Analysis on Changes of Aquaporin and Intestinal Flora in a Rat Model with Spleen-Yin Deficiency Syndrome / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo investigate the effects of spleen-Yin deficiency on gastrointestinal absorption， water metabolism and intestinal flora in rats with spleen-Yin deficiency syndrome. MethodA 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 D-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. ResultCompared 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（P<0.05， P<0.01）， and the duration of swimming was significantly decreased（P<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 D-xylose excretion rate of rats in the model group was significantly decreased（P<0.01）， and the serum GAS content was significantly decreased（P<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（P<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（P<0.01）， and the relative mRNA expression levels of AQP3 and AQP4 were significantly increased（P<0.05， P<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 β-diversity decreased significantly in the model group， the abundance of Porphyromonadaceae was increased significantly， and the abundance of Oscillibacter_ruminantium was decreased significantly（P<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（P<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（P<0.05）. ConclusionSpleen-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.