Jin Yang, Sun Meng, Jiang Xuemei, Zhang Qingqing, Feng Di, Wen Zongmei
Department of Anesthesiology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200433, China. Corresponding author: Wen Zongmei, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2019 Nov;31(11):1357-1362. doi: 10.3760/cma.j.issn.2095-4352.2019.11.009.
To explore whether extracellular histones aggravate acute respiratory distress syndrome (ARDS) by inducing peripheral blood mononuclear cell (PBMC) pyroptosis.
Twenty patients with ARDS admitted to Shanghai Pulmonary Hospital, Tongji University School of Medicine from April to September in 2019 were enrolled, and 20 healthy volunteers were enrolled as controls. In vivo experiment: peripheral blood samples of patients with ARDS within 24 hours after diagnosis and healthy volunteers were collected, and the levels of plasma extracellular histone, interleukins (IL-1β and IL-18) and lactic dehydrogenase (LDH) were determined by enzyme-linked immunosorbent assay (ELISA). PBMC were harvested, the expression levels of the pyroptosis associated N terminal-gasdermin-D (GSDMD-N) protein were determined by Western Blot. In vitro experiment: PBMC isolated from healthy volunteers were divided into four groups. Blank control group without any treatment; lipopolysaccharide (LPS) group was treated with 1 mg/L LPS for 4 hours; LPS+histones group was treated with 100 mg/L exogenous histones for 24 hours after LPS treatment; LPS+histone+heparin group was treated with 200 U heparin for 24 hours after LPS and exogenous histones treatment. The GSDMD-N protein expression was determined by Western Blot, and the levels of IL-1β, IL-18 and LDH in cell supernatant were determined by ELISA. Spearman test was used to test the correlation among the parameters.
In vivo experiment results: compared with healthy control group, the GSDMD-N protein expression in PBMC of patients with ARDS was significantly increased [GSDMD-N/GAPDH: 0.136 (0.062, 0.246) vs. 0.026 (0.018, 0.036), P < 0.01], as well as the plasma levels of IL-1β, IL-18, LDH and extracellular histones [IL-1β (ng/L): 120.0 (94.2, 213.0) vs. 88.5 (82.3, 105.3), IL-18 (ng/L): 164.5 (70.8, 236.3) vs. 60.5 (52.0, 89.0), LDH (U/L): 30.9 (24.7, 39.5) vs. 19.8 (17.2, 21.5), extracellular histones (mg/L): 73.0 (42.8, 112.9) vs. 12.2 (9.6, 16.9), all P < 0.01], indicating that the PBMC of ARDS patients had significant pyroptosis and release of a large number of inflammatory factors. The oxygenation index (PaO/FiO) of ARDS patients was 135.5 (94.5, 196.0) mmHg (1 mmHg = 0.133 kPa). Correlation analysis showed that the expression of GSDMD-N protein in patients with ARDS was negatively correlated with PaO/FiO (r = -0.935, P < 0.01) and positively correlated with IL-1β, IL-18, LDH and extracellular histones (r value was 0.844, 0.843, 0.887, 0.899, respectively, all P < 0.01). In vitro experiment results: compared with blank control group, the expression of GSDMD-N protein in PBMC and the levels of inflammatory mediators in the supernatant of the LPS group were significantly increased [GSDMD-N/GAPDH: 0.035±0.006 vs. 0.028±0.006, IL-1β (ng/L): 39.8±5.5 vs. 22.6±4.7, IL-18 (ng/L): 31.2±4.4 vs. 20.0±2.2, LDH (U/L): 51.2±7.3 vs. 36.6±7.6, all P < 0.05], indicating that LPS stimulation could increase PBMC pyroptosis and the release of inflammatory mediators. Compared with LPS group, the expression of GSDMD-N protein and the levels of inflammatory mediators of the LPS+histones group were further increased [GSDMD-N/GAPDH: 0.114±0.009 vs. 0.035±0.006, IL-1β (ng/L): 119.0±18.7 vs. 39.8±5.5, IL-18 (ng/L): 49.2±8.5 vs. 31.2±4.4, LDH (U/L): 127.8±19.8 vs. 51.2±7.3, all P < 0.01], indicating that the stimulation of LPS on PBMC could be significantly amplified by exogenous histone treatment, GSDMD-N protein expression could be up-regulated and inflammatory factor release could be promoted to further induce PBMC pyroptosis. These adverse effects of exogenous histones on PBMC could be abrogated by heparin, the expression of GSDMD-N protein and the levels of inflammatory mediators were significantly lower than those of LPS+histones group [GSDMD-N/GAPDH: 0.063±0.004 vs. 0.114±0.009, IL-1β (ng/L): 46.8±8.6 vs. 119.0±18.7, IL-18 (ng/L): 33.0±5.1 vs. 49.2±8.5, LDH (U/L): 65.4±11.0 vs. 127.8±19.8, all P < 0.05].
