研究背景 肝纤维化是指肝脏损伤后，损伤部位被瘢痕替代的病理状态。肝纤维化可由 多种慢性肝病引发，包括胆汁淤积性肝病，慢性乙肝感染，非酒精性脂肪肝炎， 酒精性肝炎等。如果得不到有效控制，肝纤维化可发展成为肝硬化，甚至肝癌。 由于肝脏具有强大的再生功能，能够在组织受损后重建原始结构和功能而不会造 成瘢痕，因此肝脏对各种损伤都有着较强的抵抗能力。然而当肝脏处于慢性肝病 中时，肝再生减弱，肝星型细胞被激活并分泌细胞外基质(Extra-cellular matrix， ECM)蛋白，造成组织纤维化。 促进肝脏再生被认为能够缓解肝脏纤维化。在肝脏中，Id1 可以被部分肝切 除术(Partial hepatectomy，PH)激活，并促进 WNT2(Wnt family member 2)和肝细 胞生长因子(Hepatocyte growth factor，HGF)的分泌。ID1-WNT2/HGF 通路的活化 能够促进肝再生并对抗多种原因引起的肝纤维化。 熊胆是我国的传统中药，用于热盛惊风，癫痫，子痫抽搐等证。熊去氧胆酸 (Ursodeoxycholic acid，UDCA)来源于传统中药熊胆，目前已可通过合成来制备。 同时，UDCA 也被中国，美国等国家批准用于治疗胆汁淤积性肝病。以往的研究 表明，UDCA 能够改善原发性胆汁性胆管炎患者的临床指标，缓解纤维化、提高 生存率。然而，关于 UDCA 的作用机制还未完全阐明。由于具有保肝作用，UDCA 被许多国家和地区的临床指南推荐用于改善不同疾病中的肝纤维化。 研究目的 通过利用胆总管结扎(Bile duct ligation，BDL)模型，PH 模型，Id1 敲低模型 与体外细胞模型等，研究 Id1、肝再生与 UDCA 抗肝纤维化作用的关系，探索 UDCA 对肝纤维化治疗的作用机制。 研究方法 首先，本研究建立 BDL 小鼠模型，采用肝体重比；血清 ALT，AST，ALP， TBil 水平；病理染色(H&E、Masson 染色等)；I 型胶原表达分析；F4/80 表达分 析；肝星型细胞活化基因(Acta2，Coll1a1，Loxl2)与 Id1 基因表达分析；Western blot 检测 α-SMA (Alpha-smooth muscle actin)蛋白表达等指标，评价 UDCA 对肝纤维 化的保护作用。其次，本研究建立 PH 小鼠模型，检测血清 ALT，AST 水平，评 价 UDCA 对 PH 肝脏的保护作用。利用肝脏细胞周期分析；肝脏细胞凋亡分析；2 Ki67 表达分析；qPCR 检测细胞周期相关基因表达等指标，评价 UDCA 对肝再 生的促进作用。利用 Western bolt 检测 ID1，WNT2，HGF，磷酸化 c-MET 以及 磷酸化 GSK-3β 蛋白表达，探索 UDCA 对 ID1-WNT2/HGF 通路的活化作用。第 三，本研究通过细胞活率实验确定 UDCA 促进 HepG2 细胞增殖的浓度，并通过 qPCR 与 Western blot 检测该浓度下 UDCA 对 ID1 基因与蛋白表达的影响。利用 细胞热转移实验以及分子对接实验，研究 UDCA 与 ID1 蛋白间的相互作用。最 后，本研究建立 Id1 敲低模型，采用肝体重比；血清 ALT，AST，ALP，TBil 水 平；Masson 染色；免疫组织化学染色检测 F4/80 表达；qPCR 检测肝星型细胞活 化基因(Acta2，Coll1a1，Loxl2)表达；Western blot 检测 α-SMA 表达等指标，探 索 Id1 敲低对 UDCA 抗纤维化的影响。在促进肝再生方面，本研究采用肝脏细 胞周期分析；Ki67 表达分析；肝脏细胞凋亡分析；细胞周期相关基因表达；Western blot 检测 ID1，WNT2，HGF，磷酸化 c-MET 以及磷酸化 GSK-3β 蛋白表达等指 标，探索 Id1 敲低对 UDCA 促进肝再生以及 ID1-WNT2/HGF 通路活化的影响。 实验结果 1. UDCA 能够改善 BDL 模型的肝脏系数，降低 ALT，AST，TBil 和 ALP 的水平，改善肝脏病理，缓解炎症，降低肝星型细胞活化基因(Acta2，Coll1a1， Loxl2)的表达。此外，UDCA 显著升高 BDL 肝脏中 Id1 基因的表达。 2. UDCA 降低 PH 模型中 ALT 与 AST 水平；促进 PH 模型中肝细胞进入细 胞周期；增加 Ki67 的表达；抑制肝细胞凋亡；增加细胞周期蛋白基因的表达； 降低细胞周期抑制因子的基因表达。UDCA 促进 PH 模型中 Id1，Wnt2，Hgf 基 因和蛋白表达，并促进 c-MET 磷酸化及 GSK-3β 磷酸化。 3. UDCA 能够促进 HepG2 细胞增殖；增加 ID1 基因与蛋白的表达。同时， UDCA 还可以增强 ID1 蛋白的热稳定性。分子对接实验预测 UDCA 与 ID1 之间 存在氢键连接。 4. 在对抗肝纤维化作用方面，UDCA 改善 Id1 未敲低组中小鼠的肝体重比； 改善血清生化指标；降低 F4/80 表达；抑制肝星型细胞活化基因(Acta2，Coll1a1， Loxl2)的表达；抑制 α-SMA 表达。在 Id1 敲低组中，UDCA 对纤维化的改善作用 被抑制。在促进肝再生方面，UDCA 促进 Id1 未敲低组中肝细胞进入细胞周期； 增加 Ki67 表达；抑制肝细胞凋亡；增加细胞周期蛋白基因的表达；降低细胞周 期抑制因子的基因表达；促进 ID1，WNT2，HGF，磷酸化 c-MET 以及磷酸化 GSK-3β 蛋白表达。在 Id1 敲低组中，UDCA 对再生与 ID1-WNT2/HGF 信号通路 的促进作用被抑制。 3 结论 1. UDCA 在 BDL 小鼠模型中具有显著的抗肝纤维化作用； 2. UDCA 可通过激活 ID1-WNT2/HGF 信号通路增强 PH 小鼠中的肝再生； 3. 通过热稳定实验发现，UDCA 能够增强 ID1 的热稳定性，提示 UDCA 可 能与 ID1 存在相互作用； 4. 通过 Id1 敲低模型，确认 UDCA 通过促进肝脏再生对抗 BDL 模型中的肝 纤维化，其分子机制为活化 ID1-WNT2/HGF 信号通路。 综上所述，本研究发现 UDCA 的新作用机制为通过激活 ID1-WNT2/HGF 信 号通路促进肝再生，对抗 BDL 造成的肝纤维化。 

