| Purpose:1.Stool samples of healthy controls and gout patients before and after treatment were collected,analyzing the difference in intestinal flora between gout patients and healthy controls,and the effect of QingreLishi Prescriptions on the intestinal flora of gout patients.2.To investigate the effect of HuShenTongFengTai(HSTFT)on intestinal flora in hyperuricemia rats,and its mechanism of inhibiting the toll-like receptor 4(TLR4)pathway,activating PI3K/AKT signaling pathway,regulating adenosine triphosphate binding cassette transporter G2(ABCG2)expression and reducing uric acid.3.The chemical composition of HSTFT was identified and its metabolic characterization in vivo was analyzed,and the main chemical components were docking analysis with TLR4 protein,which laid the foundation for the subsequent mechanism research.Methods:1.We recruited gout patients and healthy age-matched controls from Shenzhen Traditional Chinese Medical Hospital,Patients with gout were treated with QingreLishi Prescriptions of "HSTFT"(group H)and "TongFengTai"(group T)for 2 weeks,and stool samples were collected twice for patients before and after treatment and only once for controls.The samples were analyzed by metagenomic high-throughput sequencing technology.2.we established the rat model of hyperuricemia induced by potassium oxalate administration combined with an intraperitoneal injection of hypoxanthine,which was divided into a blank group,model group,allopurinol group,and Chinese medicine group(HSTFT).The model was treated for 2 weeks,and samples of blood,liver and intestinal contents were collected for detection.Uric acid and creatinine were detected by visible fraction spectrophotometry,XOD and ADA were detected by ELISA,the mRNA expressions of TLR4,PI3K,AKT and ABCG2 were detected by qPCR,and the intestinal contents of rats were analyzed by macrogene sequencing.3.we divided Male Wistar rats into blank group and drug administration groups(HSTFT).Blood,urine,feces and bile were collected at multiple time points after drug administration.Tissues(heart,kidney,liver,lung,and spleen were obtained after the rats execution.The main chemical components and metabolic distribution of HSTFT in vivo were analyzed based on UPLC QTOF/MS.By using the relevant database,the core chemical components of HSTFT and TLR4 protein were molecularly-docked to provide a basis for further investigation of the mechanism of HSTFT.Results:1.Therapeutic effect:The blood uric acid level,liver and kidney function,erythrocyte sedimentation rate,and CRP of patients before and after treatment were compared,and the results indicated that the blood uric acid level of patients was significantly decreased after treatment,with a statistically significant difference(P<0.05).There were no significant differences in ALT,AST,Cr,BUN,CHOL,LDL,TC,ESR,and CRP(P>0.05).2.Differences in intestinal flora of gout patients before and after treatment with Qingre Lishi Prescriptions:Stool samples were collected from 29 healthy controls,and 30 gout patients before and after treatment(group A/Group B).Analysis of the data reveals that:(1)In the analysis of β diversity,Firmicutes,Bacteroidetes,and Proteobacteria had the highest proportion of genes at both phyla and genus levels.Through PCoA analysis,it was found that there was little difference between group A and group B,and the difference between group A and the control group was more obvious,indicating that there were differences in intestinal flora between gout patients and healthy controls.In the α-diversity analysis,no significant difference was also found between groups A and B,but the gene richness of groups A and B was significantly lower than that of the control group,indicating that the gut flora diversity of gout patients was lower than that of healthy controls.(2)Differential gene or KOs enrichment analysis revealed large differences in microbial abundance between gout patients and healthy controls,with significant enrichment pathways including glutathione metabolism,ubiquinone,and other terpenoid quinones biosynthesis,and tryptophan and pyruvate metabolism.153 distinct KOs were detected between groups A and B,with significant enrichment in glutathione metabolism,and 153 KEGG were significantly different between groups A and B,including taurine and hypotaurine metabolism,folate biosynthesis,and fructose and mannose metabolism.(3)The relative abundance results showed that the microbial composition of patients treated with QinreLishi Prescriptions(group H and group T)were similar after treatment,but the abundance of Verrucomicrobia and Firmiculus in group T was significantly higher than that in group H.Desulfitobacterium and Photorhabdus in group H are higher than in group T,and in the number of genera with significant differences,group T is significantly higher than group H.(4)By constructing A boxplot of the B/F ratio,the expression of six phyla in different individuals before and after treatment was compared,and the A-B-control group was compared between the lower group and the upper group,respectively.It was found that Bacteroidetes and Firmicutes were significantly different,and there was an opposite trend,and their abundance changed after treatment.(5)Correlation analysis was conducted between laboratory indicators and species with significant differences,and an interactive network diagram was constructed.It was found that the correlation between clinical laboratory indicators in the up-regulated group was stronger than that in the down-regulated group.Bacteroides were positively correlated with UA and Cr,and Bacteroides,bifidobacterium,Clostridium,Haemophilus and Lactobacillus were significantly correlated with BUN.3.Results of study on the mechanism of lowering uric acid by HSTFT:(1)The levels of UA in each group were detected:Compared with the blank group,the serum uric acid levels in the model group,Chinese medicine group(HSTFT),and positive drug control group were significantly increased,with statistical significance among all groups(P<0.05);Compared with model group,the serum uric acid level in TCM group was significantly decreased,the difference was statistically significant(P<0.05).Compared with the positive control group,the serum uric acid level in the Chinese medicine group decreased,but there was no significant difference(P>0.05).