【作者】 刘娜；

【导师】 史艳秋；

【作者基本信息】 山东大学 ， 药理学， 2017， 硕士

【摘要】 复方痛风颗粒由知母、桂枝、生姜、防风等中草药组成。依据山东省某中医院知名老专家在临床上运用了多年的对痛风性关节炎有较好疗效的经验方,而研制的中药颗粒制剂。本课题采用多种动物模型对该药进行了药效学及作用机制初步研究。目的复方痛风颗粒通过家兔、大鼠两种急性痛风性关节炎模型观察对急性痛风性关节炎的作用,采用大鼠急性炎症模型、小鼠耳肿胀、小鼠热板、扭体法镇痛四种方法考察其抗炎镇痛效果,通过以上六种模型来初步探讨复方痛风颗粒的抗痛风作用机制,为临床用药提供实验依据。方法1.复方痛风颗粒对微晶型尿酸钠(Microcrystal sodium urate,MSU)致家兔急性痛风性关节炎(Acute gouty arthritis,AGA)的作用:复方痛风颗粒连续给药21d,第20天药后2h家兔关节腔注射80 mg/ml MSU混悬液0.4 ml/只制备AGA模型,第21d药后同法造模。于第1次致炎后1、3、5、24、29 h测关节肿胀度;末次致炎5h,测定关节液中WBC数目和PGE2含量,并测定关节滑膜组织中5-羟色胺(5-hydroxytryptamine,5-HT)、白细胞介素1 β(Interleukin 1 beta,IL-1 β)、白细胞介素8水平(Interleukin 8,IL-8),同时进行病理组织学检查。2.复方痛风颗粒对MSU致大鼠AGA的作用:复方痛风颗粒连续给药2天,首次药后即刻大鼠踝关节注射2.5 mg/ml MSU混悬液0.2 ml/只制备AGA模型。测定造模前和造模后1、3、5及24 h大鼠右后足容积;麻醉取血,测定血清中尿酸及前列腺素E2(Prostaglandin E2,PGE2)水平。3.复方痛风颗粒对角叉菜胶致大鼠足跖肿胀的作用:复方痛风颗粒连续21d。末次药后2h,大鼠足跖皮下注射1%角叉菜胶0.1 ml/只致足跖肿胀,测定造模前和造模后1、2、3、4及5h大鼠右后足容积。4.复方痛风颗粒对二甲苯致小鼠耳肿胀的作用:复方痛风颗粒连续给药14天。末次给药后2h,用微量注射器吸取25μl 100%二甲苯涂于右耳廓内面,左耳作为空白对照。15min后脱臼处死小鼠,剪下双耳,用8mm打孔器在同一部位打下圆耳片,称重,计算肿胀度、肿胀抑制率,从而评价药物对急性炎症反应的影响。5.复方痛风颗粒对小鼠热板法镇痛的作用:复方痛风颗粒连续给药14d。各组于末次给药后2h,放在事先加热到(55±0.5℃)的热板上,以舔后足为痛反应的指标,记录痛阈值。每鼠测定痛阈值2次(间隔1Omin),取其平均值。6.复方痛风颗粒对醋酸致小鼠扭体的镇痛作用:复方痛风颗粒连续给药15d。各组于给药2h后每鼠腹腔注射0.65%醋酸溶液(0.1 ml/10 g),注射后记录15 min内各组小鼠发生扭体反应次数。结果1.复方痛风颗粒对MSU致家兔AGA的作用:复方痛风颗粒高、中剂量3.0、1.5 g/kg/d与模型对照组比较,造模1、3、5h时家兔关节肿胀度明显减轻(P<0.01,P<0.05),关节液白细胞数目显著降低(P<0.01,P<0.05),炎症因子IL-1 β明显降低(P<0.05),PGE2、5-HT、IL-8未见明显影响(P>0.05)。复方痛风颗粒高、中、低剂量(3.0、1.5、0.75 g/kg)滑膜病理分值低于模型对照组,病变有所减轻,但统计学上未见显著性差异(P>0.05)。中剂量1.5 g/kg抗炎作用略优于痛风定0.18 g/kg,略逊于秋水仙碱4.0×10-5g/kg,均未见统计学差异(P>0.05)。2.复方痛风颗粒对MSU致大鼠AGA的作用:复方痛风颗粒高、中、低剂量6.8、3.4、1.7 g/kg/d与模型对照组比较,高剂量组动物在造模后3、5、24h关节肿胀度明显减轻(P<0.01,P<0.05),造模3h时抑制率达最大值为59.6%;中剂量组动物在造模24h时关节肿胀度明显减轻(P<0.05),抑制率达最大为35.9%;低剂量组关节肿胀度可见减轻但未显示统计学差异(P>0.05)。高、中剂量可明显降低血清中尿酸水平和炎症因子PGE2(P<0.01,P<0.05),秋水仙碱阳性对照组可明显降低炎症因子PGE2(P<0.05),秋水仙碱和痛风定阳性对照组对血清尿酸均未见明显影响(P>0.05)。中剂量3.4 g/kg降低血清中尿酸水平和PGE2略优于痛风定0.4 g/kg和秋水仙碱8.0×10-5 g/kg,均未见统计学差异(P>0.05)。3.复方痛风颗粒对角叉菜胶致大鼠足跖肿胀的作用:复方痛风颗粒高剂量6.8 g/kg/d与模型对照组比较,在造模后1、5h显示可显著抑制足跖肿胀度(P<0.01,P<0.05),造模2、3、4h时足跖肿胀度可见减轻但未见统计学差异(P>0.05),造模1h时抑制率最高,为77.3%。中剂量3.4 g/kg抗炎效果与痛风定0.4 g/kg相当,4h时抗炎效果弱于布洛芬0.07 g/kg(P<0.05),其余时间抗炎效果略逊于布洛芬0.07 g/kg但未见统计学差异(P>0.05)。4.复方痛风颗粒对二甲苯致小鼠耳肿胀的作用:高、中剂量13.4、6.7 g/kg/d与模型对照组比较,可显著降低耳廓肿胀度(P<0.01,P<0.05),抑制率分别为60.0%和45.9%。中剂量6.7 g/kg作用效果与痛风定0.8 g/kg、布洛芬0.13 g/kg相当。5.复方痛风颗粒对小鼠热板法镇痛的作用:高、中剂量13.4、6.7 g/kg/d与模型对照组比较,具有明显镇痛作用,热板小鼠的痛阈值明显升高(P<0.01,P<0.05)。中剂量6.7g/kg作用效果与痛风定0.8 g/kg、布洛芬0.13 g/kg相当。6.复方痛风颗粒对醋酸致小鼠扭体的镇痛作用:高、中剂量13.4、6.7 g/kg/d与模型对照组比较,具有明显镇痛作用,醋酸致小鼠扭体次数明显减少(P<0.01,P<0.05)。中剂量6.7 g/kg作用效果与布洛芬0.13 g/kg相当,略逊于痛风定0.8 g/kg,未见明显统计学差异(P>0.05)。结论:1、复方痛风颗粒(相当于人用剂量80、40g/d)对MSU诱导的家兔、大鼠AGA模型、二甲苯致小鼠耳部急性炎症均具有.显著的抗炎作用,对热板、醋酸致小鼠疼痛模型具有明显镇痛作用。复方痛风颗粒相当于人用量80g/d对角叉菜胶致大鼠足跖肿胀急性炎症模型具有显著抗炎作用。研究表明复方痛风颗粒是有效的治疗急性痛风性关节炎的药物。2、作用机制:复方痛风颗粒(相当于人用剂量80、40g/d)通过降低血清中尿酸水平、减少炎性细胞因子PEG2、IL-1 β的生成,发挥其良好的抗炎、镇痛作用。3、与同类药物的比较:MSU致家兔、大鼠AGA实验表明本品(家兔:1.5 g/kg,大鼠:3.4 g/kg)作用略逊于秋水仙碱(家兔:4.O×1 0-5 g/kg,大鼠:8.0×10-5g/kg)略优于痛风定(家兔:0.18 g/kg,大鼠:0.4 g/kg)。角叉菜胶致大鼠足跖急性炎症实验表明本品3.4 g/kg抗炎作用弱于布洛芬0.07 g/kg,与痛风定0.4 g/kg相当。二甲苯致小鼠耳肿胀实验显示本品抗炎效果与布洛芬和痛风定相当,小鼠镇痛实验显示本品作用与布洛芬相当,略逊于痛风定(小鼠抗炎镇痛实验中,本品:6.7 g/kg,布洛芬:0.13 g/kg,痛风定:0.8 g/kg)。更多还原

