L001治疗非酒精性脂肪性肝病的功效及机制探索

Hepatic steatosis poses a serious threat to public health. Hepatic steatosis is currently one of the leading causes of liver cirrhosis and a prevalent form of liver dysfunction, being second only to viral hepatitis. Studies have also shown that hepatic steatosis significantly raises the risk for liver failure, diabetes, as well as cancer. Dysregulation in lipid metabolism represents a hallmark symptom of hepatic steatosis. An in-depth investigation into the biochemical perturbations during the process of steatosis in hepatocytes from a cellular and molecular perspective, and the corresponding corrective measures that can revert this process, would confer critical insights to a better understanding of the pathophysiology of liver steatosis. Furthermore, research in this area would undoubtedly facilitate early disease diagnosis and the selection of specific treatment regimes. The applicant has validated using animal model and cell culture that the clinical drug L001 could mitigate aberrant lipid accumulation in liver tissues and hepatocytes, respectively. Preliminary data have demonstrated that cholesterol treatment to hepatocytes or a diet with elevated cholesterols in animals leads to enhanced expression of membrane lipid-microdomains. Remarkably, treatment with L001 at optimal concentration can notably suppress the cholesterol-induced overexpression of membrane lipid microdomains. The proposed study aims to systematically analyse the global lipidomes of liver tissues and hepatocytes during progression of hepatic steatosis and its corresponding treatment by L001, and to decipher relevant key factors in disease pathophysiology via extensive comparative lipidomic analysis, so as to conduct in-depth functional and expression analyses of the identified factors. The study will integrate and harness skills from bioinformatics, genetics, molecular and cellular biology together with biochemical methods to synergistically elucidate corrective mechanism(s) that could lead to alleviation of liver steatosis.

脂肪性肝病正严重威胁国人的健康，是仅次于病毒性肝炎的第二大肝病和隐蔽性肝硬化的常见原因。脂肪肝增加了肝功能衰竭、糖尿病和癌症的风险。脂代谢异常是脂肪肝的典型特征之一，从细胞分子水平深入探索肝细胞脂肪化过程的生化变化及探索有效治疗方案对于了解脂肪肝发病机理、进行早期诊断、选取针对性的治疗方案有着重大意义。 申请人分别在动物实验及肝细胞实验中发现临床用药L001能改善肝组织损伤和减少肝细胞中的脂肪沉积。 前期的实验数据还显示胆固醇处理或高胆固醇饮食导致质膜脂微结构域显著过表达，而L001处理显著地抑制了胆固醇引入的质膜脂微结构域过表达。 本申请拟通过系统分析肝组织脂肪化过程中的肝组织和肝细胞的全脂谱图，计划在大规模脂组学数据对比分析的基础上，选取相关的关键因子，对它们进行深入的功能分析和表达调控的研究。集合生物信息学、遗传、分子和细胞生物学、生物化学手段，剖析L001改善脂肪肝的机制。

非酒精性脂肪肝(NAFLD)代表了在无酒精滥用情况下与肝脏内脂肪变性相关的一系列肝脏疾病。肝脏内胆固醇的积累被认为是非酒精性脂肪肝的主要表征，并同疾病的严重程度密切相关。在本研究中我们使用高胆固醇饮食(HCD)诱导小鼠产生非酒精性脂肪肝作为非酒精性脂肪肝研究的主要模型，辅以对HepG2细胞及小鼠肝原代细胞进行胆固醇处理建立的细胞模型。利用脂质组学结合分子生物学和细胞生物学手段对L001缓解非酒精性脂肪肝的作用进行了探究，通过组织染色和血浆转氨酶活性分析等手段，我们发现L001可以有效减少高胆固醇喂食小鼠肝脏内的脂质积累和肝脏损伤。蛋白免疫印迹也显示L001可以有效降低参与非酒精性脂肪肝中脂肪酸和胆固醇合成与吸收的相关蛋白的表达水平。同位素标记油酸和胆固醇吸收实验证实了L001在减少肝细胞对脂肪酸和胆固醇吸收上的作用。结合蛋白免疫印迹和同位素标记油酸和胆固醇吸收实验的结果，推断CD36可能同时参与了肝细胞对脂肪酸和胆固醇的吸收。深入的脂质组学分析发现，脂肪变性的主要成分中性脂的变化可能同L001引起的定位于细胞膜微区上的鞘糖脂GM3的变化有关。在HepG2细胞中对GM3合酶进行敲降可以减少胆固醇处理所引起的脂肪酸和胆固醇吸收的增加，以及CD36蛋白表达水平的减少。进一步在HepG2细胞中对CD36进行敲降可以减少胆固醇处理所引起的胆固醇吸收增加，表明其同时参与了肝细胞对胆固醇的吸收。这些发现表明L001可以通过影响肝脏细胞微区相关的脂质和蛋白含量来调控脂肪酸和胆固醇的吸收，从而改善非酒精性脂肪肝中肝细胞内的脂肪积累。凋亡相关的脂质和蛋白的研究结果揭示L001对脂肪积累的改善进一步减少了肝细胞的凋亡。在联合治疗策略(DFO+饮食控制)下，L001能够在饮食控制策略的基础上进一步改善非酒精性脂肪肝中失调的脂质变化。综上，该研究揭示了L001对非酒精性脂肪肝的治疗作用，指示细胞膜微区作为其改善肝脏细胞脂质积累的潜在靶点，为进一步认识和治疗非酒精性脂肪肝提供了新的方法和思路。