Zhonghui Zhu

Bone morphogenetic protein-7 inhibits silica-induced pulmonary fibrosis in rats.

Bone morphogenetic protein-7 (BMP-7) has been shown to inhibit liver and renal fibrosis in in vivo and vitro studies. There is no study to investigate BMP-7s role in the development of pulmonary fibrosis induced by silica. In the current study, we used the rat model to explore the potential antifibrotic role of BMP-7 and its underlying mechanism in silica-induced pulmonary fibrosis. Sixty Wistar rats were randomly assigned into three groups. Control group received saline, silica group received silica and BMP-7 treated group received silica and BMP-7. BMP-7 was administered to silica-treated rats intraperitoneally at a dose of 300μg/kg/injection from day 8 to day 30 every other day. After the animals were sacrificed on day 15 and 30, hydroxyproline levels, the protein expressions of BMP/Smad and TGF-β/Smad signaling, and histopathology in lung tissues were analyzed. The hydroxyproline contents in BMP-7 treated groups were significantly lower than the silica groups (P<0.05). Histopathological results showed BMP-7 could reduce the progression of silica induced fibrosis. Furthermore, the expression of p-Smad1/5/8, a marker of BMP/Smad signaling, was significantly up-regulated in BMP-7 treated groups (P<0.05) compared with the silica groups. On the contrary, the expression of p-Smad2/3, a marker for TGF-β/Smad signaling, reduced significantly in BMP-7-treated groups compared with silica groups (P<0.05). In conclusion, the pulmonary fibrosis induced by silica in rats was significantly reduced with the therapeutic treatment of BMP-7. The antifibrotic effect of BMP-7 could be related to the activation of BMP/Smad signaling and inhibition of TGF-β/Smad pathways.

Suppression of thioredoxin system contributes to silica-induced oxidative stress and pulmonary fibrogenesis in rats.

Silicosis is one of the most prevalent occupational lung diseases worldwide. This study aimed to investigate the possible mechanism that silica affected thioredoxin (Trx) system during the development of silicosis in vivo. Male Wistar rats were randomly divided into saline group and silica group in which rats were intratracheally instilled with a single dose of silica suspension (50mg in 1ml saline/rat). After 7, 15 or 30 days instillation, rats were sacrificed. Biochemical parameters and histopathology were assessed. Our results demonstrated that silica could significantly cause the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) as well as activate antioxidative protein Nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream protein Trx in the early exposure to silica. The inhibition of Trx activity and the down-regulated expression of thioredoxin reductase (TrxR), suggesting that the function of Trx system may be suppressive induced by silica. Content of lung hydroxyproline and histopathological results showed significant fibrosis development with time. In conclusion, our study demonstrated that silica could suppress the Trx system to perturb the redox balance, elicit oxidative stress, and eventually induce pulmonary fibrosis.

Bone morphogenetic protein 7 attenuates epithelial–mesenchymal transition induced by silica

The epithelial–mesenchymal transition (EMT) is a critical process in the pulmonary fibrosis. It has been reported that bone morphogenetic protein 7 (BMP-7) was able to reverse EMT in proximal tubular cells. Therefore, we test the hypothesis that EMT contributes to silica-induced pulmonary fibrosis and BMP-7 inhibits EMT in silica-induced pulmonary fibrosis. Progressive silica-induced pulmonary fibrosis in the rat was used as a model of silicosis. Epithelial and mesenchymal markers were measured from rat fibrotic lungs. Then the effects of BMP-7 on the EMT were further confirmed in A549 cells. There are increases of vimentin as a mesenchymal marker and decreases of E-cadherin as an epithelial marker in the silica-exposed rat lungs, which is in agreement with the A549 cells data. However, BMP-7 treatment significantly reduced expression of vimentin in the rat pulmonary fibrosis model and in A549 cells. In conclusion, EMT contributes to silica-induced pulmonary fibrosis. Meanwhile, the treatment of BMP-7 can inhibit silica-induced EMT in vitro and in vivo.

Effect of bone morphogenic protein-7 on the expression of epithelial-mesenchymal transition markers in silicosis model

This study presented the effect of bone morphogenic protein-7 (BMP-7) inhibiting epithelial-mesenchymal transition (EMT) in silicosis model. In vivo, Wistar rats were exposed to silica by intratracheal instillation. Seven days later rats were treated with BMP-7. Rats were sacrificed at 15 and 30days after exposure of silica. The results demonstrated vimentin expression was down-regulated; and E-cadherin was up-regulated after intervention with BMP-7. The TGF-β expression and phosphorylation-p38 were lower in BMP-7 treated group than in silica group. In vitro, p38 MAPK/Snail signaling pathway was involved in the occurrence of EMT in A549 cells treated by silica. EMT was inhibited by BMP-7. The data showed BMP-7 inhibited EMT induced by silica associated with inhibition of p38 MAPK/Snail pathway.

