~非酒精性脂肪肝病（Nonalcoholic fatty liver disease，NAFLD）是一种慢性进展性肝病，包括以脂质沉积为主要特征的脂肪肝（Nonalcoholic fatty liver，NAFL）和以炎症为主要特征的非酒精性脂肪肝炎（Nonalcoholic steatohepatitis，NASH），并可进展为终末期肝病，如肝硬化、肝癌，给社会造成很大的医疗和经济压力。目前，全球大约25.2%的人口受NAFLD影响。然而，尚未有批准用于临床的治疗性药物。近年来，不少单靶点药物陆续进入临床试验阶段。由于对NAFLD复杂性预估不足，除了法尼酯X受体（Farnesoid X receptor，FXR）激动剂奥贝胆酸（Obecholic acid，OCA）以23%的有效率勉强达到Ⅲ期临床试验治疗终点，其他Ⅲ期临床试验全部失败。因此，不少学者将目光转向多靶点药物的联合应用上，以期找到干预甚至逆转NAFLD的治疗策略。双环醇（Bicyclol，Bic）是中国医学科学院药物研究所开发的、具有我国自主知识产权的创新药物，临床用于各种原因导致的肝损伤。临床荟萃分析显示，Bic可用于NAFLD的治疗。小檗碱（Berberine，BBR）是从中药黄连中提取出来的异喹啉季铵盐，临床主要用于腹泻和肠道感染。临床前的研究显示，BBR具有降脂、降糖、抗炎、抗NAFLD等多种药理活性。由于Bic和BBR的药理作用范围广，且作用机制不同，本研究探索Bic和BBR联合抗NAFLD，以期找到更好的干预NAFLD的策略和方法。
本研究采用西方饮食（Western diet，WD）和四氯化碳（Carbon tetrachloride，CCl4）注射（WD/CCl4）的方法诱导C57BL/6J小鼠构建NAFLD模型，用于Bic联合BBR治疗实验研究。WD/CCl4诱导4周后，给予 Bic（50、200 mg/kg/d）、BBR（50、200 mg/kg/d）及两者各剂量联合（Bic 50 mg/kg/d+BBR 50 mg/kg/d、Bic 50 mg/kg/d+BBR 200 mg/kg/d、Bic 200 mg/kg/d+BBR 50 mg/kg/d和Bic 200 mg/kg/d+BBR 200 mg/kg/d）处理8周。通过对小鼠体重、血清谷丙转氨酶（Alanine aminotransferase，ALT）和谷草转氨酶（Aspartate aminotransferase，AST）、肝脏甘油三酯（Triglyceride，TG）和胆固醇（Cholesterol，CHO）检测显示，各组小鼠体重没有显著差异；各给药组均能显著降低WD/CCl4导致的血清ALT和AST、肝脏TG和CHO升高。该结果提示Bic、BBR及两者各剂量联合安全性好，均能改善肝损伤和脂代谢异常。H&E、油红O和Masson染色的组织病理学结果显示，WD/CCl4导致小鼠肝脏出现脂肪空泡、气球样变和炎性细胞浸润，并出现脂质和胶原沉积，各给药组均能显著改善组织病理学异常，各剂量联合组的改善作用更显著。提示Bic、BBR及两者各剂量联合治疗NAFLD均能改善组织病理学异常，且Bic联合BBR具有优于Bic和BBR单独应用的效果。
为了评价Bic联合BBR对NAFLD的预防作用，本论文应用WD诱导NAFLD模型。WD诱导C57BL/6J小鼠，同时给予Bic（50、200 mg/kg/d）、BBR（50、200 mg/kg/d）及两者各剂量联合处理，进行Bic联合BBR预防实验研究。16周后，进行血清ALT和AST、肝脏TG和CHO检测，同时进行H&E和油红O染色。结果发现，WD诱导导致小鼠血清ALT和AST、肝脏TG和CHO显著升高，肝脏出现大面积脂肪空泡、炎症和脂质沉积，各给药组均显著改善WD导致的组织病理学损伤和脂质沉积，联合给药组对其的改善作用更好。提示Bic联合BBR治疗NAFLD具有改善代谢异常和组织病理学的作用，且改善的效果优于Bic和BBR单独应用的效果。
接着，本论文探索了Bic和BBR联合防治NAFLD的机制研究。通过Western Blot实验检测肝脏组织脂质分解代谢相关酶发现，WD/CCl4和WD诱导显著下调β氧化调节因子过氧化物酶体增殖物激活受体α（Peroxisome proliferator activated receptor alpha，PPARα）、脂质裂解酶羧酸酯酶2（Carboxylesterase 2，CES2）及PPARα和CES2的上游分子自噬受体蛋白（Sequestosome 1，p62）和核因子红系2相关因子2（Nuclear factor erythroid 2-related factor 2，Nrf2），Bic显著升高PPARα、CES2、p62和Nrf2，BBR对其没有影响且不干扰Bic的作用。提示p62-Nrf2-CES2促进脂质裂解和p62-Nrf2-PPARα增强β氧化是Bic而非BBR缓解NAFLD的机制。本研究进而对肝脏脂质合成代谢相关酶检测发现，WD/CCl4和WD诱导显著上调脂质从头合成关键酶乙酰辅酶A羧化酶（Acetyl-CoA carboxylase，ACC）和脂肪酸合成酶（Fatty acid synthase，FAS）的表达，BBR显著降低模型导致的ACC和FAS升高，Bic对其没有作用且不影响BBR作用的发挥，表明通过下调ACC和FAS的表达抑制脂质从头合成可能是BBR缓解NAFLD的机制之一。本研究通过对治疗实验小鼠肠道微生物进行α多样性分析、聚类分析、主成分分析（Principal component analysis，PCoA）、群落组成分析、丰度分析和京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）分析，结果显示，BBR和联合组的肠道菌群种类和菌群结构均发生了显著变化，主要集中在科和属水平，BBR处理显著增加类杆菌科（Bacteroidaceae）和类杆菌属（Bacteroide）水平，增加的菌群与脂代谢相关，而Bic对此没有显著作用，且不干扰BBR对肠道微生物的调节作用。提示BBR通过调节脂代谢相关肠道微生物发挥抗NAFLD作用。
