加味茵陈蒿汤通过抑制TLR4信号通路对大鼠急性肝损伤的保护作用

Protective effect of modified Yinchenhao decoction against acute liver injury in rats by inhibiting TLR4 signaling pathway

目的探讨加味茵陈蒿汤对急性肝损伤大鼠的保护机制。方法将42只SPF级雌性SD大鼠,随机分为空白组、模型组、西药组、中药组、TLR4过表达组、西药+TLR4过表达组和中药+TLR4过表达组,每组6只。空白组大鼠给予0.9%生理盐水灌胃;模型组大鼠造模后灌胃同体积生理盐水;西药组大鼠造模后灌胃复方甘草酸苷15.75 mg/kg;中药组大鼠造模后灌胃加味茵陈蒿汤58.8 g/kg;TLR4过表达组大鼠造模后静脉注射0.5 ml腺病毒包装的TLR4过表达载体(pDC-TLR4,1×10^(11) PFU);西药+TLR4过表达组大鼠造模后灌胃同体积复方甘草酸苷,并给予等量pDC-TLR4静脉注射;中药+TLR4过表达组大鼠造模后灌胃同体积加味茵陈蒿汤,并给予等量pDC-TLR4静脉注射。各组大鼠持续给药2周后,利用生化测定仪测定各组大鼠血清谷丙转氨酶(ALT)和谷草转氨酶(AST)的水平;Western blot分析肝脏组织TNF-α、p-NF-κB、MyD88和TLR4的蛋白表达;另取肝脏行病理学检测。结果与空白组比较,模型组大鼠血浆ALT、AST及肝组织中TNF-α、p-NF-κB、MyD88、TLR4水平均显著升高(P<0.01),肝细胞呈弥漫性大片状坏死,肝窦结构被破坏,汇管区明显可见炎症细胞浸润。与模型组比较,中药组和西药组大鼠血浆ALT、AST及肝组织TNF-α、TLR4、MyD88、p-NF-κB的水平均显著降低(P<0.01),且中药组与西药组相比,差异虽无统计学意义,但中药对ALT和AST表达的抑制作用更明显。与中药组和西药组相比,中药+TLR4过表达组和西药+TLR4过表达组大鼠血浆ALT、AST及肝组织TNF-α、TLR4、MyD88、p-NF-κB的水平均显著升高(P<0.01)。另外,与中药组和西药组相比,中药+TLR4过表达组和西药+TLR4过表达组大鼠肝脏损伤程度明显加重。结论TNF-α、MyD88、p-NF-κB及TLR4在急性肝损伤中起着重要作用,加味茵陈蒿汤可通过降低TLR4信号通路中TNF-α、MyD88及p-NF-κB等重要细胞因子水平缓解肝细胞损伤。

Objective To explore the protective mechanism of modified Yinchenhao decoction against acute liver injury in rats.Methods Forty-two SPF-grade female SD rats were randomly divided into blank group,model group,western medicine group,traditional Chinese medicine(TCM)group,TLR4 overexpression group,western medicine+TLR4 overexpression group and TCM+TLR4 overexpression group,with 6 rats in each group.The rats in blank group were gavaged by 0.9%normal saline.The rats in model group were gavaged with the same volume of normal saline after modeling.The rats in western medicine group were gavaged with 15.75 mg/kg compound glycyrrhizin tablets after modeling.The rats in traditional Chinese medicine(TCM)group were gavaged with 58.8 g/kg modified Yinchenhao decoction after modeling.The rats in TLR4 overexpression group were intravenously injected with 0.5 ml of adenovirus-packaged TLR4 overexpression vector(pDC-TLR4,1×10^(11) PFU)after modeling.The rats in western medicine+TLR4 overexpression group were treated with the same volume of compound glycyrrhizin tablets and pDC-TLR4 after modeling.The rats in TCM+TLR4 overexpression group were treated with the same volume of modified Yinchenhao decoction and pDC-TLR4 after modeling.After two weeks of continuous administration,serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in each group were measured using an automatic blood biochemical analyzer.The expression levels of TNF-α,p-NF-κB,MyD88,and TLR4 in liver tissues were detected by Western blotting.Results Compared with blank group,the levels of ALT,AST in serum and TNF-α,p-NF-κB,MyD88 and TLR4 expression in liver tissue were significantly increased in model group(P<0.01).Meanwhile,HE staining showed that there were liver cell diffuse necrosis and inflammatory cell infiltration in liver tissues.Compared with model group,the levels of ALT,AST in serum and TNF-α,TLR4,MyD88,and p-NF-κB in liver tissue were significantly reduced in TCM group and western medicine group(P<0.01).Compared with TCM group and western medicine group,ALT,AST in serum and TNF-α,TLR4,MyD88,and p-NF-κB levels in liver tissue were significantly increased in TCM+TLR4 overexpression group and western medicine+TLR4 overexpression group(P<0.01).In addition,the degree of liver injury in rats was significantly increased in TCM+TLR4 overexpression group and western medicine+TLR4 overexpression group compared with TCM group and western medicine group.Conclusion Modified Yinchenhao decoction could alleviate the liver damage by reducing the levels of important cytokines such as TNF-α,MyD88 and p-NF-κB in the TLR4 signaling pathway.