[关键词]
[摘要]
目的 通过构建肝性脑病大鼠模型,探究安宫牛黄丸对肝性脑病大鼠肿瘤坏死因子α/核因子κB(TNF-α/NF-κB)信号通路及神经功能的影响。方法 将60只SPF级SD大鼠分为对照组,模型组,乳果糖组(0.2 mL/200 g乳果糖)、安宫牛黄低剂量组(1.0 g/kg)、中剂量(2.0 g/kg)、高剂量(3.0 g/kg),每组10只。通过硫代乙酰胺(TAA)法构建肝性脑病模型(连续3 d对模型大鼠注射300 mg/kg TAA溶液),造模前4 d开始各组每天连续ig相应剂量乳果糖及安宫牛黄丸直至造模结束后2 d;对照组注射等量生理盐水。水迷宫实验检测大鼠学习记忆能力;ELISA法检测大鼠血清生化指标及海马组织乙酰胆碱(Ach)、谷氨酸(Glu)含量;苏木精-伊红染色(HE)观察肝组织和脑组织病理学变化;实时荧光定量PCR检测各组样本中TNF-α、NF-κB mRNA的表达情况;Western blotting检测通路相关蛋白表达情况。结果 与对照组相比,模型组寻台潜伏期、血氨、ALT、AST水平、肝组织损伤、脑组织海马CA1区损伤程度、Glu含量、TNF-α和NF-κB的mRNA及蛋白表达水平显著增加,而穿台次数、神经学评分、Ach含量显著降低(P<0.05)。与模型组相比,乳果糖组和安宫牛黄低、中、高剂量组大鼠寻台潜伏期、血氨、ALT、AST水平、肝组织损伤、脑组织海马CA1区损伤程度、Glu含量、TNF-α和NF-κB的mRNA及蛋白表达水平显著降低,而穿台次数、神经学评分、Ach含量显著增加(P<0.05),且安宫牛黄丸各组间存在一定剂量相关性。结论 安宫牛黄丸能抑制肝性脑病大鼠TNF-α/NF-κB信号通路,对大鼠神经功能具有保护作用。
[Key word]
[Abstract]
Objective To explore the effects of Angong Niuhuang Pill on tumor necrosis factor-α/nuclear factor-κB (TNF-α/NF-κB) signal pathway and neurological function in rats with hepatic encephalopathy by constructing a HE rat model. Methods Sixty SPF SD rats (10 rats in each group) were divided into control group, model group, lactulose group (0.2 mL/200 g lactulose), Angong Niuhuang low-dose group (1.0 g/kg), medium-dose group (2.0 g/kg), and high-dose group (3.0 g/kg). Hepatic encephalopathy model was established by thioacetamide (TAA) method (300 mg/kg TAA solution was injected into the model rats for 3 consecutive days). From 4 days before modeling, each treatment group was given a corresponding dose of lactulose and Angong Niuhuang Pill by gavage until 2 days after modeling; the control group was injected with the same amount of normal saline. Water maze test was used to test the ability of learning and memory, enzyme linked immunosorbent assay (ELISA) was used to detect the serum biochemical indexes and the contents of acetylcholine (ACh) and glutamic acid (Glu) in hippocampus, hematoxylin eosin staining (HE) was used to observe the pathological changes of liver and brain, real-time fluorescence quantitative PCR was used to detect the mRNA expressions of TNF-α and NF-κB, Western blot was used to detect the expression of pathway related proteins. Results Compared with the control group, the latency of platform searching, levels of blood ammonia, ALT, AST, the injury degree of liver tissue, injury degree of hippocampal CA1area in brain tissue, the content of Glu, the mRNA and protein expression levels of TNF-α and NF-κB in the model group were significantly increased, however, the times of crossing platform, neurological score and content of ACh were significantly decreased (P<0.05). Compared with those in model group, the latency of platform searching, the levels of blood ammonia, ALT, AST, the injury degree of liver tissue, injury degree of hippocampal CA1 area in brain tissue, the content of Glu, the mRNA and protein expression levels of TNF-α and NF-κB in lactulose group, low-dose group, medium-dose group and high-dose group were significantly decreased, however, the times of crossing platform, neurological score and content of ACh were significantly increased (P<0.05), there was a dose-dependent relationship between the Angong Niuhuang Pill groups. Conclusion Angong Niuhuang Pill can inhibit TNF-α/NF-κB signaling pathway in hepatic encephalopathy rats, and has protective effect on neurological function of rats.
[中图分类号]
R285.5
[基金项目]
河南省中医药科学研究专项课题(2019YZ2052)