Pharmacological Efficacy And Molecular Mechanism Of Stewed Rhubarb In The Treatment Of Chronic Renal Failure In Mice Through Regulation Of Intestinal Flora Disorders

Research Objectives:This study aimed to investigate the effects of Stewed Rhubarb(SR)on renal function,renal pathological changes,renal inflammation,renal fluid metabolism,renal fibrosis,intestinal barrier integrity,intestinal flora structure,and intestinal flora metabolites in mice with chronic renal failure(CRF)induced by 0.2%adenine diet.This study investigates the therapeutic effects of SR on CRF and provides insight into the role of intestinal flora in the treatment of CRF.This study provides a scientific basis for the clinical use of SR in CRF treatment.Research methods:1.Based on the understanding of the etiology,pathogenesis,and treatment of CRF and the theory of"intestine-kidney axis",the TCM theory of SR on CRF were analyzed and discussed.2.In this study,male C57BL/6 mice were selected as experimental animals and 0.2%adenine feed was used as the modeling drug to construct the CRF model.3.In this study,there were 36 male C57BL/6 mice randomly divided into four groups(nine mice each).1)Ctrl group,fed with normal chow diet and administered with saline by gavage for two weeks;2)CRF group,fed with 0.2%adenine diet(w/w)and administered with saline by gavage for two weeks;3)Ctrl+SR group,fed with normal chow diet and administered with SR decoction(2.0 g crude SR/kg)for two weeks by gavage;4)CRF+SR group,fed with 0.2%adenine diet(w/w)and administered with SR decoction(2.0 g/kg)by gavage for two weeks.During the animal experiment,the body weight and water drinking of mice were monitored.The changes in renal appearance,blood urea,blood creatinine,and renal pathology(H?E staining,PAS staining,and MASSON staining)were observed in each group at the end of the experiment.The effects of SR on renal inflammation(NLRP3,TLR4,IL-1?,TNF-?),renal aqueous metabolism(AQP2-4),renal fibrosis-related factors(E-Cadherin,Col1a1,and Fibronectin)in CRF mice were further examined by RT-PCR.The effect of SR on GSK-3?/?-catenin signaling pathway was examined by Western Blotting to confirm the therapeutic effect of SR on renal fibrosis.4.In this study,the colon length and colon pathological sections(H?E staining,Alcian blue staining,and WGA staining)in each group of mice were observed.Further,the changes in intestinal barrier-related factors were detected by RT-PCR technique.Bioinformatics analysis of mouse intestinal flora was performed using 16S r DNA sequencing technology to explore the changes in intestinal flora diversity,abundance,and metabolic pathways in CRF mice after SR intervention.The changes in the intestinal metabolites(short-chain fatty acids,bile acids)were further determined using GC-MS and LC-MS to investigate the regulatory effects of SR on the key intestinal metabolites.5.In this study,there were 36 male C57BL/6 mice randomly divided into 4 groups(9 mice each group).An antibiotic-treated gut microbial depletion CRF mouse model was established.The Ctrl and CRF groups were given sterilized water until the end of the experiment,and the CRF+AB and CRF+AB+SR groups were given mixed antibiotic drinking water until the end of the experiment.After 4 weeks of pre-treatment with antibiotics,the levels of major genera were detected by RT-PCR.After gut microbial depletion was observed(5^th week),the Ctrl group was given normal chow and the CRF group,CRF+AB group and CRF+AB+SR group were given 0.2%adenine diet.The modeling period was 6 weeks.The administration methods were as follows:the Ctrl group,CRF group and CRF+AB group were given saline gavage from the 9^th week,and the CRF+AB+SR group was given SR decoction(2 g/kg^-d)gavage from the9^th week.The body weight,diet and water intake of mice were recorded during the experiment.At the end of the experiment,the appearance of kidney,length of colon,blood urea,blood creatinine,pathological changes of kidney and intestine in each group of mice were observed and examined.The effects of SR on renal inflammation,renal aqueous metabolism,renal fibrosis,and factors related to intestinal barrier integrity in gut microbial depleted CRF mice were also detected by RT-PCR.Research Results:1.This study believes that SR has a comprehensive and profound theoretical origin