Division of Gastroenterology and Hepatology, Department of Medicine, Indiana University School of Medicine Research, Indianapolis, IN.
Richard L. Roudebush VA Medical Center, Indiana University School of Medicine Research, Indianapolis, IN.
Hepatology. 2021 Jun;73(6):2397-2410. doi: 10.1002/hep.31497. Epub 2021 Apr 19.
Following liver injury, mast cells (MCs) migrate into the liver and are activated in patients with cholestasis. Inhibition of MC mediators decreases ductular reaction (DR) and liver fibrosis. Transforming growth factor beta 1 (TGF-β1) contributes to fibrosis and promotes liver disease. Our aim was to demonstrate that reintroduction of MCs induces cholestatic injury through TGF-β1.
Wild-type, Kit (MC-deficient), and multidrug resistance transporter 2/ABC transporter B family member 2 knockout mice lacking l-histidine decarboxylase were injected with vehicle or PKH26-tagged murine MCs pretreated with 0.01% dimethyl sulfoxide (DMSO) or the TGF-β1 receptor inhibitor (TGF-βRi), LY2109761 (10 μM) 3 days before sacrifice. Hepatic damage was assessed by hematoxylin and eosin (H&E) and serum chemistry. Injected MCs were detected in liver, spleen, and lung by immunofluorescence (IF). DR was measured by cytokeratin 19 (CK-19) immunohistochemistry and F4/80 staining coupled with real-time quantitative PCR (qPCR) for interleukin (IL)-1β, IL-33, and F4/80; biliary senescence was evaluated by IF or qPCR for p16, p18, and p21. Fibrosis was evaluated by sirius red/fast green staining and IF for synaptophysin 9 (SYP-9), desmin, and alpha smooth muscle actin (α-SMA). TGF-β1 secretion/expression was measured by enzyme immunoassay and qPCR. Angiogenesis was detected by IF for von Willebrand factor and vascular endothelial growth factor C qPCR. In vitro, MC-TGF-β1 expression/secretion were measured after TGF-βRi treatment; conditioned medium was collected. Cholangiocytes and hepatic stellate cells (HSCs) were treated with MC-conditioned medium, and biliary proliferation/senescence was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and qPCR; HSC activation evaluated for α-SMA, SYP-9, and collagen type-1a expression. MC injection recapitulates cholestatic liver injury characterized by increased DR, fibrosis/TGF-β1 secretion, and angiogenesis. Injection of MC-TGF-βRi reversed these parameters. In vitro, MCs induce biliary proliferation/senescence and HSC activation that was reversed with MCs lacking TGF-β1.
Our study demonstrates that reintroduction of MCs mimics cholestatic liver injury and that MC-derived TGF-β1 may be a target in chronic cholestatic liver disease.
在肝损伤后，肥大细胞（MCs）迁移到肝脏并在胆汁淤积患者中被激活。MC 介质的抑制可减少胆管反应（DR）和肝纤维化。转化生长因子β 1（TGF-β1）有助于纤维化并促进肝病。我们的目的是证明通过 TGF-β1 重新引入 MC 可诱导胆汁淤积性损伤。
野生型、Kit（MC 缺陷型）和多药耐药转运蛋白 2/ABC 转运体 B 家族成员 2 敲除小鼠缺乏 l-组氨酸脱羧酶，在处死前 3 天用载体或预处理过的 PKH26 标记的鼠 MC 进行注射，预处理方法为 0.01%二甲基亚砜（DMSO）或 TGF-β1 受体抑制剂（TGF-βRi）LY2109761（10 μM）。通过苏木精和伊红（H&E）和血清化学评估肝损伤。通过免疫荧光（IF）检测肝、脾和肺中的注射 MC。通过细胞角蛋白 19（CK-19）免疫组化和 F4/80 染色与白细胞介素（IL）-1β、IL-33 和 F4/80 的实时定量 PCR（qPCR）测量 DR；通过 IF 或 qPCR 测量 p16、p18 和 p21 评估胆汁上皮细胞衰老。通过天狼星红/快绿染色和突触素 9（SYP-9）、结蛋白和α平滑肌肌动蛋白（α-SMA）的 IF 评估纤维化。通过酶联免疫吸附试验和 qPCR 测量 TGF-β1 的分泌/表达。通过 IF 检测血管内皮生长因子 C 的 von Willebrand 因子和 qPCR 检测血管生成。在体外，用 TGF-βRi 处理后测量 MC-TGF-β1 的表达/分泌；收集条件培养基。用 MC 条件培养基处理胆管细胞和肝星状细胞（HSCs），并用 3-（4,5-二甲基噻唑-2-基）-5-（3-羧基甲氧基苯基）-2-（4-磺基苯基）-2H-四唑和 qPCR 测量胆管增殖/衰老；通过α-SMA、SYP-9 和胶原 1a 表达评估 HSC 激活。MC 注射重现了以 DR、纤维化/TGF-β1 分泌和血管生成增加为特征的胆汁淤积性肝损伤。注射 MC-TGF-βRi 逆转了这些参数。在体外，MC 诱导胆管增殖/衰老和 HSC 激活，缺乏 TGF-β1 的 MC 可逆转这些作用。
我们的研究表明，重新引入 MC 可模拟胆汁淤积性肝损伤，而 MC 衍生的 TGF-β1 可能是慢性胆汁淤积性肝病的治疗靶点。
