祛风宣痹方抑制气道上皮细胞凋亡的研究

Study on Qufeng Xuanbi Formula Inhibiting Apoptosis of Airway Epithelial Cells

目的:探究祛风宣痹方对气道上皮细胞氧化损伤的保护作用。方法:体内研究采用OVA诱导哮喘小鼠模型,体外研究采用不同浓度的H_(2)O_(2)及祛风宣痹方干预人气道上皮细胞系16HBE 48 h。DCFH-DA活性氧探针检测细胞中活性氧(ROS)的生成水平;MTT(噻唑蓝)法检测细胞活力;Western blot法检测凋亡相关蛋白Bcl-2、Bax的表达水平;Annexin V-FITC/PI双染法检测细胞凋亡水平。结果:ROS结果显示,H_(2)O_(2)作用后细胞内ROS生成明显增加,祛风宣痹方提取物可降低ROS水平。MTT结果显示,H_(2)O_(2)作用后对气道上皮细胞的抑制率随浓度增加而增大(P<0.01),祛风宣痹方水提取物和祛风宣痹方醇提取物均可减少H_(2)O_(2)诱导的气道上皮细胞损伤(P<0.01)。Western blot结果提示,H_(2)O_(2)刺激后气道上皮细胞Bcl-2蛋白表达水平显著降低,Bax蛋白表达水平增高(P<0.05,P<0.01),祛风宣痹方提取物作用后可以回调H_(2)O_(2)诱导的Bcl-2蛋白表达水平降低,抑制H_(2)O_(2)诱导的Bax蛋白表达水平增加(P<0.05)。Annexin V-FITC/PI双染法结果显示,H_(2)O_(2)作用后细胞凋亡和坏死水平增高,祛风宣痹方提取物可减少细胞发生凋亡和坏死的比例。结论:祛风宣痹方可以抗氧化,减少哮喘气道上皮细胞凋亡。

Objective:To explore the protective effect of Qufeng Xuanbi Formula on oxidative damage of human airway epithelial cells.Methods:OVA-induced asthma mice model was applied in the in vivo study.16HBE cells were intervened by different concentrations of H_(2)O_(2)and the extract of Qufeng Xuanbi Formula for 48 h in the in vitro study.The DCFH-DA active oxygen probe was used for detecting the generation level of ROS in cells.The effect of H_(2)O_(2)on the cell viability of 16HBE was detected by the method of MTT.Western blot was used to detect the protein expressions of Bcl-2 and Bax.The apoptotic level was detected by Annexin V-FITC/PI double-staining.Results:The intracellular ROS production increased obviously after H_(2)O_(2)intervention,and the extract of Qufeng Xuanbi Formula could reduce the ROS level.The inhibition rate of 16HBE cells increased with the increase of H_(2)O_(2)concentration(P<0.01).Both water extract and alcohol extract of Qufeng Xuanbi Formula could reduce the injury of airway epithelial cells induced by H_(2)O_(2)(P<0.01).The protein expression of Bcl-2 in airway epithelial cells decreased significantly,and the protein expression of Bax increased after H_(2)O_(2)stimulation.The extract of Qufeng Xuanbi Formula could reverse the decrease of Bcl-2 protein expression induced by H_(2)O_(2)and inhibit the increase of Bax protein expression induced by H_(2)O_(2).The Annexin V-FITC/PI double staining showed that the level of apoptosis and necrosis increased after H_(2)O_(2)intervention,and the extract of Qufeng Xuanbi Formula could reduce the degree of apoptosis and necrosis.Conclusion:Qufeng Xuanbi Formula can inhibit the apoptosis of airway epithelial cells induced by oxidative stress.