沙棘多糖协同益生菌对NAFLD中TLR和NLRP3介导的炎症反应的抑制作用研究

Nonalcoholic fatty liver disease (NAFLD) is a worldwide concern of public health, there is currently no effective therapeutic drugs. NRLP3 inflammasome plays an important role in the incidence and progression of NAFLD, but the role of the extrahepatic NLRP3, in particular intestinal NLRP3, is less understood. In previous studies, we found that seabuckthorn polysaccharides and probiotics L. Casei Zhang have anti-inflammatory and anti-oxidation function. Both of them protect the liver injury induced by LPS/D-GalN via regulating the TLR4 signaling pathways. We hypothesized that the combination of seabuckthorn polysaccharides and probiotic L. casei Zhang have protective effect against NAFLD. This project adopts in vitro and in vivo studies. Using high fat diet induced NAFLD model to study the combined effects of seabuckthorn polysaccharides and probiotics against TLR and NLRP3-mediated inflammatory reaction in liver and intestine. The change of its downstream signaling pathways will be studied, too. The change of the intestinal flora will be studied by high-throughout sequencing method. Using microarray analysis and protein analysis to clarify the network regulation on the TLR and NLRP3 signaling pathways. This study will not only help to elucidate the pathogenesis of NAFLD, but also help to develop high efficient and low toxic drugs.

非酒精性脂肪肝病（NAFLD）是世界性关注的公共卫生问题，目前尚无有效治疗药物。NLRP3炎性小体在NAFLD发病和进展中扮演着重要角色，然而对于肝外尤其是肠道NLRP3炎性小体的作用知之甚少。我们以前的研究发现，沙棘多糖和益生菌L.casei Zhang都具有抗炎和抗氧化功能，通过调控TLR4信号通路保护LPS/D-GalN诱导的肝损伤，我们推测二者联合对NAFLD会有保护作用。本项目采用体外和体内研究相结合。拟采用高脂饮食诱导NAFLD，研究沙棘多糖联合益生菌后对大鼠肝脏和肠道TLR和NLRP3介导的炎性反应的调控，研究其下游信号通路的变化。高通量测序的方法研究肠道菌群的变化，基因芯片辅助蛋白分析来阐明沙棘多糖联合益生菌对TLR和NLRP3信号通路的网络调控。本研究不仅有助于阐明NAFLD的发病机理，而且为开发高效低毒治疗药物奠定基础。

肝脏炎症反应和氧化应激不仅是NAFLD 发生和发展的始动因素，还贯穿了NAFLD 整个病变过程。我们在之前的研究中表明，沙棘多糖和益生菌L.Casei Zhang都具有抗炎和抗氧化效果，我们推测二者单独或者联合使用都能够预防NAFLD的进展。本项目利用高脂饮食诱导大鼠非酒精性肝病构建实验模型，研究沙棘多糖（Seabuckthorn polysaccharide，SP）及益生菌Lactobacillus casei Zhang（Lcz）对NAFLD的保护作用及其机制。将6-8周的雄性Wistar 大鼠随机分为两组，即空白对照组（n=10）和的高脂饮食模型组（HFD）（n=45）。6周后确定造模成功后，将模型组随机分为5组（n=8），即高脂饮食模型组；阳性药物组：黄连素50mg/kg灌胃；沙棘多糖组：SP 200mg/kg灌胃；益生菌组：Lcz 1*109CFU/ml灌胃；沙棘多糖和益生菌联合作用组：200mg/kg SP及1*109CFU/ml Lcz混合灌胃（HFD+SP+Lcz），同时将高脂饲料换成普通维持饲料。连续灌胃4周后处死大鼠收集血清、肝脏和粪便样本用以后续实验的进行。检测血清中肝功以及血糖血脂指标；HE染色观察肝脏组织的脂肪变性情况；检测细胞因子TNF-α、IL-6、IL-1β在血清中和肝脏中的表达。Western blot法检测肝组织中TLR4、NLRP3、ASC、IL-1β、MAPKs在蛋白水平的表达含量；通过16S rRNA技术分析大鼠粪便中菌群组份的变化情况。结果表明，沙棘多糖和益生菌都抑制了HFD大鼠血清中ALT、AST水平的升高；降低血清中血糖、MDA、SOD和NO的含量； 沙棘多糖和益生菌组中脂肪变性程度显著降低；血清中炎性因子表达降低。沙棘多糖和益生菌能够抑制肝脏组织中TLR4、NLRP3和IL-1β在蛋白水平的表达，并且能够降低MAPKs在蛋白水平的磷酸化。与模型组相比，沙棘多糖和益生菌作用后Clostridia、Firmicutes、Ruminococcaceae的相对丰度分别在门、纲、目、科、属、种水平上显著升高，相反的是，Bacteroidaceae与Prevotellaceae的相对丰度在沙棘多糖和益生菌作用后显著低于模型组。结果证明SP和Lcz可能通过抗炎、抗氧化、调节糖脂代谢，改变菌群成分等途径对NAFLD起到保护作用。此外