养心通脉方对冠心病血瘀证大鼠小肠黏膜菌群和血清炎症因子、内毒素的影响及相关性分析

Effect and correlation analysis of Yangxin Tongmai Prescription on intestinal mucosal flora,serum inflammatory factors and endotoxin in coronary heart disease rats with blood stasis syndrome

目的:养心通脉方对冠心病血瘀证大鼠小肠黏膜菌群变化和血清炎症因子、内毒素的影响及相关性分析。方法:将40只SD雄性大鼠随机分为正常组8只和造模组32只。造模组采用高脂饲料喂养3个月,结合维生素D_(3)灌胃和异丙肾上腺素皮下注射的方式构建冠心病血瘀证大鼠,然后随机分为模型组、养心通脉方组(12 g·kg^(-1)·d^(-1))、阿托伐他汀组(1.8 mg·kg^(-1)·d^(-1)),均灌胃2周。干预结束后,对小肠黏膜组织进行16S rRNA测序和透射电镜检测;HE染色观察心肌组织;ELISA试验检测血清肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、内毒素含量。结果:与正常组比较,模型组心电图T波高尖,ST段显著抬高(P<0.01),心率降低(P<0.01),HE染色心肌纤维坏死溶解,大量炎症细胞浸润。养心通脉方组和阿托伐他汀组病变均较模型组减轻。与正常组比较,模型组血清TNF-α、IL-6、内毒素含量升高(P<0.01),IL-10含量下降(P<0.01);养心通脉方组TNF-α、内毒素含量较模型组下降(P<0.01),IL-10升高(P<0.01)。模型组有害菌罗尔斯通菌属(Ralstonia)相对丰度升高(P<0.05),养心通脉方组有下降趋势,但差异无统计学意义(P=0.06);模型组有益菌未鉴别的消化链球菌科菌属(Unspecified_Peptostreptococcaceae)丰度降低(P<0.01)。Pearson相关性分析结果,罗尔斯通菌属丰度与总胆固醇(TCHO)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、血流变指标、TNF-α均正相关性(P<0.05),与IL-10负相关(P<0.05);未鉴别的消化链球菌科菌属、未鉴别的梭菌目菌属(Unspecified_Clostridiales)丰度与TCHO、HDL-C、LDL-C、血流变指标、TNF-α、内毒素具负相关性(P<0.05,P<0.01),与IL-10正相关(P<0.05)。回肠组织电镜观察显示,模型组线粒体病变,胞核碎裂,微绒毛稀疏;养心通脉方组和阿托伐他汀组未见明显病变。结论:冠心病血瘀证大鼠小肠黏膜菌群与TNF-α、IL-10、内毒素、血脂、血流变具有相关性;养心通脉方可能通过调节小肠黏膜菌群,降低血清TNF-α及内毒素含量改善冠心病血瘀证。

Objective:To investigate the effect and correlation analysis of Yangxin Tongmai Prescription on changes of intestinal mucosal flora,serum inflammatory factors and endotoxin in coronary heart disease with blood stasis syndrome rats.Methods:A total of 40 male SD rats were randomly divided into normal group(n=8)and model building group(n=32).The model group were fed with high-fat diet for 3 months,combined with vitamin D_(3) intragastric administration and isoproterenol subcutaneous injection to construct coronary heart disease with blood stasis syndrome rats.Then the model building group were randomly divided into 3 groups:model group,Yangxin Tongmai Prescription group(12 g·kg^(-1)·d^(-1)),atorvastatin group(1.8 mg·kg^(-1)·d^(-1)),were given intragastric administration for 2 weeks.After the intervention,the small intestinal mucosal tissues were sequenced by 16S rRNA and detected by transmission electron microscopy;HE staining was used to observe the myocardium;Serum Tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-10(IL-10)and endotoxin were detected by ELISA.Results:Compared with the normal group,the electrocardiograph T-wave of the model building group was higher and the ST segment was significantly elevated(P<0.01),heart rate decreased(P<0.01).HE staining myocardial fiber necrosis dissolution,a large number of inflammatory cells infiltrated.The lesions in the Yangxin Tongmai Prescription group and atorvastatin group were less than those in the model group.Compared with the normal group,the contents of TNF-α,IL-6,endotoxinin serum of model group were increased(P<0.01),but IL-10 was decreased(P<0.01).Compared with the model group,the contents of TNF-αand endotoxin in Yangxin Tongmai Prescription group were decreased(P<0.01),while IL-10 was increased(P<0.01).The relative abundance of Ralstonia increased in the model group(P<0.05),but decreased in Yangxin Tongmai Prescription group,although the difference was not statistically significant(P=0.06).The abundance of Unspecified_Peptostreptococcaceae in model group decreased(P<0.01).Pearson correlation analysis showed that the abundance of Ralstonia was positively correlated with TCHO,HDL-C,LDL-C,hemorheology,TNF-α(P<0.05),and negatively correlated with IL-10(P<0.05).The abundance of Unspecifed_Peptostreptococcaceae and Unspecifed_Clostridiales were negatively correlated with TCHO,HDL-C,LDL-C,hemorheological indexes,TNF-αand endotoxin(P<0.05,P<0.01),positively correlation with IL-10(P<0.05).Transmission electron microscopy of ileum tissue showed that mitochondria lesion,cell nucleus fragmentation and microvilli sparsity were observed in model group.No obvious lesions were observed in Yangxin Tongmai Prescription group and atorvastatin group.Conclusion:The microflora of small intestinal mucosa was correlated with TNF-α,IL-10,endotoxin,blood lipid and hemorheology in rats with blood stasis syndrome of coronary heart disease.Yangxin Tongmai Prescription can improve the blood stasis syndrome of coronary heart disease by possibly regulating the intestinal mucosal microflora and reducing the levels of TNF-αand endotoxin in serum.