祖师麻膏药对胶原诱导型关节炎小鼠炎症及骨破坏的抑制作用

Inhibition of inflammation and bone destruction in mice with collagen-induced arthritis by Zushima Plaster

目的:探讨祖师麻膏药对CIA小鼠炎症及骨破坏的抑制作用。方法:采用SPF级雄性DBA/1小鼠,随机选取6只为正常组,将造模成功的18只CIA小鼠按照随机数字表法分为模型组、膏药组(1.0 g/kg)、扶他林组(0.12 g/次),每组各6只,各个给药组按照体质量予以药物贴敷治疗,正常组和模型组给予生理盐水同时贴敷透气胶纸,每日一次,4 h/次,连续给药4周。采用关节炎评分指数观测各组小鼠关节炎症状变化及关节炎指数评分,Micro-CT观察小鼠后爪损伤情况,实时荧光PCR法检测踝关节组织IL-17、IL-1β、TNF-αmRNA表达,免疫组织化学法检测踝关节组织OPG、RANKL蛋白的表达,苏木素-伊红(HE)染色后观察滑膜组织病理改变,抗酒石酸酸性磷酸酶(TRAP)法观察踝关节组织破骨细胞变化。结果:与正常组比较,模型组小鼠关节炎指数评分明显升高(P<0.05)。Micro-CT示小鼠后爪骨侵蚀严重,骨面破坏,骨量减少;踝关节组织IL-17、IL-1β、TNF-αmRNA(PCR)表达明显升高(P<0.05)。免疫组化显示踝关节组织OPG蛋白相对表达量降低(P<0.01),RANKL蛋白相对表达量升高(P<0.01)。HE结果显示中度的炎性细胞浸润,滑膜细胞肿大,大量的血管翳形成及滑膜增生;破骨细胞数量增加,关节软骨组织表面粗糙,出现严重的骨破坏,软骨层变薄。与模型组比较,膏药组和扶他林组小鼠关节炎症状缓解,关节炎指数评分降低;小鼠后爪骨质密度改善,有效地缓解骨质疏松;踝关节组织IL-17、IL-1β、TNF-αmRNA(PCR)表达明显降低(P<0.05);免疫组化结果示OPG蛋白相对表达量升高(P<0.05),RANKL蛋白相对表达量降低(P<0.01)。HE结果示滑膜细胞肿大明显改善,轻度炎性细胞浸润,滑膜增生不明显;破骨数量减少,关节软骨破坏明显改善缓解,软骨层厚度显著增加。结论:祖师麻膏药可以缓解RA局部症状,降低炎症因子表达,减轻炎症反应,可能是通过调控OPG/RANKL信号轴抑制破骨细胞的分化及活化,进一步改善关节骨及软骨破坏的生物学效应。

AIM:To investigate the inhibitory effect of Zushima ointment on inflammation and bone destruction in CIA mice.METHODS:SPF grade male DBA/1 mice were used,6 were randomly selected as the normal group,and 18 CIA mice that were successfully modelled were randomly divided into the model group,the plaster group(1.0 g/kg),and the fuselage group(0.12 g per time)according to the random number table method,6 mice in each group,and each administered group was given medication according to the body mass,and saline was given to both the normal and model groups.The normal group and the model group were given saline,and breathable adhesive paper was applied once a day for 4 h/session for 4 consecutive weeks.The arthritis scoring index was used to observe the changes of arthritis symptoms and arthritis index scores of mice in each group.Micro-CT was used to observe the damage of hind paw of mice,real-time fluorescence PCR was used to detect the mRNA expression of IL-17,IL-1βand TNF-αin ankle joint tissues,and immunohistochemistry was used to detect the expression of OPG and RANKL proteins in ankle joint tissues,and hematoxylin-eosin(HE)staining was used to observe the pathological changes of synovial tissues after the treatment.The pathological changes of synovial tissue were observed after hematoxylin-eosin(HE)staining,and the changes of osteoclasts in ankle joint tissue were observed by anti-tartaric acid phosphatase(TRAP)method.RESULTS:Compared with the normal group,the arthritis index score of the model mice was significantly higher(P<0.05).Micro-CT showed severe bone erosion in the hind paws of the mice,destruction of the bone surface and reduction of bone volume.The expression of IL-17,IL-1βand TNF-αmRNA(PCR)in the ankle joint tissues was significantly higher(P<0.05).Immunohistochemistry showed that the relative expression of OPG protein in the ankle joint tissues was reduced(P<0.05).Immunohistochemistry showed a decrease in the relative expression of OPG protein(P<0.01)and an increase in the relative expression of RANKL protein(P<0.01).HE results showed moderate inflammatory cell infiltration,swelling of synovial cells,massive formation of vascular opacities and synovial hyperplasia;an increase in the number of osteoclasts,roughness of the surface of articular cartilage tissue,severe bone destruction and thinning of the cartilage layer.Compared with the model group,the arthritic symptoms of mice in the cream group and the futalin group were relieved and the arthritis index score was reduced;the bone density of the mice's hind paws improved,effectively relieving osteoporosis;the expression of IL-17,IL-1βand TNF-αmRNA(PCR)in the ankle joint tissue was significantly reduced(P<0.05);the immunohistochemical results showed that the relative expression of OPG protein was increased(P<0.05),the relative expression of RANKL protein decreased(P<0.01).HE results showed that synovial cell enlargement was significantly improved,mild inflammatory cell infiltration,synovial hyperplasia was not obvious;the number of broken bone was reduced,articular cartilage destruction was significantly improved and relieved,and the thickness of cartilage layer was significantly increased.CONCLUSION:Ancestral hemp poultice relieves local symptoms of RA,reduces the expression of inflammatory factors and attenuates the inflammatory response,possibly by inhibiting osteoclast differentiation and activation through modulation of the OPG/RANKL signalling axis,which further ameliorates the biological effects of articular bone and cartilage destruction.