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 (n=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 (P<0.05 or P<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 (P<0.05 or P<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.
Keywords: Notch1; calcitonin gene-related peptide; eIF3a; epithelial-mesenchymal transition; rat; rutaecarpine.
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