基于16S rDNA测序探讨地黄饮子对帕金森病肾虚证大鼠肠道菌群的影响

The Effect of Dihuang Yinzi on Gut Microbiota of Parkinson’s Disease Kidney Deficiency Syndrome Rats Based on 16S rDNA Sequencing

目的探讨地黄饮子对D-半乳糖所致肾虚证及鱼藤酮诱导帕金森病(Parkinson’s disease,PD)大鼠的疗效及其对肠道菌群(Gut Microbiota,GM)的影响。方法选取90只6-8周龄雄性健康SD大鼠,随机分为6组:正常组、模型组、左旋多巴组、地黄饮子高、中、低剂量组。造模采用腹腔注射D-半乳糖(150 mg·kg^(-1))6周及背部皮下注射鱼藤酮(0.5 mg·kg^(-1))4周,联合诱导PD肾虚证病证结合模型,观察地黄饮子高、中、低剂量对PD肾虚模型大鼠的疗效评价。通过地黄饮子治疗,对大鼠粪便标本中的细菌16S rDNAV3-V4区域扩增子测序,比较PD肾虚模型大鼠GM特点及地黄饮子干预后GM变化。结果本研究共得到448个OTU。PD肾虚模型大鼠存在GM失衡的情况,主要与厚壁菌门、拟杆菌门和放线菌门及其下属部分菌群丰度水平的变化相关。物种注释结果方面,相较于模型组,地黄饮子中、高剂量组厚壁菌门显著性升高(P<0.01)、拟杆菌门降低(P<0.05),而放线菌门地黄饮子低剂量组显著性升高(P<0.01),高剂量组则显著性降低(P<0.01)。相较于模型组,地黄饮子中剂量组ACE指数、Chao1指数、Simpson指数、Shannon指数等Alpha多样性指数均增加(P<0.05),可见地黄饮子干预后物种丰富度和多样性有显著改变。通过Beta多样性分析可见,地黄饮子中、高剂量组分别与空白组大鼠的GM存在关联。而地黄饮子低、中剂量组大鼠GM的LDA评分下多样性较为突出,且有相应关联菌群相富集。结论本研究运用16S rDNA测序技术预测地黄饮子治疗PD肾虚证的作用机制,初步揭示了地黄饮子通过保护PD大鼠肠道黏膜屏障,改善肠道屏障通透性,调节厚壁菌门、拟杆菌门和放线菌门及其下属部分菌群丰度水平的变化等作用,改善PD大鼠运动障碍,而发挥防治PD的作用。研究结果预测了地黄饮子治疗PD的作用机制可能与调节GM动态的相对平衡有关,可为从肠道微环境角度揭示补肾法改善PD作用机制的研究提供参考。

Objective To investigate the effect of Dihuang Yinzi on kidney deficiency syndrome induced by D-galactose and rotenone induced Parkinson’s disease(PD)rats and its effect on gut microbiota(GM).Methods 90 male healthy SD rats aged 6-8 weeks were randomly divided into 6 groups:normal group,model group,levodopa group and Dihuang Yinzi high,medium and low dose groups.The model was established by intraperitoneal injection of D-galactose(150 mg·kg^(-1))for 6 weeks and subcutaneous injection of rotenone(0.5 mg·kg^(-1))for 4 weeks.The combined model of kidney deficiency syndrome and disease syndrome of PD was induced.The therapeutic effects of high,medium and low doses of Dihuang Yinzi on PD kidney deficiency model rats were observed.Through the treatment of Dihuang Yinzi,the bacterial 16S rDNAV3-V4 region amplicon in rat feces samples was sequenced to compare the GM characteristics of PD kidney deficiency model rats and the GM changes after Dihuang Yinzi intervention.Results A total of 448 OTUs were obtained in this study.The imbalance of GM in PD kidney deficiency model rats is mainly related to the changes of the abundance of Firmicutes,Bacteroidetes,Actinomycetes and their subordinate flora.In terms of species annotation results,compared with the model group,the medium and high dose groups of Dihuang Yinzi of Firmicutes increased significantly(P<0.01)and Bacteroidetes decreased significantly(P<0.05),while the low dose group of Actinomycetes increased significantly(P<0.01)and the high dose group decreased significantly(P<0.01).Compared with the model group,the Alpha indexes such as ACE index,Chao1 index,Simpson index and Shannon index in the medium dose group of Dihuang Yinzi increased(P<0.05).It can be seen that the species richness and diversity changed significantly after the intervention of Dihuang Yinzi.Through the Beta diversity analysis,it can be seen that the medium and high dose groups of Dihuang Yinzi are associated with the GM of the blank group rats respectively.The low and medium dose groups of Dihuang Yinzi had prominent diversity under the LDA score of GM,and the associated GM was enriched.Conclusion The results predict that the mechanism of Dihuang Yinzi in the treatment of PD may be related to the regulation of the relative balance of GM dynamics,which can provide a reference for revealing the mechanism of Kidney tonifying method in improving PD from the perspective of intestinal microenvironment.