Extracellular histones in plasma may aggravate ARDS by mediating PBMC pyroptosis.
探讨细胞外组蛋白是否通过诱导外周血单个核细胞（PBMC）焦亡加重急性呼吸窘迫综合征（ARDS）。
选取2019年4月至9月在同济大学医学院附属上海市肺科医院收治的20例ARDS患者，并选取20名健康志愿者作为对照。体内实验：采集ARDS患者确诊后24小时内的外周血样本及健康志愿者的外周血样本，采用酶联免疫吸附测定（ELISA）法检测血浆细胞外组蛋白、白细胞介素（IL-1β和IL-18）及乳酸脱氢酶（LDH）水平。采集PBMC，采用蛋白质免疫印迹法检测焦亡相关的N端gasdermin-D（GSDMD-N）蛋白表达水平。体外实验：将从健康志愿者分离的PBMC分为四组。空白对照组不做任何处理；脂多糖（LPS）组用1 mg/L LPS处理4小时；LPS+组蛋白组在LPS处理后用100 mg/L外源性组蛋白处理24小时；LPS+组蛋白+肝素组在LPS和外源性组蛋白处理后用200 U肝素处理24小时。采用蛋白质免疫印迹法检测GSDMD-N蛋白表达，采用ELISA法检测细胞上清液中IL-1β、IL-18及LDH水平。采用Spearman检验分析各参数之间的相关性。
体内实验结果：与健康对照组相比，ARDS患者PBMC中GSDMD-N蛋白表达显著升高[GSDMD-N/GAPDH：0.136（0.062，0.246）比0.026（0.018，0.036），P<0.01]，血浆IL-1β、IL-18、LDH及细胞外组蛋白水平也显著升高[IL-1β（ng/L）：120.0（94.2，213.0）比88.5（82.3，105.3），IL-18（ng/L）：164.5（70.8，236.3）比60.5（52.0，89.0），LDH（U/L）：30.9（24.7，39.5）比19.8（17.2，21.5），细胞外组蛋白（mg/L）：73.0（42.8，112.9）比12.2（9.6，16.9），均P<0.01]，提示ARDS患者PBMC发生明显焦亡并释放大量炎性因子。ARDS患者的氧合指数（PaO/FiO）为135.5（94.5，196.0）mmHg（1 mmHg = 0.133 kPa）。相关性分析显示，ARDS患者GSDMD-N蛋白表达与PaO/FiO呈负相关（r = -0.935，P<0.01），与IL-1β、IL-18、LDH及细胞外组蛋白呈正相关（r值分别为0.844、0.843、0.887、0.899，均P<0.01）。体外实验结果：与空白对照组相比，LPS组PBMC中GSDMD-N蛋白表达及上清液中炎性介质水平显著升高[GSDMD-N/GAPDH：0.035±0.006比0.028±0.006，IL-1β（ng/L）：39.8±5.5比22.6±4.7，IL-18（ng/L）：31.2±4.4比20.0±2.2，LDH（U/L）：51.2±7.3比36.6±7.6，均P<0.05]，提示LPS刺激可增加PBMC焦亡及炎性介质释放。与LPS组相比，LPS+组蛋白组GSDMD-N蛋白表达及炎性介质水平进一步升高[GSDMD-N/GAPDH：0.114±0.009比0.035±0.006，IL-1β（ng/L）：119.0±18.7比39.8±5.5，IL-18（ng/L）：49.2±8.5比31.2±4.4，LDH（U/L）：127.8±19.8比51.2±7.3，均P<0.01]，提示外源性组蛋白处理可显著放大LPS对PBMC的刺激，上调GSDMD-N蛋白表达，促进炎性因子释放，进一步诱导PBMC焦亡。肝素可消除外源性组蛋白对PBMC的这些不利影响，GSDMD-N蛋白表达及炎性介质水平均显著低于LPS+组蛋白组[GSDMD-N/GAPDH：0.063±0.004比0.114±0.009，IL-1β（ng/L）：46.8±8.6比119.0±18.7，IL-18（ng/L）：33.0±5.1比49.2±8.5，LDH（U/L）：65.4±11.0比127.8±19.8，均P<0.05]。
血浆中的细胞外组蛋白可能通过介导PBMC焦亡加重ARDS。
