| ObjectiveHyperuricemia(HUA)and gout are metabolic diseases in which the metabolism of uric acid(UA)is deranged due to disorders of purine metabolism in the body.Phyllanthus emblica L.exhibits pharmacological effects such as anti-oxidative stress,anti-inflammatory and anti-tumor due to its rich content of various bioactive components.In this study,three animal models of HUA,gouty nephropathy(GN)and acute gouty arthritis(AGA)were according to different pathological stages of gout formation,including serum UA elevation,monosodium urate(MSU)deposition and acute inflammation,combined with the common clinical manifestations of gout.By evaluating the antioxidant activity of PEFP in vitro,exploring the uric acid-lowering and hepatorenal protective effects of PEFP in HUA mice,observing the uric acid-lowering and renal protective effects of PEFP in GN mice and the anti-inflammatory effect in AGA mice,so as to assess the uric acid-lowering and anti-gout effects of PEFP,which can provide reference for the development of natural drugs against HUA and gout.Methods1.Antioxidant activity of PEFP in vitro : The iron-reducing ability of PEFP was determined by FRAP method,the hydroxyl radical scavenging ability of PEFP was determined using salicylic acid method,and the 1,1-diphenyl-2-picrylhydrazyl free radical(DPPH)free radical scavenging ability of PEFP was determined by spectrophotometric method.2.Period of elevated blood UA level: Acute and chronic HUA mouse models were established by intraperitoneal injection of hypoxanthine and potassium oxazinate,respectively.Meanwhile,PEFP at 75,150 or 300 mg/kg were given to the mice by gavage as intervention substances.The levels of uric acid(UA),creatinine(CRE),urea nitrogen(BUN),aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in serum,the activities of xanthine oxidase(XOD)and adenosine deaminase(ADA)in serum and liver were detected.The hepatic coefficient and renal coefficient of mice were calculated,and the pathological characteristics of liver and kidney tissues were observed by HE staining.3.Period of renal injury induced by MSU deposition: GN mouse model was established by combined gavage of adenine and ethambutol hydrochloride,allopurinol and benzbromarone were used as positive control drugs.The grouping of PEFP intervention group was the same as that of HUA mouse model,and the corresponding drugs were gavaged separately.Serum uric acid(UA),creatinine(CRE),urea nitrogen(BUN)and serum and liver xanthine oxidase(XOD)activity levels were measured after 21 days.The levels of urate transporter 1(URAT1),glucose transporter 9(GLUT9)and organic anion transporter 1(OAT1)in kidney tissues were measured by immunohistochemistry.The expression of URAT1,GLUT9 and OAT1 mRNA in kidney tissues was detected by fluorescent quantitative PCR,and the pathological changes in renal tissue was observed by HE staining.4.Period of arthritis induced by MSU deposition: AGA mouse model was constructed by injecting MSU to the right ankle joint of mice.Colchicine was used as the positive control drug,and the grouping of PEFP intervention group was the same as that of HUA mouse model.The drug intervention was modeled on the 6th day and continued by gavage on the 1st day after modeling.The effects of PEFP on joint swelling,interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-10(IL-10),tumor necrosis factor-α(TNF-α),superoxide dismutase(SOD),malondialdehyde(MDA)and nitric oxide(NO)of AGA mice were evaluated.The expression of nuclear factor-E2-related factor 2(Nrf2)in synovial tissue of AGA mice was detected by immunohistochemistry.The pathological characteristics of synovial tissue were observed by HE staining.Results1.In vitro antioxidant activity of PEFP: The iron reduction capacity of PEFP was stronger than that of VC at all concentrations under the present experimental conditions.The hydroxyl radical scavenging capacity of PEFP is slightly lower than that of VC above a certain concentration.PEFP exhibited better DPPH radical scavenging ability,reaching 0.8 g/L basically on par with VC.2.Period of elevated blood UA level: Compared with the model group,150 or 300mg/kg PEFP could reduce the hepatic and renal coefficient,lower serum UA,BUN,CRE,and AST content in acute hyperuricemia mice,and 75,150 or 300 mg/kg PEFP can inhibit serum and liver XOD and ADA activities in acute hyperuricemia mice,and300 mg/kg PEFP could lower serum.ALT content in acute hyperuricemia mice Meanwhile,75,150 or 300 mg/kg PEFP can reduce the hepatic coefficient and renal coefficient,lower serum UA,BUN and ALT content,inhibit XOD in serum and liver and ADA activities in serum of chronic hyperuricemia mice,at the same time 150 or300 mg/kg PEFP could lower CRE and AST content in serum as well as ADA activity in liver of chronic hyperuricemia mice.The pathological results suggested that PEFP could ameliorate the pathological changes of liver and kidney in acute and chronic HUA mice.3.Period of renal injury induced by MSU deposition: Compared with the model group,150 mg/kg PEFP significantly reduced UA,CRE and BUN levels,in serum of GN mice with gouty nephropathy and their XOD activity in serum and liver.The expression of URAT1,GLUT9 and URAT1 mRNA,GLUT9 mRNA was significantly down-regulated by 150 mg/kg PEFP.150 and 300 mg/kg PEFP significantly upregulated OAT1 and OAT1 mRNA expression.The pathological results suggested that the PEFP could improve the renal pathological changes of GN mice with gouty nephropathy.4.Period of arthritis induced by MSU deposition: In AGA model,150 and 300 mg/kg PEFP significantly improved joint swelling of mice compared with the model group at each observed time point during the experiment.The levels of IL-1β,IL-6,TNF-α,MDA and NO were significantly decreased and the levels of IL-10 and SOD were significantly up-regulated by 150 and 300 mg/kg PEFP.The expression of Nrf2 was significantly up-regulated by 150 mg/kg PEFP.The pathological results suggested that PEFP could improve the pathological changes of synovial tissue of AGA mice Conclusion1.PEFP has certain antioxidant activity in vitro.2.PEPP can effectively decrease the serum UA level of acute and chronic HUA mice,which be related to the inhibition of XOD and ADA activities in serum and liver,and the promotion of renal uric acid excretion,and play a certain protective role in renal and liver.3.PEFP can effectively reduce serum uric acid level in GN mice and has a nephroprotective effect on GN mice.The mechanism is to inhibit serum and liver XOD activity,up-regulating OAT1 and OAT1 mRNA,down-regulating URAT1 and GLUT9 and URAT1 and GLUT9 mRNA,promoting uric acid excretion,and mitigating MSU deposition.4.PEFP is effective in reducing the inflammatory response in AGA mice and has a protective effect on their ankle joints.The mechanism is to inhibits the pro-inflammatory factors IL-1β,IL-6 and TNF-α and increases the anti-inflammatory factor IL-10,up-regulates Nrf2,increases the activity of SOD,decreases the levels of MDA and NO to alleviate oxidative stress. |
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