Tanshinone IIA attenuates silica-induced pulmonary fibrosis via inhibition of TGF-β1-Smad signaling pathway.

Transforming growth factor-β 1 (TGF-β1) is a key mediator in fibrogenesis, and is upregulated and activated in fibrotic diseases. The exact role of TGF-β1-Smad signaling in the progression of silicosis fibrosis is yet to be conclusively determined. Using a Wistar rat silicosis model, we examined whether tanshinone IIA (Tan IIA) could meliorate silicosis fibrosis. The pulmonary fibroblasts of rats from the normal control group and silicosis-induced model group were extracted and examined so as to further explore the disruption of TGF-β1-Smad signaling pathway in silicosis pathogenesis and the intervention of Tan IIA in this pathway. Using RT-PCR, immunohistochemical staining, and immunofluorescence analysis, we determined that Tan IIA could ameliorate silicosis fibrosis, downregulate collagen I, collagen III, and α-SMA expression both, in vivo and in vitro. In silicosis fibroblasts, TGF-β1 induced phosphorylation of Smad2, Smad3, and negative feedback Smad7 inhibition in a dose dependent manner, and the phosphorylation of Smad3 persisted when the upstream signal was blocked. Tan IIA treatment effectively inhibited the TGF-β1-induced phosphorylation of Smads, especially the persistent phosphorylation of Smad3 in the nucleus, and upregulated the expression of Smad7 in silicosis fibroblasts, leading to a reduction in ECM deposition. Our findings indicate that dysregulation of the TGF-β1-Smad signaling pathway may play an important role in the pathological process of silicosis. Tan IIA thus ameliorates silicosis fibrosis partially by suppressing activation of TGF-β1-Smad signaling pathway, which may turn out to be a potential therapeutic approach to prevent silicosis fibrosis.

Feng F ，Li N ，Cheng P ，Zhang H ，Wang H ，Wang Y ，Wang W ... -  《-》

Tanshinone IIA attenuates silica-induced pulmonary fibrosis via Nrf2-mediated inhibition of EMT and TGF-β1/Smad signaling.

Silicosis is an occupational pulmonary fibrosis that is caused by inhalation of silica (SiO2), and there are no effective drugs to treat this disease. Tanshinone IIA (Tan IIA), a natural product, has been reported to possess antioxidant and anti-fibrotic properties in various diseases. The purpose of the current study was to examine Tan IIA's protective effects against silica-induced pulmonary fibrosis and to explore the underlying mechanisms. We found that in vivo treatment with Tan IIA significantly relieved silica-induced lung fibrosis in a silicosis rat model by histological and immunohistochemical analyses. Further, in vitro mechanistic investigations, mainly using western blot and immunofluorescence analyses, revealed that Tan IIA administration markedly inhibited the silica-induced epithelial-mesenchymal transition (EMT) and transforming growth factor-β1 (TGF-β1)/Smad signaling pathway and also reduced silica-induced oxidative stress and activated the nuclear factor erythroid 2-related factor-2 (Nrf2) signaling pathway in A549 and human bronchial epithelial (HBE) cells. Furthermore, through transfection with siRNA, we demonstrate that Nrf2 activation partially mediates the suppression effects of Tan IIA on EMT and TGF-β1/Smad signaling pathway activation induced by silica exposure, thus mediating the anti-fibrotic effects of Tan IIA against silica-induced pulmonary fibrosis. In our study, Tan IIA has been identified as a possible anti-oxidative and anti-fibrotic drug for silicosis.

Feng F ，Cheng P ，Xu S ，Li N ，Wang H ，Zhang Y ，Wang W ... -  《-》

The Protective Role of Tanshinone IIA in Silicosis Rat Model via TGF-β1/Smad Signaling Suppression, NOX4 Inhibition and Nrf2/ARE Signaling Activation.

Silicosis is an occupational disease caused by inhalation of silica and there are no effective drugs to treat this disease. Tanshinone IIA (Tan IIA), a traditional natural component, has been reported to possess anti-inflammatory, antioxidant, and anti-fibrotic properties. The current study's purpose was to examine Tan IIA's protective effects against silica-induced pulmonary fibrosis and to explore the underlying mechanisms. 48 male SD rats were randomly divided into four groups (n=12): i) Control group; ii) Silicosis group; iii) Tan IIA group; iv) Silicosis +Tan IIA group. Two days after modeling, the rats of Tan IIA group and Silicosis +Tan IIA group were given intraperitoneal administration 25 mg/kg/d Tan IIA for 40 days. Then, the four groups of rats were sacrificed and the lung inflammatory responses were measured by ELISA, lung damage and fibrosis were analyzed by hematoxylin and eosin (H&E) staining and Masson staining, the expression levels of collagen I, fibronectin and α-smooth muscle actin (α-SMA) were measured by immunohistochemistry. The markers of oxidative stress were measured by commercial kits, and the activity of the TGF-β1/Smad and NOX4, Nrf2/ARE signaling pathways were measured by RT-PCR and Western blotting. The silica-induced pulmonary inflammtory responses, structural damage and fibrosis were significantly attenuated by Tan IIA treatment. In addition, treatment with Tan IIA decreased collagen I, fibronectin and α-SMA expression, and inhibited TGF-β1/Smad signaling in the lung tissue. The upregulated levels of oxidative stress markers in silicosis rats were also markedly restored following Tan IIA treatment. Furthermore, treatment with Tan IIA reduced NOX4 expression and enhanced activation of the Nrf2/ARE pathway in the lung tissue of silicosis rats. These findings suggest that Tan IIA may protect lung from silica damage via the suppression of TGF-β1/Smad signaling, inhibition of NOX4 expression and activation of the Nrf2/ARE pathway.

