桂枝芍药知母汤对胶原诱导性关节炎小鼠软骨破坏及JAK2/STAT3信号通路的影响

Effect of Guizhi Shaoyao Zhimutang on Cartilage Destruction and JAK2/STAT3 Signaling Pathway in Mice with Collagen-induced Arthritis

目的:研究桂枝芍药知母汤在胶原诱导性关节炎(CIA)小鼠软骨破坏中的作用及其作用机制。方法:取SPF级DBA/1小鼠36只,随机分组为正常组、模型组、甲氨蝶呤组(MTX)、桂枝芍药知母汤低、中、高剂量组6组。除正常组外,其余5组小鼠采用二次免疫法构建CIA模型,并在小鼠开始出现后肢肿胀当天分别予生理盐水,MTX(1.5 mg·kg^(-1),每周2次)和桂枝芍药知母汤低、中、高剂量(6.3、12.6、25.2 g·kg^(-1)·d^(-1))灌胃,共给药4周。观察并记录各组小鼠足肿胀度变化,酶联免疫吸附测定法(ELISA)检测血清中基质金属蛋白酶-1(MMP-1)、MMP-3、MMP-9、MMP-13含量,苏木素-伊红(HE)染色观察踝关节病理改变,番红固绿染色法观察软骨破坏,蛋白免疫印迹法(Western blot)检测小鼠踝关节酪氨酸激酶2(JAK2)/转录激活因子3(STAT3)信号通路蛋白表达。结果:与正常组比较,模型组足肿胀度,血清MMP-1、MMP-3、MMP-9和MMP-13含量和踝关节磷酸化(p)-JAK2/JAK2、p-STAT3/STAT3的表达水平均显著上升(P<0.01),关节破坏加重。与模型组比较,MTX组和桂枝芍药知母汤低、中、高剂量组明显减轻小鼠足肿胀度(P<0.05,P<0.01),明显降低小鼠血清MMP-1、MMP-3、MMP-9和MMP-13含量(P<0.05,P<0.01),关节病理破坏减轻,明显降低踝关节p-JAK2/JAK2和p-STAT3/STAT3的表达水平(P<0.05,P<0.01)。结论:桂枝芍药知母汤能减轻CIA小鼠的关节肿胀程度,抑制MMP-1、MMP-3、MMP-9和MMP-13的表达,改善关节软骨破坏,其作用机制与JAK2/STAT3信号通路有关。

Objective:To study the effect of Guizhi Shaoyao Zhimutang(GSZMD)on cartilage destruction in mice with collagen-induced arthritis(CIA)and its mechanism.Method:Thirty-six DBA/1 mice in SPF grades were randomly divided into 6 groups,namely,the normal group,the model group,the methotrexate(MTX)group,the low-dose GSZMD group,the medium-dose GSZMD group,and the high-dose GSZMD group.Except the normal group,mice in the other 5 groups were used to establish the model of CIA by secondary immunization.The mice were given normal saline,MTX(1.5 mg·kg^(-1),2 times a week),and low,medium,and high-doses GSZMD(6.3,12.6,25.2 g·kg^(-1)·d^(-1))by intragastric administration on the day of the onset of hind limb swelling for 4 weeks.The changes in the degree of foot swelling of mice in each group were observed and recorded.The content of matrix metalloproteinase(MMP)-1,MMP-3,MMP-9,and MMP-13 in serum was determined by enzyme-linked immunosorbent assay(ELISA).The pathological changes in the ankle joint were observed by hematoxylin-eosin(HE)staining,and the cartilage destruction was observed by red fast green staining.The protein expression of the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway in ankle joints were detected by Western blot.Result:As compared with the normal group,the degree of foot swelling,the content of MMP-1,MMP-3,MMP-9,and MMP-13 in serum and the expression levels of phosphorylation(p)-JAK2/JAK2 and p-STAT3/STAT3 in ankle joints of the model group were increased(P<0.01),and the joint damage was aggravated.As compared with the model group,the degrees of foot swelling of the mice in the MTX group and the low,medium,and high-dose GSZMD group were reduced(P<0.05,P<0.01),and the content of MMP-1,MMP-3,MMP-9,and MMP-13 in serum was decreased(P<0.05,P<0.01).The pathological joint damage was alleviated,and the expression levels of p-JAK2/JAK2 and p-STAT3/STAT3 in ankle joints were decreased in the MTX group and GSZMD groups(P<0.05,P<0.01).Conclusion:GSZMD can reduce the degree of joint swelling in mice with CIA,inhibit the expressions of MMP-1,MMP-3,MMP-9,and MMP-13,and alleviate the destruction of articular cartilage.Its mechanism is related to the JAK2/STAT3 signaling pathway.