To Investigate The Mechanism Of Yishen Qingli Heluo Granule In The Treatment Of CKD Based On Gut Microbiota-SCFAs Mediated Intestinal Barrier Repair

BackgroundChronic kidney disease(CKD)is often accompanied with imbalanced gut microbiota and impaired intestinal barrier.Hence,efforts to ameliorate renal dysfunction by manipulating gut microbial ecosystem are underway.Yishen Qingli Heluo granule(YQHG)is a representative traditional Chinese medicine(TCM)prescription for clinical treatment of CKD.However,its underlying mechanism has not been well elucidated.ObjectiveThis study aimed to investigate the therapeutic mechanism of YQHG in 5/6 nephrectomized rats by targeting gut microbiota and intestinal barrier.MethodsClinical trials:Thirty controls and 60 CKD patients were included,and fecal samples from all subjects were collected for 16S rRNA-based high throughput sequencing.①To reveal the profile of the gut microbiota in CKD patients.② To explore the dominant and marker microbiota corresponding to different enterotypes in CKD patients based on enterotype analysis.Animal experiment:SD rats aged 6-8 weeks were randomly divided into Sham group,Model group and YQHG group.The CKD rat model of Model group and YQHG group was established by 5/6 nephrectomy.The daily doses of gavage substances were as follows:① Sham group(0.9%sterile water);②Model group(0.9%sterile water);③YQHG group(5.6g/kg YQHG).Detailed grouping and drug intervention regimen will be further elaborated.At the end of the experiment,urine and fecal samples were collected from rats before sacrificed.Blood samples,renal and colon tissues were collected from rats on the day of sampling.The above samples were used for the following studies:①A comprehensive assessment of renal appearance(colour,capsule,border),related parameters(Scr,BUN,urinary protein,glomerular fibrosis area,tubulointerstitial fibrosis area),and renal tissue histopathology(H&E,Masson)were conducted to explore the potential therapeutic value of YQHG on CKD.②The collected fecal samples were analyzed for gut microbiota,and the concentration of SCFAs was quantified by gas chromatography-mass spectrometry(GC-MS),all of which were used to investigate the regulatory effect of YQHG on the bacterial communities and the abundance of SCFA-producing bacteria in 5/6 nephrectomized rats.③Colon tissue histopathology(H&E),intestinal permeability(FITC-dextran assay),microbial translocation(FISH analysis),and renal tissue inflammation(IL-6 detection)were comprehensively assessed to explore the improvement of intestinal barrier by YQHG.④The microbiota-transfer study(co-housing and FMT)was used to verify that the therapeutic effect of YQHG was partly mediated by gut microbiota.ResultsClinical trials:① The gut microbiota dysbiosis was observed in CKD patients,and nine CKD-risk related gut microbiota were identified.②The gut microbiota of CKD patients could be classified into Enterotype 1(E 1),Enterotype 2(E2)and Enterotype 3(E3).(1)Analysis of dominant gut microbiota:the dominant bacteria in El were Blautia,Bacteroides,Klebsiella.The dominant bacteria in E2 were Escherichia-Shigella,Blautia,Ruminococcus gnavus.The dominant bacteria in E3 were Blautia,Bifidobacterium,Faecalibacterium.(2)Analysis of marker gut microbiota:the marker bacteria in El,E2 and E3 were Bacteroides,Erysipelatoclostridium,Alistipes,respectively.Animal experiment:①YQHG had nephroprotective effect in 5/6 nephrectomized rats.The renal appearance(colour,capsule,border),related parameters(Scr,BUN,urinary protein,glomerular fibrosis area,tubulointerstitial fibrosis area)and renal histopathology were improved.②YQHG could altered bacterial communities and increased the abundance of SCFA-producing bacteria(ie,Lactobacillaceae,Lactobacillus,Lactobacillus gasseri)in 5/6 nephrectomized rats.③YQHG could increased the concentrations of fecal SCFAs(ie,acetic and butyric acid).④YQHG could improve intestinal barrier damage in 5/6 nephrectomized rats.It was characterized by improved colonic histopathology,decreased intestinal permeability,microbial translocation and IL-6 expression.⑤After receiving gut microbiota from YQHG-treated rats,the renal appearance(colour,capsule,border),related parameters(Scr,BUN,urinary protein,glomerular fibrosis area),and renal tissue histopathology were improved.⑥After receiving gut microbiota from YQHG-treated rats,the abundance of SCFA-producing bacteria(ie,Lactobacillaceae,Lactobacillus,Lactobacillus gasseri)and the concentrations of fecal SCFAs(ie,acetic and butyric acid)were increased.⑦Receiving gut microbiota from YQHG-treated rats improved intestinal barrier damage.It was characterized by improved colonic histopathology,decreased intestinal permeability,microbial translocation and IL-6 expression.ConclusionThe clinical trial revealed gut microbiota dysbiosis in CKD patients.The animal experiments showed that YQHG provided significant renal protection in 5/6 nephrectomized rats by reducing renal fibrosis and inflammation,reestablishing bacterial communities,and improving intestinal barrier.The microbiota-transfer study showed that the protective effect of YQHG was partly attributed to the mediation of the gut microbiota,especially the SCFA-producing bacteria.