鹰嘴豆芽素A对LPS诱导的肺泡上皮细胞氧化应激损伤的影响

Effects of Biochanin A on oxidative stress injury of alveolar epithelial cells induced by LPS

目的 探讨鹰嘴豆芽素A(BCA)调节核因子E2相关因子2(Nrf2)/血红素加氧酶1(HO-1)信号通路对脂多糖(LPS)诱导的肺泡上皮细胞氧化应激损伤的影响。方法 将肺泡上皮细胞BEAS-2B分为ctrl组(正常培养的BEAS-2B细胞)、LPS组(10μg/mL浓度的LPS)、BCA低剂量组(5μmol/L BCA)、BCA中剂量组(10μmol/L BCA)、BCA高剂量组(20μmol/L BCA)、抑制剂组(20μmol/L BCA+5μmol/L Nrf2抑制剂ML385),除ctrl组外,其余组均给予10μg/mL LPS刺激24 h。采用CCK-8法检测细胞增殖,流式细胞仪检测细胞凋亡,DCFH-DA荧光探针法检测细胞内活性氧(ROS)水平,分光光度法检测细胞上清液中超氧化物歧化酶(SOD)活性、丙二醛(MDA)水平,试剂盒检测肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6水平,免疫印迹法(Western blotting)检测细胞Nrf2、HO-1、增殖细胞核抗原(PCNA)、Bcl-2相关X蛋白(Bax)、激活型半胱氨酸蛋白酶-3(cleaved caspase-3)/半胱氨酸蛋白酶3(caspase-3)蛋白表达。结果 与ctrl组比较,LPS组BEAS-2B细胞的吸光度(A_(450))、SOD活性、Nrf2、HO-1、PCNA表达明显降低(P<0.05),细胞凋亡率、ROS和MDA水平、TNF-α、IL-6、Bax、cleaved caspase-3/caspase-3表达明显上升(P<0.05);与LPS组比较,BCA低、中、高剂量组BEAS-2B细胞的A_(450)、SOD活性、Nrf2、HO-1、PCNA表达显著升高(P<0.05),细胞凋亡率、ROS和MDA水平、TNF-α、IL-6、Bax、cleaved caspase-3/caspase-3表达明显降低(P<0.05);BCA高剂量处理细胞并加入Nrf2抑制剂ML385后,BCA对BEAS-2B细胞增殖的促进作用减弱,细胞凋亡率、炎症反应和氧化应激损伤增强。结论 BCA可通过激活Nrf2/HO-1信号通路抑制LPS诱导的肺泡上皮细胞氧化应激损伤。

Objective To investigate the influence of Biochanin A(BCA)on lipopolysaccharide(LPS)-induced oxidative stress injury in alveolar epithelial cells by regulating nuclear factor erythroid-2-related factor 2(Nrf2)/heme oxygenase 1(HO-1)signaling pathway.Methods Alveolar epithelial cells BEAS-2B were divided into control group(ctrl group,normally cultured BEAS-2B cells),LPS group(10μg/mL LPS),BCA low-dose group(5μmol/L BCA),BCA medium-dose group(10μmol/L BCA),BCA high-dose group(20μmol/L BCA)and inhibitor group(20μmol/L BCA+5μmol/L Nrf2 inhibitor ML385),all groups except ctrl group were stimulated with 10μg/mL LPS for 24 h.Cell proliferation was detected by CCK-8 method,cell apoptosis was detected by flow cytometry,intracellular reactive oxygen species(ROS)levels were detected by DCFH-DA fluorescent probe method,and the activity of superoxide dismutase(SOD)and malondialdehyde(MDA)levels in the supernatant of cells were detected by spectrophotometry.Tumor necrosis factor-α(TNF-α)and interleukin(IL-6)expression were detected by the kit.The expressions of Nrf2,HO-1,proliferating cell nuclear antigen(PCNA),Bcl-2 associated X protein(Bax)and activated cleaved caspase-3/caspase-3 were detected by Western blotting.Results Compared with the ctrl group,the A 450,SOD activity,expressions of Nrf2,HO-1,and PCNA of BEAS-2B cells in the LPS group significantly decreased(P<0.05),while the apoptosis rate,levels of ROS and MDA,expressions of TNF-α,IL-6,Bax,cleaved caspase-3/caspase-3 significantly increased(P<0.05).Compared with the LPS group,the A 450 value,SOD activity,expressions of Nrf2,HO-1,and PCNA of BEAS-2B cells in the BCA low,medium and high dose groups significantly increased(P<0.05),the apoptosis rate,ROS and MDA level,expressions of TNF-α,IL-6,Bax,cleaved caspase-3/caspase-3 significantly decreased(P<0.05).After high-dose treatment of cells with BCA and the addition of Nrf2 inhibitor ML385,the promoting effect of BCA on BEAS-2B cell proliferation was weakened,and the apoptosis rate,inflammatory response and oxidative stress damage of BEAS-2B cells were enhanced.Conclusion BCA may inhibit LPS-induced oxidative stress injury of alveolar epithelial cells by activating the Nrf2/HO-1 signaling pathway.