HMGB1对脂多糖诱导的急性肺损伤中中性粒细胞凋亡的影响。
Effects of HMGB1 on PMN apoptosis during LPS-induced acute lung injury.
作者信息
Feng Yingkai, Yang Qinghua, Xu Jiancheng, Qian Guisheng, Liu Yousheng
机构信息
Institute of Pathology, Southwest Hospital Affiliated to Third Military Medical University, Chongqing 400038, China.
出版信息
Exp Mol Pathol. 2008 Dec;85(3):214-22. doi: 10.1016/j.yexmp.2008.09.002. Epub 2008 Oct 8.
OBJECTIVE
To observe the effects of intravenous injection of HMGB1 inhibitor sodium butyrate on changes in apoptosis of PMN during LPS-induced acute lung injury in rats and HMGB1 in vitro on human circulating PMN apoptosis, in order to clarify the role of HMGB1 in the pathogenesis of acute lung injury.
METHODS
(1) LPS-induced acute lung injury rat model was developed by LPS infusion. At different time-points after LPS challenge in the presence or absence of sodium butyrate (SB), the rat tissue sample, peripheral blood PMNs and BALF were collected. RT-PCR was applied to examining rat lung tissue HMGB1 mRNA expression level, and Western blotting analysis was adopted to determine expression of rat lung tissue HMGB1 protein. PMN apoptotic changes were determined by flow cytometric (FCM) analysis, Giemsa staining and TdT-mediated dUTP nick end labeling (TUNEL) method. (2) Separated and purified human circulating PMN were coincubated for 24 h with different doses of HMGB1 (0, 10, 100, 1000 ng/ml, respectively) at 37 degrees C in 5% CO(2). PMN apoptosis rate was determined by flow cytometric (FCM) analysis and by TdT-mediated dUTP nick end labeling (TUNEL) method.
RESULTS
(1) The percentage of apoptosis of PMN in rat model of LPS-induced ALI was gradually decreased as compared with that of normal control. The PMN apoptosis-initiation time and non-survival time in rat BALF prolonged significantly as compared with that of normal control. The injured rat lung tissue HMGB1 mRNA and protein expression was upregulated 6-24 h after LPS exposure; SB intervention significantly ameliorated the upregulation. In addition, the morphologic examination indicated that the edema severity and pathological changes of lung tissues were excessively aggravated in rats after LPS administration. By comparison, SB treatment diminished the severity of lung damage. Combined with lung HMGB1 expression level, the above changes indicate that the pathological changes of lung tissue were related to the injured lung HMGB1 expression, as well as apoptotic changes in PMN. (2) After coincubation of HMGB1 with human circulating PMNs, TUNEL and flow cytometry were performed. The study revealed that PMN apoptosis ratios was (40.53+/-4.12) % in control group (PMNs+RPMI 1640 medium), (19.05+/-2.44) % in LPS group (PMNs+RPMI 1640 medium+10 microg/ml LPS), (40.52+/-2.73) % in HMGB1-1 group (PMNs+RPMI 1640 medium+10 ng/ml HMGB1), (34.89+/-1.15) % in HMGB1-2 group (PMNs+RPMI 1640 medium+100 ng/ml HMGB1), and (18.77+/-3.02) % in HMGB1-3 group (PMNs+RPMI 1640 medium+1 000 ng/ml HMGB1). There was statistical significance. Meanwhile, PMN TUNEL positive rate was (31.42+/-4.40) %, (31.39+/-3.80) %, (25.62+/-2.46) %, and (17.98+/-3.20) % in control group, HMGB1-1 group, HMGB1-2 group and HMGB1-3 group, respectively. The inhibitory effect was HMGB1 dose-depended as compared with that of control group.
