Effect of Huazhuo Jiedu Huoxue Tongluo Prescription on Brain-gut Axis of Rats with Cerebral Ischemia-reperfusion Injury Based on 16S rRNA Sequencing of Intestinal Flora

Rui-qing HUO; Min-han ZHAO; Fang-zhao LI; Yu-fan HAN; Kuo SUN; Jun-biao TIAN

doi:10.13422/j.cnki.syfjx.20220194

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Effect of Huazhuo Jiedu Huoxue Tongluo Prescription on Brain-gut Axis of Rats with Cerebral Ischemia-reperfusion Injury Based on 16S rRNA Sequencing of Intestinal Flora

更新时间：2022-01-04

- Effect of Huazhuo Jiedu Huoxue Tongluo Prescription on Brain-gut Axis of Rats with Cerebral Ischemia-reperfusion Injury Based on 16S rRNA Sequencing of Intestinal Flora
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 1, Pages: 121-130(2022)
- 作者机构：
  
  1.河北中医学院，石家庄 050200
  2.河北省中医院，石家庄 050011
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20220194
  CLC： R22;R242;R2-031;R287;R743.3
- Received：16 March 2021，
  
  Published Online：09 November 2021，
  
  Published：05 January 2022
- 稿件说明：
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霍瑞卿,赵敏菡,李芳钊等.基于16S rRNA测序的肠道菌群探讨化浊解毒活血通络方对脑缺血再灌注损伤大鼠脑-肠轴的影响[J].中国实验方剂学杂志,2022,28(01):121-130.

HUO Rui-qing,ZHAO Min-han,LI Fang-zhao,et al.Effect of Huazhuo Jiedu Huoxue Tongluo Prescription on Brain-gut Axis of Rats with Cerebral Ischemia-reperfusion Injury Based on 16S rRNA Sequencing of Intestinal Flora[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(01):121-130.
霍瑞卿,赵敏菡,李芳钊等.基于16S rRNA测序的肠道菌群探讨化浊解毒活血通络方对脑缺血再灌注损伤大鼠脑-肠轴的影响[J].中国实验方剂学杂志,2022,28(01):121-130. DOI： 10.13422/j.cnki.syfjx.20220194.

HUO Rui-qing,ZHAO Min-han,LI Fang-zhao,et al.Effect of Huazhuo Jiedu Huoxue Tongluo Prescription on Brain-gut Axis of Rats with Cerebral Ischemia-reperfusion Injury Based on 16S rRNA Sequencing of Intestinal Flora[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(01):121-130. DOI： 10.13422/j.cnki.syfjx.20220194.

摘要

目的

研究化浊解毒活血通络方对脑缺血再灌注损伤大鼠肠道菌群的影响，探讨中药调控肠道微生物群进而恢复脑-肠轴平衡的机制。

方法

将50只SPF级雄性SD大鼠随机分为假手术组，模型组，化浊解毒活血通络方高（25.0 g·kg

-1

），中（12.5 g·kg

-1

），低（6.25 g·kg

-1

）剂量组，每组10只。参照Longa法并结合既往研究经验制备大脑中动脉梗塞大鼠模型，缺血2 h后再灌注。于脑缺血再灌注损伤72 h检测大鼠神经功能缺损情况、脑梗死范围百分比；采用实时荧光定量聚合酶链式反应（Real-time PCR）检测大鼠结肠闭锁小带蛋白-1（ZO-1），闭锁蛋白（Occludin） mRNA表达；苏木素-伊红（HE）染色观察各组大鼠肠道组织、脑组织损坏程度；每组收集6只大鼠粪便进行16S rRNA测序；免疫组化、酶联免疫吸附测定试验（ELISA）检测肠道组织、外周血、脑组织调节性T细胞（Treg），辅助性T细胞17（Th17）的表达。

