Rupatadine Protects against Pulmonary Fibrosis by Attenuating PAF-Mediated Senescence in Rodents

<div><p>A similar immune response is implicated in the pathogenesis of pulmonary fibrosis and allergic disorders. We investigated the potential therapeutic efficacy and mechanism of rupatadine, a dual antagonist of histamine and platelet-activation factor (PAF), in bleomycin- (BLM-) and silica-induced pulmonary fibrosis. The indicated dosages of rupatadine were administered in rodents with bleomycin or silica-induced pulmonary fibrosis. The tissue injury, fibrosis, inflammatory cells and cytokines, and lung function were examined to evaluate the therapeutic efficacy of rupatadine. The anti-fibrosis effect of rupatadine was compared with an H1 or PAF receptor antagonist, and efforts were made to reveal rupatadine’s anti-fibrotic mechanism. Rupatadine promoted the resolution of pulmonary inflammation and fibrosis in a dose-dependent manner, as indicated by the reductions in inflammation score, collagen deposition and epithelial-mesenchymal transformation, and infiltration or expression of inflammatory cells or cytokines in the fibrotic lung tissue. Thus, rupatadine treatment improved the declined lung function and significantly decreased animal death. Moreover, rupatadine was able not only to attenuate silica-induced silicosis but also to produce a superior therapeutic efficacy compared to pirfenidone, histamine H1 antagonist loratadine, or PAF antagonist CV-3988. The anti-fibrotic action of rupatadine might relate to its attenuation of BLM- or PAF-induced premature senescence because rupatadine treatment protected against the <i>in vivo</i> and <i>in vitro</i> activation of the p53/p21-dependent senescence pathway. Our studies indicate that rupatadine promotes the resolution of pulmonary inflammation and fibrosis by attenuating the PAF-mediated senescence response. Rupatadine holds promise as a novel drug to treat the devastating disease of pulmonary fibrosis.</p> </div

PFC reduces the expression of CD11b on circulating PMNs from rats treated with LPS.

<p>(A) Representative flow cytometry plots detecting CD11b on PMN cells at 6 h after NS or LPS exposure are shown. (B) Mean fluorescence intensity was measured to evaluate the expression of CD11b on PMN cells in different groups. Results are given as mean ± SD (n = 6). * <i>P</i><0.05, ** <i>P</i><0.01 between the indicated groups.</p

Rupatadine reduces enhanced lung density and improves lung functions in fibrotic mice.

<p>(<b>A</b>–<b>C</b>) Rupatadine treatment reduced the BLM-induced lung density shown by micro-CT. Representative micro-CT of main pulmonary lesions of Sham- (A, left), BLM instilled- (A, middle) and rupatadine-treated mice (6.0 mg/kg per day) (A, right) were shown at different slices. Quantification of lung parenchyma density was measured in upper, central and lower pulmonary regions excluding the hilum and bronchi. Scale bar in images = 1 cm. The data are expressed as the mean Hounsfield units (HU) ± SEM of 8 mice per group (B). Rupatadine (6.0 mg/kg per day) reduced parenchymal loss in the fibrotic mice (C). (<b>D</b>) Rupatadine (6.0 mg/kg per day) improved lung functions in the fibrotic mice. Mice were anesthetized with 50 mg/kg i.p. pentobarbital and placed on the flexivent system at the indicated times after bleomycin administration. Mice were mechanically ventilated with a tidal volume of 10 ml/kg and a respiratory rate of 150 breaths/min. The parameters of lung function were calculated by measuring total lung capacity, Snapshot, Quickprime-3, and pressure-volume loops. All perturbations were performed until three acceptable measurements with a coefficient of determination (COD) ≥ 0.9 were recorded for each individual subject. The data are expressed as the mean ± SEM of 6 mice per group. ^#<i>P</i><0.05, ^{# #}<i>P</i><0.01 vs. Sham group; ^*<i>P</i><0.05, ^**<i>P</i><0.01 vs. BLM treated group.</p

Jatrophacine, a 4,5-<i>seco</i>-rhamnofolane diterpenoid with potent anti-inflammatory activity from <i>Jatropha curcas</i>

A new diterpenoid named jatrophacine (1), with an unusual 4,5-seco- rhamnofolane skeleton, was isolated from the roots of Jatropha curcas, together with eleven known diterpenoids. The structure of the new compound was elucidated through a detailed analysis of its 1 D- and 2 D-NMR spectra. The X-ray structure of jatrophol (2) is also presented. Anti-inflammatory activity with LPS-induced RAW 264.7 macrophages revealed that compound 1 strongly inhibited the production of nitric oxide (IC50 = 0.53 μM). </p

Rupatadine attenuates BLM-induced pulmonary fibrosis.

