组蛋白甲基化在持续营养过剩引发脂肪肝传代性恶化中的关键作用研究

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of hepatocelluar carcinoma and hepatic cirrhosis in many counties. The number of newly diagnosed NAFLD patients is dramatically increased in China. Our previous study has demonstrated that there are transgenerational exacerbation of NAFLD and downregulation of histone methylation in the liver of the offspring after continuous high fat diet (HFD) feeding. To address whether this downregulation of histone methylation is the key factor that regulates the transgenerational exacerbation of NAFLD in the offspring, high fat and normal diets will give to mice for three generations as we previously reported. In this model of continuous over-nutrition, ChIP-seq will be performed to investigate the genes or signaling pathways that regulated by H3k9me2 and H3k9me1 in the liver of the offspring. The possible roles of other histone modifications involved in this transgenerational effects will also be investigated. To prevent this transgenerational effects, nutrients enriched in methyl-groups will be given to the continuous HFD-fed mice. The effects of these methyl-group enriched nutrients on histone methylation and pathogenesis of NAFLD in the offspring of continuous over-nutrition will be investigated. To manipulate the expression level of G9a, one of the key histone methyltransferases, transgenic G9a mice and liver specific G9a knock-out mice will be created. The histone methylation levels and pathogenesis of NAFLD will be investigated in the offspring of these new mouse lines after continuous HFD- and normal diet-feeding. This proposal will help us to understand the significant role of histone methylation in the transgenerational accumulation of metabolic diseases, including NAFLD. Through these studies, we may find new therapeutic targets to inhibit these transgenerational effects.

非酒精性脂肪肝（NAFLD）是肝硬化和肝癌的主要病因，其发病率在我国急剧上升。申请人前期研究显示在持续营养过剩的条件下，NAFLD病变有显著的传代性恶化效应，并伴随着组蛋白甲基化程度传代性下降。据此申请人提出"在持续营养过剩条件下，组蛋白甲基化修饰积累是引发后代NAFLD传代性恶化的关键"的假说。研究拟采用高脂和正常食物持续喂养的多代小鼠模型，①运用ChIP-seq，在肝脏中研究H3k9me2和H3k9me1调控的传代性变化基因和通路，并探讨其它组蛋白修饰参与NAFLD传代性恶化的可能性；②研究能否通过补充食物中甲基供体来抑制组蛋白甲基化下降，从而改善后代NAFLD的传代性恶化；③研究能否通过改变组蛋白甲基化酶G9a的表达水平来调控后代NAFLD病变进程。本项目将揭示组蛋白甲基化在传代性NAFLD以及相关代谢疾病中的重要生理和病理作用，为预防和治疗该疾病提供理论指导。

随着人类社会进步和生活方式的改善，慢性疾病也随之而来，如肥胖和非酒精性脂肪肝。更严重的是，所影响的人群已从老年人向中、青少年转移。一般，处于怀孕和哺乳期的母亲以及儿童的营养都极其丰富，因此孕期和儿童的营养过剩现象十分普遍。那么代谢疾病，如脂肪肝等，在持续营养过剩的条件下，是否有累积，表观遗传修饰是否在其中起到重要的作用呢？.本项目主要的研究内容是：.（1）高脂和正常食物喂养的多代（F0，F1和F2代）小鼠中代谢疾病的变化程度，以及表观遗传修饰的改变和检测。.（2）在同样的高脂食物喂养条件下，雄性和雌性后代相关生理指标、脂肪肝病变、代谢通路变化的异同。.（3）药物是否能通过调控组蛋白修饰来影响脂肪肝病变。.（4）建立肝脏特异性G9a敲除小鼠，研究调控该组蛋白甲基化酶的表达，对基因水平的影响；进一步研究G9a的缺失是否能影响高脂喂养条件下脂肪肝病变进程，以及LPS刺激下的肝损伤情况。.重要研究结果：.1）在持续高脂食物喂养多代条件下，小鼠的脂肪肝和肥胖传代累积。脂肪肝的传代积累，是由于组蛋白甲基化，尤其是H3K9me2的传代性下降，引起内质网应激反应和脂合成酶的代与代之间积累。而白色脂肪病变的形成与DNA甲基化在炎症基因上的传代性下降引起炎症的持续增加相关。.2）在高脂食物连续刺激下，雄性与雌性后代的肝脏病变没有明显差异，但雄鼠后代的肝脏更容易产生炎症和增殖，而且与脂合成途径持续激活。.3）给予肥胖小鼠熊果酸能显著减轻脂肪肝的形成，同时该药物也可以调控H3K18乙酰化水平。.4）组蛋白甲基化酶G9a可以在体内和体外调控肝脏中胰岛素信号通路，从而改善小鼠血糖水平。肝脏特异性G9a敲除小鼠在正常饮食下，肝脏会积累更多的脂类，同时可加重LPS刺激下的炎症反应，增加肝损伤。. 整个项目执行期间，共发表19篇SCI论文，其中通讯作者16篇。还有4篇SCI文章在投稿和修回中。.总之，本项目揭示了一定的持续营养过剩条件下后代NAFLD病变传代性累积的表观遗传机制，为预防和治疗脂肪肝，特别是青少年脂肪肝提供新思路和理论依据。