Study On The Anti-gout Molecular Mechanism Of Three Flavonoids From Lagotis Brachystachys Maxim

Objective:(1)To study the effects of luteolin,luteolin-7-O-β-D-glucopyranoside and apigenin on urate transporters in renal tubular epithelial cells(HK-2);(2)To study the effects of luteolin,luteolin-7-O-β-D-glucopyranoside and apigenin on the MSU-induced inflammation in RAW264.7 macrophages,an in vitro inflammation model;(3)To study the effects of luteolin,luteolin-7-O-β-D-glucopyranoside and apigenin on the potassium oxazinate(OA)stimulated hyperuricemia in mice,as an in vivo model.Methods :(1)The effects of luteolin,luteolin-7-O-β-D-glucopyranoside,apigenin,benzbromarone,and uric acid on the viability of HK-2 cells were measured by sulforhodamine B(SRB)colorimetry,without intervention and selection because uric acid interferes with HK-2 cells.Western-Blot was used to detect the effects of luteolin,luteolin-7-O-β-D-glucopyranoside,and apigenin on glucose transporter 9(GLUT9)and urate anion transporter 1(URAT1)in HK-2 cells with and without modeling and also protein expression of organic anion transporter(OAT1).(2)For establishing an inflammatory model,RAW264.7 macrophages were stimulated using monosodium urate crystals to generate an inflammatory response.The effects of luteolin,luteolin-7-O-β-D-glucopyranoside,apigenin,and colchicine on the viability of RAW264.7 cells were detected by the SRB method,and interleukin-1β(IL-1β)was determined by Enzyme-linked immunosorbent assay(ELISA)kit.The expression levels of tumor necrosis factor-α(TNF-α),malondialdehyde(MDA),reduced glutathione(GSH),and superoxide dismutase(SOD)levels were screened for modeling conditions and measured after modeling.While TOLL-like receptor(TLR4,TLR2),NOD-like receptor protein 3(NLRP3),nuclear transcription factor-κB(NF-κB),myeloid differentiation factor(My D88),and IL-1β protein expression levelswere detected by Western-Blot.(3)A hyperuricemia(HUA)mouse model was established by intraperitoneal injection of potassium oxazinate,and kits were used to measure serum uric acid(UA),urea nitrogen(BUN),and adenosine deaminase(ADA).The liver xanthine oxidase(XOD)level and activity,serum levels of IL-1β,and TNF-α were measured by ELISA method.Moreover,pathological kidney section slides of hyperuricemia mice were made and observed for kidney damage.Western blot method was used for determining the expression levels of GLUT9,URAT1,and OAT1 proteins in kidney tissues and the expression levels of TLR4,TLR2,NLRP3,NF-κB,My D88,and IL-1β protein in liver tissue of mice.Results:(1)In the absence of modeling,it was found that the expression levels of OAT1 proteins were increased in both the luteolin-low and high-dose groups,and luteolin-7-O-β-D-glucopyranoside-low and high-dose groups,while the expression levels of other proteins did not change significantly.When modeling was performed,it was found that the low-apigenin and high-dose groups could significantly down-regulate the expression levels of GLUT9 and URAT1 while low and high-dose groups of luteolin and luteolin-7-O-β-D-glucopyranoside could significantly increase the expression of OAT1 protein.(2)By establishing an inflammation model,it was found that the groups of low and high-dose luteolin,high-dose luteolin-7-O-β-D-glucopyranoside,and low and high-dose apigenin groups can significantly reduce TNF-α levels;While low and high-dose luteolin,luteolin-7-O-β-D-glucopyranoside and apigenin groups can significantly reduce MDA levels;whereas low and high-dose luteolin,high-dose luteolin-7-O-β-D-glucopyranoside and low and high-dose apigenin groups can significantly increase GSH levels;the high-dose luteolin,luteolin-7-O-β-D-glucopyranoside,and low and high-dose apigenin can significantly increase SOD levels.Additionally,low-and high-dose luteolin and high-dose apigenin groups can significantly reduce TLR4 protein levels,the low and high-dose luteolin,luteolin-7-O-β-D-glucopyranoside and apigenin groups can significantly reduce NLRP3,NF-κB,and IL-1β protein levels;and also the high-dose luteolin and apigenin can significantly reduce My D88 protein levels.(3)From HUA establishedmodel in mice,it was found that low and high-dose luteolin,luteolin-7-O-β-D-glucopyranoside and low and high-dose apigenin groups can significantly reduce the levels of UA,BUN,XOD,IL-1β,and TNF-α in mice;the low and high-dose luteolin and apigenin groups can significantly reduce ADA levels in mice.Histopathological sections of mouse kidney showed that luteolin,luteolin-7-O-β-D-glucopyranoside,and apigenin all improved the inflammatory cell infiltration and renal tubular dilatation of renal tissue in hyperuricemia mice,and the degree of renal damage was significantly reduced as a whole.It was also found that the levels of GLUT9 and URAT1 were significantly reduced in the low and high-dose apigenin groups;the OAT1 protein levels were significantly increased in the low and high-dose luteolin,and also apigenin groups can significantly reduce TLR4 protein levels;the groups of low and high-dose luteolin,luteolin-7-O-β-D-glucopyranoside and apigenin can significantly reduce NLRP3,NF-κB,and IL-1β proteins levels;the group of low-dose luteolin and low and high-dose apigenin group can significantly reduce My D88 protein levels.Conclusion:Luteolin,luteolin-7-O-β-D-glucopyranoside,and apigenin have anti-gout and anti-gouty arthritis effects,and can regulate the levels of MDA,GSH,SOD,UA,BUN,XOD,IL-1β,TNF-α,and ADA.The effect of luteolin may be related to OAT1,TLR4,NLRP3,NF-κB,My D88,and IL-1β;the effect of luteolin-7-O-β-D-glucopyranoside could be related to OAT1,NLRP3,NF-κB,and IL-1β;Whereas the effect of apigenin may be associated to GLUT9,URAT1,TLR4,NLRP3,NF-κB,My D88,and IL-1β.