基于CRISPR/cas9系统分析化橘红防治酒精性脂肪肝的生物靶标与物质基础

Alcoholic liver disease is a common healthcare problem, Traditional Chinese herb Huajuhong(Exocarpium Citri Grandis, ECG) has a unique role in the prevention of alcoholic liver disease, however, its effective active ingredient and related target mechanism still not fully understood. Base on our previous studies, we raise the hypothesis that Caveolin-1 is an critical biological target in alcoholic fatty liver injury, and intend to investigate that Caveolin-1 as an alcohol-induced fatty liver central regulator associated with reactive nitrogen stress, lipid metabolism and inflammation. In the study, alcoholic hepatocyte steatosis model and alcoholic fatty liver zebrafish model will be used to establish the quick verification analysis platform for ECG active ingredients efficacy evaluation and Caveolin-1 biological role in alcoholic fatty liver injury in vitro and in vivo. Moreover, CRISPR/Cas9 technologies and Lentiviral will be used to mutate Caveolin-1 gene for identification and screening of the Caveolin-1 signal pathway and ECG active ingredients biological targets. Simultaneously, combined with Caveolin-1 knockout mice, to further verification the detail endpoints of Caveolin-1 and the resistant target of ECG active ingredients in the alcoholic hepatic steatosis. Finally, the underlying mechanism of Caveolin-1 and ECG biological effects in alcoholic fatty liver disease will be clearly elucidated in vivo and in vitro. Findings from the study will generate the potential values of ECG as a further-developed new drugs for alcoholic liver disease, and Caveolin-1, a novel target protein for Traditional Chinese Medicine drug discovery, which will open a new therapeutic window for liver diseases.

酒精性肝病严重威胁人类的健康，传统中药化橘红具有独特的防治酒精性肝病作用，然而其有效活性成分和药物靶标机制至今尚不完全清楚。基于前期研究基础，我们提出“Caveolin-1是酒精性脂肪肝病的关键生物调控靶标”的假说，并拟以Caveolin-1作为酒精诱导的脂肪肝形成的调控“中心”，以其介导的活性氮自由基应激以及脂质代谢作为切入点，利用体外肝细胞模型、体内斑马鱼模型，建立化橘红有效物质快速验证分析平台；并通过第三代基因编辑技术(CRISPR/cas9)结合慢病毒体系修饰Caveolin-1基因进行靶标调控机制探索及药物靶向性分析，最后结合正常野生型和Caveolin-1基因敲除小鼠模型进一步验证，明确化橘红防治酒精性脂肪肝的活性物质基础及Caveolin-1靶标调控机制。本研究的开展将为进一步开发作用机制清楚、靶点明确的新型抗酒精性肝病中药奠定基础。

酒精性肝病严重威胁人类的健康，传统中药化橘红具有独特的防治酒精性肝病作用，然而其有效活性成分和药物靶标机制至今尚不完全清楚。基于前期研究基础，我们提出“Caveolin-1是酒精性脂肪肝病的关键生物调控靶标”的假说，并以Caveolin-1作为酒精诱导的脂肪肝形成的调控“中心”，以其介导的活性氮自由基应激以及脂质代谢作为研究切入点。利用斑马鱼酒精性脂肪肝模型，建立化橘红有效物质快速验证分析平台，并成功筛选出三种有效活性成分：柚皮素、柚皮苷、橙皮苷，初步确定了化橘红抗酒精性脂肪肝的物质基础。同时我们通过第三代基因编辑技术(CRISPR/cas9)体系对斑马鱼进行Caveolin-1基因修饰，成功构建了Caveolin-1基因突变体斑马鱼；应用慢病毒进行L02细胞Caveolin-1基因修饰并开展体外脂肪肝靶标调控机制探索及分析，结合正常野生型和Caveolin-1基因敲除小鼠模型进一步验证，最终明确了Caveolin-1能够通过保护酒精诱导的肝细胞损伤、对抗氮自由基应激及脂质代谢异常，进而发挥对酒精性脂肪肝的关键调控作用，同时阐明了化橘红活性物质柚皮苷靶向Caveolin-1防治酒精性脂肪肝的作用机制。本项目研究结果有助于岭南中药化橘红保肝降脂疗效和机制的进一步认知，并促进其深度开发及临床有效应用，为开发作用机制清楚、靶点明确的新型抗酒精性肝病中药奠定基础。