SYA通过下调ARNTL调控自噬抑制HSC活化参与蒙药德都红花-7味散治疗肝纤维化的机理研究

Liver fibrosis is inevitable stage that chronic liver diseases develop to liver cirrhosis and liver cancer. Effective intervention in liver fibrosis in its early stage is important in its repairmen. The efficacy of traditional Mongolian medicine Deduhonghua-7(DDHH-7) on liver fibrosis is exactly. Our research showed that mechanism of DDHH-7 was modulation of TGF－β1/smad pathway, stable condition of MMP/TIMP, function of hepatic stellate cells (HSCs) and revert HSC to its quiescent state. Especially, discovery of downregulating function of DDHH-7 and its component safflower yellow A on ARNTL gene provided evidence first time to study on its autophagy regulating function on ARNTL and activating function on HSC. In this research, the liver fibrosis animal model was established to research the preventing and curing function of DDHH-7 according its autophagy regulating mechanism, observe the HSC inhibiting function of safflower yellow A according regulation of ARNTL. The expressions of type I, type Ⅲ collagen, a-SMA, TGF-β1, autophagy-related Beclin1, LC3B-Ⅱ / LC3B-Ⅰ and ARNTL were detected by immunohistochemistry, Western blotting and fluorescence quantitative PCR. The autophagy communication was detected by mRFP-GFP-LC3 tandem fluorescence method, which provided the scientific basis for the clinical usage of the Mongolian medicine DDHH-7, and provided new targets for the development of innovative drugs for liver fibrosis.

肝纤维化（HF）是慢性肝病发展到肝硬化、肝癌必经阶段，早期有效干预可逆转。德都红花－7味散治疗HF疗效确切，与调节TGF－β1/smad通路、MMP/TIMP的稳态、HSC细胞功能，使HSC恢复到静止态相关。组方优化首次发现该方和红花黄色素A(SYA)有下调ARNTL作用。本课题用动物模型研究德都红花-7味散通过自噬防治HF的功能，用细胞模型研究SYA抑制HSC活化的功能，研究ARNTL通过调控自噬促进HSC活化的功能，探讨SYA通过ARNTL调控自噬而抑制HSC活化作用。用免疫组化、蛋白印迹、PT-PCR法检测HSC活化标志物I、Ⅲ型胶原、a-SMA、TGF-β1,自噬相关Beclin1、LC3B-Ⅱ/LC3B-Ⅰ, ARNTL的表达，用mRFP- GFP-LC3串联荧光法检测自噬流，为德都红花-7味散的临床使用提供依据，为肝纤维化研发创新药提供新靶点。

本项目采用TGF-β1诱导HSC-T6建立肝纤维化细胞模型，采用红花黄色素A、羟基红花黄色素A、木犀草素、芹菜素干预，Collagen I、Collagen III、α-SMA蛋白表达，自噬相关基因AKT、AMPK、ULK1、Map1lc3a、Map1lc3b、Beclin1、ATG7、p62、ATG5、ATG12、LAMP1、LAMP2mRNA表达；芹菜素、木犀草素对凋亡影响；木犀草素干预的差异蛋白研究。动物实验验证了德都红花-7味散及木犀草素抗肝纤维化作用。.研究结果：①采用TGF-β1 100ng处理HSC-T6细胞48h建立肝纤维化细胞模型。②4种单体药物对TGF-β1处理HSC-T6细胞的增殖有所抑制，随着药物浓度的升高，抑制作用越显著。其中，高浓度10 μM的木犀草素的抑制作用更加明显。羟基红花黄色素A、芹菜素、木犀草素可降低a-sma、Collagen I含量，芹菜素低剂降低TIMP-1mRNA表达，③红花黄色素A、羟基红花黄色素A作用模型细胞后，自噬点数与模型组无明显差异。芹菜素、木犀草素作用模型细胞后，对自噬的调控较明显，调控作用与药物剂量呈正相关。红花黄色素A、芹菜素抑制Map1lc3b 、羟基红花黄色素抑制ATG17、木犀草素抑制ATG12、LAMP1基因表达相关。④芹菜素对早期TGF-β1诱导HSC-T6凋亡细胞增加作用；木犀草素对早期凋亡和晚期凋亡细胞均增加作用。⑤蛋白质组学研究表明木犀草素主要参与mitotic spindle midzone assembly，collagen fibril organization等生物学过程，执行层粘连蛋白结合，核糖核苷二磷酸还原酶活性等功能，iron ion transport，glycoside catabolic process等生物学过程，执行NADH脱氢酶（泛醌）活性，单链DNA解旋酶活性等活性。KEGG分析表明主要参与萜类主链生物合成，ECM受体相互作用等通路，糖胺聚糖降解，氧化磷酸化等通路相关。PRM验证结果发现：Mki67、LOC100359539、Cd59、 Naga 、Ccdc80、 Apob 、Ccr1、Dnmt1 等蛋白表达差异显著且和4D label free趋势一致。⑥动物实验研究表明德都红花-7味散及其主要蓝盆花和木犀草素具有明显的抗肝纤维化作用。