最后，本研究在NAFLD细胞模型中验证Bic和BBR的联合作用。用0.1 mM游离脂肪酸（Free fatty acid，FFA）诱导HepG2细胞构建NAFLD细胞模型，同时用Bic（0.5、2 μM）、BBR（0.5、2 μM）及两者各剂量联合（Bic 0.5 μM+BBR 0.5 μM、Bic 0.5 μM+BBR 2 μM、Bic 2 μM+BBR 0.5 μM和Bic 2 μM+BBR 2 μM）处理24 h。MTT染色实验显示，在该药物处理条件下对细胞无明显毒性。尼罗红染色实验显示，Bic、BBR和各剂量联合均可降低细胞内脂滴聚集。Western Blot结果发现，Bic联合BBR通过降低ACC和FAS等脂质合成相关蛋白的水平，提高CES2和PPARα等脂质裂解相关蛋白的水平。在细胞水平上显示两者联合可降低脂质合成和加速脂质降解，从而发挥降低细胞内脂滴聚集的作用。
综上，Bic和BBR通过不同的机制显著降低NAFLD相关的肝损伤和脂代谢异常，Bic和BBR联合具有较单独应用更好的治疗和预防NAFLD的作用。Bic联合BBR通过不同的机制发挥抗NAFLD作用，Bic通过p62-Nrf2-CES2/PPARα信号通路促进脂质裂解和氧化，BBR则通过ACC和FAS抑制脂质从头合成和调节肠道微生物发挥抗NAFLD的作用，两者联合互不影响。Bic联合BBR用药研究结果为临床抗NAFLD提供一种可能的用药选择，也为后续抗NAFLD的药物研发提供策略和方向。

~Nonalcoholic fatty liver disease (NAFLD) is a chronic progressive liver disease, which includes nonalcoholic fatty liver (NAFL) mainly characterized by lipid deposition and nonalcoholic steatohepatitis (NASH) markedly by inflammation. It can progress to end-stage liver disease, such as cirrhosis and hepatocellular carcinoma, which causes great medical and economic pressure to the society. At present, about 25.2% of the global population is affected by NAFLD. However, no therapeutic drugs have been approved in clinic. In recent years, many single-target drugs have entered clinical trials with different stages. Due to the insufficient prediction of the complexity of NAFLD, all phase III clinical trials failed except that the farnesoid X receptor (FXR) agonist obecholic acid (OCA) narrowly reached the end point of treatment in phase III clinical trial with effective rate of 23%. Therefore, many scholars turn their attention to the combined application with multi-target drugs in order to find a treatment strategy to intervene or even reverse NAFLD. Bicyclol (Bic) is an innovative drug with independent intellectual property rights developed by the Institute of Pharmacy, Chinese Academy of Medical Sciences. It is clinically used for liver injury caused by various causes. Patients meta-analysis showed that Bic could be used in the treatment of NAFLD. Berberine (BBR) is an isoquinoline quaternary ammonium salt extracted from coptis chinensis. It is mainly used in diarrhea and intestinal infection in clinic. Preclinical studies have shown that BBR has many pharmacological activities, such as lipid-lowering, hypoglycemic, anti-inflammatory, anti-NAFLD. Considering the wide range of pharmacological effects and different mechanisms of Bic and BBR, we combined Bic and BBR in the study of resisting NAFLD in order to find a better strategy and method to intervene NAFLD.
