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.

Objective To analyze the morphological and structural changes to pulmonary arterioles in rats induced by smoke exposure combined with Klebsiella infection,and to evaluate the severity of the pulmonary arteriolar lesions.Methods Pulmonary arteriolar images from lung sections of control and model rats treated with smoke exposure combined with Klebsiella infection were analyzed by qualitative and quantitative method.Victorian-blue-stained sections were used for the detection of pulmonary arteriolar muscularization,vascular wall thickness,vascular occlusion score,the intima thickness and media thickness of muscular arterioles,and neointima proliferation.Hematoxylin and eosin-stained sections were used for the observation and detection of inflammatory cell infiltration and plexiform lesions around arterioles.Van Gieson-stained sections were used for the observation of collagen fibers in the intima and detection of the percentage of collagen fiber area in the arteriolar wall.Based on the above analyses,the degree of pulmonary arteriolar pathology was rated according to Heath-Edwards criteria.Results For≤50 μm diameter arterioles,the percentage of non-muscular vessels was significantly decreased(P＜0.01),the percentage of muscular vessels was increased(P＜0.01),the percentage of partial muscular vessels was not significantly different(P＞0.05),the thicknesses of the non-muscular vessel walls and muscular vessel walls were significantly increased(P＜0.05,P＜0.01),and the occlusion scores of both non-muscular and muscular pulmonary arterioles were significantly increased in the model group compared with the Control group(P＜0.05,P＜0.01).For 50 μm＜diameter≤100 μm arterioles,the percentage of non-muscular vessels was significantly decreased(P＜0.05),the percentages of muscular vessels and partial muscular vessels were not significantly different(P＞0.05),the wall thickness and occlusion score of muscular vessels were significantly increased(P＜0.05),and the wall thickness and occlusion score of non-muscular vessels were not significantly different in the model group compared with the Control group(P＞0.05).Compared with the Control group,the model group showed significantly increased intimal thickness and media thickness and significantly increased perivascular inflammatory infiltration score in both muscular arterioles of≤50 μm diameter and 50 μm＜diameter≤100 μm(P＜0.05,P＜0.01).In the Control group(n=9),only one section with two neointimal lesions was found,and the degree of neointima proliferation was 1.61%.In the model group(n=10),five sections had neointima lesions,and the degree of neointima proliferation was 1.04%to 17.14%.No plexiform lesions were found in any section.For pulmonary arterioles with a diameter of≤100 μm,there was no change in the expression of intimal collagen fibers in the model group compared with the Control group,and there was no significant difference in the percentage of collagen fiber area in the vessel walls(P＞0.05).According to Heath-Edwards criteria,the pulmonary arteriole lesions in the model rats did not reach grade Ⅲ.Conclusions The model rats showed pathological manifestations such as pulmonary arteriolar muscularization,thickening of the intima and media,and mild to moderate inflammatory reactions around arterioles.The low amount of neointimal proliferation and collagen fibers in the vascular wall and the absence of plexiform lesions suggest that the model may be up to grade Ⅱ lesions,according to the Heath-Edwards criteria.