二甲双胍通过激活线粒体自噬抑制人肺成纤维细胞的活化^*

Metformin inhibits the activation of human lung fibroblasts through mitophagy

摘要

目的 探讨二甲双胍对肺纤维化的作用,阐明二甲双胍调节线粒体自噬抑制人肺成纤维细胞活化的作用机制。方法 采用10 ng/mL转化生长因子-β1(TGF-β1)诱导人肺成纤维细胞建立纤维化细胞模型,给予2 mmol/L二甲双胍干预24 h,通过Western blot、ELISA实验检测纤维化标志物α-平滑肌动蛋白(α-SMA)、Ⅰ型胶原蛋白α1链(Col1α1)和白细胞介素-6(IL-6)。结合转录组数据分析(GSE264038)确定线粒体自噬通路,使用Western blot实验检测线粒体自噬相关蛋白PTEN诱导激酶1(PINK1)、Parkin、p62。利用自噬抑制剂SBI-0206965预处理后再次检测以上纤维化标志物。结果 Western blot与ELISA实验结果显示,TGF-β1成功诱导了人肺成纤维细胞的纤维化模型,表现为α-SMA、Col1α1及IL-6表达水平的显著上调。与TGF-β1模型组比较,TGF-β1联合二甲双胍处理组的α-SMA表达、Col1α1和IL-6分泌明显降低,表明二甲双胍干预能显著逆转上述纤维化表型。转录数据组分析显示二甲双胍与线粒体自噬通路激活相关。Western blot与ELISA实验结果进一步验证二甲双胍激活了线粒体自噬通路,表现为PINK1、Parkin及p62蛋白表达的同步升高。在使用自噬抑制剂SBI-0206965预处理人肺成纤维细胞抑制其线粒体自噬后,与TGF-β1联合二甲双胍处理组比较,SBI-0206965预处理组的α-SMA、Col1α1和IL-6表达水平升高,表明二甲双胍的抗纤维化作用被部分抵消。结论 二甲双胍通过激活PINK1/Parkin介导的线粒体自噬来抑制人肺成纤维细胞活化,为抗肺纤维化治疗提供新靶点。

Abstract

Objective To explore the effect of metformin on pulmonary fibrosis and clarify the mechanism by which metformin regulates mitophagy to inhibit the activation of human lung fibroblasts. Methods Human lung fibroblasts were induced by 10 ng/mL transforming growth factor beta 1(TGF-β1) to establish a fibrosis cell model, and then treated with 2 mmol/L metformin for 24 hours. Western blot and ELISA experiment were used to detect fibrosis markers alpha-smooth muscle actin (α-SMA),Alpha-1 type Ⅰcollagen(Col1α1), and interleukin-6 (IL-6). The mitophagy pathway was determined by transcriptome data analysis (GSE264038), and the mitophagy-related proteins PTEN-induced kinase 1 (PINK1), Parkin and p62 were detected by Western blot. After pretreatment with the autophagy inhibitor SBI-0206965, fibrosis markers were detected again. Results Western blot and ELISA experiment results demonstrated that TGF-β1 successfully induced a fibrotic model in human lung fibroblasts, as evidenced by significantly upregulated expression levels of α-SMA, Col1α1, and IL-6. Compared with the TGF-β1 model group, the group treated with TGF-β1 combined with metformin showed markedly reduced α-SMA expression, Col1α1 and IL-6 secretion, indicating that metformin intervention significantly reversed the fibrotic phenotype. Transcriptomic analysis revealed an association between metformin and the activation of the mitophagy pathway. Further validation by Western blot and ELISA experiments confirmed that metformin activated the mitophagy pathway, manifested by synchronous increases in the protein expression of PINK1, Parkin, and p62. After inhibiting mitophagy in human lung fibroblasts by pretreatment with the autophagy inhibitor SBI-0206965, the expression levels of α-SMA, Col1α1, and IL-6 were increased in the SBI-0206965 pretreatment group compared to the TGF-β1 plus metformin group, suggesting that the anti-fibrotic effect of metformin was partially attenuated. Conclusion Metformin inhibits the activation of human pulmonary fibroblasts by activating PINK1/Parkin mediated mitophagy, which may provide a new target for the treatment of pulmonary fibrosis.

关键词

肺纤维化 / 二甲双胍 / 线粒体自噬 / 转化生长因子-β1 / PTEN诱导激酶1/Parkin信号通路

Key words

Pulmonary fibrosis / Metformin / Mitophagy / Transforming growth factor beta 1 / PTEN-induced kinase 1(PINK1)/Parkin signaling pathway

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基金资助

^*吉林省科学技术厅重大疾病防治重大科技专项(20220303003SF);吉林省科学技术厅科技发展计划项目(20220505030ZP);长春医学高等专科学校2024年度科学研究项目(2024KJ04)