南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (5): 775-782.doi: 10.12122/j.issn.1673-4254.2021.05.20

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富血小板血浆可减轻大鼠急性心肌缺血-再灌注损伤

王德奖,李 挺,徐颖怡,杨雪雯,何铭垣,张智勇,吴 炜,燕 翼   

  • 出版日期:2021-05-20 发布日期:2021-06-11

Platelet-rich plasma alleviates myocardial ischemia-reperfusion injury in rats

  • Online:2021-05-20 Published:2021-06-11

摘要: 目的 探讨富血小板血浆(PRP)对急性缺血-再灌注损伤心肌的保护作用及其机制。方法 制备PRP并测量PDGF-BB和TGF-β1的浓度;SD大鼠乳鼠心肌细胞缺氧3 h后,在复氧条件下分别用1%、5%、10%、20%体积的PRP共培养细胞12 h,检测细胞活力,同时对心肌细胞进行免疫荧光染色,提取蛋白检测AMP依赖的蛋白激酶(AMPK)及其磷酸化水平;检测PRP对大鼠主动脉内皮细胞增殖及迁移的影响;以SD大鼠建立心肌缺血-再灌注损伤模型,在恢复灌注后立刻在结扎部位周围四个部位注射100 μL的PRP(PRP组,n=20)或生理盐水(Control组,n=20),1 d后各组随机选6只大鼠取材心脏行TTC染色并测量肌钙蛋白I的浓度,其余动物1周后通过超声心动图检测心功能,并取材心脏进行HE染色;体外对RAW264.7细胞缺氧3 h,分别给予PRP或生理盐水作用24 h后行流式细胞仪检测M1和M2型巨噬细胞的极化情况。结果 PRP中PDGF-BB和TGF-β1的浓度显著高于全血,1%体积PRP可以显著减少缺氧-复氧所导致的心肌细胞死亡,其保护作用与AMPK的激活密切相关;PRP促进内皮细胞的增殖与迁移;动物实验中PRP组梗死面积和肌钙蛋白I浓度均小于生理盐水组(P<0.001),超声显示PRP组心功能明显改善,HE染色示缺血区域炎症反应较生理盐水组明显减轻;RAW264.7细胞经PRP处理M1型巨噬细胞减少,M2型巨噬细胞增 多。结论 PRP通过促进AMPK磷酸化及巨噬细胞免疫调节作用来改善大鼠心肌急性缺血-再灌注损伤区域的炎症微环境,从而起到心肌保护的作用。

关键词: 富血小板血浆;心肌缺血-再灌注;AMPK;免疫调节;炎症反应

Abstract: Objective To investigate the protective effect of platelet-rich plasma (PRP) against acute myocardial ischemia-reperfusion (IR) injury and the possible mechanism. Method Aortic blood samples were collected from 10 SD rats to prepare PRP, in which the concentrations of platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) were measured. Cell models of IR injury were established in primary cultures of neonatal SD rat cardiomyocytes by exposing the cells to 3 h of hypoxia. The cells were then reoxygenated and co-cultured with 1%, 5%, 10%, and 20% volume of PRP for 12 h, and the changes in cell viability was assessed. Immunofluorescence staining of the cardiomyocytes was performed, and the cellular expression of AMPK and its phosphorylation level were detected. The effects of PRP on the proliferation and migration of rat aortic endothelial cells (RAOECs) were examined. In a SD rat model of myocardial IR injury, 100 μL of PRP (n= 20) or normal saline (n=20) was injected at 4 sites around the ligation site immediately after cardiac reperfusion. One day after the injection, 6 rats were selected from each group for TTC staining of the myocardial tissues and measurement of troponin I content. One week later, the cardiac function of the remaining rats was assessed by echocardiography, and HE staining of the myocardial tissues was performed. The effect of PRP treatment for 24 h on polarization of M1 and M2 macrophages was also examined by flow cytometry in RAW264.7 cells after hypoxic exposure for 3 h. Results The concentrations of PDGF-BB and TGF-β1 were significantly higher in PRP than in whole blood. Addition of 1% volume of PRP significantly reduced death of the cardiomyocytes following reoxygenation, and this effect was closely related with the activation of AMPK. Treatment with PRP obviously promoted the proliferation and migration of RAOECs. In rat models of acute myocardial IR injury, injections of PRP significantly reduced the infarct size and troponin I concentration as compared with saline injection (P<0.001). One week after PRP injection, the rats showed significantly improved cardiac function with a lowered level of inflammatory response in comparison with the rats with saline injection. In RAW264.7 cells with hypoxic exposure, treatment with PRP obviously decreased the number of M1 macrophages and increase the number of M2 macrophages. Conclusion PRP can improve acute myocardial IR injury in rats by phosphorylating AMPK and regulating macrophage polarization, which produces a protective immunomodulatory effect on the ischemic myocardial tissues.

Key words: platelet- rich plasma; myocardial ischemia-reperfusion; AMPK; immunomodulation; inflammation