in the treatment of CRF.SR treats CRF from the intestine mainly by regulating the relationship among"spleen,intestine and kidney"through the method of Catharisis Decoction and Activate blood and resolve stasis.Combined with the hot topics of intestinal microecology,it is speculated that SR may play a role in the treatment of CRF by regulating intestinal microflora and its metabolites.2.In this study,a CRF model was constructed by using 0.2%adenine diet.The result shows that body weight and kidney indices of CRF mice was gradually decreased(P<0.01).And the kidney morphology of CRF mice was characterized by obvious shrinkage and paleness.Plasma creatinine and urea were significantly increased(P<0.01),and renal H?E sections showed glomerulosclerosis and tubular damage.The above results show that CRF's model was established.In addition,because the adenine feed was administered directly to the intestine,the changes in the colon were also observed.The colon of CRF mice was found to be significantly shortened(P<0.01),and the colon H?E sections also showed pathological changes such as edema of the intestinal wall and thinning of the muscular layer in CRF mice.This also indicated a certain degree of intestinal damage in CRF mice,and provided ideas for the next experiment.3.SR intervention in CRF mice improved related indexes,including mouse body weight,blood urea,blood creatinine,mouse kidney appearance and morphology,water consumption,and kidney index(P<0.05 or 0.01).H?E staining,PAS staining,and MASSON staining showed that SR could significantly improve the pathological changes of the kidney including renal fibrosis,glomerulosclerosis,tubular dilatation,and atrophy.Moreover,it was confirmed by RT-PCR that SR could effectively improve the changes of renal inflammation,aqueous metabolism,renal fibrosis at the m RNA level.And SR could inhibit renal fibrosis by inhibiting GSK-3?/?-Catenin signaling pathway.4.SR improved colon shortening in CRF mice(P<0.05).H?E staining,Alcin blue staining and WGA staining showed that SR decoction could significantly improve the pathological changes of the colon and protect the integrity of the intestinal barrier(P<0.05 or 0.01).And SR improved intestinal barrier related factors at m RNA levels.Alpha diversity was calculated by a Shannon index,which represented the richness of gut microbiota.It was shown that SR decoction reversed the up-regulation of bacterial richness in CRF mice(P<0.05,vs CRF group).The primary component analysis(PCA)and non-metric multidimensional scaling(NMDS)plot revealed four distinct clusters,suggesting the different structure of gut microbiota among four experimental groups.At phylum levels,as compared to the Ctrl group,the abundances of Firmicutes and Tenericutes phylum were up-regulated,while the contents of Verrucomicrobia and Bacteroidetes phyla were down-regulated in the CRF group.SR did not affect these phylum changes of CRF mice.However,it reversed the increase of Bacteroidaceae and the decrease of Rikenellaceae and Erysipelotrichaceae at family levels in the CRF group.The abundances of a series of bacteria were significantly altered in the CRF group at genus levels.Among them,the content of Parvibacter was promoted after SR decoction treatment.In contrast,some other bacteria were decreased in abundances,including Odoribacter,Acetatifactor,Alistipes,Anaerotruncus,Blautia,Clostridium,Desulfovibrio,Enterococcus,Lachnospiraceae,Marvinbryantia,Rikenellaceae,Rikenella,Ruminiclostridium,and Roseburia.The contents of gut microbiota metabolites in fecal samples among four experimental groups were quantified by LC/GC-MS analysis.The level of acetic acid was increased in the feces of CRF mice but remarkably decreased after SR treatment(P<0.05).In contrast,the fecal levels of propionic acid and valeric acid in CRF mice were reduced,whereas SR statistically reversed these changes(P<0.05 or 0.01).Notably,the contents of four SCFAs were also increased in control mice with SR treatment.Consistently,the relative proportions of four SCFAs in feces were changed as indicated by the increased abundance of acetic acid and decreased abundance of pentanoic acid in CRF mice,which were markedly curbed by SR.Meanwhile,SR decoction suppressed the reduction of fecal indole in CRF mice(P<0.01).Compared to the Ctrl group,the total BAs in feces were significantly increased in CRF mice(P<0.01).Although SR failed to block the increment of total BAs in CRF mice,it inhibited the changed proportions of several individual BAs,like UCDCA and CA.The absolute contents of individual BAs in feces among experimental groups were determined.Except for T-?