Purine-induced IFN-γ Promotes Uric Acid Production By Upregulating Xanthine Oxidoreductase Expression

Objective:The prevalence of hyperuricemia and gout were gradually increasing worldwide.In recent years,the incidence of hyperuricemia and gout has been soaring in China,and the incidence of gout in some regions has been close to the European and American,which may be related to the economic development,lifestyle and dietary changes in China.Intake of red meat,seafood,alcohol and fructose was positively correlated with the risk of hyperuricemia and gout.It is well known that high-purine intake is the most common trigger for an acute attack of gout.Exogenous purines are mainly metabolized and oxidized by the liver to the end product uric acid(UA).The enzyme xanthine oxidoreductase(XOR)is a rate-limiting enzyme that produces UA during purine metabolism.XOR inhibitors,such as allopurinol and febuxostat have almost become the consensus of medical circles for reducing UA levels in the treatment of gout.Abnormal XOR expression and activity can lead to elevated UA levels and increase the risk of gout.XOR is often considered a drug target for the treatment of gout and hyperuricemia.XOR inhibitors such as allopurinol and febuxostat are routinely used in the treatment of gout.Animal experiments showed that the expression and activity of XOR in liver of hyperuricemia rats were significantly increased.Although XOR is closely related to the pathogenesis of hyperuricemia and gout,the causes of XOR abnormal expression and activation remains unclear.In the process of metabolism,excess substrate usually induces expression of its catalytic enzyme.Therefore,this study initially hypothesized that excessive intake of purine as a metabolic substrate might directly induce XOR expression in hepatocytes.However,the results were unexpected in that excess purine,although able to induce increased UA production,did not directly induce an increase in XOR expression.Dietary Inflammatory Index(DII)is a scoring mechanism used to evaluate the Dietary proinflammatory potential.There are studies have shown that dietary factors play an important role in regulating chronic inflammation in the body.Red meat,seafood,sugar and other dietary structure can affect the body inflammation,resulting in a higher DII score.Studies have shown that intake of red meat,seafood,alcohol and fructose is not only positively associated with the risk of hyperuricemia and gout,but also closely associated with inflammation.These results suggest that a high purine diet may not only increase UA levels,but also be closely related to chronic inflammation.However,the causal relationship between inflammation and UA levels has not been universally established.It is not clear whether elevated UA levels are a sign of an inflammatory state or a trigger for systemic inflammation.Some researchers have speculated that the relationship between high UA and systemic inflammation may be related to a separate mediating system that both drives inflammation and increases serum UA levels.Therefore,we hypothesized that purines,in addition to serving as metabolic substrates to produce UA,may also regulating UA production through dietary inflammation mechanism,thus participating in the molecular mechanism of hyperuricemia.Subsequently,it was experimentally verified that high purines can induce increased IFN-γexpression in lymphocytes.More interestingly,purine-induced IFN-γ production indirectly upregulated XOR expression and promoted UA production.Cytokines(such as IL-1,IL-6,TNF-α and IFN-γ)promote UA production through up-regulation of XOR expression and have been which have been reported in various tissue types including lung,breast and renal endothelial cells and neutrophils,especially IFN-γactivation of XOR protein expression.However,the mechanism of IFN-γ-induced XOR expression has not been studied in depth,and the effect of cytokines on XOR expression in hepatocytes has not been reported.JAK/STAT,as a classical signaling pathway of IFN-γactivation,is activated in many diseases,including metabolism,inflammation and tumor.Studies have reported that the JAK/STAT pathway is activated in diet-induced hyperuricemic rat,and inhibition of the JAK/STAT pathway can effectively improve renal uric acid excretion and alleviate renal injury caused by UA.This is suggested that the JAK/STAT signaling pathway may be involved in the regulation of hyperuricemia.Therefore,this study intends