满天星异荭草苷对肝纤维化NF-κB信号通路的干预作用及其机制研究

Nuclear factor-kappa B (NF-κB) is the focus of regulation network for various pathogeny of liver fibrosis. Based on the NF-κB target, the drug targeting therapy is one of the effective ways for anti-liver fibrosis. We have endeavored to find ethnic herbs or their active principles against chronic hepatic fibrosis for years, and have extracted a flavonoid compound (isoorientin-2″- O-α-L- arabinopyranosyl, isoorientin) from the Gypsophila elegans Bieb. Our pilot study revealed that isoorientin had obvious inhibitory effects on liver fibrosis, which might be via regulating the NF-κB signaling pathway. The results would lend valuable reference for the investigation of the intervention and its mechanism of isoorientin on NF-κB signaling pathway in liver fibrosis. In this study, the effects of isoorientin on proliferation, apoptosis, synthesis and secretion of collagen tape III and expression of p-IκBα and NF-κB p65 induced by IL-1β in rat hepatic stellate cells (HSCs) will be investigated. Besides, the effects of isoorientin on serum biochemical indices, pathology changes in the liver tissue, NF-κB binding activity, phosphorylation of IκB and related key cytokines of rats with chemical-, or immunological-induced hepatic fibrosis will also be analysed, respectively. This research will elucidate the molecular mechanisms of isoorientin on NF-κB signaling pathway in liver fibrosis, and provide the experimental evidence and theoretical support for its therapy of liver fibrosis.

转录因子NF-κB 是肝纤维化各种病因调节的网络焦点，基于该靶点研究药物对肝纤维化的靶向治疗，是抑制肝纤维化的一个有效途径。多年来，我们一直致力于抗肝纤维化民族药研究，前期高通量筛选试验中偶然发现满天星活性成分- - 异荭草苷具有良好的活性，且其机制可能与调控NF-кB信号通路有关，激起了我们极大兴趣和深入研究。本申请拟采用原代肝星状细胞(HSCs)模型，观察异荭草苷对IL-1β刺激的HSCs增殖、凋亡和III型胶原的抑制作用，及对HSCs胞浆p-IκBα、胞核NF-κBp65的表达和NF-κB核转位的影响；并利用化学性和免疫性肝纤维化动物模型，了解异荭草苷对肝纤维化大鼠血清生化指标、肝脏组织病理、NF-κB结合活性、IκB磷酸化水平和相关细胞因子的影响。本研究将阐明满天星异荭草苷介导NF-кB信号途径对肝纤维化发生和发展的干预作用及其机制，为其治疗肝纤维化提供实验依据和理论支持。

核因子-κB （nuclear transcription factor-kappa B, NF-κB）作为一种广泛存在体内细胞的核转录调节因子，参与免疫、炎症及细胞增殖的调控。目前认为，抑制NF-κB信号传导是治疗肝纤维化的一条有效途径。本研究采用体外肝星状细胞模型（包括大鼠HSC-T6、人LX-2和人7702细胞系），观察异荭草苷对肝星状细胞活化增殖、凋亡、胞内胶原的抑制作用，及对胞浆p-IκBα、胞核NF-κBp65的表达及NF-κB核转位的影响；另外，分别利用CCl4和猪血清诱导化学性和免疫性肝纤维化大鼠模型，观察异荭草苷对肝纤维化大鼠血清生化指标、肝脏组织病理、胶原分布及沉积的影响；检测其对炎症细胞因子（TNF-α、IL-1和IL-10）和基质金属蛋白酶的抑制作用；并探讨其对NF-кB信号通路相关蛋白的结合活性、核转位和磷酸化水平的调控作用。我们发现，异荭草苷能明显抑制肝星状细胞的活化增殖，阻滞细胞于G2期，诱导细胞凋亡，减少胶原的合成和分泌，降低炎症细胞因子水平，抑制基质金属蛋白酶MMP-9活性，降低NF-κB p65磷酸化、NF-κB p50表达，上调IκBα的表达，同时伴随 IκBα磷酸化、NF-κBp65 表达和核转位降低。此外，异荭草苷能明显减轻CCl4或猪血清诱导的大鼠肝损伤和肝纤维化程度，抑制胶原形成与沉积，降低炎症细胞因子的释放，减轻脂质过氧化，降低NF-κB磷酸化水平。总之，我们的实验表明，异荭草苷能减轻肝纤维化的发生发展，其机制与抑制NF-κB信号通路有关。本研究初步阐明了异荭草苷抗肝纤维化作用及其机制，为其治疗肝纤维化提供了前期实验依据和理论支持。