Effect And Mechanism Of Polydatin On Hyperuricemia And Gouty Arthritis

[Objective]Gout is a metabolic disease in which monosodium urate(MSU)deposited in joints due to elevated serum uric acid levels.Hyperuricemia(HUA)and gouty arthritis(GA)are two different stages in the development of gout,the patients with HUA combined with GA account for about 10%of the gout population.In recent years,the incidence of gout has increased significantly,and there are many adverse reactions of the drugs used in clinical treatment of gout.Furthermore,HUA combined with GA model is rarely reported.Therefore,potassium oxonate,xanthine and MSU were used to establish the mouse model of HUA combined with GA to investigate the therapeutic effect of PD.Then,the mechanism of polydatin on HUA and GA was explored using adenosine triphosphate-binding cassette transporter G2(ABCG2)inhibitor Ko143,nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome inhibitor MCC950 and nuclear factor kappa-B(NF-κB)pathway inhibitor QNZ through MSU-induced HK-2 cell damage and lipopolysaccharides(LPS)+MSU-stimulated THP-1 cell inflammatory response.[Methods]1.Animal experiment:Male C57BL/6 mice were randomly divided into control group,model group,PD groups(low,medium and high dose),colchicine group and benzbromarone group.The mice in control group and model group were given sterile normal saline,and the corresponding administration group was given corresponding drugs successively for 14 days.During this period,potassium oxonate and xanthine suspension(280 mg/kg)were injected into the abdominal cavity of mice every day to induce HUA mouse model except control group,while mice in control group was injected with an equal volume of normal saline solution.On the 13th day,the GA mouse model was induced by performing intra-articular injection of MSU suspension(50 mg/mL,0.025 mL per mouse)into the ankle of mice in all groups except control group.MSU suspension in control group was replaced by sterile normal saline solution.To explore the efficacy and mechanism of PD in treating HUA combined with GA,after intervention with different concentrations of PD,the levels of uric acid and inflammatory cytokines,the degree of ankle joint swelling,the inflammatory cell infiltration of kidneys and ankle joints in mice were measured,and the protein expressions of urate transporters,NLRP3 inflammasome and NF-κB pathway were analyzed by Western blotting.2.Cell experiment:(1)The effects of different concentrations of PD,benzbromarone,Ko143 and MSU on the viability of HK-2 cell were determined by CCK-8,and the concentrations of benzbromarone,Ko143 and MSU were determined.Next,according to the manufacturer’s instructions,the levels of oxidative stress related indexes were detected to determine the concentration of PD.Then Western blotting was used to detect the expression of ABCG2 protein to explore the mechanism of PD on MSU-induced HK-2 cell damage.(2)The effects of different concentrations of PD,MCC950,QNZ on THP-1 cell viability were detected by CCK-8,and the concentrations of MCC950 and QNZ were determined.Then the levels of inflammatory cytokines were detected by ELISA to determine the concentrations of PD.The expressions of NLRP3 inflammasome and NF-κB pathway related protein were detected by Western blotting to investigate the mechanism of PD on LPS+MSU-induced inflammatory response of THP-1 cell.[Results]1.Animal experiment:After PD treatment,the levels of uric acid and inflammatory cytokines in HUA combined with GA mice were significantly reduced,and the ankle swelling,the inflammatory cell infiltration of kidneys and ankle joints in mice were obviously improved.In addition,PD alleviated HUA combined with GA via regulating urate transporters,NLRP3 inflammasome and NF-κB pathway.2.Cell experiment:(1)It was determined by CCK-8 that the concentration of benzbromarone,MSU and Ko143 were 0.4 μM,400 μM and 1μM,respectively.The results showed that 10 μM PD can significantly improve MSU-induced HK-2 cell damage.Then,1μM Ko143 and 10 μM PD simultaneously intervened in HK-2 cell,and PD significantly up-regulated the expression of ABCG2 protein.(2)The concentrations of MCC950 and QNZ were determined by CCK-8,which were 1μM and 10 nM,respectively.PD decreased the levels of inflammatory cytokines in THP-1 cell in a dose-dependent manner,and 40 μM PD had the best effect.After the intervention of 1 μM MCC950,10 nM QNZ and 40 μM PD simultaneously,the expressions of NLRP3 inflammasome and NF-κB pathway related proteins in THP-1 cell were significantly decreased.[Conclusion]1.The mouse model of HUA combined with GA was established by potassium oxonate,xanthine and MSU,which proved that PD had the effect of reducing uric acid and anti-GA.2.Based on damage of HK-2 cell stimulated by MSU and inflammatory response of THP-1 cell induced by LPS+MSU,then the inhibitors of ABCG2,NLRP3 inflammasome and NF-κB pathway(Ko143,MCC950 and QNZ)were used to demonstrate that PD played a role in reducing uric acid and anti-GA via regulating urate transporters,NLRP3 inflammasome and NF-κB pathway.