to use cytokines to stimulate hepatocytes to explore their effects on XOR expression and UA levels,as well as the possible molecular mechanisms.This study targeted the molecular mechanism of purine-induced elevation of UA with the aim of investigating whether high exogenous purine intake increased expression of XOR,and whether high purine regulates XOR expression by affecting the inflammatory,thereby exacerbating UA production,and its possible molecular mechanism.Our study is expected to provide direct evidence and new ideas for the involvement of purines and XOR in the pathogenesis of hyperuricemia,and may provide a theoretical basis for the prevention and treatment of hyperuricemia and gout.Materials and Methods:1.Hep G2 and Bel-7402 cells were stimulated by xanthine(Xan)and hypoxanthine(h Xan)at different concentrations and at different times to detect UA levels and explore the effect of high-purine intake on UA production in hepatocytes;2.Hep G2 and Bel-7402 cells were stimulated by Xan and h Xan,and detected XOR m RNA and protein expression by q PCR and Western blotting to explore whether excessive purine substrate induced XOR expression increase;XOR enzyme activity assay kit was used to detect XOR activity and explore the effect of high-purine intake on XOR activity;3.Jurkat cells were stimulated by Xan and h Xan,and the production of IFN-γ was detected by ELISA to investigate whether purine affected the synthesis and secretion of IFN-γ by T lymphocytes;4.Hep G2 and Bel-7402 cells were exposed to the medium from purine-stimulated T lymphocytes,and detect XOR expression and UA generation in the hepatocytes.Non-contact co-culture was used to explore the effect of purine-induced T lymphocyte secretion cytokines on XOR expression in hepatocytes,and to verify whether exogenous purine activated purine metabolism pathway to promote UA production by inflammatory mechanisms;5.Hep G2 and Bel-7402 cells were stimulated by IL-1,IL-6 or IFN-γ to detect XOR expression,enzyme activity and UA production,and to investigate whether IL-1,IL-6 or IFN-γ induced XOR activation and promoted UA production in hepatocytes;6.After STAT1 expression was knocked down with si RNA,Hep G2 cells were stimulated by human recombinant IFN-γ.XOR expression and the STAT1 enrichment in XDH promoter was detected to verify whether IFN-γ upregulated XOR expression through the JAK/STAT1 signaling pathway;7.IRF1 was found to be a potential transcriptional activator of XDH(XOR encoding gene)by transcription factor prediction.The enrichment of STAT1,IRF1 and CBP in the XDH promoter region were detected by Ch IP-q PCR;Co-IP were used to analyze whether there was a direct interaction between STAT1,IRF1 and CBP to co-activate XDH transcription expression;8.To verify the correlation between hyperuricemia and IFN-γ in the body,we collected plasma samples from patients with hyperuricemia and normal control population,and analyzed the correlation between IFN-γ,purine and UA.Results:1.Exogenous purine stimulated UA production in hepatocytes without affecting XOR expression and activity;2.Exogenous purine increased IFN-γ expression in T lymphocytes,thereby promoting XOR expression and UA production in hepatocytes;3.IFN-γ increased the expression and activity of XOR and then promotes the production of UA;4.IFN-γ activates the JAK/STAT1 signaling pathway in hepatocytes,and synergistic with transcription factor IRF1 to recruit CBP,and co-activate XDH transcriptional regulation;5.Serum UA is positively correlated with IFN-γ in patients with hyperuricemia,and there was also a positive correlation between purine and IFN-γ.Conclusion:It is well known that exogenous purine is mainly metabolized by the liver to UA.Here we provide new in vitro evidence that excessive purine does not directly promote XOR expression in the human liver cells,but rather acts on lymphocytes,for enhanced production and release of IFN-γ,which stimulates XOR expression by activating the hepatocyte JAK/STAT1 pathway in hepatocytes resulting in elevation of UA production.Activated STAT1 and IRF1 bind to XDH promoter and recruit CBP to co-activate XDH transcription and up-regulate XOR expression,thereby enhancing uric acid production.Purine not only acts as a metabolic substrate to produce UA,but also induces inflammatory response to promote UA production,which may be one of the molecular mechanisms by which exogenous purine participate in the pathogenesis of hyperuricemia.