Anti-fibrotic effects of bone morphogenetic protein-7-modified bone marrow mesenchymal stem cells on silica-induced pulmonary fibrosis

Silicosis is an occupational lung disease caused by exposure to small particles of crystalline silica, which ultimately results in diffuse pulmonary fibrosis. Evidence indicates an anti-fibrotic role of bone morphogenetic protein-7 (BMP-7) and bone marrow mesenchymal stem cells (BMSCs) in lung diseases. Therefore, strategies incorporating genetic engineering and stem cell biology might have a tremendous potential to treat critical injuries and diseases. Therefore, we modified BMSCs to overexpress the BMP-7 gene (BMP-7-BMSCs) by lentivirus transduction, and then evaluated whether fibrotic processes were inhibited by these cells in vivo. Wistar rats were divided into four groups: control, silica, BMSCs, and BMP-7-BMSCs. The control group received saline, the silica group received silica and saline, the BMSCs group received silica and BMSCs, and the BMP-7-BMSCs group received silica and BMP-7-BMSCs. Rats were sacrificed on days 15 or 30 after silica instillation. Hematoxylin and eosin, and Massons trichrome staining were performed for histological examination. The severity of fibrosis was evaluated by the levels of hydroxyproline, fibronectin (FN), and transforming growth factor (TGF)-β1. Restoration of the alveolar epithelium was detected by the epithelial marker surfactant protein (SP)-C and aquaporin (AQP)-5. Histopathological results showed that BMP-7-BMSCs could remarkably block the progression of silica-induced fibrosis. Hydroxyproline, FN, and TGF-β1 contents in the BMP-7-BMSCs-treated group were significantly lower than those in the BMSCs group (P<0.05). Furthermore, the expression of SP-C and AQP-5 in the BMP-7-BMSCs-treated group was significantly higher than those in the BMSCs group (P<0.05). In conclusion, the pulmonary fibrosis induced by silica in rats was significantly reduced by treatment with BMP-7-BMSCs and BMSCs. The anti-fibrotic effect of BMSCs can be strengthened by BMP-7. Treatment with BMP-7-BMSCs might be a potential therapeutic intervention for silicosis.

Bone marrow mesenchymal stem cells attenuate silica-induced pulmonary fibrosis via paracrine mechanisms

The purpose of this study was to investigate the anti-fibrotic effect and possible mechanism of bone marrow mesenchymal stem cells (BMSCs) in silica-induced lung injury and fibrosis in vivo and in vitro. In vivo, rats were exposed to 50mg/ml silica intratracheally. The rats were sacrificed on day 15 or day 30 after intravenous injection of BMSCs. Histopathological examination demonstrated that BMSCs decreased the blue areas of collagen fibers and the number of nodules. Alveolar epithelium was damaged by silica, but it was restored by BMSCs. In vitro, BMSCs co-cultured with RLE-6TN cells in 6-Transwell plates were evaluated to determine the possible mechanism. The results demonstrated that BMSCs downregulated the expression of collagen I and III. BMSCs reversed morphological abnormalities and reduced the proliferation of RLE-6TN cells. These data showed that BMSCs did not give rise to alveolar epithelial cells directly, while the levels of hepatocyte growth factor, keratinocyte growth factor and bone morphogenetic protein -7 increased and expression of tumor necrosis factor-α and transforming growth factor-β1 decreased in the 6TN+Silica+BMSCs group compared with the 6TN+Silica group. Our results revealed that BMSCs exerted anti-fibrotic effects on silica-induced pulmonary fibrosis, which might be associated with paracrine mechanisms rather than differentiation.

Increased expression of bone morphogenetic protein-7 and its related pathway provides an anti-fibrotic effect on silica induced fibrosis in vitro

Objective: to better understand the effect and mechanism of modulating endogenous BMP-7 expression on silica induced fibrosis, and to investigate whether the fibrotic processes were regulated by pretreatment with small organic molecules in vitro. Methods: we established BMP-7 over-expressing (BMP-7(+)/RLE-6TN) and BMP-7 silent (BMP-7(−)/RLE-6TN) cell lines by lentivirus mediated gene transduction in RLE-6TN, a kind of alveolar type II epithelial cells. For inhibition studies, the inhibitor of the TGF-β/activin type I receptor (SB-431542) or BMP receptor (LDN-193189) was used to pretreat the RLE-6TN cells. The progress of fibrosis was assessed by the content of hydroxyproline (Hyp). Using qPCR and western blotting, the mRNA and protein levels of BMP-7, fibronectin (FN), collagen I and collagen III were detected. The Smad signaling pathway proteins, including phosphorylated Smad1/5 (P-Smad1/5) and phosphorylated Smad2/3 (P-Smad2/3) were detected using western blotting. In addition, the MTT assay was used to explore the toxic effects of SB-431542 and LDN-193189. Results: results showed that pretreating with SB-431542 or over-expressed BMP-7 could attenuate the decrease of P-Smad1/5 and the increase of P-Smad2/3 and fibrosis-related markers (collagen I, collagen III, and FN). On the contrary, using LDN-193189 or small hairpin RNA (shRNA) against BMP-7 could promote the secretion of Hyp and fibrosis-related mRNA and protein expression by increasing P-Smad2/3 and decreasing P-Smad1/5. Conclusion: BMP-7 exerts an inhibitory effect on silica induced fibrosis in RLE-6TN cells via a blockade of TGF-β signaling and a restoration of BMP signaling. BMP-7 can encourage the development of new therapeutic agents in silica induced fibrosis.

