睡莲花有效成分抗肝纤维化作用及其作用机制研究

Nymphaea candida possessed the efficacy of clearing away heat, tranquilization, nourishing the liver, and relieving inflammation, and was used to treat various disease in Uighur medicine. Previous studies have shown that some extracts and constituents from N. Candida have significantly preventive and therapeutic effect against chemical and immunological liver injury. Though biological activities screening experiment in vitro, it was found that these extracts and constituents not only could inhibit proliferation of hepatitis stella cell (HSC), but also inhibit the expression of Smad 2/3 protein and promote the expression of Smad 7 protein. However, anti-hepatic fibrosis effective of this plant was rarely reported. Therefore, This project aims to investigate anti-hepatic fibrosis effect of N. candida against CCl4 induced hepatic fibrosis in rat and its mechanism on the basement of isolation and purification of active fraction/compounds. Moreover, the anti-hepatic fibrosis effective target of N. candida and its molecular mechanism were further studied by apoptosis of HSC-T6, secretion of collagen as well as NF-κB、PDGF and TGF-β1/Smad signal transduction pathways in HSC-T6. The results will provide a theoretical basis for the further expansion of N. candida in the medical field as well as development of new drugs on prevention and treatment of liver fibrosis.

维吾尔药睡莲花具有降热养肝、消炎止咳之功效，用于干热性肝虚等病症的治疗。前期实验显示睡莲花有效部位/成分对小鼠化学性和免疫性肝损伤均有较为显著的防治作用；通过体外试验还发现其有效部位/成分对肝星状细胞的增殖具有显著的抑制作用。然而，该药材抗肝纤维化作用的研究却鲜有文献报道。因此，本项目拟通过各种色谱技术分离纯化睡莲花中的有效部位/成分，并进一步采用四氯化碳诱导的大鼠肝纤维化模型，通过生化指标检测及病理组织学观察，在整体水平上考察有效部位/成分对大鼠肝纤维化的作用及其作用机制。体外选用HSC-T6细胞株，通过观察效有效部位/成分对HSC-T6增殖、凋亡和胶原分泌的作用，以及对NF-κB、PDGF和TGF-β1/Smad信号传导通路的影响，揭示睡莲花有效部位/成分的作用靶点；并从细胞和分子水平上阐明其抗肝纤维化的分子机制。结果可为维吾尔药睡莲花的深入研究及其新药开发提供基础数据。

传统中药睡莲花具有降热养肝、消炎止咳之功效，用于干热性肝虚等病症的治疗。前期实验显示睡莲花有效部位及其成分烟花苷对小鼠化学性和免疫性肝损伤均有较为显著的防治作用。本项目建立了睡莲花主要标识性成分之一睡莲酚的制备工艺，并发现其对四氯化碳、刀豆蛋白A诱导的小鼠肝损伤均有较好的的保护作用。在此基础上，体外通过肝星状细胞（HSC-T6）开展有效部位/成分抗肝纤维化活性研究，结果发现睡莲花总黄酮有效部位NCTF及主要标识性成分烟花苷和睡莲酚对HSC细胞增殖以及胶原分泌均有较好的抑制作用，三者对TGF-β1诱导HSC 细胞的Col-I、α-SMA、Smad2及Smad3的蛋白表达也有显著的抑制作用，并能上调smad7的蛋白表达。采用四氯化碳诱导的大鼠肝纤维化模型，进一步考察了NCTF的抗肝纤维化作用。与模型组相比，NCTF组能改善肝组织病理损伤及纤维化状态；能显著降低大鼠血清ALT、AST、ALP水平及大鼠肝组织Hyp的含量，提高血清TP和ALB含量；能显著抑制HA、LN、PCⅢ和CIV型蛋白的表达；NCTF均能明显降低血清MDA、NO水平，提高大鼠血清GSH、SOD水平，能显著降低大鼠血清中IL-1β、IL-6、TNF-α及肝组织中α-SMA的蛋白表达水平；能明显降低TGF-β1、Smad2、Smad3 mRNA及其蛋白表达水平，升高Smad7 mRNA及其蛋白水平。NCTF及标识性成分睡莲酚对四氯化碳诱导的小鼠肝纤维化也有较好的防治作用。NCTF和睡莲酚能明显改善肝纤维化症状，并能降低肝纤维化组织IKKα、IҡBα和NF-ҡB mRNA含量，降低IKKα、NF-ҡB p65和促进IҡBα的蛋白表达。以上数据揭示睡莲花总黄酮及有效成分睡莲酚是睡莲花抗肝纤维化的主要药效物质基础，作用机制与其抗氧化、免疫调节、抑制胶原合成以及对TGF-β1/Smad、NF-ҡB信号通路的调控相关。研究结果可为睡莲花的开发利用及进一步的创新药物研发提供理论依据。