[Bufei Yishen formula attenuates cigarette smoke extract-induced airway mucus hypersecretion by regulating Notch signaling pathway].

OBJECTIVE: To explore the mechanism of Bufei Yishen formula (BYF) on attenuating cigarette smoke extract (CSE)-induced airway mucus hypersecretion by regulating Notch signaling pathway. METHODS: The human airway epithelial cell 16HBE was cultured in vitro, and the cells in logarithmic growth phase were used for the experiments. (1) Intervention condition screening experiment: the 16HBE cells were grouped, methylthiazolyldiphenyl-tetrazolium (MTT) method and enzyme-linked immunosorbent assay (ELISA) were used to detect the effects of different concentrations of CSE (2.5%, 5%, 10%, 20%, 40%), different concentrations of BYF drug-containing serum (5%, 10%, 20%, 40%), and different concentrations of Notch signal pathway blocker DAPT (5, 10, 20, 40 µmol/L) on cell activity and secretion of mucin 5AC (MUC5AC) levels. In addition, a blank control group was set up to screen out the best conditions for preparing CSE-induced cell mucus hypersecretion model and BYF and DAPT intervention. (2) Intervention experiment: the 16HBE cells were divided into four groups. The blank control group was not given any treatment; the 16HBE cells were induced by 10% CSE for 24 hours to prepare mucus hypersecretion model in the CSE model group; the cells in the CSE+BYF group and CSE+DAPT group were given 10% BYF or 20 µmol/L DAPT, respectively, for intervention at the same time for 24 hours. Real-time fluorescent quantitative polymerase chain reaction (qPCR) was used to detect the mRNA expressions of MUC5AC, Notch3 and hairy and enhancer of split 1 (HES1) in the cells. Western blotting was used to detect the protein expressions of Notch3 and HES1 in the cells. RESULTS: (1) Results of the screening experiment of intervention conditions: compared with the blank control group, 10% CSE induction for 24 hours was the best condition for establishing cell mucus hypersecretion model that neither affected cell viability nor increased the secretion of MUC5AC; while 10% BYF and 20 µmol/L DAPT was the optimal intervention condition. (2) Intervention experiment results: compared with the blank control group, the mRNA expressions of MUC5AC, Notch3, and HES1 and the protein expressions of Notch3 and HES1 in the CSE model group were significantly increased, indicating that CSE activated Notch3 and HES1 signal activation and induced 16HBE cells to secrete mucus protein. Compared with the CSE model group, BYF and DAPT could significantly down-regulate the mRNA and protein expressions of MUC5AC, Notch3, and HES1 in cells [MUC5AC mRNA (2-ΔΔCT): 1.03±0.13, 0.96±0.05 vs. 1.35±0.07, Notch3 mRNA (2-ΔΔCT): 1.10±0.14, 1.10±0.02 vs. 1.31±0.15, HES1 mRNA (2-ΔΔCT): 1.26±0.10, 1.14±0.15 vs. 1.45±0.08, Notch3 protein (Notch3/GAPDH): 0.10±0.03, 0.16±0.03 vs. 0.31±0.09, HES1 protein (HES1/GAPDH): 0.37±0.06, 0.34±0.08 vs. 0.50±0.05, all P < 0.05]. CONCLUSIONS: The mechanism of BYF attenuating mucus hypersecretion of 16HBE cells induced by CSE was associated with the inhibition of Notch signaling pathway activation.

Bufei Yishen Formula Restores Th17/Treg Balance and Attenuates Chronic Obstructive Pulmonary Disease via Activation of the Adenosine 2a Receptor.

Bufei Yishen formula (BYF) is a Traditional Chinese Medicine (TCM) reported to ameliorate chronic obstructive pulmonary disease (COPD) by regulating the balance between T helper (Th) 17 and regulatory T (Treg) cells. However, its mechanism remains unknown. Therefore, this study aimed to explore the underlying mechanisms of BYF. Naïve CD4+ T cells were exposed to anti-CD3, anti-CD28, transforming growth factor (TGF)-ß, and/or interleukin (IL)-6 to promote their differentiation into Th17 or Treg cells. A rat model of cigarette smoke- and bacterial infection-induced COPD was established and orally treated with BYF and/or an adenosine 2a receptor (A2aR) antagonist. Then, the rats were sacrificed, their lung tissues were removed for histological analysis, and their spleens were collected to evaluate Th17 and Treg cells. The results showed that BYF significantly suppressed Th17 cell differentiation and its related cytokines and enhanced Treg cell differentiation and its related cytokines. In addition, BYF activated the A2aR, increased the levels of p-signal transducer and activator of transcription (STAT)5, and decreased the level of p-STAT3 in Treg and Th17 cells. The A2aR antagonist suppressed the changes induced by BYF treatment in Th17 and Treg cells. Furthermore, the A2aR antagonist diminished the therapeutic effect of BYF on COPD, as indicated by the lung injury scores, bronchiole wall thickness, small pulmonary vessels wall thickness, bronchiole stenosis, alveolar diameters, decrease in inflammatory cytokines, increase in alveolar number, and lung functions. Similarly, the A2aR antagonist reversed the effects of BYF on the proportion of Th17 and Treg cells in the spleen. Additionally, BYF increased the protein and mRNA levels of A2aR and regulated the phosphorylation of STAT3 and STAT5 in spleen and lung tissues, which were inhibited by cotreatment with the A2aR antagonist. In conclusion, this study suggested that BYF exhibited its anti-COPD efficacy by restoring the Th17/Treg balance via activating A2aR, which may provide evidence for the clinical application of BYF in the treatment of COPD.

