Thorax:吸入糖皮质激素延缓COPD患者内皮祖细胞衰老

2022-03-17 从医路漫漫 MedSci原创

细胞衰老是包括心血管疾病(CVD)和慢性阻塞性肺疾病(COPD)在内的衰老相关疾病的基本病理生理机制。氧化应激激活的DNA损伤反应(DDR)导致细胞周期停滞、衰老或凋亡。

背景:细胞衰老是包括心血管疾病(CVD)和慢性阻塞性肺疾病(COPD)在内的衰老相关疾病的基本病理生理机制。氧化应激激活的DNA损伤反应(DDR)导致细胞周期停滞、衰老或凋亡。衰老的内皮细胞功能紊乱,表现出促炎症的“衰老相关分泌表型”(SASP),促进血管炎症、动脉粥样硬化形成和血栓形成。使用循环内皮祖细胞,称为内皮集落形成细胞(ECFC)或血液生长内皮细胞,我们证明吸烟者和COPD患者由于表观遗传功能障碍而加速内皮衰老,支持内皮加速老化是引起CVD的一个因素的概念。

吸入性皮质类固醇(ICS)广泛应用于病情严重、反复的慢性阻塞性肺疾病(COPD)患者。ICS可能对COPD患者的心血管并发症有保护作用,尽管这一点一直存在争议,其机制尚不清楚。在这里,我们证明ICS可以延缓COPD患者ECFC中的衰老和SASP,提示皮质类固醇对内皮的保护作用是一种新的机制。

方法及结果:利用内皮细胞集落形成细胞(ECFC),我们发现吸烟者和COPD患者与非吸烟者相比衰老加速.亚组分析表明,COPD患者吸入糖皮质激素治疗的ECFC(n=14,8例)与非吸入糖皮质激素治疗的COPD患者相比,衰老显著降低(衰老相关β半乳糖苷酶活性,p21CIP1)、γ损伤反应标志物和干扰素诱导蛋白-10。如前所述,健康吸烟者和慢性阻塞性肺病患者的细胞外周血单个核细胞衰老程度和DDR标志物均高于健康不吸烟者,通过衰老相关β-半乳糖苷酶(SA-β-GAL)活性、p21CIP1、p16INK4、53BP1和γ-H2AX((图1A)和已发表的队列)进行测量。亚组分析的一个意外发现是,与非慢性阻塞性肺病患者相比,慢性阻塞性肺病患者的细胞外周血单个核细胞衰老程度降低。其他衰老标记物如p21CIP1(mRNA(6例COPD-NO ICS与5例COPD-ICS)、免疫印迹(n=3例COPD-ICS与4例COPD-ICS)、免疫荧光(n=3例COPD-NO ICS与4例COPD-ICS)和p16INK4(n=2例)等衰老标志物进一步证实了COPD组ICS衰老程度的降低(图1B-D)。我们接下来研究DDR信令。γ-H2AX和53BP1是DNA修复的介质,它们调节下游效应分子,促进衰老和凋亡。COPD组ICS(n=3)DDR标志物减少(图1E),提示糖皮质激素对DDR和内皮衰老有保护作用。

图1减少了ICS上COPD患者的衰老和DNA损伤反应)。(A)衰老相关β半乳糖苷酶活性作为细胞衰老的标志,分别来自健康非吸烟者(n=11)、健康吸烟者(n=6)和慢性阻塞性肺疾病患者(n=14,8)。吸烟者和COPD患者的ECFC与不吸烟者相比衰老程度增加;Kruskal-Wallis检验和Dunn‘s多重比较检验(图A-左图)。接受ICS治疗的COPD患者的ECFC与未接受ICS治疗的COPD患者的ECFC相比,衰老程度较低;Mann-Whitney U检验(A图右面板);(刻度条100µm)。(B)实时荧光定量PCR检测11例ICS患者ECFC中p21mRNA水平。核糖体蛋白L13a用于正常化。(C)Western blotting检测p21蛋白水平。测定微管蛋白(α-Tubulin)正常化(每组3例)。(D)COPD-ICS与COPD-非ICS患者ECFC免疫荧光染色的代表性图像:p21(青色,左图)和p16(青色,右图)。DAPI(蓝色)作为细胞核染色,VE-cadherin(洋红)作为内皮标记物。(E)用免疫荧光染色法检测53BP1(绿色)和γ-H2AX(红色)的DNA损伤程度(每组3例)。DAPI(蓝色)作为核标记,VE-cadherin(品红)作为内皮标记。使用63倍物镜(比例尺=20µm),在至少5个z堆叠图像和20个细胞中计数每个核的不同核免疫荧光病灶(见箭头)的数量。Mann-Whitney U检验;*P<0.0001;慢性阻塞性肺疾病;内皮细胞集落形成细胞;ICS,吸入糖皮质激素。

图2糖皮质激素可能对吸入糖皮质激素的慢性阻塞性肺疾病患者因干扰素-γ(IFN-γ)诱导蛋白-10(IP-10)相关的氧化应激减少衰老相关的分泌表型引起的内皮提前衰老起到保护作用。(A)人脐静脉内皮细胞(HUVECs)在不同浓度的布地奈德(10−10−10−6mol/L)或对照溶剂(二甲基亚砜)存在或不存在的情况下培养。预处理1h后,将HUVECs暴露于50µM的H2O2中1.5h,诱导应激诱导的HUVECs提前衰老。H_2O_2处理72小时后测定SA-β-GAL活性;n=3(标尺100µm)。(B)通过测定过氧化氢处理24小时后的caspase-3/7-Glo活性来定量细胞凋亡,如(A)所述;n=3(样本一式三份)。(C)如(A)所述,从H2O2处理24小时后的γ-H2AX蛋白;n=3。(A-C)弗里德曼检验和邓恩多重比较检验。(D)HUVECs在H2O2处理后60min、240min、24h或48h分别进行53BP1和DRAQ5(核标记)染色。计数53BP1阳性细胞数和每细胞病灶数(标尺20µm)。(E)用Luminex法检测非吸烟者(n=5)和慢性阻塞性肺疾病(n=5)患者外周血单个核细胞培养上清液中IL-8和干扰素-γ诱导蛋白10(IP-10或CXCL10)的含量。IL-8、IP-10与SA-b-GAL活性的Pearson相关系数。与未接受ICS的COPD患者相比,ICS的COPD患者的样本中IP-10的表达降低;Kruskal-Wallis试验和Dunn的多重比较试验。(F)免疫荧光染色IP-10(绿色)和p21(青色)。DAPI(蓝色)作为核标记,VE-cadherin(品红)作为内皮标记。使用来自COPD-ICS(n=4)和COPD-no ICS(n=3)(标尺=20µm)的63倍物镜,对至少5个z堆叠图像和每个ECFC样本20个细胞进行IP-10和p21的分析;Mann-Whitney U检验;*p<0.0001。二甲基亚砜;ECFC,内皮细胞集落形成细胞;ICS,吸入皮质类固醇;IL-8,白细胞介素-8。

结果:体外人脐静脉内皮细胞研究表明,ICS对氧化应激引起的DDR、衰老和凋亡具有保护作用,提示糖皮质激素对内皮细胞有保护作用的分子机制。

原文出处:Paschalaki K,  Rossios C,  Pericleous C,et al.Inhaled corticosteroids reduce senescence in endothelial progenitor cells from patients with COPD.Thorax 2022 Jan 13

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    2022-03-16 gous
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