平喘宁通过TRPM8/PKC/NF-κB信号通路缓解冷刺激加重的人支气管上皮细胞炎症作用机制研究  

Study on the Mechanism of Pingchuanning(平喘宁)in Alleviating the Inflammation of Human Bronchial Epithelial Cells Aggravated by Cold Stimulation through TRPM8/PKC/NF-κB Signaling Pathway

作　　者：丁鹤影 方向明[1] 程悦 叶卫东 袁亚美[1] 李秋慧[1] DING Heying;FANG Xiangming;CHENG Yue;YE Weidong;YUAN Yamei;LI Qiuhui(Anhui University of Chinese Medicine,Hefei 230012,Anhui,China)

机构地区：[1]安徽中医药大学,安徽合肥230012

出　　处：《辽宁中医药大学学报》2024年第12期17-23,共7页Journal of Liaoning University of Traditional Chinese Medicine

基　　金：安徽省高等学校科学研究重大项目(KJ2021ZD0064);安徽省教育厅科学研究重点项目(2022AH050446);安徽中医药大学自然科学研究重点项目(2021zrzd18)。

摘　　要：目的探讨平喘宁(PCN)通过瞬时受体电位melastatin家族成员8/蛋白激酶C/核因子-κB(TRPM8/PKC/NF-κB)信号通路对缓解冷刺激加重的人支气管上皮细胞炎症的作用机制,以此阐发平喘宁治疗寒哮的科学内涵。方法选用人支气管上皮细胞16HBE作为研究对象,用流式细胞术检测18℃培养不同时间的16HBE细胞的Ca^(2+)表达水平,选择Ca^(2+)水平较高的时间段组作为模型组;采用细胞增殖与活性检测试剂盒(CCK-8)法检测TRPM8离子通道的特异性抑制剂(BCTC)和PCN冻干粉对16HBE细胞的活性影响,以筛选出作用于16HBE细胞的适宜条件的浓度和时间。将细胞分为对照组、18℃冷刺激组(模型组)、18℃+BCTC组(BCTC组)、18℃+PCN冻干粉组(PCN组)、18℃+BCTC+PCN冻干粉组(BCTC+PCN组)。流式细胞术测量各组细胞内Ca^(2+)浓度变化;qRT-PCR测定各组TRPM8、PKC、NF-κB mRNA的表达水平;免疫荧光标记法检测各组磷酸化PKC、磷酸化NF-κB抑制因子(IκB)的蛋白含量;ELISA法检测各组细胞炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素-13(IL-13)的含量变化。结果与对照组比较,模型组的Ca^(2+)浓度显著升高(P<0.01),TRPM8、PKC、NF-κB p65 mRNA表达水平显著升高(P<0.01),磷酸化IκB、磷酸化PKC表达水平显著升高(P<0.01),促炎因子TNF-α、IL-13表达水平显著升高(P<0.01)。与模型组比较,PCN组的Ca^(2+)浓度显著降低(P<0.01),TRPM8、PKC、NF-κB p65 mRNA表达水平显著降低(P<0.01),磷酸化IκB、磷酸化PKC表达水平显著降低(P<0.01),促炎因子TNF-α、IL-13表达水平显著降低(P<0.01)。与BCTC组相比,PCN组的Ca^(2+)浓度均显著降低(P<0.01),NF-κB p65 mRNA表达水平显著降低(P<0.05),TRPM8、PKC mRNA表达水平降低,但差异无统计学意义(P>0.05),磷酸化IκB、磷酸化PKC表达水平显著降低(P<0.01),促炎因子TNF-α、IL-13表达水平显著降低(P<0.01);BCTC+PCN组的Ca^(2+)浓度均显著降低(P<0.01),TRPM8、PKC、NF-κB p65 mRNA表达水平Objective To investigate the mechanism of Pingchuanning(平喘宁,PCN)in relieving the inflammation of human bronchial epithelial cells with increased cold stimulation through transient receptor potential melastatin family member 8/protein kinase C/nuclear factor kappa B(TRPM8/PKC/NF-κB)signaling pathway,and to elucidate the scientific connotation of PCN in the treatment of cold asthma.Methods Human bronchial epithelial cells 16HBE were selected as the study object,and the Ca^(2+)concentration of 16HBE cells cultured at 18℃at different time was detected by flow cytometry.The time group with high Ca^(2+)level was selected as the model group.The effect of TRPM8 ion channel specific inhibitor(BCTC)and PCN freeze-dried powder on the activity of 16HBE cells were detected by cell proliferation and activity detection kit(CCK-8)method,in order to screen out the appropriate concentration and time of 16HBE cells.The cells were divided into control group,18℃cold stimulation group(model group),18℃+BCTC group(BCTC group),18℃+PCN freezedried powder group(PCN group)and 18℃+BCTC+PCN freeze-dried powder group(BCTC+PCN group).Intracellular Ca^(2+)concentration was measured by flow cytometry.The expression levels of TRPM8,PKC and NF-κB mRNA in each group were detected by qRT-PCR.The contents of phosphorylated PKC and phosphorylated NF-κB inhibitor(IκB)protein in each group were detected by immunofluorescence method.The contents of inflammatory cytokines TNF-αand IL-13 were detected by ELISA.Results Compared with the control group,in the model group:the concentration of Ca^(2+)was significantly increased(P<0.01),the mRNA expression levels of TRPM8,PKC and NF-κB p65 were significantly increased(P<0.01),the expression levels of phosphorylated IκB and phosphorylated PKC were significantly increased(P<0.01),and the expression levels of pro-inflammatory factors TNF-αand IL-13 were significantly increased(P<0.01).Compared with the model group,in the PCN group:the concentration of Ca^(2+)was significantly decreased(P<0.01

关 键 词：平喘宁 瞬时受体电位melastatin家族成员8 蛋白激酶C 核因子-κB 哮喘 

分 类 号：R289.5[医药卫生—方剂学]