氯喹对脂多糖致急性肺损伤大鼠的保护性作用

Protective Mechanism of Chloroquine on Lipopolysaccharide-Induced Acute Lung Injury in Rat

目的探讨氯喹对脂多糖(LPS)所致急性肺损伤(ALI)的保护性作用及机制。方法将日龄40～50 d SD雄性大鼠96只随机分为对照组、模型组、氯喹干预组,每组各分为注射后2、4、8、12 h 4个时间点亚组。静脉注射LPS(6 mg/kg)建立ALI动物模型,氯喹干预组是在注射LPS后立即予以氯喹(30 mg/kg)腹腔注射,对照组予以静脉注射9 g/L盐水,3组注射的液体总量相等。在各观察时间点处死。测定肺湿/干质量比值(W/D),石蜡包埋切片,HE染色行肺组织病理评分,末端脱氧核苷酰基转移酶介导性dUTP切口末端标记(TUNEL)法检测肺泡巨噬细胞(AM)凋亡率,免疫组织化学法检测核因子-κBp65(NF-κBp65)表达,酶联免疫吸附法(ELISA)检测血浆IL-6水平,中性红法检测AM吞噬功能,半定量反转录-PCR(RT-PCR)法检测AM的分泌型磷脂酶A2ⅡA型(sPLA2ⅡA)、Bcl-2、Bax基因的表达。采用SPSS12.0统计软件进行分析。结果与对照组比较,模型组肺W/D、肺组织病理评分、AM的NF-κBp65表达及凋亡、AM的sPLA2ⅡA基因、Bax基因表达均显著增加(Pa<0.01),AM吞噬功能、Bcl-2基因的表达均显著下降(Pa<0.01)。与对照组比较,模型组血浆IL-6水平在注射LPS后明显升高(P<0.01)。与模型组比较,氯喹干预组上述改变得以逆转,肺损伤程度明显减轻(Pa<0.01)。结论ALI早期使用氯喹可下调AM表达sPLA2ⅡA和Bax基因,上调AM表达Bcl-2基因,减少AM凋亡,抑制NF-κB活化,增强AM吞噬功能,对LPS所致ALI有一定的保护作用。

Objective To explore the protective effects and mechanisms of chloroquine on lipopolysacchaIide （ LPS） - induced acute lung injury （ALI） in rat. Methods Ninety - six 40 - 50 days age SD male rats were randomly assigned to 3 groups ： control group , model group and chloroquine treatment group. All rats in each group were divided into injected 2,4,8,12 hours subgroups. ALl model was induced by intravenous injection of LPS （6 mg/kg） , chloroquine treatment group was immediately administered ehloroquine （30 mg/kg） by intraperitoneal injection after they had been injected LPS, control group was intravenous injected of physiological saline, the injected fluid volume of 3 groups was equal. These animal were killed at the each observation time point. The lung wet/dry weight ratio（W/D） ,lung pathologic tissue score were detected with hematoxylin and eosin （HE） stain ,alveolar maerophages （AM） apoptosis ratio was determined with terminal deoxynueleotidyl transferase - mediated dUTP nick end labeling （TUNEL） method, nuclear factor - kBp65 （ NF - kBp65 ） expression was measured with immunohistochemistry technique, plasma level of IL - 6 was determined with enzyme /inked immunosorbent assay （ELISA） , AM phagotrophic function with neutral red, and secretory phospholipase A2 type ⅡA （ sPLA2 ⅡA）, Bcl - 2, Bax genes expression were measured with reverse transcription polymerase chain reation （RT- PCR） technique. Statistics analysis was carried out by SPSS 12.0 statistics software. Results Compared with control group, the lung W/D,lung pathologic tissue score, AM apoptosis ratio, AM NF - kBp65 expression, sPLA2 ⅡA and Bax genes expression were significantly increased and AM phagutrophic function, Bel - 2 genes expression were significantly decreased in model group rats （Pa 〈 0.01 ）. Compared with control group, plasma level of IL -6 obviously heightened after injected LPS （P 〈 0.01 ）. These changes were inverted in chloroquine treatment group rats, the degree of lung injury was markedly relieved （Pa〈 0.01 ）. Conclusions In early ALI, chloroquine potentially down regulation expression of sPLA2 ⅡA and Bax gene, up - regulation Bcl - 2 genes expression of AM ,inhibit activation of NF -kB in AM ,enhance AM phagotrophie function, decrease AM apoptosis, which has protective function in LPS- induced ALI rats.