[Differential effects of Qingqiao and Laoqiao on bleomycin-induced pulmonary fibrosis in mice by principal component analysis].

ABSTRACT

The mechanism of alleviating bleomycin-induced pulmonary fibrosis in mice was compared between Qingqiao(Forsythiae Fructus produced with immature fruits) and Laoqiao(Forsythiae Fructus produced with mature fruits) from the pharmacodynamic correlation and composition differences. Mice were randomized into normal, model, pirfenidone(50 mg·kg~(-1)), low-and high-dose(1.3, 2.6 g·kg~(-1), respectively) Qingqiao, and low-and high-dose(1.3, 2.6 g·kg~(-1), respectively) Laoqiao groups. The mouse model of pulmonary fibrosis was established by intratracheal instillation of bleomycin, during which the survival rate and body weight changes of the mice were measured. After modeling, the lung index was calculated, and the pathological changes in the lung tissue were evaluated by hematoxylin-eosin(HE), Masson, and Sirius red staining. Transmission electron microscopy was employed to observe the ultrastructure of the lung tissue. The biochemical assay was employed to measure the levels of transforming growth factor-ß1(TGF-ß1), α-smooth muscle actin(α-SMA), E-cadherin, and hydroxyproline(HYP) in the lung tissue and interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) in the bronchoalveolar lavage fluid(BALF). The mRNA and protein levels of matrix metalloproteinase 7(MMP7), collagen Ⅰ, E-cadherin, TNF-α, vimentin, TGF-ß1, and α-SMA in the lung tissue were determined by RT-qPCR and Western blot, respectively. The expression of α-SMA in the lung tissue was detected by the immunofluorescence assay. Principal component analysis was performed to compare the effects of Qingqiao and Laoqiao in ameliorating pulmonary fibrosis. Molecular docking was employed to analyze the binding between the compounds with high content in Laoqiao and TGF-ß1. The cell-counting kit(CCK-8) assay was used to examine the effects of the active compounds on TGF-ß1-induced BEAS-2B and HFL1 cell models. The results showed that Qingqiao and Laoqiao increased the survival rate, reduced the lung index, alleviated the pathological damage and collagen deposition in the lung tissue, ameliorated the damage of lamellar bodies in alveolar epithelial type Ⅱ cells, lowered the level of IL-6 and TNF-α in the BALF, down-regulated the expression of HYP, MMP7, vimentin, collagen Ⅰ, TGF-ß1, and α-SMA, and up-regulated the expression of E-cadherin in the lung tissue of the mouse model of pulmonary fibrosis. The collagen deposition in the mouse model of pulmonary fibrosis was comprehensively evaluated by principal component analysis, and the effects of different treatments followed the trend of high-dose Laoqiao>low-dose Laoqiao>high-dose Qingqiao>low-dose Qingqiao. Molecular docking showed that hydroxytyrosol, caffeic acid, phillygenin, and(-)-lariciresinol had strong binding affinity with TGF-ß1 receptor. The results of cell experiments showed that these compounds significantly attenuated the TGF-ß1-induced damage in BEAS-2B cells and inhibited the TGF-ß1-induced proliferation of HFL1 cells. In conclusion, both Qingqiao and Laoqiao were effective in ameliorating bleomycin-induced pulmonary fibrosis in mice. Laoqiao was superior to Qingqiao in reducing collagen deposition, which might be attributed to the higher content of hydroxytyrosol, caffeic acid, phillygenin, and(-)-lariciresinol in Laoqiao than in Qingqiao.