ObjectiveTo investigate the effect of the Buyang Huanwutang （BHT）-containing serum on the bleomycin-induced senescence of A549 cells and explore the potential mechanism by which BHT ameliorates pulmonary fibrosis. MethodsA549 cells were cultured in vitro and modeled for senescence through the application of bleomycin. SD rats were administrated with BHT by gavage for the preparation of BHT-containing serum， and the effect of BHT-containing serum on the viability of the cell model was studied through a cell experiment designed with the following groups： blank group， model group， 2.5%， 5%， and 10% BHT-containing serumgroups. The effect of BHT-containing serum on the BLM-induced senescence of A549 cells was studied by the experiment designed with blank group， model group， positive group （PC，pirfenidone，600 mg·L^-1）， 2.5%， 5%， 10% BHT-containing serum groups. SA-β-Gal staining was used to reveal the area of senescence-positive cells， and flow cytometry was employed to analyze the cell cycle distribution. Real-time PCR and the immunofluorescence assay were adopted to determine the mRNA and protein levels of cell senescence markers p16 and p21. Western blot was employed to quantify the protein levels of senescence-associated secretory phenotype （SASP） molecules interleukin （IL）-1β， IL-6， matrix metalloproteinase （MMP）-3， chemokine ligand （CCL）2， tumor necrosis factor （TNF）-α， and transforming growth factor-β （TGF-β）/Smad pathway molecules TGF-β₁， Smad2， phosphorylated （p）-Smad2， Smad3， and p-Smad3. ResultsCompared with the blank group， the model group showed a decrease in cell survival rate （P<0.01）， increases in the SA-β-Gal-positive area， the proportion of cells in G₀/G₁ phase， the mRNA and protein levels of P16 and P21， and the protein levels of IL-1β， IL-6， TNF-α， MMP-3， CCL2， TGF-β₁， p-Smad2， and p-Smad3 （P<0.05，P<0.01）. Compared with the model group， the BHT-containing serum at each dose increased the cell survival rate （P<0.01） and decreased the SA-β-Gal-positive area， the proportion of cells in G₀/G₁ phase， the mRNA and protein levels of p16 and p21， and the protein levels of IL-1β， IL-6， TNF-α， MMP-3， and CCL2 （P<0.05， P<0.01）. Moreover， the inhibitory effect of BHT-containing serum on cell senescence showed a dose-dependent manner， with the 10% BHT-containing serum showing the most obvious effects and inhibiting the expression of TGF-β₁， p-Smad2， and p-Smad3 （P<0.01）. The effect of 10% BHT-containing serum was comparable to that of pirfenidone， and the serum even outperformed pirfenidone in inhibiting the expression of TNF-α and Smad2 （P<0.05）. ConclusionThe BHT-containing serum can inhibit BLM-induced senescence of A549 cells in a dose-dependent manner by regulating the TGF-β/Smad signaling pathway.

OBJECTIVE To investigate the improvement effect and mechanism of Buyang huanwu tang on idiopathic pulmonary fibrosis （IPF） model rats. METHODS The rats were randomly divided into normal group， model group， pirfenidone group （positive control， 0.162 g/kg）， Buyang huanwu tang low-， medium- and high-dose groups （6.435， 12.87， 25.74 g/kg）， with 6 rats in each group. Except for the normal group， IPF model was established in the remaining groups by intratracheal injection of bleomycin sulfate. On the second day after successful modeling， medication was administered once a day for 28 consecutive days. After the last medication， the appearance and morphology， pathological changes， and fibrosis status of the lung tissues in rats were observed. cyclin-dependent kinase inhibitor 2A （P16）， P21， transforming growth factor-β1 （TGF-β1）， Smad3， Smad7， epidermal growth factor （EGF）， epidermal growth factor receptor （EGFR）， matrix metalloproteinase 12 （MMP-12）， chemokine ligand 2 （CCL2） and interleukin-4 （IL-4） protein expression in lung tissue were all determined. RESULTS Compared with the normal group， the lung tissue of rats in the model group exhibited gray-white color， harder texture， obvious bruising and cysts. Additionally， alveolar septa were significantly thickened， their structural integrity severely compromised， accompanied by pronounced infiltration of inflammatory cells and severe pulmonary fibrosis. Collagen volume fraction， protein expressions of P16， P21， TGF-β1， Smad3， EGF， EGFR and （IL-4） in lung tissue significantly increased （P＜0.05 or P＜0.01）， while Smad7， MMP-12 and CCL2 protein expressions were significantly decreased （P＜0.05 or P＜0.01）. Compared with the model group， the appearance， pathological morphology， and fibrosis degree of rat lung tissue in Buyang huanwu tang groups were significantly improved， and the above quantitative indicators were significantly reversed （P＜0.05 or P＜0.01）. CONCLUSIONS Buyang huanwu tang can ameliorate IPF in rats， and its underlying mechanism may be associated with the inhibition of TGF-β1/Smad signaling pathway activity and the attenuation of cellular senescence.

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.

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.