Changes in Diversity of Intestinal Butyrate-producing Bacteria During Treatment with Shenling Baizhusan and Lizhongtang in Animal Model of AAD

Meng-lin MAO; Ping LIN; Lin-lin XIONG; Yong FENG; Qing-long SHU

doi:10.13422/j.cnki.syfjx.20212106

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Changes in Diversity of Intestinal Butyrate-producing Bacteria During Treatment with Shenling Baizhusan and Lizhongtang in Animal Model of AAD

更新时间：2021-10-21

- Changes in Diversity of Intestinal Butyrate-producing Bacteria During Treatment with Shenling Baizhusan and Lizhongtang in Animal Model of AAD
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 27, Issue 22, Pages: 23-30(2021)
- 作者机构：
  
  江西中医药大学中医学院，南昌 330004
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20212106
  CLC： R2-0;R22;R285.5;R33
- Received：11 July 2021，
  
  Published Online：28 September 2021，
  
  Published：20 November 2021
- 稿件说明：
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毛梦琳,林萍,熊林林等.参苓白术散和理中汤对AAD动物模型肠道产丁酸菌群多样性变化的影响[J].中国实验方剂学杂志,2021,27(22):23-30.

MAO Meng-lin,LIN Ping,XIONG Lin-lin,et al.Changes in Diversity of Intestinal Butyrate-producing Bacteria During Treatment with Shenling Baizhusan and Lizhongtang in Animal Model of AAD[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(22):23-30.
毛梦琳,林萍,熊林林等.参苓白术散和理中汤对AAD动物模型肠道产丁酸菌群多样性变化的影响[J].中国实验方剂学杂志,2021,27(22):23-30. DOI： 10.13422/j.cnki.syfjx.20212106.

MAO Meng-lin,LIN Ping,XIONG Lin-lin,et al.Changes in Diversity of Intestinal Butyrate-producing Bacteria During Treatment with Shenling Baizhusan and Lizhongtang in Animal Model of AAD[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(22):23-30. DOI： 10.13422/j.cnki.syfjx.20212106.

摘要

目的

基于肠道产丁酸菌群多样性变化，研究参苓白术散和理中汤治疗抗生素相关性腹泻（AAD）微生态机制。

方法

将SD大鼠随机分为参苓白术散汤剂治疗组（SPS）和理中汤汤剂治疗组（LPT），灌胃盐酸克林霉素（315 mg·kg

-1

·d

-1

）建立菌群紊乱模型，灌胃致病菌艰难梭菌构建AAD模型，分别予以参苓白术散汤剂（5.5 g·kg

-1

·d

-1

）和理中汤汤剂（5.5 g·kg

-1

·d

-1

）进行治疗，收集不同阶段粪便样本，提取粪便总DNA，利用丁酰辅酶A-辅酶A转移酶基因引物进行聚合酶链式反应（PCR）扩增，再对PCR产物进行克隆测序，分析产丁酸菌群多样性响应。

结果

治疗后两组大鼠饮食逐渐增加，粪便成形，被毛光泽和顺滑度增加，活动量增加，灵敏度提高。以丁酰辅酶A-辅酶A转移酶基因为分子标记的扩增分别获得SPS 297条序列，LPT 300条序列确定为产丁酸菌群多样性结果，SPS正常阶段、造模阶段、治疗阶段分别获得98，100，99条产丁酸菌序列，分别属于8，3，6个运算分类单位（OTU），相似性范围分别为78%～97%，86%～99%，81%～97%，治疗后OTU数量恢复至正常阶段的75%；LPT正常阶段、造模阶段、治疗阶段均获得100条产丁酸菌序列，分别属于6，2，4个OTU，相似性范围为83%～97%，92%～99%，85%～99%，治疗后OTU数量恢复至正常阶段的80%。产丁酸菌均存在于两组大鼠的所有阶段，都以厚壁菌门产丁酸菌为主，占总数量98%以上。SPS在属水平对产丁酸菌菌群的影响主要集中在

Clostridium

属丰度显著降低，

Eubacterium

属丰度显著升高。LPT主要集中在

Roseburia

属，并且增加了

Eubacterium

属，

Lacrimi

ora

属和

Clostridium

属的丰度。根据系统发育树结果，SPS治疗后，产丁酸菌菌群的分布由造模后的5个cluster增加到治疗后的7个cluster；LPT分布由造模后的3个Cluster增加到治疗后的9个Cluster。

结论

参苓白术散和理中汤治疗AAD病过程中，能够调节肠道产丁酸菌的结构和丰度，恢复产丁酸菌菌群多样性，改善肠道微生态环境失稳状况。

Abstract

Objective

To explore the microecological mechanisms of Shenling Baizhusan （SLBZ） and Lizhongtang （LZ） in treating antibiotic-associated diarrhea （AAD） based on changes in the diversity of intestinal butyrate-producing bacteria.

Method

SD rats were randomly divided into an SLBZ group （5.5 g·kg

-1

·d

-1

） and an LZ group （5.5 g·kg

-1

·d

-1

）. The gut microbiota disturbance model was induced by intragastric administration of clindamycin hydrochloride （315 mg·kg

-1

·d

-1

） and AAD model by

Clostridium difficile

. Subsequently， the rats were treated correspondingly. Fecal samples at different stages were collected and the total DNA was extracted. Polymerase chain reaction （PCR） amplification was performed with the primers of butyryl coenzyme A （CoA）-CoA transferase genes. The PCR products were cloned and sequenced to analyze the diversity response of butyrate-producing bacteria.

Result

After treatment， both groups showed increased food uptake， formed feces， glossy and smooth fur， and improved activity and sensitivity. With the butyryl CoA-CoA transferase gene as the molecular marker， 297 sequences of butyrate-producing bacteria in the SLBZ group （SPD for short） and 300 sequences of butyrate-producing bacteria in the LZ group （LPD for short） were obtained. In the SLBZ group， 98， 100， and 99 sequences of SPD were obtained at the normal stage， the modeling stage， and the treatment stage， respectively， belonging to 8， 3， and 6 operational taxonomic units （OTUs）， with similarity ranges of 78%-97%， 86%-99% ， and 81%-97%. The number of OTUs recovered to 75% of the normal level after treatment. In the LZ group， 100 sequences of LPD were obtained at the normal stage， the modeling stage， and the treatment stage， respectively， belonging to 6， 2， and 4 OTUs， with similarity ranges of 83%-97%， 92%-99%， and 85%-99%. The number of OTUs recovered to 80% of the normal level after treatment. Butyrate-producing bacteria were present in all stages of the two groups， dominated by Firmicutes， accounting for more than 98% of the total number. The effects of SLBZ on SPD at the genus level were observed in the significant decrease in

Clostridium

abundance and the significant increase in

Eubacterium

abundance. The effect of LZ on LPD was mainly concentrated on the

Roseburia

at the genus level， and LZ also increased the abundance of

Eubacterium

，

Lacrimi

ora

， and

Clostridium

. According to the phylogenetic tree， the classification of butyrate-producing bacteria increased from five clusters to seven clusters after SLBZ treatment， while that increased from three clusters to nine clusters after LZ treatment.

Conclusion

In the treatment of AAD， SLBZ and LZ can regulate the structure and abundance of butyrate-producing bacteria in the intestine， restore their diversity， and improve the instability of the intestinal microecological environment.

关键词

Keywords

references

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