白虎追风丸抗RA作用机理研究——对AA大鼠滑膜细胞功能的影响

【作者】 张声鹏；

【导师】 赖天松；

【作者基本信息】 广州中医药大学 ， 方剂学， 2001， 硕士

【摘要】 类风湿关节炎是一种以周围关节的对称性多关节炎为主要特征的慢性全身性疾病，具有病程长，致残率高等特点。RA的病因未明，发病机制尚未完全清楚。已知由滑膜细胞、软骨细胞、巨噬细胞等分泌的单核因子如IL-1、TNF等在RA的急性滑膜炎症、纤维化，骨及软骨破坏中起主导作用。NO与PGE₂是重要的炎症介质，在RA的发病机制中起具有广泛的效应。因此，通过抑制RA滑膜分泌过多的细胞因子IL-1、TNF及释放过多的炎症介质NO、PGE₂应是治疗RA的可取途径之一。 白虎追风丸是在白虎加桂枝汤基础上加减而成，具有清热祛湿、驱风通络止痛的功效，经多年临床运用证实本方对早期RA具有较好疗效。为了深入探讨其抗RA的作用机理，我们以大鼠AA作为病理模型，观察了白虎追风丸对AA大鼠的治疗作用，及其对原代培养滑膜细胞分泌IL-1β、TNF-α及释放NO、PGE₂的影响。 本课题的研究结果表明： 1、大鼠滑膜细胞原代培养方法学研究 滑膜组织通过消化法与组织块培养法均能获得理想的培养体系，但消化法可获得三种典型的细胞形态：巨噬细胞样细胞（MCs）、成纤维样细胞（FCs）、树突样细胞（DCs），而组织块培养法只能获得FCs一种细胞形态。最佳消化分离条件为0.1％胶原酶（出含10％小牛血清DMEM完全培养液配制）消化2.5小时，再由0.25％胰蛋白酶（D-hanks液配制）消化0.5小时。 2、白虎追风丸对AA大鼠的治疗作用 白虎追风丸中、低剂量组使大鼠体重增加且增加量明显高于正常组，高剂量组与雷公藤多甙组体重增加量明显低于正常组，显示本方剂量相关性地对大鼠体重的增加量呈双向调节作用。白虎追风丸各剂量组对AA大鼠致炎侧的足趾肿胀尤其是继发性肿胀显示了良好的抑制作用，但与雷公藤多贰相比无显著性差异。改良玻璃容器测量法与足趾厚度法的足趾肿胀率测量结果呈正相关。踝关节病理切片显示，白虎追风丸高、中、低剂量组均可在不同程度上减轻滑膜水肿及炎性细胞浸h)］，尤以高剂量组最为明显。雷公藤多贰组也可以减轻滑膜的炎性细胞浸润，但滑膜血管扩张较为明显。 3、白虎追风丸对原代培养滑膜细胞功能的影响 白虎追风丸各剂量组均可降低AA 大鼠原代滑膜细胞分泌的几－！0、TNF－日水平，也可降低培养上清液中 NO、PGEz水平，其作用与雷公膝多贰相比无显著性差异。 本课题以白虎追风丸为处理因素，以AA大鼠为受试对象，通过消化法原代培养滑膜细胞，从细胞药理学的角度探讨了本方抗RA作用机理，实验结果初步揭示其治疗RA的机理与抑制滑膜细胞分泌炎症性细胞因子 IL－16、TNF－口及释放炎症介质 NO、PGEz有关，并对今后的进一步研究提出了引入中药血清药理学实验方法的设想。更多还原

【Abstract】 ABSTACTRheumatoid arthritis is a chronic disease mainly characterizedas peripheral symmetric and polyarticular inflammation, it alwayslasting long time and having high cripple rate. Neither the causenor the mechanism of RA is entirely clear. It has been known thatmonokines including IL- l 0 and TNF- Q secreted bysynoviocytes, chondrocytes, macrophage have a main effect in themany processes of RA, such as acute synovial inflammation,fibrosis, distruction of bone and cartilage, etc. As two kinds ofimportant inflammatory mediums, nitric oxide (NO) andprosglandin E 2 (PGE2) have a widely effect in the mechanism ofRA. So, it must be a reasonable way to treat RA by restrain thesynovial membrane secreting too many cytokines such as IL-l 6,TNF- Q or releasing too many inflammatory mediums such as NO,PGE2.Baihu Zhuifeng Wan (BFZFW) was created on the base ofBaihu JiaGuizhi Tang. It has the effects of clearing heat andremoving dampness, dispe1ling wind and activating channel,alleviating pain. lt has been proved having a good therapeuticeffect for early RA through clinic operations for many years. Tostudy the mechanism of this effect deeply, we observed thetherapeutic effect on adjuvant arthritis(AA) rats by BHZFW, andthe effect on the secretion level of IL-l 6,TNF- Q and releasinglevel of NO, PGE2 by primary cultured synoviocytes derived fromthose AA rats.The results of this discussion indicate that:1. Although we can get primary cultured synviocytes by bothdigesting method and tissue method, just digesting method makes itSure that we can observe three typical kinds of cell shapes:3Macrophagelike cells (MCs), fibroblastlike cells (FCs), dendriticcells (DCs). At the same time, we get only one kind of cell shapeby tissue method: FCs. The best condition for digestingsynoviocyte is digested by 0. l% coIIagenase for 2.5 hours, and thedigested by 0.25% tripsin for 0.5 hour.2.The therapeutic effects of BHZFW on AA rats.The middle and low dose of BHZFW obviously make the addedweight of AA rats larger than the control. High dose andtriterpenoids make the added weight smaller than the control. Suchresults showe a two way regulation effect to the weight adding ofAA rats. Each dose of BHZFW showe a good restraining effect onthe sweIling rate of AA rats’ paws, especially the continuedsweIIing, but there is not obviousIy difference from the effect oftriterpenoids. There is a positive corre1ation between the pawvolume method and paw thickness method on measuring theswelling rate. Pathological study indicate that all the doses ofBHZFW can Iighten the synovium sweIling and inflammatory ceIlsinfiltration, high dose is more obviousIy. Triterpenoids also canlighten the infiltration of inflammatory cells but always aIong withenl arged .vessel s.3.The effect of BHZFW to primary synviocyte function.Each dose of BHZFW can decrease the IL-l or TNF- Q levelsecreted by primary cultured synviocytes derived’from AA rats, sodoes NO and PGE2. There is not any obvious difference betweenBHZFW and triterpenoids.Making BHZFW as treating factor and AA rats as tested object,we researched the anti-RA mechanism of BHZFW from the metl1odof cytopharmcological by primary cu1tured digested synoviocytesin this discussion. The resu1ts of this experiment showe that the4mechanism is concerned with decreased lL- 1 P, TNF- Q secretionlevel and NO, PGE2 releasing level by synoviocytes. In this articlewe also consider the seropharmcological experimental method asa usefuI approach tbr fLlture study.更多还原