Electroacupuncture Influences Macrophage M2 Polarization and TLR4 and MyD88 Expression in Myocardial Tissue of Myocardial Ischemia Injury Mice
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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. -
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 -
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