| Objective:This topic is based on intestinal kidney axis theory and previous research,observation the adjustment of intestinal flora and the improvement of the intestinal mucosa barrier inflammation after rhubarb enema on chronic kidney disease(CKD)model of rat,and to further explore the correlation between the mucosal barrier and intestinal flora after rhubarb enema,to provide a new experimental basis for colon administration of traditional Chinese medicine to delay the progression of chronic renal failure.Methods:In this study,the 5/6 nephrectomy model was used as the chronic kidney disease model,and 50 SD rats were divided into 5 groups,including model group,rhubarb enema low dose group,rhubarb enema medium dose group,rhubarb enema high dose group,and sham operation group.Model group was given sterile water enema as contrast treatment group,radix et rhizoma rhei enema enema each dose group as the experimental group,once-daily given rhubarb granule retention enema,enema four consecutive weeks,collecting blood,urine,kidney,colon,and organization of excrement and urine,detection of biochemical indicators,kidney pathological HE staining,Western blot method to detect colon tissue TLR4,the nf-kappa B,MyD88 protein,pathological colon HE staining,ELISA to detect serum IL-1 beta,IL-6,LPS level,the second generation sequencing of testing fecal intestinal flora,bioinformatics methods to analyze the second generation sequencing results.Results:1)Compared with sham control group(sham),the serum creatinine and urea nitrogen in model group and low,medium and high dose rhubarb enema group were significantly increased(P<0.05).Compared with the model group,serum creatinine was significantly decreased in the high-dose rhubarb enema group(P<0.05).2)kidney and colon HE staining showed in the model group,in the model group,chronic kidney disease(CKD)in the rat kidney tissue appeared obvious increase renal small tube cavity,renal tubular atrophy and associated with many mononuclear lymphocyte infiltration and fibrosis,rhubarb enema for low,medium and high group after treatment all can improve the histopathological changes,with high dose of rhubarb enema group improved the most obvious.3)the quantitative results of western-blot showed that,compared with the sham operation group,the expressions of occludin,claudin-1 and Z01 in the colon tissues of CKD rats in the model group and the rhubarb enema group were significantly reduced(P<0.05),and the expressions of occludin,claudin-1 and Z01 in the colon tissues of the rhubarb enema group were significantly higher than those in the model group(P<0.05).Compared with the sham group,the expressions of TLR4,nf-b and MyD88 in the colon tissues of CKD rats in the model group and rhubarb enema group were significantly increased(P<0.05),while the expressions of TLR4,nf-b and MyD88 in the colon tissues of rhubarb enema group were lower than those in the model group(P<0.05).4)LPS,il-1,and il-6 levels in serum and kidney of rats after enema were detected by ELISA,and the results showed that LPS,i1-1,and il-6 levels in serum and/or kidney of rats in model group and rhubarb enema group were significantly increased compared with the sham operation group(P<0.05).LPS,il-1 and il-6 levels in serum and/or renal tissues of rats in the high-dose rhubarb enema group were significantly lower than those in the model group(P<0.05).5)the second-generation sequencing results showed that,based on the genus level,Shannon index of the sham operation group,model group and rhubarb enema group showed statistical differences(p=0.015).Intestinal microbial diversity was different in different groups.Bacteroidetes(Prevotella,Parabacteroides)and Firmicutes(Lactobacillus)were the major differences between groups.In the model group,Firmicutes(Clostridia,Erysipelotrichi)and Actinobacteria(Actinobacteria,Coriobacteriia)were the major differences.The Rhubarb group consisted mainly of Bacteroidetes(class Bacteroidia),Firmicutes(class Clostridia),and Proteobacteria(class Betaproteobacteria and Gammaproteobacteria).Correlation analysis of intestinal microorganism with intestinal barrier and inflammation showed that Z01,occludin and claudin-1 were positively correlated with intestinal microflora diversity of the sham operation group,and other indicators were positively correlated with intestinal microflora diversity of the Model group.Clostridia,Erysipelotrichi and Clostridium were positively correlated with p-nf-kB and LPS,respectively,while Lactobacillus was negatively correlated with p-NF-kB.Conclusion:Rhubarb enema may down-regulation the intestinal TLR4 pathway by regulating the intestinal flora and intestinal barrier function of CKD model rats,thereby reducing the intestinal and systemic inflammatory state and ultimately delaying the progression of chronic kidney disease. |
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