| Background Chronic Obstructive Pulmonary Disease(COPD)is associated with Th1/Th2,Th17/Treg imbalance.Fine particle matters(PM2.5)can lead to dysfunctions and pathological states in various systems and organs,including the respiratory system,aggravating the COPD.Notch signaling pathway plays an important role in the differentiation and development of T lymphocytes,and its activity can be blocked by γ-secretase inhibitor(GSI).At present,the study on the mechanism of PM2.5 on the immune imbalance of COPD is still rarely reported.Objective In this study,PM2.5 was collected and the proportion of Th1,Th2,Th17 and Treg subgroups of spleen T lymphocytes in COPD mice were observed before and after PM2.5 exposure.we also observed the influence Notch1/2/3/4 receptor and its downstream target genes Hes1/5,Hey1 on splenic T lymphocytes in COPD,and the cytokines IFN-γ,IL-4,IL-17 and IL-10 to explose the role of Notch signaling pathways in inflammation in COPD.The aim of this study was to investigate the mechanism by which PM2.5 influences the Notch pathway leading to worsening immune function and accelerating COPD development.Methods 80 SPF male BALB/c mice were randomly assigned into the Healthy group,Healthy+PM2.5 group,Healthy+γ-secretase inhibitor(GSI)group,Healthy+PM2.5+GSI group,COPD group,COPD+PM2.5 group,COPD+GSI group and COPD+PM2.5+GSI group.A mouse model of COPD was established by cigarette smoke exposure.PM2.5 exposure was performed by aerosol inhalation.GSI was given using intraperitoneal injection.Splenic T lymphocytes were purified using a density gradient centrifugation method and nylon wool column separation.The proportion of mouse helper T cells Th1,Th2,Th17,and Treg subsets were measured using flow cytometry and Th1/Th2 and Th17/Treg ratios were calculated.Real-time quantitative polymerase chain reaction(RT-PCR)was used to detect T lymphocytes Notch1/2/3/4,Hes(hairy/enhancer of split)1/5,Hey(hairy/enhancer-of-split related with YRPW motif family members)1 m RNA.The expression of T lymphocyte Notch1/2/3/4,Hes1/5,Hey1 protein was determined by Western blot.ELISA was used to detect serum INF-γ,IL-4,IL-17,and IL-10 concentrations.Result(1)After exposure to cigarette smoke,the body weight in the COPD group mice(26.17±3.11)g was significantly lower than that in the healthy group mice(38.53±3.65)g,the body weight in the COPD+PM2.5 group mice(21.58±1.11)g was significantly lower than those in the healthy+PM2.5 group mice and COPD group mice(13.76±1.64,26.17±3.11)g,the difference was statistically significant(all P <0.01).The body weight in the COPD+PM2.5+GSI group mice(25.94±4.06)g was significantly higher than that in the COPD+PM2.5 group mice(21.58±1.11)g,and the difference was statistically significant(both P <0.01).COPD group mice peak inspiratory flow(PIF)and peak expiratory flow(PEF)(7.57±0.31,4.81±0.32)ml/s was significantly lower than Healthy group(9.75±0.45,7.77±0.33)ml/s,the difference was statistically significant(all P < 0.01).The COPD+PM2.5 group mice PIF and PEF(5.80±0.30,3.89±0.29)ml/s were lower than the Healthy+PM2.5 group(8.42±0.38,6.11±0.22)ml/s and COPD group(7.57±0.31,4.81±0.32)ml/s,the difference was statistically significant(all P <0.01).The COPD+PM2.5+GSI group mice PIF and PEF(6.65±0.36,4.27±0.31)ml/s were higher than the COPD+PM2.5 group(5.80±0.30,3.89±0.29)ml/s,the difference was statistically significant(all P <0.01).COPD mice lung tissue can be seen bronchitis and emphysema clearly.The bronchitis and emphysema in the COPD+PM2.5 group were significantly higher than those in the COPD group.The changes of bronchitis,emphysema and inflammatory cell infiltration in COPD+PM2.5+GSI were lower than those in the control group.While the lung tissue of healthy group mice had no such pathological changes.