Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Sapienza University of Rome, 00161 Rome, Italy.
Division of Gastroenterology and Hepatology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Cells. 2022 May 9;11(9):1591. doi: 10.3390/cells11091591.
BACKGROUND & AIMS: Cholangiocytes are the target cells of liver diseases that are characterized by biliary senescence (evidenced by enhanced levels of senescence-associated secretory phenotype, SASP, e.g., TGF-β1), and liver inflammation and fibrosis accompanied by altered bile acid (BA) homeostasis. Taurocholic acid (TC) stimulates biliary hyperplasia by activation of 3',5'-cyclic cyclic adenosine monophosphate (cAMP) signaling, thereby preventing biliary damage (caused by cholinergic/adrenergic denervation) through enhanced liver angiogenesis. Also: (i) α-calcitonin gene-related peptide (α-CGRP, which activates the calcitonin receptor-like receptor, CRLR), stimulates biliary proliferation/senescence and liver fibrosis by enhanced biliary secretion of SASPs; and (ii) knock-out of α-CGRP reduces these phenotypes by decreased cAMP levels in cholestatic models. We aimed to demonstrate that TC effects on liver phenotypes are dependent on changes in the α-CGRP/CALCRL/cAMP/PKA/ERK1/2/TGF-β1/VEGF axis.
Wild-type and mice were fed with a control (BAC) or TC diet for 1 or 2 wk. We measured: (i) CGRP levels by both ELISA kits in serum and by PCR in isolated cholangiocytes (CALCA gene for α-CGRP); (ii) CALCRL immunoreactivity by immunohistochemistry (IHC) in liver sections; (iii) liver histology, intrahepatic biliary mass, biliary senescence (by β-GAL staining and double immunofluorescence (IF) for p16/CK19), and liver fibrosis (by Red Sirius staining and double IF for collagen/CK19 in liver sections), as well as by PCR for senescence markers in isolated cholangiocytes; and (iv) phosphorylation of PKA/ERK1/2, immunoreactivity of TGF-β1/TGF- βRI and angiogenic factors by IHC/immunofluorescence in liver sections and PCR in isolated cholangiocytes. We measured changes in BA composition in total liver by liquid chromatography/mass spectrometry.
TC feeding increased CALCA expression, biliary damage, and liver inflammation and fibrosis, as well as phenotypes that were associated with enhanced immunoreactivity of the PKA/ERK1/2/TGF-β1/TGF-βRI/VEGF axis compared to BAC-fed mice and phenotypes that were reversed in α-CGRP mice fed TC coupled with changes in hepatic BA composition.
Modulation of the TC/ α-CGRP/CALCRL/PKA/ERK1/2/TGF-β1/VEGF axis may be important in the management of cholangiopathies characterized by BA accumulation.
胆管细胞是肝脏疾病的靶细胞，其特征是胆汁衰老（表现为衰老相关分泌表型（SASP）水平升高，例如 TGF-β1），以及伴有胆汁酸（BA）稳态改变的肝炎症和纤维化。牛磺胆酸（TC）通过激活 3'，5'-环磷酸腺苷（cAMP）信号刺激胆汁增生，从而通过增强肝血管生成来防止胆汁损伤（由胆碱能/肾上腺素能去神经支配引起）。此外：（i）α-降钙素基因相关肽（α-CGRP，其激活降钙素受体样受体，CRLR）通过增强 SASP 的胆汁分泌来刺激胆汁增殖/衰老和肝纤维化；和（ii）在胆淤积模型中，α-CGRP 的敲除通过降低 cAMP 水平来减少这些表型。我们旨在证明 TC 对肝脏表型的影响取决于 α-CGRP/CALCRL/cAMP/PKA/ERK1/2/TGF-β1/VEGF 轴的变化。
用对照（BAC）或 TC 饮食喂养野生型和 α-CGRP 基因敲除（α-CGRP-/-）小鼠 1 或 2 周。我们测量了：（i）通过 ELISA 试剂盒在血清中以及通过 PCR 在分离的胆管细胞（CALCA 基因用于 α-CGRP）中测量 CGRP 水平；（ii）通过免疫组化（IHC）在肝切片中测量 CALCRL 免疫反应性；（iii）肝组织学、肝内胆管质量、胆汁衰老（通过β-GAL 染色和 p16/CK19 的双重免疫荧光（IF））和肝纤维化（通过 Red Sirius 染色和胶原/CK19 在肝切片中的双重 IF），以及分离的胆管细胞中衰老标志物的 PCR；和（iv）通过 IHC/免疫荧光在肝切片和分离的胆管细胞中的 PCR 测量 PKA/ERK1/2、TGF-β1/TGF-βRI 和血管生成因子的磷酸化。我们通过液相色谱/质谱法测量总肝中 BA 组成的变化。
与 BAC 喂养的小鼠相比，TC 喂养增加了 CALCA 表达、胆汁损伤和肝炎症和纤维化，以及与增强的 PKA/ERK1/2/TGF-β1/TGF-βRI/VEGF 轴的免疫反应性相关的表型，并且在 TC 喂养的 α-CGRP 基因敲除小鼠中，这些表型发生了逆转，并伴有肝 BA 组成的变化。
TC/α-CGRP/CALCRL/PKA/ERK1/2/TGF-β1/VEGF 轴的调节可能在以 BA 积累为特征的胆管病的治疗中很重要。
