青蒿琥酯上调血红素氧合酶-1对脓毒症小鼠急性肺损伤的作用研究

Protective effects of artesunate on septic lung injury in mice via modulation of heme oxygenase-1

目的 探讨青蒿琥酯（artesunate,AS）能否上调血红素氧合酶-1（heme oxygenase-1,HO-1）对脓毒症小鼠急性肺损伤（ALI）起到保护作用.方法 本实验于哈尔滨医科大学附属第一医院外科中心实验室完成.60只雄性昆明小白鼠,随机（随机数字法）分为假手术组（Sham组,n=15）、模型组（CLP组,n=15）、青蒿琥酯干预组（AS＋CLP组,n=15）和HO-1抑制剂组（AS＋ ZnPP＋CLP组,n=15）.采用改良盲肠结扎穿孔（CLP）法制备脓毒症模型,青蒿琥酯干预组,造模前2h经腹腔注射青蒿琥酯（15 mg/kg）,HO-1抑制剂ZnPP（40μmol/kg）则在给予青蒿琥酯1h后经腹腔注射,其余各组小鼠给予等体积生理盐水.于术后24h处死小鼠,采用酶联免疫吸附法（ELISA）检测血清炎症介质（TNF-α、IL-6）变化情况.通过测定肺脏湿/干（W/D）质量比检测肺水肿情况,采用HE染色观察肺组织病理损伤情况.采用蛋白印迹以及免疫组化法检测肺组织HO-1的表达情况,并且通过蛋白印迹法检测HO-1的上游调节转录因子核因子E2相关因子2（Nrf-2）的表达情况.组间多重比较采用单因素方差分析,两两比较采用SNK-q（Student-Newman-Keuls）法,以P＜0.05为差异具有统计学意义.结果 与Sham组比较,CLP组血清炎症介质TNF-α （pg/mL）（54.37±15.59 vs.627.45±117.03,P＜0.05）、IL-6 （pg/mL） （81.53±26.89vs.898.52±222.78,P＜0.05）释放增加,蛋白渗出明显,肺水肿（肺脏W/D比值：4.27±0.22vs.6.78±0.73,P＜0.05）和肺脏病理损伤（肺损伤评分：2.20±0.2 vs.13.25±2.67,P＜0.05）均加重.肺组织中HO-1、Nrf-2蛋白表达含量增加（P＜0.05）.给予AS干预后,与CLP组比较,血清炎症介质TNF-α（pg/mL）（627.45±117.03 vs.307.88±72.33,P＜0.05）、IL-6 （pg/mL）（898.52±222.78 vs.413.47±115.14,P＜0.05）释放减少,蛋白渗出减少,肺水肿减轻（肺脏W/D比值：6.78±0.73vs.5.05±0.61,P＜0.05）,肺脏病理损伤减轻（肺损伤评分：13.25±2.67vs.4.95±1.46,P＜0.05）.肺组织HO-1、Nrf-2蛋白表达含量明显增加（P＜0.05）,而AS对于脓毒症小鼠肺组织的上述保护作用被HO-1抑制剂ZnPP所逆转,HO-1抑制剂组肺脏损伤情况与CLP组相当（肺损伤评分：12.15±2.95vs.13.25±2.67,P＞0.05;肺W/D比值：6.78±0.73 vs.6.29±0.82,P＞0.05）.结论 AS对脓毒症诱发的ALI小鼠具有保护作用,其作用通路可能与上调HO-1减轻肺组织炎症反应,继而减轻肺损伤有关.

Objective To investigate the effects of artesunate （AS） on septic lung injury in mice and to study the modulation of heme oxygenase-1 （HO-1） in lung in order to clarify the mechanism of AS action.Methods Sixty male Kunming mice were randomly （random number） divided into four groups： Sham group （n =15）, CLP group （n =15）, AS ＋ CLP group （n =15） and AS ＋ ZnPP ＋ CLP group （n =15）.Cecal ligation and puncture （CLP） method was employed to induce septic lung injury.AS （15 mg/ kg） was injected into the abdomen of mice 2 hours before the CLP procedures, and ZnPP Ⅸ, an inhibitor of HO-1, was intraperitoneally injected in dose of 40 μmol/kg 1 hour after the AS injection.The equivalent volume of normal saline was intraperitoneally injected instead in mice of Sham group and CLP group.The mice were sacrificed 24 hours after the CLP procedures.The TNF-α, IL-6 in serum were assayed by ELISA method.The lung injury score and wet/dry ratio were measured.The western blotting and immunohistochemistry methods were used to determine HO-1 protein expression in lung tissue.The protein level of nuclear factor-E2-related factor-2 （Nrf-2）, an important transcriptional factor of HO-1 in lung tissue was also analyzed by western blotting.One-way analysis of variance （ANOVA） was used for comparisons among the groups, and SNK-q （Student-Newman-Keuls） test was performed for further comparison, and difference was statistically significant at P 〈 0.05.Results The TNF-α （pg/mL） （54.37 ± 15.59 vs.627.45 ± 117.03, P 〈 0.05）, IL-6 （pg/mL） （81.53 ± 26.89 vs.898.52 ± 222.78, P 〈 0.05） in serum was increased, and the lung protein exudation, pulmonary edema （wet/dry weight ratio： 4.27 ± 0.22 vs.6.78 ±0.73, P 〈 0.05）, pulmonary pathology injury （lung injury score： 2.20 ± 0.2 vs.13.25 ± 2.67, P 〈 0.05） were aggravated by CLP.The HO-1 and Nrf-2 were up-regulated in lung tissue in CLP group compared with the sham group （P 〈 0.05）.After the intervention of AS, the HO-1 and Nrf-2 were further increased （P〈0.05）, theTNF-α （pg/mL） （627.45 ±117.03 vs.307.88 ±72.33, P〈0.05）, IL-6 （pg/mL） （898.52 ± 222.78 vs.413.47 ± 115.14, P 〈 0.05） in serum, lung protein exudation, pulmonary edema （wet/dry weight ratio： 6.78 ± 0.73 vs.5.05 ± 0.61, P 〈 0.05）, pulmonary pathology injury （lung injury score： 13.25 ± 2.67 vs.4.95 ± 1.46, P 〈 0.05） were attenuated compared with the CLP group.However, the protective role of AS in the septic lung injury in mice was partly reversed by ZnPP, and no significant difference was detected between the AS ＋ CLP ＋ ZnPP and CLP group （lung injury score： 12.15 ± 2.95 vs.13.25 ± 2.67, P 〉 0.05;wet/dry weight ratio： 6.78 ± 0.73 vs.6.29 ± 0.82, P 〉 0.05）.Conclusions AS plays protective roles in septic lung injury, and it is attributed to limiting lung inflammation via up-regulation of HO-1.