Electroacupuncture Influences Macrophage M2 Polarization and TLR4 and MyD88 Expression in Myocardial Tissue of Myocardial Ischemia Injury Mice
-
摘要:
目的 观察电针对心肌缺血损伤小鼠心功能及心脏组织中巨噬细胞极化、TLR4、MyD88等表达的影响, 探讨电针促进心肌损伤保护的可能机制。 方法 27只雄性C57BL/6J小鼠随机分为假手术组、模型组、电针组, 每组9只。模型组、电针组通过结扎心脏冠状动脉左前降支建立心肌缺血模型。电针组于造模成功后, 电针小鼠双侧“内关”穴治疗: 2/15 Hz, 疏密波, 1 mA, 20 min·次-1, 每日1次, 共治疗7 d。运用超声心动图评价小鼠心脏射血分数(EF) 及短轴收缩率(FS), 天狼星红染色观察小鼠心脏纤维化情况, 流式细胞术检测心肌组织中中性粒细胞、巨噬细胞数量及各表型巨噬细胞占比, qPCR法检测心肌组织中TNF-α、IL-1β的mRNA表达量, Western blot法检测心肌组织中TLR4、MyD88、IL-1β、IL-17A的蛋白表达水平。 结果 与假手术组相比, 模型组小鼠EF、FS值下降(P < 0.0001), 胶原容积分数升高(P < 0.001), 心脏组织中中性粒细胞、巨噬细胞数增多(P < 0.0001, P < 0.001), 同时M1型巨噬细胞占比增高(P < 0.05), 心肌组织中TNF-α、IL-1β的mRNA表达量上升(P < 0.01, P < 0.001), TLR4、MyD88、IL-1β、IL-17A蛋白表达水平上升(P < 0.05, P < 0.01);与模型组相比, 电针组小鼠EF、FS值升高(P < 0.01, P < 0.001), 胶原容积分数降低(P < 0.001), 心脏组织中中性粒细胞、巨噬细胞数减少(P < 0.0001, P < 0.01), 其中M2型巨噬细胞占比增高(P < 0.05), 心肌组织中TNF-α、IL-1β的mRNA表达量下降(P < 0.001), TLR4、MyD88、IL-1β、IL-17A蛋白表达水平降低(P < 0.05, P < 0.001)。 结论 电针可能通过降低TLR4、MyD88在心肌组织中的表达, 促进心肌缺血损伤后心脏巨噬细胞向M2型极化, 减轻局部炎症, 抑制心肌纤维化, 实现心肌保护效应。 Abstract:OBJECTIVE To observe the effect of electroacupuncture on cardiac function and its effect on macrophage polarization, TLR4 and MyD88 expression in the heart of mice with myocardial infarction, and to explore the possible mechanism of electroacupuncture against cardioprotection. METHODS 27 male C57BL/6J mice were randomly divided into the sham-operated group, model group, and electroacupuncture group, with 9 mice in each group. The myocardial ischemia model was established by ligating the left anterior descending branch of the coronary artery in the model group and electroacupuncture group. In the electroacupuncture group, after successful modeling, the mice were treated with electroacupuncture at the bilateral Neiguan points: 2/15 Hz, dense and sparse waves, 1 mA, 20 min/time, once a day for 7 days. The ejection fraction (EF) and short-axis systolic rate (FS) were calculated by echocardiography; the fibrosis of mice heart was observed by Sirius red staining; the number of neutrophils and macrophages in myocardial tissue and the percentage of macrophages in each phenotype was detected by flow cytometry; the mRNA expression of TNF-α and IL-1β in myocardial tissue were detected by real-time quantitative fluorescence PCR; the protein expression levels of TLR4, MyD88, IL-1β, and IL-17A in myocardial tissues were detected by Western blot. RESULTS Compared with the sham-operated group, mice in the model group showed decreased EF and FS values (P < 0.0001), increased collagen volume fraction (P < 0.001), increased numbers of neutrophils and macrophages in cardiac tissues (P < 0.0001, P < 0.001), as well as increased percentage of M1-type macrophages (P < 0.05). The mRNA expression of TNF-α, IL-1β in myocardial tissues expression increased (P < 0.01, P < 0.001); TLR4, MyD88, IL-1β, IL-17A protein expression levels increased (P < 0.05, P < 0.01); compared with the model group, mice in