切换至 "中华医学电子期刊资源库"

中华关节外科杂志(电子版) ›› 2022, Vol. 16 ›› Issue (03) : 315 -320. doi: 10.3877/cma.j.issn.1674-134X.2022.03.009

基础论著

桃红四物汤调控巨噬细胞极化对肩袖损伤的机制研究
陈建发1(), 马思雯2, 向孝兵1,()   
  1. 1. 510405 广州中医药大学第一附属医院四骨科
    2. 510405 广州中医药大学第一临床医学院
  • 收稿日期:2021-09-22 出版日期:2022-06-01
  • 通信作者: 陈建发, 向孝兵
  • 基金资助:
    广东省中医药局科研课题(20191101)

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

Jianfa Chen1,(), Siwen Ma2, Xiaobing Xiang1()   

  1. 1. The Fourth Department of Orthopedics, The First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, Guangzhou 510405, China
    2. The First Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Guangzhou 510405, China
  • Received:2021-09-22 Published:2022-06-01
  • Corresponding author: Jianfa Chen, Xiaobing Xiang
引用本文:

陈建发, 马思雯, 向孝兵. 桃红四物汤调控巨噬细胞极化对肩袖损伤的机制研究[J/OL]. 中华关节外科杂志(电子版), 2022, 16(03): 315-320.

Jianfa Chen, Siwen Ma, Xiaobing Xiang. Mechanism of Taohong Siwu decoction regulating macrophage polarization on rotator cuff injury[J/OL]. Chinese Journal of Joint Surgery(Electronic Edition), 2022, 16(03): 315-320.

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-1β, tumor necrosis factor(TNF)-α, IL-10, and inflammatory zone molecule 1 (Fizz1) 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 ANOVA 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 (M1 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-1β and TNF mRNA (M1 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 Fizz1 mRNA (M2 polarization marker) were up-regulated (F=41.731, 67.431, both P<0.05). The protein expressions of arg1, 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.

