2025 年第 4 期 第 20 卷

胰高血糖素样肽1（28-36）酰胺靶向线粒体相关途径抑制转化生长因子β介导的肺成纤维细胞活化

Glucagon like peptide 1 (28-36) amide targets mitochondrial related pathways to inhibit transforming growth factor β-mediated activation of pulmonary fibroblasts

作者：董泽海1侯思彤1赏霖骄2刘雨欣2吕晓希2于洋1王晓霞1

英文作者：Dong Zehai1 Hou Sitong1 Shang Linjiao2 Liu Yuxin2 Lyu Xiaoxi2 Yu Yang1 Wang Xiaoxia1

单位：1北京医院内分泌科国家老年医学中心中国医学科学院北京协和医学院研究生院，北京100730；2呼吸和共病全国重点实验室中国医学科学院北京协和医学院药物研究所，北京100050

英文单位：1Department of Endocrinology Beijing Hospital National Center of Gerontology Institute of Geriatric Medicine Chinese Academy of Medical Sciences Graduate School of Peking Union Medical College Beijing 100730 China; 2State Key Laboratory of Respiratory Health and Multimorbidity Institute of Materia Medica Chinese Academy of Medical Sciences & Peking Union Medical College Beijing 100050 China

关键词：胰高血糖素样肽1（28-36）酰胺；肺纤维化；转化生长因子β；线粒体；配体垂钓

英文关键词：Glucagonlikepeptide1(28-36)amide;Pulmonaryfibrosis;Transforminggrowthfactor-β;Mitochondria;Ligandfishing

• 摘要：

• 目的 探讨胰高血糖素样肽1（GLP-1）代谢片段GLP-1（28-36）酰胺对转化生长因子β（TGF-β）介导的肺成纤维细胞增殖和活化的影响及可能的作用机制。方法 取小鼠肺成纤维细胞（MLg细胞）分为对照组、TGF-β组、TGF-β+利拉鲁肽组、TGF-β+GLP-1（28-36）酰胺组，通过蛋白质印迹法检测MLg细胞Smad同源物3(SMAD3)磷酸化水平；细胞计数试剂盒8法检测MLg细胞增殖情况；通过带生物素标记的GLP-1（28-36）酰胺［BIOT（28-36）］和链霉素琼脂糖对MLg细胞裂解物进行配体垂钓；对垂钓得到的蛋白样品进行高效液相色谱-串联质谱分析；根据质谱分析数据筛选丰度前100的蛋白进行生物信息学分析，进一步筛选可能与GLP-1（28-36）酰胺相互结合的蛋白；通过Autodock vina1.5.6软件进行分子对接初步验证蛋白之间的结合。结果 与对照组比较，TGF-β组MLg细胞SMAD3磷酸化水平升高（P＜0.01）、细胞增殖率上升（P＜0.05）。与TGF-β组比较，TGF-β+GLP-1（28-36）酰胺组MLg细胞SMAD3磷酸化水平下降［（0.677±0.046）比（1.414±0.037）］（P＜0.05），增殖率差异无统计学意义（P＞0.05）。生物信息学分析筛选得到线粒体蛋白YME1样1ATP酶（YME1L1）、磷酸甘油酸变位酶5（PGAM5）、线粒体翻译延伸因子Tu（TUFM）、热休克蛋白家族D成员1（HSPD1）、溶质载体家族25成员5（SLC25A5）可能与GLP-1（28-36）酰胺相互结合。Autodock vina1.5.6软件进行分子对接并使用Pymol 3.1软件可视化处理，验证上述蛋白与GLP-1（28-36）酰胺的Docking Score均＜-4 kcal/mol。结论 GLP-1（28-36）酰胺能抑制TGF-β介导的肺成纤维细胞活化，其可能是通过线粒体相关途径发挥作用的，能与GLP-1（28-36）酰胺直接结合的蛋白可能是YME1L1、PGAM5、TUFM、HSPD1、SLC25A5等线粒体蛋白。

• Objective To investigate the effect of glucagon like peptide 1 (GLP-1) metabolite GLP-1(28-36) amide on transforming growth factor-β (TGF-β) -mediated proliferation and activation of pulmonary fibroblasts and its possible mechanism. Methods Mouse lung fibroblasts (MLg cells) were divided into control group, TGF-β group, TGF-β+ liraglutide group, TGF-β+GLP-1(28-36) amide group. The phosphorylation level of Smad homolog 3(SMAD3) in MLg cells was detected by Western blotting. Cell counting kit-8 was used to detect the proliferation of MLg cells. Ligand fishing of MLg cell lysates was performed by biotin-labeled GLP-1(28-36) amide ［BIOT(28-36)］ and streptomycin agarose. The protein samples obtained from fishing were analyzed by high performance liquid chromatography-tandem mass spectrometry. According to the mass spectrometry data, the top 100 abundant proteins were screened for bioinformatics analysis to further screen the proteins that might interact with GLP-1(28-36) amide. Autodock vina1.5.6 software was used to verify the binding between proteins. Results Compared with the control group, the phosphorylation level of SMAD3 in MLg cells was increased (P<0.01) and the cell proliferation rate was increased (P<0.05) in TGF-β group. Compared with TGF-β group, the phosphorylation level of SMAD3 in TGF-β+GLP-1(28-36) amide group was decreased ［(0.677±0.046) vs (1.414±0.037)］(P<0.05), and there was no significant difference in proliferation rate (P>0.05). Bioinformatics analysis showed that mitochondrial protein YME1-like 1atpase (YME1L1), phosphoglycerate mutase 5 (PGAM5), mitochondrial translation elongation factor Tu (TUFM), heat shock protein family D member 1 (HSPD1), solute carrier family 25 member 5 (SLC25A5) may be related to GLP-1(28 -36) amides bind to each other. Autodock vina1.5.6 software was used for molecular Docking and Pymol 3.1 software was used for visualization. The docking scores of the above-mentioned proteins with GLP-1(28-36) amide were all <-4 kcal/mol. Conclusion GLP-1(28-36) amide can inhibit TGF-β-mediated activation of pulmonary fibroblasts, which may act through mitochondrial related pathways. The proteins that can directly bind to GLP-1(28-36) amide may be mitochondrial proteins such as YME1L1, PGAM5, TUFM, HSPD1, and SLC25A5.