br Materials and methods br Results br Discussion
Materials and methods
Discussion Pulmonary fibrosis is a progressive and chronic lung disease and its typical features include BAMB-4 proliferation, epithelial injury, chronic interstitial inflammation, increased extracellular matrix (ECM) production, and collapse of alveoli, all of which contribute to progressive fibrosis and severe lung dysfunction . Therefore, PF is generally associated with high morbidity and mortality . Although considerable research efforts have been devoted to experimental and clinical studies, there is still no therapy available for this disorder partly due to the lack of understanding of its pathogenesis . Resveratrol is a newly discovered drug that could potentially be used to treat PF. In the present study, we explored the mechanisms underlying how Res reduces bleomycin-induced pulmonary fibrosis, in an effort to lay the foundation for clinical treatment of PF. MiRNAs are involved in many biological cellular processes, such as early development , proliferation, differentiation, tumorigenesis , cell fate determination , apoptosis , signal transduction , organ development , and the regulation of endothelial function [27,28]. MicroRNA-21 (miR-21) has been shown to play an important role in the development of lung fibrosis and may serve as a promising therapeutic target for pulmonary fibrosis disease . Based on this evidence, we hypothesized that the positive effects of Res treatment on lung fibrosis are associated with the regulation of miR-21 levels. In this study, we found that BLM treatment in rats significantly increased miR-21 expression, but this upregulation was significantly reversed by Res, suggesting that Res inhibits miR-21 expression. Our results were partially agreed with a previous study that confirmed miR-21 as a direct target of Res in the inhibition of prostate cancer growth, survival, and invasiveness . The deposition of extracellular matrix and collagen are common features of lung fibrosis . Representative images of HE- and Masson-stained lung tissues showed that BLM treatment caused morphological changes and severe damage to lung tissues that included pneumonitis, hyporrhea and edema, fibroblastic foci, collagen deposition, inflammatory exudates, and structural distortion. While the above symptoms were significantly alleviated by Res treatment, they reappeared with administration of agomiR-21, indicating that miR-21 was able to augment the extent and intensity of collagen deposition. We also measured the hydroxyproline level in lung tissues, which is an important fibrotic indicator of collagen deposition , as well as the levels of ECM-related proteins including Fn, Col1 A1, Col3 A1, and the cytoskeletal protein α-SMA that is considered to be a marker of myofibroblast phenotype and ongoing fibroblast differentiation. Not surprisingly, agomiR-21 treatment increased collagen deposition and significantly elevated the expression levels of Fn, Col1 A1, Col3 A1, and α-SMA. In addition, the decreased mRNA levels of MMP-2, MMP-9, and MMP13 in lung tissues that were induced by Res were significantly increased after treatment with miR-21, a finding which is supported by several prior reports to some extent [30,31]. MiR-21 agomir also resulted in a significant increase in TIMP-1 levels, which is an inhibitor of MMP-2 and 9 and is associated with chronic impaired tissue remodeling and collagen deposition . Collectively, the above findings demonstrated that Res inhibits PF through the regulation of fibrotic-related protein levels, including Fn, COL1 A1, COL3 A1, α-SMA, TIMP-1, as well as MMP-2, 9, and 13. MiR-21 has been shown to participate in the TGF-β1 signaling pathway and aggravate the process of lung fibrosis by magnifying signals in a feed-forward loop . TGF-β1 increases the expression levels of miR-21 in primary lung fibroblasts, which subsequently promotes the pro-fibrogenic effects of TGF-β1 and contributes to fibroblast activation, collagen deposition, and ultimately caused fibrosis, whereas miR-21 silencing weakens the profibrotic effects of TGF-β1 . In the in vivo experiments of our study, abnormal upregulations of TGF-β1 and p-Smad-2/3, and the downregulation of Smad7 in rats with PF were obvious, while these deviations were markedly inhibited by Res but then again induced by miR-21 agomir. In in vitro experiments, TGF-β treatment contributed to an increase in total collagen content, an increase in the levels of Fn, α-SMA, and p-Smad2, and a reduction in Smad7 levels. Res administration efficiently reversed the above observations while agomiR-21 again induced them. Several previous studies have demonstrated the important role of the TGF-β1/Smad signaling pathway in the pathogenesis of pulmonary fibrosis [, , ]. Liu et al.  reported that miR-21 can aggravate collagen deposition by targeting Smad family members. In agreement, we conclude that the positive effects of Res in pulmonary fibrosis are related to the inhibition of the TGF-β/Smad signaling pathway. Furthermore, the TargetScan database predicts that Smad 7 is a direct target of miR-21-5p, which reveals that miR-21-5p exerts its anti-fibrotic effect by regulating the TGF-β1/Smad pathway via Smad7 targeting.