Study On The Therapeutic Effect Of Geranium Wilfordii Maxim On Hyperuricemia Rats Based On Transcriptome And Metabolomics Analysis

Objective:Optimize the extraction process of xanthine oxidase inhibitor in Geranium wilfordii Maxim（GWM）.Analyze the inhibitor chemical constituent and the components absorbed into blood.Investigate the effect of GWM on hyperuricemia rats by combining pharmacodynamics,transcriptomics and non-targeted metabolomics,which provides experimental bases for GWM application in the treatment of hyperuricemia.Methods:1.Using xanthine oxidase inhibitory activity as the index of investigation,one-way investigation and response surface test were used to optimize the extraction process of GWM,and to determine the optimal extraction process parameters.2.Determine the chemical constituent and the components constituents absorbed into blood of GWM extracts by using UPLC-Q-Orbitrap MS/MS.3.The rat model of hyperuricaemia was constructed by gavage of hypoxanthine and potassium oxonate,and was randomly divided into a blank group,a model group,an allopurinol group（positive group）,a low dose group of GWM,a medium dose group of GWM,and a high dose group of GWM.Body weight and liver and kidney weight were measured,and organ index was calculated.24-hour urine was collected and volume was recorded.Biochemical kits were used to determine the content of uric acid,creatinine,urea nitrogen and xanthine oxidase,and to calculate the fraction excretion of uric acid.The content of pro-inflammatory factors TNF-αand IL-6 was determined by ELISA method in rat kidneys.Rat kidneys were observed for the appearance of the morphology,and the damage of glomeruli in rats was observed by HE staining.Western-blot method was used to detect the expression of related transporter proteins URAT1,GLUT9,ABCG2,NPT1 and AQP1 in rat kidney tissues.4.Based on the RNA-seq transcriptomics technology,the effect of the alcoholic extract of GWM on the renal gene expression of rats with hyperuricemia was determined,differentially expressed genes that could improve hyperuricemia was screened,and the functional categories of the differentially expressed genes and the metabolic pathways they were involved in by GO enrichment and KEGG enrichment was searched.5.UPLC-Q-Orbitrap MS/MS technique combined with multivariate statistical analysis was used to investigate the effects of the alcoholic extract of GWM on metabolites in the kidneys of hyperuricemic rats,to screen for differential metabolites that can ameliorate hyperuricemia,and to search for metabolic pathways related to them through KEGG enrichment.Results:1.The optimized extraction process of GWM is as follows,ethanol concentration:70%,extraction time:1.5 h,extraction temperature:60℃,IC₅₀ value:0.3991 mg/m L.2.37 compounds,including quercetin and rutin,were identified from the extract of GWM,which can be classified as flavonoids,phenolic acids and alkaloids.19 of the 37 identified compounds,including gallic acid and ellagic acid,were the prototypical constituents absorbed into blood.3.GWM reversed the elevation of renal index due to impairment,lowered the levels of uric acid and creatinine in serum,and increased the fraction excretion of uric acid,lowered the activity of xanthine oxidase in serum and liver,reduced the synthesis of uric acid,lowered the levels of TNF-αand IL-6 in the kidneys,and alleviated the pathological damage of renal tissues,lowered the expressions of uric acid reabsorption proteins URAT1 and GLUT9,as well as AQP1,increase the expression of uric acid secretion protein ABCG2 and NPT1,and increase uric acid excretion.4.Combined with bioinformatics analysis,a total of 185 renal differential genes were obtained including Xdh,Abcg2,Slc22a8,Slc22a12,Ccl2,IL-6,IL-1βand S100a9,ect.These genes are involved purine metabolism,arginine and proline metabolism,phenylalanine metabolism,NF-κB signaling pathway,IL-17 signaling pathway,and glycolysis/glycolysis,etc.The q-PCR method was used to further verify that the alcoholic extract of GWM has a regulatory effect on the expression of key differential gene m RNAs,such as Xdh,Abcg2,Ccl2and S100a9 in the kidney.5.A total of 25 differential metabolites were screened in combination with multivariate statistical analysis,mainly including uric acid,taurine,riboflavin,anserine,xanthine and Glutamine.The differential metabolite enrichment analysis showed that the extracts of GWM could regulate 17 metabolic pathways,including purine metabolism,arginine biosynthesis,pyrimidine metabolism,tryptophan metabolism,and phenylalanine metabolism.Conclusion:GWM can play a uric acid-lowering role in terms of reducing uric acid synthesis,promoting uric acid excretion,improving renal injury,and diuresis,which provides a scientific basis for the development of the drug of old stork for the treatment of hyperuricaemia,and expands the use of the drug.