桂皮醛对关节炎大鼠JAK/STAT信号通路的作用机制研究

Effect of Cinnamaldehyde on JAK/STAT signaling pathway in synoviocytes and arthritis rats

目的:探讨桂皮醛(Cin)通过调控JAK/STAT信号通路对人类风湿性关节炎成纤维样滑膜细胞MH7A和Ⅱ型胶原诱导的大鼠关节炎的作用及可能的机制。方法:体外培养MH7A细胞,CCK-8法检测不同浓度Cin对MH7A细胞活性的影响,筛选合适的药物浓度;将MH7A细胞分为对照组、IL-1β组和IL-1β+Cin组,IL-1β组使用白介素-1β(IL-1β)20 ng/ml处理,IL-1β+Cin组使用IL-1β20 ng/ml和不同浓度Cin(40、60、80 nmol/L)处理,对照组未进行药物干预,24 h后检测各组细胞培养上清液中IL-6、IL-8、TNF-α的含量;Western blot法检测各组细胞中JAK2、p-JAK2、STAT3、p-STAT3蛋白表达情况。体内实验选取40只SD大鼠,随机分为对照组、模型组、Cin组(75 mg/kg)、甲氨蝶呤组(MTX,1 mg/kg),除对照组外,其余3组建立Ⅱ型胶原诱导的大鼠关节炎(CIA)模型,造模成功后连续给药21 d,模型组和对照组给予等量蒸馏水,观察Cin对CIA大鼠关节炎评分(AI)和足跖肿胀度的影响,检测各组大鼠血清中IL-6、IL-8、IL-1β和TNF-α的水平;HE染色观察各组大鼠关节组织病理改变。结果:体外实验结果显示,干预24 h后,IL-1β可促进MH7A细胞释放IL-6、IL-6、TNF-α等炎症因子,增加JAK2、p-JAK2、STAT3、p-STAT3的蛋白相对表达水平,Cin处理后可抑制MH7A细胞释放炎症因子,降低JAK2、p-JAK2、STAT3、p-STAT3的蛋白表达,呈剂量依赖性。体内实验结果显示,与对照组比较,模型组大鼠AI评分和足跖肿胀程度显著升高,血清中IL-6、IL-8、IL-1β和TNF-α的水平显著升高,关节组织出现明显的炎症细胞浸润和骨质破坏;与模型组比较,Cin和MTX能够降低大鼠AI评分和足跖肿胀程度,显著降低血清中IL-6、IL-8、IL-1β和TNF-α的水平,并减轻关节组织的病理损伤。上述指标差异均具有统计学意义(P<0.05)。结论:Cin能够抑制IL-1β诱导的MH7A细胞释放炎症因子,改善Ⅱ型胶原诱导型CIA大鼠的AI值和足跖肿胀程度,减轻CIA大鼠关节组织的炎症反应和骨质破坏,其机制可能与降低JAK2和STAT3的磷酸化水平,抑制JAK/STAT信号通路活化有关。

Objective:To investigate the effect and possible mechanism of cinnamaldehyde(Cin)on fibroblast-like synoviocytes MH7 A and type Ⅱ collagen induced rat arthritis by regulating JAK/STAT signaling pathway.Methods:MH7 A cells were cultured in vitro,CCK-8 method was used to detect the effect of different concentrations of Cin on the activity of MH7 A cells,and the appropriate drug concentration was screened. MH7 A cells were divided into control group,IL-1β group and IL-1β+Cin group. The IL-1βgroup was treated with interleukin-1β(IL-1β)20 ng/ml and the IL-1β+Cin group was treated with IL-1β 20 ng/ml and different concentrations of Cin(40,60,80 nmol/L). No drug intervention was performed in the control group. After 24 h,the content of IL-6,IL-8 and TNF-α in the cell culture fluid of each group was detected. Western blot method was used to detect the expression of JAK2,pJAK2,STAT3 and p-STAT3 protein in the cells of each group. In the in vivo experiment,we selected 40 SD rats and randomly divided them into control group,model group,Cin group(75 mg/kg),and methotrexate group(MTX,1 mg/kg). In addition to the control group,the remaining 3 groups established a Collagen-induced arthritis(CIA)model,which was administered continuously for 21 days after successful modeling. Rats in the model group and control group were given the same amount of distilled water by gavage. Observed the effects of Cin on the arthritis index(AI)and the plantar swelling of CIA rats,and detected the levels of IL-6,IL-8,IL-1βand TNF-α in the serum of rats in each group. HE staining was used to observe the pathological changes of joint tissues of rats in each group.Results:In vitro experiments:the results show that after 24 h of intervention,IL-1β could promote the release of IL-6,IL-8,TNF-α inflammatory factors from MH7 A cells,increase the relative expression levels of JAK2,p-JAK2,STAT3,p-STAT3 protein.Treatment with Cin could inhibit the release of inflammatory factors from MH7 A cells and reduce the protein expression of JAK2,pJAK2,STAT3,and p-STAT3 in a dose-dependent manner. In vivo experiments:compared with the control group,the AI score and the degree of paw swelling of the model group were significantly increased,the levels of IL-6,IL-8,IL-1β and TNF-α in the serum were significantly increased,and the joint tissue appeared;obvious inflammatory cell infiltration and bone destruction. Compared with the model group,Cin and MTX could reduce the rat AI score and the degree of paw swelling,and significantly reduce serum IL-6,IL-8,IL-1β and TNF-α levels,and reduce the pathological damage of joint tissue. The differences in the above indicators were statistically significant(P<0.05).Conclusion:Cin can inhibit the release of inflammatory factors induced by IL-1β in MH7 A cells,improve the AI value and plantar swelling of type Ⅱ collagen-induced CIA rats,and reduce the inflammatory response and bone destruction of CIA rats. The mechanism may be related to reducing the phosphorylation level of JAK2 and STAT3 and inhibiting the activation of JAK/STAT signaling pathway.