Inhibition of mast cell-derived histamine secretion by cromolyn sodium treatment decreases biliary hyperplasia in cholestatic rodents
Inhibition of mast cell-derived histamine secretion by cromolyn sodium treatment decreases biliary hyperplasia in cholestatic rodents
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DOI：
10.1038/labinvest.2014.129
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发表时间：
2014-12-01
影响因子：
5
通讯作者：
Francis, Heather L.
中科院分区：
文献类型：
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作者：
Kennedy, Lindsey L.;Hargrove, Laura A.;Francis, Heather L.
胆管病的特征是胆管生长和丢失之间的平衡失调。我们已经证明组胺(HA)通过自分泌机制刺激胆管生长。为评价肥大细胞(MC)稳定对胆管增殖的旁分泌作用，用生理盐水或色甘酸钠(24 mg/kgbw/d，抑制肥大细胞组胺释放)给假手术大鼠或BDL大鼠灌胃1周。收集血清、肝块和胆管细胞。实时定量聚合酶链式反应检测胆管细胞组氨酸脱羧酶(HDC)的表达。肝内胆管质量(IBDM)采用免疫组织化学方法检测CK-19。甲苯胺蓝染色测定MC数，并与IBDM相关。用肝切片和纯化的胆管细胞中增殖细胞核抗原的表达来评价细胞增殖情况。我们用实时荧光定量聚合酶链式反应和免疫组织化学检测Bax来评估细胞的凋亡。用实时荧光定量聚合酶链式反应检测MC干细胞因子受体c-kit、蛋白水解酶、类胰淀粉酶的表达。用EIA法测定血清透明质酸水平。在体外，用0.1%牛血清白蛋白(基础)或色甘露(25mU M)处理MC和胆管细胞48h，然后检测HDC的表达、HA水平以及糜酶和类胰蛋白酶的表达。将含或不含色甘膦的MC培养上清液加入胆管细胞，用MU比色法检测细胞增殖，用实时荧光定量聚合酶链式反应检测HDC基因的表达，用EIA法检测HA的释放。在体内，色甘露治疗降低了BDL诱导的：(I)IBDM、MC数量和胆管增殖；(Ii)HDC和MC标记物的表达；以及(Iii)HA水平。克罗莫林治疗后胆管细胞凋亡率增加。体外实验中，色甘露可降低系膜细胞HA的释放、糜酶和类胰蛋白酶的表达，但对胆管细胞无明显影响。色甘油处理的MC上清液可抑制胆管细胞增殖和HA释放。这些研究提供的证据表明，MC组胺是胆管增殖的关键，并可能成为治疗胆管病的靶点。
Cholangiopathies are characterized by dysregulation of the balance between biliary growth and loss. We have shown that histamine (HA) stimulates biliary growth via autocrine mechanisms. To evaluate the paracrine effects of mast cell (MC) stabilization on biliary proliferation, sham or BDL rats were treated by IP-implanted osmotic pumps filled with saline or cromolyn sodium (24 mg/kg BW/day (inhibits MC histamine release)) for 1 week. Serum, liver blocks and cholangiocytes were collected. Histidine decarboxylase (HDC) expression was measured using real-time PCR in cholangiocytes. Intrahepatic bile duct mass (IBDM) was evaluated by IHC for CK-19. MC number was determined using toluidine blue staining and correlated to IBDM. Proliferation was evaluated by PCNA expression in liver sections and purified cholangiocytes. We assessed apoptosis using real-time PCR and IHC for BAX. Expression of MC stem factor receptor, c-kit, and the proteases chymase and tryptase were measured by real-time PCR. HA levels were measured in serum by EIA. In vitro, MCs and cholangiocytes were treated with 0.1% BSA (basal) or cromolyn (25 mu M) for up to 48 h prior to assessing HDC expression, HA levels and chymase and tryptase expression. Supernatants from MCs treated with or without cromolyn were added to cholangiocytes before measuring (i) proliferation by MU assays, (ii) HDC gene expression by real-time PCR and (iii) HA release by EIA. In vivo, cromolyn treatment decreased BDL-induced: (i) IBDM, MC number, and biliary proliferation; (ii) HDC and MC marker expression; and (iii) HA levels. Cromolyn treatment increased cholangiocyte apoptosis. In vitro, cromolyn decreased HA release and chymase and tryptase expression in MCs but not in cholangiocytes. Cromolyn-treated MC supernatants decreased biliary proliferation and HA release. These studies provide evidence that MC histamine is key to biliary proliferation and may be a therapeutic target for the treatment of cholangiopathies.