Apigenin Inhibits Bleomycin-induced Mouse Pulmonary Fibrosis And Its Mechanisms

Objective: To investigate the inhibitory effect of apigenin on bleomycin-induced pulmonary fibrosis in mice and its potential mechanisms.Methods: Male ICR mice were used in the vivo study. Except from the control mice which were intratracheal instillation of the same volume of normal saline, the other mice were intratracheal instillation of the bleomycin. Subsequently, bleomycin-treated mice were further divided into the model, apigenin 150 and 300 mg/kg, and positive drug dexamethasone 0.125 mg/kg groups. The medicine-treated mice were orally given apigenin or dexamethasone by gavage once daily for 28 days. Finally, all of mice were sacrificed. The oxidant and antioxidant indexes in the serum and lung tissue, hydroxyproline content in the lung tissue, and lung morphological changes were examined. The protein levels of lung peroxisome proliferator-activated receptor γ(PPAR γ), nuclear factor kappa-B p65(NF-κB p65), transforming growth factor-β1(TGF-β1), matrix metalloproteinase-9(MMP-9), Smad-7, E-cadherin, and Vimentin expressions were determined by Western blot method. The alveolar epithelial cell line A549 was used in the vitro study and divided into control, TGF-β1 stimulation, apigenin(2.5, 5, 10 μM), and positive drug rosiglitazone(10 μM) groups. After incubation of A549 cells with drugs for 24 h, the expression level of epithelial marker protein E-cadherin was examined, and those of PPARγ and NF-κB p65 proteins were also measured.Results: In the vivo study, after administration of apigenin 150-300 mg/kg for 28 days, the lung weight and its weight coefficient and hydroxyproline content were significantly decreased. Under the light microscope, the infiltration of inflammatory cells, thickness of alveolar wall, and degree of fibrosis in the lung tissue were reduced, especially in the apigenin 300 mg/kg group. Apigenin treatment could decrease the serum malondialdehyde(MDA) level, and increase the serum superoxide dismutase(SOD) level. Meanwhile, apigenin also increased the lung reduced glutathione(GSH) and superoxide dismutase(SOD) levels. Western blot assay showed that in the apigenin-treated mice, the expression levels of lung PPARγ, Smad-7, and E-cadherin proteins increased, whereas those of lung NF-κB p65, TGF-β1, MMP-9, and Vimentin proteins decreased. In vitro, the experimental results showed during the epithelial-mesenchymal transition(EMT) of A549 cells induced by TGF-β1, apigenin treatment at 5-10 μM for 24 h could significantly increase the expression level of epithelial marker protein E-cadherin. Also, apigenin increased the expression level of PPARγ protein and decreased the expression level of NF-κB p65 protein.Conclusion: Apigenin may inhibit the bleomycin-induced pulmonary fibrosis in mice, and its mechanism may be related to the increments of lung antioxidant ability and PPARγ expression. The former may scavenge the reactive oxygen species produced by bleomycin, and consequently inhibit the oxidative stress-mediated NF-κB signal pathway, while the latter may further inhibit the expression of NF-κB protein. The synergic effects of the both on NF-κB may finally lead to the reductions of TGF-β/Smad pathway-mediated lung EMT and collagen production, and contribute to the amelioration of pulmonary fibrosis.