| Objective Notch signaling pathway involved in the immune imbalance of asthma and other respiratory diseases by regulating the growth and differentiation of T cells, but the roles in chronic obstructive pulmonary disease (COPD) is unclear. In this study, We observed the influence of the Notch signaling pathway Notchl receptor, Jaggedl ligand and its downstream products Hesl in COPD mouse model splenic-derived CD4+ T lymphocyte Th1, Th2, Th17, Treg subsets and Th1/Th2 and Thl7/Treg ratio to explore the roles of Notch signaling pathway on immune imbalance in COPD.Methods 120 of SPF 8-week-old male Balb/c mice were randomly assigned into the healthy control group, COPD group, COPD Dimethyl sulfoxide (DMSO) group and COPD Gamma secretase inhibitors (GSI) group. Using Cigarette smoke exposure 90 days to establish mouse model of chronic obstructive pulmonary disease, pulmonary function and pathological changes were observed. Flow cytometry was used to detect the proportion of the mouse spleen-derived Th1 cells, Th2 cells, Th17 cells, Treg cells; Using real-time quantitative polymerase chain reaction (RT-PCR) and Western blot (WB) to detect the Alveolar macrophages ligand Jagged1, splenic T lymphocyte’s Notch1 and its downstream Hes1 mRNA and protein expression.Results (1) The establishment of COPD model:Smoke exposure 90 days alone, successfully established mouse model of chronic obstructive pulmonary disease. COPD mice lung tissue can be seen bronchitis and emphysema clearly, while no such changes in healthy control mice. COPD group peak inspiratory flow (PIF) and peak expiratory flow (PEF) (6.82 ± 0.46), (3.87 ± 0.44) was significantly lower than the healthy control group (9.84 ± 0.46), (7.96 ± 0.52) (all P< 0.01);(2) COPD mice Th1, Th17 and Treg cell percentage (13.20 ± 0.95,10.22 ± 0.45,0.41 ± 0.09)% compared with the healthy control group, the proportion of cells in mice (8.07 ± 0.44,5.98 ± 0.26,0.26 ± 0.05)% significantly increased, the differences were statistically significant (P 0.01); No significant increase in the proportion of Th2 cells in COPD mice (0.72 ± 0.12)% compared with healthy mice (0.65 ± 0.08)%, the difference was not statistically significant (P> 0.05); and the proportion of Thl and Th17 cells in COPD GSI group mice (9.48 ± 0.66,7.70 ± 0.39)% was significantly reduced compared with COPD group (13.20 ± 0.95,10.22 ± 0.45)%, the difference was statistically significant (P<0.01), No significant increase in the ratio of Th2 and Treg cells in COPD GSI mice (0.71 ± 0.09,0.45 ± 0.07)% compared with COPD group (0.72 ± 0.12,0.41 ± 0.09)%, the difference was not statistically significant (P> 0.05). COPD group Thl/Th2 ratio (18.70 ± 4.12) compared with the healthy control group (12.63 ± 1.91) was significantly increased, the difference was statistically significant (P<0.01); the ratio of Thl/Th2 in COPD GSI Group (13.63 ± 2.56) was significantly lower than COPD group (18.70 ± 4.12), the difference was statistically significant (P<0.01). No significant difference in the ratio of Thl/Th2 in the COPD group and COPD DMSO group. COPD group Th17/Treg ratio (26.27 ± 7.09) compared with the healthy control group (24.07 ± 6.01) was increased, but the difference was not statistically significant (P> 0.05); and the ratio of Thl7/Treg in COPD GSI Group (17.55 ±3.17) lower than COPD group (26.27 ± 7.09), the difference was statistically significant (P<0.01). Thl7/Treg ratio in COPD group and COPD DMSO group had no significant difference.(3) COPD mice spleen T lymphocyte surface Notchl receptor and its downstream Hesl mRNA expression (5.15 ± 0.77,1.92 ± 0.32) compared with the healthy group (1.00 ± 0.00,1.00 ± 0.00) were significantly increased, COPD alveolar macrophages JaggedlmRNA expression (2.23 ± 0.36) was significantly higher than the healthy group (1.00 ± 0.00), the difference was statistical significance (P<0.01); COPD GSI group Notchl and its downstream Hesl mRNA expression (3.47 ±0.41,1.34 ± 0.04) compared with the COPD group (5.15 ± 0.77,1.92 ± 0.32) decreased significantly, COPD GSI group alveolar macrophages Jaggedl mRNA expression (1.80 ± 0.21) compared with COPD group (2.23 ± 0.36) was significantly reduced, the difference was statistical significance (P<0.01); No significant difference in the expression of Notchl, Jagged1 and its downstream Hesl mRNA in the COPD group and COPD DMSO group.(4) COPD mice spleen T lymphocyte surface Notchl receptor and its downstream Hesl protein expression (0.85±0.04,0.16±0.02) compared with the healthy group (0.17+0.01,0.09 ±0.01) were significantly increased, COPD alveolar macrophages Jaggedl protein expression (0.37 ± 0.01) was significantly higher than the healthy group (0.21 ± 0.01), the difference was statistical significance(P<0.01); COPD GSI group Notchl and its downstream Hesl group protein expression (0.53±0.04,0.12±0.01) compared with the COPD group (0.85±0.04,0.16± 0.02) decreased significantly, COPD GSI group alveolar macrophages Jagged1 protein expression (0.28 ± 0.01) compared with COPD group (0.37 ± 0.01) was significantly reduced, the difference was statistical significance(P<0.01); No significant difference in the expression of Notchl, Jagged1 and its downstream Hesl protein in the COPD group and COPD DMSO group.(5) Correlation analysis:COPD group and COPD GSI group Notchl and Hesl mRNA and protein expression was positively correlated with Thl and Th17 cells and negatively correlated with Th2 and Treg cells, while, COPD group and COPD GSI group Jaggedl mRNA and protein expression was negatively correlated with Th1 and Th17 cells and positively correlated with Th2 and Treg cells, the difference was statistically significant.Conclusion 1.The method of Cigarette smoke exposure can establish a mouse model of chronic obstructive pulmonary disease; 2. The Th1/Th2, Th17/Treg immune disorders existed in mice with Chronic obstructive pulmonary disease mice; 3. Chronic obstructive pulmonary disease mice mice Notch 1 receptor, Jagged1 ligand and its downstream Hes1 molecule mRNA and protein levels elevated, and with the Th1/Th2 and Th17/Treg related; 4. GSI could partially inhibit Notch1, Jagged1, Hes1 and T cell subsets level, suggesting that Notch signaling pathway involved in the immune disorder of Chronic obstructive pulmonary disease mice. |
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