| Background and objective:Cigarette smoke (CS) is a risk factor of chronic obstructive pulmonary disease (COPD), it contains a variety of oxidative components that were implicated in the regulation of Shp2activity. Shp2plays an important role in acute cigarette smoke-mediated lung inflammation. However, the contribution of Shp2enzyme to COPD pathogenesis remains unclear. We investigated the role of Shp2enzyme in blockading the CS-induced pulmonary fibrosis.Methods:In vivo, PHPS1, a specific inhibitor of Shp2enzyme, was used to treat pulmonary fibrosis in CS-exposed mice. Endogenous inactivation of Shp2was achieved using the genetic strategy that the Shp2gene was selectively abolished in pulmonary epithelia. Mice were exposed to cigarette smoke to induce epithelial injury and pulmonary fibrosis. The bronchoalveolar lavage fluids (BALFs) and lung tissues were collected15h after the last CS exposure.1) Shp2, MMP-9, TGF-β1, S100A4were measured by Q-PCR, WB, Elisa and IHC.2) Lung tissues were stained with Masson.In vitro,1) Cell viability was assessed by MTT.2) The activity of p-Shp2and Shp2induced by CSE were measured by WB.3) The effect of CSE on MMP-9level was measured by Q-PCR and WB in MLE-12cells.4) To explore the role of Shp2by inhibiting either the activity or expression of Shp2in CSE-induced MMP-9level in MLE-12cells.5) The effects of CSE on TGF-β1level was measured by RT-PCR and Elisa in A549cells.6) To explore the role of Shp2by inhibiting the activity of Shp2in CSE-induced TGF-β1level by Q-PCR in A549cells.7) The mechanisms, to investigative the signaling events that underlie pulmonary fibrosis, we tested the Erkl/2inhibitor U0126, JNK inhibitor SP600125, p38inhibitor SB203580in CSE-stimulated epithelial cells.Results:In vivo,1) We found that the MMP-9, TGF-β1and S100A4release were significantly increased in mice after CS exposure.2) To assess the potential role of Shp2in regulating pulmonary fibrosis, the MMP-9and TGF-β1release were significantly reduced in PHPS1-pretreated mice.3) To further probe the effect of Shp2, lung-specific Shp2knockout mice (Shp2KO) mice were exposed to CS for10consecutive days showed a remarkable reduction of MMP-9and TGF-β1expression.4) Mice either injected with a Shp2inhibitor PHPS1or Shp2gene knockdown show the reduction of collagen fibers after CS exposure.In vitro,1) CSE caused cell injury in MLE-12cells. The cells were exposed to different concentrations of CSE (0.5%-5%) for24h,48h,72h. CSE differently induced toxicity in a concentration-and time-dependent manner.2) CSE increased p-Shp2and Shp2activity in a concentration-and time-dependent manner.3) The increased in MMP-9mRNA and protein expression induced by CSE in epithelial cells in a concentration-and time-dependent manner.4) To assess the potential role of Shp2in regulating pulmonary fibrosis, we found MMP-9gene expression and production decreased in pulmonary epithelial cells with the administration of PHPS1.5) Using siRNA-mediated knockdown of Shp2in pulmonary epithelial cells, we found inactivation of Shp2decreased CSE-induced MMP-9release.6) CSE increased TGF-β1mRNA and protein production in epithelial cells in a concentration-and time-dependent manner.7) We also found TGF-β1gene expression decreased in pulmonary epithelial cells with the administration of PHPS1, a Shp2inhibitor.8) The mechanisms, CSE differently induced JNK and p38protein expression in a concentration dependent manner in MLE-12cell line. Inhibition of the activity of Shp2suppressed CSE-induced JNK activation.Conclusions:1) Pulmonary fibrosis was alleviated in PHPS1-pretreated mice and lung-specific Shp2knockout mice which exposed to CS.2) MMP-9and TGF-β1gene expression and production was decreased in pulmonary epithelial cells with the administration of PHPS1or siRNA-mediated knockdown of Shp2in pulmonary epithelial cells.3) Our results demonstrated that Shp2/JNK may be an important signaling pathway or target, which potentially contributes to the development of a novel therapeutic target for pulmonary fibrosis. |
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