从Fas/FasL-PI3K/Akt通路探讨壮肝逐瘀煎调控肝纤维化肝星状细胞凋亡机制

In the previous studies, we proposed the general deficiency-stasis-colorectal pathogenesis of hepatofibrosis from traditional Chinese medicine (TCM ) aspect. Based on this, we formulated a corresponding TCM therapeutic principle and invented Fuzhengzhuyujian decotion. We accompished a certain number of research subjects including the National Natural Science Funds (№：30460158), and demonstrated that the anti-fibrosis effect of Zhuangganzhuyujian decotion was closely related to its inhibiting effects on activation and proliferation of hepatic stellate cell (HSC) by regulating TGF-β1/smads signaling pathway. The results of this research has woned the second prize of Scientific Development Award from the Guangxi Province Academy of Sciences.The current study is designed to further explore the molecular mechanisms underlying the anti-fibrosis effect of Zhuangganzhuyujian decotion using both in vivo and in vitro experiments by establishing rat liver fibrosis model and subculture of rat HSC. Immunohistochemical studies along with image analysis technique, Western blot, real-time PCR, MTT, flow cytometry, and ELISA methods would be employed to explore the signal transduction of Fas/FasL-PI3K/Akt pathway which responsible for the mechanisms of the regulation effects of Fuzhengzhuyujian decotion on hepatic stellate cell apoptosis in hepatofibrosis. The scanning electron microscope technique and histopathological morphology analysis woulde be also introduced to study the pathological changes of hepatic tissue and cell apoptosis of HSC. The expected results of current research would be beneficial to the treatments of hepatofibrosis and would contribute to the developments and utilizations of herbal medicines of Guangxi province.

前期工作中，提出肝纤维化中医肝虚瘀结病机，制定扶正逐瘀治法，创制壮肝逐瘀煎。完成包括前一个基金（№：30460158）在内的多个课题，证实了该方抗肝纤维化作用与调控TGF-β1/smads信号传导，抑制HSC的活化和增殖有关。获得省级科技进步二等奖1项。本项目拟以体内实验与体外实验相结合，建立CCL4复合因素大鼠肝纤维化模型及体外大鼠传代HSC培养，应用免疫组织化学染色图像分析、Western blot、real-time PCR、MTT、流式细胞术、ELISA等方法，从调控Fas/FasL-PI3K/Akt传导通路，影响HSC细胞凋亡角度，结合电镜超微形态学分析，从组织、细胞、蛋白等方面，探讨壮肝逐瘀煎对HSC细胞凋亡和肝组织病理学的影响。深入探讨壮肝逐瘀煎多靶点抗肝纤维化的机理，进一步求证扶正逐瘀治法。预期成果将有益于肝纤维化患者的治疗，并有利于广西道地中药进一步开发利用。

本项目通过备制大鼠HF模型与HSC细胞模型，观察壮肝逐瘀煎对大鼠肝纤维化的治疗作用。应用常规病理与扫描电镜对HF大鼠肝组织的病理形态进行了观察，对HSC细胞的超微结构进行了观察；应用流式细胞术测定了HSC细胞凋亡的情况；应用组织化学染色、LELISA法、real-time PCR方法、Western blot方法等，检测了对HF大鼠影响HSC凋亡的相关蛋白Fas/FasL；对影响HSC凋亡相关蛋白的PI3K/Akt通道相关因子以及关键酶进行了测定。PI3K/Akt通道相关因子包括：PI3K、Akt、PDK1、XIAP、P53、Bcl-2、Bax等。结果表明，壮肝逐瘀煎能够显著改善HF大鼠肝组织的病理变化，壮肝逐瘀煎能够显著抑制HF大鼠肝组织与血清及HSC-T6细胞的Fas、FasL表达，显著抑制HF大鼠肝脏肝组织及HSC-T6细胞PI3K、Akt、PDK1、XIAP、P53、Bcl-2的表达，增强P53、Bax的表达。能够显著改善HSC-T6的凋亡。初步证实了壮肝逐瘀煎抗肝纤维化作用的机制，与通过影响对HSC细胞凋亡有调控作用的Fas/FasL-PI3K/Akt传导通路有关这一科学假设。在信息调控分子水平上进一步阐述“扶正逐瘀”了治法的科学内涵，为壮肝逐瘀煎的深入研究与开发提供理论和基础实验依据，并有利于广西道地药物资源的开发利用。