The role of 4-octyl itaconate and related mechanisms in lung fibroblast-to-myofibroblast differentiation
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 of α-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 of α-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 of α-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 of α-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.
Lung fibrosis4-octyl itaconateOxidative stressNuclear factor-E2 related factor 2
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