南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (1): 60-69.doi: 10.12122/j.issn.1673-4254.2024.01.08

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桔梗素D通过下调成纤维细胞TRPC6表达改善小鼠肺纤维化

梁梓琛，余常辉，梁世秀，周梓聪，周子丽，孟晓静，邹 飞，蔡绍曦   

  1. 南方医科大学南方医院呼吸与危重症医学科，广东 广州 510515；南方医科大学公共卫生与热带医学学院，广东 广州 510515
  • 发布日期:2024-01-24

Platycodin D improves pulmonary fibrosis in mice by down-regulating TRPC6 expression and reducing ROS production in lung fibroblasts

LIANG Zichen, YU Changhui, LIANG Shixiu, ZHOU Zicong, ZHOU Zili, MENG Xiaojing, ZOU Fei, CAI Shaoxi   

  1. Department of Respiratory and Critical Care Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China
  • Published:2024-01-24

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摘要： 目的 探究桔梗素D（PD）对肺纤维化的影响及其机制。方法 博来霉素（BLM）气道内滴入构建C57BL/6J小鼠肺纤维化模型，再予PD（10 mg/kg）灌胃，28 d后处死小鼠。分组如下：对照组（control）、BLM（10 mg/kg）模型组、BLM（10 mg/kg）+PD（10 mg/kg）组。肺组织石蜡切片HE染色、免疫组化和天狼星红染色评估小鼠肺组织纤维化程度和瞬时受体电位离子通道亚族C成员6（TRPC6）的表达与分布。Western blot检测肺组织α-平滑肌肌动蛋白（α-SMA）的表达水平。小鼠原代肺成纤维细胞体外培养，分别用PD、TRPC6抑制剂醋酸落叶松酯（LA）预处理后加入转化生长因子-β1（TGF-β1）刺激细胞，根据加入的TGF-β1和 PD 浓度不同分组如下：control 组、TGF-β1（10 ng/mL）组、PD（2.5 μmol/L）+TGF-β1 组、PD（5 μmol/L）+TGF-β1 组和 PD（10 μmol/L）+TGF-β1组，LA处理分组如下：control组、TGF-β1（10 ng/mL）组和LA（10 μmol/L）+TGF-β1组，测定细胞存活率、collagen Ⅰ、α-SMA和TRPC6的表达水平、活性氧（ROS）生成水平、线粒体膜电位、细胞增殖能力等指标。利用网络药理学方法结合文献分析PD作用于肺纤维化可能通过的机制。结果 PD可明显减轻BLM诱导小鼠模型的肺部纤维化程度、减少α-SMA表达（P<0.05）。CCK-8结果显示PD、TGF-β1和LA的最适宜刺激浓度分别是10 μmol/L、10 ng/mL和10 μmol/L；5-乙炔基-2'-脱氧尿苷（EdU）染色结果显示PD能够有效抑制肺成纤维细胞增殖；2,7-二氯荧光素二乙酸酯（DCFH-DA）染色显示PD能有效减少TGF-β1诱导的细胞ROS生成（P<0.0001）；线粒体膜电位检测（Jc-1染色）结果显示PD能有效缓解TGF-β1诱导的细胞线粒体膜电位下降（P<0.001）；蛋白电泳结果显示PD能显著抑制TGF-β1诱导的α-SMA和collagen Ⅰ的表达（P<0.05）。网络药理学结合文献分析发现PD可能通过TRPC6作用于肺纤维化。免疫组织化学结果显示PD明显减轻了BLM诱导的肺组织TRPC6表达。蛋白电泳结果显示PD可明显抑制TGF-β1诱导的TRPC6表达（P<0.05）；使用LA可明显抑制细胞TRPC6、α-SMA、collagen Ⅰ的表达（P<0.05）。结论 PD可降低小鼠肺纤维化，其机制可能与下调TRPC6并减少ROS产生有关。

关键词: 肺纤维化；桔梗素D；瞬时受体电位离子通道亚族C成员6；活性氧

Abstract: Objective To assess the effect of platycodin D (PD) for alleviating pulmonary fibrosis in mice and explore the underlying mechanism. Methods C57BL/6J mouse models of pulmonary fibrosis induced by bleomycin injection into the airway were treated with daily intragastric administration of 10 mg/kg PD for 28 days. The changes of pulmonary fibrosis and the expression and distribution of transient receptor potential cation channel subfamily C member 6 (TRPC6) were evaluated with immunohistochemistry, HE staining and Sirius Red staining. Western blotting was used to detect α- SMA expression in the lung tissues of the mice. Primary cultures of mouse lung fibroblasts were pretreated with PD (2.5, 5.0, and 10 μmol/L) or larixyl acetate (LA; 10 μmol/L) before exposure to 10 ng/mL transforming growth factor-β1 (TGF-β1), and the changes in cell survival rate, expressions of collagen I, α-SMA and TRPC6, reactive oxygen species (ROS) production, mitochondrial membrane potential, and cell proliferation capacity were assessed. Network pharmacology analysis was performed to explore the mechanism by which PD alleviated pulmonary fibrosis. Results PD treatment significantly alleviated pulmonary fibrosis and reduced α-SMA expression in BLM-induced mouse models (P<0.05). In TGF-β1-induced primary mouse lung fibroblasts, PD effectively inhibited the cell proliferation, reduced ROS production (P<0.0001), rescued the reduction of mitochondrial membrane potential (P<0.001), and inhibited the expressions of α- SMA and collagen Ⅰ (P<0.05). Network pharmacology analysis suggested that TRPC6 mediated the effect of PD for alleviating pulmonary fibrosis. Immunohistochemistry showed that PD significantly reduced TRPC6 expression in the lung tissues of BLM-induced mice. In primary mouse lung fibroblasts, PD significantly inhibited TGF-β1-induced TRPC6 expression (P<0.05), and LA treatment obviously lowered the expression levels of TRPC6, α-SMA and collagenⅠ (P<0.05). Conclusion PD alleviated pulmonary fibrosis in mice possibly by down-regulating TRPC6 and reducing ROS production.

Key words: pulmonary fibrosis; platycodin D; transient receptor potential cation channel subfamily C member 6; reactive oxygen species