目的 探究积雪草苷抑制转化生长因子-β1（transforming growth factor-β1，TGF-β1）诱导的心肌成纤维细胞活化的作用机制。方法 采用TGF-β1诱导建立心肌成纤维细胞活化模型，给予积雪草苷处理后，采用CCK-8法检测积雪草苷对TGF-β1诱导的心肌成纤维细胞活力的影响；采用免疫荧光检测积雪草苷对TGF-β1诱导的心肌成纤维细胞活化的影响；采用划痕实验检测积雪草苷对TGF-β1诱导的心肌成纤维细胞迁移能力的影响。通过GEO数据库筛选纤维化相关转录组数据，对差异表达基因进行基因本体（gene ontology，GO）功能及京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路富集分析，采用分子对接和分子动力学模拟检测积雪草苷与核心靶点的结合能力。通过qRT-PCR检测积雪草苷对核心靶点mRNA表达的影响，采用ELISA试剂盒检测积雪草苷对TGF-β1诱导的心肌成纤维细胞上清液中软骨寡聚蛋白（cartilage oligomeric protein，COMP）和甘露糖结合凝集素2（mannose-binding lectin 2，MBL2）水平的影响。结果 积雪草苷显著抑制TGF-β1诱导的心肌成纤维细胞活化（P＜0.05、0.01），并提高细胞迁移能力（P＜0.01）。GEO数据库筛选出542个差异表达基因，主要影响MHC Ⅱ类蛋白复合物结合、浓缩染色体、超分子纤维组织等生物功能和吞噬体、细胞黏附分子、哮喘等信号通路。分子对接结果显示，积雪草苷与吞噬体通路中的磷脂酰肌醇3-激酶催化亚基3（phosphatidylinositol 3-kinase catalytic subunit 3，PIK3C3）、动力蛋白细胞质2重链1（dynein cytoplasmic 2 heavy chain 1，DYNC2H1）、微管蛋白（tubulin，TUB）结合密切。分子动力学模拟结果显示，积雪草苷可以促进PIK3C3的结构稳定。qRT-PCR结果显示，积雪草苷显著下调PIK3C3、DYNC2H1、TUB的mRNA表达（P＜0.01）。ELISA结果显示，积雪草苷显著降低COMP和MBL2的释放（P＜0.01）。结论 积雪草苷可以通过抑制PIK3C3、DYNC2H1、TUB的表达，抑制吞噬体的形成，进而减少COMP、MBL2等促纤维化因子的释放，降低心肌成纤维细胞活化，减轻TGF-β1诱导的细胞损伤。

Objective To investigate the mechanism of asiaticoside (ASS) in inhibiting transforming growth factor-β1 (TGF-β1)-induced activation of cardiac fibroblasts.Methods A cardiac fibroblasts activation model was established by TGF-β1 induction. After treatment with ASS, CCK-8 method was used to detect the effect of ASS on viability of TGF-β1-induced cardiac fibroblast. Immunofluorescence was used to detect the effect of ASS on activation of TGF-β1-induced cardiac fibroblasts. Scratch assay was used to detect the effect of ASS on migration ability of TGF-β1-induced cardiac fibroblasts. Fibrosis-related transcriptomic data were screened from GEO database, gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed on differentially expressed genes, molecular docking and molecular dynamics simulation were used to detect the binding ability of ASS to core targets. The effect of ASS on mRNA expressions of core targets was detected by qRT-PCR, and the effect of ASS on levels of cartilage oligomeric protein (COMP) and mannose binding lectin 2 (MBL2) in supernatant of TGF-β1-induced cardiac fibroblasts was detected by ELISA.Results ASS significantly inhibited TGF-β1-induced activation of cardiac fibroblasts (P < 0.05, 0.01), and increased cell migration ability (P < 0.01). The GEO database identified 542 differentially expressed genes, which mainly affected biological functions such as MHC class II protein complex binding, chromosome condensation and supramolecular fibrous tissue, as well as signaling pathways such as phagosomes, cell adhesion molecules and asthma. The molecular docking results showed that ASS closely bind to phosphatidylinositol 3-kinase catalytic subunit 3 (PIK3C3), dynein cytoplasmic heavy chain 1 (DYNC2H1) and tubulin (TUB) in phagosome pathway. The molecular dynamics simulation results showed that ASS could promote the structural stability of PIK3C3. qRT-PCR results showed that ASS significantly down-regulated the mRNA expressions of PIK3C3, DYNC2H1 and TUB (P < 0.01). ELISA results showed that ASS significantly reduced the release of COMP and MBL2 (P < 0.01).Conclusion ASS could inhibit the expressions of PIK3C3, DYNC2H1 and TUB, suppress the formation of phagosomes, reduce the release of pro-fibrotic factors such as COMP and MBL2, decrease myocardial fibroblast activation, and alleviate TGF-β1-induced cell damage.

R285.5

国家自然科学基金重点资助项目（82030124）；国家自然科学基金重点资助项目（82174015）；中国中医科学院西苑医院能力提升项目（XYZX0303-03）