【Abstract】 Compound gout granules are composed of Anemarrhena,cassia twig,ginger,wind and other Chinese herbal medicine composition.A well-known old expert in a Chinese medicine hospital in Shandong province has used the experience prescription for the treatment of gouty arthritis for many years,Chinese medicine granule preparation is de-veloped according to this experience prescription.In this study,a variety of animal models were used to study the pharmacodynamics and mechanism of the drug.Objective.The effect of compound Gout Granules on acute gouty arthritis was observed in two models of acute gouty arthritis in rabbits and rats,The antiphlogistic analgesic effect was investigated by four methods of the acute inflammatory model,mice ear swelling,mouse hot plate and writhing analgesia,Through the above six models,the anti-gout mechanism of compound gout particles was discussed and the experimental basis was provided for the clinical application.Method 1.Effect of compound gout granules on acute GA in rabbits induced by MSU:Compound gout granules were continuously administered 21d,The 20th day two hours after of administration,The GA model was prepared by injection of 0.4 ml of 80 mg/ml MSU suspension in the joint cavity of rabbits,The same model was prepared in 21d.The degree of joint swelling was measured at 1,3,5,24,and 29h after the first inflammatory;at the last time of inflammatory 5h,the number of WBC and PGE2 content in the joint fluid were measured,determine the level of 5-HT、IL-1β、IL-8 in joint synovial tissues,histopathological examination was performed at the same time.2.Effect of compound gout granules on acute GA induced by MSU in rats:the compound gout granules were continuously administered 2 days.The GA model was prepared by the 0.2 ml of 2.5 mg/ml MSU suspension was injected into the ankle joint of rats immediately.The right posterior foot volume of rats at 1,3,5 and 24h were measured before and after modeling.Rats were anesthetized and blood was drawn,serum uric acid and PGE2 levels were measured.3.Effect of compound gout granules on carrageenin induced paw swelling in rats:Compound gout granules were continuously administered 21d,2h after the last dose,rats foot metatarsal were subcutaneously injected with 1%carrageenan 0.1 ml,1%carrageenan induced swelling of sole plantar.The right hind foot volume of rats was measured in 1,2,3,4 and 5h before and after the model establishment.4.Effect of compound gout granules on ear swelling induced by xylene in mice:Compound gout granules were continuously administered for 14 days.2h after the last administration,Use a microsyringe to absorb 25μl 100%xylene on the inner surface of the right auricle,left ear as a blank control.After 15min,the mice were killed by dislocation.The ears were cut.Make the circular ear slices at the same place with 8mm punch and weigh it.The swelling degree and the inhibition rate of swelling were calculated,and