Effective-constituent compatibility-based analysis of Bufei Yishen formula, a traditional herbal compound as an effective treatment for chronic obstructive pulmonary disease.

OBJECTIVE: Critical effective constituents were identified from Bufei Yishen formula (BYF), a traditional herbal compound and combined as effective-constituent compatibility (ECC) of BYF I, which may have potential bioactive equivalence to BYF. METHODS: The active constituents of BYF were identified using four cellular models and categorised into Groups 1 (Bufeiqi), 2 (Bushen), 3 (Huatan) and 4 (Huoxue) according to Chinese medicinal theory. An orthogonal design and a combination method were used to determine the optimal ratios of effective constituents in each group and the ratios of "Groups 1 to 4" according to their pharmacological activity. We also comprehensively assessed bioactive equivalence between the BYF and the ECC of BYF I in a rat model of chronic obstructive pulmonary disease (COPD). RESULTS: We identified 12 active constituents in BYF. The numbers of constituents in Groups 1 to 4 were 3, 2, 5 and 2, respectively. We identified the optimal ratios of effective constituents within each group. In Group 1, total ginsenosides:Astragalus polysaccharide:astragaloside IV ratio was 9:5:2. In Group 2, icariin:schisandrin B ratio was 100:12.5. In Group 3, nobiletin:hesperidin:peimine:peiminine:kaempferol ratio was 4:30:6.25:0:0. In Group 4, paeoniflorin:paeonol ratio was 4:1. An orthogonal design was then used to establish the optimal ratios of Group 1, Group 2, Group 3 and Group 4 in ECC of BYF I. The ratio for total ginsenosides:Astragalus polysaccharide:astragaloside IV:icariin:schisandrin B:nobiletin:hesperidin:peimine:paeoniflorin:paeonol was determined to be 22.5:12.5:5:100:12.5:4:30:6.25:25:6.25. A comprehensive evaluation confirmed that ECC of BYF I presented with bioactive equivalence to the original BYF. CONCLUSION: Based on the ECC of traditional Chinese medicine formula method, the effective constituents of BYF were identified and combined in a fixed ratio as ECC of BYF I that was as effective as BYF itself in treating rats with COPD.

[Inhibitory effect of Panax notoginseng saponins on alveolar epithelial to mesenchymal transition].

In the study, the effects of Panax notoginseng saponins (PNS) on alveolar epithelial to mesenchymal transition (EMT) and extracellular matrix degradation were observed in a type of human alveolar epithelial cell, A549 cells, stimulated by TGF-beta1. Firstly, MTT method was applied to evaluation of cellular proliferation and found that PNS from 12.5 mg x L(-1) to 200 mg x L(-1) dosage could not inhibit significantly cellular proliferation. Then, cells were divided into five groups, normal group, TGF-beta1 group, TGF-beta1 + 50 mg x L(-1) PNS group, TGF-beta1 + 100 mg x L(-1) PNS group and TGF-beta1 + 200 mg x L(-1) PNS group. Normal cells were not stimulatec by TGF-beta1; TGF-beta1 cells were only stimulated by TGF-beta1 and the other cells were stimulated by TGF-beta1 with different doses of PNS, respectively. After stimulation, cells and supernatants were collected for assays. Cellular roundness was applied to quantitative evaluation of morphological change. Immunocytochemistry was applied to examine E-cadherion, a-SMA and FN proteins expression in the cells. Enzyme linked-immunosorbent assay was applied to MMP-9 and TIMP-1 levels. The results showed that EMT of A549 cells was induced by TGF-beta1, showing significant change of roundness, E-cadherion, alpha-SMA and FN (P < 0.05, P < 0.01). Compared to TGF-beta1, PNS significantly inhibited the changes of roundness (P < 0.05), FN and alpha-SMA (P < 0.05, P < 0.01) and not significantly inhibited the change of E-cadherion. Furthermore, MMP-9 levels were significantly increased by TGFbeta1 stimulation (P < 0.05), without significant change of TIMP-1. Compared with TGF-beta1, PNS could significantly increase MMP-9 level (P < 0.05) and decrease TIMP-1 levels (P < 0.05, P < 0.01). In conclusion, PNS could inhibit alveolar epithelial cell EMT induced by TGF-beta1, with increase of extracellular matrix degradation ability, which showed anti-fibrosis of lung ability.