Background Liver fibrosis refers to a pathological state in which the injury part of liver is replaced by scars. Liver fibrosis can cause liver function damage, and even develop into cirrhosis, leading to liver failure. Chronic liver diseases accounts for cirrhosis, such as non-alcoholic fatty liver disease (NAFLD) etc. Liver possesses a remarkable regeneration function, it can rebuild the original structure and function after tissue damage without leaving scars. Therefore, the liver recovers from various injuries. When the liver suffers from chronic damage, such as various chronic liver diseases, its regenerative capacity is usually impaired, and it is not enough to fulfill self-repair. Therefore, another mechanism (wound healing) began to participate in the repair of chronic injuries. In this case, hepatic stellate cells will be activated and transdifferentiate into myofibroblast-like cells, resulting in increased secretion of extracellular matrix protein (ECM), and excessive deposition of ECM protein can cause tissue fibrosis. Promoting liver regeneration is believed to alleviate liver fibrosis. In the partial hepatectomy (PH) model for studying liver regeneration, the liver can recover its mass and function even 70% of liver is removed, which is considered as a consequence of unaffected liver regeneration capacity. The activation of ID1-WNT2/HGF pathway is also believed to inhibite liver fibrosis by promoting hepatocytes regeneration. Bear gall is a traditional Chinese medicine in China. It is used for syndromes such as fever, epilepsy, and eclampsia. Ursodeoxycholic acid (UDCA) is one of the main active ingredients of bear gall, and it is also a hydrolysate of tauroursodeoxycholic acid. Previous studies have shown that UDCA can improve the biochemical indicators, delay histological progress, and improve survival in primary biliary cholangitis patients. However, the mechanism of UDCA has not yet been fully elucidated. Objective Through the use of bile duct ligation (BDL) model, PH model, Id1 knockdown model and in vitro cell model, this research explores the relationship between Id1, liver regeneration and UDCA's anti-hepatic fibrosis effect, and explore the effect of UDCA on the treatment of liver fibrosis. Mechanism. Methods First, we use blood biochemistry indicators, histological staining, type I collagen expression, F4/80 expression, hepatic stellate cell activation genes (Acta2, Coll1a1, Loxl2) and Id1 gene expression, α-SMA expression and so on to evaluate the protective effect of UDCA on BDL-induced liver fibrosis. Second, we use blood biochemistry indicators to assess UDCA’s protection on liver hepatectomy. With use cell cycle assay, TUNEL assay, Ki67 stain, cell cycle-related gene expression, we evaluate the effects of UDCA on liver regeneration. We detect the expression of ID1, WNT2, HGF, phosphorylated c-MET and phosphorylated GSK-3β proteins to explore the activation of ID1-WNT2/HGF pathway by UDCA. Third, the cell viability experiment was used to determine the optimal concentration of UDCA to promote the proliferation of HepG2 cells. The effects of UDCA on the expression of ID1 gene and protein at this concentration are then detected by qPCR and Western blot. Using cellular thermal shift assay and