【作者】 杨锦华；

【导师】 熊昌源；

【作者基本信息】 湖北中医学院 ， 中医骨伤科学， 2009， 博士

【摘要】 目的：探讨刺五加关节腔注射治疗原发性膝关节骨性关节炎的作用机制。方法：进行实验研究，64只新西兰白兔随机分为4组(每组16只)，16只不造模作为正常组，其余48只用内侧半月板部分切除的方法造模，最后分为正常组、刺五加组、透明质酸钠组，模型组，正常组既不造模也不给药，刺五加组给予刺五加注射液0．5ml／只术侧关节腔注射，透明质酸钠组给予0．3ml／只术侧关节腔注射，模型组只造模。术后2周开始用药干预，直到处死前1周。分6周和10周两个时相点采集血清和关节液，测定血液和关节滑液中NO、iNOs、SOD、MDA水平。同样，分两个时相点处死动物取材，肉眼观察关节软骨及滑膜，骨赘和软骨溃疡，拍膝关节X线片，观察软骨下骨的硬化情况。制备软骨及滑膜石蜡切片HE及AB-PAS染色，对软骨和滑膜分别在光镜下进行病理评分。用末端原位标记检测软骨细胞凋亡状况，免疫组化法检测bcl-2在软骨中的表达，并检测尿激酶型纤溶酶原激活物(uPA)在滑膜上的表达。结果：正常组关节面光滑有光泽，软骨透明，无明显缺损和新生物，滑膜无充血。6周时，可见刺五加组和HA膝关节有轻度病变，模型组病变较前者稍重。1 0周时，模型组关节面不光滑，无光泽，软骨不透明，有明显缺损和新生物，滑膜色暗充血肿胀；刺五加组软骨色泽较暗，可见溃疡形成较表浅，透明度差，有少到中等量骨赘形成．透明质酸钠组关节面见溃疡生成较表浅，滑膜肿胀，骨赘形成较多。随时间增长病变有加重的趋势，病理学评分分值增高。X线检查显示手术3组动物在6周时内侧关节间隙狭窄，胫骨平台软骨下骨密度轻微增高，可有少许骨赘生成；10周时上述变化更加明显。HE及AB-PAS染色可见：除正常组外，软骨的完整性均不同程度的遭到破坏。AB-PAS不同程度的基质失染，表示各手术造模组蛋白多糖有不同程度的丢失。还可见软骨中不同程度的新生血管和纤维化变，其中以模型组最严重，且10周组较6周组病变严重。6周时模型组血清及滑液中的NO较正常组有明显的升高(P＜0．05)，刺五加组和HA组的NO值无明显升高(P＞0．05)，HA组和模型组iNOs较正常组有所升高(P＜0．05)，刺五加组未见明显变化(P＞0．05)，HA组iNOs较刺五加组上升明显(P＜0．05)。各手术造模组的SOD较正常组都有明显的降低(P＜0．05)，各手术造模组MDA较正常组有明显增高。10周时各手术造模组的血清及关节滑液中的NO、iNOs、MDA水平较正常组有明显的升高，以模型组升高最明显(P＜0．05)。HA组NO水平较刺五加组上升明显(P＜0．05)。各手术造模组的SOD水平较正常组有明显的降低(P＜0．05)，刺五加组SOD水平又高于HA组(P＜0．05)。HA组MDA和iNOS水平似乎略高于刺五加组，但是无统计学意义。6周与10周两个时相点相比较，血清和关节滑液中的NO、iNOS及MDA水平有较明显的上升趋势(P＜0．05)，SOD水平有明显的下降趋势(P＜0．05)。两个时相点，刺五加组和HA组的凋亡率低于模型组(P＜0．05)，bcl-2的表达高与模型组(P＜0．05)，刺五加组与HA组之间无差别(p＞0．05)。在6周时，手术造模各组滑膜中uPA表达强于正常组(P＜0．05)，其中HA组和刺五加组的表达要弱于模型组(P＜0．05)。到1 0周时，HA组、刺五加组和正常组uPA表达较6周时未见明显变化，只有模型组的可以见有增加趋势但不具有统计学意义。结论：刺五加注射液可以减缓实验性兔膝关节骨性关节炎的病程进展，其机理可能是：1、刺五加注射液通过对滑膜与软骨两个途径减缓关节退变。2、刺五加有可能通过其活血化瘀，免疫稳定和糖皮质激素样作用，减轻滑膜病变，抑制巨噬细胞活化，从而下调iNOS从而减少NO的生成量，并且下调uPA在滑膜中的表达，减少炎症细胞因子的生成，从而降低细胞凋亡指数，减轻软骨的病变。3、刺五加是抗氧化剂，可升高关节液中SOD和软骨中的bcl-2水平，降低MDA水平，从而减少氧化应激对软骨细胞的损害，抑制软骨细胞凋亡。更多还原

