| PurposeChronic kidney disease(CKD)has become a global public health problem.Professor Xusheng Liu from Guangdong Provincial Hospital of Traditional Chinese Medicine developed Bupi Yishen Formula(BYF)and a multi-center,double-blind,randomized controlled trial has demonstrated that the BYF group experienced slower renal function decline compared to the losartan group over 48 weeks,without significant group differences in the incidence rates of adverse events.However,the underlying mechanism of the effect of BYF on CKD is unclear.Based on the "gut-kidney axis" theory,microbiome-metabolomic analysis and cell experiments were applied to explore the the underlying mechanism of the effect of BYF on CKD.Methods1.Animal experiment:The 5/6 nephrectomy(5/6Nx)model was used as CKD model,and 40 rats were randomly divided into four groups:Sham Group),5/6Nx Group,Low-dose BYF(L-BYF Group)and High-dose BYF(H-BYF Group).Sham Group and 5/6Nx group were fed with sterile water,while L-BYF Group and H-BYF Group were given low-dose and high-dose BYF,once per day for 4 weeks.At the end of the study,serum,urine,feces,kidney and colon samples were collected for renal function test,urine protein quantification,kidney and colon pathology,western-blot method to detect ZO-1,Occludin,Claudin-1,IL-22 in colon tissue and AhR,CyP1Al,CyP1B1 kidney tissue,ELISA to detect serum IL-1? and IL-6 levels,Second-generation sequencing to detect fecal intestinal flora and non-targeted metabonomics to analysis metabolic changes.2.Cell experiment:Human proximal tubular epithelial cells(HK-2)was induced for injury by to induce injury indoxyl sulfate(IS).Astragaloside?(AS?)was used as an intervention medicine.Western-blot,flow cytometry,immunofluorescence and siRNA transient transfection were applied to discover the mechanism of inhibiting AhR expression in IS-induced oxidative damage and apoptosis of HK-2 cells,thereby clarify the role of BYF in inhibiting AhR signaling pathway to protect against kidney injury.Results1.Compared with Sham Group,serum creatinine,urea nitrogen,24h urine protein quantification,IL-6 and IL-1? in 5/6Nx Group were significantly decreased(P<0.05).Compared with 5/6Nx Group,serum creatinine,urea nitrogen,24h urine protein quantification and IL-6 in H-BYF Group were significantly decreased(P<0.05)and IL-? showed a downward trend,but there was no statistical difference(P>0.05)).HE staining showed there were obvious glomerular structural disorders,enlarged renal tubule lumen,renal tubule atrophy,renal interstitial fibrosis and mononuclear lymphocyte infiltration in the renal tissue of 5/6Nx Group.Above pathological changes have been improved in L-BYF Group and H-BYF Group,and H-BYF Group showed more obvious improvement.Masson staining showed that there was no fiber deposition in the Sham group;a large amount of collagen fiber were deposited in the renal interstitium in 5/6Nx Group,and the collagen fiber deposition in the kidney tissue of rats decreased in L-BYF and H-BYF Group.PAS staining showed that there were no obvious pathological changes in the Sham group;5/6Nx rats showed mesangial cell and mechanism proliferation,renal tubule lumen enlargement,interstitial fibrosis,renal tubule atrophy,and mononuclear lymphocytes infiltration.In BYF treatment groups,the pathological damage of the kidney was improved,especially in H-BYF Group.In Western-blot analysis,compared with Sham group,the expression of AhR,CyP1A1 and CyPIBl in kidney tissues of 5/6Nx Group significantly increased(P<0.05);compared with 5/6Nx Group,the expression of AhR,CyP1A1 and CyP1B1 in the kidney of H-BYF Group tissue decreased significantly(P<0.05).2.Colon tissue pathology showed that the colonic mucosal tissue of rats in Sham Group was compact and there was few inflammatory cell;in the colon tissue of rats in 5/6Nx Group,there was mucosal edema,loose lamina propria tissue and severe inflammatory cell infiltration.Compared with 5/6Nx Group,the edema of the mucosal layer and lamina propria and the infiltration of inflammatory cells in the mucosal layer of rats in L-BYF Group and H-BYF Group was significantly improved.The results of western-blot showed that compared with Sham group,the expressions of ZO-1,Occludin,Claudin-1 and IL-22 in the colon tissue of 5/6Nx Group were significantly decreased(P<0.05);compared with 5/6Nx Group,the expressions of ZO-1,Occludin,Claudin-1 and IL-22 in the H-BYF Group were significantly increased(P<0.05).3.The analysis of intestinal flora:As for ?