桃红四物汤调控巨噬细胞极化对肩袖损伤的机制研究

Mechanism of Taohong Siwu decoction regulating macrophage polarization on rotator cuff injury

目的:基于白介素(IL)-4/信号传导及转录激活蛋白(STAT)6信号通路调控巨噬细胞极化,利用小鼠动物模型探讨桃红四物汤对肩袖撕裂术后愈合的影响及其作用机制。方法:将50只C57BL/6J小鼠随机分为空白对照组、肩袖损伤组(模型组)及桃红四物汤低、中、高剂量组,每组10只。除对照组外,其他组小鼠均构建肩袖撕裂重建模型。术后预防感染并正常饲养12周,桃红四物汤组小鼠灌胃相应药液,每次每只小鼠灌胃量均为2 ml;对照组每日清晨予等体积生理盐水;连续给药12周,1次/d。末次给药24 h后,进行冈上肌腱生物力学刚度测试;采用酶联免疫吸附测定法(ELISA)检测小鼠血清一氧化氮合酶(iNOS)、IL-6、甘露糖受体(CD206)的含量;采用实时定量PCR(RT-PCR)技术检测小鼠腱骨界面组织IL-1β、肿瘤坏死因(TNF)子-α、IL-10、炎症区域分子1(Fizz1)mRNA表达;采用Western Blot法检测腱骨界面组织iNOS、精氨酸酶-1(Arg-1)、IL-4、STAT6、磷酸化-STAT 6(p-STAT6)蛋白含量。多组间数据比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果:用药干预后与肩袖损伤组比较,桃红四物汤各个剂量组拉断载荷、刚度测试数值上调(F=64.822、58.431,均为P<0.05);iNOS、IL-6(M1极化标志物)水平下调(F=82.618、61.372,均为P<0.05),CD206(M2极化标志物)水平上调(F=58.942,P<0.05);桃红四物汤中、高剂量组IL-1β、TNF-mRNA(M1极化标志物)的表达下调(F=38.631、33.714,均为P<0.05),而IL-10、Fizz1 mRNA(M2极化标志物)的表达上调(F=41.731、67.431,均为P<0.05)。桃红四物汤低、中、高剂量组Arg1、IL-4、p-STAT6的蛋白表达上调(F=26.841、29.750、26.863,均为P<0.05)。结论:桃红四物汤可促进肩袖撕裂小鼠的肌腱愈合,其机制可能与IL-4/STAT6信号通路调控巨噬细胞极化有关。

Objective To explore the effect of TaoHong Siwu decoction on rotator cuff tear in mice,so as to clarify its mechanism of the prevention and treatment in rotator cuff injury based on interleukin(IL)-4/transcriptional activator protein(STAT)6.Methods Fifty C57BL/6J mice were randomly divided into the control group,the rotator cuff injury group(model group),low,middle and high dose Taohong Siwu decoction groups with 10 mice in each group.Except for the control group,the rotator cuff tear reconstruction model was established in the other groups.The mice were fed with normal diet for 12 weeks and given the infection prevention treatment.After that,each mouse in the Taohong Siwu decoction group was intragastrically infused with corresponding solution each time,and the control group were given the same volume of normal saline every morning;the volume was 0.5 ml/20 g once per day,for the consecutive 12 weeks.Twenty-four hours after the last administration,the biomechanical stiffness of supraspinatus tendon was tested,the contents of serum nitric oxide synthase(iNOS),interleukin-6(IL-6)and mannose receptor(cluster of differentiation 206,CD206)in mice were detected by enzyme-linked immunosorbent assay(ELISA).Real time PCR(RT-PCR)detected mRNA expressions of IL-10,tumor necrosis factor(TNF)-α,IL-10,and inflammatory zone molecule 1(Fizzl)in mouse tendon-bone interface tissue.Western Blot was used to detect iNOS,arginase-1(Arg-1),IL-4,STAT6,and phosphorylation-STAT6(p-STAT6)protein content.One-way AN OVA was used for comparison among multiple groups,and LSD-t test was used for pairwise comparison between groups.Results After drug intervention,compared with the rotator cuff injury group,the tensile load and stiffness test values of each dose group of Taohong Siwu decoction were up-regulated(F=64.822,58.431,both P<0.05);iNOS and IL-6(Ml polarization marker)decreased(F=82.618,61.372,both P<0.05),and CD206(M2 polarization marker)increased(F=58.942,P<0.05).The expression sof IL-10 and TNF mRNA(Ml polarization marker)in the medium and high dose groups of Taohong Siwu decoction,were down-regulated(F=38.631,33.714,both P<0.05),while the expressions of IL-10 and Fizzl mRNA(M2 polarization marker)were up-regulated(F=41.731,67.431,both P<0.05).The protein expressions of argl,IL-4 and p-stat6 were up-regulated in low,medium and high dose groups of Taohong Siwu Decoction(F=26.841,29.750,26.863,all P<0.05).Conclusion Taohong Siwu decoction can accelerate tendon remodeling in mice with rotator cuff tear,and its mechanism may be related to the regulation of macrophage polarization by IL-4/STAT6 signal pathway.