摘要
目的观察参附注射液对NLRP3/Caspase-1/GSDMD介导的J774A.1细胞焦亡的影响并探讨其机制。方法采用MTT法确定低、中及高剂量参附注射液组药物浓度分别为2.5、5、10μL/mL。将J774A.1细胞分为5组,每组6孔。空白组在细胞接种后,常规培养细胞;模型组细胞在100 ng/mL LPS提前处理4 h之后加入10μmol/L Nigericin处理2 h;低、中和高参附注射液组细胞在模型组的基础上加入上述相应浓度的参附注射液进行共刺激。倒置显微镜观察各组细胞形态改变;LDH释放法检测各组细胞活性;Western blotting检测细胞及上清液中Pro-Caspase-1、Caspase-1、GSDMD及GSDMD-N蛋白水平;ELISA检测细胞培养上清中白细胞介素-1β(IL-1β)水平。结果在模型组细胞中观察到膨胀球形改变及胞膜破裂,向胞外释放的LDH明显增多(P<0.05),经参附注射液干预后形态改变有减轻,LDH释放显著减少(P<0.05);Western blotting结果显示,模型组Caspase-1及GSDMD-N蛋白水平显著升高(P<0.05),经参附注射液干预后,上述蛋白水平明显下降(P<0.05);ELISA测得的细胞培养上清IL-1β水平,在模型组中亦明显升高(P<0.05),而在各参附注射液组中均有显著下降(P<0.05)。结论参附注射液能抑制NLRP3/Caspase-1/GSDMD介导的J774A.1细胞焦亡改变,机制可能与其抑制Pro-Caspase-1与GSDMD的活化过程相关。
Objective:To observe the effect of Shenfu Injection(SFI)on NLRP3/Caspase-1/GSDMD-mediated J774A.1 cell pyroptosis and to explore the mechanism.Methods:MTT method was used to determine the drug concentration of 2.5,5 and 10μL/mL in the low,medium and high dose SFI groups,respectively.J774A.1 cells were divided into 5 groups with 6 wells in each group.In the normal group,the cells were routinely cultured after cell inoculation.In the model group,cells were treated with 10μL/mL Nigericin for 2 h,following a 4 h treatment with 100 ng/mL LPS.The cells in the low,medium and high SFI groups were co-stimulated by adding the corre⁃sponding concentration of SFI mentioned above to that in the model group.Inverted microscope was used to ob⁃serve the morphological changes of the cells in each group.LDH release assay was used to detect the activity of the cells in each group.Western blotting was used to detect the levels of Pro-Caspase-1,Caspase-1,GSDMD and GSDMD-N proteins in the cells and the supernatant.ELISA was used to detect the levels of IL-1βin the superna⁃tant of the cell culture.Results:In the cells of the model group,swelling,spherical shape change,and cell mem⁃brane rupture were observed,and the release of LDH into the extracellular space was significantly increased(P<0.05),and the morphological change was alleviated and the release of LDH was significantly reduced after the in⁃tervention of SFI(P<0.05).Western blotting results showed that the levels of Caspase-1 and GSDMD-N proteins were significantly increased in the model group(P<0.05),and the levels of the above proteins were significantly decreased after SFI intervention(P<0.05).IL-1βlevels in cell culture supernatants measured by ELISA were al⁃so significantly increased in the model group(P<0.05),and significantly decreased in all SFI groups(P<0.05).Conclusion:SFI inhibits NLRP3/Caspase-1/GSDMD-mediated pyroptosis of J774A.1 by a mechanism that may be related to its inhibition of the activation process of Pro-Caspase-1 and GSDMD.
作者
陈腾飞
张玲梅
王施玮
周晓
任园园
陈波
施小伟
叶成坚
郑文贺
Chen Tengfei;Zhang Lingmei;Wang Shiwei;Zhou Xiao;Ren Yuanyuan;Chen Bo;Shi Xiaowei;Ye Chengjian;Zheng Wenhe(The Second People′s Hospital Affiliated to Fujian University of Traditional Chinese Medicine,Fujian,Fuzhou 350003,China)
出处
《中国中医急症》
2023年第12期2103-2106,共4页
Journal of Emergency in Traditional Chinese Medicine
基金
福建省自然科学基金项目(2023J01816)
福建省卫生健康青年科研课题(2020QNA063)。