-diversity,based on the OTU level,the Shannon index of 5/6Nx Group was higher than that of H-BYF Group,and the Simpson index of 5/6Nx Group was lower than that of H-BYF Group,both showing significant difference(P=0.0011 and P=0.0006).As for?-diversity,the results of PCoA analyses further revealed difference in the colonic bacterial communities between 5/6Nx Group and H-BYF Group.In the phylum level analysis,the Bacteroidete and Firmicutes were the two major microbiome members.F/B ratio was higher in 5/6Nx Group than that in Sham group and decreased in H-BYF Group.Compared with 5/6Nx Group,the relative abundance of Prevotella,Phascolarctobacterium and Megamonas was significantly increased while the relative abundance of Clostridiaceae,Haloplasmataceae,Micrococcaceae,and Pseudomonadaceae was decreased in H-BYF Group.4.In metabolomics analysis,405 and 195 metabolites were identified in negative and positive ion modes,respectively.Metabolic pathway enrichment analysis of differential metabolites based on the Kyoto Encyclopedia of Genes and Genomes database identified 35 metabolic pathways,among which three pathways(tyrosine and tryptophan biosynthesis,tryptophan metabolism,and tryptophan-mediated regulation of inflammatory factors)were related to tryptophan.Compared with 5/6Nx Group,the level of indoxyl sulfate(IS)and kynurenic acid(KA)were decreased significant while the level of melatonin increased obviously(P<0.05).The results of western-blot showed that compared with Sham group,the expressions of AhR,CyP1A1 and CyP1B1 in the kidney tissue of 5/6Nx Group were significantly increased(P<0.05);compared with 5/6Nx Group,the expressions of AhR,CyP1A1 and CyP1B1 in the H-BYF Group were significantly decreased(P<0.05).5.Cell experiment:Compared with Normal Group,abnormal cell morphology,decreased mitochondrial membrane potential and increased ROS was observed in IS Group,accompanying with increased expression of AhR signaling pathway molecules including AhR,CyP1A1 and CyP1B1(P<0.05).Compared with IS Group,HK-2 in AhR-siRNA+IS Group showed inhibited AhR expression,increased mitochondrial membrane potential,decreased ROS content,and decreased cell apoptosis.Compared with AhR-siRNA+IS Group,AhR-siRNA+IS+ASIV Group that added with ASIV inhibited the expression of AhR in the cells,but the number of apoptosis cells did not further decrease.The results of molecular docking simulations showed that ASIV binded to the surface of AhR closely and perfectly.ASIV binded to the active site of AhR protein,and was close to the active site of TYP 239,LEU 253,ASN 244,GLU 243 and HIS 153 to form a hydrogen bond interaction.Conclusion1.In this study,it was confirmed that BYF could reduce serum creatinine,urea nitrogen and proteinuria level in 5/6 nephrectomy rats,and alleviate kidney pathological damage.2.Based on the intestinal flora,the study found that BYF regulated the intestinal flora,reduces the relative abundance of toxin-producing bacteria and other pathogenic bacteria,increased the relative abundance of probiotics,and promotes the production of short-chain fatty acids,promote the expression of tight junction protein and IL-22,improve the intestinal barrier function.3.Based on the non-targeted metabolomics method of LC-MS/MS,the analysis of the KEGG enrichment pathway suggested that tryptophan metabolism is one of the metabolic pathways of BYF to improve renal function.BYF regulated tryptophan metabolism,inhibited the kynurenine pathway to produce kynurenic acid and the indole pathway to produce indoxyl sulfate,and promotes the serotonin pathway to produce more melatonin,which also confirmed the reduction of uremic toxins in CKD rats of the first part study.At the same time,BYF inhibited the expression of AhR,CyP1A1 and CyP1B1 in the kidney.4.In in vitro experiments,the expression of AhR was an important cause of indoxyl sulfate-induced oxidative stress in renal tubular epithelial cells.Astragaloside ? is the main component of BYF,and its role in inhibiting oxidative stress is AhR is the target of action.Therefore,BYF may play a role in protecting kidney function by inhibiting the AhR signaling pathway.5.The full text of the study constitutes the potential mechanism that BYF may regulate intestinal flora,protect the intestinal barrier/regulate tryptophan metabolism,reduce uremic toxins,inhibit AhR signaling pathway,thereby improving renal function. |
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