Inhibition of nuclear thioredoxin aggregation attenuates PM2.5-induced NF-κB activation and pro-inflammatory responses

ABSTRACT Exposure to fine particulate matter (PM2.5) can induce oxidative stress and proinflammatory cytokine production, which are central for the induction of PM2.5‐mediated adverse effects on public health. Nuclear factor kappa B (NF‐&kgr;B) signaling is essential for inflammation. The subcellular distribution of thioredoxin (Trx) is related to the activation of NF‐&kgr;B, but the mechanism involved is unclear. In the current study, we focused on the relationship between the antioxidant Trx and NF‐&kgr;B in human bronchial epithelial cells (BEAS‐2B) after PM2.5 exposure. We inhibited the nuclear translocation of Trx by cHCEU (4‐cyclohexyl‐[3‐(2‐chloroethyl)ureido]benzene) and subsequently increased the transcriptional activity of Nrf2 to upregulate the expression of Trx by t‐BHQ. Our data suggest that PM2.5 exposure induces the activation of NF‐&kgr;B and the expression of the downstream proinflammatory cytokines IL‐1, IL‐6, IL‐8 and TNF‐&agr; in BEAS‐2B cells. CHCEU alleviates inflammatory cytokines by blocking Trx nuclear translocation and inhibits the DNA binding activity of NF‐&kgr;B. T‐BHQ could promote the transcriptional activity of Nrf2 but failed to alleviate the production of inflammatory cytokines. Furthermore, the synergistic effect of t‐BHQ and cHCEU on alleviating PM2.5‐induced inflammation is more effective than the use of cHCEU alone. Our findings characterize the underlying molecular mechanisms of proinflammatory responses induced by PM2.5 and show that the nuclear translocation and accumulation of Trx in nuclei play important roles in PM2.5‐induced NF‐&kgr;B activation and proinflammatory responses. Graphical abstract Figure. No Caption available. HighlightsPM2.5 induces oxidative stress and pro‐inflammatory cytokine production in BEAS‐2B cell.PM2.5 promotes nuclear translocation of Trx and enhances DNA binding activity of NF‐&kgr;B.CHCEU alleviates the inflammatory cytokines by inhibiting Trx nuclear translocation.

The protective effects of bone morphogenetic protein-7 against epithelial injury and matrix metalloproteases upregulation induced by silica in vitro

Objective: We investigate the effects of bone morphogenetic protein-7 (BMP-7) on models with silica-induced and macrophage-mediated fibrosis and its possible mechanisms in vitro. Methods: Rat alveolar II epithelial (RLE-6TN) cells were incubated with the supernatant of mouse macrophage-like cells (RAW264.7) and treated with 0, 25, 50, and 100 μg/mL silica. Using Western blotting, the epithelial markers (surfactant proteins-C and E-cadherin) and the mesenchymal markers (fibronectin (FN) and viminten (Vim)) were detected. After neutralizing the BMP-7, the progress of fibrosis was assessed by the content of hydroxyproline (Hyp) and collagen I, III protein levels as well as the Smad signaling pathway proteins, including phosphorylated Smad1/5(P-Smad1/5) and phosphorylated Smad2/3(P-Smad2/3). Collagen I was also identified by immunofluorescence and pretreated with SB-431542, LDN-193189, or anti-BMP-7-neutralizing antibody. In addition, the levels of matrix metalloproteinase-2 (MMP-2) and MMP-9 were detected using Western blotting. Results: The model of RLE-6TN cells was established successfully, the expressions of Vim, FN, MMP-2, and MMP-9 were upregulated, while the concentration of silica is increased. Neutralizing BMP-7 stimulated the decrease of P-Smad1/5 and the increase of P-Smad2/3, as well as the collagen I, collagen III, FN, and Hyp via Smad signaling pathway. Furthermore, pretreated with LDN-193189 or anti-BMP-7-neutralizing antibody, the expression of collagen I was increased, yet it was decreased with SB-431542 intervention. Conclusion: The activated BMP/Smad and suppressed transforming growth factor-β/Smad pathways could suppress silica-induced fibrosis via a MMP-dependent mechanism. BMP-7 is expected to be the optimized strategy of delaying the interstitial changes.