沉默FABP3基因对脂多糖诱导的肺泡上皮细胞凋亡和内质网应激的影响  

Effect of silencing FABP3 gene on LPS-induced apoptosis and endoplasmic reticulum stress in alveolar epithelial cells

作　　者：吴星[1] 史长松[1] 刘爽[1] 周长江[1] 苗永红[1] 曹振锋[1] Wu Xing;Shi Changsong;Liu Shuang;Zhou Changjiang;Miao Yonghong;Cao Zhenfeng(Department of Pediatrics,Henan Provincial People′s Hospital,Zhengzhou 450000,China)

机构地区：[1]河南省人民医院儿科,郑州450000

出　　处：《中华医学杂志》2019年第48期3808-3813,共6页National Medical Journal of China

基　　金：河南省科技厅科研项目(182102310133)。

摘　　要：目的探讨沉默脂肪酸结合蛋白3(FABP3)基因对脂多糖(LPS)诱导的肺泡上皮细胞A549凋亡和内质网应激的影响。方法根据处理方法,将A549细胞分为对照组(A549细胞正常培养24 h)、LPS组(10 mg/L的LPS处理A549细胞24 h)、LPS+乱序无意义阴性序列(si-con)组(LPS+si-con组,10 mg/L的LPS处理转染si-con的A549细胞24 h)和LPS+FABP3的小干扰RNA(si-FABP3)组(LPS+si-FABP3组,LPS作用转染si-FABP3的A549细胞24 h),实时荧光定量PCR检测FABP3 mRNA的相对表达量,四甲基噻唑蓝染色法(MTT)检测细胞增殖,流式细胞仪检测细胞凋亡,Western印迹法检测FABP3、细胞周期蛋白D1(CyclinD1)、活化的caspase-3、葡萄糖调节蛋白78(GRP78)、激活转录因子4(ATF4)和CCAAT增强子结合蛋白同源蛋白(CHOP)、活化的caspase-12和磷酸化的蛋白激酶激酶B(p-Akt)和PI3K催化亚基p110α(PI3Kp110α)蛋白的相对表达量,酶联免疫吸附法检测白细胞介素(IL)-6、IL-8、肿瘤坏死因子α(TNF-α)水平。结果LPS组FABP3蛋白和mRNA相对表达量、细胞凋亡率和IL-6、IL-8、TNF-α水平以及活化的caspase-3、GRP78、ATF4、CHOP、活化的caspase-12、p-Akt和PI3Kp110α蛋白相对表达量均显著高于对照组[(1.00±0.09)比(0.53±0.05)、(2.15±0.22)比(1.00±0.10)、(26.1±2.6)%比(4.5±0.5)%、(554.4±55.4)比(75.4±7.5)ng/L、(389.3±38.5)比(25.2±2.5)ng/L、(601.3±60.0)比(66.5±6.7)ng/L、(1.00±0.11)比(0.34±0.05)、(1.05±0.11)比(0.35±0.05)、(1.20±0.12)比(0.43±0.05)、(1.05±0.10)比(0.37±0.04)、(1.10±0.11)比(0.45±0.05)、(0.88±0.08)比(0.16±0.04)、(0.75±0.08)比(0.35±0.05),均P<0.05]。LPS组细胞存活率[(50.1±5.4)%]和CyclinD1蛋白相对表达量(0.40±0.05)均显著低于对照组[(100.1±12.4)%、(1.25±0.12)](均P<0.05)。LPS+si-FABP3组细胞存活率[(89.1±8.5)%]和CyclinD1蛋白相对表达量(1.15±0.11)均显著高于LPS+si-con组[(53.1±5.4)%、(0.42±0.05)](均P<0.05)。LPS+si-FABP3组细胞凋亡率[(10.5±1.1)%]、IL-6[(301.3±30.0)ng/L]、IL-8[(189Objective To investigate the effect of silencing fatty acid binding protein 3(FABP3)gene on lipopolysaccharide(LPS)-induced apoptosis and endoplasmic reticulum stress in alveolar epithelial cells A549.Methods According to the processing method,A549 cells were divided into control group(A549 cells cultured for 24 h),LPS group(10 mg/L LPS treated A549 cells for 24 h),LPS+si-con group(10 mg/L LPS was used to treat A549 cells transfected with si-con for 24 h)and LPS+si-FABP3 group(10 mg/L LPS was used to treat A549 cells transfected with si-FABP3 for 24 h).Then quantitative real-time PCR was used to detect the level of FABP3,methylthiazoletrazolium was used to detect the cell proliferation,flow cytometry was used to detect the apoptosis,and Western Blot was used to detect the levels of FABP3,CyclinD1,cleaved-caspase-3,GRP78,ATF4,CHOP,cleaved-caspase-12 and p-Akt and PI3Kp110αprotein expression.Enzyme-linked immunosorbent assay was used to detect the levels of IL-6,IL-8 and TNF-αlevels.Results In the LPS group,FABP3 protein level(1.00±0.09)and mRNA(2.15±0.22),apoptosis rate[(26.1±2.6)%],inflammatory factor IL-6[(554.4±55.4)ng/L],IL-8[(389.3±38.5)ng/L]and TNF-α[(601.3±60.0)ng/L],cleaved-caspase-3(1.00±0.11),GRP78(1.05±0.11),ATF4(1.20±0.12)),CHOP(1.05±0.10),cleaved-caspase-12(1.10±0.11),p-Akt(0.88±0.08)and PI3Kp110α(0.75±0.08)protein levels were significantly higher than the control group[(0.53±0.05),(1.00±0.10),(4.5±0.5)%,(75.4±7.5)ng/L,(25.2±2.5)ng/L,(66.5±6.7)ng/L,(0.34±0.05),(0.35±0.05),(0.43±0.05),(0.37±0.04),(0.45±0.05),(0.16±0.04),(0.35±0.05)](all P<0.05).Cell viability[(50.1±5.4)%]and CyclinD1 protein level(0.40±0.05)in LPS group were significantly lower than those in the control group[(100.1±12.4)%,(1.25±0.12)](both P<0.05).Cell viability[(89.1±8.5)%]and CyclinD1 protein level(1.15±0.11)in LPS+si-FABP3 group were significantly higher than those in LPS+si-con group[(53.1±5.4)%,(0.42±0.05)](both P<0.05).Apoptosis rate[(10.5±1.1)%],IL-6[(301.3±30.0)ng/L],IL-8[(189.4±19.0)ng

关 键 词：肺泡 上皮细胞 脂肪酸结合蛋白质类 细胞凋亡 内质网应激 

分 类 号：R563[医药卫生—呼吸系统]