Study On The Changes Of Intestinal Mucosal Barrier In Spleen-yin Deficiency Rats And The Effect Of ZiBu PiYin Recipe

Objective:The purpose of this study was to investigate the changes of intestinal mucosal barrier in spleen-yin deficiency(SYD)model rat,and to observe the effects of ZiBu Pi Yin Recipe(ZBPYR).Methods:After 3 days of adaptive feeding,SPF male Wistar rats were divided into normal group(n=6)and model group(n=14)according to the principle of randomization.SYD rats model were established by the method of classical compound factors,which are abnormal diet and fatigue combined with injured-yin herbs.The experiment lasted for 38 days and was divided into 3 stages.Day 1-14 was the first stage,which was the spleen-qi deficiency modeling stage.Rats in the model group were fed for 1 day and fasted for 2 days(3 days for a cycle).And these rats were forced to swim until they were exhausted once a day.The second stage was SYD modeling stage,from the 15th to 24th day.One rat which did not meet the standard of SYD and one dead rat were excluded.The remaining 12 rats were included in the stage of SYD.These rats were treated with 1 mL/100 g of injured-yin herbs once a day in this stage.The third stage is ZBPYR administration stage,from the 25th to 38th day.The model group was divided into SYD group(n=6)and ZBPYR group(n=6).Rats in ZBPYR group was given the intervention of ZBPYR intragastrically every day with 1 mL/100 g per rat.And the normal group and SYD group were given the same volume of normal saline intragastrically.Rat feces were collected on the 7th,14th,21st,28th and 35th day of this experiment.The diversity of intestinal flora and the abundance of different flora at the level of phylum and genus in rats were analyzed by 16S rRNA V3-V4 region sequencing.Results:1.Study on the changes of intestinal mucosal biological barrier in SYD rats and the effect of ZBPYR.(1)The results of apha diversity showed that Simpson and Shannon index in model group were higher than those in normal group at the end of spleen-qi deficiency stage.But the changes of Simpson and Shannon index in SYD group were not significant at the stage of SYD.Compared with the normal group,Simpson and Shannon index in SYD group were decreased at the end of ZBPYR intervention stage.Simpson and Shannon index in ZBPYR group were higher than those in SYD group.There was no significant difference between ZBPYR group and normal group.(2)Based on results of differential flora analysis at the phylum level,a total of 8 main bacterias were detected in intestinal microflora of these rats.Firmicutes,Bacteroides and Actinobacteria are three major components of fecal bacteria in SYD rats.Among them,Firmicutes was the most abundant phylum.Here,the changes of dominant intestinal flora of rats in each group from the 1st to 5th week were compared.The results showed that changes were significant in each stage.At the first week of spleen-qi deficiency stage,compared with the normal group,the ratio of Firmicutes and F/B in spleen-qi deficiency group was significantly increased,while Bacteroides and Actinomycetes decreased.At the 2nd week,compared with the normal group,the proportion of Firmicutes and F/B in spleen-qi deficiency group decreased,while Actinobacteria increased significantly.Compared with the normal group,the abundance of Bacteroides had an upward trend,but there was no statistical significance.At SYD stage,the proportion of Firmicutes,F/B and Actinobacteria in intestinal flora of SYD rats have an increasing tendency,while the proportion of Bacteroides have a downward tendency.But those differences both have not statistically significant.At the end of ZBPYR intervention stage,the levels of Firmicutes and F/B in SYD group increased compared with the normal group,while Bacteroides and Actinobacteria decreased in the 5th week.Compared with the SYD group,Firmicutes and F/B in ZBPYR group were significantly lower than those in SYD group,while the proportion of Bacteroides and Actinobacteria increased.