基于TLR4/NF-κB信号通路研究三叶香茶菜抗肝纤维化作用机制

The Isodon ternifolia( D. Don)Kudo（三叶香茶菜）is Chinese folk medicine, it is applicated on Lan Han disease(acute and chronic hepatitis) and Lan yan disease(liver cirrhosis) widely among the Yao in Guangxi province of China. .Our previous studies demonstrated Isodon ternifolia( D. Don)Kudo have anti-liver fibrosis functions，and demonstrated it can inhibit inflammation through decreasing the production of TLR4mRNA、NF-kappa B p65、IL-1β、TNF-α、IL-6 and inhibiting liver tissue apoptosis of rats with hepatic fibrosis induced by CCL4. These indicated Isodon ternifolia(D. Don)Kudo can anti-hepatic fibrosis through down-regulation of TLR4/NF-kappa B signal pathway. But, its mechanisms of inhibiting this pathway are not fully understood. Therefore, in the present project, we will further investigate the mechanisms of Isodon ternifolia(D. Don) Kudo how to regulate the TLR4/NF-kappa B signal pathway . The CCL4-induced hepatic fibrosis rats, hepatocyte, macrophage and HSC-T6 stimulated by LPS, ELISA assay, Real-time PCR assay, Western blot assay and immunohistochemistry assay will be applied to investigate the effect of Isodon ternifolia(D. Don) Kudo on TLR4/NF-kappa B signaling. The present project will identify the mechanisms of anti-liver fibrosis of Isodon ternifolia(D. Don) Kudo and provide the new rational theory for the application of Isodon ternifolia(D. Don) Kudo.

三叶香茶菜是广西瑶药材，属瑶医风打相兼药，能清热解毒，利湿疏肝，用于望胆篮虷（急慢性肝炎）和篮严（早期肝硬化）等。课题组研究证实三叶香茶菜能抑制CCL4致大鼠肝组织纤维化和细胞凋亡，下调TLR4mRNA、NF-κBp65蛋白、IL-1β、TNF-α、IL-6、TGF-β表达。表明三叶香茶菜能下调TLR4/NF-κB信号，达抗肝纤维化作用，但三叶香茶菜如何下调TLR4/NF-κB信号途径活化确切机制仍不甚明了。为此，本课题拟在前期研究基础上，以TLR4/NF-κB信号途径为靶点，采用CCL4致肝纤维化大鼠、大鼠原代肝细胞、原代KC、原代HSC，ELISA、定量PCR、Western blot、免疫荧光和免疫组化，以TLR4/NF-κB信号途径活化相关分子和效应分子为指标，研究三叶香茶菜对TLR4/NF-κB信号通路活化调控作用，揭示三叶香茶菜抗肝纤维化分子机制，进一步丰富三叶香茶菜药理理论。

本项目采用 CCl4肝纤维化大鼠模型、大鼠原代肝细胞、原代肝 KC 细胞和原代 HSC 细胞模型，研究三叶香茶菜对TLR4/NF-κB信号的影响。本项目研究证实了三叶香茶菜能抑制CCL4致大鼠肝组织纤维化，抑制KC细胞、HSC细胞及肝实质细胞活化，下调肝组织、KC细胞、HSC细胞及肝实质细胞TLR4、IκBα、NF-κB、p-IκBα、IL-1β、IL-18、TNF-α、IL-6、的表过，抑制肝组织及HSC细胞TGF-β、HYP、α-SMA、HA、LN及胶原表达。表明三叶香茶菜能通过下调TLR4/NF-κB信号通路的多个相关分子表达活化，从而抑制TLR4/NF-κB信号通路的活化，抑制肝纤维化效应细胞HSC的活化，抑制胶原的表达，达到抗肝纤维化作用。本研究阐明三叶香茶菜抑制TLR4/NF-κB信号通路活化是其抗肝纤维化的分子机制，进一步丰富三叶香茶菜药理理论。