[The mechanism of nicotine on human bronchial smooth muscle cell contraction].

Objective: To investigate the molecular mechanism of contractility dysfunction of human bronchial smooth muscle cells induced by nicotine. Methods: Primary human bronchial smooth muscle cells were cultured in vitro. The cells were divided into a control group and a nicotine group which was treated with 10(-5) mol/L nicotine for 48 h and transfected with or without α7nAChR-siRNA (The siNC group, siNC + nicotine group and siα7nAChR + nicotine group). The effects of nicotine on the cell contractile function were examined by collagen gel shrinkage assay. The expressions of α7nAChR and TRPC6 protein in nicotine-treated human bronchial smooth muscle cells were detected by Western blotting. The change of intracellular calcium concentration by nicotine was detected by calcium ion imaging system.Data were analyzed by t test or single factor analysis of variance. Results: The area of collagen gel in the nicotine group (24±8)% was significantly lower than that in the control group (59±14)% (t=3.78, P<0.05). Compared with the control group, the expression of α7nAChR protein in nicotine-induced group (173±16)% was significantly higher than that of controls 100±0)%, t=-6.848, P<0.05. Compared with the siNC group [(72±10)%, (0.79±0.07), (0.41±0.04) and (0.17±0.02) respectively], the collagen gel area of siNC + nicotine group was significantly reduced by (37±10)%. However, the basal calcium level (1.04±0.02), store operated calcium entry level (SOCE, 0.68±0.03) and receptor operated calcium entry level (ROCE, 0.36±0.02) were remarkably elevated in the nicotine treated group (all P<0.05). Furthermore, compared with siNC + nicotine group, the area of collagen gel in siα7nAChR + nicotine group was significantly increased (62±10)%, and the basal calcium level (0.78±0.06), SOCE level (0.39±0.05) and ROCE level (0.15±0.02) were significantly reduced (all P<0.05). Conclusions: Nicotine can increase the expression of TRPC6 protein, SOCE and ROCE level, and increase the intracellular calcium concentration by upregulating the expression of α7nAChR protein, thereby promoting smooth muscle cell contraction.

[Preliminary analysis of lung function of population with biofuel smoke exposure].

Objective: To analyse the impaired lung functions of people with biofuel smoke exposure. Methods: Nonsmokers with biofuel smoke exposure were selected as research objects in a mountainous area of northern Guangdong where the families used biofuels as main energies and the nonsmokers without biofuel smoke exposure in the same area as control. Questionnaire interviews and spirometry tests were performed on all subjects. To analyse the differences of lung functions in both. Results: Seventy hundred and seventeen subjects were enro1led in this study.There were 530 nonsmokers with biofuel smoke exposure(observation group) including 442 women and 88 men, average age 54±10. There were 187 nonsmokers without biofuel smoke exposure(control group) including 141 women and 46 men, average age 54±10. There was no significant difference between two groups in age, height, weight, BMI, waist circumference, hip circumference and waist/hip ratio(P>0.05). The pulmonary ventilation function index(FEV(1)%Pred, FEV(1)/FVC) in the observation group was significantly less than that in control group [(100±18) vs.(106±25); (80±10) vs.(83±6) respectively, P<0.05]. Small airway function index(PEF25, PEF50, PEF75, MMPEF and MMPEF%Pred) was significantly less than that in control group(P<0.01). According to the univariate regression analysis and multivariate regression analysis, regression coefficients between BIOFUEL-INDEX and FEV/FVC was -0.1, 95%CI(-0.1, -0.1, P<0.01). According to the threshold analysis, the vertice of BIOFUEL-INDEX was 46.0, where the predicted Y value was 81.76, 95%CI (80.2, 83.33). When BIOFUEL-INDEX<46.0, the regression coefficient was 0, 95%CI (-0.1, 0.0)(P>0.05); when BIOFUEL-INDEX> 46.0, the regression coefficient 2 was -0.1, 95%CI (-0.2, -0.1)(P<0.01). The difference between coefficient 2 and 1 was -0.1, 95%CI (-0.2, 0.0), which was statistically significant (P<0.05). The Log-Likelihood ratio between Model I and Model Ⅱ had statistical significance (P=0.019). Conclusions: The biofuel smokes exposure causes damages in lung function.

[PM2.5 from traffic-related ambient air and wood smoke induces epithelial-mesenchymal transition in human bronchial epithelial cells].

Objective: To observe if arterial traffic ambient PM2.5 (TAPM2.5) and wood smoke PM2.5(WSPM2.5) exposure can induce epithelial-mesenchymal transition (EMT) in human bronchial cells (HBEC). Methods: PM2.5 was collected from an arterial traffic road and a typical southern kitchen, and then the collections were extracted by DMSO. The viability of HBEC was measured by Cell Counting Kit (CCK-8) after culture with PM2.5-DMSO extracts for 24 hours. The expressions of EMT markers, including E-cadherin, cytokeratin, α-smooth muscle actin (α-SMA), vimentin and collagen typeⅠ (COL-Ⅰ) in HBEC were assayed by cell immunofluorescence and Western blot analysis after exposed to two different sources of PM2.5-DMSO extracts for 14 days. Results: The cell viability of HBEC increased at low concentrations (1, 2, 10 µg/ml and 1, 5, 10 µg/ml, corresponding to [(118.4±13.7)%, (118.2±8.0)%, (123.0±19.6)% and (112.4±4.1)%, (120±5.4)%, (117.8±7.0)%, respectively, all P<0.05], and then declined at high levels [20, 100, 200 µg/ml and 15, 20, 30, 40 µg/ml, corresponding to (100.7±12.1)%, (53.4±15.3)%, (9.4±1.7)% and (106.8±10.0)%, (93.8±7.9)%, (60.9±9.5)%, (46.2±3.6)%, respectively, P values were 0.923, 0.000, 0.000 and 0.231, 0.278, 0.000, 0.000, respectively] in both TAPM2.5-DMSO and WSPM2.5-DMSO incubation. After exposure for 14 days, the cells lost their typical cobblestone-like shape which implied that EMT might occur. The same treatment caused decreased positive signals of E-cadherin and cytokeratin in a small proportion of the cells. The decreased expressions of cytokeratin were verified by Western blot (TAPM2.5 and WSPM2.5 were 0.063±0.109 and 0.039±0.313, P values were 0.033 and 0.030, respectively), while α-SMA was only significantly upregulated in the WSPM2.5-DMSO group (7.853±4.784, P=0.049). The expressions of E-cadherin decreased in both groups but not statistically significant in Western blot (0.862±0.096 and 0.817±0.212, P values were 0.228 and 0.117, respectively). Another marker of EMT, COL-I, markedly increased in both PM2.5 treatment groups (2.549±1.037 and 3.658±1.207, P values were 0.034 and 0.001). Conclusions: Both PM2.5 from arterial traffic ambient air and wood smoke could induce EMT in human bronchial epithelial cells, while WSPM2.5 appeared to have a more significant influence on EMT in HBEC.