高尿酸血症动物模型的建立及抗痛风中药的筛选
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摘要
痛风(gout)是长期嘌呤代谢障碍、血尿酸增高引起组织损伤的一组异质性疾病。临床特点是:高尿酸血症(hyperuricemia)、痛风性急性关节炎反复发作、痛风石沉积、特征性慢性关节炎和关节畸形,常累及肾脏引起慢性间质性肾炎和肾尿酸结石形成。痛风的生化标志是高尿酸血症,指细胞外液的尿酸盐呈超饱和状态,一般认为血尿酸≥416μmol/L时为高尿酸血症。约5%-12%高尿酸血症患者会发展成为痛风。痛风的急性发作是尿酸钠(monosodium urate crystal,MSU)在关节及关节周围组织以结晶形式沉积引起的急性炎症反应。痛风不仅可以侵犯骨和关节,而且还容易累及肾脏和心血管系统。高尿酸血症及原发性痛风与肥胖症、高脂血症、高血压病、糖尿病、动脉粥样硬化等疾病呈显著正相关。因此,痛风是危害人类健康的一种严重的代谢性疾病。
近年来,随着人们生活水平的提高,饮食结构发生变化,糖、脂肪、蛋白质的摄入量明显增加,高尿酸血症和痛风的发病率日益增高,已成为一种常见病。为了避免关节炎反复发作和肾脏病变形成,痛风患者需要长期服用有降低血尿酸(uric acid,UA)作用的药物,但目前安全、有效、经济的降UA药物较少。我国用中草药治疗痛风病有悠久的历史,但迄今尚无能够有效降低血UA的中药制剂用于临床。因此,寻找新型降UA药物,特别是借鉴中医经验开发有降UA作用的中草药制剂具有重要的意义。
本博士学位论文共分为四部分,第一部分建立可靠的高尿酸血症动物模型;第二部分运用建立的高尿酸血症动物模型,筛选具有降尿酸作用的中药并初步探讨其降尿酸机制;第三部分建立急性痛风性关节炎动物模型,观察中药对急性痛风性关节炎的治疗效果并分析其作用机制;第四部分应用表面增强激光解吸电离飞行时间质谱技术(surface-enhanced laser desorption/ionization time of flight massspectrometry SELDI-TOF MS)分析急性痛风性关节炎动物模型与正常小鼠之间及模型小鼠中药治疗前后血清蛋白质谱的变化。本研究主要目的、方法、结果和结论概述如下:
研究目的:建立高尿酸血症及急性痛风性关节炎动物模型,筛选具有降尿酸及抗炎镇痛作用的中药并分析其可能作用机制,应用蛋白组学方法寻找急性痛风性关节炎动物模型的血清蛋白标志物,分析急性痛风性关节炎动物中药治疗前后血清蛋白质谱的变化。
研究方法:1.雄性昆明小鼠随机分组,分别用酵母膏、尿酸、氧嗪酸钾盐、酵母膏+乙胺丁醇、氧嗪酸钾盐+乙胺丁醇给药制备动物模型。酵母膏按30g/kg/d灌胃给药,每日2次,乙胺丁醇按250mg/kg灌胃给药,每日1次,均连续用药7天。尿酸、氧嗪酸钾盐分别按250mg/kg及300mg/kg一次性腹腔注射给药。检测建模前后各组小鼠血尿酸、血尿素氮、血肌酐水平的变化,电镜观察各组小鼠肾组织超微结构改变。2.选择腹腔注射氧嗪酸钾盐法造模,雄性昆明小鼠随机分组,分别予苯溴马隆20mg/kg和别嘌呤醇40mg/kg灌胃3天,观察各组小鼠血尿酸及尿尿酸水平变化。3.雄性昆明小鼠随机分为9组,采用腹腔注射氧嗪酸钾盐法建模,分别给予生理盐水、黄柏、萆薢、土茯苓、泽泻、车前子水煎液及别嘌呤醇、苯溴马隆灌胃,每目1次,7日后观察血尿酸水平及小鼠肝脏黄嘌呤氧化酶活性的变化。4.雄性Wistar大鼠36只,随机分为6组,氧嗪酸钾盐腹腔注射建立高尿酸血症动物模型,分别给予土茯苓、泽泻、车前子水煎液及别嘌呤醇、苯溴马隆灌胃,每日1次,连续7天,收集第6-7天24h尿液,观察各组大鼠尿量及尿尿酸浓度的变化。5.