Gao Yun-Xing, Jiang Li-Li, Zhang Qian, Zuo Dong-Ze, Li Xian-Wei
Departments of Immunology, Wannan Medical College, Wuhu 241002, China.
Anhui Provincial Engineering Technology Research Center for Polysaccharide Drugs, Department of Pharmacology, Wannan Medical College, Wuhu 241002, China.
Zhongguo Zhong Yao Za Zhi. 2018 Sep;43(17):3530-3538. doi: 10.19540/j.cnki.cjcmm.20180530.005.
To investigate whether the protection of rutaecarpine against bleomycin-induced pulmonary fibrosis is mediated by inhibiting Notch1/eukaryotic initiation factor 3a (eIF3a) signaling pathway, and whether these effects are related to the synthesis and release of calcitonin gene-related peptide (CGRP) and inhibition of epithelial-mesenchymal transition (EMT) of alveolar epithelial cells, male Sprague-Dawley rats were randomly divided into five groups (=12), respectively, Control group, bleomycin group, rutaecarpine (100, 300 mg·kg⁻¹) group and capsaicin plus rutaecarpine (300 mg·kg⁻¹) group. Bleomycin (5 mg·kg⁻¹) was used to induce pulmonary fibrosis rat model. Rats were given capsaicin (50 mg·kg⁻¹) by subcutaneous injections 1 days before and 7, 14, 21 days after induce pulmonary fibrosis rat model to deplete endogenous CGRP. At the end of experiments, blood was collected from carotid artery to determinate the plasma levels of CGRP by ELISA. Pulmonary tissue change was observed by HE staining. Masson's trichrome stain was used to demonstration collagen deposition. The collagen I expression in pulmonary tissue was measured by immunohistochemisty. The expression of CGRP, Notch1, eIF3a, collagen I, vimentin, alpha-smooth muscle actin (α-SMA), E-cadherin and zonula occludens-1 (ZO-1) was detected by qPCR or Western blot. Compared with the control group, the pulmonary tissue of the bleomycin group showed significant fibrosis, including significant disturbed alveolar structure, marked thickening of the interalveolar septa and dense interstitial infiltration by inflammatory cells and fibroblasts, and concomitantly with the decrease in plasma CGRP and expression of CGRP. Importantly the expression of E-cadherin and ZO-1 was decreased and expression of Notch1, eIF3a, collagen I, vimentin and α-SMA was increased in bleomycin group (<0.05 or <0.01). Compared with the bleomycin group, rutaecarpine (100, 300 mg·kg⁻¹) group significantly reduced bleomycin-induced pulmonary injury concomitantly with the increase in plasma CGRP and expression of CGRP. Importantly the expression of E-cadherin and ZO-1 was increased and expression of Notch1, eIF3a, collagen I, vimentin and α-SMA was decreased by rutaecarpine treatment (<0.05 or <0.01). All these effects of rutaecarpine were abolished by capsaicin.These results suggest that rutaecarpine protects against bleomycin-induced pulmonary fibrosis by inhibiting Notch1/eIF3a signaling pathway, alleviating EMT process, which is related to the increased synthesis and release of CGRP.
为研究吴茱萸碱对博来霉素诱导的肺纤维化的保护作用是否通过抑制Notch1/真核起始因子3a（eIF3a）信号通路介导，以及这些作用是否与降钙素基因相关肽（CGRP）的合成与释放及抑制肺泡上皮细胞上皮-间质转化（EMT）有关，将雄性Sprague-Dawley大鼠随机分为五组（每组n = 12），分别为对照组、博来霉素组、吴茱萸碱（100、300 mg·kg⁻¹）组和辣椒素加吴茱萸碱（300 mg·kg⁻¹）组。采用博来霉素（5 mg·kg⁻¹）诱导大鼠肺纤维化模型。在诱导肺纤维化模型前1天及诱导后7、14、21天，通过皮下注射给予大鼠辣椒素（50 mg·kg⁻¹）以耗尽内源性CGRP。实验结束时，从颈动脉取血，采用酶联免疫吸附测定法（ELISA）测定血浆CGRP水平。通过苏木精-伊红（HE）染色观察肺组织变化。采用Masson三色染色法显示胶原沉积。通过免疫组织化学法检测肺组织中Ⅰ型胶原的表达。采用实时荧光定量聚合酶链反应（qPCR）或蛋白质免疫印迹法（Western blot）检测CGRP、Notch1、eIF3a、Ⅰ型胶原、波形蛋白、α-平滑肌肌动蛋白（α-SMA）、E-钙黏蛋白和紧密连接蛋白1（ZO-1）的表达。与对照组相比，博来霉素组肺组织出现明显纤维化，包括肺泡结构明显紊乱、肺泡间隔显著增厚以及炎性细胞和成纤维细胞密集的间质浸润，同时血浆CGRP及CGRP表达降低。重要的是，博来霉素组E-钙黏蛋白和ZO-1表达降低，Notch1、eIF3a、Ⅰ型胶原、波形蛋白和α-SMA表达增加（P < 0.05或P < 0.01）。与博来霉素组相比，吴茱萸碱（100、300 mg·kg⁻¹）组显著减轻博来霉素诱导的肺损伤，同时血浆CGRP及CGRP表达增加。重要的是，吴茱萸碱治疗使E-钙黏蛋白和ZO-1表达增加，Notch1、eIF3a、Ⅰ型胶原、波形蛋白和α-SMA表达降低（P < 0.05或P < 0.01）。吴茱萸碱的所有这些作用均被辣椒素消除。这些结果表明，吴茱萸碱通过抑制Notch1/eIF3a信号通路、减轻EMT过程来保护大鼠免受博来霉素诱导的肺纤维化，这与CGRP合成和释放增加有关。
