葶苈大枣泻肺汤通过Caspase-1诱导A549细胞焦亡与凋亡的机制

Mechanism of Tingli Dazao Xiefeitang on Cell Pyrolysis and Apoptosis Induced by Caspase-1 in A549 Cells

目的:探讨葶苈大枣泻肺汤诱导A549细胞焦亡和凋亡的机制。方法:将A549细胞随机分为空白组、葶苈大枣泻肺汤低、中、高质量浓度组(中药低、中、高质量浓度组),中药低、中、高质量浓度组分别以葶苈大枣泻肺汤20、40、60 mg·L^(-1)处理,空白组以等体积培养液处理。药物作用48 h后,划痕实验检测细胞迁移能力,流式细胞仪检测细胞凋亡,蛋白免疫印迹法(Western blot)检测半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)、NOD样受体蛋白3(NLRP3)、消皮素D(GSDMD)、细胞凋亡抑制因子(Survivin)蛋白及核转录因子-κB(NF-κB)通路蛋白相对表达量,DCFH-DA荧光探针法检测细胞内活性氧(ROS)水平,酶联免疫吸附测定法(ELISA)检测细胞上清液肿瘤坏死因子-β(TNF-β)和白细胞介素-1β(IL-1β)含量。结果:与空白组比较,中药低、中、高质量浓度组细胞划痕愈合率明显降低,细胞凋亡率明显增加,Survivin蛋白相对表达量明显降低,Caspase-1、GSDMD、NLRP3相对表达量明显增加,ROS和NF-κB磷酸化水平明显上升,细胞上清液TNF-β和IL-1β含量明显增加(P<0.05);与中药低质量浓度组比较,中药中、高质量浓度组细胞划痕愈合率明显降低,细胞凋亡率明显增加,Survivin蛋白相对表达量明显降低,Caspase-1、GSDMD、NLRP3相对表达量明显增加,ROS和NF-κB磷酸化水平明显上升,细胞上清液TNF-β和IL-1β含量明显增加(P<0.05);与中药中质量浓度组比较,中药高质量浓度组细胞划痕愈合率明显降低,细胞凋亡率明显增加,Survivin蛋白相对表达量明显降低,Caspase-1、GSDMD、NLRP3相对表达量明显增加,ROS和NF-κB磷酸化水平明显上升,细胞上清液TNF-β和IL-1β含量明显增加(P<0.05)。结论:葶苈大枣泻肺汤可提高NLRP3蛋白表达,抑制Survivin蛋白表达,促进A549细胞凋亡;同时,可激活NF-κB通路和ROS系统,上调细胞焦亡因子Caspase-1,GSDMD的表达,介导A549细胞焦亡。

Objective:To explore the mechanism of coking death and apoptosis of A549 cells induced by Tingli Dazao Xiefeitang. Method: A549 cells were randomized into blank group, traditional Chinese medicine(TCM)low,medium,and high concentration groups,which were treated with 20,40,60 mg·L^(-1) Tingli Dazao Xiefeitang,and TCM low,medium,and high concentration groups,respectively,and blank group was treated with equal volume culture medium. After 48 h of treatment,cell migration was detected by scratch assay and cell apoptosis was detected by flow cytometry. The relative expression levels of cysteine aspartate protease-1(Caspase-1),NOD-like receptor protein 3(NLRP3),dermoderin D(GSDMD),Survivin protein and nuclear transcription factor-κB(NF-κB)pathway proteins were detected by Western blot. The levels of intracellular reactive oxygen species(ROS)were determined by DCFH-DA fluorescence probe,and the contents of tumor necrosis factor-β(TNF-β)and interleukin-1β(IL-1β)in supernatant were determined by enzyme-linked immunosorbent assay(ELISA). Result: Compared with blank group,the scratch healing rate,apoptosis rate, relative expression of Survivin protein, Caspase-1, GSDMD, NLRP3, ROS and NF-κB phosphorylation levels were significantly increased in low,medium and high concentration groups. The contents of TNF-β and IL-1β in supernatant were significantly increased(P<0.05). Compared with the low concentration group,the scratch healing rate,apoptosis rate,Survivin protein relative expression,Caspase-1,GSDMD,NLRP3 relative expression,ROS and NF-κB phosphorylation levels were significantly increased in the medium and high concentration groups. The contents of TNF-β and IL-1β in supernatant were significantly increased(P<0.05). Compared with the TCM group,the scratch healing rate,apoptosis rate,Survivin protein relative expression,Caspase-1,GSDMD,NLRP3 relative expression,ROS and NF-κB phosphorylation levels were significantly increased in the high concentration group. The contents of TNF-β and IL-1β in supernatant were significantly increased(P<0.05). Conclusion: Tingli Dazao Xiefeitang can improve NLRP3 protein expression,inhibit Survivin protein expression and promote apoptosis of A549 cells. At the same time,it can activate NF-κB pathway and ROS system,up-regulate the expression of Caspase-1 and GSDMD,mediate scortosis of A549 cells.