(2)The levels of BUN and CR in each group were detected:Compared with the blank group,the serum creatinine levels in the model group,Chinese medicine group and positive drug control group had an increasing trend,but the data distribution of each group had no statistical significance(P>0.05).Compared with the blank group,the BUN level of the model group,Chinese medicine group,and the allopurine control group had no significant difference,and the data distribution of each group had no statistical significance(P>0.05).(3)The activities of XOD and ADA in liver tissues of all groups were detected:compared with the blank group,model group,and Chinese medicine group,ADA level in the positive control group(allopurinol)was significantly increased,and the differences were statistically significant(P<0.05).Compared with the blank group,XOD level of the positive control group(allopurinol)was increased,and the difference of data distribution between the two groups was statistically significant(P<0.05).Compared with the blank group and model group,there was no difference in XOD and ADA levels in the Chinese medicine group,and no significant difference in data distribution among all groups(P>0.05).(4)The mRNA expression levels of TLR4,PI3K,AKT2,and ABCG2 in each group were detected:compared with the blank group,the mRNA expression of ABCG2 in the Chinese medicine group was significantly increased,and the difference in data distribution between the two groups was statistically significant(P<0.05).The positive control group(allopurinol)had no significant difference(P>0.05).Compared with blank group,the expression of TLR4,PI3K,and AKT2 in the Chinese medicine group were significantly different,and the difference of data distribution among all groups was statistically significant(P<0.05).However,compared with the model group,the expression of TLR4mRNA in the TCM group was significantly decreased,and the expressions of PI3K and AKT2 were higher than those in the model group,with no significant difference in data distribution among all groups(P>0.05).(5)The intestinal contents of rats were collected and analyzed by metagenomic sequencing:It was found that the microbial abundance of the normal group and positive control group(allopurinol)was higher(65.5%and 69.7%,respectively),while that of the model group(48.7%)and Chinese medicine group(46.8%)was lower.Compared with the normal group and the model group,the proportion of bacteria in the Chinese medicine group was the lowest,only 32.6%,suggesting that Chinese medicine had a greater effect on the composition of bacteria.Through various genus metagenomic annotation analyses,it was found that compared with the normal group,the bacterial nodes of the other three histones were significantly reduced,the verruca subdivision was interrupted in the model group,the lactobacillus YL32 could not be separated nodes in the allopurinol group,and the flow direction of enterobacterium was reduced in the Chinese medicine group,suggesting that each group may play a role by affecting the abundance of different species.4.Results of chemical composition and in vivo characterization analysis by HSTFT:(1)We identified 156 components in HSTFT,including 26 flavonoids,15 phenylpropanes,24 coumarins,6 tryptamines,16 iridoid terpenes,13 alkaloids,10 monoterpenes,6 steroids and steroidal saponins,11 triterpenes saponins,and other glycosides,purines,furans,amino acids and other compounds.(2)According to the HSTFT identification results,51 prototypes were extracted in plasma,urine,feces,or bile.Of these,25 could be detected in plasma,46 in urine,13 in feces,and 12 in bile.(3)Metabolite analysis was performed on the above 51 prototype compounds,and 87 metabolites were matched.Among them,16 metabolites could be detected in plasma,87 in urine,27 in bile,and no corresponding metabolites could be detected in stool.The tissue distribution of the above compounds was analyzed,and the results showed that the kidney and liver were the organs with the largest distribution of chemical components.(4)Based on a systematic analysis of the chemical composition structure of HSTFT,the main components of HSTFT were interlinked with TLR4 protein,and it was found that 5-O-Methylvisammioside,Albiflorin,Gentiopicroside,Hypoxanthine,Inosine,Loganin,Morroniside,Prim-O-glucosylcimifugin could be formed Strong docking and combination ability can be used for subsequent experimental analysis.Conclusion:1.There were differences in intestinal flora between gout patients and healthy controls.The diversity of intestinal flora was significantly lower than that of healthy controls.There was no significant change in intestinal flora abundance before and after treatment with QinreLishi Prescriptions,but there were differences in phylum and genus level between the two groups(H and T),and there were great differences in metabolic pathways.Through enrichment analysis,it was found that glutathione was significantly enriched before and after treatment,suggesting that it might play a role through related pathways.2.HSTFT can significantly reduce the blood uric acid level of hyperuricemia rats,affect the intestinal flora of rats,inhibit the expression of TLR4 mRNA,and promote the expression of PI3K,AKT,and ABCG2 mRNA,suggesting that it may regulate the intestinal flora composition of hyperuricemia rats.It can affect the expression of the TL4/PI3K/AKT signaling pathway and uric acid transporter ABCG2,and play a role in lowering uric acid.3.There are 156 main chemical components of HSTFT in vivo,51 prototype compounds,and 87 metabolites,which are mainly distributed in the kidney and liver.Its main components also have a strong binding ability for molecular docking with TLR4 protein. |
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