(2)The Th1%,Th17%,Th1/Th2 and Th17/Treg in the Healthy+PM2.5 group(19.61±1.22,18.16±1.37)%,(30.50±3.56,34.81±5.15)were increased when compared to those in the Healthy group(7.84±0.73,5.59±1.49)%,(9.65±2.01,5.81±3.03),The Th2% and Treg% in the Healthy+PM2.5 group(0.65±0.09,0.53±0.08)% were lower than those in the Healthy group(1.13±0.39,1.18±0.60)%,the differences were statistically significant(P <0.01 or P <0.05).The Th1%,Th17%,Th1/Th2 and Th17/Treg in the COPD group(19.40±3.03,17.69±5.08)%,(31.85±3.67,22.50±7.72)were higher than those in the Healthy group(7.84±0.73,5.59±1.49)%、(9.65±2.01,5.81±3.03),The Th2% and Treg% in the COPD group(0.75±0.17,0.84±0.29)% were lower than those in the Healthy group(1.13±0.39,1.18±0.60)%,the differences were statistically significant(P <0.01 or P <0.05).Th1%,Th17%,Th1/Th2 and Th17/Treg in the COPD+PM2.5 group(37.60±2.86,32.16±6.06)%,(39.02±8.73,77.32±2.21)were higher than the those in Healthy+PM2.5 group(19.61±1.22,18.16±1.37)%,(30.50±3.56、34.81±5.15)and COPD group(19.40±3.03,17.69±5.08)%,(31.85±3.67,22.50±7.72),The Th2% and Treg% in the COPD+PM2.5 group(0.51±0.14、0.45±0.16)% were lower than those in the Healthy+PM2.5 group(0.65±0.09,0.53±0.08)% and COPD group(0.75±0.17,0.84±0.29),the differences were statistically significant(P <0.01 or P <0.05).The Th1%,Th17%,Th1/Th2 and Th17/Treg in the COPD+PM2.5+GSI group(22.75±3.88,27.57±7.38)%,(33.53±5.07,38.72±1.50)were lower than that in the COPD+PM2.5 group(37.60±2.86,32.16±6.06)%,(39.02±8.73,77.32±2.21),The Th2% and Treg% in the COPD+PM2.5+GSI group(0.71±0.10、0.77±0.23)%were higher than those in the COPD+PM2.5 group(0.51±0.14、0.45±0.16)%,the differences were statistically significant(P <0.01 or P <0.05).(3)In the Healthy+PM2.5 group the T lymphocytes’ Notch1/2/3/4,Hes1/5and Hey1 m RNA(5.87±1.22,6.52±2.49,6.49±2.26,7.06±1.12,5.57±1.82,5.32±1.42,8.44±3.84)was higher than in the Healthy group(1.03±0.29,1.09±0.46,1.01±0.13,1.06±0.38,1.10±0.55,1.08±0.44,1.06±0.43),the differences were statistically significant(all P <0.01).In the COPD group the T lymphocytes’ Notch1/2/3/4,Hes1/5and Hey1 m RNA(11.56±1.39,11.89±3.42,11.25±3.19,9.70±2.38,6.98±1.97,9.49±1.89,13.20±3.89)was higher than the Health group(1.03±0.29,1.09±0.46,1.01±0.13,1.06±0.38,1.10±0.55,1.08±0.44,1.06±0.43),the differences were statistically significant(all P <0.01).The COPD+PM2.5 mice T lymphocytes’ Notch1/2/3/4,Hes1/5 and Hey1 m RNA(23.11±5.98,20.14±1.89,21.80±4.30,21.99±4.30,16.59±1.30,17.98±7.08,23.57±2.90)were higher than in the Healthy+PM2.5 group(5.87±1.22,6.52±2.49,6.49±2.26,7.06±1.12,5.57±1.82,5.32±1.42,8.44±3.84)and COPD group(11.56±1.39,11.89±3.42,11.25±3.19,9.70±2.38,6.98±1.97,9.49±1.89,13.20±3.89),the differences were statistically significant(all P <0.01).COPD+GSI mice T cells’ Hes1/5 and Hey1 m RNA(4.27±1.60,6.40±1.52,8.62±2.59)was lower than in the COPD group(6.98±1.97,9.49±1.89,13.20±3.89),the differences were statistically significant(all P <0.01).The COPD+PM2.5+GSI group mice T lymphocytes’ Notch1/2/3/4,Hes1/5 and Hey1 m RNA(17.05±2.40,16.48±2.41,16.09±5.13,15.02±1.38,10.28±0.26,13.96±3.63,18.65±4.53)were lower than the COPD+PM2.5 group(23.11±5.98,20.14±1.89,21.80±4.30,21.99±4.30,16.59±1.30,17.98±7.08,23.57±2.90),the differences were statistically significant(all P <0.01).(4)In the Healthy+PM2.5 group,the T lymphocytes’ Notch1/2/3/4,Hes1/5and Hey1 proteins(0.78±0.04,0.88±0.06,0.76±0.05,0.93±0.07,0.89±0.07,0.83±0.09,0.87±0.04)were higher than in the Healthy group(0.30±0.04,0.30±0.04,0.35±0.08,0.24±0.02,0.54±0.04,0.38±0.06,0.53±0.04),the differences were statistically significant(all P <0.01).COPD mice T lymphocytes’ Notch1/2/3/4,Hes1/5 and Hey1 proteins(1.11±0.13,1.06±0.05,0.89±0.05,0.99±0.14,0.97±0.05,1.16±0.07,0.95±0.07)were higher than in the Healthy group(0.30±0.04,0.30±0.04,0.35±0.08,0.24±0.02,0.54±0.04,0.38±0.06,0.53±0.04),the differences were statistically significant(all P <0.01).COPD+PM2.5 mice T lymphocytes’ Notch1/2/3/4,Hes1/5 and Hey1 