the electroacupuncture group had higher EF and FS values (P < 0.01, P < 0.001), lower collagen volume fraction (P < 0.001), the numbers of neutrophils and macrophages in cardiac tissues decreased (P < 0.0001, P < 0.01), while the percentage of M2-type macrophages increased (P < 0.05), the mRNA expression of TNF-α and IL-1β in myocardial tissues decreased (P < 0.001), and the TLR4, MyD88, IL-1β, IL-17A protein expression levels were decreased (P < 0.05, P < 0.001). CONCLUSION Electroacupuncture may achieve myocardial protective effects by reducing the expression of TLR4 and MyD88 in myocardial tissue, promoting the polarization of cardiac macrophages to M2 type after MI, reducing local inflammation, and inhibiting myocardial fibrosis. -
图 1 心肌缺血损伤模型制作前后心电图变化
注：Ⅱ导联, 箭头示ST段抬高
Figure 1. Changes in electrocardiogram before and after the establishment of myocardial ischemia injury model
图 2 中性粒细胞、巨噬细胞流式圈门策略
Figure 2. Flow cytometry gate strategy for neutrophils and macrophages
图 3 各组小鼠FS及EF比较
注: 与假手术组比较, ****P < 0.0001；与模型组比较, ####P < 0.0001。x±s, n=9。
Figure 3. Comparision of FS and EF in each group
图 4 各组小鼠心脏纤维化比较
注: 与假手术组比较, ***P < 0.001；与模型组比较, ###P < 0.001。x±s, n=3。
Figure 4. Comparision of cardiac fibrosis in each group
图 5 各组小鼠心肌组织中TNF-α、IL-1β的mRNA表达量比较
注: 与假手术组比较, **P < 0.01，***P < 0.001；与模型组比较, ###P < 0.001。x±s, n=3。
Figure 5. TNF-α, IL-1β mRNA expression in myocardial tissue of each group
图 6 各组小鼠心肌组织中IL-1β、IL-17A蛋白表达水平比较
注: 与假手术组比较, *P < 0.05，**P < 0.01；与模型组比较, #P < 0.05，###P < 0.001。x±s, n=3。
Figure 6. IL-1β, IL-17A protein levels in myocardial tissue of each group
图 7 各组小鼠心肌组织中中性粒细胞数量、巨噬细胞数量及各表型占比比较
注: 与假手术组比较, *P < 0.05，***P < 0.001；与模型组比较, #P < 0.05，###P < 0.001。x±s, n=3。
Figure 7. Comparision of the numbers of neutrophil and macrophage as well as proportions of different phenotypes in myocardial tissue of each group
图 8 电针对各组小鼠心脏组织中TLR4、MyD88的蛋白表达水平的影响
注: 与假手术组比较, *P < 0.05；与模型组比较, #P < 0.05。x±s, n=3。
Figure 8. Effects of electroacupuncture on protein expression of TLR4, MyD88 in myocardial tissue of each group
表 1 引物序列
Table 1. Primer sequences
基因名称 序列(5′→3′) TNF-α 上游: TCTTCTCATTCCTGCTTGTGG
下游: GGTCTGGGCCATAGAACTGAIL-1β 上游: AGAAGCTGTGGCAGCTACCTG
下游: GGAAAAGAAGGTGCTCATGTCCGAPDH 上游: AAATGGTGAAGGTCGGTGTGAAC
下游: CAACAATCTCCACTTTGCCACTG
下载: 导出CSV
-
[1] 马丽媛, 王增武, 樊静, 等. 《中国心血管健康与疾病报告2021》概要[J]. 中国介入心脏病学杂志, 2022, 30(7): 481-496. https://www.cnki.com.cn/Article/CJFDTOTAL-ZJXB202207001.htmMA LY, WANG ZW, FAN J, et al. Summary of China cardiovascular health and disease report2021[J]. Chin J Interv Cardiol, 2022, 30(7): 481-496. https://www.cnki.com.cn/Article/CJFDTOTAL-ZJXB202207001.htm [2] MAHTTA D, SUDHAKAR D, KONERU S, et al. Targeting inflammation after myocardial infarction[J]. Curr Cardiol Rep, 2020, 22(10): 110. doi: 10.1007/s11886-020-01358-2 [3] ZHAO L, LI DH, ZHENG H, et al. Acupuncture as adjunctive therapy for chronic stable angina: A randomized clinical trial[J]. JAMA Intern Med, 2019, 179(10): 1388-1397. doi: 10.1001/jamainternmed.2019.2407 [4] 徐森磊, 顾任钧, 杨雪莹, 等. 