Regulation Mechanism Of Notch Signaling Pathway On Secretion Of Muc5ac Induced By Atmospheric PM_2.5 In Asthma

An increase in the concentration of suspended particulate matter such as PM_2.5 can cause damage to the human airways,leading to the onset and acute attack of asthma.It is of great significance for the diagnosis,treatment and prognosis of asthma to identify the key molecules and signal pathways that PM_2.5 influenced the occurrence and progression of asthma and to take corresponding intervention measures.Muc5ac is a major mucin in human respiratory tract,and its production and secretion is closely related to the pathogenesis,treatment and prognosis of asthma.The proper secretion of Muc5 ac was helpful to remove foreign substance and maintain the health of the body.Overexpression and oversecretion of Muc5 ac can form mucus plug,aggravate asthma and even induce death.Thus,the expression and secretion of Muc5 ac is an important factor affecting asthma.When PM_2.5 enters the airway,it can affect the expression and secretion of Mucus and Mucin.However,the mechanisms of air pollutants regulating mucin secretion have not been fully elucidated.In order to investigate the effects of atmospheric PM_2.5 on Notch signaling pathway and Muc5 ac expression,after different concentrations of atmospheric PM_2.5 were exposed to Beas-2B cells and healthy and asthma rats,the levels of m RNA and protein of receptors（Notch1,Notch2,Notch3,Notch4）and ligands（Jadgged1,Jadgged2,Dll3,Dll4）,downstream target gene Hes1 and Muc5 ac in the Notch signaling pathway were examined.Then Beas-2B cells and normal and asthma rats were given the Notch signaling inhibitor DAPT,the expression of Muc5 ac in the Notch signaling inhibitor group and control group was detected after they were exposed to PM_2.5 of different concentrations.Thus,the regulation mechanism of Notch signaling pathway on secretion of Muc5 ac induced by atmospheric PM_2.5 in asthma has been explored.Objects: To investigate the regulation mechanism of Notch signaling pathway on secretion of Muc5 ac induced by atmospheric PM_2.5 in Beas-2B cells and asthma rats,and to provide treatment targets for asthma and clinical diseases related to Mucin hypersecretion.Methods: Bea S-2B cells were used as research subjects,which were exposed to PM_2.5 of different concentrations（0 μg / ml,100 μg / ml,200 μg / ml,400 μg / ml）for 12 hours,24 hours and 48 hours respectively,cell survival rates were measured by MTT,the levels of m RNA and protein of the receptors,Ligands,downstream target gene Hes1 and Muc5 ac in the Notch signaling pathway were detected by q PCR and western.Then Beas-2B cells were exposed to PM_2.5 of different concentrations（0 μg / ml,100 μg / ml,200 μg / ml,400 μg / ml）for 12 hours,24 hours and 48 hours respectively after given the Notch signal pathway inhibitor DAPT 48 h.cell survival rates were measured by MTT,the levels of m RNA and protein of the receptors,ligands,downstream target gene Hes1 and Muc5 ac in the Notch signaling pathway were detected by q PCR and western.The rats of asthma model were given to PM_2.5 and DAPT respectively,the pathological changes of lung tissues were observed by HE staining,the mucus of trachea was observed by AB-PAS staining,and the Muc5 ac level of airway and lung tissues was detected by ELISA,the expression level of Notch signaling gene and Hes1 were detected by q PCR and Western.The data were analyzed with IBM SPSS 24.0.Results:1.The survival rate of Beas-2B cells exposed to 100 μg/ml and 200 μg/ml PM_2.5 for 12 hours was significantly higher than that in the control group（P < 0.05）,and the survival rate of Beas-2B cells decreased significantly with the increase of exposure time and concentration of PM_2.5（P < 0.05）.2.The relative expressions of Muc5 ac m RNA in Beas-2B cells exposed to PM_2.5 for 12 hours were significantly higher than that in control group（P < 0.05）,and the relative expressions of Muc5 ac m RNA were significantly decreased with the increase of exposure time and concentration of PM_2.5（P < 0.05）.The expressions of Muc5 ac protein were significantly lower in all dose groups except for 100 μg/ml PM_2.5 exposure 24 hours than in the control group,the expressions of Muc5 ac protein in 100 μg/ml PM_2.5 exposure group were the highest,and the expressions of Muc5 ac protein in 200 μg/ml PM_2.5 exposure group were the lowest（P < 0.05）.As the exposure time of PM_2.5 prolonged,the expressions of Muc5 ac protein increased at first and then decreased（P < 0.05）.3.The relative expressions of Hes1 m RNA in Beas-2B cells were significantly higher in 100 μg/ml and 200 μg/ml PM_2.5 exposure group than that in control group（P < 0.05）,and the relative expressions of Hes1 m RNA were significantly decreased with the increased exposure time of PM_2.5（P < 0.05）.The expressions of Hes1 protein in 400 μg/ml PM_2.5 exposure group were significantly higher than that in control group（P < 0.05）.4.The expressions of genes in the Notch signaling pathway were different in Beas-2B cells exposed to different concentration of PM_2.5 for different times.The expressions of Notch1,Notch2 and Notch3 m RNA in Beas-2B cells exposed to PM_2.5 were significantly lower than those in control group（P < 0.05）,the relative expressions of Notch1 and Notch2 m RNA in Beas-2B cells decreased with the increase of exposure time and concentration of PM_2.5（P < 0.05）,the relative expressions of Notch3 m RNA decreased at first and then increased with the exposure time of PM_2.5（P < 