黄芩苷对脂多糖联合三磷酸腺苷刺激BEAS-2B细胞氧化应激的影响

Effects of baicalin on oxidative stress in BEAS-2B cells stimulated by lipopolysaccharide combined with adenosine triphosphate

背景:氧化应激及炎症反应在急性肺损伤的发生发展过程中发挥了重要的作用,研究表明黄芩苷具有抗氧化、抗炎等生物活性。目的:探讨黄芩苷对脂多糖联合三磷酸腺苷刺激人正常支气管上皮细胞BEAS-2B氧化应激和TXNIP/NLRP3通路的影响。方法:采用脂多糖/三磷酸腺苷联合刺激BEAS-2B细胞建立炎症模型,将细胞随机分为空白对照组、模型组和黄芩苷低、中、高剂量组(2.5,5,10 mg/L);采用MTT法检测细胞活力,ELISA法检测细胞因子白细胞介素1β、白细胞介素6、白细胞介素18和肿瘤坏死因子ɑ水平,荧光探针DCFH-DA法检测各组细胞内活性氧水平,酶标仪检测超氧化物歧化酶和丙二醛水平,实时荧光半定量PCR检测TXNIP、NLRP3、ASC、Caspase-1、白细胞介素1β和白细胞介素18 mRNA的表达水平,Western blot检测细胞中TXNIP、Caspase-1、NLRP3蛋白表达量。结果与结论:与空白对照组相比,模型组细胞内活性氧和丙二醛水平升高,超氧化物歧化酶活性降低,细胞活力下降,细胞因子白细胞介素1β、白细胞介素6、白细胞介素18、肿瘤坏死因子ɑ的分泌量和TXNIP、NLRP3、ASC、Caspase-1、白细胞介素1β、白细胞介素18的基因表达量以及TXNIP、Caspase-1、NLRP3蛋白表达量均显著升高。与模型组比较,黄芩苷组可显著逆转上述指标,且呈浓度依赖性。结果表明,黄芩苷可通过减轻氧化应激反应,进而抑制TXNIP/NLRP3炎症通路的激活,来发挥其对脂多糖/三磷酸腺苷联合诱导BEAS-2B细胞损伤的保护作用。

BACKGROUND:Oxidative stress and inflammatory reaction play important roles in the occurrence and development of acute lung injury.Studies have shown that baicalin has biological activities such as antioxidant and anti-inflammatory.OBJECTIVE:To investigate the effects of baicalin on oxidative stress and TXNIP/NLRP3 pathway in BEAS-2B cells induced by lipopolysaccharide combined with adenosine triphosphate.METHODS:The inflammatory model of BEAS-2B cells was established by lipopolysaccharide combined with adenosine triphosphate stimulation.The cells were randomly divided into blank control group,model group and baicalin group(2.5,5,and 10 mg/L).MTT method was used to detect the cell vitality.ELISA was used to detect the levels of interleukin-1β,interleukin-6,interleukin-18 and tumor necrosis factor-ɑ.The changes of intracellular reactive oxygen species,superoxide dismutase and malondialdehyde were measured by fluorescent probe DCFH-DA and microplate reader,respectively.qRT-PCR was used to detect the mRNA expression levels of TXNIP,NLRP3,ASC,Caspase-1,interleukin-1βand interleukin-18.Western blot was used to detect the protein expression of TXNIP,Caspase-1 and NLRP3 in the cells.RESULTS AND CONCLUSION:Compared with the blank control group,intracellular reactive oxygen species and malondialdehyde levels were significantly increased in the model group,while the activity of superoxide dismutase and cell viability were significantly decreased.There was a significant increase in the secretion levels of interleukin-1β,interleukin-6,interleukin-18,tumor necrosis factor-ɑas well as the gene expression levels of TXNIP,NLRP3,ASC,Caspase-1,interleukin-1β,interleukin-18 and the protein expression levels of TXNIP,Caspase-1 and NLRP3 in the model group compared with the blank control group.Baicalin could considerably reverse the above indicators in a concentration-dependent manner.To conclude,baicalin has a protective effect on the damage in BEAS-2B cells stimulated by lipopolysaccharide combined with adenosine triphosphate,by reducing oxidative stress and inhibiting the activation of the TXNIP/NLRP3 signaling pathway.