基于肠道菌群探讨黄精水提物改善高脂高糖饮食所致大鼠认知功能损伤的作用机制

Action Mechanism of Aqueous Extract of Polygonatum sibiricum in Improving Cognitive Function Impairment Induced by High-Fat and High-Sugar Rats Based on Gut Microbiota

目的:研究黄精水提物通过靶向肠道菌群对高脂高糖大鼠认知功能损伤改善的作用机制。方法:40只SPF级SD雄性大鼠随机分为正常对照组(Control组)、模型组(Model组)、黄精水提物低剂量组(HJL组)和黄精水提物高剂量组(HJH组),每组10只。除正常对照组外,对其余大鼠通过摄食高脂高糖饲料建立认知功能损伤模型。建模成功后,Control组和Model组大鼠给予生理盐水灌胃,HJL、HJH组大鼠分别给予1、1.5 g/kg黄精水提物灌胃,共干预6周,监测体质量变化。治疗结束后采用Morris水迷宫(MWM)评估大鼠认知功能情况,测定血清中葡萄糖(GLU)及总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)水平。苏木精-伊红染色观察肠道形态学变化,肠道粪便使用16s RNA测序分析菌群多样性变化。ELISA法检测血清及粪便中脂多糖(LPS)水平,透射电镜下观察海马区超微结构改变。WesternBlot法检测海马中Toll样受体4(TLR4)、磷酸化核因子κBp65(NF-κBp65)、含NLR家族PYRIN域蛋白3(NOD-,LRR-andpyrindomain-containing 3,NLRP3)和胱冬肽酶-1(Caspase-1)炎症相关蛋白表达。结果:与Model组相比,水迷宫实验中HJH组大鼠潜伏期明显缩短,穿越平台次数明显增加(P<0.05)。黄精水提物能有效调控高脂高糖大鼠体质量(P<0.01),降低血糖及血脂水平(GLU、TC、TG、LDL-C)(P均<0.05)。HE染色显示,黄精水提物可以减少结肠组织炎细胞浸润,抑制肠腺萎缩。16s RNA测序分析提示黄精水提物可提高拟杆菌门丰度,逆转厚壁菌门(Firmicutes)/拟杆菌门(Bacteroides)比值,对肠道菌群产生的内毒素脂多糖(LPS)具有抑制作用。同时,电镜下观察黄精水提物改善海马组织神经细胞病理损害情况显示,海马内TLR4、NF-κBp65、NLRP3蛋白表达均降低。结论:黄精水提物对高脂高糖饮食所致大鼠认知功能损伤具有改善作用,其作用机制可能是通过靶向肠道菌群,重塑肠壁黏膜屏障,降低内毒素表达,抑制脑内TLR4/NF-κB炎症信号通路,从而改善认知功能损伤。

Objective:To investigate the mechanism of aqueous extract of Polygonatum sibiricum on improving cognitive function induced by high-fat and high-sugar in rats based on targeting gut microbiota.Methods:40 SPF SD rats were randomly divided into the control group,the model group,HJL group and HJH group,with 10 rats in each group.The cognitive function injury model was established by high-fat and high sugar diet.The control group and the model group were given intragastric administration of normal saline,HJL group and HJH group were treated with aqueous extract of Polygonatum sibiricum at the dose of 1g/kg and 1.5g/kg,respectively.After 6 weeks of intervention,the changes of body mass were monitored.The cognitive function was detected by behavioral tests.The serum levels of GLU,TC,TG,LDL-C and HDL-C were determined.The intestinal morphology was observed by HE staining.The bacterial diversity was analyzed by 16sRNA sequencing in intestinal feces,The serum and feces levels of LPS were detected by ELISA,and ultrastructural changes of hippocampus were observed under transmission electron microscope.The protein expressions of TLR4,NF-κB-p65,NLRP3 and Caspase-1 were detected by Western Blot method.Results:Compared with those in the model group,the incubation period was significantly shortened and the number of crossing platform increased significantly in HJH group(P<0.05).Aqueous extract of Polygonatum sibiricum effectively regulated body mass of rats with high-fat and high-glucose(P<0.01),and it decreased the levels of blood glucose and blood lipid of GLU,TC,TG and LDL-C(P<0.05).HE staining showed that aqueous extract of Polygonatum sibiricum could reduce inflammatory cell infiltration of colon tissues and inhibit intestinal gland atrophy.16sRNA suggested that aqueous extract of Polygonatum sibiricum could increase the abundance of Bacteroides,reverse the ratio of Firmicutes/Bacteroides,and inhibit the production of endotoxin lipolysaccharides(LPS).At the same time,electron microscopy observation showed that the pathological damage of nerve cells in hippocampal tissues was improved and the protein expressions of TLR4,NF-κB-p65 and NLRP3 in hippocampal tissues were decreased by aqueous extract of Polygonatum sibiricum.Conclusion:The aqueous extract of Polygonatum sibiricum can ameliorate the cognitive impairment of rats with high-fat and high-sugar by targeting intestinal flora,which mechanism may be related to reshaping the intestinal wall mucosal barrier,reducing the expression of endotoxin,inhibiting the TLR4/NF-κB inflammatory signaling pathway in the brain.