结果

与假手术组比较，模型组大鼠神经功能缺损症状加重（

0.05），脑梗死面积增加（

0.05）；与模型组比较，化浊解毒活血通络方高、中剂量组大鼠神经功能缺损症状明显减轻（

0.05），脑梗死面积减少（

0.05）。与假手术组比较，模型组大鼠结肠黏膜结构破坏，上皮细胞、杯状细胞结构不完整；与模型比较，化浊解毒活血通络方高、中剂量组大鼠结肠黏膜破坏较少，上皮细胞、杯状细胞结构完整。与假手术组比较，模型组大鼠Occludin，ZO-1 mRNA表达降低（

0.05），与模型组比较，化浊解毒活血通络方高剂量组Occludin，ZO-1 mRNA水平均有所增加（

0.05）。模型组与假手术组肠道菌群微生态结构存在差异，化浊解毒活血通络方高剂量组与假手术组组群落结果更相近。与假手术组比较，模型组大鼠肠道组织、外周血、脑组织Treg细胞表达下降，Th17细胞表达升高（

0.05）；与模型比较，化浊解毒活血通络方高、中剂量组大鼠肠道组织、外周血、脑组织Treg细胞表达升高，Th17细胞表达下降（

0.05）。

结论

化浊解毒活血通络方可能通过调整肠道微生物的丰度和多样性，改善肠壁通透性，减少肠道Th17细胞迁移至缺血侧脑组织，减少炎症反应，进而减轻大鼠脑缺血再灌注损伤。

Abstract

Objective

To study the effect of Huazhuo Jiedu Huoxue Tongluo （HJHT） prescription on the intestinal flora in rats with cerebral ischemia-reperfusion injury， and to explore the mechanism of Chinese medicinal prescription regulating intestinal flora to restore the balance of brain-gut axis.

Method

Fifty male SPF SD rats were randomly assigned into sham group， model group， high-dose HJHT group （25.0 g·kg

-1

）， medium-dose HJHT group （12.5 g·kg

-1

）， and low-dose HJHT group （6.25 g·kg

-1

）， with 10 rats in each group. The rat model of permanent middle cerebral artery infarction was established according to Longa method and previous research experience， and reperfusion was performed 2 h after ischemia. The recovery of neurological function deficit and the percentage of cerebral infarction area were detected 72 h after administration. Real-time PCR was performed to detect the mRNA levels of Occludin and zonula occludens-1 （ZO-1） in rat colon. Hematoxylin-eosin （HE） staining was conducted to reveal the intestinal damage. The feces of 6 rats in each group were collected for 16S rRNA sequencing. The expression of Treg and Th17 in intestinal tissue， peripheral blood， and brain tissue were detected.

Result

Compared with the sham group， the model group showed obvious neurological deficit （

0.05） and large cerebral infarction area （

0.05）. High-dose and medium-doses HJHT alleviated the symptoms of neurological impairment （

0.05） and reduce the cerebral infarction area （

0.05） compared with the model group. Compared with the sham group， the model group showed destroyed structure of colonic mucosa and incomplete epithelial cells and goblet cells， while high-dose and medium-doses HJHT alleviated such changes. The mRNA levels of Occludin and ZO-1 in the model group were lower than those in the sham group （

0.05），and the high-dose HJHT groups were higher than the model group （

0.05）. The intestinal flora structure was significantly different between the model group and the sham group while similar between the high-dose HJHT group and sham group. Compared with the sham group， the model group showed down-regulated expression of Treg and up-regulated expression of Th17 in the intestinal tissue， peripheral blood， and brain tissue， and high-dose and medium-dose HJHT alleviated the changes in the expression of Treg and Th17 in the model group （

0.05）.

Conclusion

Huazhuo Jiedu Huoxue Tongluo prescription may improve the permeability of intestinal wall by adjusting the abundance and diversity of intestinal microorganisms to reduce the migration of intestinal Th17 cells toward the ischemic lateral brain tissue， mitigate the inflammatory response， and thus alleviate the cerebral ischemia-reperfusion injury in rats.

关键词

Keywords

references

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