靶向活化Ⅱ型大麻素受体抑制RA炎症性骨侵蚀的实验研究

发布时间：2018-06-07 06:56

本文选题：类风湿性关节炎 + 炎症　；参考：《苏州大学》2015年硕士论文

【摘要】：目的:观察Ⅱ型大麻素受体(cannabinoid receptor 2,CB2)在类风湿性关节炎(rheumatoid arthritis,RA)疾病进程中的表达变化,分析CB2选择性激动剂JWH-133对RA炎症性骨侵蚀的治疗作用。方法:雄性DBA/1小鼠50只,采用随机数字表法分为5组,即正常对照组(Control)、模型组(Vehicle)、JWH-133低剂量治疗组(L-JWH-133)、JWH-133高剂量治疗组(H-JWH-133)和阳性对照组(甲氨蝶呤治疗组,MTX),每组10只。采用牛Ⅱ胶原诱导的小鼠CIA模型,药物治疗组于初次免疫后第28天(D28)经腹腔分别给予JWH133(1mg/kg/d和10mg/kg/d)和MTX(20mg/kg,每周2次),连续给药6周。模型组经腹腔注射生理盐水0.1ml/kg/d,空白组小鼠常规饲养,不予处理。分别观察各组小鼠的体重、后足足掌厚度变化,并对关节炎的肿胀程度进行评分。Micro-CT检查评估骨组织破坏程度,HE染色评估滑膜增生和炎性细胞浸润程度,番红O染色检测软骨破坏程度,抗酒石酸酸性磷酸酶(TRAP)染色检测成熟破骨细胞,免疫组织化学染色检测CB2、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白介素(interleukin,IL)-1β、白介素-6(IL-6)、基质金属蛋白酶-3(matrix metallo proteinases-3,MMP-3)、环氧化酶-2(Cyclooxygenase-2,COX-2)、诱导性一氧化氮合酶(hadueed-nitric oxide syathase,i NOS)以及核因子-κB受体活化因子配体(receptor activators of nuclear factor-kappa B ligand,RANKL)和骨保护素(osteoprotegerin,OPG)的表达变化。结果:DBA/1小鼠初次免疫21天(D21)开始,体重逐渐下降,最低为(D35,17.99±1.44)g,明显低于Control组(D35,21.81±1.23)g比较差异有统计学意义(P0.05)给予JWH-133后,小鼠体重逐渐增加,与Vehicle组比较,差异有统计学意义(P0.05)。CIA模型小鼠后足于D21开始出现肿胀,靶向激活CB2后小鼠足掌厚度开始减少,H-JWH-133组D42足掌厚度为(2.52±0.12)mm与Vehicle组(2.63±0.07)mm比较,差异有统计学意义(P0.05);D42时H-JWH-133组关节炎评分(13.6±1.14)与Vehicle组(15.6±0.55)比较,差异有统计学意义(P0.05)。Micro-CT扫描结果显示Vehicle组骨质破坏严重,关节结构消失,经选择性CB2受体激动剂治疗后骨质破坏逐渐减轻,关节结构得到恢复;Control组骨体积(BV)、骨体积分数(BV/TV)、骨面积分数(BS/BV)、骨小梁厚度(Tb.Th)分别为(5.90±0.12)mm3、(32.52±0.89)%、(9.26±0.62)mm、(0.36±0.02)mm;Vehicle组为(3.77±0.22)mm3、(17.81±0.95)%、(17.78±1.03)mm、(0.21±0.01)mm,H-JWH-133组为(3.91±0.23)mm3、(19.45±0.98)%、(16.82±0.56)mm、(0.24±0.01)mm;H-JWH-133组为(5.22±0.19)mm3、(24.94±1.12)%、(11.81±0.60)mm、(0.30±0.02)mm,与Vehicle组相比差异有统计学意义(P0.01)。HE染色显示Vehicle组滑膜组织滑膜组织显著增生,大量炎症细胞浸润,靶向活化CB2后滑膜组织炎症明显减轻。番红O染色显示Vehicle组软骨细胞大量坏死,局部软骨层完全消失,H-JWH-133组软骨细胞显著增多,软骨层结构明显恢复。TRAP染色结果显示,CIA模型组关节有大片紫红色区域,H-JWH-133治疗后破骨细胞数量明显减少。免疫组织化学染色结果显示CB2在CIA小鼠的滑膜组织和骨组织均有表达;Vehicle组TNF-?、IL-1?、IL-6、MMP-3、COX-2和i NOS的表达明显增高,选择性CB2受体激动剂治疗后上述因子表达显著降低;H-JWH-133组RANKL表达强度较Vehicle组显著降低,OPG表达则高于Vehicle组。结论:CB2选择性激动剂JWH-133能抑制炎性细胞浸润,减少炎性因子分泌;JWH-133能下调RANKL表达,促进OPG表达,抑制软骨和骨组织的破坏。CB2有望成为治疗RA炎症性骨侵蚀的新靶点。
[Abstract]:Objective: To observe the expression changes of cannabinoid receptor 2 (CB2) in the process of rheumatoid arthritis (rheumatoid arthritis, RA), and to analyze the therapeutic effect of CB2 selective agonist JWH-133 on RA inflammatory bone erosion. Methods: 50 male DBA/1 rats were divided into 5 groups by random digital table, that is, normal control group (normal control group). Control), model group (Vehicle), JWH-133 low dose treatment group (L-JWH-133), JWH-133 high dose treatment group (H-JWH-133) and positive control group (methotrexate treatment group, MTX), 10 rats in each group. The mouse CIA model induced by bovine collagen was used, and the drug treatment group was given JWH133 (1mg/kg/d and 10mg/kg/d) in the abdominal cavity twenty-eighth days after primary immunization (D28). And MTX (20mg/kg, 2 times a week) for 6 weeks. The model group was injected with saline 0.1ml/kg/d by intraperitoneal injection, and the blank group was fed with normal feeding, no treatment. The weight of the mice in each group and the thickness of hind foot palmar were observed, and the degree of swelling of the arthritis was evaluated by.Micro-CT examination to evaluate the destruction of bone tissue, and HE staining was used to evaluate the increase of synovial membrane. The degree of infiltration of raw and inflammatory cells, the damage degree of cartilage by O staining, anti