TXNIP通过诱导氧化应激促进心肌纤维化的发生

Thioredoxin-interacting protein contributes to cardiac fibrosis by elevating oxidative stress in cardiac fibroblasts

目的:探讨硫氧还蛋白-互作蛋白(TXNIP)在心肌纤维化发生过程中的表达变化及其潜在调节机制。方法:通过皮下泵入血管紧张素(Ang)Ⅱ建立心肌纤维化小鼠模型,Western blot检测TXNIP在心肌纤维化小鼠和假手术组小鼠心肌组织中的表达差异。分离和培养新生大鼠的心肌成纤维细胞(CFs),外源性使用人重组Ang Ⅱ和(或)losartan处理CFs,用Westernblot和细胞免疫荧光检测TXNIP表达的变化。构建TXNIP的小干扰RNA(si RNA),利用细胞划痕实验、结晶紫染色、Western blot以及细胞活性氧簇探针等多种方法检测TXNIP对Ang Ⅱ刺激状态下CFs迁移、纤维激活相关蛋白[Ⅰ型胶原蛋白(ColⅠ)、Ⅲ型胶原蛋白(ColⅢ)、波形蛋白(vimentin)和α-平滑肌肌动蛋白(α-SMA)]表达、氧化应激及MAPK信号通路的影响。结果:TXNIP在心肌纤维化小鼠的心肌组织中表达上调。Ang Ⅱ可浓度和时间依赖性地促进CFs中TXNIP表达上调,这种效应可以被losartan完全抵消。敲减TXNIP可以显著抑制Ang Ⅱ诱导的CFs迁移及ColⅠ、ColⅢ、vimentin和α-SMA表达升高。此外,敲减TXNIP可以抑制Ang Ⅱ诱导的细胞氧化应激和MAPK信号通路激活。结论:TXNIP可能通过MAPK信号通路诱导CFs发生氧化应激,进而促进心肌纤维化的发生;TXNIP可能成为治疗心肌纤维化的新靶点。

AIM:To explore the role of thioredoxin-interacting protein(TXNIP)in mediating cardiac fibro-sis.METHODS:In vivo,4 weeks of angiotensin(Ang)Ⅱ infusion was used to induced fibrogenesis in the heart of the mice.In vitro,cardiac fibroblasts(CFs)isolated from neonatal rats were treated with Ang Ⅱ at different concentrations for different durations.The protein expression of TXNIP in the hearts of the mice and CFs was detected by Western blot.The CFs were pretreated with losartan(an Ang Ⅱ type 1 receptor blocker)prior to Ang Ⅱ stimulation,and then TXNIP expres-sion was detected by immunofluorescence and Western blot.Subsequently,after knock-down of TXNIP by si RNA,multi-ple methods including wound healing assay,crystal violet staining,Western blot and ROS detection were used to analyze the changes in cell migration,expression of fibrosis-related proteins collagen typeⅠ(ColⅠ),collagen typeⅢ(ColⅢ),vi-mentin andα-smooth muscle actin(α-SMA),oxidative stress and MAPK signaling in CFs exposed to Ang Ⅱ.RESULTS:In vivo,TXNIP was up-regulated in the hearts of mice with cardiac fibrosis.In vitro,TXNIP was induced by Ang Ⅱ in a dose-and time-dependent manner,but this increase was reversed by losartan.TXNIP knock-down inhibited Ang Ⅱ induced cell migration and decreased the protein levels ofα-SMA,vimentin,ColⅠand ColⅢin Ang Ⅱ-treated CFs.Nota-bly,TXNIP knock-down alleviated cellular oxidative stress and suppressed the activated MAPK signaling pathway in CFs under Ang Ⅱ stimulation.CONCLUSION:TXNIP may participate in the pathogenesis of cardiac fibrosis through pro-moting oxidative stress via MAPK signaling pathway,suggesting that TXNIP may be a promising therapeutic target for treatment of cardiac fibrosis.