proteins(1.51±0.09,1.63±0.10,1.34±0.13,1.54±0.14,1.51±0.06,1.75±0.14,1.81±0.17)were higher than in the Healthy+PM2.5 group(0.78±0.04,0.88±0.06,0.76±0.05,0.93±0.07,0.89±0.07,0.83±0.09,0.87±0.04)and in the COPD group(1.11±0.13,1.06±0.05,0.89±0.05,0.99±0.14,0.97±0.05,1.16±0.07,0.95±0.07),the differences were statistically significant(all P <0.01).COPD+GSI mice T cells’ Hes1/5 and Hey1 protein(0.81±0.06,0.59±0.05,0.79±0.05)were significantly lower than the COPD group(0.97±0.05,1.16±0.07,0.95±0.07),the differences were statistically significant(all P <0.01).COPD+PM2.5+GSI mice T lymphocytes’ Notch1/2/3/4,Hes1/5 and Hey1 proteins(1.36±0.10,1.35±0.07,1.11±0.07,1.23±0.10,1.28±0.07,1.47±0.08,1.40±0.13)were lower than in the COPD+PM2.5 group(1.51±0.09,1.63±0.10,1.34±0.13,1.54±0.14,1.51±0.06,1.75±0.14,1.81±0.17),the differences were statistically significant(all P <0.01).(5)The Healthy+PM2.5 mice serum IFN-γ and IL-17 secretion(26.70±1.59,55.69±3.19)pg/ml was higher than in the Healthy group(17.67±1.71,44.14±3.86)pg/ml,the Healthy+PM2.5 mice serum IL-4 and IL-10 secretion(20.20±1.39)pg/ml,(3.23±0.68)ng/ml was reduced compared with the Healthy group(25.45±2.29)pg/ml,(4.15±1.40)ng/ml,the differences were statistically significant(all P <0.01).COPD mice serum IFN-γ and IL-17 secretion(55.46±1.49,60.70±2.57)pg/ml was higher than in the Healthy group(17.67±1.71,44.14±3.86)pg/ml,COPD mice serum IL-4 and IL-10 secretion(15.86±1.84)pg/ml,(2.60±0.47)ng/ml was reduced compared with the Healthy group(25.45±2.29)pg/ml,(4.15±1.40)ng/ml(all P <0.01).COPD+PM2.5 mice serum IFN-γ and IL-17 secretion(71.27±1.57,71.15±3.69)pg/ml was increased when compared with the Healthy+PM2.5 group(26.70±1.59,55.69±3.19)pg/ml and the COPD group(55.46±1.49,60.70±2.57)pg/ml,COPD+PM2.5 mice serum IL-4 and IL-10 secretion(12.66±1.37)pg/ml,(0.57±0.19)ng/ml was reduced compared to the Healthy+PM2.5 group(20.20±1.39)pg/ml 、(3.23±0.68)ng/ml and the COPD mice(15.86±1.84)pg/ml,(2.60±0.47)ng/ml,the differences were statistically significant(all P <0.01).COPD+GSI mice serum IFN-γ and IL-17 secretion(49.83±2.20,54.30±3.00)pg/ml was reduced compared with the COPD group(71.27±1.57,71.15±3.69)pg/ml,COPD+GSI mice serum IL-4 and IL-10 secretion(16.82±1.19)pg/ml,(3.32±0.14)ng/ml was increased compared with the COPD mice(15.86±1.84)pg/ml,(2.60±0.47)ng/ml,the differences were statistically significant(both P <0.01).COPD+PM2.5+GSI mice serum IFN-γ and IL-17 secretion(60.95±3.15,65.19±2.64)pg/ml was reduced compared to the COPD+PM2.5 group(71.27±1.57,71.15±3.69)pg/ml,COPD+PM2.5+GSI mice serum IL-4 and IL-10 secretion(14.40±1.29)pg/ml,(1.51±0.12)ng/ml was increased when compared to the COPD+PM2.5 group(12.66±1.37)pg/ml,(0.57±0.19)ng/ml,the differences were statistically significant(P <0.01 or P <0.05).(6)Correlation analysis: The Notch1/2/3/4,Hes1/5 and Hey1 proteins were positively correlated with Th1/Th2 and Th17/Treg(r = 0.854～0.867,P < 0.01 or P < 0.05).The Th1% was positively correlated with IFN-γ content(r = 0.666 ～ 0.915,P < 0.01 or P < 0.05).The Th2% was positively correlated with IL-4 levels(r = 0.650 ～ 0.804,P < 0.01 or P < 0.05).The Th17% was positively correlated with IL-17 content(r = 0.638 ～ 0.840,P < 0.01 or P < 0.05).The Treg% was positively correlated with IL-10 content(r = 0.652 ～ 0.804,P < 0.01 or P < 0.05).Conclusion Exposed to cigarette smoke can be used to establish the model of COPD mice.The COPD group demonstrated Th1/Th2 and Th17/Treg immune imbalance.PM2.5 exposure leads Healthy mice to an immune disorder evidenced by an abnormal increase in Th1 and Th17.It also aggravates the Th1/Th2 and Th17/Treg ratio immune imbalance in COPD mice.The Notch pathway participates in the immune imbalance of COPD.PM2.5 causes immune system dysfunction via Notch signaling pathway,which is involved in the pathogenesis of COPD. |
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