表1 引物序列及长度
表2 肩袖损伤愈合腱骨界面拉断载荷和刚度的测试[N/mm,(±s)]
表3 桃红四物汤对肩袖损伤模型小鼠血清中iNOS、IL-6、CD206水平的影响[μg/L,(±s)]
表4 桃红四物汤对肩袖损伤模型小鼠IL-1β、TNF-α、IL-10、Fizz1mRNA表达水平的影响(±s)
表5 桃红四物汤对肩袖损伤模型小鼠iNOS、Arg1、IL-4、STAT6、p-STAT6蛋白表达的影响(±s)
图1 桃红四物汤对肩袖损伤模型小鼠蛋白表达的影响注:A-对照组;B-肩袖损伤组;C-桃红四物汤低剂量组;D-桃红四物汤中剂量组;E-桃红四物汤高剂量组;INOS-一氧化氮合酶;Arg-精氨酸酶-1;IL-白介素;p-STAT-磷酸化-信号传导及转录激活蛋白;STAT-信号传导及转录激活蛋白;GAPDH-内参
[1]
李敏,Badshah Shaikh Atik,彭赓,等.合并肩袖损伤的肱二头肌长头腱病变固定与切断比较[J].中国矫形外科杂志202129(24):2272-2274+2278.
[2]
张顶顶,张涛,涂振兴,等.关节镜下双排技术与缝线桥技术治疗肩袖损伤疗效比较Meta分析[J].华北理工大学学报(医学版)202224(1):59-67,71.
[3]
赖圳登,赵建宁,包倪荣.肩袖损伤产生疼痛的信号通路研究进展[J].中国骨与关节损伤杂志202035(11):1230-1232.
[4]
张泽坤,陈兴真,李振宇,等.不可修复肩袖撕裂的治疗研究进展[J].中国骨与关节损伤杂志202136(5):552-554.
[5]
刘爽,刘希斗,闫茹,等.肩袖损伤发病机制的研究进展[J].中医正骨202032(8):34-38.
[6]
徐美光,陈保军,韩潜,等.肩袖肌腱损伤病理机制的研究进展[J].中华创伤杂志202137(11):1042-1047.
[7]
万云峰,曹寅生,易强,等.玻璃酸钠联合桃红四物汤合六味地黄丸在肩袖修补术后的应用[J].中国当代医药202027(5):162-165,169.
[8]
Liu Y, Wang X, Pang J, et al. Attenuation of atherosclerosis by protocatechuic acid via inhibition of M1 and promotion of M2 macrophage polarization[J]. J Agric Food Chem, 2019, 67(3): 807-818.
[9]
陈鹏,方斌,易浪,等.桃红四物汤含药血清对巨噬细胞的影响[J].中国药理学通报201329(4):582-584.
[10]
Lebaschi AH, Deng XH, Camp CL, et al. Biomechanical,histologic,and molecular evaluation of tendon healing in a new murine model of rotator cuff repair[J]. Arthroscopy, 2018, 16(1): 1-11.
[11]
Abraham AC, Shah SA, Thomopoulos S. Targeting inflammation in rotator cuff tendon degeneration and repair[J]. Tech Shoulder Elb Surg, 2017, 18(3): 84-90.
[12]
Krieger JR, Tellier LE, Ollukaren MT, et al. Quantitative analysis of immune cell subset infiltration of supraspinatus muscle after severe rotator cuff injury[J]. Regen Eng Transl Med, 2017, 3(2): 82-93.
[13]
Ji JH, Kim YY, Patel K, et al. Dexamethasone facilitates NF-κB signal pathway in TNF-α stimulated rotator cuff tenocytes[J]. J Microbiol Biotechnol, 2019, 29(2): 297-303.
[14]
Man SM, Karki R, Kanneganti TD. Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases[J]. Immunol Rev, 2017, 277(1): 61-75.
[15]
Moore EM, West JL. Harnessing macrophages for vascularization in tissue engineering[J]. Ann Biomed Eng, 2019, 47(2): 354-365.
[16]
Orecchioni M, Ghosheh Y, Pramod AB, et al. Macrophage polarization: different gene signatures in M1(LPS+) vs. classically and M2(LPS-) vs. alternatively activated macrophages[J/OL]. Front Immunology, DOI:10.3389/fimmu.2019.01084.
[17]
Wang LX, Zhang SX, Wu HJ, et al. M2b macrophage polarization and its roles in diseases[J]. J Leukoc Biol, 2019, 106(2): 345-358.
[18]
Oettgen HC. Regulation of the IgE isotype switch: new insights on cytokine signals and the functions of epsilon germline transcripts[J]. Curr Opin Immunol, 2000, 12: 618-623.
[19]
Waqas S F H, Ampem G, Röszer T. Analysis of IL-4/STAT6 Signaling in Macrophages[J]. Methods Mol Biol, 20191966(6): 211-224.
[20]
殷胜骏.乌梅丸加减对溃疡性结肠炎大鼠IL-4/STAT6信号通路的调节作用[D].北京:北京中医药大学,2009.
[21]
Goenka S, Kaplan MH. Transcriptional regulation by STAT6[J]. Immunol Res, 2011, 50(1): 87-96.
[1] 纪小孟, 刘璠, 唐晓波, 卞为伟, 董佩龙, 刘振鲁. 两种手术方式治疗肩袖撕裂合并粘连性肩关节囊炎[J/OL]. 中华关节外科杂志(电子版), 2024, 18(05): 561-567.
[2] 张凯, 乔永杰, 林志强, 刘健, 邓泽群, 谭飞, 曾健康, 李嘉欢, 李培杰, 周胜虎. 假体周围骨溶解中巨噬细胞极化的机制研究进展[J/OL]. 中华关节外科杂志(电子版), 2024, 18(05): 618-625.
[3] 李程, 朱梁, 庞勇, 查国春, 仇尚, 孙伟, 冯硕. 侧侧缝合联合无结缝线桥技术治疗大型L型肩袖撕裂[J/OL]. 中华关节外科杂志(电子版), 2024, 18(03): 301-306.
[4] 韩燕鸿, 潘建科, 罗明辉, 黄和涛, 曹厚然, 李想, 曾令烽, 梁桂洪, 陈红云, 刘军, 杨伟毅. 肩袖撕裂修复术后腱-骨愈合影响因素的研究进展[J/OL]. 中华关节外科杂志(电子版), 2024, 18(02): 239-244.
[5] 李帝均, 王斌. 肩袖损伤修复的生物材料选择及动物模型评价[J/OL]. 中华关节外科杂志(电子版), 2024, 18(01): 118-124.
[6] 张骏, 李强, 孟柏屹, 李成宇. 关节镜松解联合肩袖修复治疗肩袖损伤合并继发冻结肩[J/OL]. 中华关节外科杂志(电子版), 2024, 18(01): 1-7.
[7] 杨瑾, 刘雪克, 张媛媛, 金钧, 韦瑶. 肠道微生物来源石胆酸对脓毒症相关肝损伤的保护作用[J/OL]. 中华危重症医学杂志(电子版), 2024, 17(04): 265-274.
[8] 李璐璐, 马利红, 金佳佳, 谷伟. 干扰素基因刺激因子通过肺巨噬细胞胞葬功能调控急性肺损伤小鼠修复的研究[J/OL]. 中华危重症医学杂志(电子版), 2024, 17(02): 97-103.
[9] 狄静怿, 陈禹江, 陈欣欣, 陈文霞. 基质细胞衍生因子1通过PI3K/AKT1信号通路对巨噬细胞极化的影响[J/OL]. 中华口腔医学研究杂志(电子版), 2024, 18(02): 89-95.
[10] 李卓骋, 陈羽翔, 高亮, 张宇, 朱许源, 马晓杰, 李涛, 赵甜甜, 蒋鸿涛. 巨噬细胞-肌成纤维细胞转化在肾纤维化过程中的作用[J/OL]. 中华移植杂志(电子版), 2024, 18(03): 181-185.
[11] 曹飞, 庞俊. 前列腺癌免疫微环境中免疫抑制性细胞分类及其作用机制[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(02): 121-125.
[12] 朱军, 宋家伟, 乔一桓, 郭雅婕, 刘帅, 姜玉, 李纪鹏. M2型巨噬细胞特征基因与结肠癌免疫微环境研究[J/OL]. 中华结直肠疾病电子杂志, 2024, 13(04): 303-311.
[13] 李松栗, 黄蔚, 巢杰, 杨毅, 邱海波. 单核/巨噬细胞来源的细胞外囊泡在急性呼吸窘迫综合征中的研究进展[J/OL]. 中华重症医学电子杂志, 2024, 10(03): 253-257.
[14] 陈含冰, 储翠林, 邱海波. 急性呼吸窘迫综合征中巨噬细胞死亡方式的研究进展[J/OL]. 中华重症医学电子杂志, 2024, 10(01): 79-84.
[15] 李仔祥, 王苏贵, 张先云, 卢建文, 嵇宏声, 姜福金. 肿瘤相关性巨噬细胞通过TNF-α/B7H3调节人膀胱癌细胞增殖的研究[J/OL]. 中华临床医师杂志(电子版), 2024, 18(01): 64-71.
阅读次数
全文


摘要