molecular docking experiments, we study the interaction between UDCA and ID1 protein. At last, serum ALT, AST, ALP, TBil levels, Masson staining, F4/80 expression, hepatic stellate cell activation genes (Acta2, Coll1a1, Loxl2) expression, α-SMA expression are used to explore the effect of Id1 knockdown on anti-fibrosis effects of UDCA. Then, cell cycle assay, Ki67 expression, TUNEL assay, cell cycle-related gene expression, ID1, WNT2, HGF, and phosphorylation of c-MET and GSK-3β are used to explore the effects of Id1 knockdown on UDCA's promotion of liver regeneration. Results 1. UDCA improves the liver to body weight ratio of the BDL model, reduce the levels of ALT, AST, TBil and ALP, improve liver histology, relieve inflammation, and reduce the expression of hepatic stellate cell activation genes (Acta2, Coll1a1, Loxl2). In addition, UDCA significantly increases the expression of Id1 gene in BDL liver. 2. UDCA reduces the levels of ALT and AST in the PH model, promotes hepatocytes in the PH model to enter the cell cycle, increases the expression of Ki67, inhibits hepatocyte apoptosis, increases the expression of cyclin genes, inhibits the gene expression of cell cycle inhibitors. UDCA promotes the expression of Id1, Wnt2, Hgf genes and proteins in the PH model, and promotes phosphorylations of c-MET and GSK-3β. 3. UDCA promote the proliferation of HepG2 cells, increase the expression of ID1 gene and protein. UDCA can also enhance the thermal stability of ID1. Molecular docking experiments predicted the interaction model between UDCA and ID1. 4. UDCA improves the liver coefficient of BDL mice in knockdown control group, reduces serum ALT, AST, TBil and ALP levels, reduces F4/80 expression, inhibits hepatic stellate cell activation genes (Acta2, Coll1a1, Loxl2) expressions, inhibits α-SMA expression. Id1 knockdown inhibits the improving effect of UDCA on fibrosis. UDCA promotes liver regeneration by promoting cell cycle entry, increasing Ki67 expression, inhibiting hepatocyte apoptosis, increaseing cyclin gene expression, inhibiting cell cycle inhibitor genes expression, promoting ID1, WNT2, HGF, and phosphorylation c-MET and phosphorylated GSK-3β protein expression. Conclusion Our Research indicates: 1. UDCA has significant anti-liver fibrosis effect in BDL mice model; 2. UDCA can significantly enhance liver regeneration in PH mice, and play a role by activating the ID1-WNT2/HGF signaling pathway; 3. Through thermal stability experiments, it is found that UDCA can enhance the thermal stability of ID1, suggesting that UDCA may interact with ID1; 4. Through the Id1 knockdown model, confirm that UDCA can fight liver fibrosis in the BDL model by promoting liver regeneration, and its molecular mechanism is to activate the ID1-WNT2/HGF signaling pathway. In summary, our study found that the new mechanism of UDCA is to promote liver regeneration by activating the ID1-WNT2/HGF signaling pathway, and to combat liver fibrosis caused by BDL.