昆明小鼠随机分为4组,采用MSU关节腔内注射诱导小鼠急性痛风性关节炎模型,土茯苓、泽泻、车前子水煎液和吲哚美辛分别灌胃给药,每日1次,连续6天。观察给药后关节肿胀度以及关节周围组织炎症介质IL-1β和TNF-α水平的变化,并进行关节病理学检查。6.分别采用醋酸腹腔注射诱导小鼠扭体反应法和热板法的镇痛实验及蛋清、二甲苯诱导小鼠足跖肿胀、耳肿胀的抗炎实验,观察土茯苓、泽泻、车前子和吲哚美辛的镇痛抗炎作用。7.用CM10芯片结合SELDI-TOF MS技术检测正常组、模型组、土茯苓组各12只小鼠血清,Ciphergen ProteinChip 3.1软件进行数据分析,比较模型组与正常组、模型组与土茯苓组间的蛋白质谱峰差异,用判别分析方法建立急性痛风性关节炎小鼠成模判断模型,观察模型小鼠中药治疗后蛋白质峰的动态变化。
研究结果:1.尿酸组、氧嗪酸钾盐组、氧嗪酸钾盐+乙胺丁醇组用药后血尿酸水平较正常对照组均显著升高(尿酸组从108.9±10.1μmol/L升高到301.8±24.7μmol/L,P＜0.01:氧嗪酸钾盐组从119.1±20.6μmol/L升高到276.3+32.5μmol/L,P＜0.01;氧嗪酸钾盐+乙胺丁醇组从115.7±17.4μmol/L升高到282.2±40.7μmol/L,P＜0.01)。但酵母膏组、酵母膏+乙胺丁醇组血尿酸水平与正常对照组比较无显著差异。电镜观察发现,酵母膏组及酵母膏+乙胺丁醇组肾组织超微结构发生改变,酵母膏组肾近曲小管上皮细胞线粒体多处出现局部嵴缺失、空化;酵母膏+乙胺丁醇组肾近曲小管上皮细胞核周池肿胀、内质网扩张,形成大小不等的空泡。余用药组肾组织超微结构未发现明显改变。2.采用氧嗪酸钾盐法建立小鼠高尿酸血症动物模型,别嘌呤醇组和苯溴马隆组小鼠用药后血尿酸水平均明显低于模型组。别嘌呤醇组小鼠尿尿酸浓度低于模型组,苯溴马隆组尿尿酸浓度明显高于模性组,说明此模型可用于筛选降UA药物。3.采用氧嗪酸钾盐法建模,黄柏、土茯苓、泽泻组小鼠用药后血尿酸水平下降,分别为171.2±28.8μmol/L、163.0±48.8μmol/L、158.7±45.2μmol/L,与模型组(250.44±55.8μmol/L)比较有明显差异;萆薢、车前子组小鼠血尿酸水平与模型组比较无明显差异。黄柏、土茯苓组小鼠肝脏黄嘌呤氧化酶活性分别为73.23±7.21U/L、69.39±5.84U/L,与模型组(89.07±8.34 U/L)比较明显下降,抑制率分别为20.0%和22.5%,但较别嘌呤醇29.2%的抑制率低。萆薜、泽泻、车前子组小鼠肝脏黄嘌呤氧化酶活性与模型组比较无明显差异。4.采用氧嗪酸钾盐法建立高尿酸血症大鼠动物模型,泽泻组大鼠尿尿酸浓度为187.50±46.81μmol/L,较模型组(136.02±11.55μmol/L)明显增加,但低于苯溴马隆组(231.55±29.32μmol/L)尿尿酸浓度,同时,泽泻组大鼠24h尿量较模型组有增加趋势。土茯苓组大鼠尿量及尿尿酸浓度与模型组比较无明显差异。5.小鼠关节内注射MSU溶液,24h后关节明显肿胀,模型组小鼠肿胀关节周围组织中炎症介质IL-1β和TNF-α水平明显增高,病理学检查发现模型组小鼠软骨破坏,滑膜组织充血、坏死伴炎细胞浸润。土茯苓组小鼠关节肿胀度明显减轻,关节周围组织中炎症介质IL-1β和TNF-α水平显著降低,关节组织病理学改变显著改善。泽泻、车前子组小鼠关节肿胀度、病理检查结果与模型组比较无明显差异。6.土茯苓12g/kg灌胃给药能显著减轻蛋清致小鼠足跖肿胀度及二甲苯致耳肿胀度:延长醋酸腹腔注射致小鼠扭体反应的潜伏期,减少扭体次数;对热板致小鼠疼痛反应潜伏期与模型组无明显差异,但有延长趋势。泽泻、车前子组小鼠镇痛抗炎实验结果与模型组比较无明显差异。7.急性痛风性关节炎模型组与正常组间表达差异有统计学意义的蛋白质峰27个。