4-辛基衣康酸在肺成纤维细胞分化中的作用及机制初步探讨  

The role of 4-octyl itaconate and related mechanisms in lung fibroblast-to-myofibroblast differentiation

作　　者：李世贞 龚辉 谭胜玉[1] 张湘瑜[1] Li Shizhen;Gong Hui;Tan Shengyu;Zhang Xiangyu(Department of Geriatrics,The Second Xiangya Hospital,Central South University,Changsha 410011,China)

机构地区：[1]中南大学湘雅二医院老年医学科,长沙410011

出　　处：《中华老年医学杂志》2024年第5期603-608,共6页Chinese Journal of Geriatrics

基　　金：国家自然科学基金面上项目(82271625);湖南省重点研发项目(2022SK2013);湖南省自然科学基金(2022JJ70063、2020JJ4808)。

摘　　要：目的探讨4-辛基衣康酸(4-OI)在转化生长因子β1(TGF-β1)诱导肺成纤维细胞分化过程中的作用及机制。方法采用TGF-β1诱导肺成纤维细胞MRC-5分化;观察4-OI预处理对肺成纤维细胞分化的影响。采用细胞计数试剂盒(CCK-8)检测4-OI的细胞毒性;蛋白免疫印迹法(Western blot)检测α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白α1(COL1A1)、纤维粘连蛋白(FN)、磷酸化Smad2/3(p-Smad2/3)蛋白、总Smad2/3蛋白,以及核因子E2相关因子2(Nrf2)蛋白的表达水平;实时荧光定量聚合酶链反应法(real-time PCR)检测α-SMA、COL1A1和FN的mRNA表达水平;荧光显微镜及流式细胞仪检测细胞活性氧(ROS)水平变化;分光光度法检测细胞中谷胱甘肽(GSH)含量。结果4-OI预处理可以抑制TGF-β1诱导增加的α-SMA、COL1A1和FN蛋白水平(F=122.8、51.5、27.2,均P<0.05)及mRNA表达(F=29.83、51.62、94.82,均P<0.01)。4-OI可以抑制TGF-β1介导的p-Smad2/3蛋白水平并呈浓度依赖性(F=21.80、36.69,均P<0.01)。4-OI可以降低TGF-β1诱导增加的ROS水平(P<0.01),并且提高TGF-β1抑制的GSH含量(P<0.05);减轻TGF-β1对Nrf2表达的抑制作用,并促进Nrf2核转位(P<0.05)。沉默Nrf2表达后,4-OI不能抑制TGF-β1诱导增加的COL1A1蛋白水平,但仍可抑制TGF-β1诱导增加的α-SMA、FN蛋白表达水平(P<0.05)。结论4-OI部分通过Nrf2途径抑制TGF-β1诱导的肺成纤维细胞分化。Objective To investigate the effect of 4-octyl itaconate(4-OI)on transforming growth factor-β1(TGF-β1)-induced lung fibroblast-to-myofibroblast differentiation and related mechanisms.Methods TGF-β1 was employed to induce the differentiation of the human embryonic lung fibroblast cell line MRC-5,and the effect of 4-OI on lung fibroblast-to-myofibroblast differentiation was examined.Cytotoxicity of 4-OI on MRC-5 cells was detected by the CCK-8 assay.Western blot was used to detect the protein levels ofα-smooth muscle actin(α-SMA),collagen 1α1(COL1A1),fibronectin(FN),phosphorylated and total Smad2/3,and nuclear facor-E2 related factor 2(Nrf2).Real-time fluorescence quantitative PCR was used to detect the mRNA expression ofα-SMA,COL1A1 and FN.Reactive oxygen species(ROS)levels were assessed by fluorescence microscopy and flow cytometry.Intracellular glutathione(GSH)concentrations were measured by spectrophotometry.Results Pretreatment with 4-OI was able to inhibit TGF-β1-induced protein overexpression ofα-SMA,COL1A1 and FN(F=122.8,51.5,27.2,all P<0.05),and increased mRNA levels(F=29.83,51.62,94.82,all P<0.01).In addition,4-OI inhibited TGF-β1-mediated phosphorylation of Smad2/3 proteins in a dose-dependent manner(F=21.80,36.69,P<0.01 for both).Pretreatment with 4-OI also reversed increased ROS levels(P<0.01)induced by TGF-β1 and enhanced GSH concentrations via disinhibition of TGF-β1(P<0.05).The inhibitory effect of TGF-β1 on Nrf2 expression was alleviated and Nrf2 nuclear translocation was uplifted by 4-OI pretreatment(P<0.05).After silencing Nrf2,4-OI was unable to inhibit the increased protein expression of COL1A1 induced by TGF-β1,but was still able to inhibit the increased expression ofα-SMA and FN protein induced by TGF-β1(P<0.05).Conclusions 4-OI could inhibit lung fibroblast-to-myofibroblast differentiation partially via Nrf2 activation.

关 键 词：肺纤维化 4-辛基衣康酸 氧化应激 核因子E2相关因子2 

分 类 号：R563[医药卫生—呼吸系统]