[Ameliorative effects and mechanisms of Buyang Huanwu Decoction and its different ethanol elution fractions on pulmonary fibrosis model rats].

This study aimed to investigate the differences in the ameliorative effects and underlying mechanisms of Buyang Huanwu Decoction(BHD) and its different ethanol elution fractions on pulmonary fibrosis(PF). The content of amygdalin, formononetin, kaempferol, ferulic acid and paeoniflorin in the drug-containing sera of BHD and its different fractions were determined using ultra-high-performance liquid chromatography-tandem quadrupole mass spectrometry. A rat model of PF was established by intratracheal instillation of bleomycin(BLM). The rats were divided into seven groups: a control group, a BLM group, a prednisone acetate(PA) group, a BHD group, a 30% ethanol elution fraction(Fr30) group, a 50% ethanol elution fraction(Fr50) group, and a 90% ethanol elution fraction(Fr90) group. All treatments were administered continuously for 21 days. The degrees of lung inflammation and fibrosis were observed by HE and Masson staining. The hydroxyproline(HYP) levels in lung tissue were determined using a HYP assay kit. The levels of interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-10(IL-10), tumor necrosis factor-α(TNF-α), and high mobility group box 1 protein(HMGB1) in lung tissue were determined using ELISA kits. The levels of total superoxide dismutase(T-SOD), malondialdehyde(MDA) and glutathione peroxidase(GSH-PX) in lung tissue were determined using corresponding commercial kits. Immunofluorescence staining and Western blot were used to detect the levels of epithelial-mesenchymal transition(EMT)-related proteins in lung tissue. RT-qPCR and Western blot were employed to determine the mRNA and protein levels of factors associated with the transforming growth factor-β1(TGF-β1)/Sma and Mad-related protein(Smad) pathway in lung tissue. The results showed that amygdalin was detected in the drug-containing sera of all groups, with the highest content in the Fr30 group and the lowest in the Fr90 group. Formononetin and kaempferol were detected in the BHD, Fr50, and Fr90 groups, with the highest content in the Fr50 group and the lowest in the Fr90 group. Ferulic acid was detected in all groups, with the highest content in the BHD group and the lowest in the Fr30 group. Paeoniflorin was only detected in the BHD and Fr30 groups, with similar content. Compared with the BLM group, the levels of HYP, TNF-α, IL-6, IL-1β, HMGB1, and MDA decreased in all treatment groups, while the levels of IL-10, T-SOD, and GSH-PX increased. However, there was no statistical significance in the changes of T-SOD level in the Fr30 group and HYP and IL-10 levels in the Fr90 group compared with the BLM group. Immunofluorescence staining showed a marked decrease in the protein expression of α-smooth muscle actin(α-SMA) in the lung tissue of the BHD, Fr30, Fr50, and Fr90 groups. Western blot analysis showed that the expression of epithelial cadherin(E-cadherin) significantly increased, while α-SMA and vimentin protein expressions significantly decreased in the lung tissue of the BHD, Fr30, Fr50, and Fr90 groups. RT-qPCR and Western blot analysis revealed that the mRNA and protein levels of TGF-β1 in the BHD and Fr30 groups significantly decreased, the phosphorylation(p)-Smad3/Smad3 ratio in all treatment groups significantly decreased, and the mRNA and protein levels of Smad7 in the BHD, Fr30 and Fr50 groups significantly increased. In conclusion, BHD and its different ethanol elution fractions may ameliorate BLM-induced PF in rats by inhibiting the TGF-β1/Smad pathway, with the Fr30 and Fr50 fractions showing better ameliorative effects.