RESUMO
ObjectiveTo investigate the role of the Wnt1/β-catenin signaling pathway in the intervention of medicated serum of Buyang Huanwutang （BYHWT） in endothelial-to-mesenchymal transition （EndMT） of human pulmonary artery endothelial cells （HPAECs） as well as its related mechanisms. MethodMedicated serum of BYHWT was prepared by gavage to New Zealand rabbits with a dosage of 53.36 g·kg-1·d-1 after decocting the medicine as usual. In addition， the same volume of normal saline was used to prepare blank serum. The HPAECs were cultured in vitro， and then induced by the transforming growth factor-β1 （TGF-β1） to establish the EndMT model. Five groups were established： blank group （10% blank serum）， model group （TGF-β1+10% blank serum）， low-dose BYHWT group （TGF-β1+2.5% medicated serum+7.5% blank serum）， medium-dose BYHWT group （TGF-β1+5% medicated serum+5% blank serum） and high-dose BYHWT group （TGF-β1+10% medicated serum）. Through Western blot， the expressions of Wnt1， β-catenin， and glycogen synthase kinase-3β （GSK-3β） were detected. In order to further clarify the mechanism of the Wnt1/β-catenin signaling pathway in the intervention of the medicated serum of BYHWT in inhibiting EndMT， the overexpression of β-catenin was confirmed by polymerase chain reaction after plasmid of overexpression β-catenin was constructed and transfected into the HPAECs. The HPAECs were intervened by 10% medicated serum with the optimal effect in previous studies. Then， they were divided into another five groups： the blank group （10% blank serum）， the model group （TGF-β1+10% blank serum）， the BYHWT group （TGF-β1+10% medicated serum）， the BYHWT+overexpression plasmid control group （TGF-β1+10% medicated serum+blank plasmid） and the BYHWT+β-catenin overexpression plasmid group （TGF-β1+10% medicated serum+β-catenin）. Apart from that， cell proliferation ability was detected by the methyl thiazolyl tetrazolium （MTT） method and cell migration ability by scratch assay and Transwell assay together. Immunofluorescence was adopted to detect the expressions of platelet endothelial cell adhesion molecule （PECAM-1/CD31）， vascular endothelial cadherin （VE-cadherin）， fibroblast-specific protein 1 （FSP1）， and α-smooth muscle actin （α-SMA）. ResultIn comparison to the blank group， the expressions of Wnt1 and β-catenin were significantly increased （P<0.01） while the expression of GSK-3β significantly decreased （P<0.01） in the model group. In comparison to the model group， the expressions of Wnt1 and β-catenin were significantly decreased （P<0.01） while the expression of GSK-3β was significantly increased （P<0.01） in the high-dose BYHWT group. The expression of β-catenin was significantly decreased （P<0.01） while the expression of GSK-3β was significantly increased （P<0.01） in the medium-dose BYHWT group. There was no significant difference in these indexes of the low-dose BYHWT group. In comparison to the blank group， proliferation and migration abilities were remarkably increased （P<0.01） and the immunofluorescence intensities of CD31 and VE-cadherin were decreased， while those of FSP1 and α-SMA were increased in the model group. In comparison to the model group， proliferation and migration abilities were significantly decreased （P<0.01） and the immunofluorescence intensities of CD31 and VE-cadherin were increased， while those of FSP1 and α-SMA diminished in the BYHWT group. Beyond that， the change trend of those indexes in the BYHWT+β-catenin overexpression plasmid group was consistent with that in the model group. In comparison to the BYHWT+overexpression plasmid control group， proliferation and migration abilities were significantly increased （P<0.01） and the immunofluorescence intensities of CD31 and VE-cadherin were decreased， while those of FSP1 and α-SMA were increased in the BYHWT+β-catenin overexpression plasmid group. ConclusionMedicated serum of BYHWT can inhibit EndMT of HPAECs by the Wnt1/β-catenin signaling pathway.
RESUMO
OBJECTIVE： To investigate the effects of Buyang huanwu decoction on endothelial-mesenchymal transformation （EndMT） of lung tissue in idiopathic pulmonary fibrosis （IPF） model rats， and to explore its potential mechanism. METHODS： Male SD rats were randomly divided into normal group， model gorup， dexamethasone group [0.405 mg/（kg·d）]， Buyang huanwu decoction low-dose， medium-dose and high-dose groups [6.435， 12.87， 25.74 g/（kg·d）， by raw material]， with 8 rats in each group. Except for normal group， other groups were given endotracheal injection of bleomycin to induce IPF model. On the second day after modeling， normal group and model group were given water intrgastrically [10 mL/（kg·d）]； administration groups were given relevant medicine intragastrically， once a day， for consecutive 28 days. 24 h after last medication， the expression of endothelial cell markers [platelet endothelial cell adhesion molecule 1， vascular endothelial cell cadherin] and interstitial cell markers [α-smooth muscle actin， fibroblast specific protein 1] were detected by immunohistochemistry method. The expression of Notch4 and DLL4 in lung tissue of rats were detected by Western blotting assay. RESULTS： Compared with normal group， the expression of endothelial cell markers were decreased significantly in lung tissue of model group， while the expressipon of interstitial cell markers， Notch4 and DLL4 were increased significantly （P＜0.01）. Compared with model group， the expression of endothelial cell markers in lung tissue of rats were increased significantly in administration groups， while Buyang huanwu decoction low-dose group was significantly lower than dexamethasone group； the expression of interstitial cell markers， Notch4 and DLL4 were decreased significantly， while Buyang huanwu decoction low-dose group was significantly higher than dexamethasone group （P＜0.05 or P＜0.01）. CONCLUSIONS： Buyang huanwu decoction can relieve IPF of model rats by intervening in EndMT， the mechanism of which may be associated with inhibiting DLL4/Notch4 singaling pathway.