the effect of the medicine on the acute inflammatory reaction was evaluated.5.Effect of compound gout granules on hot plate method in mice:Compound gout granules continuous administration of 14d.Each group was treated with 2h at the end of the last dose,and placed on the hot plate(55+0.5 ℃)before heating,and the pain threshold was recorded by licking the foot as the index of pain response.The pain threshold of each mouse was measured 2 times(interval 10min),and the average value was determined.6.Analgesic effect of compound gout granules on writhing test in mice:Compound gout granules were continuously administered 15d.2h after each group was given,0.65%acetic acid solution(0.1 ml/10 g)was injected into abdominal cavity,and the number of writhing reactions of mice in each group was recorded after injection of 15min.Result 1.MSU induced rabbit acute GA test:Compound gout granules high dose 3.0 g/kg/d,medium dose 1.5 g/kg/d,compared with the model control group,model 1,3 and 5h of rabbit articular swelling significantly reduced(P<0.01,0.05),synovial fluid leukocyte number was significantly reduce(P<0.01,0.05),inflammatory cytokines decreased IL-1β significantly(P<0.05),PGE2,5-HT,IL-8(P>0.05)has no obvious effect.The pathological scores of the high,middle and low doses(3,1.5 and 0.75 g/kg)of the compound gout granules were lower than that of the model control group,the lesions were alleviated,but there was no statistically significant difference(P>0.05).anti-inflammatory effect of medium dose 1.5 g/kg/d is slightly better than Tongfengding 0.18 g/kg,slightly lower than the colchicine 4×10-5 g/kg,showed no significant difference(P>0.05).2.MSU induced acute GA test in rats:Compound gout granules high,medium and low dose of 6.8,3.4 and 1.7 g/kg/d compared with the model control group,high dose group of the animal at 3,5,24h after building model significantly reduce joint swelling degree(P<0.01,0.05),model 3h inhibition rate reached a maximum value of 59.6%;in the medium dose group in the model 24h joint swelling was significantly alleviated(P<0.05),the maximum of inhibition rate reached 35.9%;low dose group joint swelling visible reduce but no significant difference was found(P>0.05).High and medium dosage can significantly reduce serum uric acid level and inflammatory factor PGE 2(p<0.01,0.05),colchicine positive control group can significantly reduce inflammation factor PGE 2(p<0.05),colchicine and gout positive control group has no significant effect on serum uric acid(p>0.05).The medium dose of 3.4 g/kg decreased the serum uric acid levels and PGE2 slightly better than the gouty 0.4 g/kg and colchicine 8.0×10-5 g/kg,showed no significant difference(P>0.05).3.Carrageenan induced rat paw edema test:Compound gout granules with high doses of 6.8 g/kg/d compared with the control group,1h and 5h after building model showed significantly inhibited paw swelling(P<0.01,0.05),model 2,3 and 4h swelling visible alleviate but there was no statistical difference(P>0.05),1h after model the highest