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3-吲哚乙酸通过抑制Wnt/β-catenin通路减少肝星状细胞活化并减轻肝脏类器官MASH相关肝纤维化

Indole-3-acetic acid reduces hepatic stellate cell activation by inhibiting the Wnt/β-catenin pathway and alleviates MASH-associated liver fibrosis in liver organoids

    摘要
  • 摘要:
    目的 探讨3-吲哚乙酸（indole-3-acetic acid, IAA）在代谢功能障碍相关脂肪性肝炎（metabolic dysfunction associated steatohepatitis, MASH）相关肝纤维化中的作用及可能的分子机制。
    方法 建立人源3D肝脏类器官，使用含游离脂肪酸（free fatty acid, FFA）的培养基诱导MASH模型，共分为5组：空白对照组（Ctrl组）、MASH组（FFA处理）、IAA低剂量组（FFA＋4 μmol/L IAA）、IAA中剂量组（FFA＋20 μmol/L IAA）、IAA高剂量组（FFA＋100 μmol/L IAA）。使用转化生长因子β1（transforming growth factor-β1, TGF-β1）处理肝星状细胞系LX-2，共分为4组：空白对照组（Ctrl组）、TGF-β1处理组、IAA中剂量组（TGF-β1＋20 μmol/L IAA）、IAA高剂量组（TGF-β1＋100 μmol/L IAA）。通过苏木精-伊红染色与天狼星红染色评估病理学改变；采用免疫荧光检测平滑肌肌动蛋白α（smooth muscle actin α, α-SMA）、COL1A1、β-catenin表达；实时荧光定量反转录聚合酶链式反应检测COL1A1、TGFB、CTNNB1、CCND1、MMP7、ACTA2等基因表达；Western印迹法检测α-SMA、β-catenin表达；CCK-8法检测细胞增殖水平。应用Wnt通路激动剂CHIR99021进行挽救实验。
    结果 FFA处理成功诱导3D肝脏类器官MASH表型。与MASH组相比，IAA各剂量组胶原面积百分比降低（P＜0.05），肝星状细胞活化标志物α-SMA及COL1A1表达水平下降（P＜0.05）。细胞实验中，IAA中剂量组和高剂量组肝星状细胞活化相关基因（TGFB、ACTA2、COL1A1）的表达均低于TGF-β1处理组（P＜0.05），IAA高剂量组细胞增殖水平低于TGF-β1处理组（P＜0.05）。与TGF-β1处理组相比，IAA可下调β-catenin的水平，并抑制Wnt通路下游基因表达（P＜0.05）；激动Wnt通路可部分逆转IAA的作用。
    结论 IAA通过抑制Wnt/β-catenin信号通路减少肝星状细胞的增殖和活化，并减轻MASH相关肝纤维化。

     

    Abstract:
    Objective To explore the effects of indole-3-acetic acid (IAA) on metabolic dysfunction associated steatohepatitis (MASH) related liver fibrosis and its molecular mechanism.
    Methods A human-derived 3D liver organoid was established and free fatty acid (FFA) was added to induce MASH model. The organoids were divided into five groups: control group, MASH group, low dose IAA group (FFA＋4 μmol/L IAA), medium dose IAA group (FFA＋20 μmol/L IAA) and high dose IAA group (FFA＋100 μmol/L IAA). Transforming growth factor-β1 (TGF-β1) was administrated to LX-2 cell lines. LX-2 cells were divided into four groups: control group, TGF-β1 group, medium dose IAA group (TGF-β1＋20 μmol/L IAA) and high dose IAA group (TGF-β1＋100 μmol/L IAA). Hematoxylin and eosin staining and Sirius red staining were performed to estimate pathological change. Immunofluorescence was performed to evaluate smooth muscle actin α (α-SMA), COL1A1, β-catenin level. Real-time quantitative reverse transcription polymerase chain reaction was performed to evaluate COL1A1, TGFB, CTNNB1, CCND1, MMP7, ACTA2 levels. Western blotting was performed to evaluate α-SMA and β-catenin level. CCK-8 was performed to evaluate cell proliferation. Wnt pathway agonist CHIR99021 was administrated in rescue experiments.
    Results FFA treatment successfully induced the MASH phenotype in 3D liver organoids. Compared with the MASH group, the percentage of collagen area was reduced in all IAA dose groups (P＜0.05), and the expression levels of hepatic stellate cell activation markers α-SMA and COL1A1 were decreased (P＜0.05). In cell experiments, the expression of hepatic stellate cell activation-related genes (TGFB, ACTA2, COL1A1) in medium dose and high dose IAA groups was lower than that in the TGF-β1 group (P＜0.05), and the cell proliferation level in the high dose IAA group was lower than that in the TGF-β1 group (P＜0.05). Compared with the TGF-β1 group, IAA downregulated β-catenin levels and inhibited the expression of downstream genes in the Wnt pathway (P＜0.05); activation of the Wnt pathway could partially reverse the effects of IAA.
    Conclusions IAA may inhibit the proliferation and activation of hepatic stellate cells by suppressing the Wnt/β-catenin signaling pathway and alleviate MASH-related liver fibrosis.

     

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