9个蛋白质峰的t检验或Mann-Whitney秩和检验所得P值在10~(-4)数量级以下。M/Z位于4612、12915、5637、3018和23103Da的5个差异蛋白峰在模型组高表达,M/Z位于6456、4973、3923和15090Da的4个蛋白质峰在模型组血清低表达;急性痛风性关节炎模型组与土茯苓组间表达差异有统计学意义的蛋白质峰15个。其中6个蛋白质峰的t检验或Mann-Whitney秩和检验所得P值在10~(-3)数量级以下。M/Z位于2598、4614、3444、1968 6和3018Da的5个蛋白峰在模型组高表达,M/Z为2937的1个蛋白质峰在中药组血清高表达。4612Da和6456Da两个蛋白质峰组合所构建的模型能达到鉴别急性痛风性关节炎小鼠和正常小鼠的最佳检测效果,总准确率75.0%。4612Da在正常组低表达,模型组高表达;4614Da在模型组高表达,中药治疗后低表达,软件分析二者为同一个融合峰,提示该峰可能为急性痛风性关节炎的候选标志蛋白质峰。
研究结论:1.腹腔注射氧嗪酸钾盐法可作为建立高尿酸血症动物模型、筛选降尿酸药物的可靠方法。2.黄柏、土茯苓、泽泻能降低高尿酸血症小鼠血清尿酸水平;土茯苓降低高尿酸血症小鼠血尿酸水平的机制至少部分在于对肝脏黄嘌呤氧化酶的抑制作用;泽泻降低高尿酸血症小鼠血尿酸水平的机制可能主要在于促进尿酸排泄及增加尿量。3.土茯苓能减轻急性痛风性关节炎的关节肿胀,减少炎细胞浸润,降低局部组织炎症介质IL-1β和TNF-α的水平表明土茯苓有较强的抗炎及镇痛作用。4.SELDI-TOF MS技术检测到急性痛风性关节炎动物模型与正常小鼠及模型小鼠与中药治疗组间血清蛋白质谱的差异,对寻找急性痛风性关节炎模型小鼠血清蛋白标志物提供了有用的信息。
研究意义:1.确立了简便可靠的高尿酸血症动物模型。2.研究中发现中药土茯苓在高尿酸血症动物模型表现出降尿酸效果,对急性痛风性关节炎模型有抗炎镇痛作用,对研制安全、经济、有效的新型抗痛风药制剂有重要意义。3.运用SELDI-TOF MS蛋白芯片技术,检测到急性痛风性关节炎动物模型血清中特异性标志蛋白质峰,为进一步发现痛风性关节炎的特异性诊断标志物提供实验依据。
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.
近年来,随着人们生活水平的提高,饮食结构发生变化,糖、脂肪、蛋白质的摄入量明显增加,高尿酸血症和痛风的发病率日益增高,已成为一种常见病。为了避免关节炎反复发作和肾脏病变形成,痛风患者需要长期服用有降低血尿酸(uric acid,UA)作用的药物,但目前安全、有效、经济的降UA药物较少。我国用中草药治疗痛风病有悠久的历史,但迄今尚无能够有效降低血UA的中药制剂用于临床。因此,寻找新型降UA药物,特别是借鉴中医经验开发有降UA作用的中草药制剂具有重要的意义。
本博士学位论文共分为四部分,第一部分建立可靠的高尿酸血症动物模型;第二部分运用建立的高尿酸血症动物模型,筛选具有降尿酸作用的中药并初步探讨其降尿酸机制;第三部分建立急性痛风性关节炎动物模型,观察中药对急性痛风性关节炎的治疗效果并分析其作用机制;第四部分应用表面增强激光解吸电离飞行时间质谱技术(surface-enhanced laser desorption/ionization time of flight massspectrometry SELDI-TOF MS)分析急性痛风性关节炎动物模型与正常小鼠之间及模型小鼠中药治疗前后血清蛋白质谱的变化。本研究主要目的、方法、结果和结论概述如下:
研究目的:建立高尿酸血症及急性痛风性关节炎动物模型,筛选具有降尿酸及抗炎镇痛作用的中药并分析其可能作用机制,应用蛋白组学方法寻找急性痛风性关节炎动物模型的血清蛋白标志物,分析急性痛风性关节炎动物中药治疗前后血清蛋白质谱的变化。
