清肺通络方调控Caspase-1对肺炎支原体诱导人正常肺上皮细胞BEAS-2B焦亡的干预作用

Effect of Qingfei Tongluo Prescription Regulating Caspase-1 in the Intervention of Pyroptosis of Mycoplasma Pneumoniae-Induced Human Pulmonary Epithelial Cells BEAS-2B

【目的】探讨清肺通络方(桑白皮、地骨皮等)对肺炎支原体(MP)诱导人正常肺上皮细胞BEAS-2B由半胱氨酸天冬氨酸蛋白酶1(Caspase-1)/消皮素D(GSDMD)通路介导的细胞焦亡的干预作用。【方法】建立MP感染的BEAS-2B细胞模型,分别给予清肺通络方、Caspase-1抑制剂干预。制备过表达Caspase-1质粒,转染MP感染的BEAS-2B细胞,给予清肺通络方干预。采用细胞计数试剂盒8(CCK-8)检测细胞增殖活性,Western Blot法检测细胞焦亡关键蛋白pro-Caspase-1、Caspase-1、消皮素D(GSDMD)、消皮素D-N端片段(GSDMD-N)的表达水平,酶联免疫吸附分析(ELISA)检测细胞因子白细胞介素(IL)-1β和IL-18水平,生化法检测乳酸脱氢酶(LDH)和Na+-K+-ATP酶活性,流式细胞术检测细胞焦亡。【结果】MP感染BEAS-2B细胞增殖活性明显降低,细胞焦亡关键蛋白pro-Caspase-1、Caspase-1、GSDMD、GSDMD-N表达水平均明显升高,下游细胞因子IL-1β和IL-18水平也显著升高,LDH活性明显升高,Na+-K+-ATP酶活性明显下降,细胞焦亡率显著上升。应用清肺通络方、Caspase-1抑制剂干预MP感染BEAS-2B细胞,及应用清肺通络方干预过表达Caspase-1质粒转染的MP感染BEAS-2B细胞后,细胞增殖活性均升高,细胞焦亡关键蛋白pro-Caspase-1、Caspase-1、GSDMD、GSDMD-N表达水平降低,下游细胞因子IL-1β和IL-18水平均显著降低,LDH活性明显降低、Na+-K+-ATP酶活性明显上升,细胞焦亡率明显下降。【结论】MP感染BEAS-2B细胞后,激活Caspase-1/GSDMD通路,促进炎症因子释放,使细胞结构受损,最终导致细胞焦亡。清肺通络方可以抑制Caspase-1/GSDMD通路的激活,减轻炎症反应,减轻细胞损伤,抑制细胞焦亡。

Objective To investigate the effect of Qinglung Tongluo Prescription(a herbal compound mainly with the actions of clearing lung to unblock collaterals composed of Mori Cortex, Lycii Cortex, etc.)on pyroptosis mediated by Caspase-1/GSDMD pathway in human normal lung epithelial cells BEAS-2B induced by mycoplasma pneumoniae. Methods A model of mycoplasma pneumoniae(MP)-infected BEAS-2B cells was established,and Qinglung Tongluo Prescription and Caspase-1 interventions were respectively administered. MP-infected BEAS-2B cells were transfected with Caspase-1 plasmids prepared, and then were treated by Qinglung Tongluo Prescription. Cell proliferation activity was detected by cell counting kit 8(CCK-8), expression levels of pyroptosis-related key proteins pro-Caspase-1,Caspase-1, Gasdermin-D(GSDMD), and GSDMD-N were detected by Western Blot. The expression levels of interleukin(IL)-1β and IL-18 were detected by enzyme-linked immunosorbent assay(ELISA). The expression of lactate dehydrogenase(LDH)and Na+-K+-ATPase activity were detected by biochemical assay, and the pyroptosis was detected by flow cytometry. Results The proliferative activity of MP-infected BEAS-2B cells was significantly reduced,the expression levels of pyroptosis-related key proteins pro-Caspase-1,Caspase-1,GSDMD and GSDMD-N were increased,and the levels of downstream cytokines IL-1β and IL-18 were also significantly increased,LDH activity was significantly raised,Na+-K+-ATPase activity was significantly decreased,pyroptosis rate was significantly increased. After the application of Qinglung Tongluo Prescription and Caspase-1 inhibitor to treat MP-infected BEAS-2B cells and the application of Qinglung Tongluo Prescription to treat MP-infected BEAS-2B cells transfected with overexpressed Caspase-1plasmids,the cell proliferation activity was increased and the expression levels of pyroptosis-related key proteins pro-Caspase-1,Caspase-1,GSDMD and GSDMD-N were decreased,the levels of downstream cytokines IL-1β and IL-18 were significantly reduced,LDH activity was significantly reduced,Na+-K+-ATPase activity was significantly increased,and the pyroptosis rate was significantly reduced. Conclusion MP infection of BEAS-2B cells activates the Caspase-1/GSDMD pathway,promote the release of inflammatory factors,cause damage to cell structure and eventually lead to pyroptosis. Qinglung Tongluo Prescription has effects on inhibiting the activation of Caspase-1/GSDMD pathway, reducing the inflammatory response, mitigating cell damage and inhibiting pyroptosis.