GLP-1受体在脂多糖诱导的大鼠急性肺损伤中的作用及其机制

Role of glucagon-like peptide-1 receptor in lipopolysaccharide-induced acute lung injury in rats and its mechanism

摘要:

目的 研究glucagon like peptide-1(GLP-1)受体在急性肺损伤(ALI)中的作用及相关机制。方法 通过气管内注射脂多糖(LPS)法建立ALI模型。采用随机数表法将30只大鼠随机分为5组,每组6只。对照组大鼠建立假模型后接受生理盐水(NS)处理；空白药物组大鼠建立假模型后接受GLP-1受体激动剂利拉鲁肽(2mg/kg)处理；模型组大鼠建立ALI模型后接受NS处理；治疗组大鼠建立ALI模型后接受利拉鲁肽(2mg/kg)处理；抑制剂组大鼠建立ALI模型后接受利拉鲁肽(2mg/kg)及自噬抑制剂3-methyladenine(3MA,10mg/kg)处理。蛋白免疫印迹实验法检测各组大鼠肺中LC3II、Beclin-1、P62、Bax、Bcl-2蛋白的表达。采用TUNEL染色法检测细胞凋亡。应用SPSS 20.0软件进行数据处理。组间比较采用单因素方差分析和LSD多重比较。结果 模型组LC3II、Beclin-1及P62表达分别为100.0%±5.3%、100.0%±5.6%、100.0%±6.1%,治疗组中LC3II及Beclin-1表达分别显著增加至214.0%±12.3%及329.0%±33.8%,P62表达显著下降至50.0%±7.0%(均P<0.01)。对照组肺损伤分数、湿/干重比(W/D)、Bax、Bcl-2及凋亡细胞分别为0.3±0.5、4.08±0.14、100.0%±7.0%、100.0%±3.6%、1.0±0.9,模型组肺损伤分数、湿/干重比(W/D)、Bax及凋亡细胞分别显著增加为4.6±0.8、4.83±0.23、284.0%±25.6%及42.5±8.2,Bcl-2显著下降至63.0%±6.2%(均P<0.001)；与模型组比较,治疗组肺损伤分数、W/D、Bax、及凋亡细胞分别显著下降至2.5±1.0、4.42±0.15、198.0%±24.8%及17.0±3.4,Bcl-2显著增加至83.0%±5.3%(均P<0.01)；与治疗组比较,抑制剂组肺损伤分数、W/D、Bax及凋亡细胞分别显著增加至4.3±0.8、4.77±0.25、277.0%±25.5%及39.5±6.2,Bcl-2下降至54.0%±5.0%(均P<0.01)。结论 激活GLP-1受体可以显著改善脂多糖介导的急性肺损伤,其机制与其通过上调细胞自噬、进而减轻细胞凋亡有关。

Abstract:

Objective To investigate the role of glucagon-like peptide-1 (GLP-1) receptor in the lipopolysaccharide(LPS)-induced acute lung injury (ALI) and its mechanism. Methods ALI in rats was induced by intratracheal administration of LPS. A total of 30 rats were randomly divided into 5 groups, with 6 in each:the control group, sham model treated with normal saline (NS); the drug-blank group, sham model treated with liraglutide (2mg/kg); the model group, ALI model treated with NS; the treatment group, ALI model treated with liraglutide (2mg/kg); and the inhibitor group, ALI model treated with liraglutide (2mg/kg) and 3-methyladenine (10mg/kg). Expression of LC3II, Beclin-1, P62, Bax and Bcl-2 were detected by Western blotting. Cell apoptosis was determined by the terminal deoxyribonucleotidyl transferase-mediated deoxyuridine 5-Triphosphate-Digoxigenin nick end labeling (TUNEL) assay. SPSS statistics 20.0 was used for data processing, and one-way analysis of variance and LSD multiple comparisons were used for comparison between groups. Results The expression of LC3II, Beclin-1 and P62 in the model group were 100.0%±5.3%, 100.0%±5.6%, 100.0%±6.1%, respectively. The expression of LC3II and Beclin-1 in treatment group increased respectively to 214.0%±12.3% and 329.0%±33.8%, and that of P62 decreased to 50.0%±7.0% (P<0.01). The control group had a lung injury score of 0.3±0.5, a wet/dry weight ratio (W/D) of 4.08±0.14, a Bax of 100.0%±7.0%, a Bcl-2 of 100.0%±3.6%, and apoptotic cells of 1.0±0.9. In the model group, the lung injury score, wet/dry weight (W/D), expression of Bax and apoptotic cells increased respectively to 4.6±0.8,4.83±0.23,284.0%±25.6% and 42.5±8.2, and expression of Bcl-2 decreased to 63.0%±6.2% (all P<0.01). Compared with the model group, the lung injury score, W/D, expression of Bax and apoptotic cells in treatment group decreased respectively to 2.5±1.0,4.42±0.15,198.0%±24.8% and 17.0±3.4, and the expression of Bcl-2 in treatment group increased to 83.0%±5.3% (all P<0.01). Compared with the treatment group, the lung injury score, W/D, expression of Bax and apoptotic cells in inhibitor group increased respectively to 4.3±0.8, 4.77±0.25,277.0%±25.5% and 39.5±6.2, and expression of Bcl-2 in inhibitor group decreased to 54.0%±5.0% (all P<0.01). Conclusion Activation of GLP-1 receptor significantly alleviates LPS-induced ALI, and the mechanism is associated with the up-regulation of autophagy that prevents cell apoptosis.