腺苷介导循环机械按压内关对心肌缺血大鼠的保护效应[J]. 南京中医药大学学报, 2021, 37(5): 682-687. http://xbs.njucm.edu.cn/jnutcmns/ch/reader/view_abstract.aspx?file_no=zr20210510&flag=1XU SL, GU RJ, YANG XY, et al. Adenosine-mediated cardioprotection of local cyclical compression on Neiguan on myocardial ischemic rats[J]. J Nanjing Univ Tradit Chin Med, 2021, 37(5): 682-687. http://xbs.njucm.edu.cn/jnutcmns/ch/reader/view_abstract.aspx?file_no=zr20210510&flag=1 [5] 卢圣锋, 袁璟, 丁亚娟, 等. 电针对大鼠缺血心肌组织中炎性物质和交感神经活性物质表达的影响[J]. 针刺研究, 2019, 44(5): 313-318. https://www.cnki.com.cn/Article/CJFDTOTAL-XCYJ201905002.htmLU SF, YUAN J, DING YJ, et al. Effect of electroacupuncture on the expression of inflammatory substances and sympathetic active substances in ischemic myocardium of rats[J]. Acupunct Res, 2019, 44(5): 313-318. https://www.cnki.com.cn/Article/CJFDTOTAL-XCYJ201905002.htm [6] 张娟, 尚德静. Toll样受体4(TLR4) 信号通路及其靶向药物的研究进展[J]. 细胞与分子免疫学杂志, 2021, 37(7): 657-662. https://www.cnki.com.cn/Article/CJFDTOTAL-XBFM202107014.htmZHANG J, SHANG DJ. Research progress of TLR4 signaling pathway and its targeted drugs[J]. Chin J Cell Mol Immunol, 2021, 37(7): 657-662. https://www.cnki.com.cn/Article/CJFDTOTAL-XBFM202107014.htm [7] CAI SS, ZHAO MY, ZHOU B, et al. Mitochondrial dysfunction in macrophages promotes inflammation and suppresses repair after myocardial infarction[J]. J Clin Invest, 2023, 133(4): e159498. doi: 10.1172/JCI159498 [8] ITALIANI P, BORASCHI D. From monocytes to M1/M2 macrophages: Phenotypical vs. functional differentiation[J]. Front Immunol, 2014, 5: 514. [9] JING R, LONG TY, PAN W, et al. IL-6 knockout ameliorates myocardial remodeling after myocardial infarction by regulating activation of M2 macrophages and fibroblast cells[J]. Eur Rev Med Pharmacol Sci, 2019, 23(14): 6283-6291. [10] 杨文秀, 陈栎遥, 杨嘉丽, 等. 不同强度电针预处理对急性心肌缺血小鼠心功能及巨噬细胞极化的影响[J]. 针刺研究, 2022, 47(11): 955-961. https://www.cnki.com.cn/Article/CJFDTOTAL-XCYJ202211003.htmYANG WX, CHEN LY, YANG JL, et al. Effect of different electrical current intensities of electroacupuncture preconditioning on cardiac function and macrophage polarization in mice with acute myocardial ischemia[J]. Acupunct Res, 2022, 47(11): 955-961. https://www.cnki.com.cn/Article/CJFDTOTAL-XCYJ202211003.htm [11] 唐勇. 实验针灸学[M]. 3版. 上海: 上海科学技术出版社, 2021: 144.TANG Y. Experimental Acupuncture[M]. 3rd ed. Shanghai: Shanghai scientific & technical publishers, 2021: 144. [12] ZHANG ZW, CHEN Y, ZHENG LX, et al. A DUSP6 inhibitor suppresses inflammatory cardiac remodeling and improves heart function after myocardial infarction[J]. Dis Model Mech, 2023, 16(5): dmm049662. doi: 10.1242/dmm.049662 [13] ZHANG T, YANG WX, WANG YL, et al. Electroacupuncture preconditioning attenuates acute myocardial ischemia injury through inhibiting NLRP3 inflammasome activation in mice[J]. Life Sci, 2020, 248: 117451. doi: 10.1016/j.lfs.2020.117451 [14] LU SF, WANG JM, YUAN J, et al. Electroacupuncture improves cardiac function and reduces infarct size by modulating cardiac autonomic remodeling in a mouse model of myocardial ischemia[J]. Acupunct Med, 2021, 39(6): 681-690. [15] 向延卫, 范斌, 徐蓉, 等. 