The Molecular Mechanisms Of Rhubarb And Its Active Substance Enema Treatment In Rats With Chronic Kidney Disease

Objective:chronic kidney disease(CKD)has become a global health issue.Currently,the underlying mechanisms of the development and progression of CKD remain largely unknown.In clinical practice,CKD patients mainly receive symptomatic treatment and dialysis treatment.The duration for the treatments is very long,which causes heavy burden to the patients,families and whole society.Therefore,it is under intensive investigation to clarify the molecular mechanisms for CKD progression and to identify novel therapeutic option to prevent CKD progression.During the last 50 years,the rhubarb-based enema treatment,as a traditional Chinese medicine therapy,is widely used in clinical practice,and can effectively improve CKD-related constipation and other intestinal dysfunction and promote the removal of uremic toxins,which will slow down the progression of CKD.However,the mechanisms for the therapeutic effects of rhubarb-based enema treatment on CKD is not clear,Very few study has focused on this unknown area.Recently,accumulating studies demonstrate that intestinal microecology,especially gut microbiota,is closely asscoiated with the development of chronic kidney disease.Therefore,based on the model of gut-kidney axis theory,this study explored the molecular mechanisms by which rhubarb and its active substance emodin delay the progression of CKD from the perspective of gut microbiota as the center of intestinal microecology.The study will further promote application of traditional Chinese medicine enema treatment and will lay a solid foundation for the identification of novel strategy to prevent CKD progression.Methods:This study was divided into two parts:1)the effect and mechanisms of rhubarb enema on CKD in rats;2)The mechanisms underlying the functional effect of emodin enema in rats with CKD.5/6 nephrectomy model was used to establish the CKD model in rats,rats after modeling for 8 weeks were randomly divided into control treatment group and experimentaltreatment group,sham operation model was performed for the blank control group.1)In the experiment of rhubarb enema,selecting rhubarb granules as the experiment drug,using ultra-performance liquid chromatography-tandem mass spectrometer mass spectrometry(UPLS-MS/MS)to detect the fingerprints of rhubarb granules and the active content of emodin was quantitatively analyzed using ultra performance liquid chromatography.The rats in the control treatment group were treated with sterile water enema(5ml/day);the rhubarb enema group received 0.lg/ml rhubarb granules solution(5ml/day,as 0.3g/ml raw rhubarb)for 2min.4 weeks after continuous enema,the blood,urine,kidney,colon and fecal tissues were respectively collected.Biochemical indexes measurement(whole blood analysis,serum creatinine,urine creatinine ratio,creatinine clearance rate and hs-CRP),HE staining and Masson staining for renal pathology,immunofluorescence for 8-OHdG,NF-?B protein in the kidney tissues,Western blot for NF-?B protein in the kidney tissues,HE staining for colon pathology,immunohistochemistry staining for claudin-1 and occludin in the colon tissues,the domain bacteria culture(including E.coli,Enteroroccus,Staphylococcus aureus,Fungi,Lacto.bacillus,Bifidbacteria)for fresh fecal samples and serum indoxyl sulfate(IS)using high-performace liquid chromatography-mass spectrometry(HPLC-MS)and limulus reagent method for serum endotoxin lipopolysaccharide(LPS)level were respectively performed to evaluate the effect of rhubarb enema on the pathological changes of kidney tissues and intestinal microecology in rats with CKD.(2)In the emodin enema experiment,the CKD rats in controltreatment group received 0.4%CMC-Na enema(5ml/day),the CKD rats in experimental group received emodin enema(5ml per day,0.215mg/ml)with emodin retention enema 2min.After 4 weeks of continuous enema,the blood,urine and fecal tissues were collected and the biochemical indexes(serum creatinine,urea,whole blood analysis,urine creatinine ratio,creatinine clearance rate),real-time quantitative PCR for fecal 16S rDNA to measure the number of major intestinal bacteria in feces(including E.coli,Lactobacillus,Bifidbacteria,B.fragilis,Enteroroccus,C.perfringens),HPLC-MS method for serum IS level,serum endotoxin lipopolysaccharide(LPS)level using limulus reagent method,second-generation high-throughput sequencing.for gut microbiota with bioinformatics analysis using Mothur 1.29 and QIIME pipelines were respectively performed.The measurement data in this study were expressed as mean ± standard deviation.GraphPad Prism 5.0 software was used for data analysis.When the data follow the normal distribution,one-way ANOVA analysis for groups,and Student-Newman-Kueuls Q test for comparative analysis;nonparametric test,and Cruskal-Wallis H test were used when the data do not follow the normal distribution.P<0.05 was regarded as a statistically significant difference.Results:1)the results in the study of the effect and mechanisms of rhubarb enema on CKD in rats showed:the content ratio of emodin in the rhubarb granules was 0.215%.Compared with the sham operation group,the creatinine,urinary creatinine ratio and LPS level in the sterile water enema group and the rhubarb group were significantly increased(P<0.05),while the creatinine clearance and body weight were significantly decreased(P<0.05).Compared with sham operation group,the levels of IS and hs-CRP in the sterile water enema group were significantly increased(P<.05).Compared with the sterile water enema group,the levels of creatinine,IS and hs-CRP were significantly decreased in the rhubarb enema group(P<0.05),and the creatinine clearance rate was significantly increased(P<0.05).HE staining showed that CKD rats in the control enema group had significant tubular enlargement,protein cast,tubular atrophy and interstitial dilatation associated with many mononuclear cell infiltration and interstitial