Analysis on Changes of Aquaporin and Intestinal Flora in a Rat Model with Spleen-Yin Deficiency Syndrome

HUANG Hongxia; LU Xuefeng; ZHOU Quan; FANG Hong; DENG Xiaxia; SUN Youzhi; ZHAO Yi

doi:10.13422/j.cnki.syfjx.20230864

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Analysis on Changes of Aquaporin and Intestinal Flora in a Rat Model with Spleen-Yin Deficiency Syndrome

更新时间：2023-11-07

- Analysis on Changes of Aquaporin and Intestinal Flora in a Rat Model with Spleen-Yin Deficiency Syndrome
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 23, Pages: 131-139(2023)
- 作者机构：
  
  江西中医药大学中医基础理论分化发展研究中心，江西省中医病因生物学重点实验室，方-证研究中心，南昌 330004
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20230864
  CLC： R22;R574.4;R28;Q939
- Published：05 December 2023，
  
  Published Online：23 April 2023，
  
  Received：09 February 2023，
- 稿件说明：
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黄红侠,卢雪凤,周权等.脾阴虚证大鼠模型中水通道蛋白及肠道菌群的变化分析[J].中国实验方剂学杂志,2023,29(23):131-139.

HUANG Hongxia,LU Xuefeng,ZHOU Quan,et al.Analysis on Changes of Aquaporin and Intestinal Flora in a Rat Model with Spleen-Yin Deficiency Syndrome[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(23):131-139.
黄红侠,卢雪凤,周权等.脾阴虚证大鼠模型中水通道蛋白及肠道菌群的变化分析[J].中国实验方剂学杂志,2023,29(23):131-139. DOI： 10.13422/j.cnki.syfjx.20230864.

HUANG Hongxia,LU Xuefeng,ZHOU Quan,et al.Analysis on Changes of Aquaporin and Intestinal Flora in a Rat Model with Spleen-Yin Deficiency Syndrome[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(23):131-139. DOI： 10.13422/j.cnki.syfjx.20230864.

摘要

目的

以脾阴虚证模型大鼠为研究对象，探析脾阴虚对胃肠吸收、水液代谢及肠道菌群的影响。

方法

采用荤素饮食不节、负重疲劳游泳和温热伤阴药灌胃复合因素建立脾阴虚证大鼠模型，观察空白组和模型组大鼠的体质量、进食量、饮水量、游泳持续时间的变化；苏木素-伊红（HE）染色观察胃和结肠组织病理损伤情况；间苯三酚法检测尿

-木糖排泄率；酶联免疫吸附测定法（ELISA）检测血清中胃泌素（GAS）含量；蛋白免疫印迹法（Western blot）检测胃组织中血管活性肠肽（VIP）、水通道蛋白3（AQP3）、AQP4的相对表达水平；实时荧光定量聚合酶链式反应（Real-time PCR）检测胃组织中VIP、AQP3、AQP4的mRNA相对表达水平；16S rDNA测序分析其肠道菌群的变化。

结果

与空白组比较，一般体征结果显示，模型组大鼠体质量和进食量均明显降低，饮水量明显增多（

0.05，

0.01），游泳持续时间显著减少（

0.01）；病理检测结果显示，模型组大鼠胃组织黏膜出现排列不齐，结肠组织黏膜可见明显变薄或有残缺，上皮细胞出现坏死甚至脱落现象；与空白组比较，模型组大鼠尿

-木糖排泄率显著降低（

0.01），血清GAS含量明显降低（

0.05）；Western blot结果显示，与空白组比较，模型组大鼠胃组织中VIP蛋白的相对表达水平显著降低（

0.01），AQP4、AQP3蛋白的相对表达水平显著增加（

0.01）；Real-time PCR结果显示，与空白组比较，模型组大鼠胃组织中VIP mRNA的相对表达水平显著降低（

0.01），AQP3和AQP4 mRNA的相对表达水平明显增加（

0.05，

0.01）；肠道菌群分析结果显示，与空白组比较，模型组操作分类单元（OTU）数目和

多样性增加，

多样性明显降低，紫单胞菌科丰度明显增加，产丁酸菌种丰度明显降低（

0.05）；斯皮尔曼计算法（Spearman）相关分析显示，紫单胞菌科与AQP4蛋白水平呈明显正相关（

0.05），产丁酸菌种与VIP蛋白水平呈明显正相关（

0.05），与AQP3、AQP4蛋白水平呈明显负相关（

0.05）；线性判别分析（LDA）效应量（LEfSe）结果显示，组间多种肠道菌存在明显差异，模型组大鼠肠道中迷踪菌门/目/科/属、

-变形菌纲、伯克氏菌目、萨特菌科、副萨特氏菌属被明显富集（

0.05）。

结论

脾阴虚证可以减弱胃肠道的消化和吸收能力，引起水液代谢和肠道菌群的紊乱，胃黏膜VIP、AQP3、AQP4蛋白水平与紫单胞菌科、产丁酸菌种密切相关，VIP、AQP3、AQP4可能参与调节肠道菌群以影响脾主运化的生理功能。