0.05）,the relative expressions of Notch4 m RNA decreased significantly with the increase of PM_2.5 exposure time（P < 0.05）.The expressions of Notch1 protein in Beas-2B cells exposed to PM_2.5 decreased with the increase of exposure concentration of PM_2.5 and increased with the increase of exposure time of PM_2.5（P < 0.05）,the expressions of Notch2 protein increased at first and then decreased with the increase of exposure concentration and time of PM_2.5（P < 0.05）,the expressions of Notch3 and Notch4 decreased at first and then increased with the increase of exposure concentration and time of PM_2.5（P < 0.05）.5.The relative expressions of Muc5 ac m RNA were positively correlated with the relative expressions of Hes1 m RNA in Beas-2B cells of all groups（P﹤0.01）.The relative expressions of Muc5 ac m RNA were negatively correlated with the relative expressions of Notch2 and Jaddged2 m RNA,after Beas-2B cells were exposed to PM_2.5 of low concentrate for short time（P﹤0.05）.The relative expressions of Muc5 ac m RNA were positively correlated with the relative expressions of Notch3 m RNA in Beas-2B cells exposed to PM_2.5 of high concentrate for 24h（P﹤0.01）.6.After the Notch signaling pathway was inhibited,the relative expressions of Muc5 ac m RNA in Beas-2B cells decreased with the increase of exposure concentration and time of PM_2.5（P < 0.05）,while the expressions of Muc5 ac protein in Beas-2B cells exposed to PM_2.5 were lower than those in control group,but increased with the increase of PM_2.5 exposure time（P < 0.05）.The relative expressions of Hes1 m RNA and protein decreased with the increase of PM_2.5 exposure time（P < 0.05）.7.More eosinophil granulocytes were shown in alveolar lavage fluid of asthma rats than in normal rats,more monocytes were shown in alveolar lavage fluid in rats exposed to 1.5 mg / kg PM_2.5,and more eosinophil granulocytes were shown in alveolar lavage fluid in rats exposed to 7.5 mg / kg PM_2.5.The alveolar lavage fluid of rats exposed to 37.5 mg / kg PM_2.5 was full of cells,mainly eosinophil granulocyte,monocytes and lymphocyte.8.In asthma model rats of the low dose group,the infiltration of lymphocyte,neutrophil and eosinophil granulocyte increased in lung,with irregular alveolar expansion.A lot of eosinophil granulocyte cells and macrophages were observed in alveolar tissue in asthma model rats of the middle dose Group,and a large number of eosinophil granulocyte cells and lymphocyte cells were observed in alveolar cavity in asthma model rats of the high dose group.9.With the increase of PM_2.5 exposure,the level of Hes1 protein in lung tissue increased,the expressions of Hes1 protein of lung tissue in asthma model rats of the high dose group and middle dose group were higher significantly than those in the control group and asthma model rats of the low dose group（P < 0.05）.10.The goblet cell proliferation and mucin secretion in asthma model rats of the low and middle dose groups were significantly higher than those in the control group,but the mucin secretion in the high dose group was not significantly increased.11.The expressions of muc5 ac in asthma model rats of the high dose group were significantly higher than those in the low and middle dose groups（P<0.05）.However,there was no significant difference in the expression of muc5 ac between groups.12.In Notch signaling inhibition experiments,the expressions of Hes1 of rats in pathway inhibition group of the healthy control were significantly lower than that in the healthy control group（P<0.05）,and the expressions of Hes1 in pathway inhibition group of the asthma model were significantly lower than that in the asthma model group（P<0.05）.Moreover,the expressions of muc5 ac of the airway and lung tissue in pathway inhibition group of the asthma model were significantly lower than those of the asthma model group（P<0.05）.Results:1.PM_2.5 has obvious injury effect on lung epithelium.2.Exposure to PM_2.5 for a short time may result in the secretion of mucin to remove pollutants from the airway.However,with the increase of exposure time and concentration of PM_2.5,the cells may be damaged,and the secretion of mucin in the airway may decrease,which could lead to the occurrence and development respiratory disease.3.The Notch signaling pathway may positively regulate the influence of PM_2.5 in Muc5 ac secretion through Hes1 in Bear-2B cells.4.In the level of m RNA,Jadgged2 and Notch2 may negatively regulate the expressions of Muc5 ac,and Notch3 may positively regulate the expressions of Muc5 ac.In the level of protein,Notch2,Notch4,and Jadgedd2 may positively regulate the expressions of Muc5 ac,and Notch3 may negatively regulate the expressions of Muc5 ac.5.PM_2.5 of low dose can stimulate monocyte infiltration in alveoli of rats to help the body to remove PM_2.5,the rats exposed to PM_2.5 of middle dose mainly show allergic reaction,rats exposed to PM_2.5 of high doses are in a completely disordered state,which may lead to the occurrence and development of asthma.6.PM_2.5 can induce the increase of Hes1 expression in asthma rats in a dose-dependent manner to activate Notch signal,and the level of Hes1 decrease after the Notch signal is blocked.7.PM_2.5 can induce a strong inflammatory response and an increase in mucin and Muc5 ac secretion in asthma rats,and inflammation and mucus secretion are reduced after the Notch signal is blocked.8.The Notch signaling pathway may regulate the influence of PM_2.5 in the development and progression of asthma by Hes1.