研究方法:1.雄性昆明小鼠随机分组,分别用酵母膏、尿酸、氧嗪酸钾盐、酵母膏+乙胺丁醇、氧嗪酸钾盐+乙胺丁醇给药制备动物模型。酵母膏按30g/kg/d灌胃给药,每日2次,乙胺丁醇按250mg/kg灌胃给药,每日1次,均连续用药7天。尿酸、氧嗪酸钾盐分别按250mg/kg及300mg/kg一次性腹腔注射给药。检测建模前后各组小鼠血尿酸、血尿素氮、血肌酐水平的变化,电镜观察各组小鼠肾组织超微结构改变。2.选择腹腔注射氧嗪酸钾盐法造模,雄性昆明小鼠随机分组,分别予苯溴马隆20mg/kg和别嘌呤醇40mg/kg灌胃3天,观察各组小鼠血尿酸及尿尿酸水平变化。3.雄性昆明小鼠随机分为9组,采用腹腔注射氧嗪酸钾盐法建模,分别给予生理盐水、黄柏、萆薢、土茯苓、泽泻、车前子水煎液及别嘌呤醇、苯溴马隆灌胃,每目1次,7日后观察血尿酸水平及小鼠肝脏黄嘌呤氧化酶活性的变化。4.雄性Wistar大鼠36只,随机分为6组,氧嗪酸钾盐腹腔注射建立高尿酸血症动物模型,分别给予土茯苓、泽泻、车前子水煎液及别嘌呤醇、苯溴马隆灌胃,每日1次,连续7天,收集第6-7天24h尿液,观察各组大鼠尿量及尿尿酸浓度的变化。5.昆明小鼠随机分为4组,采用MSU关节腔内注射诱导小鼠急性痛风性关节炎模型,土茯苓、泽泻、车前子水煎液和吲哚美辛分别灌胃给药,每日1次,连续6天。观察给药后关节肿胀度以及关节周围组织炎症介质IL-1β和TNF-α水平的变化,并进行关节病理学检查。6.分别采用醋酸腹腔注射诱导小鼠扭体反应法和热板法的镇痛实验及蛋清、二甲苯诱导小鼠足跖肿胀、耳肿胀的抗炎实验,观察土茯苓、泽泻、车前子和吲哚美辛的镇痛抗炎作用。7.用CM10芯片结合SELDI-TOF MS技术检测正常组、模型组、土茯苓组各12只小鼠血清,Ciphergen ProteinChip 3.1软件进行数据分析,比较模型组与正常组、模型组与土茯苓组间的蛋白质谱峰差异,用判别分析方法建立急性痛风性关节炎小鼠成模判断模型,观察模型小鼠中药治疗后蛋白质峰的动态变化。
研究结果:1.尿酸组、氧嗪酸钾盐组、氧嗪酸钾盐+乙胺丁醇组用药后血尿酸水平较正常对照组均显著升高(尿酸组从108.9±10.1μmol/L升高到301.8±24.7μmol/L,P＜0.01:氧嗪酸钾盐组从119.1±20.6μmol/L升高到276.3+32.5μmol/L,P＜0.01;氧嗪酸钾盐+乙胺丁醇组从115.7±17.4μmol/L升高到282.2±40.7μmol/L,P＜0.01)。但酵母膏组、酵母膏+乙胺丁醇组血尿酸水平与正常对照组比较无显著差异。电镜观察发现,酵母膏组及酵母膏+乙胺丁醇组肾组织超微结构发生改变,酵母膏组肾近曲小管上皮细胞线粒体多处出现局部嵴缺失、空化;酵母膏+乙胺丁醇组肾近曲小管上皮细胞核周池肿胀、内质网扩张,形成大小不等的空泡。余用药组肾组织超微结构未发现明显改变。2.采用氧嗪酸钾盐法建立小鼠高尿酸血症动物模型,别嘌呤醇组和苯溴马隆组小鼠用药后血尿酸水平均明显低于模型组。别嘌呤醇组小鼠尿尿酸浓度低于模型组,苯溴马隆组尿尿酸浓度明显高于模性组,说明此模型可用于筛选降UA药物。3.采用氧嗪酸钾盐法建模,黄柏、土茯苓、泽泻组小鼠用药后血尿酸水平下降,分别为171.2±28.8μmol/L、163.0±48.8μmol/L、158.7±45.2μmol/L,与模型组(250.44±55.8μmol/L)比较有明显差异;萆薢、车前子组小鼠血尿酸水平与模型组比较无明显差异。黄柏、土茯苓组小鼠肝脏黄嘌呤氧化酶活性分别为73.23±7.21U/L、69.39±5.84U/L,与模型组(89.07±8.34 U/L)比较明显下降,抑制率分别为20.0%和22.5%,但较别嘌呤醇29.2%的抑制率低。