分子生物钟调控肠道zonulin的机制及其在NAFLD进展中的作用

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and the leading cause of chronic liver disease in China. Twenty percent of NAFLD individuals develop non-alcoholic steatohepatitis(NASH) associated with cirrhosis, portal hypertension, hepatocellular carcinoma and increased cardiovascular risk, yet causes of progression from NAFLD to NASH remain obscure. . Circadian rhythms refer to physiological processes that occur with a repeating period of approximately 24 h and ensure that internal physiology is synchronized with the external environment. Mounting research evidence demonstrates a significant negative impact of circadian disruption on human health. Shift work, chronic jet lag and sleep disturbances are associated with increased incidence of metabolic syndrome, and consequently result in obesity, type 2 diabetes and dyslipidemia. Recent studies have suggested that circadian disruption is pathological in the progression of NAFLD, but the molecular mechanisms are unclear.. Zonulin is the only physiological mediator to regulate gut permeability reversibly by disassembling intestinal tight junctions. Our previous studies have showed that increased intestinal zonulin is involved in the circadian disruption-induced progression of NAFLD. And zonulin can be regulated by circadian clock nuclear receptors, RORα and Rev-erbα. Based on these, we hypothesized that: circadian disruption , e.g. imbalance between RORα and Rev-erbα, up-regulates intestinal expression of zonulin and increases intestinal permeability, and promotes the progression from NAFLD to NASH. . In the following studies, a series of ROR and Rev-erbα agonists, ROR and Rev-erbα over-expression and siRNA plasmids will be used to investigate the effects of ROR and Rev-erbα on zonulin expression and intestinal epithelial cell permeability in vitro. In order to verify the hypothesis in vivo, we will use animal models of circadian disruption, such as continuous light HFD mice and Rev-erbα KO mice, combined with zonulin antagonist (AT1001) treatment, to investigate the roles of circadian disruption, RORα/Rev-erbα imbalance, and increased expression of intestinal zonulin in the progression of NAFLD. . This project may supply new ideas for prevention the progression from NAFLD to NASH.

生物钟是生物适应环境昼夜周期性变化的一种内在机制，其紊乱严重影响健康。研究表明，生物钟紊乱促进非酒精性脂肪肝病（NAFLD）进展为脂肪性肝炎（NASH），但具体分子机制不明。我们发现：介导肠道通透性的关键因子zonulin与之有关，且zonulin受生物钟关键转录因子RORα和Rev-erbα的调控。推测：分子生物钟紊乱，如RORα/Rev-erbα失衡，引起肠道zonulin表达上调，使肠道通透性增加，从而导致NAFLD进展、NASH发生。本项目通过过表达、siRNA干扰等手段，研究RORα/Rev-erbα对肠道zonulin的调控作用及机制；并利用已建立的、持续光照结合高脂饮食诱导的NAFLD动物模型，结合基因敲除和zonulin拮抗剂等，研究RORα/Rev-erbα失衡与肠道zonulin表达及通透性的关联，明确这一过程在NAFLD进展中的作用。本项目的研究将为NAFLD的防治提供新思路、新视角。

生物钟是生物适应环境昼夜周期性变化的一种内在机制，其紊乱严重影响健康。研究表明，生物钟紊乱促进非酒精性脂肪肝病(NAFLD)进展为脂肪性肝炎(NASH)，但具体机制不明。本研究建立了持续光照改变生物钟的动物模型，研究了持续光照对高脂饮食大鼠肥胖、NAFLD进展等的影响及其相关机制，发现（1）持续光照加重高脂饮食大鼠的肥胖、糖脂代谢紊乱、慢性炎症和胰岛素抵抗等；（2）持续光照导致NAFLD的进展，即引起NASH和肝纤维化加重；（3）该过程与肠道通透性增加、肠道菌群紊乱有关，我们发现，持续光照的高脂饮食大鼠较正常光照的高脂饮食大鼠，zonulin表达增加，结肠组织occludin-1、zonula occluden−1 (ZO-1)表达增加，血清 LPS 和肝组织 LBP mRNA 表达增加，即肠道通透性增加；进一步发现，持续光照的高脂饮食大鼠较正常光照的高脂饮食大鼠其肠道 丁酸球菌（Butyricicoccus）, 梭菌属（Clostridium） 和Turicibacte 菌属减少，结肠内容物丁酸盐下降；（4）该过程与肝脏鞘磷脂代谢失衡有关，我们发现，持续光照的高脂饮食大鼠较正常光照的高脂饮食大鼠，其血浆 sphingosine-1-phosphate 水平下降，肝组织总神经酰胺、神经酰胺d18:0/24:0、神经酰胺d18:1/22:0、神经酰胺 d18:1/24:0和神经酰胺 d18:1/24:1等升高，并与肝细胞凋亡增加有关；（5）体外研究发现，生物钟分子Rev-erbα 可下调zonulin的表达，RORα对zonulin的表达无显著影响。随着人们生活方式的改变，生物钟紊乱（如时差、轮班工作、睡眠时间不规律等）已成为代谢性疾病危险因素中不可忽视的一部分，并且有时也无法避免（如轮班工作的医务人员、安保人员等等），本研究将可能对肥胖、NAFLD乃至代谢综合征的防治提供新思路。