝组织肝细胞的病理损伤,提高小鼠生存率。与对照组相比,D-GalN/LPS组肝组织LC3-Ⅱ、Beclin1表达下降;而与D-GalN/LPS组相比,大黄素+D-GalN/LPS组肝组织LC3-Ⅱ、Beclin1表达升高。联合自噬抑制剂3-MA后大黄素的肝保护效应被逆转,AST、ALT、TNF-α、IL-6水平和MPO活性[(2398.78±233.57)U/L、(242.79±43.46)U/L、(505.07±67.89)pg/mL、(151.46±14.11)pg/mL、(1.27±0.15)U/mg]升高(P<0.05),肝组织病理损伤加重。结论大黄素减轻D-GalN/LPS诱导的急性肝损伤,这可能与激活LC3-Ⅱ、Beclin1蛋白恢复自噬有关。

Objective To explore the protective effect of emodin on D-galactosamine(D-GalN)/lipopolysaccharide(LPS)-induced acute liver injury and its mechanism.Methods A total of 40 male BALB/c mice were randomly(random number)divided into 5 groups(n=8 in each group):the control group,the emodin group,the D-GalN/LPS group,the emodin+D-GalN/LPS group and the 3-MA+emodin+D-GalN/LPS group.D-GalN(700 mg/kg)and LPS(10μg/kg)were intraperitoneally injected to induce acute liver injury in mice.Autophagy inhibitor 3-MA(15 mg/kg)and/or emodin(20 mg/kg)were intraperitoneally injected 30 min before the liver injury model.The animals were sacrificed under anaesthesia 6 h after D-GalN/LPS challenge,blood samples and liver tissues were collected.The levels of alanineaminotransferase(ALT)and aspartateaminotransferase(AST)in serum,and myeloperoxidase(MPO)activity of liver tissues were determined by colorimetric quantitative method;the levels of tumor necrosis factor-α(TNF-α),and interleukin-6(IL-6)were measured by ELISA;the expression of LC3-II and Beclin 1 in the liver tissues were evaluated by Western blot;the pathological changes of liver was evaluated by HE staining.Animal survival rate was also analyzed.The one-way ANOVA was use to compare quantitative data,SNK-q test was used for pairwise comparison between two groups,and Games-Howell test was used when homogeneity of variance were not met.Results Compared with the control group,the levels of ALT,AST,TNF-α,IL-6 and MPO activity[(2476.80±263.14)U/L,(271.71±47.15)U/L,(537.92±89.35)pg/mL,(169.74±25.52)pg/mL,and(1.37±0.22)U/mg]were obviously increased in the D-GalN/LPS group(P<0.05).Compared with the D-GalN/LPS group,the levels of ALT,AST,TNF-α,IL-6 and MPO activity[(1248.01±380.70)U/L,(142.59±34.63)U/L,(288.91±67.21)pg/mL,(61.83±13.64)pg/mL,and(0.80±0.21)U/mg]were obviously decreased in the emodin+D-GalN/LPS group(P<0.05).Compared with the D-GalN/LPS group,the histopathological abnormalities in liver tissue were significantly alleviated and the survival rate of mice was improved in the emodin+D-GalN/LPS group.Compared with the control group,the expression of LC3-II and Beclin1 was decreased in the liver tissue in the D-GalN/LPS group,while compared with the D-GalN/LPS group,the expression of LC3-II and Beclin1 was increased in the emodin+D-GalN/LPS group.With co-administration of 3-MA,the protective effects of emodin in acute liver injury were reversed,the levels of AST,ALT,TNF-α,IL-6,and MPO[(2398.78±233.57)U/L,(242.79±43.46)U/L,(505.07±67.89)pg/mL,(151.46±14.11)pg/mL,and(1.27±0.15)U/mg]were increased,and the pathological damage of liver tissue was aggravated.Conclusions Emodin alleviates D-GalN/LPS-induced acute liver injury in mice,which may be related to the activation of protein LC3-II,Beclin1 and restored autophagy.