The Evaluation Of The Efficacy And Regulatory Mechanisms Of Uric Acid-reducing Peptides And Flavonoids Based On A New High Uric Acid Model

Hyperuricemia,a metabolic disease caused by purine metabolism disorder,is closely related to the occurrence of metabolic syndromes such as hypertension,hyperlipidemia and diabetes.It has become one of the important diseases threatening human health.At present,most drugs aimed at lowering uric acid have toxic side effects,and in vitro screening of uric acid-lowering active substances are inefficient or the evaluated activities differs greatly from the physiological conditions of the body.Food-derived functional factors with natural activities and additional advantage of less or reduced side effects are gradually emerging.In this paper,a new high-uric acid organoid model for screening and evaluating the efficacy of uric acid-lowering functional factors was constructed based on a stable differentiated three-dimensional organoid system.Then,gene editing technology was used to construct a spontaneously high uric acid mouse model,which simulates the purine metabolism disorder.This was then used to clarified the uric acid-lowering efficacy of food-derived functional factors in vivo and its regulatory mechanism from different dimensions.Using a three-dimensional culture technology,a liver organoid culture system with stable morphological characteristics was obtained.Ed U and Ki67 methods were used to evaluate the proliferation characteristics of the liver organoids,and the results suggested that liver organoids have high proliferation ability.The differentiation results showed that the organoids ALB,HNF4?,E-cadherin,ZO-1,CFTR and KRT19 genes were positively regulated.It is consistent with the detection results of the key liver markers HNF4?and E-Ca co-stained,ZO1 and CK19 co-stained,which suggested that the system had the characteristic properties of liver tissues.In addition,the transcriptome sequencing results of the liver organoids showed that liver organoids had stable proliferation and differentiation functions,and expressed key purine metabolism genes.The above results provided an important research basis for the subsequent construction of a high uric acid model.A new high uric acid organ model was established.First,uric acid detection conditions and stability analysis was optimized.The results showed that the HPLC detection conditions had high stability and sensitivity,and the uric acid solution prepared with 1?mol/L Na OH shows high stability.Based on the stably differentiated organoid system,the gene expression of key enzymes in the purine metabolism pathway of organoids was evaluated,and the results suggested that liver organoids showed a complete purine metabolism pathway at the m RNA level.The xanthine oxidase(XO)gene XDH was significantly up-regulated with a prolonged differentiation time(p<0.001).Western blot results also demonstrated that the organoid differentiation time was prolonged and XO expression increased.Using highly differentiated organoids as the modeling system and uric acid production as the evaluation index,the optimal conditions for screening high uric acid modeling were as follows,0.5 mmol/L inducer xanthine concentration,5×10~4cells/m L organoid plating density and an induction time of 24h.Allopurinol significantly reduced the uric acid content of organoids(p<0.01),and showed a certain dose-effect relationship.The immunofluorescence results showed that the morphological structure of the high uric acid organoid model was complete,and the cell survival rate was high.The above results suggested that a new type of organoid model with high uric acid was successfully constructed.This allowed for the in vitro rapid evaluation of the efficacy of food-derived functional factors and the exploration of their regulatory mechanisms.In the high uric acid organoid model,histidine dipeptide(carnosine,anserine)and flavonoids(puerarin,quercetin)had significant uric acid-lowering activity within a certain concentration range(p<0.05).The results of the immunofluorescence HNF4?and E-Ca co-staining,ZO1 and CK19 co-staining showed that carnosine and puerarin were less toxic to organoid cells.At the same time,the studies on the regulation of purine metabolism genes showed that carnosine negatively regulated the key enzyme genes for de novo synthesis of GART and PAICS.Furthermore,anserine negatively regulated the key enzyme genes for salvage synthesis of APRT and the key enzyme gene for uric acid synthesis of XDH.Puerarin negatively regulated the key enzyme genes for de novo synthesis of PPAT,PAICS,ATIC,and salvage synthesis of HPRT.Using CRISPR/Cas9 gene knockout technology,a spontaneous high uric acid(Uox-ko)mouse model was developed,with stable genetic properties which conforms to Mendelian inheritance laws.Phenotypic analysis of mice showed that the Uox-ko homozygous mice did not express uricase,blood uric acid content was significantly higher than that of wild-type mice,and mean uric acid was significantly higher in male compared to female mice.Mice homozygous after 8 weeks of birth had a higher survival more than 50%.Comparative analysis of Uox-ko mice and high-fructose,potassium oxonate and high uric acid mice showed that the uric acid level of the Uox-ko group was significantly higher than that of the high-fructose and potassium oxonate groups(p<0.01).Glucose tolerance,HE staining of kidney tissue and blood routine results suggested that high uric acid content had varying degrees of influence on glucose metabolism disorders,kidney damage,red blood cell destruction and the immune system.In the in vivo evaluation of the uric acid-lowering efficacy of puerarin and the exploration of the regulatory mechanism,it was found that Uox-ko mice were sensitive to uric acid lowering-positive drugs.After two weeks of puerarin intervention,uric acid level was significantly reduced(p<0.05),which was consistent with the results of the high uric acid organ model.The results of HE staining of tissue organs of the Uox-ko mice showed that the model and allopurinol groups had certain level of liver and kidney damage.Allopurinol intervention caused damage to the intestinal mucosa of the mice.After the puerarin intervention,the tissue morphology and cell structure of mice liver and kidney were significantly restored.From the results of endogenous purine and xanthine oxidase content in various organs and tissues,puerarin could increase renal uric acid excretion and/or reduce the generation of uric acid content through the reduction of purine precursors.The purpose of this study was to lower uric acid level using food-derived functional factors.The construction of a new high uric acid organoid model is an upgrade and a breakthrough for the two-dimensional high uric acid cell model.Furthermore,gene editing technology was used to construct a spontaneously high uric acid mouse model to simulate the process of purine metabolism,forming a comprehensive evaluation system of multi-dimensional in vitro high-throughput screening and in-vivo deep-level simulation.Based on the evaluation system of the high uric acid model,the effect and mechanism of histidine dipeptide(carnosine,anserine)and flavonoids(puerarin,quercetin)in lowering uric acid were demonstrated and confirmed.This provides theoretical guidance for further research into similar uric acid-lowering active substances in the food and related fields.