-MCA,TCA,and TDCA,the levels of other BAs were elevated in the feces of the CRF group(P<0.01).Among them,the contents of TUDCA and CDCA were statistically reduced after SR intervention(P<0.05 or 0.01),while the contents of T-?-MCA and T-?-MCA were further promoted by SR decoction(P<0.05or 0.01).The PICRUST analysis was employed to assess the impact of SR on metabolic pathways of gut microbiota in CRF mice.Seven evidently changed ones were screened for comparison among four experimental groups.Compared to the Ctrl group,there were five pathways found to be affected in the CRF group(P<0.01).Among them,the metabolic activity of two pathways was upregulated,including Tryptophan metabolism and synthesis/degradation of ketone bodies;three pathways were downregulated,including pentose and glucuronate interconversions,amino sugar and nucleotide sugar metabolism,and fructose and mannose metabolism.Conversely,most of these altered pathways were recovered after SR treatment(P<0.01 or 0.05).To explore the association between gut microbiota dysbiosis and CRF occurrence,Spearman's correlation coefficient between physiochemical indexes and 23 genera with the greatest changes in abundance among four experimental groups were calculated.17 bacteria(Anaerotruncus,Blautia,Clostridium,etc.)were negatively correlated with body weight,kidney index,E-cadherin level,and metabolism of SCFA and indole,while positively related to renal function,Col1a1,and acetic acid levels,and BA metabolism.By contrast,totally different correlations were observed between the other six genera and physiochemical parameters of CRF mice,such as Akkermansia,Alistipes,and Coriobacteriaceae,etc.The modification of these genera by SR was confirmed to be beneficial in mitigating CRF progression.5.After AB treatment for 4 weeks,the intestinal bacteria of mice were almost depleted as indicated by the undetectable abundances of all bacteria and major phyla in comparison with those of the Ctrl and CRF group.Most of the deteriorated physiochemical parameters in the CRF group were not improved in either the CRF+AB group or the CRF+AB+SR group,including the growth curve,weight gain,renal fibrosis,and inflammation,disorders of water metabolism in kidneys.As compared to the CRF group,the change of kidney index and levels of urea and creatinine in plasma were statistically suppressed in mice of the CRF+AB group due to the depletion of gut microbiota(P<0.05).However,SR decoction failed to further strengthen the AB-initiated protective effect.Meanwhile,SR decoction also did not rescue the intestinal damage to CRF mice,as can be seen by the worsened colonic structure in the CRF+AB+SR group.The above results imply that SR decoction had no alleviating effect on the occurrence of CRF in mice with gut microbiota depletion.But fecal transplantation experiments are needed to confirm this conclusion since antibiotics can retard the progression of CRF.Research Conclusions:1.SR treats CRF from the intestine mainly by regulating the relationship among"spleen,intestine and kidney"through the method of Catharisis Decoction and Activate blood and resolve stasis,which provides theoretical guidance for further exploration.2.A CRF mouse model can be successfully constructed by a 0.2%adenine diet,which has the advantages of easy molding,model stability,and no mortality.3.The study confirmed that SR could improve renal function,renal inflammation,renal water metabolism,and renal fibrosis.The potential molecular mechanisms underlying the therapeutic effects of SR decoction were related to the reshaping of imbalanced gut microbiota and suppression of abnormal intestinal metabolite production,mainly including the protection of intestinal barrier by SR,modulating the structure of intestinal microbiota,decreasing the abundance of pathogenic bacteria(e.g.Clostridium spp.),increasing the abundance of probiotic bacteria(e.g.Parvibacter spp.)and the beneficial metabolites of the intestinal flora(e.g.short-chain fatty acids and indole).