<p>The mice were intragastrically administered with solvent alone (sham group) or rupatadine at 1.5, 3.0, or 6.0 mg/kg per day from day 10 to 28 after BLM administration (5 U/kg). On day 28, mice were sacrificed and a lung was obtained for histological analysis and other studies. (<b>A</b>–<b>D</b>) Rupatadine attenuated pulmonary fibrosis and inflammation. Representative H&E staining data are shown (A, top), and the expression of α-SMA in fibrotic lungs. The lung sections were stained with an anti-α-SMA antibody for immunohistochemistry analysis (A, bottom). The IOD of each section was analyzed by Image-Pro Plus image analysis software (D) (n=10 per group). Inflammatory score was evaluated by a professional pathologist who was blind to the animal groups (B) (n=15 per group). Rupatadine treatment reduced the lung index in a dose-dependent manner (C) (n=10 per group). (<b>E</b>–<b>G</b>) Rupatadine decreased collagen deposition in the lungs. The lung tissue sections were stained with Sirius Red (SR) (normal light and polarized light) to indicate the collagen deposition (E and F). Additionally, rupatadine decreased hydroxyproline contents in fibrotic mice (<b>G</b>). Scale bar in images = 200 μm. The representative images in A and E were obtained from animals treated with rupatadine of 6.0 mg/kg per day. Data are expressed as the mean ± SEM of 8 mice per group. Rupatadine treatment inhibited the fibrosis-associated molecules in the fibrotic lung tissue (<b>H</b>). Western blot analysis was performed on lung lysates and detected the expression of α-SMA, E-cadherin and collagen-I in lung tissue. Data were expressed as folds of the sham group ± SEM of 8 mice per group. Rupatadine decreased animal death in BLM-injured mice (<b>I</b>). The cumulative survival rates of mice were analyzed by the Kaplan-Meier method (n=40 per group which were at the start of the experiment). ^#<i>P</i><0.05, ^{# #}<i>P</i><0.01 vs. Sham group; ^*<i>P</i><0.05, ^**<i>P</i><0.01 vs. BLM treated group.</p

Rupatadine inhibits BLM- and PAF-induced epithelial cellular senescence.

<p>(<b>A</b>) Rupatadine inhibited the expression or phosphorylation of the BLM-induced senescence-related molecules in MLE-12 cells. The data are representative immune blots and expressed as the mean ± SEM of four independent assays. (<b>B</b>) Rupatadine inhibited the secretion of IL-6 and PAF from the BLM- and PAF-induced senescent MLE-12 cells. The content of IL-6 and PAF in supernatant solutions was detected by ELISA kits. The data are expressed as the mean ± SEM of four independent assays with triplicates. (<b>C</b>) Rupatadine inhibited the expression of SA β-gal induced by BLM and C-PAF. MLE-12 cells were planted on coverglass-bottom dishes and treated with BLM (3 μg/ml), rupatadine (25 μg/ml), histamine (10 μg/ml), or C-PAF (5 μg/ml) for 96 hours. MLE-12 cells were stained by a senescence kit and examined for SA β-gal activity (blue). Scale bar in images = 50 μm. (<b>D</b> and <b>E</b>) Recovery from BLM and C-PAF induced growth arrest by rupatadine. MLE-12 cells were cultured in the presence or absence of BLM (3 μg/ml), rupatadine (25 μg/ml), histamine (10 μg/ml), and C-PAF (5 μg/ml) for 10 days. Population doubling (D) and representative DNA profile for the treated cells at day 10 were examined by flow cytometry (E). ^#<i>P</i><0.05, ^{# #}<i>P</i><0.01 vs. Untreated group; ^*<i>P</i><0.05, ^**<i>P</i><0.01 vs. BLM treated group; † † <i>P</i><0.01 vs. C-PAF group.</p

Histological examination of the lung sections from the rats treated by NS, LPS or LPS+PFC.

<p>The rats were treated with NS (n = 6) or LPS (n = 36) by intratracheal instillation. 18 rats received PFC through femoral vein prior to LPS instillation (LPS+PFC). Lung tissues were collected at the indicated time points after exposure to LPS and stained with HE. Scale bar, 25 µm.</p

Rupatadine attenuates cellular senescence in fibrotic mice.

<p>(<b>A</b>) Rupatadine (6.0 mg/kg per day) attenuated the expression or activity of senescence-related molecules in fibrotic lung tissue. Lung tissue extract was prepared for western blotting. The data are representative immune blots and expressed as the mean ± SEM of 8 mice per group. (<b>B</b>) Rupatadine (6 mg/kg) reduced the number of senescent cell in fibrotic mouse lung tissue. Representative images of lung sections were acquired by confocal microscopy for DAPI (blue)/ γ-H2A.X (green) or immunohistochemistry for p21. Scale bar in images = 10 μm or 50 μm. The data are expressed as the mean ± SEM of 5 mice per group. ^#<i>P</i><0.05, ^{# #}<i>P</i><0.01 vs. Sham group; ^*<i>P</i><0.05, ^**<i>P</i><0.01 vs. BLM treated group.</p