fibrosis in the kidney tissues,colonic mucosal edema and lamina propria inflammatary infiltration in the colon tissues,and rhubarb enema significantly alleviated these pathological changes occuring in control treatment group.Kidney Masson staining showed significant tubulointerstitial fibrosis in CKD rats from controlenema group.However,after receiving rhubarb enema for 4 weeks,the progression of tubulointerstitial fibrosis with CKD rats was significantly delayed and the tubulointerstitial fibers score was significantly reduced about 18.75%(P<0.05).The expression of NF-?B in the renal glomeruli and tubules of the CKD rats showed a significant increase of 8-OHdG positive region,and the expression of NF-?B was significantly increased in the renal glomeruli and renal tubules suggested by western-blot(P<0.05).After rhubarb enema treatment,the expression of 8-OHdG in glomeruli was not significantly reduced,but the expression of 8-OHdG in renal tubules was significantly decreased(P<0.05)along with the significant decrese of NF-?B expression in residual kidney(P<0.05)which was also,supported by the western blot results(P<0.05).The results of bacteria culture showed that there was no significant difference regarding the number of Enteroroccvs,Staphylococcus aureus and Fungi between the three groups.Compared with sham operation group,E.coli in the sterile water enema control group was significantly increased(P<0.05),E.coli,Lactobacillus,Bifidbacteria in the rhubarb enema group was significantly increased(P<0.05).Compared with the sterile water enema group,the number of Bifidbacteria was significantly increased(P<0.05)in rhubarb enema group.Compared with sham operation group,the expression of claudin-1 and occludin in intestine epithelial was significantly reduced in the control enema group while was increased in rhubarb enema group.2)the results in the study of the effect and mechanisms of emodin enema on CKD in rats showed:compared with the sham operation group,the serum creatinine,urea concentration and creatinine ratio in 0.40%CMC-Na enema group and emodin enema group were significantly increased(P<0.05)while the hemoglobin,hematocrit level,creatinine clearance rate and body weight were significantly decreased(P<0.05).Compared with sham operation group,the levels of IS and LPS in the 0.4%CMC-Na enema group were significantly increased(P<0.05).Compared with CMC-Na enema group,the concentration of urea and creatinine ratio,IS and LPS were significantly decreased in the emodin enema group(P<0.05).Real-time qPCR showed no significant change in the total number of all bacteria between the three groups.C.perfringens increased significantly(P<0.05),while Lactobacillus was significantly decreased in the CMC-Na enema control group compared with the sham group(P<0.05).Compared with CMC-Na enema group,Lactobacillus was significantly increased in the emodin enema group(P<0.05),E.coli,Enteroroccus and C.perfringens were significantly decreased(P<0.05).The second-generation sequencing generated 128,238 sequences,3,744 OTUs in total,and 488 ± 120 OTUs per sample.The rarefaction curves and the alpha diversity analysis had shown the most characters of the samples,with most diversities being captured.Weighted UniFrac PCoA analysis revealed that the gut microbiota structure of the three groups exhibited a deviation away from one another.At the phylum level,Tenericutes was also very abundant and significantly increased in ECI group compared with CMC-Na enema group or sham operation control group(P<0.05).At the genus level,compared with the sham operation group,Lactobacillus in the CMC-Na enema control group was significantly decreased(P<0.05).Compared with the CMC-Na enema group,the the unclassified genera from S24-7 family and the Clostridium were significantly decreased(P<0.05)in the emodin enema group,whereas unclassified genus from RF39 order and Bacteroides were markedly increased(P<0.05)in the emodin enema group with a slight improvement of the Lactobacillus without statistical significance.Clostridium exhibited a significant positive correlation with urea and IS(P<0.05),while Prevotella and Lactobacillus had a significant negative correlation with Urea(P<0.05).Conclusions:Based on the data from this study,we concluded that rhubarb enema can delay the progression of CKD.The possible molecular mechanism is mainly through the regulation of intestinal microecology mainly including the regulation of gut microbiota mainly increasing the number of probiotics Bifidobacteria,restore the mucosal protein claudin-1,occludin and immunoregulation of reducing endotoxin LPS levels,reduce uremic IS production,thereby relieving the system of inflammation,further improving renal function and renal inflammation and oxidative stress and renal fibrosis in CKD.Rhubarb enema can be seen with multi-target multi-level role in CKD rats.We further carried out the effect of emodin monomeric substances in rhubarb on the CKD rat model and its results show that emodin enema treatment can reduce uremic toxoid urea and intestinal uremia IS,reduce serum LPS levels,thereby reducing systemic inflammation and kidney damage,the possible important mechanism is to regulate the structure of gut microbio.ta,in particular reducing the number of harmful bacteria(e.g.,Clostridium,including C.perfringens)and increasing the number of beneficial bacteria such as Lactobacillus in the intestine,and further found that the harmful Clostridium was positively correlated with the levels of urea and IS uremic toxins,while the probiotics Prevotella and Lactobacillus were negatively correlated with urea.The rhubarb active substance emodin could be directly treated to the target of gut microbiota,which was mainly involved in the most important role of mechanism of rhubarb delaying CKD progression.It will improve the progression of the pharmacological mechanism of rhubarb and the the molecular mechanism of related delay CKD progression from the intestinal microecological point.