Effects Of Short-term High-salt Diet On Th1/Th2,Th17/Treg Balance In Asthmatic Mice

background:As a chronic airway inflammatory disease,the incidence rate of asthma is increasing year by year,which not only endangers the life and health of patients,but also increases the economic burden on families and society.Environment and genetics are indispensable in the occurrence and progression of asthma.In recent years,the impact of diet on health has received widespread attention.Research has found that high salt diets can trigger excessive immune responses and exacerbate inflammation,which has been proven to have adverse effects on the cardiovascular system,kidneys,skin,brain,and bones.objective:In this study,we established an asthma mouse model,administered a short-term high salt diet,and tested Th related cells(Th1,Th2,Th17,Treg cells)and their molecular levels(IFN-γ、IL-4、IL-17、TGF-β 、IL-10)to investigate the effects of short-term high salt diet on Th1/Th2,Th17/Treg cell balance and airway inflammation in asthmatic mice.Methods:1.Twenty female BABL/c mice aged 6 to 8 weeks and weighing 18 to 22 g were randomly divided into four groups using a double blind method: control group,high salt group,asthma group,and asthma+high salt group.Mouse asthma models were established using ovalbumin(OVA),and mice were sensitized by intraperitoneal injection of OVA on the 14 th,21st,and 28 th days;Starting from the 35 th day,mice were given 2.5% OVA solution for atomization for half an hour each time,once a day,for a continuous week,to observe the behavior and temperament changes of the mice.The high salt group and asthma+high salt group were fed with 1% saline and 8% high salt feed throughout the entire process.The study group mice were killed the next day after the end of atomization.2.Mice in each group were stained with hematoxylin eosin(HE)and Schiff periodic acid shiff(PAS)to observe the pathological changes of lung tissue and evaluate the inflammation score.The right lung of mice was subjected to bronchoalveolar lavage with phosphate buffered solution(PBS).Cells in the bronchoalveolar lavage fluid(BALF)were counted under a microscope.3.Take mouse eyeball blood and use enzyme linked immunosorbent assay(ELISA)to detect the expression level of immunoglobulin E(Ig E)in the serum of mice in each group.4.Prepare mouse spleen single cell suspension and detect the expression of Th1,Th2,Th17,and Treg cells in spleen tissue using flow cytometry.5.Detection the relative expression of Th cell related cytokines IFN-γ、IL-4、IL-17、TGF-β in mouse lung tissue using real-time fluorescence quantitative PCR.The relative expression of IL-10.6.Immunohistochemistry(IHC)detection of IFN-γ、IL-4、IL-17、FOXP3、TGF-β、IL-10 protein expression in mouse lung tissue.Results:1.Compared with control,the mice in the high salt group showed no significant changes in behavior and temperament;the mice in the asthma group exhibited significant acute asthma episodes such as nasal scratching,shallow and fast breathing,irritability,excitement,restlessness,arch back,and forelimb lifting during the later stage of atomization.However,the symptoms of acute asthma attacks such as scratching in mice in the asthma+high salt group were significantly alleviated compared to those in the asthma group.2.Compared with control,HE and PAS staining in the lung tissue of mice in the the mice in the high salt group showed no significant inflammatory infiltration in the lung tissue and no difference in the sum number of cells in BALF(P>0.05);asthma group showed significant inflammatory infiltration,and the count of BALF showed a significant increase in the total cells number(P<0.001);Compared with the asthma group,the inflammatory infiltration in the lung tissue of mice in the asthma+high salt group was significantly reduced,with a lower inflammatory score and a notably decreased sum number of cells in the BALF(P<0.01).3.Compared with control,the amount of eosinophils in BALF of mice in the asthma group significantly increased(P<0.0001);The amount of eosinophils in BALF of mice in the asthma+high salt group was higher than that in the normal group(P<0.05),but significantly decreased compared to the asthma group(P<0.0001);There wasn’t difference in the number of eosinophils in the alveolar lavage fluid between the high-salt group and the control group(P>0.05).4.Compared with control,the serum Ig E level of mice in the asthma group was significantly raised(P<0.01);There wasn’t difference in Ig E levels between the asthma+high salt group and the asthma group(P>0.05).There was no significant difference in Ig E levels between the high salt group mice and the control group(P>0.05).5.The flow cytometry results suggested that compared with control,the ratio of Th2 and Th17 cells in the spleen of mice in the asthma group conspicuously raised(P<0.01),yet the scale of Th1 cells and Treg cells decreased(P<0.05);Compared with the asthma group,the scale of Th2 and Th17 cells in the asthma+high salt group descended significantly(P<0.01),while the scale of Treg cells raised(P<0.05).There wasn’t difference in the proportion of Th1 cells(P>0.05);There wasn’t difference in the scale of spleen cells between the high salt group and the control group(P>0.05).6.The PCR results showed that compared with control,the expression of IL-4,IL-17 and TGF-βin the lung tissue of mice in the asthma group raised(P<0.01),the expression of IL-10descended(P<0.001).There wasn’t difference in the expression of IFN-γ(P>0.05);Compared with the asthma group,the expression of IL-4,IL-17 and TGF-βin the lung tissue of mice in the asthma+high salt group descended(P<0.01),the expression of IL-10 increased(P<0.05).There wasn’t difference in the expression of IFN-γ(P>0.05).Compared with control,there wasn’t difference in the expression of the above cytokines m RNA in the lung tissue of mice in the high salt group(P>0.05).7.Immunohistochemical results showed that compared with control,the expression of IL-4,IL-17 and TGF-βprotein in the lung tissue of mice in the asthma group raised(P<0.05),The expression of IFN-γ,FOXP3 and IL-10 proteins descended(P<0.05);Compared with the asthma group,the expression of IL-4 protein in the lung tissue of mice in the asthma+high salt group descended(P<0.01),the expression of IFN-γ,FOXP3,IL-10 protein raised(P<0.05).There wasn’t difference in the expression of IL-17,TGF-βprotein(P>0.05).Compared with control,there wasn’t difference in the expression of the above proteins in the lung tissue of mice in the high salt group(P>0.05).Conclusion:Th cell imbalance exists in asthmatic mice.A short-term high salt diet can inhibit airway inflammation in asthmatic mice by regulating the balance of Th cells in the spleen tissue and regulating the expression of Th cell related cytokines and proteins in the lung tissue of asthmatic mice.Short term high salt diet can effectively interfere with OVA mediated asthma progression in mice.