芩珠凉血方对银屑病血热证患者血清IL-17、IL-23及皮损中相关转录因子的影响[J]. 南京中医药大学学报, 2016, 32(4): 326-329. http://xbs.njucm.edu.cn/jnutcmns/ch/reader/view_abstract.aspx?file_no=zr20160407&flag=1XIANG YW, FAN B, XU R, et al. Effect of Qinzhu Liangxue recipe on serum IL-17, IL-23 and related transcription factors in skin lesions of patients with blood heat syndrome of psoriasis[J]. J Nanjing Univ Tradit Chin Med, 2016, 32(4): 326-329. http://xbs.njucm.edu.cn/jnutcmns/ch/reader/view_abstract.aspx?file_no=zr20160407&flag=1 [16] 李梦瑶, 王倩怡, 张璐莎, 等. 中性粒细胞在心肌梗死中作用的文献计量学和可视化分析[J]. 中国循证心血管医学杂志, 2022, 14(6): 664-668. https://www.cnki.com.cn/Article/CJFDTOTAL-PZXX202206006.htmLI MY, WANG QY, ZHANG LS, et al. Bibliometric and visual analysis of the role of neutrophils in myocardial infarction[J]. Chin J Evid Based Cardiovasc Med, 2022, 14(6): 664-668. https://www.cnki.com.cn/Article/CJFDTOTAL-PZXX202206006.htm [17] BÄCK M, WEBER C, LUTGENS E. Regulation of atherosclerotic plaque inflammation[J]. J Intern Med, 2015, 278(5): 462-482. [18] HENEIN MY, VANCHERI S, LONGO G, et al. The role of inflammation in cardiovascular disease[J]. Int J Mol Sci, 2022, 23(21): 12906. [19] PEET C, IVETIC A, BROMAGE DI, et al. Cardiac monocytes and macrophages after myocardial infarction[J]. Cardiovasc Res, 2020, 116(6): 1101-1112. [20] ZHOU MM, LI DW, XU L, et al. Propionate alleviated post-infarction cardiac dysfunction by macrophage polarization in a rat model[J]. Int Immunopharmacol, 2023, 115: 109618. [21] FAROOQ M, BATOOL M, KIM MS, et al. Toll-like receptors as a therapeutic target in the era of immunotherapies[J]. Front Cell Dev Biol, 2021, 9: 756315. [22] BUCCINI DF, RORIZ BC, RODRIGUES JM, et al. Antimicrobial peptides could antagonize uncontrolled inflammation via Toll-like 4 receptor[J]. Front Bioeng Biotechnol, 2022, 10: 1037147. [23] ROH JS, SOHN DH. Damage-associated molecular patterns in inflammatory diseases[J]. Immune Netw, 2018, 18(4): e27. http://www.ncbi.nlm.nih.gov/pubmed/30181915 [24] FALLACH R, SHAINBERG A, AVLAS O, et al. Cardiomyocyte Toll-like receptor 4 is involved in heart dysfunction following septic shock or myocardial ischemia[J]. J Mol Cell Cardiol, 2010, 48(6): 1236-1244. [25] KAWASAKI T, KAWAI T. Toll-like receptor signaling pathways[J]. Front Immunol, 2014, 5: 461. [26] LIU SB, WANG ZF, SU YS, et al. A neuroanatomical basis for electroacupuncture to drive the vagal-adrenal axis[J]. Nature, 2021, 598(7882): 641-645. [27] 柳钶, 姜劲峰, 卢圣锋. 针灸调节自主神经研究的效应特点及其途径分析[J]. 针刺研究, 2021, 46(4): 335-341. https://www.cnki.com.cn/Article/CJFDTOTAL-XCYJ202104014.htmLIU K, JIANG JF, LU SF. Effect characteristics and mechanism of acupuncture in autonomic nerve regulation[J]. Acupunct Res, 2021, 46(4): 335-341. https://www.cnki.com.cn/Article/CJFDTOTAL-XCYJ202104014.htm -
点击查看大图
计量
- 文章访问数: 251
- HTML全文浏览量: 25
- PDF下载量: 17
- 被引次数: 0
