摘要
目的探究解旋酶样转录因子(HLTF)对血管紧张素Ⅱ(AngⅡ)诱导的心脏成纤维细胞(CF)中纤维化功能和表型的影响及作用机制。方法分离培养原代小鼠CF,用浓度为1μmol/L的AngⅡ处理CF,分为正常组和AngⅡ组。同时分别用腺病毒转染下调或上调HLTF的表达,将细胞进一步分为下调对照+AngⅡ组和下调HLTF+AngⅡ组及上调对照+AngⅡ组和上调HLTF+AngⅡ组。采用CCK-8法和细胞划痕实验检测细胞的增殖和迁移能力。RNA测序筛选相关差异基因。蛋白质印迹法(Western blotting)检测HLTF、平滑肌肌动蛋白α(α-SMA)和R-spondin1(Rspo1)蛋白水平。实时荧光定量聚合酶链反应检测Ⅰ型胶原蛋白α1(COL1A1)mRNA水平。免疫荧光染色检测α-SMA水平。结果AngⅡ诱导CF后HLTF蛋白及COL1A1 mRNA水平升高,CF增殖及迁移能力增强,且免疫荧光染色显示α-SMA表达增强(P<0.05)。下调HLTF+AngⅡ组与下调对照组比,CF中HLTF、α-SMA蛋白及COL1A1 mRNA均降低,CF增殖及迁移能力减弱(P<0.05)。相反上调HLTF进一步促进AngⅡ诱导CF中HLTF、α-SMA蛋白及COL1A1 mRNA表达,促进CF的增殖和迁移能力(P<0.05)。此外,RNA测序显示在下调HLTF后差异基因中Rspo1改变最显著。同时Western blotting结果表明,AngⅡ诱导的CF中HLTF下调或上调,Rspo1蛋白水平相应降低或升高。结论下调HLTF可缓解AngⅡ诱导的CF纤维化功能和表型转换,且可能通过下调Rspo1改善心脏纤维化。
Objective To investigate the effect and mechanisms of helicase-like transcription factor(HLTF)on fibrosis function and phenotype in angiotensinⅡ(AngⅡ)-induced cardiac fibroblasts(CF).Methods Primary mouse CF were isolated and cultured,and treated with 1μmol/L AngⅡ.Cells were divided into normal group and AngⅡgroup.Additionally,adenoviral transfection was used to modulate HLTF expression,resulting in additional groups:downregulation control+AngⅡgroup,HLTF downregulation+AngⅡgroup,upregulation control+AngⅡgroup and HLTF upregulation+AngⅡgroup.Cell proliferation and migration were assessed using the CCK-8 assay and wound healing assay.RNA sequencing identified differentially expressed genes.Western blotting was used to analyze the protein levels of HLTF,smooth muscle actinα(α-SMA)and R-spondin1(Rspo1).Quantitative real-time PCR assessed mRNA levels of collagen typeⅠ(COL1A1).Immunofluorescence staining was used to assessα-SMA expression.Results AngⅡstimulation increased HLTF protein and COL1A1 mRNA levels,enhanced CF proliferation and migration,and elevatedα-SMA expression as shown by immunofluorescence staining(P<0.05).Compared with the control group,the HLTF downregulation+AngⅡgroup showed reduced levels of HLTF,α-SMA protein,and COL1A1 mRNA,as well as decreased CF proliferation and migration(P<0.05).Conversely,HLTF upregulation promoted AngⅡ-induced HLTF,α-SMA protein,and COL1A1 mRNA expression,further enhancing CF proliferation and migration(P<0.05).RNA sequencing revealed that Rspo1 was the most significantly altered gene after HLTF downregulation.Western blotting confirmed that HLTF downregulation or upregulation correspondingly decreased or increased Rspo1 protein levels in AngⅡ-treated CF.Conclusion HLTF downregulation can alleviate AngⅡ-induced fibrosis function and phenotype transformation in CF,potentially by suppressing Rspo1,thereby improving cardiac fibrosis.
作者
刘双
张博方
陈静
LIU Shuang;ZHANG Bofang;CHEN Jing(Department of Cardiology,Renmin Hospital of Wuhan University,Cardiovascular Research Institute,Wuhan University,Hubei Key Laboratory of Cardiology,Wuhan 430060,Hubei,China)
出处
《心血管病学进展》
2025年第2期166-172,共7页
Advances in Cardiovascular Diseases
基金
国家自然科学基金(82470336,82100287)。