CONCLUSION
After LPS challenge, high expression of rats' lung HMGB1 mRNA occurs at a later phase, but keeps for a long time. Sodium butyrate (SB) treatment attenuated LPS-induced PMN apoptosis delay and inhibition, and down-regulated HMGB1 mRNA expression of injured lung. HMGB1 in vitro inhibited human circulating PMN apoptosis markedly, and the inhibitory effect was HMGB1 dose-depended. The results demonstrated that HMGB1 may play an important role as a modulator in apoptotic changes in PMN during LPS-induced ALI. It concludes that HMGB1 may contribute to the development of PMN apoptotic changes during LPS-induced acute lung injury.
目的
观察静脉注射HMGB1抑制剂丁酸钠对脂多糖（LPS）诱导的大鼠急性肺损伤过程中中性粒细胞（PMN）凋亡变化的影响以及HMGB1体外对人循环PMN凋亡的影响，以阐明HMGB1在急性肺损伤发病机制中的作用。
方法
（1）通过注入LPS建立LPS诱导的急性肺损伤大鼠模型。在LPS攻击后不同时间点，在有或无丁酸钠（SB）的情况下，收集大鼠组织样本、外周血PMN和支气管肺泡灌洗液（BALF）。采用逆转录聚合酶链反应（RT-PCR）检测大鼠肺组织HMGB1 mRNA表达水平，采用蛋白质免疫印迹分析确定大鼠肺组织HMGB1蛋白表达。通过流式细胞术（FCM）分析、吉姆萨染色和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）法测定PMN凋亡变化。（2）将分离纯化的人循环PMN在37℃、5%二氧化碳条件下与不同剂量的HMGB1（分别为0、10、100、1000 ng/ml）共孵育24 h。通过流式细胞术（FCM）分析和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）法测定PMN凋亡率。
结果
（1）与正常对照组相比，LPS诱导的ALI大鼠模型中PMN凋亡百分比逐渐降低。大鼠BALF中PMN凋亡起始时间和非存活时间与正常对照组相比显著延长。LPS暴露后6 - 24 h，损伤的大鼠肺组织HMGB1 mRNA和蛋白表达上调；SB干预显著改善了这种上调。此外，形态学检查表明，LPS给药后大鼠肺组织水肿严重程度和病理变化过度加重。相比之下，SB治疗减轻了肺损伤的严重程度。结合肺HMGB1表达水平，上述变化表明肺组织的病理变化与损伤的肺HMGB1表达以及PMN凋亡变化有关。（2）HMGB1与人循环PMN共孵育后，进行TUNEL和流式细胞术检测。研究显示，对照组（PMN + RPMI 1640培养基）中PMN凋亡率为（40.53±4.12）%，LPS组（PMN + RPMI 1640培养基 + 10 μg/ml LPS）中为（19.05±2.44）%，HMGB1 - 1组（PMN + RPMI 1640培养基 + 10 ng/ml HMGB1）中为（40.52±2.73）%，HMGB1 - 2组（PMN + RPMI 1640培养基 + 100 ng/ml HMGB1）中为（34.89±1.15）%，HMGB1 - 3组（PMN + RPMI 1640培养基 + 1000 ng/ml HMGB1）中为（18.77±3.02）%。差异具有统计学意义。同时，对照组、HMGB1 - 1组、HMGB1 - 2组和HMGB1 - 3组中PMN TUNEL阳性率分别为（31.42±4.40）%、（31.39±3.80）%、（25.62±2.46）%和（17.98±3.20）%。与对照组相比，抑制作用呈HMGB1剂量依赖性。
结论
LPS攻击后，大鼠肺HMGB1 mRNA后期高表达，但持续时间长管。丁酸钠（SB）治疗减轻了LPS诱导的PMN凋亡延迟和抑制，并下调了损伤肺组织的HMGB1 mRNA表达。HMGB1体外显著抑制人循环PMN凋亡，且抑制作用呈HMGB1剂量依赖性。结果表明，HMGB1可能作为一种调节因子在LPS诱导的ALI过程中PMN凋亡变化中起重要作用。结论是，HMGB1可能在LPS诱导的急性肺损伤过程中PMN凋亡变化的发生发展中起作用。
Zotero 插件