基于肠道菌群对膈下逐瘀汤治疗毒瘀内结证晚期结直肠癌患者作用机制的研究

Study on mechanism of the infradiaphragmatic stasis-expelling decoction on advanced colorectal cancer patients with toxin and blood stasis syndrome based on intestinal microflora

目的:运用16S rDNA基因测序方法,明确膈下逐瘀汤治疗晚期毒瘀内结证结直肠癌(colorectal cancer,CRC)对肠道微生物的影响,以探明该处方对肠道菌群相关作用机制。方法:纳入10例毒瘀内结证晚期CRC患者(CRC组),口服膈下逐瘀汤,治疗前后收集粪便标本,与10例健康对照人群(健康对照组)的粪便使用Silva数据库做比较,并将序列聚类到操作分类单元(OTU),通过Qiime 2平台对OTU进行注释,使用GraphPad Prism 8.0.1软件制作主要细菌的相对丰度图,采用Mothur软件进行组内α多样性分析,作主坐标分析(PCoA)图反映肠道菌群β多样性,进一步依托线性判别影响力(LEfSe)分析发现各组间的差异菌群。结果:CRC组患者拟杆菌门、拟杆菌属比例增加,而厚壁菌门和放线菌门、双歧杆菌属的比例减少,经过中药治疗,厚壁菌门、双歧杆菌属的比例增加,但差异无统计学意义(P>0.05);CRC组治疗前菌群丰度在observed species、Chao1、ACE指数上均显著低于健康对照组(P<0.05),而CRC组治疗后较治疗前的菌群丰度在observed species、Chao1、ACE指数上有升高趋势,但差异无统计学意义(P>0.05);与健康对照组比较,CRC组在Shannon指数、Simpson指数上的菌群多样性均降低,但差异无统计学意义(P>0.05),CRC组治疗后菌群均匀度有下降趋势,差异无统计学意义(P>0.05);β多样性方面,健康对照组聚类较好,而CRC组与健康对照组肠道菌群物种组成结构具有较大差异;LEfSe分析结果显示(LDA值>3):CRC组患者治疗前与健康对照组在噬纤维菌目(Order Cytophagales)、橙色粘球菌门(Phylum Myxococcota)和Species Bacteroides_vulgatus等方面差异有统计学意义(P<0.05),治疗前后比较,发现中药汤剂对Order Cytophagales、AKYH767细菌、Genus Escherichia_Shigella等影响最大。结论:膈下逐瘀汤治疗晚期毒瘀内结证CRC患者时,可能通过增加厚壁菌门、双歧杆菌属的比例,提高肠道菌群的多样性和丰富性,并作用于噬纤维菌目细菌等差异菌而达到治疗效果。

Objective:This research has made clear that infradiaphragmatic stasis-expelling decoction effects on gut microbes of advanced CRC patients with toxin and blood stasis through syndrome 16S rDNA gene sequencing method,to ascertain the gut microbiota mechanism of this prescription.Methods:This research included 10 advanced CRC patients with toxin and blood stasis,who take orally infra-diaphragmatic stasis-expelling decoction combined with preliminary core prescription,compared with 10 healthy control populations.Fecal samples of CRC patients were collected before and after treatment,compared with fecal samples of 10 healthy controls.We clustered the above sequences to operational taxonomic units(OTU) compared OTU against Silva database annotated OTU by Qiime 2 Platform and drew finally the figure of relative abundance of main bacteria by GraphPad Prism 8.0.1 software.Moreover,α diversity analysis intra-group was necessary to implement by Mothur software,and beta diversity analysis was observed by Weighted UniFrac distance of PCoA figure.In the end,differential floras inter groups were sought based on Linear discriminant analysis Effect Size(LEfSe).Results:The result of the relative abundance of bacteria indicated that CRC disease itself increased Phylum Bacteroidota and Genus Bacteroides,but decreased the proportion of Phylum Firmicutes,Phylum Actinobacteriota,and Genus Bifidobacterium.However,the proportion of Phylum Firmicutes and Genus Bifidobacterium is gradually increasing after TCM treatment.The bacterial richness estimators CRC patients before treatment were significantly lower than the healthy control group in observed species,Chao1,ACE estimators(P<0.05).α diversity analysis also reflects that the bacterial richness estimators(observed species,Chao1,ACE estimators).CRC patients after treatment seem to have an upward trend than before treatment,but the difference was not statistically significant(P>0.05).In terms of gut microbiota diversity,the bacterial diversity of CRC patients after treatment was higher than that before treatment in Shannon index and Simpson index(P>0.05).The degree of gut microbiota homogeneity of CRC patients before treatment has a downward trend compared with the healthy control group(P>0.05).The beta diversity analysis shows that the cluster of the healthy control group was obvious,while advanced CRC patients with toxin and blood stasis have greater differences in species composition and structure of gut microbiota compared with the healthy control group,which is caused by the influence of disease itself or intervention of Traditional Chinese Medicine.The result of LEfSe(LDA score>3) indicates that the most discrepancyof gut microbiota of advanced CRC patients with toxin and blood stasis before treatment on Order Cytophagales,Phylum Myxococcota,and Species Bacteroides_vulgatus compared with healthy control group.The main effects of TCM decoction on Order Cytophagale,AKYH767 bacteria,and Genus Escherichia_Shigella,et al through comparing between before and after treatment.Conclusion:The research of gut microbiota concludes that advanced CRC patients with toxin and blood stasis were treated effectively with infra-diaphragmatic stasis-expelling decoction attributed to that increasing the proportion of Phylum Firmicutes and Genus Bifidobacterium and improving the diversity and richness of gut microbiota and exerting differential bacterias such as Order Cytophagales at the same time.