蛋白酶激活受体2在人中性粒细胞弹性蛋白酶诱发气道黏液速发式分泌中的作用及机制被引量：5

Role of Protease-activated Receptor 2 in Neutrophil Elastase-induced Fast Exocytosis of Mucus in the Airway

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摘要目的研究蛋白酶激活受体2(protease-activated receptor 2,PAR2)在人中性粒细胞弹性蛋白酶(neutrophil elastase,NE)诱发气道黏蛋白(mucin,MUC)5AC速发式分泌中的作用及机制。方法用Western blot法检测对照组及NE刺激组Calu-3细胞中PARs的表达。NE分别干预野生型(空白对照组)及转染PARs siRNA的Calu-3细胞,通过ELISA法检测其上清液中MUC5AC的表达,采用Fluo-3AM荧光探针法检测细胞内Ca2+浓度、Western blot法检测胞膜及胞质中蛋白激酶C(protein kinase C,PKC)水平。NE分别刺激空白对照组、转染PAR2siRNA组、细胞内钙离子螯合剂BAPTA-AM预处理组及PKC抑制剂Chelerythrine预处理组,再通过ELISA法检测其上清液中MUC5AC浓度、细胞免疫荧光法测定细胞质内滞留的MUC5AC。结果 Calu-3细胞系中PAR1、PAR2、PAR3蛋白均呈阳性表达,其中以PAR2为主,而PAR4则呈近似阴性表达。NE刺激Calu-3细胞,其PAR1、PAR3表达较空白对照组无明显变化(P>0.05),而PAR2则较空白对照组明显增加(P<0.05)。NE刺激野生型Calu-3细胞,其上清液中MUC5AC分泌量及细胞内Ca2+浓度较刺激前明显增加(均P<0.05);而转染PAR2siRNA组,其MUC5AC分泌量增加不明显(P>0.05),细胞内Ca2+浓度升高水平也不及空白对照组(P<0.05)。同时,空白对照组PKC主要存在于胞质中,当给予NE刺激以后,PKC被引导至胞膜。转染PAR2siRNA,细胞内Ca2+螯合剂BAPTA-AM及PKC选择性抑制剂Chelerythrine预处理,均可部分削弱NE刺激后细胞上清液中MUC5AC水平的升高(均P<0.05)。结论 NE刺激可导致Calu-3细胞PAR2表达上调,并通过激活PAR2、增加细胞内Ca2+浓度、PKC激活及膜移位导致MUC5AC的速发式分泌。 Objective To investigate the role of protease-activated receptor 2 （PAR2） in the neutrophil elastase （NE）-in duced fast exocytosis of mucin 5AC （MUCSAC） in the airway and the possible mechanism. Methods The expression levels of PARs were detected in Calu-3 cells in the control group and NE-stimulated group by Western blotting. The wild-type and PAR2 siRNA-transfected Calu-3 cells were treated by NE. The expression level of MUC5AC in the supernatant was detected by ELISA. The concentration of intracellular calcium was measured by using fluorescence probe Fluo-3AM and the expression level of protein kinase C （PKC） in the cytomembrane and cytoplasm by Western blotting. Cells were treated with NE in the wild-type Calu-3 cell group,PAR2-siRNA-transfected group, the calcium chelating （BAPTA-AM）-pre-treated group and the PKC-selec- tive inhibitor （chelerythrine）-treated group, respectively. The concentration of MUC5AC was detected in the supernatant by ELISA and in the cytoplasm by iramumofluorescence. Results Calu-3 cells expressed PAR1,PARZ and PAR3 with a predomi- nant level of PAR2 and nearly negative expression of PAR4. The expression levels of PAR1 and PAR3 in NE-stimulated Calu-3 cells were not significantly different from those in control group （P〉0. 05）, whereas those of PAR2 were increased markedly in NE-stimulated Calu-3 cells when compared with control group （P〈0.05）. The level of MUC5AC and the concentration of in- tracellular calcium were profoundly increased in the supernatant of NE stimulated wild-type Calu-3 cell group rather than in the PAR2-siRNA-transfected group when compared ＇with the control group （P〈0. 05）. PKC in Calu-3 cells was translocated from the cytoplasm to the cell membrane after NE stimulation. The NE induced increase in the level of MUC5AC in the supernatant was weakened partly by PAR2 siRNA transfection,the intracellular calcium chelating agent BAPTA-AM and PKC-selective in- hibitor chelerythrine（P〈0.05）. Conclusion NE stimulation can enhance the PAR2 expression and lead to fast exocvtosis ofMUC5AC via the activation of PAR2 ,increase of cytosolic calcium concentration, and activation and translocation of PKC to the cell membrane.

作者周佳周向东许瑞唐小葵

机构地区重庆医科大学附属第一医院呼吸内科

出处《华中科技大学学报（医学版）》 CAS CSCD 北大核心 2015年第6期634-639,共6页 Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基金国家临床重点专科专项经费资助项目(卫办医政函[2012]949号)

关键词中性粒细胞弹性蛋白酶蛋白酶激活受体2 信号通路黏蛋白5AC neutrophil elastase protease-actived receptor 2 signal pathmay mucin 5AC

分类号 R562.25 [医药卫生—呼吸系统]

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