附子汤对佐剂性关节炎大鼠的治疗作用

Therapeutic effect of Fuzi decoction on adjuvant arthritis rats

目的观察附子汤对佐剂性关节炎(AA)大鼠的治疗作用,并探讨附子汤对炎性细胞因子肿瘤坏死因子α(TNF⁃α)、白介素-17A(IL⁃17A)和磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(AKT/PKB)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路基因蛋白表达的影响,为临床应用提供实验依据。方法在大鼠右后足跖皮内注射弗氏完全佐剂建立佐剂性关节炎模型,并随机分为空白组、模型组、硫酸羟氯喹阳性药对照组(阳性对照组,42 mg·kg^(⁃1)·d^(⁃1))、附子汤低剂量组(2.678 g·kg^(⁃1)·d^(⁃1))、附子汤中剂量组(5.355 g·kg^(⁃1)·d^(⁃1))、附子汤高剂量组(10.71 g·kg^(⁃1)·d^(⁃1))。造模成功后开始进行每日灌胃给药,持续28 d。观察致炎前后大鼠一般情况和致炎足足趾容积变化。实验结束后采用苏木精-伊红染色法(HE染色法)观察大鼠踝关节病理组织学改变情况,ELISA检测血清中TNF⁃α、IL⁃6、IL⁃2、IL⁃17A水平,免疫组织化学染色法检测踝关节滑膜组织PI3K、AKT、mTOR的蛋白表达。结果造模后第6天,与空白组原发性足趾肿胀度(0.11±0.02)mL比较,模型组原发性足趾肿胀度(2.20±0.66)mL显著升高,差异有统计学意义(P<0.01)。给药后,在第33天时,与模型组原发性足趾肿胀度(2.75±0.32)mL比较,阳性对照组和附子汤低、中、高剂量组原发性足趾肿胀度分别为(0.58±0.18)、(0.85±0.22)、(0.65±0.17)、(0.46±0.13)mL,差异均有统计学意义(P均<0.05)。踝关节组织病理学观察显示,模型组出现踝关节滑膜结构破坏,滑膜细胞增殖,炎性细胞浸润,血管翳形成,广泛纤维组织增生,骨侵蚀等现象,阳性对照组和附子汤各治疗组踝关节组织病变有一定程度的改善。模型组TNF⁃α水平为90.47(79.59~91.65)pg/mL,附子汤低、中、高剂量组TNF⁃α均下降,分别为43.72(23.73~64.54)、32.12(24.45~36.93)、29.39(22.41~39.73)pg/mL,差异均有统计学意义(P均<0.05);模型组IL⁃17A水平为2.20(2.03~2.45)pg/mL,附子汤高剂量组IL⁃17A水平1.01(0.59~1.44)pg/mL显著下降,差异有统计学意义(P<0.01)。与模型组比较,阳性对照组及附子汤低、中、高剂量组踝关节滑膜组织PI3K、AKT蛋白表达水平均显著下调,差异均有统计学意义(F=11.435、11.104,P均<0.05);附子汤低、高剂量组踝关节滑膜mTOR蛋白表达水平与模型组比较显著下调,差异均有统计学意义(F=8.063,P均<0.05)。结论附子汤对AA大鼠具有较好的治疗作用,可以明显缓解关节的肿胀程度,关节病理改变得到明显改善,作用机制与减少炎症细胞因子TNF⁃α、IL⁃17A的产生和负调控PI3K/AKT/mTOR信号通路基因的蛋白表达有关。

Objective To observe the therapeutic effect of Fuzi decoction on adjuvant arthritis(AA)rats,and explore the influence of Fuzi decoction on the protein expression of inflammatory cytokines,tumor necrosis factorα(TNF⁃α),interleukin⁃17A(IL⁃17A)and phosphatidylinositol 3 kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)signaling pathway genes,so as to provide experimental basis for its clinical application.Methods The arthritis model was established by intradermal injection of Freund's complete adjuvant in the right hind paw of rats,and they were randomly divided into blank group,model group,hydroxychloroquine sulfate positive drug control group(42 mg·kg^(⁃1)·d^(⁃1)),low dose of Fuzi decoction group(2.678 g·kg^(⁃1)·d^(⁃1)),medium dose of Fuzi decoction group(5.355 g·kg^(⁃1)·d^(⁃1)),high dose of Fuzi decoction group(10.71 g·kg^(⁃1)·d^(⁃1)).After the success of modeling,the drug was given orally every day for 28 days.The general situation of rats before and after inflammation and the change of toe volume were observed.After the experiment,the HE staining method was used to observe the histopathological changes of the ankle.The contents of serum TNF⁃α,IL⁃6,IL⁃2 and IL⁃17A were detected by ELISA.The protein expressions of PI3K,AKT and mTOR in the synovial tissue of the ankle joint were detected by immunohistochemistry staining.Results On the 6th day after modeling,compared with the primary toe swelling(0.11±0.02)mL in the blank group,the primary toe swelling(2.20±0.66)mL in the model group was significantly increased,the difference was statistically significant(P<0.01).On the 33rd day after administration,compared with the primary toe swelling degree of the model group(2.75±0.32)mL,the primary toe swelling degrees of the hydroxychloroquine sulfate positive drug control group and the low,medium and high dosage groups of Fuzi decoction were(0.58±0.18),(0.85±0.22),(0.65±0.17),and(0.46±0.13)mL,respectively;the differences were statistically significant(all P<0.05).Histopathological observation of ankle joint showed that in the model group,the synovial membrane structure of ankle joint was destroyed,synovial cells proliferated,inflammatory cells infiltrated,pannus formed,extensive fibrous tissue hyperplasia,bone erosion and other phenomena.The pathological changes of ankle joint in the group with hydroxychloroquine sulfate and Fuzi decoction were improved to some extent.The level of TNF⁃αin the model group was 90.47(79.59-91.65)pg/mL,while that in the low,middle and high dosage groups of Fuzi decoction decreased to 43.72(23.73-64.54),32.12(24.45-36.93)and 29.39(22.41-39.73)pg/mL,respectively;the differences were statistically significant(all P<0.05).The level of IL⁃17A in the model group was 2.20(2.03-2.45)pg/mL,while the level of IL⁃17A in the high dose group of Fuzi decoction was 1.01(0.59-1.44)pg/mL,and the difference was statistically significant(P<0.01).Compared with the model group,the expression levels of PI3K and AKT protein in the synovial tissue of ankle joint in hydroxychloroquine sulfate positive drug control group and low,medium and high dose groups of Fuzi decoction were significantly decreased(F=11.435,11.104;all P<0.05).Compared with the model group,the expression level of mTOR protein in ankle joint synovium of low and high doses of Fuzi decoction was significantly decreased and the differences were statistically significant(F=8.063,P<0.05).Conclusion Fuzi decoction had a good therapeutic effect on AA rats;in that,it could significantly alleviate the degree of joint swelling and significantly improve the pathological changes of joints,which was related to the reduction of the production of inflammatory cytokines,TNF⁃αand IL⁃17A and the negative regulation of protein expression in PI3K/AKT/mTOR signaling pathway.