tartaric acid phosphatase (TRAP) staining to detect mature osteoclasts, immunohistochemical staining for detection of CB2, tumor necrosis factor - alpha (tumor necrosis factor- alpha, TNF- a), interleukin, IL -1 beta, interleukin--6 (IL-6), matrix metalloproteinase X metallo proteinases-3, MMP-3), cyclooxygenase -2 (Cyclooxygenase-2, COX-2), inducible nitric oxide synthase (hadueed-nitric oxide Syathase, I NOS), and the expression changes of nuclear factor kappa activation factor ligand and osteoprotegerin. /1 mice first immunized 21 days (D21), the body weight gradually decreased, the lowest was (D35,17.99 + 1.44) g, obviously lower than the Control group (D35,21.81 1.23) g, the difference was statistically significant (P0.05), the mice weight gradually increased, and compared with the Vehicle group, the difference has statistical significance (P0.05).CIA model mice began to appear swelling after the onset. The thickness of the foot of the mouse was reduced after the target activation of CB2, and the thickness of D42 foot palms in group H-JWH-133 was (2.52 + 0.12) mm and Vehicle group (2.63 + 0.07) mm, the difference was statistically significant (P0.05); at D42, the score of H-JWH-133 (13.6 + 1.14) was compared with the Vehicle group (15.6 + 0.55), and the difference was statistically significant (P0.05). In group ehicle, bone destruction was serious, joint structure disappeared, bone destruction gradually lessen and joint structure recovered after selective CB2 receptor agonist; Control group bone volume (BV), bone volume fraction (BV/TV), bone area fraction (BS/BV), bone small Liang Houdu (Tb.Th) were (5.90 + 0.12) mm3, (32.52 + 0.89)%, (9.26 + 0.62) mm, (0.36 + 0.02) mm; V Group ehicle was (3.77 + 0.22) mm3, (17.81 + 0.95)%, (17.78 + 1.03) mm, (0.21 + 0.01) mm, H-JWH-133 group was (3.91 + 0.23) mm3, (19.45 + 0.98)%, (16.82 + 0.98) mm, mm, H-JWH-133 +% mm3, mm, mm, and mm. The synovial tissue of the synovium of group E was significantly proliferated, a large number of inflammatory cells infiltrated, and the inflammation of the synovial tissue was obviously reduced after the target activation of CB2. The red O staining showed that the chondrocytes in the group Vehicle were necrotic, the local cartilaginous layer completely disappeared, the cartilage cells in the H-JWH-133 group increased significantly, the cartilage structure was obviously restored to the.TRAP staining results, and the CIA model group was displayed. The number of osteoclasts in the joint was significantly reduced after H-JWH-133 treatment. The results of immunohistochemical staining showed that CB2 was expressed in the synovium and bone tissue of CIA mice, and the expression of TNF-? IL-1?, IL-6, MMP-3, COX-2 and I NOS increased in the Vehicle group, and the expression of the above factors was significant after the selective CB2 agonist. The expression intensity of RANKL in group H-JWH-133 was significantly lower than that in group Vehicle, and the expression of OPG was higher than that in group Vehicle. Conclusion: CB2 selective agonist JWH-133 can inhibit inflammatory cell infiltration and reduce inflammatory factor secretion; JWH-133 can down regulate the expression of RANKL, promote OPG expression, and inhibit the destruction of cartilage and bone tissue..CB2 is expected to be the treatment of inflammatory bone erosion of RA. New targets.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.22

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