磷酸川芎嗪片联合甲氨蝶呤干预对胶原诱导关节炎大鼠炎症反应及血液流变的影响

Effect of Combination Therapy of Tetramethylpyrazine with Methotrexate on Inflammatory Reactions and Hemorheology in Collagen-induced Arthritis Rats

目的探讨磷酸川芎嗪片联合甲氨蝶呤干预对胶原诱导关节炎大鼠(collagen induced arthritis,CIA)炎症反应及血液流变的影响。方法将55只雄性SD大鼠按体重分层,随机选取9只作为正常对照组,其余46只采用牛Ⅱ型胶原诱导法制备大鼠关节炎模型。对成模大鼠(40只),按足趾肿胀程度分层随机方法分为模型(CIA)组、甲氨蝶呤(MTX)组、磷酸川芎嗪(TMP)组及联用(MTX+TMP)组,每组10只。各药物干预组分别给予以下药物灌胃:MTX组给予MTX 1.2 mg/kg,每周1次,连续4周;TMP组予TMP 40 mg/kg,每日1次,先连续给药10天,停药7天,再继续给药10天;MTX+TMP组分别给予等剂量两种药物,即在两药都给予时,给予两药的等剂量混合药物;在MTX停药时只给予TMP,则按照TMP组方法给药;正常对照组及CIA组给予等量生理盐水。分别于给药第0、4、1 1、18、26天检测大鼠脚掌厚度和踝关节直径。4周后杀鼠取材,观察各组大鼠关节病理变化;采用E LISA方法检测各组大鼠血清细胞因子IL-1β、IL-6及IL-17A变化,检测血浆血流变学指标纤维蛋白原(FIB)含量及血小板聚集率(PAg)变化。结果与正常对照组比较,CIA组大鼠脚掌厚度与踝关节直径明显增加(P<0.01),FIB含量及PAg均升高(P<0.05,P<0.01);血清IL-1β、IL-6及IL-17A水平亦明显升高(P<0.01);关节滑膜出现明显的细胞增生、炎细胞浸润、组织充血水肿,血管翳形成浸入软骨,出现明显的坏死。与CIA组比较,各药物干预组大鼠脚掌厚度与踝关节直径均减小(P<0.05,P<0.01),其中MTX组效果优于TMP组及MTX+TMP组(P<0.05,P<0.01);MTX组FIB含量降低(P<0.05),血清IL-1β、IL-6水平亦降低(P<0.05);TMP组和MTX+TMP组FIB含量、血清IL-1β、IL-6水平降低,且MTX+TMP组FIB和IL-6水平明显低于MTX组及TMP组,差异有统计学意义(P<0.01);在降低PAg和血清IL-17A水平方面,TMP组优于MTX组及MTX+TMP组;各药物均可显著改善关节病理,联用效果最佳。结论 TMP联合MTX可以显著改善胶原诱导关节炎大鼠的炎症反应及全血FIB含量。

Objective To explore the effect of combination therapy of tetramethylpyrazine（TMP） with methotrexate（MTX） on collagen induced arthritis（CIA） rats.Methods Totally 55 male SD rats were stratified by body weight.Nine of them were randomly recruited as the normal control group.The rest 46 were immunized with type Ⅱ bovine collagen（CⅡ） for establishing rheumatoid arthritis（RA）model.Forty successfully modeled rats were randomly divided into 4 groups according to swollen toe degree,i.e.,the CIA group,the TMP group,the MTX group,and the TMP plus MTX group,10 in each group.Rats in the MTX group were administered with MTX（1.2 mg/kg）,once per week for 4 continuous weeks.Those in the TMP group were administered with 40 mg/kg TMP,once per day for 10 continuous days,and then discontinued for 7 successive days,and continued for another 10 successive days.Rats in the TMP plus MTX group were administered with a mixture of equal dose MTX and TMP,and when MTX was discontinue,TMP was administered according to the way in the TMP group.Equal volume of saline solution was given to rats in the normal control group and the CIA group.Clinical parameters including ankle width（mediolateral diameter） and hindpaw swelling were measured at day 0,4,11,18,and 26 after treatment.Rats were sacrificed 28 days after treatment,their knee joints and ankle joints were collected for pathological analyses.Serum levels of IL-1β,IL-6,and IL-17 A were detected by ELISA.Changes of fibrinogen（FIB） and platelet aggregation rate（PAg） were detected.Results Compared with the normal control group,the ankle width and hindpaw swelling increased significantly（P 〈0.01）,contents of FIB and PAg increased obviously（P〈0.05,P 〈0.01）,serum levels of IL-1β,IL-6,and IL-17 increased remarkably（P〈0.01） in the CIA group.Obvious cell proliferation,inflammatory cell infiltration,hyperemia and edema of synovial tissues could be seen.Pannus formed and immerged in cartilages,resulting in necrosis.Compared with the model group,changes of ankle width and hindpaw swelling were all alleviated in each medicated group（P 〈0.05,P 〈0.01）.Of them,the effect was superior in the MTX group to that of the TMP group and the MTX plus TMP group（P 〈0.05,P 〈0.01）.Contents of FIB,serum levels of IL-1βand IL-6 decreased significantly in the MTX group（P 〈0.05）.Contents of FIB,serum levels of IL-1β and IL-6 decreased significantly in the TMP group and the MTX plus TMP group（P 〈0.05）.Besides,serum levels of FIB and IL-6 were obviously lower in the MTX plus TMP group than in the TMP group and the MTX group（P 〈0.01）.Levels of PAg and IL-17 A were more significantly lowered in the TMP group than in the MTX plus TMP group and the MTX group.Pathological changes could be alleviated in each medicated group,with the optimal effect obtained in the MTX plus TMP group.Conclusion Combination of TMP with MTX could significantly ameliorate inflammatory reactions and FIB contents of CIA rats.