心肌缺氧状态下间充质干细胞对巨噬细胞 M2 极化的影响及其机制研究

Effect of mesenchymal cells on the M2 polarization of macrophages under myocardial hypoxia and its mechanism

摘要:

目的 探讨心肌缺氧状态下间充质干细胞（MSC）对巨噬细胞极化的影响，并揭示其调控巨噬 细胞极化的机制。方法 在M0 型巨噬细胞培养液中加入无糖无氧培养的心肌细胞上清液，与MSC 共培养。 采用流式细胞术、Western blotting 及逆转录聚合酶链反应检测M1/M2 型巨噬细胞生物学标志，以判断MSC 在模拟心肌缺氧状态下对巨噬细胞极化的影响，并观察MSC 对巨噬细胞髓样分化因子88 及其上游TLR2、 TLR4 受体表达的影响，以及其下游的细胞核转录因子-κB（NF-κB）和丝裂原活化蛋白激酶（MAPK） 信号通路磷酸化的水平。结果 与M0 组比较，模拟心肌缺氧状态下培养的M0 巨噬细胞成功诱导M1 极化 （P <0.05）；与MSC 共培养促进M1 向M2 极化（P <0.05）。同等条件下加入不同浓度髓样分化因子88 抑制剂后， 与M0+OGD 组比较，M2 型巨噬细胞比例随着ST2825 剂量的增加逐渐升高（P <0.05）。与M0+OGD 组比较， M0+OGD+MSC 组白细胞介素10、转化生成因子-β1 表达水平升高（P <0.05）。Western blotting 检测显示 MSC 共培养后，巨噬细胞TLR2、TLR4 及髓样分化因子88 表达下调，其下游的NF-κB 和MAPK 信号通 路标志分子P65、IKKAα/β、JNK1/2、P38、ERK1/2 等磷酸化水平降低。结论 在心肌缺氧状态下，MSC 促进巨噬细胞M2 极化。其机制之一可能是MSC 下调巨噬细胞TLR2、TLR4 及髓样分化因子88 表达，抑制 TLR2/4- 髓样分化因子88 信号及其下游的NF-κB 和MAPK 通路。

Abstract:

Objective To investigate the effects of mesenchymal stem cells (MSC) on the polarization of macrophages under myocardial hypoxia condition, and reveal the underlying the mechanisms for polarization. Methods The supernatant of cardiomyocytes cultured in oxygen and glucose deprivation (OGD) was added into the culture medium of M0 macrophages to simulate myocardial hypoxia. Then the macrophages were directly co-cultured with MSC. To determine the effect of MSC on the M2 polarization of macrophages, the biological markers of M1/M2 macrophages were detected by flow cytometry, western blot and RT-PCR, respectively. And the expression of MyD88 (myeloid differentiation factor 88) and its upstream factors TLR2 (Toll-like receptor 2) and TLR4 (Toll-like receptor 4) as well as the phosphorylation level of NF-κB (nuclear factor-kappa B) and MAPK (mitogen-activated protein kinase) signaling pathway were measured. Results Compared with the control group, flow cytometry showed that M1 polarization were successfully induced in M0 macrophages cultured under myocardial hypoxia condition (P<0.05). Co-cultured with MSC, M0 was promoted significantly to M2 polarization (P<0.05). After adding different concentrations of MyD88 inhibitor, the proportion of M2 macrophages gradually increased with the dosage of ST2825 (P<0.05) compared with the control group. RT-qPCR showed that compared with the control group, the M2 macrophage-associated inflammatory factors IL-10 and TGF-β1 levels were increased after co-cultured with MSC (P<0.05). Western blot showed that the expression of TLR 2/4 and MyD88 in macrophages was down-regulated after co-cultured with MSC, and the phosphorylation level of downstream factors including P65, IKKAα/β, JNK1/2, P38, ERK1/2, the markers of the downstream signaling pathway of TLR2/4- MyD88, were reduced. Conclusions MSC promote the M2 polarization from M0 macrophages under myocardial hypoxia condition. One of the main mechanisms might be that MSC down-regulate the expression of TLR2, TLR4 and MyD88 in macrophages, and inhibit TLR2/4-MyD88 signaling and its downstream NF-κB and MAPK pathways.