Sun Meng, Jiang Xuemei, Jin Yang, Yang Hua, Chen Chang, Lyu Xin, Wen Zongmei
Nanchang University, Nanchang 330006, Jiangxi, China.
Department of Anesthesiology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200433, China.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2020 Feb;32(2):194-198. doi: 10.3760/cma.j.cn121430-20191113-00036.
To explore the role and mechanism of extracellular histones involved in lipopolysaccharide (LPS)-induced alveolar macrophage injury.
The mouse alveolar macrophage cell line (MH-S) was cultured in vitro and passaged, and the cells were cultured to 80% of cells for cell proliferation. The cells were stimulated with 1 mg/L LPS for 3 hours and 50 mg/L exogenous histones for 3, 6, 12, and 24 hours, respectively (LPS+histones 3, 6, 12, 24 h groups), and other groups included phosphate buffered saline (PBS) control group (PBS group), LPS alone stimulation group (LPS group), the exogenous histones alone stimulation group (histones group) and heparin pretreatment histones group (heparin+LPS+histones group). The cells in each group were challenged with different reagent, the expression of lactate dehydrogenase (LDH) and inflammatory factors in the supernatant were detected by enzyme linked immunosorbent assay (ELISA), and the change of intracellular K concentration was detected by FluxOR II green potassium channel. The proteins such as potassium channel protein (TWIK2), inflammasome (NLRP3), and apoptosis associated speck like protein containing a CARD (ASC) were determined by Western Blot.
Compared with the PBS group, the levels of LDH and inflammatory factors such as interleukin (IL-1β, IL-18) and tumor necrosis factor-α (TNF-α) were significantly increased after LPS stimulation group. Compared with the LPS group, the levels of LDH and inflammatory factors were significantly increased after the treatment with exogenous histones, and reached a peak after 3 hours of the histones stimulation [LDH (U/L): 123.10±1.83 vs. 85.32±1.66, IL-1β (mg/L): 40.75±2.60 vs. 18.78±1.37, IL-18 (mg/L): 49.94±2.45 vs. 30.19±1.82, TNF-α (mg/L): 36.51±1.56 vs. 20.84±1.61, all P < 0.01]. Western Blot results showed that compared with the LPS group, NLRP3, ASC and TWIK2 protein expression were significantly up-regulated in the LPS+histones group (NLRP3/GAPDH: 0.80±0.02 vs. 0.57±0.02, ASC/GAPDH: 0.57±0.02 vs. 0.38±0.01, TWIK2/GAPDH: 0.65±0.01 vs. 0.41±0.01, all P < 0.01), and the expression of the above proteins were significantly down-regulated after heparin pretreatment (NLRP3/GAPDH: 0.28±0.02 vs. 0.80±0.02, ASC/GAPDH: 0.25±0.02 vs. 0.57±0.02, TWIK2/GAPDH: 0.35±0.01 vs. 0.65±0.01, all P < 0.01), indicating that histones could activate NLRP3 through TWIK2 to participate in inflammatory reaction. In addition, intracellular K concentration in LPS+histones group decreased significantly compared with the LPS group (fluorescence intensity: 35.48±2.53 vs. 83.92±3.11, P < 0.01). Compared with LPS+histones group, K concentration increased significantly after pretreatment with heparin (fluorescence intensity: 72.10±1.78 vs. 35.48±2.53, P < 0.01), indicating that extracellular histones could cause K massive efflux through TWIK2, and thus mediate NLRP3 activation and participate in inflammatory injury of alveolar macrophages.
Extracellular histones can cause inflammatory damage in alveolar macrophages, and its mechanism may be related to the activation of NLRP3 by extracellular histones activation of TWIK2 channel to promote K efflux.
探讨细胞外组蛋白在脂多糖（LPS）诱导的肺泡巨噬细胞损伤中的作用及机制。
体外培养并传代小鼠肺泡巨噬细胞系（MH-S），培养至细胞融合度达80%用于细胞增殖实验。分别用1 mg/L LPS刺激细胞3小时，50 mg/L外源性组蛋白刺激细胞3、6、12和24小时（LPS + 组蛋白3、6、12、24小时组），其他组包括磷酸盐缓冲液（PBS）对照组（PBS组）、单独LPS刺激组（LPS组）、单独外源性组蛋白刺激组（组蛋白组）和肝素预处理组蛋白组（肝素 + LPS + 组蛋白组）。对每组细胞给予不同试剂处理，采用酶联免疫吸附测定（ELISA）法检测上清液中乳酸脱氢酶（LDH）和炎性因子的表达，通过FluxOR II绿色钾通道检测细胞内K⁺浓度变化。采用蛋白质免疫印迹法检测钾通道蛋白（TWIK2）、炎性小体（NLRP3）和含半胱天冬酶招募结构域的凋亡相关斑点样蛋白（ASC）等蛋白。
与PBS组相比，LPS刺激组细胞上清液中LDH及白细胞介素（IL - 1β、IL - 18）和肿瘤坏死因子 - α（TNF - α）等炎性因子水平显著升高。与LPS组相比，外源性组蛋白处理后LDH及炎性因子水平显著升高，且在组蛋白刺激3小时后达到峰值[LDH（U/L）：123.10±1.83 vs. 85.32±1.66，IL - 1β（mg/L）：40.75±2.60 vs. 18.78±1.37，IL - 18（mg/L）：49.94±2.45 vs. 30.19±1.82，TNF - α（mg/L）：36.51±1.56 vs. 20.84±1.61，均P < 0.01]。蛋白质免疫印迹结果显示，与LPS组相比，LPS + 组蛋白组中NLRP3、ASC和TWIK2蛋白表达显著上调（NLRP3/GAPDH：0.80±0.02 vs. 0.57±0.02，ASC/GAPDH：0.57±0.02 vs. 0.38±0.01，TWIK2/GAPDH：0.65±0.01 vs. 0.41±0.01，均P < 0.01），肝素预处理后上述蛋白表达显著下调（NLRP3/GAPDH：0.28±0.02 vs. 0.80±0.02，ASC/GAPDH：0.25±0.02 vs. 0.57±0.02，TWIK2/GAPDH：0.35±0.01 vs. 0.65±0.01，均P < 0.01），表明组蛋白可通过TWIK2激活NLRP3参与炎症反应。此外，LPS + 组蛋白组细胞内K⁺浓度较LPS组显著降低（荧光强度：35.48±2.53 vs. 83.92±3.11，P < 0.01）。与LPS + 组蛋白组相比，肝素预处理后K⁺浓度显著升高（荧光强度：72.10±1.78 vs. 35.48±2.53，P < 0.01），表明细胞外组蛋白可通过TWIK2导致K⁺大量外流，进而介导NLRP3激活并参与肺泡巨噬细胞的炎性损伤。
细胞外组蛋白可引起肺泡巨噬细胞炎性损伤，其机制可能与细胞外组蛋白激活TWIK2通道促进K⁺外流从而激活NLRP3有关。
