雷帕霉素调控巨噬细胞表型改善肺动脉高压

doi:10.6040/j.issn.1671-7554.0.2017.850

摘要/Abstract

摘要： 目的研究雷帕霉素(RAP)调控巨噬细胞表型对肺动脉高压(PAH)的影响及其作用机制。方法建立PAH大鼠模型,随机分为对照组、PAH组和PAH+RAP组,每组10只。造模后5 d分别给予对照组溶媒和PAH+RAP组RAP肌肉注射。通过右心导管检测平均肺动脉压(mPAP),并称量左右心室、室间隔以计算右心室肥厚指数(RVHI)。通过免疫组化技术评价巨噬细胞浸润程度和肺小动脉平滑肌细胞增殖程度,免疫印迹技术检测α-SMA蛋白表达水平,免疫荧光检测M2型巨噬细胞表达水平。利用RT-PCR检测各组TNF-α、IL-1β、IL-6和IL-10 mRNA表达水平。体外培养巨噬细胞,分别给予LPS+INF-γ、IL-4和RAP干预,并将细胞分为空白对照组、LPS+INF-γ干预组、IL-4干预组和LPS+INF-γ+RAP干预组;流式细胞术检测巨噬细胞表型,RT-PCR检测以上各炎性因子mRNA表达水平。结果成功建立了PAH大鼠模型。PAH组的mPAP、RVHI、α-SMA蛋白表达水平、巨噬细胞浸润程度以及炎性因子TNF-α、IL-1β、IL-6、IL-10 mRNA的表达水平均高于对照组(P<0.05)。PAH+RAP组的mPAP、RVHI、α-SMA蛋白表达水平以及炎性因子TNF-α、IL-1β、IL-6、IL-10 mRNA的表达水平均低于PAH组(P<0.05)。PAH+RAP组M2型巨噬细胞相对表达水平高于PAH组(P<0.05)。体外实验中,LPS+INF-γ干预组的CD86阳性巨噬细胞百分比及TNF-α、IL-1β、IL-6、IL-10 mRNA相对表达水平均高于空白对照组(P<0.05);LPS+IFN-γ+RAP干预组的CD86阳性巨噬细胞百分比及TNF-α、IL-1β、IL-6、IL-10 mRNA相对表达水平均低于LPS+INF-γ干预组(P<0.05)。结论 PAH的发生伴随着巨噬细胞浸润及炎性因子表达水平的升高;RAP可通过诱导巨噬细胞向M2型转化,从而降低炎性因子表达水平,进而改善PAH。

关键词: 雷帕霉素, 肺动脉高压, 巨噬细胞, 肺动脉平滑肌细胞, 表型转换, 白介素-1β, 白介素-6

Abstract: Objective To study the effect of rapamycin(RAP)modulating macrophage phenotypes on pulmonary artery hypertension(PAH)and the correlated mechanism. Methods Thirty male Sprague-Dawley rats were randomly divided into the control, PAH and PAH+RAP groups, ten in each group. PAH rat model was induced by left pneumonectomy(PE)and monocrotaline(MCT). Five days after PAH model establishment, the control group and the PAH+RAP group received an intramuscular injection of solvent and RAP for thirty days, respectively. The mean pulmonary artery pressure(mPAP)and right ventricular hypertrophy index(RVHI)were measured in each group via the right cardiac catheter. The degrees of macrophages infiltration and the proliferation of pulmonary arteriolar smooth muscle cells 山东大学学报 (医学版)56卷4期 -王波,等.雷帕霉素调控巨噬细胞表型改善肺动脉高压 \=-were evaluated by immunohistochemistry, the expression of α-SMA protein was detected by Western blotting, and the expression levels of M2 macrophages was detected by immunofluorescence. The expression levels of TNF-α, IL-1, IL-6 and IL-10 mRNA in each group were detected by RT-PCR. Macrophages were cultured in vitro and treated with LPS+INF-γ, IL-4, and LPS+INF-γ+RAP, respectively; the cells were divided into the blank control group, LPS+INF-γ group, IL-4 group, and LPS+INF-γ+RAP group accordingly. The phenotypes of macrophages were detected by flow cytometry, and the mRNA expression levels of the inflammatory factors were dected by RT-PCR. Results PHA rat models were established successfully. The mPAP, RVHI, α-SMA protein expression level, macrophage infiltration degree and mRNA expression levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-10 in PAH group were higher than those in the control group(all P<0.05). The mPAP, RVHI, α-SMA protein expression level, and mRNA expression levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-10 in PAH+RAP group were lower than those in PAH group(all P<0.05). The expression level of M2-type macrophages in PAH+RAP group was higher than that in PAH group(P<0.05). CD86 positive macrophages percentage and mRNA expression levels of TNF-α, IL-1β, IL-6, and IL-10 in LPS+INF-γ group were higher than those in the blank control group in vitro(all P<0.05). CD86 positive macrophages percentage and mRNA expression levels of TNF-α, IL-1β, IL-6, and IL-10 in LPS+IFN-γ+RAP group were lower than those in LPS+INF-γ group(all P<0.05). Conclusion PAH occurrence is accompanied with macrophage infiltration and expression level increase of inflammatory factors. RAP improves PAH via reducing inflammatory response partly depending on inducing the macrophages transformation into M2.

Key words: Rapamycin, Pulmonary arterial hypertension, Macrophages, Pulmonary arterial smooth muscle cells, Phenotype transformation, Interleukin-lβ, Interleukin-6

中图分类号:

R574

王波,薛江,刘爱虹,翟蕊蕊,王一彪. 雷帕霉素调控巨噬细胞表型改善肺动脉高压[J]. 山东大学学报 (医学版), 2018, 56(4): 51-57.

WANG Bo, XUE Jiang, LIU Aihong, ZHAI Ruirui, WANG Yibiao. Rapamycin improved pulmonary artery hypertension via modulating the phenotypes of macrophages[J]. Journal of Shandong University (Health Sciences), 2018, 56(4): 51-57.

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