Feng F ，Cheng P ，Zhang H ，Li N ，Qi Y ，Wang H ，Wang Y ，Wang W ... -  《Drug Design Development and Therapy》

Inhibitory effects of astragaloside IV on silica-induced pulmonary fibrosis via inactivating TGF-β1/Smad3 signaling.

To observe the effect of astragaloside ASV (ASV) on silicosis fibroblasts, and further investigate its regulatory mechanism on TGF-β1/Smad3 signaling pathway. Silica-induced rats model was established in this study. RT-qPCR was performed to detect α-SMA, Collagen I, Collagen III, Smad2, Smad3 and Smad7 expression. Immunofluorescence was conducted to detect α-SMA, Collagen I, Collagen III and p-Smad3 protein and the nucleoplasmic distribution of p-Smad3.Western-blotting was performed to detect the protein of Smad2, p-Smad2, Smad3, p-Smad3 and Smad7. 20 μg/mL ASV could effectively reduce the expression of α-SMA, Collagen I, Collagen III. TGF-β1 stimulated the proliferation of fibroblasts, promoted phosphorylation of Smad2 and Smad3, and down-regulated Smad7 expression. Among them, continuous phosphorylation of Smad3 is a major factor in causing fibrosis. Besides, ASV can inhibit silica-induced lung fibroblast fibrosis through TGF-β1/Smad3 signaling pathway, thereby inhibiting the formation of silicosis. ASV could inhibit the expression of collagen in fibroblasts and the transformation to myofibroblasts, and has an anti-silicosis fibrosis effect, which may be related to the continuous phosphorylation of Smad3 in the TGF-β1/Smad signaling pathway.

Li N ，Feng F ，Wu K ，Zhang H ，Zhang W ，Wang W ... -  《-》

Tanshinone IIA ameliorates bleomycin-induced pulmonary fibrosis and inhibits transforming growth factor-beta-β-dependent epithelial to mesenchymal transition.

Epithelial to mesenchymal transition (EMT) of alveolar epithelial cells occurs in lung fibrotic diseases. Tanshinone IIA (Tan IIA) has been reported to exert anti-inflammatory effects in pulmonary fibrosis. Nonetheless, whether Tan IIA affects lung fibrosis-related EMT remains unknown and requires for further investigations. A single intratracheal instillation of saline containing bleomycin (BLM; 5 mg/kg body weight) was performed to induce pulmonary fibrosis in Sprague-Dawley rats. Rats receiving an instillation of equivoluminal normal saline served as controls. Then, these rats were given a daily intraperitoneal administration of Tan IIA (15 mg/kg body weight) for 28 d before sacrifice. In vitro, recombinant transforming growth factor-beta 1 (TGF-β1; 10 ng/mL) was used to treat human alveolar epithelial A549 cells for 48 h. Tan IIA (10 μM) or control DMSO was used to pretreat cells for 2 h before TGF-β1 stimulation. Rat lung tissue samples and A549 cells were then subjected to further assessments. Tan IIA was noted to alleviate BLM-induced pulmonary collagen deposition and macrophage infiltration in rats. Epithelial-cadherin expression was decreased after BLM stimulation, whereas α-smooth muscle actin, fibronectin, and vimentin were increased. These expression alterations were partially reversed by Tan IIA. Moreover, Tan IIA suppressed BLM-induced increases in TGF-β1, phosphorylated Smad-2, and -3 in rats. Additionally, pretreatment of Tan IIA inhibited TGF-β1-triggered EMT, reduced collagen Ⅰ production, and blocked TGF-β signal transduction in A549 cells. Our research suggests that Tan IIA mitigates BLM-induced pulmonary fibrosis and suppresses TGF-β-dependent EMT of lung alveolar epithelial cells.

Tang H ，He H ，Ji H ，Gao L ，Mao J ，Liu J ，Lin H ，Wu T ... -  《-》