行气活血法调控枯否细胞中PPAR-γDNA甲基化干预早期肝纤维化的机制研究

In the early stage of hepatic fibrosis(HF), it is an important treatment method to cure qi stagnation with removing stasis medicinal herbs. The fruits of Euscaphisfukienensis Hsu. own the function of warming the middle jiao and regulating qi. In our previous studies, the fruits showed good effect on anti-hepatic fibrosis, and the triterpenoids(EFT) of the fruits prevent the kupffer cell(KC)to polarize to M1 macrophage. We suppose the fruit should show better effect on anti-hepatic fibrosis with Danshen, which is a good medicinal herbs with improving circulation of blood. Activation of hepatic stellate cell (HSC) is the key chain in HF. KC plays a important role in activating HSC. Under induction of LPS, KC is polarized to M1 macrophage to produce pro-imflammatory factors to activate HSC. NF-κB is closely related to KC polarization. NF-κB is the critical downstream effector protein in TLR pathway. In physiological status, TLR pathway is inhibited by PPAR-γ, but in early stage of HF, PPAR-γ gene is in silence expression. Gene expression is controlled by DNA methylation. So we assume there is a relationship between the PPAR-γ DNA methylation and KC polarization. Methylation of PPAR-γ DNA inhibits the express of PPAR-γ in KC , activates the NF-κB and promotes the polarization of KC to M1 macrophage. The fruits of Euscaphisfukienensis Hsu. and Danshen and their effective constituents (EFT and phenolic acids of Danshen)will prevent the methylation of PPAR-γ DNA , inhibit the NF-κB to stop the polarization of KC, and finally prevent the activation of HSC. The results provide the experimental basis for the fruits of Euscaphisfukienensis Hsu. and Danshen and their effective constituents in the treatment of hepatic fibrosis, and also explain the scientific intension of Xinqinhuoxue method in treatment HF.

早期肝纤维化（HF）时以疏肝理气为枢机，配合活血化瘀药是治疗其有效方法。前期研究发现，具有理气止痛的福建野鸦椿果有较好的抗HF效果，其所含三萜能抑制枯否细胞（KC）M1极化，辅以丹参将增加其抗HF效果。KC在LPS诱导下极化成M1型，分泌促炎细胞因子，促使HSC增殖活化。KC表型变化与NF-κB有关，而NF-κB是TLR信号通路下游蛋白，生理状态下TLR 信号可由PPAR-γ负调控。HF发生时PPAR-γ 表达沉默，而基因表达沉默与DNA甲基化有关。因此我们提出福建野鸦椿果与丹参及其有效组分能抑制PPAR-γ DNA甲基化，增加PPAR-γ的表达，抑制NF-κΒ活性，干预KC M1极化， 降低HSC 的增殖与激活。本项目通过体内外实验从PPAR-γ DNA甲基化对KC极化影响探讨行气活血法指导下的药物作用机制，研究结果不仅为临床用药提供参考，也将从新角度阐明行气活血法治疗HF的科学内涵。

行气活血是中医治疗早期肝纤维化的重要治法。巨噬细胞、肝细胞、肝星状细胞等细胞共同参与了肝纤维化的进程，其中巨噬细胞在肝纤维化的早期主要表现为促炎的M1型。本项目围绕理气药福建野鸦椿果和活血祛瘀药丹参的抗早期肝纤维化的有效成分及作用机制展开了一系列体内外研究。第一，在药物的物质基础研究方面，本项目对福建野鸦椿果的化学成分进行了分离和鉴定，并建立了福建野鸦椿果三萜和丹参酚酸的制备方法和质量标准。第二，在体内实验方面，本项目明确了三萜和酚酸分别是福建野鸦椿果和丹参抗早期肝纤维化的有效成分，二者配伍抗肝纤维化的机制之一在于调节肠道菌群及其代谢产物；此外，肝脏组织的转录组学为下一步的机制研究提供了思路。第三，本项目通过体外实验的活性筛选，表明了坡膜酸（PA）、齐墩果酸（OA）、熊果酸（UA）以及丹酚酸B（Sal B）等来自于上述两味药材的抑制巨噬细胞M1型极化的活性成分，并通过DNA甲基化测序、蛋白组学、代谢组学等方法探索了药物的可能作用机制；根据上述结果本项目进一步通过分子实验研究了OA和UA等抑制巨噬细胞M1型极化的作用靶点和机制。第四、本项目对课题组的化合物库中的三萜类成分进行了抑制肝细胞凋亡及肝星状细胞活化活性筛选和机制研究，最终发现甘草酸（GA）可通过下调PPAR-γ DNA甲基化水平抑制肝细胞凋亡及肝星状细胞活化。