甘露清瘟方通过调控Bcl-2/Bax/Caspase-3信号通路抑制急性肺损伤大鼠肺组织细胞凋亡的机制研究

Ganlu Qingwen formula inhibits apoptosis in lung tissue of acute lung injury rats by regulating the Bcl-2/Bax/Caspase-3 signaling pathway

摘要:

目的 探讨甘露清瘟方对急性肺损伤（ALI）/急性呼吸窘迫综合征（ARDS）大鼠B淋巴细胞瘤-2（Bcl-2）/凋亡效应蛋白Bax/Caspase-3信号通路的调节作用，以及其调控细胞凋亡的作用机制。方法 48只雄性SD大鼠随机分为6组，甘露清瘟方低、中、高剂量组，空白组，脂多糖模型组，地塞米松组，每组8只。进行ALI大鼠模型复制。模型复制前3 d，对6组大鼠进行灌胃给药，1次/d，连续3 d。空白组给予生理盐水1.0 mL/kg，脂多糖模型组给予脂多糖5 mg/kg，地塞米松组给予地塞米松溶液2 mg/kg，甘露清瘟方低、中、高、剂量组分别给予甘露清瘟方溶液0.60、1.20、2.40 g/kg；第3天给药后，将20 mg/kg 脂多糖溶于100 μL磷酸盐缓冲液（PBS）中，地塞米松组和甘露清瘟方各组大鼠气管内滴注；空白组大鼠气管内滴注等体积生理盐水。苏木精-伊红（HE）染色观察肺组织病理形态学变化；酶联免疫吸附试验（ELISA）检测各组大鼠血清及肺组织的肿瘤坏死因子-α（TNF-α）、干扰素-γ（IFN-γ）、白细胞介素-6（IL-6）水平；Western blotting检测各组大鼠肺组织Bcl-2、Bax、Caspase-3蛋白表达；实时荧光定量聚合酶链反应（qRT-PCR）检测大鼠Bcl-2、Bax、Caspase-3 mRNA表达。结果 脂多糖模型组大鼠肺组织大面积实质化，肺泡结构不清，可见弥漫性炎症细胞浸润。与脂多糖模型组比较，地塞米松组和甘露清瘟方低、中、高剂量组血清及肺组织IL-6、TNF-α、IFN-γ水平降低（P <0.05），Bcl-2蛋白表达及mRNA表达增加（P <0.05），Bax、Caspase-3蛋白表达及mRNA表达降低（P <0.05）。与甘露清瘟方低剂量组比较，甘露清瘟方中剂量组大鼠肺组织Caspase-3蛋白相对表达量下降（P <0.05），甘露清瘟方高剂量组大鼠肺组织Bcl-2蛋白相对表达量升高，Bax、Caspase-3蛋白相对表达量下降（P <0.05）。与甘露清瘟方低剂量组比较，甘露清瘟方中、高剂量组大鼠肺组织Bax、Caspase-3 mRNA相对表达量下降（P <0.05），Bcl-2 mRNA相对表达量上升（P <0.05）；与甘露清瘟方中剂量组比较，甘露清瘟方高剂量组大鼠肺组织Bax mRNA相对表达量下降（P <0.05），Bcl-2 mRNA相对表达量上升（P <0.05）。结论 甘露清瘟方可通过促进Bcl-2表达、抑制Caspase-3、Bax表达减轻肺损伤并降低肺组织细胞凋亡。

Abstract:

Objective To investigate the effect of Ganlu Qingwen Formula (GLQWF) on the Bcl-2/Bax/Caspase-3 signaling pathway and its role in inhibiting apoptosis in a rat model of lipopolysaccharide (LPS)-induced acute lung injury (ALI)/acute respiratory distress syndrome (ARDS).Methods Forty-eight male SD rats were randomized into: Blank (saline), Model (LPS), Dexamethasone (Dex, 2 mg/kg), and GLQWF Low, Medium, High dose groups (0.6, 1.2, 2.4 g/kg). Drugs were administered by gavage for 3 days. On day 3, ALI was induced by intratracheal LPS instillation (Blank: saline). Lung histopathology (H&E), serum and lung tissue cytokines (ELISA: TNF-α, IFN-γ, IL-6), and lung tissue apoptosis-related protein (Western blot) and mRNA (qRT-PCR) expression (Bcl-2, Bax, Caspase-3) were analyzed.Results Model group lungs showed extensive consolidation and inflammation. GLQWF (all doses) and Dex significantly reduced serum and lung TNF-α, IFN-γ, IL-6 levels compared to Model (P < 0.05). GLQWF (Medium/High doses) and Dex significantly increased Bcl-2 protein/mRNA and decreased Bax/Caspase-3 protein/mRNA expression compared to Model (P < 0.05). High-dose GLQWF showed greater effects on protein expression than low-dose.Conclusion Ganlu Qingwen Formula alleviates LPS-induced ALI/ARDS by modulating the Bcl-2/Bax/Caspase-3 pathway, promoting anti-apoptotic Bcl-2 expression, and inhibiting pro-apoptotic Bax and Caspase-3 expression.