【Abstract】 Objectives: To researth the mechanisms of intraarticular injecting Acanthopanax senticosus treating idiopathetic osteoarthritis of the knee joint.Methods: 64 New Zealand white rabbits were banlenced divided into 4 groups (n = 16), 16 Nonsurgical rabbits as a normal group, and the remaining 48 with resection of the partial medial meniscus model, subsequently divided into model group, Acanthopanax senticosus group and sodium hyaluronate group, the model group and the normal group were given no treatment. Each animal of Acanthopanax senticosus group was injected 0.5ml Acanthopanax senticosus parenteralia into the operative side knee weekly. The sodium hyaluronate group was given 0.3ml HA in the same way. All regimens were executed at 2 weeks after operation until 1 week before the animal sacrificed. At 6 weeks and 10 weeks both time points, all rabbits’ serum and synovial fluid was collected, then all samples of NO, iNOs, SOD and MDA levels were tested. At the same time point animals were killed, the surface of articular , synovial membrane, osteophyte size and cartilage ulcer area were estimated. In order to observe the situation of subchondral bone sclerosis and joint space, knee X-ray photo were taken. Cartilage and synovial membrane paraffin sections were made and stained with H&E and AB-PAS, respectively. By light microscope, each section was evaluated and scored. Chondrocyte apoptosis were detected by in situ terminal labeling method, the expression of bcl-2 in cartilage and urokinase-type plasminogen activators (uPA) in synovial membrane were detectived by immunohistochemistry technique.Results: At both time points, the normal group has shiny smooth articular surface, with transparent cartilage appearence, no obvious defects and osteophyte generating, as well as synovial congestive. At 6 week, there were more severe changes in model group than other group.At 10 week; there was a rough matte articular surface, markedly opaque defective cartilage, a lot of new biology formation, dark swelling congesting synovium in model group. While, Acanthopanax senticosus group has a less rough articular surface, a light gloomy cartilage, more superficial ulcers, lower degree of transparency, little to moderate amount of osteophyte formation and a light swelling synovium. Except for larger osteophyt formation in HA group than that in cwj group, there was no significant difference between the two groups. Over time, each of the rabbits’ knee OA defect has aggravated, so it pathological score increased. Animals in each group, at the end of 6 week, there had been slight subchondral bone osteosclerosis and joint space narrow happened, and, at 10 week, all these got wores. Except for the normal group, H&E and AB-PAS staining showed that the integrity of cartilage was damaged to various degrees. The extent of AB-PAS staining losing indicated that the different proteoglycan decrease in the matrix of the cartilage among the three operation groups, As well as some new born veins and fibrosis were found in the articular cartilage sections. There was the most defective cartilage in model group. Also, with the time past, neovascularization and fibrosis formation were more and more severe. At the end of 6 week, when compared with the normal group, in all surgery groups, serum and synovial fluid values of NO increased significantly (P <0. 05). However, the level of No in Acanthopanax senticosus and HA group was changed slightly. (P> 0. 05) Although iNOs value of HA group and model group increased significantly (P<0. 05), there was no significant changes in Acanthopanax senticosus group (P>0. 05). HA group had higher iNos values than that of Acanthopanax senticosus Group (P<0. 05). compared to normal group, those three surgery group have significantly lower SOD levels, but higher MDA levels (P<0. 05). At 10 weeks, in all three surgical groups, serum and synovial fluid of the NO, iNos, MDA levels were higher than that in the normal group (P <0. 05), in addition, the model group had the highest values. There was significantly lower NO levels in Acanthopanax senticosus group than that in HA group (P <0. 05). SOD levels of three surgery groups were lower than that of the normal group(P<0. 05). Also, compared to the HA group, Acanthopanax senticosus group had higher SOD level (P <0. 05). It seems that the levels of MDA and iNOS in HA group was slightly higher than that of in the Acanthopanax senticosus group, but, it was no statistical significance, compared to the end of 6 weeks, at the end of 10 weeks, the NO, iNOS and MDA level of the serum and synovial fluid of the joints appeared upward tendency (P<0. 05), however, SOD levels was declined markedly (P<0. 05). Both of those two time points, the index of apoptosis of the HA and Acanthopanax senticosus group were lower than that of the model group (P<0. 05), but, bcl-2 expression was higher than that of the model group (P < 0. 05). There was no difference between Acanthopanax senticosus group and the HA group (p>0. 05). AT 6 weeks, positive expression of synovial uPA in the three surgery group was stronger than that of the normal group(P <0. 05), positive expression of synovial uPA in the HA and the Acanthopanax senticosus group were weaker than that of the model group (P < 0.05). By the 10 weeks, compared to 6 weeks, positive expression of uPA in HA Acanthopanax senticosus and normal groups had no significant changes .Although It seemed that there was an upward trend of positive upa expression in the model group, it was no difference statistically.Conclusions: The rabbit’s experimental osteoarthritis disease progression can be slowed down by intraarticular injection of Acanthopanax senticosus. The mechanisms may be:1、Joint degeneration may be slowed by Acanthopanax senticosus by both mediators which is synovium and cartilage.2、By using Acanthopanax senticosus, blood circulation may be promoted to remove blood stasis,Synovial lesions may be improved. Because of its immunal stability and role of glucocorticoid-like, macrophage activation was inhibited, thus,iNOS expression , NO formation and uPA expression in the synovium could be reduced, as a result, the generation of inflammatory cytokines may be cut down, the apoptotic index could be reduceded, so, the cartilage lesions got lighted.3、Acanthopanax senticosus being an antioxidant, levels of SOD in both synovial fluid and blood could be elevated as well as bcl-2 expression in cartilage, however, the MDA levels could be lowered, subsequently, the cartilage cells could be prevented from oxidative stress damage, and apoptotic index of cartilage cells could be cut down.更多还原