维吾尔药“护肝布祖热”抗大鼠肝纤维化作用及其机制研究

Hepatic fibrosis (HF) is not only the pathological characteristic of chronic liver disease, but also the reason which leads to the development of chronic hepatitis and cirrhosis. Even though there are many researches in the field of control, prophylxis and treatment of HF, it has lacked a kind of safe and effective drug duing the treatment. Therefore, developing new medicine from ethno-medicine has become a hot issue. Hugan buzure was a Uyghur compound for HF treatment. Our preliminary studies were explored the hepatoprotection of Uyghur traditional medicine Cichorii glandulosum, which is one of herb in Hugan buzure compound. Therefore, the present study aim to investigate the anti-HF activities and its action mechanisms of Hugan buzure. In vivo, research were focus on proliferation, activation and apoptosis of hepatic stellate cells (HSC) . Histopathological observations, biological parameters, enzyme levels on the HF models were determined. Molecular biological techniques were applied to analysis cytokine, protein and mRNA express. Further research were focus on elucidating the actions of TGF-β/Smad, TNF-α/NF-κB and PDGF including Ras/ERK, JAK/STAT and PI3-K/Akt signal transduction pathways. The subjects would contribute to development of Uyghur drug research platform, deeper exploration of traditional medicine and support our competitions in the worldwide.

肝纤维化是多种慢性肝病共有的病理过程，抗肝纤维化是治疗慢性肝病和预防肝硬化的瓶颈。民族药防治肝纤维化效果显著，优点突出。护肝布祖热为维吾尔经典保肝复方，临床疗效确切。课题组前期研究显示该方中主药毛菊苣具有显著肝保护和抗肝纤维化作用。本项目拟采用现代药理学和分子生物学技术，分别从体外和体内实验研究护肝布祖热抗肝纤维化作用及机制：体外实验探索药物对大鼠肝星状细胞增殖、活化及凋亡的影响；体内实验以肝纤维化大鼠为研究对象，通过检测其病理切片、血清生化指标等阐明药物抗肝纤维化作用，并从分子水平研究其对各通路中关键信号分子的转录水平和蛋白表达的影响，探索药物对肝纤维化相关信号转导通路TGFβ-Smad、TNF-α/NF-κB、PDGF（Ras/ERK、 JAK/STAT、PI3-K/Akt）的影响，揭示护肝布祖热抗肝纤维化作用的分子机制，以期为阐明维药复方作用靶点、发现新的抗肝纤维化药物提供理论依据。

护肝布祖热方是中国传统医药中保肝护肝的经典方之一，主要由茴香根皮、菊苣根、菊苣子、芹菜根、芹菜子、菟丝子、小茴香七味药材组成，具有补益肝胃，散气止痛，保肝利胆的功效。1. 本研究探讨了护肝布祖热方对四氯化碳诱导的肝纤维化大鼠的保护作用，观察大鼠血清中肝纤维化相关指标及肝组织病理变化，检测肝组织中相关通路因子的表达。结果显示，给予护肝布祖热方治疗后，大鼠血清中HA、LN、IV-C、PCIII、Hyp、AST、ALT、LDL、TG、TC、MDA水平显著降低（P＜0.01），ALB、TP、HDL和SOD水平显著升高（P＜0.01），护肝布祖热方可以显著下调 samd2、Smad3、Smad4、PDGF-B、JAK2、α-SMA、TGF-β1、ERK1/2、c-Fos、c-Jun、PI3-K、AKT1、TIMP-1、COL-I、COL-III和Smad4基因和蛋白表达水平，同时上调抑制性因子Smad7基因和蛋白表达水平，表明护肝布祖热方具有抗肝纤维化作用，其作用机制可能与调控TGF-β1/Smad通路及PDGF介导的JAK/STAT、Ras/ERK、PI3-K/AKT信号通路有关。2. 考察CCl4建立体外肝损伤模型的浓度及护肝布祖热对LX-2细胞的增殖抑制及促凋亡作用。RT-PCR和Western-blot检测肝损伤标志分子及TGF-β1/Smad 通路、JAK/STAT通路、Ras/ERK 通路、PI3-K/AKT通路中相关因子基因和蛋白表达。结果显示，最终选择5mmol/L CCl4干预24h建立肝损伤模型，不同浓度的护肝布祖热干预后，细胞的增殖抑制率及凋亡率较模型组明显增加（P＜0.05）。与模型组相比，护肝布祖热组中PTP1B、Jak1、Smad7、Stat3基因和蛋白表达水平明显升高（P＜0.05），TGFβ、Smad3、Smad4、CHOP、PERK、IRE1、Jak1、ATF6基因和蛋白表达水平显著降低（P＜0.05），提示护肝布祖热能通过调控TGFβ-Smad信号通路、Jak-Stat信号通路和内质网应激（ERS）相关基因转录水平和蛋白的表达，以达到促进细胞凋亡、减轻炎症反应的目的，从而保护肝损伤。研究结果深入阐述了护肝布祖热方保肝作用及其作用机制，为发展中医药方剂研究提供参考。