补骨脂醇提物对免疫应激模型大鼠的特异质肝损伤及机制研究

Buguzhi(补骨脂) Alcohol Extract Induces Idiosyncratic Liver Injury in Rat Model of Immune Stress

目的:基于免疫应激大鼠模型,研究补骨脂特异质肝损伤属性及相关作用机制。方法:将大鼠分为正常对照组、补骨脂3.6 g/kg组、脂多糖模型对照组、补骨脂3.6 g/kg+脂多糖模型组。药物组灌胃相应药物,造模大鼠在灌胃2 h后尾静脉注射2 mg/kg的脂多糖,在尾静脉注射脂多糖10 h时麻醉取血,取大鼠肝脏。通过生化法检测血清谷丙转氨酶(ALT)及谷草转氨酶(AST)活力,采用HE染色法检测肝脏病理变化;ELISA法检测血清白介素-1α(IL-1α)、IL-1β、IL-4、IL-5、IL-6、IL-10、IL-18、表皮生长因子(EGF)、肿瘤坏死因子α(TNF-α)及干扰素γ(INF-γ)等多项指标含量;全自动生化分析仪检测血清免疫球蛋白G(IgG)、IgM及补体C3、C4等含量;流式细胞术检测全血免疫细胞亚群比例;免疫组化法检测肝脏中免疫细胞数量及能量代谢相关指标丝氨酸-苏氨酸激酶1(LKB1)、单磷酸腺苷活化蛋白激酶(AMPK)、CREB分子调节转录共激活剂1(TORC1)蛋白表达。结果:与正常对照组比较,模型对照组可明显升高血清IL-1α、IL-1β、IL-6、IL-18含量(P<0.05或P<0.01),升高全血CD4^(+)/CD3^(+)比例、CD4^(+)CD25+/CD3^(+)比例、CD4^(+)/CD8^(+)比值(P<0.05或P<0.01),升高肝脏CD3^(+)、CD8^(+)、AMPK、TORC1蛋白阳性表达(P<0.05或P<0.01);降低血清C4含量、全血淋巴细胞总数、全血CD8^(+)/CD3^(+)比例,肝脏CD4^(+)/CD8^(+)比值(P<0.01),肝脏LKB1的蛋白阳性表达(P<0.01);与脂多糖致免疫应激大鼠模型比较,补骨脂3.6 g/kg+脂多糖模型组血清ALT、AST活力明显升高(P<0.05或P<0.01),肝脏切片可见明显的病理损伤;血清IL-1α、IL-1β、IL-18和TNF-α含量明显升高,补体C4含量明显降低(P<0.05);全血CD8^(+)/CD3^(+)比例明显降低,CD4^(+)/CD8^(+)比值明显升高(P<0.05);肝脏组织中CD3^(+)、CD8^(+)、TORC1蛋白阳性表达明显升高(P<0.05)。结论:研究结果证实了补骨脂特异质肝损伤的属性,其作用机制可能与免疫应激与能量代谢相互作用有关。

Objective:To study the mechanism of idiosyncratic liver injury induced by Buguzhi(补骨脂)in the rat model of immune stress.Methods:Rats were randomly assigned into a normal control group,a Buguzhi group,a lipopolysaccharide model control group,and a Buguzhi+lipopolysaccharide model group.The rats in Buguzhi group and Buguzhi+lipopolysaccharide model group were orally administrated with the alcohol extract of Buguzhi at a single dose of 3.6 g/kg,and those in the Buguzhi+lipopolysaccharide model group were injected with lipopolysaccharide at a dose of 2.0 mg/kg via tail vein after 2 h.The rats were anesthetized 10 h after lipopolysaccharide injection,and then the blood and liver tissue samples were collected.The serum activities of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were determined by biochemical methods.Pathological changes were observed by hematoxylin-eosin(HE)staining.Enzyme-linked immunosorbent assay(ELISA)was employed to determine the serum levels of interleukin-1α(IL-1α),IL-1β,IL-4,IL-5,IL-6,IL-10,IL-18,epidermal growth factor(EGF),tumor necrosis factor-alpha(TNFα),and interferon-γ(INFγ).The levels of immunoglobulin G(IgG),IgM,IgE,and complements C3,C4 were determined by an automatic biochemical analyzer.The percentages of immune cell subpopulations in the blood and liver were detected by flow cytometry.The number of immune cells and the protein levels of serine/threonine kinase(LKB1),adenosine monophosphate-activated protein kinase(AMPK),and CREB-regulated transcription coactivator 1(TORC1)involved in energy metabolism in the liver were determined by immunohistochemistry.Results:Compared with the normal control group,the modeling with lipopolysaccharide elevated the serum levels of IL-1α,IL-1β,IL-6,and IL-18(P<0.05 or P<0.01),increased the CD4^(+)/CD3^(+),CD~(4+)CD25~+/CD3^(+),and CD4^(+)/CD8^(+)ratios in blood(P<0.05 or P<0.01),and up-regulated the protein levels of CD3^(+),CD8^(+),AMPK,and TORC1 in the liver tissue(P<0.05 or P<0.01).Meanwhile,it lowered the serum level of C4,reduced the lymphocyte count,decreased the CD8^(+)/CD3^(+)ratio in blood and the CD4^(+)/CD8^(+)ratio in the liver,and down-regulated the protein level of LKB1(P<0.05 or P<0.01).Compared with the lipopolysaccharide model control group,Buguzhi+lipopolysaccharide enhanced the activities of ALT and AST(P<0.05 or P<0.01),caused obvious pathological changes in liver sections,elevated the serum levels of IL-1α,IL-1β,IL-18,and TNFα,and reduced the level of C4(P<0.05).Furthermore,it decreased the CD8^(+)/CD3^(+)ratio in blood,increased the CD4^(+)/CD8^(+)ratio in blood(P<0.05),increased the CD3^(+)and CD8^(+)T cells in the liver tissue(P<0.05),and the up-regulated the protein level of TORC1 in the liver tissue(P<0.05).Conclusion:Buguzhi may induce idiosyncratic liver injury via the interactions with immune stress and energy metabolism.