干预TLR4/NF-κB信号通路研究荔枝核总黄酮治疗胆汁淤积性肝纤维化的机制

The stage of liver fibrosis occurred before the cholestasis liver disease. If the patient use the effective drug, liver fibrosis can delay,prevent and even reverse the disease.We found that the effective components of litchi litchi flavonoids (TFL) to cholestasis hepatic fibrosis rats have remarkable curative effect, western blot results showed that NF-kappa B protein in TFL used rat liver has effect, therefore, we assumed that"Total Flavone of litchi flavonoids through the intervention TLR4 / NF-kappa B signal path treatment cholestasis hepatic fibrosis". We used the liver fibrosis rats and two hepatic stellate cell line as the research object, through immunohistochemistry, Western blot, cell culture, flow cytometry, fluorescence quantitative PCR instrument and RNA interference and other technical methods, study the expression of TLR4, the nf-kappa B, IL - 1 in TFL used rat and human hepatic stellate cell line and rat liver. Early studies have shown that TFL for cholestasis liver fibrosis rats have significant curative effect. Preliminary test results show that TFL for rat liver NF-kappa B protein blocking effect to a certain extent, but the precise mechanism is unknown. This topic from animal models based on clinical success example, using the modern research disease model method, through the molecular signaling pathways research state of disease each protein in the cell and tissue of the distribution and expression situation, proposed by the train of thought of evidence-based medicine building cell and molecular level model, the use of specific protein inhibitor refining drug effect, si RNA technology further proof of the drug to the NF-kappa B protein may target function, multi-angle comprehensive prove the reliability of the hypothesis. Experimental results for transformation, make the development of new medicine, further clear TFL treatment of liver fibrosis drug targets and provide a basis for molecular mechanism.

在肝纤维化阶段采用有效的药物干预可以延缓、阻止、甚至逆转疾病的不良预后。本课题以肝纤维化大鼠模型和肝星状细胞系为研究对象，通过免疫组化、Western blot、荧光定量PCR、细胞培养、流式细胞仪及RNA干扰等技术方法，深入研究荔枝核总黄酮（TFL）对鼠/人肝星状细胞系及大鼠肝脏TLR4、NF-κB、IL-1表达的影响。前期研究表明TFL对胆汁淤积性肝纤维化大鼠有显著疗效。预试验结果显示：TFL对大鼠肝脏NF-κB蛋白有一定的阻断效应，但确切机制不明。为此，我们提出"荔枝核总黄酮通过干预TLR4/NF-κB信号通路治疗胆汁淤积性肝纤维化"的假说。拟通过循证医学的思路构建细胞、分子层面的模型，使用特定的蛋白抑制剂细化药物作用，RNA干扰进一步证明药物的作用靶点，多角度全方位证明假说的可靠性。为转化实验成果，变废为宝，临床开发该药治疗肝纤维化患者提供理论支持和实验依据。

前期实验中本课题组对荔枝核总黄酮(Total Flavone of Litchi Chinensis Sonn. TFL )抗肝损伤作用进行了较深入的研究，已初步证实TFL能起到抗纤维化作用。为探寻TFL治疗肝纤维化的药物靶点及其相关分子机制，本课题基于TLR4/NF-κB 信号传导通路，以肝纤维化大鼠模型和肝星状细胞系为研究对象，通过免疫组化、Western blot、荧光定量PCR、细胞培养、流式细胞仪及RNA干扰等技术方法，深入研究荔枝核总黄酮对鼠/人肝星状细胞系及大鼠肝脏TLR4、NF-κB、IL-1R I表达的影响；并通过循证医学的思路构建细胞、分子层面的模型，使用特定的蛋白抑制剂细化药物作用以及RNA干扰技术进一步证明药物的作用靶点，多角度、全方位证明荔枝核总黄酮可通过干预 TLR4/NF-κB 信号传导通路抑制肝纤维化进展，为促进中药复方的开发及单药成份开发提供理论依据与实验支持。