吴门芪藤汤对佐剂性关节炎大鼠膝骨关节滑膜、软骨组织细胞凋亡的影响

Effect of Wumen Qiteng decoction on apoptosis of synovial and cartilage tissues of knee joint in rats with adjuvant arthritis

目的观察吴门芪藤汤对佐剂性关节炎(AA)大鼠膝骨关节滑膜、软骨组织细胞凋亡的影响及其可能机制。方法从40只雄性健康SD大鼠中选30只建立AA模型,并随机分为模型组、阳性药组、中药组,每组10只;其余10只为空白组。造模成功后第2 d开始给药,模型组、空白组均予0.9%氯化钠注射液10 mL/kg灌胃;阳性药组予美洛昔康片10 mL/kg(3.4 mg/kg)灌胃;中药组予吴门芪藤汤10 mL/kg(24 g生药/kg)灌胃。每日灌胃1次,灌胃周期21 d。观察实验过程中各组大鼠一般状态变化;测定大鼠血清炎症因子白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)及基质金属蛋白酶3(MMP-3)含量;观察各组大鼠膝骨关节滑膜、软骨组织病理学变化;比较各组大鼠膝骨关节滑膜、软骨组织细胞凋亡指数变化;观察各组大鼠膝骨关节滑膜组织中c-Jun氨基末端激酶(JNK)蛋白表达水平。结果与空白组比较,其他各组大鼠10～14 d后开始出现多发性的关节炎症,病变累及到未注射弗氏完全佐剂的大鼠膝骨关节及足爪,表现为关节局部程度不同的红、肿、热及功能障碍。随着病程延长,多发性关节炎可反复出现,部分大鼠可在病程不同时期于耳、尾部出现数量不等、程度不一的红斑、炎性小结,并有脱毛、鼻黏膜充血、渗血、精神萎靡等关节外症状。与模型组比较,阳性药组、中药组大鼠关节炎症状明显减轻。模型组大鼠IL-1β、TNF-α及MMP-3水平均较空白组升高(P<0.05),说明造模成功。阳性药组、中药组IL-1β、TNF-α及MMP-3水平均低于模型组(P<0.05)。中药组IL-1β高于阳性药组(P<0.05)。模型组大鼠膝骨关节滑膜、软骨组织细胞凋亡指数均高于空白组(P<0.05)。阳性药组、中药组膝骨关节滑膜、软骨组织细胞凋亡指数均低于模型组(P<0.05)。阳性药组与中药组膝骨关节滑膜、软骨组织细胞凋亡指数组间比较差异均无统计学意义(P>0.05)。与空白组比较,模型组膝骨关节滑膜组织中JNK蛋白表达水平上调(P<0.05)。中药组膝骨关节滑膜组织中JNK蛋白表达水平低于模型组和阳性药组(P<0.05)。结论吴门芪藤汤可能通过抑制JNK信号通路,减少炎症介质释放,降低炎性刺激,从而抑制膝骨关节滑膜、软骨细胞过度凋亡,减轻膝骨关节炎症状。

Objective To observe the effect and its possible mechanism of Wumen Qiteng decoction on apoptosis of synovium and cartilage tissues of knee joint in rats with adjuvant arthritis (AA). Methods 30 male healthy SD rats were selected to establish AA model and randomly divided into model group, positive drug group and Chinese medicine group, with 10 rats in each group, and the rest of 10 rats as the blank group. On the 2nd day after the models were succeffessfully made,the model group and the blank group were given 0.9% sodium chloride injection 10 mL/kg by gavage,the positive drug group was given meloxican tablets 10 mL/kg(3.4 mg/kg)by garage,the Chinese medicine group was given Wumen Qiteng decoction 10 mL/kg(24 g crude drug/kg) by gavage, The drug was administered once a day for 21 days. The changes of general state of rats in each group during the experiment were observed. The serum levels of inflammatory factor interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α) and matrix metalloproteinase-3 (MMP-3) were measured. The histopathological changes of synovium and cartilage tissues of knee joint of rats in each group were observed. The changes of apoptotic index in synovial and cartilage tissues of knee joint of rats in each group were compared. To observe the expression of c-Jun amino terminal kinase (JNK) in synovial tissue of knee joint of rats in each group. Results Compared with the blank group, the rats in the other groups began to suffer from multiple arthritis about 10~14 days later. The lesions involved knee joints and feet without Freund's complete adjuvant injection, manifesting as local redness, swelling, fever and dysfunction of the joints. With the prolongation of the course of disease, polyarthritis can occur repeatedly. Some rats may have different amounts and degrees of erythema and inflammatory nodules in the ear and tail at different stages of the course, and have extraarticular symptoms such as hair removal, nasal mucosal congestion, bleeding and mental atrophy. Compared with the model group, the arthritis symptoms of rats in positive drug group and Chinese medicine group were significantly alleviated. The levels of IL-1β, TNF-α and MMP-3 in the model group were higher than those in the blank group ( P <0.05), indicating that the model was successful. The levels of IL-1β, TNF-α and MMP-3 in positive drug group and Chinese medicine group were lower than those in model group ( P <0.05), indicating that the treatment was effective. The IL-1β level in Chinese medicine group was higher than that in positive drug group ( P <0.05). The apoptotic index of synovium and cartilage tissue in model group was higher than that in blank group ( P <0.05). The apoptotic index of synovium and cartilage tissue in positive drug group and Chinese medicine group was lower than that in the model group ( P <0.05). There was no significant difference in apoptotic index of synovium and cartilage tissue between positive drug group and Chinese medicine group ( P >0.05). Compared with the blank group, the expression level of JNK protein in the synovial tissue of knee joint in the model group was up-regulated ( P <0.05). The expression level of JNK protein in synovial tissue of knee joint in Chinese medicine group was lower than that in model group and positive drug group ( P <0.05). Conclusion Wumen Qiteng decoction may inhibit the excessive apoptosis of synovial and chondrocyte of knee joint and relieve the symptoms of knee osteoarthritis by inhibiting JNK signaling pathway, reducing the release of inflammatory mediators and reducing inflammatory stimulation.