高迁移率族蛋白1参与脓毒症大鼠急性呼吸窘迫综合征时中性粒细胞的凋亡

High mobility group box 1 participate in polymorphonuclear neutrophil apoptosis delay of lipopolysaccharide-induced acute respiratory distress syndrome in rats

目的探究脓毒症大鼠急性呼吸窘迫综合征（acute respiratory distress syndrome，ARDS）时高迁移率族蛋白1（high mobility group box1，HMGB1）参与中性粒细胞（polymorphonuclear neutrophils，PMN）凋亡延迟的相关机制及正丁酸钠（sodium butyrate，SB）的干预效果。方法采用随机数字表法将90只健康成年雄性Sprague-Dawley（SD）大鼠随机分为正常对照组（NS组，6只）、内毒素（lipopolysaccharide，LPS）致伤组（LPS组，42只）、HMGBI抑制剂SB干预组（SB组，42只），LPS组、SB组又分为7个时相点（LPS致伤后0．5、1、2、6、12、24、48h），每个时相点6只动物。LPS组，腹腔注射LPS5mg／kg；SB组，腹腔注射LPS5mg／kg后0．5h静脉注射sB，每次剂量500mg／kg；NS组，腹腔注射生理盐水1ml。LPS组、SB组于LPS注射后各时点，颈静脉取血检查PMN，取血后左肺灌洗收集支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF），取右肺中叶行HMGB1 mRNA检测；于LPS注射12h时点（NS组大鼠留取颈静脉血、收集BALF、取右肺中叶行HMGB1 mRNA检测），3组大鼠收集左肺BALF后，取右肺下叶检测肺组织湿，干重比（wet／dryweight ratio，W／D），取右肺上叶进行病理组织学检查；于24、48h时点，LPS组、SB组取右肺下叶检测肺组织W／D、取右肺上叶进行病理组织学检查。结果与NS组比较，LPS组肺组织中HMGB1 mRNA表达量明显增高，24h达最大值，差异有统计学意义（P〈0．05）；SB组的HMGB1 mRNA表达量减少，差异有统计学意义（P〈0．05）。LPS组BALF中凋亡早期PMN表达量与NS组类似，但凋亡晚期PMN变化则不同，LPS组外周血中凋亡晚期PMN细胞高于NS组（P〈0．05），LPS组BALF中凋亡晚期PMN细胞均低于NS组（P〈0．05）.LPS12h组BALF的PMN百分比明显高于NS组[（49．1±6．8）、（2．7±0．5）]，差异有统计学意义（P〈0．01）。LPS组肺组织光镜下可见肺泡腔水肿，支气管壁中发现PMN浸润，肺泡壁毛细血管扩充血。LPS24h组肺组织的W／D明显高于NS组[（6．71±0．12）、（4．18±0．26）]，差异有统计学意义（P〈0．05）.SB干预组的HMGB1 mRNA表达量减少，早期凋亡率类似，SB组的BALF晚期凋亡率高于同时点LPS组的BALF，SB外周血组晚期凋亡低于同时点LPS外周血组，差异有统计学意义（P〈0．05）。SB组BALF的PMN百分比、肺组织病理损伤程度、W／D均低于LPS组，差异有统计学意义（P〈0．05）.结论在大鼠ARDS中，HMGB1 mRNA表达较晚，但持续时间长，SB对HMGB1有潜在的治疗作用，HMGB1可能参与PMN凋亡机制的调节。

Objective To investigate the effects of high mobility group box 1 （HMGB1） on apoptosis of polymorphonuclear neutrophil（PMN） in LPS-induced acute respiratory distress syndrome and observe the effect of sodium butyrate（SB） treatment on septic rats. Methods Ninety adult male Sprague-Dawley （SD） rats were randomly assigned to one of three groups： normal saline control group（ NS group） （n=6）, lipopolysaccharides（ LPS ） group（LPS group） （n=42）, sodium butyrate group（SB group ） （n=42）. At different time points （0.5, 1, 2, 6, 12, 24, 48 h）, rats assigned to LPS group received 5 mg/kg LPS by i.p. injection. Rats in SB groups were injected 5 mg/kg LPS and 500 mg/kg sodium butyrate after half an hour by i.p. injection. Rats in NS group received 1 ml normal saline by i.p. injection. PMNs in peripheral blood and the bronchoalveolar lavage fluid （BALF） of the left lung were collected at different time-points. The middle of the right lung was taken out to detect the expression of HMGB1 mRNA in lung tissues. At 12 h after LPS injected（peripheral blood and BALF was collected and the expression of HMGB1 mRNA was detected in NS group）, all the three groups taken the left lung to collect BALF, the inferior lobe of right lung to determinate the wet/dry weight ratio （W/D）, the superior lobe of right lung to detect the histopathologic changes of lung. At 24, 48 h after LPS injected, both LPS and SB group detected the W/D and the histopathologic changes as the same way. Results With the administration of LPS, the expression of HMGBI mRNA were significantly higher than that in NS group （P〈0.05）. The early-phase apoptosis rate are similar, the late-phase apoptosis of neutrophil in bronchoalveolar lavage fluid of LPS was decreased but in peripheral blood was increased（P〈0.05 ） in LPS group compared with NS group. The neutrophil percentage of LPS 12 h group was higher than that in NS group[ （49.1~6.8）,（2.7~0.5） ] （P〈0.01）. Pathological examination showed that the normal structure of lung was destroyed badly after LPS injection, including infra-alveolar hemorrhage,interstitial edema, alveolar collapse and massive inflammatory cells infiltration. The lung water content of LPS 12 h group was significantly increased [（6.71±0.12）, （4.18±0.26）] （P〈0.05）. However, sodium butyrate treatment can significantly reduce the expression of I-IMGBl mRNA, and all the above indicators in SB group significantly lower than LPS group （P〈O.05）. Conclusions HMGB1 mRNA expression in acute respiratory distress syndrome is late, but a long duration, SB has the potential therapeutic effect on HMGB1. HMGB1 may participate in the regulation of neutrophil mechanism.