(3)The results of intestinal flora analysis based on genus level showed that the overall level of Lactobacillus in intestinal flora of rats with SYD decreased from the 1st to 5th week.The level of Lactobacillus in the 5th week was lower than that in the 1st and 2nd week.The relative abundance of Clostridium showed a gradual upward trend from 1st to 5trh week.The level of Oscillospira was lower in SYD stage than that in the stage of spleen-qi deficiency,and increased at the 4th to 5th week.Metastats analysis showed that the intestinal differential floras of each group were constantly changing in three stages.It mainly includes Lactobacillus,Oscillospira,Bifidobacterium,Bacteroides,Adlercreutzia,Anaerostipes,Rothia,Aggregatibacter,Allobaculun,Prevotella,Butyricimonas,Cornebocterium,Actinobacillus,Blautia,Ruminococcus and Parapreivotella.Compared with the normal group,the levels of Lactobacillus and Paraprevotella in the SYD group were significantly increased,while Adlercreutzia and Ruminococcus were significantly decreased at the end of the 5th week.Compared with SYD group,the abundance of Lactobacillus decreased significantly,while the level of Adlercreutzia and Ruminococcus increased in ZBPYR group.There was no significant difference in Lactobacillus,Ruminococcus,Adlercreutzia and Paraprevotella between ZBPYR group and normal group.2.Study on the changes of intestinal mucosal mechanical barrier in SYD rats and the effect of ZBPYR.The results of Western blotting showed that the levels of Claudin-1 and Occludin protein in SYD rats jejunum were significantly lower than those in normal group,and the expression of ZO-1 protein tended to decrease.The expression of Claudin-1 and Occludin protein in ZBPYR group was higher than that in SYD group,and the expression of ZO-1 tend to increase in ZBPYR group than that in SYD group.However,there was no significant statistical significance.The mRNA expression of relative tight junction proteins in jejunum of rats were detected by qPCR.Compared with normal group,the expression of Claudin-1 and Occludin mRNA in SYD rats jejunum decreased,and the expression of ZO-1 mRNA has a downward tendency.The expressions of Claudin-1,Occludin and ZO-1 mRNA in ZBPYR group were higher than those in SYD group.While there was no significant difference in the expression of Claudin-1,Occludin and ZO-1 mRNA between ZBPYR group and the normal group.3.Study on the changes of intestinal mucosal immune barrier in SYD rats and the effect of ZBPYR.The contents of sIgA,IFN-γ,IL-4,IL-10,IL-17 and TGF-β in jejunum of SYD rats were detected by the method of ELISA.The results showed that contents of slgA,1L-4,IL-10 and TGF-β in SYD group were lower than those in normal group.The contents of IFN-y and IL-17 increased.Compared with SYD group,the expression of sIgA,IL-4,IL-10 and TGF-β increased,while the expression of IFN-y and IL-17 decreased in ZBPYR group,but there was no significant difference compared with normal group.Conclusion:1.The changes of intestinal flora diversity during SYD modeling were dynamic.At the end of spleen-qi deficiency modeling stage,the diversity of intestinal flora in model group increased,while the diversity did not change at SYD modeling stage.At ZBPYR administration stage,the diversity of intestinal flora in SYD rat was reduced,and the intervention of ZBPYR could increase the intestinal flora diversity of SYD rats.2.Under the state of SYD,the number and structure of intestinal flora were disordered.With development of SYD syndrome,the level of probiotics Lactobacillus can be reduced,and the proportion of Clostridium can be increased to a certain extent.It is speculated that Lactobacillus and Clostridium may be the specific bacteria in the development of SYD syndrome.In the state of SYD,decreased levels of butyric acid-associated bacteria(Rothia and Butyricimonas)may affect the normal function of intestinal mucosal barrier.3.ZBPYR can improve dysfunctional intestinal flora of SYD rats by down-regulating the proportion of Firmicutes,F/B and Lactobacillus,while increasing the abundance of Bacteroides,Actinobacteria,Ruminococcus and Adlercreutzia.4.The down-regulation of Claudin-1,Occludin,ZO-1 protein and mRNA expression in jejunum of SYD rats may be one of the important factors leading to the damage of intestinal mucosal mechanical barrier.ZBPYR can improve the damage of intestinal mucosal mechanical barrier in SYD rat by up-regulating the levels of Claudin-1,Occludin and ZO-1 mRNA,and increasing the expression of Claudin-1 and Occludin protein.ZBPYR can improve intestinal mechanical barrier.5.The ratio of Th1/Th2 and Th17/Treg cytokines in intestinal tract of SYD rats was unbalanced,and the immune barrier function of intestinal mucosa was significantly decreased.ZBPYR can effectively improve abnormal changes of intestinal mucosal immune function in SYD rats by regulating the proportion of cytokines secreted by Thl/Th2 and Th17/Treg cells,thereby reducing the injury of intestinal mucosal immune barrier.