共刺激分子程序性死亡配体-1在急性肺损伤中的作用及机制初探

The potential role of co-inhibitory molecule programmed death ligand-1 in acute lung injury and itsmechanism

目的探讨程序性死亡配体-1（PD-L1）表达在急性肺损伤（ALI）中的作用机制。方法清洁级健康雄性C57BL／6小鼠20只，PD～L1基因敲除雄性小鼠20只，分别按随机数字表法分为假手术组（Sham）和ALI组，每组10只。采用失血性休克／脓毒症双重打击方法制备ALI小鼠模型，Sham组仅显露双侧股动脉并结扎但不放血、仅分离盲肠但不结扎穿孔。于制模后24h处死小鼠，取肺组织和支气管肺泡灌洗液（BALF）。用实时定量反转录-聚合酶链反应（RT-qPCR）和蛋白质免疫印迹试验（WesternBlot）检测肺组织PD-LI的mRNA和蛋白表达；光镜下观察肺组织病理学改变，测定BALF中蛋白含量；用流式细胞仪检测粒细胞分化抗原1（Grl）阳性细胞数及肺组织髓过氧化物酶（MPO）活性；用酶联免疫吸附试验（ELISA）检测肺组织和BALF中促炎因子白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）及趋化因子角化细胞源性趋化因子（KC）、巨噬细胞炎性蛋白-2（MIP～2）水平。结果与Sham组相比，ALI组C57BL／6小鼠肺组织PD-L1的mRNA和蛋白表达均明显升高[PD-L1mRNA（2-△△Ct）：3．20±0．76比1．01±0．03，PD-L1蛋白（4值）：0．98±0．16比0．15±0．04，均P〈0．05]；光镜下显示，ALI组C57BL／6小鼠肺泡壁间隔增厚、充血水肿和点状出血，肺组织有大量炎性细胞浸润，BALF中有明显的蛋白渗漏（ng／L：0．18±0．06比0．05±0．01，P〈0．05）；而PD-L1基因敲除小鼠肺损伤程度及BALF中蛋白渗漏较ALIC57BL／6小鼠明显减轻（ng／L：0．11±0．02比0．18±0．06，P〈0．05）。与相应Sham组比较，ALI小鼠肺组织Grl阳性细胞数、MPO活性，肺组织和BALF中促炎因子及趋化因子水平均明显增高；但PD-L1基因敲除ALI小鼠肺组织和BALF中上述指标均较ALIC57BL／6小鼠明显降低[肺组织Grl阳性细胞数：（39．0±4．0）％比（45．0±8．0）％，MPO活性（U·μg^-1·min^-1）：2．85±0．62比4．52±1．16，IL-6（ng／g）：461±111比728±28，TNF-βα（ng／g）：1123±175比1500±327，KC（ng／g）：150±34比250±28，MIP-2（ng／g）：1263±468比1763±323；BALF中IL-6（ng／L）：134±22比258±38，TNF-α（ng／L）：598±102比889±139，KC（ng／L）：934±286比1258±336，MIP-2（ng／L）：650±130比950±256，均P〈0．05j。结论PD-L1基因缺陷可通过降低中性粒细胞趋化因子KC和MIP-2的产生，减轻肺部中性粒细胞归巢，对ALI小鼠发挥保护作用。

Objective To investigate the regulatory role of programmed death ligand-1 （PD-L1） on acute lung injury （ALl）, and its molecular mechanism. Methods Twenty C57BL/6 male mice and 20 PD-LI knock out male mice were collected, and they were divided into two groups by random number table, respectively： namely sham group and ALI group, 10 mice in each group. The model of ALI was reproduced by two-hit of hemorrhagic shock and sepsis, and the mice in sham group were only got bilateral femoral artery exposure and ligation without bleeding, cecal separation without ligation and perforation. The mice were sacrificed 24 hours after model reproduction, and the lungtissue and bronchoalveolar lavage fluid （BALF） were collected. The mRNA and protein expression levels of PD-L1 in the lungs were determined by real-time quantitative reverse transcription-polymerase chain reaction （RT-qPCR） and Western Blot. The pathological changes were observed with microscopy. The protein levels in BALF were determined. The granulocyte differentiation antigen 1 （Grl） positive cells was determined by cytometry, and myeloperoxidase （MPO） activity in lung tissue was determined. The levels of proinflammatory factors interleukin-6 （IL-6）, tumor necrosis factor-ot （TNF-α）, and chemotatic factors keratinocyte chemoattractant （KC）, macrophage inflammatory protein-2 （MIP-2） in lung homogenates and BALF were determined by enzyme-linked immunosorbent assay （ELISA）. Results Compared with sham group, the mRNA and protein levels of PD-L1 in lung tissue of C57BL/6 mice in ALI group were significantly elevated [PD-L1 mRNA （2-△△ct）： 3.20±0.76 vs. 1.01 ±0.03, PD-L1 protein （A value）： 0.98±0.16 vs. 0.15±0.04, both P 〈 0.05]. It was shown by light microscopy that the alveolar wall was thickened, congestive, edema and spot bleeding with a large number of inflammatory cell infiltration in the lung tissue of C57BL/6 mice in ALI group, and an obvious protein leakage was found in BALF （ng/L： 0.18±0.06 vs. 0.05±0.01, P 〈 0.05）. The lung injury degree of PD-L1 knockout ALI mice was significantly less than that of C57BL/6 ALI mice, and the protein leakage was significantly reduced in BALF （ng/L： 0. 11± 0.02 vs. 0. 18 ±0.06, P 〈 0.05）. Compared with corresponding sham group, the number of Grl positive cells, MPO activity in lung tissue as well as the levels proinflammatory factors and chemotaiic factors in lung tissue and BALF in ALI group were significantly increased. However, when compared with C57BL/6 ALI mice, above parameters in lung homogenates and BALF were significantly decreased in PD-L1 knockout ALI mice [number of Grl positive cells： （39.0 ± 4.0）% vs. （45.0 ± 8.0）%, MPO activity （U·μg^-1·min^-1）： 2.85±0.62 vs. 4.52± 1.16; lung IL-6 （ng/g）： 461 ±111 vs. 728±28, TNF- α （ng/g）： 1 123±175 vs. 1 500 ±327, KC （ng/g）： 150 ± 34 vs. 250 ± 28, MIP-2 （ng/g）： 1 263 ± 468 vs. 1763± 323; BALF IL-6 （ng/L）： 134 ± 22 vs. 258＋38, TNF-α （ng/L）： 598± 102 vs. 889±139, KC （ng/L）： 934＋286 vs. 1 258±336, MIP-2 （ng/L）： 650±130 vs. 950± 256; all P 〈 0.05]. Conclusion PD-L1 may play an important protective role in the immunological mechanism of ALI, which may be mediated by decreasing chemotactic factor KC and MIP-2 and mitigating neutrophil ehemotaxis in lung tissue.