Discovery And Functional Study Of Water Extract Of Rice Derived Anti-gout Peptides

Objectives:Gout,a crystal related joint disease caused by crystal deposition of monosodium urate(MSU),which is directly related to hyperuricemia(HUA)caused by purine metabolism disorder and/or reduction of uric acid excretion.HUA is the basis of gout which is related to several complications.The continuously and uncontrolled of HUA can not only induce acute attack of gout,cause severe pain,but also cause kidney disease.The patients with HUA may suffer joint injury,renal damage,long-term hyperlipidemia,hypertension,diabetes,arteriosclerosis,coronary heart disease,etc.Therefore,the treatment of gout needs not only alleviate the acute pain but also control the HUA.However,it's hard to ignore the side effects of most anti-gout and hyperuricemic drugs which can only be effective for one symptom,which also means that patients may need to use two or more drugs in combination.However,the combination of drugs may lead to unpredictable drug interactions and adverse reactions,which also limits the clinical use of drugs.Therefore,the development of new multifunctional anti-gout drugs that can play a role in the whole process of gout is the focus of current research.The purpose of this project is to discover novel peptides that can be used as the medicine candidates for the research and development of anti-gout drugs,and to explore their mechanisms and functions of them.Methods:The shelled fruits of Oryza Sativa were collected from Yunnan,China.The samples were purified and identified by G50 and RP-HPLC from the water extract of shelled fruits of O.Sativa.The primary structures of the active peptides were determined by mass spectrometry and sequence analyze and high purity samples were obtained by commercial synthesis.The hemolytic activity,acute toxicity,plasma stability and other stabilities of peptides were then tested.The HUA animal models were established to detect the lowering uric acid and kidney protecting abilities of peptides,and then a series of tests such as western blot assay,biochemical indicators in vivo and molecular docking were performed to understand the mechanism of samples in anti-hyperuricemia.The animal pain model induced by formalin and the gout model induced by MSU were established to detect the anti-inflammatory and analgesic abilities of the samples,and the role of them in relieving inflammatory pain and swelling were observed and analyzed by ELISA and H&E staining.Results:(1)Three natural peptides,named RDP1,RDP2 and RDP3,were obtained from the water extract of shelled fruits of O.Sativa,and all of them had the novel structure and was named RDP1(AAAAGAKAR),RDP2(AAAAGAMPK-NH2)and RDP3(AAAAMAGPK-NH2),respectively;(2)In the anti-hyperuricemic abilities test of these peptides,all of them showed the ability of reducing serum uric acid and creatinine.From high to low,the ability of reducing uric acid was RDP2>RDP1>RDP3,and in that of reducing creatinine was RDP3>RDP2>RDP1;(3)RDP1 had no hemolytic activity,no acute toxicity and strong stability;(4)RDP1(1 mg/kg,100?g/kg,10?g/kg)could reduce serum uric acid and creatinine and alleviate kidney damage in concentration dependently,and could also inhibit XOD in vitro and in vivo.In molecular docking simulation,the RDP1-XOD showed a good affinity(-10.2 kcal/mol);(5)RDP2 and RDP3 had no hemolytic activity,no acute toxicity and strong stability;(6)RDP2(100?g/kg,10 ?g/kg,5?g/kg)and RDP3(1 mg/kg,500 ?g/kg,100 ?g/kg)could significantly reduce serum uric acid and creatinine in HUA mice,and could also alleviated the kidney injury of HUA mice;(7)RDP2 and RDP3 inhibited the XOD activity of serum and liver,and reduced the expression of URAT1 in HUA mice kidney;(8)RDP2 and RDP3 could decrease IL-1? level of HUA mice,and might alleviate renal injury by reducing the expression of NLRP3 inflammasome(NLRP3?ASC?Caspase 1)in mice kidney;(9)RDP2 and RDP3 reduced the paw pain induced by formalin and alleviated the paw swelling caused by MSU;(10)RDP2 and RDP3 reduced the level of IL-1? and TNF-? in MSU mice,and alleviated tissue injury of paw.Conclusions:(1)RDP1 might reduce uric acid in vivo through XOD inhibition,and had the ability of alleviating renal injury which indicated the potential that RDP1 had to be developed into an anti-hyperuricemic drug;(2)RDP2 and RDP3 showed obviously inhibitory effects on the whole process of gout,which can not only reduce the serum uric acid by reducing the XOD and URAT1,but also anti-inflammatory and analgesic,indicating the potential of them to development into novel anti-gout drugs and health products;(3)The shelled fruits of O.Sativa has potential both in the development of anti-gout medicine and health product.