阿魏酸钠阻断NF-κB降低NALP3诱导的矽肺成纤维细胞增殖  

Sodium ferulate blocks NF-κB action and reduces NALP3-induced silicosis fibroblasts proliferation

作　　者：韩静茵 王淑娟 贾仰民 干晓瑜 HAN Jing-yin;WANG Shu-juan;JIANG Yang-min;GAN Xiao-yu(Department of Occupational Diseases,Hangzhou Red Cross Hospital,Hangzhou,Zhejiang 310005,China)

机构地区：[1]浙江大学医学院附属杭州市胸科医院职业病科,杭州310003

出　　处：《浙江中西医结合杂志》2023年第12期1095-1100,共6页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

基　　金：浙江省医药卫生科技计划项目(No.2020KY739)。

摘　　要：目的探讨阿魏酸钠(SF)对核苷酸结合寡聚化结构域样受体3(NALP3)诱导的矽肺模型小鼠成纤维细胞增殖的内在分子机制。方法采用C57BL/6雄性小鼠建立矽肺模型,提取的肺成纤维细胞用于后续实验。细胞被浓度为0、0.1和0.28 mg/mL的SF分别处理48 h后,采用蛋白质印迹实验(Western blot)检测核因子κB(NF-κB)通路相关蛋白表达。Control组的细胞正常培养,SF组细胞给予0.28 mg/mL SF,Prostratin组细胞给予100 nmol NF-κB激活剂(Prostratin),SF+Prostratin组细胞给予0.28 mg/mL SF和100 nmol Prostatin。处理48 h后,采用MTT、EdU、酶联免疫吸附试验(ELISA)检测细胞活力、增殖、白介素1β(IL-1β)和半胱氨酸天冬氨酸特异性蛋白酶-1(Caspase-1)的活性。通过实时荧光定量聚合酶链反应实验(QRT-PCR)确定NALP3敲减质粒(shNALP3)的转染效率后,Prostratin组细胞给予100 nmol Prostratin,Prostratin+shNALP3组细胞转染shNALP3并给予100 nmol Prostratin,Prostratin+shNC组细胞转染NALP3阴性对照(shNC)并给予100 nmol Prostratin。处理48 h后,采用上述细胞功能学实验和Western blot分析细胞功能和分子表达变化。结果0.28 mg/mL的SF抑制核转录因子p65(p65)、抑制因子κB(IkB)、NALP3、胶原蛋白-1(collagen-1)和平滑肌肌动蛋白(α-SMA)的表达[p65:(0.51±0.04)比(1.00±0.08);IkB:(0.46±0.04)比(1.00±0.09);NALP3:(0.54±0.05)比(1.00±0.08);collagen-1:(0.49±0.04)比(1.00±0.09);α-SMA:(0.62±0.05)比(1.00±0.08);P均<0.05],Prostratin逆转SF抑制的细胞活力、增殖、Caspase-1和IL-1β活性[细胞活力:(80.26±5.00)%比(56.33±3.99)%;细胞增殖:(3.80±0.25)%比(1.80±0.10)%;Caspase-1:(111.02±7.00)pg/mL比(54.28±4.00)pg/mL;IL-1β:(74.05±5.01)pg/mL比(19.28±2.01)pg/mL;P均<0.05],同时逆转SF阻断的NF-κB通路蛋白表达和降低NALP3表达[p65:(1.03±0.09)比(0.22±0.03);IkB:(1.06±0.09)比(0.34±0.03);NALP3:(1.10±0.09)比(0.33±0.03);collagen-1:(1.03±0.09)比(0.36±0.03);α-SMA:(1.12±0.09)比(0.25±0.03);P均<0.05Objective To investigate the underlying molecular mechanism of the effect of sodium ferulate(SF)on fibroblast proliferation in a mouse model of the nucleotide binding oligomerization domain-like receptor 3(NALP3)-induced silicosis.Methods C57BL/6 male mice were selected to establish an animal silicosis model and then isolated the lung fibroblasts for subsequent experiments.Fibroblasts were treated with SF at concentrations of 0,0.1,or 0.28 mg/ml for 48 h and then subjected to Western blot analysis of the nuclear factor kappa-B(NF-ession of ts were treated withFurthermore,fibroblasts were cultured regularly as a control or treated with SF(0.28 mg/ml),an NF-roblasts in Prostratin g;100 nmol),and a combination of SF+Prostratin(0.28 mg/ml SF and 100 nmol Prostatin)for 48 h;after that,the cell viability,proliferation,activities of interleukin 1F at concentrations of 0e(e detected by MTT,EdU,and enzyme linked immunosorbent assay(ELISA),respectively.After assessing the transfection efficiency of the NALP3 knockdown plasmid(shNALP3)using the quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR),Prostratin-treated fibroblasts(100 nmol)were transfected with or without shNALP3 or shNC for 48 h and cell viability and gene expression were assessed accordingly.Results SF at a dose of 0.28 mg/ml was able to inhibit expression of p65(0.51±0.04 vs.1.00±0.08),IkB(0.51±0.04 vs.1.00±0.09),NALP3(0.54±0.05 vs.1.00±0.08),collagen-1(0.49±0.04 vs.1.00±0.09),and lagen(0.62±0.05 vs.1.00±0.08;All P<0.05).Prostratin was able to reverse the SP-inhibited cell viability(80.26±5.00 vs.56.33±3.99),proliferation(3.80±0.25 vs.1.80±0.10),and Caspase-1(111.02±7.00 vs.54.28±4.00)and IL-11i(74.05±5.01 vs.19.28±2.01;All P<0.05),as well as the NF-ession of as eins,e.g.,p65(1.03±0.09 vs.0.22±0.03),IkB(1.06±0.09 vs.0.34±0.03),NALP3(1.10±0.09 vs.0.33±0.03),collagen-1(1.03±0.09 vs.0.36±0.03),andαnd 6(1.12±0.09 vs.0.25±0.03;P<0.05).However,transfection of shNALP3 reduced Prostratin-promoted fibroblast level of NALP

关 键 词：小鼠 阿魏酸钠 矽肺 NF-ΚB NALP3 成纤维细胞 

分 类 号：R73[医药卫生—肿瘤]