inhibition rate was 77.3%.The anti-inflammatory effect of the medium dose 3.4 g/kg was equal to Tongfengding 0.4 g/kg,4h anti-inflammatory effect is weaker than ibuprofen 0.07 g/kg(P<0.05),the rest time the anti-inflammatory effect is slightly weaker than of ibuprofen 0.07 g/kg but there was no statistical difference(P>0.05).4.Xylene induced ear swelling in mice:high and medium doses of 13.4,6.7 g/kg/d,compared with model control group,can significantly reduce the degree of swelling of the auricle(P<0.01,0.05),the inhibition rate was 60%and 45.9%.The effect of medium dose of 6.7 g/kg was similar to that of gouty 0.8 g/kg and ibuprofen 0.13 g/kg.5.Mice hot plate analgesia test:high and medium doses of 13.4,6.7 g/kg/d,compared with model control group,had obvious analgesic effect,and the pain threshold of hot plate mice increased significantly(P<0.01,0.05).The effect of medium dose of 6.7 g/kg was similar to that of gouty 0.8 g/kg and ibuprofen 0.13 g/kg.6.The writhing test of mice:the high and medium doses of 13.4 and 6.7 g/kg/d had obvious analgesic effect compared with the model control group,and the number of writhing times of mice caused by acetic acid was obviously reduced(P<0.01,0.05).In the medium dose effect of 6.7 g/kg was equal to the ibuprofen 0.13 g/kg,and was less than Tongfengding 0.8 g/kg,no statistically significant differences(P>0.05).Conclusion 1.Compound gout granules(equivalent human dose 80 40g/d)has significant anti inflammation effects induced by MSU in rabbits,acute GA rat model,xylene induced mouse ear acute inflammation,has obvious analgesic effect on the hot plate,the pain model of mice induced by acetic acid.When the compound Gout Granule is equivalent to the human consumption of 80g/d,it has significant anti-inflammatory effect when the acute inflammatory model of the foot swelling induced by carrageenan in rats.Studies have shown that compound Gout Granule is effective in the treatment of acute gouty arthritis.2.Mechanism of action:Compound gout granules(equivalent to human dose of 80,40g/d)by reducing uric acid levels in blood and the production of inflammatory cytokines IL-1βreduce,play a good anti-inflammatory and analgesic effects.3.Comparing with other drugs:The acute GA test in rabbits and mice induced by MSU shows that effect of this product(rabbit:1.5 g/kg,rat:3.4 g/kg)is slightly worse than that of colchicine(in rabbits:4.0×10-5 g/kg rats:8×10-5 g/kg)and slightly better than Tongfengding(rabbits:0.18 g/kg,rats:0.4 g/kg).The acute inflammation test in rats treated with carrageenin showed that the anti-inflammatory effect of 3.4 g/kg was weaker than that of ibuprofen 0.07 g/kg,which was similar to that of 0.4 g/kg.Xylene induced mouse ear swelling test showed that the anti-inflammatory effect of this products was equivalent to Bloven and Tongfengding,analgesic test showed that this function of product was equal to Bloven,and is slightly lower than Tongfengding(mice analgesic test,this product:6.7 g/kg,Bloven:0.13 g/kg,Tongfengding:0.8 g/kg).更多还原