Abstract

Objective

To investigate the effects of spleen-Yin deficiency on gastrointestinal absorption， water metabolism and intestinal flora in rats with spleen-Yin deficiency syndrome.

Method

A rat model of spleen-Yin deficiency syndrome was established by using the composite factors， including irregular meat and vegetable diet， weight-bearing fatigue swimming and gavage with warm-heat injury-Yin drugs. The changes of body weight， food intake， water intake and duration of swimming in the blank and model groups were observed. Hematoxylin-eosin（HE） staining was used to observe the histopathological damage of the stomach and colon. Urinary excretion rate of

-xylose was determined by phloroglucinol method. The content of gastrin（GAS） in serum was determined by enzyme-linked immunosorbent assay（ELISA）. The relative expression levels of vasoactive intestinal peptide（VIP）， aquaporin 3（AQP3） and AQP4 in gastric tissues were detected by Western blot. The relative mRNA expression levels of VIP， AQP3 and AQP4 in gastric tissues were detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， and the changes of intestinal flora were analyzed by 16S rDNA sequencing.

Result

Compared with the blank group， the results of general physical signs showed that the body weight and food intake of rats in the model group were significantly decreased， the water intake was significantly increased（

0.05，

0.01）， and the duration of swimming was significantly decreased（

0.01）. Pathological examination results showed that in the mucosa of gastric tissues of rats in the model group appeared to be misaligned， the mucosa of colonic tissues could be seen to be obviously thinned or mutilated， and the epithelial cells appeared to be necrotic or even exfoliated. Compared with the blank group， the urinary

-xylose excretion rate of rats in the model group was significantly decreased（

0.01）， and the serum GAS content was significantly decreased（

0.05）. Compared with the blank group， Western blot results showed that the relative expression level of VIP protein in gastric tissues of rats in the model group was significantly decreased， while the relative expression levels of AQP4 and AQP3 proteins were significantly increased（

0.01）. Compared with the blank group， Real-time PCR results showed that the relative expression level of VIP mRNA in gastric tissues of rats in the model group was significantly decreased（

0.01）， and the relative mRNA expression levels of AQP3 and AQP4 were significantly increased（

0.05，

0.01）. Compared with the blank group， the results of intestinal flora analysis showed that the number of operational taxonomic units（OTUs） and

α-

diversity increased and

-diversity decreased significantly in the model group， the abundance of Porphyromonadaceae was increased significantly， and the abundance of

Oscillibacter_ruminantium

was decreased significantly（

0.05）. Spearman correlation analysis showed that Porphyromonadaceae was significantly positively correlated with AQP4 protein level， while

Oscillibacter_ruminantium

was significantly positively correlated with VIP protein level， and negatively correlated with AQP3 and AQP4 protein levels（

0.05）. Linear discriminant analysis effect size（LEfSe） analysis results showed that there were significant differences in a variety of intestinal bacteria between groups， and the intestinal bacteria of the model group were significantly enriched in the phylum/order/family/genus of Elusimicrobia， Betaproteobacteria， Burkholderiales， Sutterellaceae and

Parasutterella

（

0.05）.

Conclusion

Spleen-Yin deficiency syndrome can weaken the digestion and absorption capacity of gastrointestinal tract， and cause the disturbance of water metabolism and intestinal flora. AQP4， AQP3 and VIP protein levels of gastric mucosa are closely related to Porphyromonadaceae and

Oscillibacter_ruminantium

. And AQP4， AQP3 and VIP may be involved in the regulation of intestinal flora in order to affect the physiological function of spleen governing transportation and transformation.

关键词

脾阴虚证血管活性肠肽（VIP）水通道蛋白（AQP）水液代谢肠道菌群相关性分析聚合酶链式反应（PCR）

Keywords

spleen-Yin deficiency syndromevasoactive intestinal peptide（VIP）aquaporin（AQP）water-fluid metabolismintestinal floracorrelation analysispolymerase chain reaction（PCR）

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