解毒降浊颗粒对代谢综合征SD大鼠肠道菌群结构及TLR-4/NF-κB信号通路的影响

Effects of Jiedu-jiangzhuo Granules on Gut Microbiota and TLR4/NF-κB Pathway of Metabolic Syndrome Rats

目的探讨解毒降浊颗粒对代谢综合征(MS)SD大鼠肠道菌群结构及TLR-4/NF-κB信号通路的影响。方法将SD大鼠随机分为空白对照组、模型组、二甲双胍组(0.103 g·kg^-1·d^-1)、受试高剂量组(解毒降浊颗粒2.4 g·kg^-1·d^-1)和受试低剂量组(解毒降浊颗粒1.2 g·kg^-1·d^-1),每组各12只。除空白对照组大鼠外,其余大鼠饲以高脂、高糖饲料,8周后,二甲双胍组和两组受试大鼠分别给予二甲双胍或解毒降浊颗粒灌胃。生化法检测血浆内毒素(LPS)水平及血脂含量,称取肝脏和附睾周围脂肪组织重量。提取粪便总DNA并扩增,文库制备,采用Illumina Miseq高通量测序法分析大鼠肠道菌群结构和多样性。采用Western blot法检测结肠组织中Toll样受体4(TLR4)和核因子-κB(NF-κB)蛋白的表达水平。结果实验结束时,受试高剂量组大鼠体重、血浆LPS、血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白-胆固醇(LDL-C)水平明显低于模型组大鼠,而高密度脂蛋白-胆固醇(HDL-C)水平高于模型组大鼠,但是与空白对照组相比仍然存在统计学差异(P<0.05);上述各指标与二甲双胍组基本一致(P>0.05)。另外,受试高剂量组大鼠肝脏系数(%)和附睾脂肪系数(%)均明显低于模型组(P<0.05);但是与空白对照组和二甲双胍组基本一致(P>0.05)。受试高剂量组大鼠NF-κB、IL-6及TNF-α水平与空白对照组基本一致(P>0.05),但是明显低于模型组、二甲双胍组和受试低剂量组(P<0.05)。经α-多样性分析,受试高剂量组大鼠肠道菌群丰度(Chaol指数)明显高于其他4组(P<0.05),但是5组大鼠肠道菌群复杂度(Shannon指数)比较无统计学差异(P>0.05)。受试高剂量组大鼠肠道菌群中厚壁菌门、变形菌门、放线菌门和软壁菌门比例以及普雷沃菌属、考拉杆菌属比例明显降低,拟杆菌门比例以及帕拉普氏菌属和拟杆菌属比例升高,与模型组相比有统计学差异(P<0.05)。最后,受试高剂量组和受试低剂量组大鼠结肠组织中TLR4和NF-κB蛋白表达量较模型组明显降低,尤其以受试高剂量组降低最为明显(P<0.05)。结论解毒降浊颗粒对代谢综合征大鼠有一定的减肥及降脂作用,并能改善肠道菌群,抑制TLR-4/NF-κB信号通路活化。

OBJECTIVE To explore the effects of Jiedu-jiangzhuo granules on gut microbiota of metabolic syndrome rats and TLR4/NF-κB pathway.METHODS SD rats were randomly divided into normal control group,model group,metformin group,experimental high-dose of Jiedu-jiangzhuo granules 2.4 g·kg^-1·d^-1 group and experimental low-dose of Jiedu-jiangzhuo granules 1.2 g·kg^-1·d^-1 group,with 12 rats in each group.Except for rats in the control group,the other rats were fed with high-fat/sugar/salt diet for 12 weeks.When fed for 8 weeks,the rats in metformin group(0.103 g·kg^-1·d^-1),experimental high-dose group(2.4 g·kg^-1·d^-1)or experimental low-dose group(1.2 g·kg^-1·d^-1)were simultaneously gavaged with metformin or Jiedu-jiangzhuo granules for 4 weeks.Body weight,serum lipid indexes and lipopolysaccharide(LPS)were determined by biochemical method,liver coefficient and epididymal fat tissue coefficient of rats were detected.Total genomic DNA of gut microbiota was extracted from fecal samples,then DNA library was built.Community structures of fecal microbes andα-diversity were sequenced on the Illumina Miseq platform.RESULTS At the end of experiment,the body weight,LPS,serum total cholesterol(TC),triglyceride(TG),low density lipoprotein-cholesterol(LDL-C)and fasting blood glucose(FBG)of rats in experimental high-dose group were lower than those in the model group and higher than those in the control group(P<0.05),and HDL-C was also higher in experimental high-dose group than that in the model group,but lower than that in the control group(P<0.05);but there was no statistical difference in TC,TG,LDLC,HDL-C and FBG levels between experimental high-dose group and metformin group(P>0.05).The liver coefficient and epididymal fat tissue coefficient of rats in experimental high-dose group were lower than those in the model group(P<0.05),but there was no statistical difference between experimental high-dose group and control group or metformin group(P>0.05).Besides,the NF-κB,IL-6,TNF-αlevels of rats inexperimental high-dose group were lower than those in the model group,metformin group or low-dose group(P<0.05),but almost the same as those in the control group(P>0.05).Chaol index of gut microbiota was higher than that in the other four groups(P<0.05),but there was no statistical difference of Shannon diversity among the five groups(P>0.05).The ratios of Firmicutes,Proteobacteria,Actinobacteria,Tenericutes and Prevotella,Phascolarctobacterium in experimental high-dose group were lower than those in model group(P<0.05),meanwhile the ratios of Bacteroidetes,Paraprevotella and Bacteroides were higher than those in model group(P<0.05).Finally,TLR4 and NF-κB proteins in colonic tissues of rats in experimental high-dose group were lower than those in model group(P<0.05).CONCLUSIONS There are significant evidences that Jiedu-jiangzhuo granules could play a role in lower of weight gain and blood lipids in metabolic syndrome rats by adjusting the gut microbiota and TLR4/NF-κB pathway.