Intervention Of Quinoa Bran Saponins On Hyperuricemia And Its Mechanism

Hyperuricemia has become the second most common metabolic disease after diabetes,and its global prevalence is increasing year after year.However,the pathogenesis of hyperuricemia is complex,and most of the existing anti-uricemia drugs will cause liver and kidney damage,allergic reaction and other side effects.Therefore,the study of dietary supplements to intervene the occurrence and development of hyperuricemia is extremely important for preserving human health.Quinoa is known as the golden grain.Studies have found that quinoa saponins have antioxidant and antibacterial activities,but there is no report on the effect of hyperuricemia.In this study,quinoa bran saponins(QBS)were isolated and purified to analyze the basic compositions of QBS4.Adenine and potassium oxyazine were used to induce hyperuricemia in animal model,and the intervention effect and mechanism of QBS4 on hyperuricemia were explored.Through co-culture of uric acid and human renal tubular epithelial cells(HK-2),a hyperuric acid cell model was constructed to learn more about QBS4’s molecular mechanism in the intervention of hyperuricemia.Specific research contents and results are as follows:1.Isolation,purification and structural identification of quinoa bran saponins.The crude saponins were extracted from quinoa bran by ultrasonic-assisted ethanol extraction.The D101 macroporous adsorption resins were chosen to purify after various macroporous adsorption resin types were evaluated using analytical and static adsorption rates.Through the process optimization of loading concentration,loading velocity and loading volume,the gradient elution method is used to optimize the elution concentration and elution velocity.Composition 4(QBS4)with the most saponins content was selected for structural identification,and the main saponins and aglygens in QBS4 were analyzed by Fourier infrared spectroscopy,ultra-high performance liquid chromatogi-mass spectrometry and high performance liquid chromatography.The results show that:(1)When the loading concentration of saponins was 5mg/mL and the loading volume was 50 mL,the loading flow rate was 1 mL/min through the D101 macroporous adsorption resin,and the gradient elution was performed at a flow rate of 1 mL/min.Fractions of 10,30,50,70 and 90%were collected to obtain fractions 1-5,respectively.The highest content of QBS4 saponins was 701.42±5.92 mg/g.(2)The saponins in QBS4 were identified by ultra-high performance liquid chromatography and mass spectrometry.Eleven saponins were found,among which 5 belonged to pentacyclic triterpene saponins,accounting for 77.12%of the total saponins content,and the remaining 6 belonged to tetracyclic triterpene saponins.The contents of saponin and oleanolic acid in QBS4 hydrolyzed samples were 2.84±0.01 and 0.90±0.03 mg/10 mg,respectively.2.The intervention effect and mechanism of quinoa bran saponins on hyperuricemia.Adenine and potassium oxazine were used to create a mouse model of hyperuricemia,and 25,50 and 100 mg/kg QBS4 were administered to investigate the effects of QBS4 on blood uric acid levels,blood glucose and lipid,as well as the activities of xanthine oxidase(XOD)and adenosine deaminase(ADA)in liver.MASSON staining and HE staining were used to observe the improvement effect of QBS4 on renal injury in hyperuricemia mice.The mRNA and protein expressions of key genes in the PI3K/AKT/NFκB pathway in kidney tissues were studied by RT-qPCR,Western Blot and immunohistochemistry,in order to explore the intervention effect of QBS4 on hyperuricemia mice and its mechanism.The results show that:(1)Compared with the negative control group,the contents of serum uric acid,urea nitrogen and creatinine in the model group were significantly increased(P<0.01),indicating successful modeling of hyperuricemia mice.The serum uric acid levels in the QBS4 group were considerably lower than those in the model group,urea nitrogen and creatinine of hyperuricemia mice(P<0.01),and improve the rise of blood glucose and blood lipid of hyperuricemia mice(P<0.01).In addition,kidney histopathology showed that compared with the model group,the renal tubular dilatation and glomerular atrophy were reduced in the QBS4 intervention group,and the renal fibrosis area in the model mice was also reduced.QBS4 could improve the renal fibrosis and tubular dilatation in hyperuricemia mice.These results indicate that QBS4 can relieve uric acid,blood glucose and lipid abnormalities and kidney injury in the dose range of 50-100 mg/kg.(2)The preliminary mechanism study showed that the activities of XOD and ADA in the liver of mice in the model group were significantly increased(P<0.05,P<0.01),and the activities of XOD and ADA in the liver of mice in the medium-high dose QBS4 group were inhibited.QBS4 can also reduce the expression of NLRP3 and caspasel inflammasome in the kidney of hyperuricemia mice(P<0.01),suggesting that QBS4 may regulate hyperuricemia by reducing the production of uric acid and alleviating the kidney inflammatory injury.(3)Compared with the negative group,the results of RT-qPCR,Western blot and immunohistochemical detection showed that the expression levels of URAT1,GLUT9,PI3K,AKT,IKKβ and NFκB in the model group were significantly increased(P<0.05,P<0.01).The expression of ABCG2 was significantly decreased(P<0.01).Compared with model group,the expressions of URAT1,GLUT9,PI3K,AKT,IKKβ and NFκB in medium-high dose QBS4 group were significantly down-regulated(P<0.05,P<0.01),and up-regulated(P<0.05,P<0.01).These results suggest that QBS4 regulates hyperuricemia by promoting uric acid excretion,inhibiting uric acid reabsorption and inhibiting PI3K/AKT/NFκB signaling pathway.3.Protective effect of quinoa bran saponins on HK-2 cells damaged by uric acid and verification of its molecular mechanism.HK-2 cells were induced by uric acid to establish a high uric acid cell model.At the same time,QBS4 was administered to determine the content of inflammatory cytokines and the expression of inflammatory bodies in the cells.The molecular mechanism of the protective effect of QBS4 on HK-2 cells was further verified by adding PI3K activator.The results show that:(1)The cytotoxicity of QBS4 was studied by CCK8 method,and the intervention doses were determined to be 100,200 and 400 μg/mL.By determining the contents of nitric oxide(NO)and lactate dehydrogenase(LDH),the molding dose of uric acid was 200μg/mL.The results of cell experiments showed that QBS4 could significantly reduce the contents of NO,LDH,IL-1β and TNF-α and the expressions of NLRP3 and caspasel inflammatosomes in uric-induced HK-2 cells compared with the high uric acid group at the dose range of 200-400 μg/mL(P<0.01).(2)Molecular biology studies showed that QBS4 could reduce the phosphorylation levels of PI3K,AKT and NFκB in HK-2 cells(P<0.01),and also significantly reduce the protein expression of IKKβ(P<0.01).After adding 10 μg/mL PI3K activator,the expression of related proteins in PI3K/AKT/NFκB signaling pathway was increased again.These results suggest that QBS4 intervenes in hyperuricemia by regulating PI3K/AKT/NFκB signaling pathway.Conclusion:QBS4 is a pentacyclic triterpenoid saponin with ivy saponin and oleanolic acid as aglycones.QBS4 can regulate the abnormal uric acid,urea nitrogen and creatinine in hyperuricemia mice,and reduce renal fibrosis and renal inflammation.Its mechanism may be to relieve hyperuricemia by regulating the expression of uric acid transporters and inhibiting the activation of PI3K/AKT/NFκB inflammatory signaling pathway.Therefore,QBS4 is promising as a natural dietary supplement for the prevention of hyperuricemia.