摘要
目的:在"心与小肠相表里"中医理论指导下,探讨松萝治疗动脉粥样硬化(AS)大鼠与回肠肠道菌群的关系。方法:48只SD大鼠随机分为正常组8只和造模组40只,采用高脂饲料联合腹腔注射维生素D3建立AS模型。造模组大鼠随机分为5组,模型组、辛伐他汀组(4 mg·kg^(-1)和松萝醇提物低(0.7 g·kg^(-1),中(1.4 g·kg^(-1),高剂量组(2.8 g·kg^(-1),每组8只。干预治疗4周后,收集各组大鼠血液、主动脉、回肠及回肠内容物。酶联免疫吸附法检测大鼠血清脂多糖(LPS),肿瘤坏死因子-α(TNF-α),白细胞介素-6(IL-6)的含量,苏木素-伊红(HE)染色法检测大鼠胸主动脉病理形态,蛋白免疫印迹法(Western blot)检测大鼠回肠组织紧密连接蛋白闭锁小带蛋白-1(ZO-1),闭合蛋白(Occludin)的蛋白表达,16S r RNA高通量测序技术检测各组大鼠回肠肠道菌群多样性和丰度的变化。结果:与正常组比较,模型组大鼠主动脉斑块沉积明显,血清LPS,TNF-α,IL-6的含量均显著升高(P<0.01),回肠组织ZO-1,Occludin的蛋白表达显著降低(P<0.01)。与模型组比较,松萝能明显改善模型大鼠主动脉的斑块沉积,降低血清LPS,TNF-α,IL-6的含量(P<0.05,P<0.01),同时提高紧密连接蛋白ZO-1,Occludin的蛋白表达(P<0.05,P<0.01)。与正常组比较,模型组变形菌门Proteobacteria和拟杆菌门Bacteroidetes的丰度发生显著变化,Bacteroidetes/厚壁菌门(Firmicutes)(B/F)明显降低(P<0.05)。Alpha和Beta多样性分析表示模型组肠道菌群物种总数较高,但丰度较低且分布不均(P<0.05,P<0.01),且富集大量致病菌。与模型组比较,松萝醇提物可显著升高B/F值,改善模型大鼠回肠菌群结构的紊乱,降低致病菌、提高益生菌的相对丰度。结论:松萝对AS具有良好的治疗效果,其作用机制可能是与改善肠道菌群结构紊乱,增强肠黏膜屏障,降低血清LPS及炎症因子水平有关。
Objective:To explore the correlation between the efficacy of Usnea diffracta in treating atherosclerosis(AS)and the altered microbial flora in rat ileum based on the interior-exterior relationship between heart and small intestine in traditional Chinese medicine(TCM).Method:Forty-eight SD rats were randomized into a normal group(n=8)and a modeling group(n=40).The AS model was established with highfat diet combined with intraperitoneal injection of vitamin D3.The successfully modeled rats were further randomly divided into the model group,positive control(simvastatin,4 mg·kg^(-1)group,and low-(0.7 g·kg^(-1),medium-(1.4 g·kg^(-1),and high-dose(2.8 g·kg^(-1)U.diffracta ethanol extract groups,with eight rats in each group.After four weeks of intervention,the blood,aorta,ileum,and ileum content of rats in each group were collected.The levels of serum lipopolysaccharide(LPS),tumor necrosis factor-α(TNF-α)and interleukin 6(IL-6)were measured by enzyme-linked immunosorbent assay(ELISA),and the pathological changes in rat thoracic aorta was detected by hematoxylin-eosin(HE)staining.Western blot was conducted to determine the protein expression levels of tight junction protein zonula occluden(ZO-1)and Occludin in rat ileum,and the high-throughput 16 S rRNA sequencing technology was employed to detect changes in microbial diversity and abundance in rat ileum of each group.Result:Compared with the normal group,the model group exhibited obvious aortic plaque deposition,increased LPS,TNF-α,and IL-6 levels(P<0.01),but decreased ZO-1 and Occludin protein expression(P<0.01).The comparison with the model group revealed that U.diffracta significantly ameliorated the aortic plaque deposition of model rats,lowered serum LPS,TNF-α,and IL-6 levels(P<0.05,P<0.01),and up-regulated ZO-1 and Occludin protein expression(P<0.05,P<0.01).The abundance of Proteobacteria and Bacteroidetes in the model group changed significantly in contrast to that in the normal group,and the Bacteroidetes/Firmicutes(B/F)value declined(P<0.05).Alpha and Beta diversity analysis indicated higher total number of intestinal flora species in the model group,but lower richness and uneven distribution(P<0.05,P<0.01),with a large number of pathogenic bacteria enriched.The ethanol extract of U.diffracta significantly increased the B/F value,corrected the structural disorder of microbial flora in ileum,reduced pathogenic bacteria,and increased the relative abundance of probiotics.Conclusion:U.diffracta exerts the therapeutic effect against AS possibly by improving the intestinal microbial communities,strengthening the intestinal mucosal barrier function,and reducing the serum LPS and inflammatory factors.
作者
张留代
熊伟
许青
刘星鸿
黄静群
张倩倩
闫智勇
ZHANG Liu-dai;XIONG Wei;XU Qing;LIU Xing-hong;HUANG Jing-qun;ZHANG Qian-qian;YAN Zhi-yong(School of Life Science and Engineering,Southwest Jiaotong University,Chengdu 610000,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2021年第14期36-46,共11页
Chinese Journal of Experimental Traditional Medical Formulae
基金
中央高校基本科研业务费专项(2682020ZT81)
大学生创新创业训练计划项目(202010613066)。
关键词
松萝
动脉粥样硬化(AS)
炎症
肠黏膜屏障
肠道菌群
Usnea diffracta
atherosclerosis(AS)
inflammation
intestinal mucosal barrier
intestinal flora
作者简介
第一作者:张留代,在读硕士,从事中药药理学研究,E-mail:2909923127@qq.com;通信作者:闫智勇,博士,教授,从事中药药理学研究,E-mail:yzhiy@swjtu.edu.cn。
