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腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)激活具有抗纤维化的作用,但其具体机制仍不十分清楚。本研究拟在心脏成纤维细胞中,明确AMPK激活对血管紧张素Ⅱ(angiotensin Ⅱ,AngⅡ)引起的转化生长因子β1(transforming growth factor-β1,TGFβ1)表达的作用及其具体机制。分离培养成年小鼠心脏成纤维细胞,酶联免疫吸附实验检测TGFβ1蛋白表达,免疫印迹实验检测AMPK活性和CCAAT/增强子结合蛋白β(CCAAT/enhancer-binding proteinβ,C/EBPβ)表达,双荧光素酶报告基因实验检测TGFβ1转录活性。结果显示,给予AMPK激活剂AICAR预处理可以抑制AngⅡ引起的TGFβ1产生增加,这一抑制作用可被AMPK抑制剂Compound C逆转。生物信息学分析显示在小鼠Tgfb1启动子区存在C/EBPβ转录因子可能的结合位点。双荧光素酶报告基因实验表明,对于转染Tgfb1启动子区序列质粒的小鼠胚胎成纤维细胞,AngⅡ可以增加TGFβ1转录活性;但是对于转染缺失C/EBPβ结合位点的Tgfb1启动子区序列质粒的细胞,AngⅡ则不能增加其转录活性,提示C/EBPβ介导了AngⅡ引起的TGFβ1转录表达。进一步免疫印迹实验显示,AICAR可以抑制AngⅡ引起的C/EBPβ增加。上述结果表明,AMPK激活可以抑制AngⅡ引起的TGFβ1表达增加,其作用机制是通过抑制转录因子C/EBPβ表达,进而抑制TGFβ1表达。该研究结果提示了AMPK抗纤维化作用的一个新机制。
AMP-activated protein kinase (AMPK) activation has anti-fibrotic effect, but the specific mechanism is still not very clear. This study aimed to clarify the effect of AMPK activation on the expression of transforming growth factor-β1 (TGFβ1) induced by angiotensin Ⅱ (AngⅡ) and its specific mechanism in cardiac fibroblasts. The cardiac fibroblasts of adult mice were isolated and cultured. The expression of TGFβ1 protein was detected by enzyme-linked immunosorbent assay (ELISA), the activity of AMPK and the expression of CCAAT / enhancer-binding proteinβ (C / EBPβ) Enzyme reporter assay was used to detect TGFβ1 transcriptional activity. The results showed that pretreatment with AMPK activator AICAR can inhibit Ang Ⅱ-induced TGFβ1 production increased, this inhibition can be reversed AMPK inhibitor Compound C. Bioinformatics analysis showed that there is a potential binding site for C / EBPβ transcription factor in mouse Tgfb1 promoter region. Dual luciferase reporter gene assay showed that AngⅡcan increase TGFβ1 transcriptional activity in mouse embryonic fibroblasts transfected with plasmid sequence of Tgfb1 promoter region. However, for the Tgfb1 promoter region sequence lacking C / EBPβ binding sites Plasmid cells, Ang Ⅱ can not increase its transcriptional activity, suggesting that C / EBPβ mediated Ang Ⅱ induced TGFβ1 transcriptional expression. Further Western blotting experiments showed that AICAR can inhibit Ang Ⅱ-induced increase in C / EBPβ. The above results show that AMPK activation can inhibit Ang Ⅱ induced TGFβ1 expression increased its mechanism by inhibiting the transcription factor C / EBPβ expression, thereby inhibiting TGFβ1 expression. The results suggest a new mechanism of AMPK anti-fibrosis.