萆薜、泽泻、车前子组小鼠肝脏黄嘌呤氧化酶活性与模型组比较无明显差异。4.采用氧嗪酸钾盐法建立高尿酸血症大鼠动物模型,泽泻组大鼠尿尿酸浓度为187.50±46.81μmol/L,较模型组(136.02±11.55μmol/L)明显增加,但低于苯溴马隆组(231.55±29.32μmol/L)尿尿酸浓度,同时,泽泻组大鼠24h尿量较模型组有增加趋势。土茯苓组大鼠尿量及尿尿酸浓度与模型组比较无明显差异。5.小鼠关节内注射MSU溶液,24h后关节明显肿胀,模型组小鼠肿胀关节周围组织中炎症介质IL-1β和TNF-α水平明显增高,病理学检查发现模型组小鼠软骨破坏,滑膜组织充血、坏死伴炎细胞浸润。土茯苓组小鼠关节肿胀度明显减轻,关节周围组织中炎症介质IL-1β和TNF-α水平显著降低,关节组织病理学改变显著改善。泽泻、车前子组小鼠关节肿胀度、病理检查结果与模型组比较无明显差异。6.土茯苓12g/kg灌胃给药能显著减轻蛋清致小鼠足跖肿胀度及二甲苯致耳肿胀度:延长醋酸腹腔注射致小鼠扭体反应的潜伏期,减少扭体次数;对热板致小鼠疼痛反应潜伏期与模型组无明显差异,但有延长趋势。泽泻、车前子组小鼠镇痛抗炎实验结果与模型组比较无明显差异。7.急性痛风性关节炎模型组与正常组间表达差异有统计学意义的蛋白质峰27个。9个蛋白质峰的t检验或Mann-Whitney秩和检验所得P值在10~(-4)数量级以下。M/Z位于4612、12915、5637、3018和23103Da的5个差异蛋白峰在模型组高表达,M/Z位于6456、4973、3923和15090Da的4个蛋白质峰在模型组血清低表达;急性痛风性关节炎模型组与土茯苓组间表达差异有统计学意义的蛋白质峰15个。其中6个蛋白质峰的t检验或Mann-Whitney秩和检验所得P值在10~(-3)数量级以下。M/Z位于2598、4614、3444、1968 6和3018Da的5个蛋白峰在模型组高表达,M/Z为2937的1个蛋白质峰在中药组血清高表达。4612Da和6456Da两个蛋白质峰组合所构建的模型能达到鉴别急性痛风性关节炎小鼠和正常小鼠的最佳检测效果,总准确率75.0%。4612Da在正常组低表达,模型组高表达;4614Da在模型组高表达,中药治疗后低表达,软件分析二者为同一个融合峰,提示该峰可能为急性痛风性关节炎的候选标志蛋白质峰。
研究结论:1.腹腔注射氧嗪酸钾盐法可作为建立高尿酸血症动物模型、筛选降尿酸药物的可靠方法。2.黄柏、土茯苓、泽泻能降低高尿酸血症小鼠血清尿酸水平;土茯苓降低高尿酸血症小鼠血尿酸水平的机制至少部分在于对肝脏黄嘌呤氧化酶的抑制作用;泽泻降低高尿酸血症小鼠血尿酸水平的机制可能主要在于促进尿酸排泄及增加尿量。3.土茯苓能减轻急性痛风性关节炎的关节肿胀,减少炎细胞浸润,降低局部组织炎症介质IL-1β和TNF-α的水平表明土茯苓有较强的抗炎及镇痛作用。4.SELDI-TOF MS技术检测到急性痛风性关节炎动物模型与正常小鼠及模型小鼠与中药治疗组间血清蛋白质谱的差异,对寻找急性痛风性关节炎模型小鼠血清蛋白标志物提供了有用的信息。
研究意义:1.确立了简便可靠的高尿酸血症动物模型。2.研究中发现中药土茯苓在高尿酸血症动物模型表现出降尿酸效果,对急性痛风性关节炎模型有抗炎镇痛作用,对研制安全、经济、有效的新型抗痛风药制剂有重要意义。3.运用SELDI-TOF MS蛋白芯片技术,检测到急性痛风性关节炎动物模型血清中特异性标志蛋白质峰,为进一步发现痛风性关节炎的特异性诊断标志物提供实验依据。
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.
引文
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