In this study, C57BL/6J mice were induced to construct NAFLD model by western diet (WD) and carbon tetrachloride (CCl4) injection (WD/CCl4). Four weeks after WD/CCl4 induction, Bic (50, 200 mg/kg/d), BBR (50, 200 mg/kg/d) and their combinations (Bic 50 mg/kg/d+BBR 50 mg/kg/d, Bic 50 mg/kg/d+BBR 200 mg/kg/d, Bic 200 mg/kg/d+BBR 50 mg/kg/d and Bic 200 mg/kg/d+BBR 200 mg/kg/d) were given for 8 weeks. Body weight, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), liver triglyceride (TG) and cholesterol (CHO) were detected. The results showed that there was no significant difference in the weight of mice in each group; All treatment groups could significantly reduced the increase of serum ALT and AST, liver TG and CHO caused by WD/CCl4. This suggested that Bic, BBR and their combinations were safe and improved liver injury and abnormal lipid metabolism. The histopathological results of H&E, oil red O and Masson staining showed that WD/CCl4 led to fat vacuoles, balloon-like degeneration, inflammatory cell infiltration, lipid and collagen deposition in the livers of mice. Each drug administration group could significantly improve the histopathological abnormalities, and the improvement in combinations was more significant than Bic or BBR applied alone. It indicated that Bic, BBR and their combinations can improve histopathological abnormalities, and the effect of Bic combined with BBR was better than that of Bic or BBR alone.
In order to evaluate the preventive effect of the combination with Bic and BBR on NAFLD, WD induced NAFLD model was used in this study. C57BL/6J mice were induced by WD and treated with Bic (50, 200 mg/kg/d), BBR (50, 200 mg/kg/d) and their combinations respectively. After 16 weeks, serum ALT and AST, liver TG and CHO were detected, and H&E and oil red O staining were performed. The results showed that WD treatment resulted in a significant increase in serum ALT and AST, liver TG and CHO, and a large area of fat vacuoles, inflammation and lipid deposition in the liver. Each drug treated group significantly improved the histopathological damage and lipid deposition caused by WD, and the combined administration group had a better effect on it. It suggested that Bic combined with BBR can improve metabolic abnormalities and histopathology, and the improvement effect was better than that of Bic or BBR alone.
Next, the mechanisms of the combination with Bic and BBR in prevention and therapy of NAFLD was explored. The enzymes related to lipid catabolism in liver were detected by Western blot. It was found that β oxidation regulator peroxisome proliferator activated receptor alpha (PPARα), lipolysis enzyme carboxylesterase 2 (CES2) and the upstream of PPARα and CES2, autophagy receptor protein (p62) and nuclear factor erythroid 2-related factor 2 (Nrf2) were significantly down regulated in WD/CCl4 and WD treatment groups, Bic significantly increased the protein level of PPARα、CES2, p62 and Nrf2, BBR had no significant effect on them and did not interfere Bic. It suggested that p62-Nrf2-CES2 promoting lipolysis and p62-Nrf2-PPARα enhancing β oxidation were the mechanisms of Bic rather than BBR alleviating NAFLD. The enzymes related to lipid anabolism were detected further and found that WD/CCl4 and WD treatment significantly increased the key enzymes of de novo lipogenesis, acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). BBR significantly decreased the increasing of ACC and FAS, and Bic had no significant effect on them and did not affect the function of BBR. It suggested that inhibiting lipogenesis by down-regulating the expression of ACC and FAS may be one of the mechanisms of BBR alleviating NAFLD. Through the analysis of intestinal microorganisms in theraputic experiment mice by α diversity analysis, cluster analysis, principal component analysis (PCoA), community composition analysis, abundance analysis and kyoto encyclopedia of genes and genomes (KEGG) analysis showed that there were significant changes in the species and structures of intestinal flora in BBR and the combination groups, mainly at the family and genus levels. BBR treatment significantly increased the levels of Bacteroideae and Bacteroides, and the increased flora was related to lipid metabolism, while Bic had no significant effect on this, and did not interfere with the regulation of BBR. It estimated that BBR resisted NAFLD by regulating intestinal microorganisms related to lipid metabolism.
Finally, this study verified the combined effect of Bic and BBR in NAFLD cell model. HepG2 cells with 0.1 mM FFA were induced to construct NAFLD cellular model, and Bic (0.5, 2 μM）、BBR（0.5, 2 μM) and the combinations (Bic 0.5 μM+BBR 0.5 μM, Bic 0.5 μM+BBR 2 μM, Bic 2 μM+BBR 0.5 μM and Bic 2 μM+BBR 2 μM) were treated for 24 h. MTT assay showed that there was no significant toxicity under this condition. Nile red staining showed that Bic, BBR and the combinations could reduce the accumulation of intracellular lipid droplets. Western blot showed that the combination with Bic and BBR reduced the protein levels related to lipid synthesis such as ACC and FAS, and elevating lipolysis proteins of CES2 and PPARα. At the cellular level, the combination of Bic and BBR reduced intracellular lipid by decreasing lipogenesis and accelerating lipid degradation.
In conclusion, Bic and BBR significantly reduced NAFLD related liver injury and abnormal lipid metabolism through different mechanisms. The combination of Bic and BBR had a better effect on the therapy and prevention of NAFLD than single application. The combination with Bic and BBR defended against NAFLD through different mechanisms. Bic resisted NAFLD through p62-Nrf2-CES2/PPARα signal pathway promoting lipolysis and oxidation, while BBR inhibited de novo lipogenesis through ACC and FAS and regulated intestinal microorganisms. The combination of Bic and BBR had no effect on each other. The research results of Bic combined with BBR not only provided a possible drug choice for clinic against NAFLD, but also offered strategy and direction for subsequent drug research and development against NAFLD.