ABSTRACT
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]. Conclusion:The mechanism of BYF attenuating mucus hypersecretion of 16HBE cells induced by CSE was associated with the inhibition of Notch signaling pathway activation.
ABSTRACT
Objective:To evaluate the compatibility laws of effective-component compatibility of Bufei Yishen formula Ⅲ (ECC-BYFⅢ) in regulating mucus hypersecretion of chronic obstructive pulmonary disease (COPD).Methods:According to the efficacy of the original Chinese medicine, the components of ECC-BYFⅢ were divided into four categories: Buqi (Ginsenoside Rh1+Astragaloside), Bushen (Icariin), Huatan (Nobiletin), and Huoxue (Paeonol). The four categories were divided into 14 groups based on the method of mathematical permutation. ① The rats were divided into control group, model group, ECC-BYFⅢ, and different components compatibility groups according to the random number table, totaling 17 groups. COPD rat model in stable phase was established by cigarette smoke exposure combined with repeated bacterial infections. The corresponding drugs were given by gavage at the 9th week of modeling, and the samples were collected at the end of the 16th week. The levels of matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of metalloproteinase 1 (TIMP-1) in serum and bronchoalveolar lavage fluid (BALF), and the levels of mucin (MUC) 5AC in lung tissue and BALF were detected by enzyme linked immunosorbent assay (ELISA). ② Human lung epithelial cells BEAS-2B were divided into blank group, model group, and different components compatibility groups. Hypoxia-induced mucus hypersecretion model of human lung epithelial cells BEAS-2B was established 4 hours after corresponding drug pretreatment. The mRNA expressions of MUC5AC, MUC5B, and MUC1 were detected by quantitative polymerase chain reaction (PCR). The mucus secretion indexes of rats and BEAS-2B cells were evaluated by Region (R) value comprehensive evaluation method.Results:① Compared with the control group, MMP-9 in serum and BALF from the model group were significantly increased, the level of TIMP-1 was significantly decreased, and MUC5AC in lung tissue and BALF were significantly increased. The results of R value comprehensive evaluation showed that except for the Buqi and Bushen groups, ECC-BYFⅢ and other components compatibility groups significantly corrected mucus hypersecretion in COPD rats, ECC-BYFⅢ, Bushen Quxie, Fuzheng Huatan, and Quxie groups were much better (R values were 2.15±0.42, 2.11±0.23, 2.16±0.23 and 2.16±0.55, respectively), compared with the model group (R value: 3.00±0.00), the differences were statistically significant (all P < 0.05). ② Compared with the blank group, the mRNA expressions of MUC5AC, MUC5B, and MUC1 increased in the model group. But different components compatibility groups had no significant effects on the mucus secretion of BEAS-2B cells. ③ The comprehensive evaluation results of R value about each in vivo and in vitro index showed that ECC-BYFⅢ, Huoxue, Quxie, Bushen Huoxue, Fuzheng Huatan, Buqi Quxie groups significantly corrected the mucus hypersecretion (R values were 2.30±0.43, 2.33±0.44, 2.12±0.68, 2.27±0.64, 2.24±0.27 and 2.29±0.47, respectively), compared with the model group (R value: 3.00±0.00), the difference was statistically significant (all P < 0.01). The order was: Quxie > Fuzheng Huatan > Bushen Huoxue > Buqi Quxie > ECC-BYFⅢ > Huoxue. Conclusions:Different components compatibility of ECC-BYFⅢ had different effects on COPD mucus secretion. The components containing Huatan (Nobiletin) or Huoxue (Paeonol) showed a better inhibitory effect on mucus secretion.
ABSTRACT
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.