Establishment Of Animal Hyperuricemia Model And Selection Of Chinese Traditional Medicine For Antigouts

Gout is a clinical syndrome of tissue damage induced by a chronic metabolism disorder of purine,characterized by marked hyperuricemia. Hyperuricemia leads to the accumulation of uric acid in the body causing gouty arthritis and uric acid nephrolithiasis.Primary gout was positively correlated with several conditions such as obesity, hyperglycemia,diabetes mellitus and atherosclerosis.As a kind of serious metabolic disease,gout does cause harm to human health. Therefore,it is greatly necessary to explore medications that can prevent and cure hyperuricemia,as well as gout.In recent years,the frequency of hyperuricemia and gout has gradually risen along with the improvement of living standards.Current treatment of hyperuricemia associated with gout entails the use of anti-inflammatory agents to relieve the symptoms of the disease as well as xanthine oxidase inhibitors to block the biosynthesis of uric acid from purine.But the use of current medication can result in a number of adverse side effects,such as hepatitis and allergic reaction,and the adverse effects limit theirs therapy.Thus,the development of new hypouricemic agents of greater effectiveness and safety is highly warranted.Although the use of medicinal plants in the prevention and treatment of hyperuricemia and gout is frequent based on the experience of traditional medicine systems,their uses in modern medicine suffer from the lack of scientific evidences.Attention has been focused on selecting effective traditional plant medicine,extracting its phytochemicals and investigating the new medicament.Aim:1.Establish animal hyperuricemia and gouty arthritis model and select Chinese traditional medicine which can decrease the uric acid level and have anti-inflammatiory and analgesic effects.Use surface-enhanced laser desorption/ionization time of flight mass spectrometry(SELDI-TOF MS)to identify the serum protein marker in animal gouty arthritis model.Methods:1.Mice hyperuricemic models were made respectively on male kunming mice by yeast extract paste ig,uric acid ip,potassium oxonate ip,yeast extract paste plus ethambutol ig,and potassium oxonate ip plus ethambutol ig.The level of uric acid was detected by blood uric acid monitoring system and the ultrastructure's alteration of nephridial tissue was observed by electron microscope.2.Allopurinol and benzbromarone were given to the mouse hyperuricemia model established with potassium oxonate for three days,and then the level of blood and urine uric acid were detectd.3.Phellodendri cortex, dioscoreaceae,smilax glabra Roxb,alismataceae,plantaginaceae, allopurinol and benzbromarone were given to mice hyperuricemia model established with potassium oxonate for seven days,and then the level of bood uric acid and xanthine oxidase(XOD)activities in mouse liver were detectd.4.Smilax glabra Roxb,alismataceae,plantaginaceae, allopurinol and benzbromarone were given to rat hyperuricemia model for seven days,collecting 24 hour urine and then the volume of urine and the level of urine uric acid were detectd.5.Mice gouty arthritis model was established by MSU intraarticular injection,smilax glabra Roxb,alismataceae,plantaginaceae and indometacin were given to the model for six days,and then the joint swell degree,inflammatory factor in the joint leachate were detected,moreover histopathologic examination was done.6.Mice feet swelling was induced by ovi albumin,mice ear swelling was induced by dimethylbenzene,the mice writhing respone was induced by acetic acid and the mice pain was induced by hot plate.In these models the swelling degree,pain response latency and pain response frequency were detected respectively.7.SELDI-TOF MS and CMI0 ProteinChip were used to detect the serum proteomic patterns of 36 mice serum specimens collected respectively from the normal group,the model group and smilax glabra Roxb.The decision model for animal gouty arthritis was established by discriminant analysis and the change of protein peaks after the treatment of medicine herb was observed.Results:1.The blood uric acid level of mice in uric acid group, potassium oxonate group and potassium oxonate plus ethambutol group all significantly increased,but the blood uric acid level of mice in yeast extract paste group and yeast extract paste plus ethambutol has no difference from that of the model group.2.Allopurinol and benzbromarone both could decrease the blood uric acid level of mice hyperuricemia model established by potassium oxonate,moreover benzbromarone could decrease the urine uric acid level of model.3. Phellodendri cortex,smilax glabra Roxb,alismataceae all could decrease the blood uric acid level of mice hyperuricemia model established by potassium oxonate,but the blood uric acid level of mice treated by dioscoreaceae or plantaginaceae has no difference from that of the model group.In the mouse hyperuricemia model induced by potassium oxonate,the serum XOD activities in phellodendri cortex group and smilax glabra Roxb group were significantly inhibited,but the serum XOD activities in alismataceae group and plantaginaceae group has no difference from that of the model group.4.In the rat hyperuricemia model induced by potassium oxonate,the urine uric acid level in alismataceae group markedly increased but lower than that of benzbromarone.The 24h urine volume of rats in alismataceae group seemed to add but has no difference from that of the model group.5.In the mouse gouty arthritis model,the joint swelling was very obvious at 24h.The level of IL-1βand TNF-αin the model group was significantly increased and histopathologic examination showed that the synovial cells were seriously injured in the model group.Smilax glabra Roxb could significantly inhibit joint swelling of mouse,reduce the level of IL-1βand TNF-αand markedly improve the histopathologic changes.6. In the model induced by ovi albumin,the swelling degree in smilax glabra Roxb group was significantly decreased compared with the model group.In the models induced by dimethylbenzen and writhing respone was induced by acetic acid,smilax glabra Roxb could significantly reduce mouse ear sweling and frequency of writhing respone.Smilax glabra Roxb seemed to prolong pain response latency on hot plate but has no difference from that of the model group.7.27 serum protein peaks are obviously different between animal gouty arthritis and normal controls.Protein peaks at the m/z of 4612,12915, 5637,3018 and 23103 Da are up-regulated in model group and protein peaks at the m/z of 6456,4973,3923 and 15090 Da are down-regulated in model group.15 serum protein peaks are obviously different between animal gouty arthritis and treatment group.Protein peaks at the m/z of 2598,4614,3444,19686 and 3018 Da are up-regulated in model group and protein peak at the m/z of 2937 Da is up-regulated in the treatment group.A decision model was built with protein peaks of 4612 Da and 6456 Da,which could discriminate gouty arthritis from normal controls with the accuracy of 75.0%.Protein peaks at the m/z of 4612 Da and 4614 Da were two parts of the same obtuse peak,of which protein content was up-regulated in model group and down-regulated in both normal group and treatment group.Protein peaks at the m/z of 4612 and 4614 Da are selected as candidate biomarkers of animal gouty arthritis.Conclusion:1.Potassium oxonate given by ip could eatablish hyperuricemia model.2.Phellodendri cortex,smilax glabra Roxb and alismataceae could decrease the blood uric acid level of mice hyperuricemia model.The mechanism for smilax glabra Roxb may be relevant in inhibiting the activities of XOD and the mechanism for alismataceae may be relevant in accelerating the excretion of uric acid. 3.Smilax glabra Roxb possessed antigout arthritis effects,and its mechanism may be related to inhibit the product of inflammatory cytokines.4.SELDI-TOF MS could discrimanate gouty arthritis from normal controls and gouty arthritis from treatment group.It is useful for finding the candidate biomarkers of animal gouty arthritis.Significance:1.Establish a reliable animal hyperuricemia model.2. Smilax glabra Roxb is found to decrease the blood uric acid level and have significant anti-inflammatiory and analgesic effects.It is useful for finding a new medicamen for gout.3.SELDI-TOF MS is a favourable tool to find the candidate biomarkers of animal gouty arthritis.