Effects Of 1α,25-dihydroxy Vitamin D₃ On Expression Of SIRT1,GATA-3 And Airway Inflammation In Asthmatic Mice

Objective:By establishing the mice model of asthma,plus 1α,25-dihydroxy vitamin D₃ intervention,by observing the expression of sile nce-regulating factor 1（SIRT1）and GATA-binding protein-3（GATA-3）in lung tissue of asthmatic mice,the changes of serum Ig E and the c oncentration of inflammatory factors（IL-4,IL-5,IL-13）in alveolar lav age fluid（BALF）,To explore the effect and possible mechanism of 1α,25-dihydroxyvitamin D3 on airway inflammation in asthmatic mice.Methods:The OVA-induced asthma mouse model was established by SPF BALB/c female mice and randomly divided into normal contro l group（NC）,asthma model group（OVA）and asthma+1α,25-dihydr oxy vitamin D₃ intervention group（OVA+VD₃）.The first intraperitonea l injection of sensitization was defined as the 1st day of modeling,an d the OVA group and OVA+VD₃ group were given 0.2ml of OVA sus pension for intraperitoneal sensitization on the 1st,8th and 15th day r espectively,among which the NC group was given 0.2ml of normal sa line.On the 22-28 days,1%OVA was used to stimulate the atomizatio n inhalation,wherein the NC group was given the normal saline atomi zation,each atomization 30min,once a day,continuous stimulation for7 days;NC group and OVA group were given 0.1ml of normal saline intraperitoneal injection 30min before each atomization,OVA+VD₃ grou p was given 1α,25-dihydroxy vitamin D₃ injection 0.1ml intraperitonea l injection.The behavioral changes of mice were observed dynamically during the experiment.Serum,BALF,and lung tissue samples were c ollected 12 hours after the last excitation.Hematoxylin-eosin（HE）stai ning and Periodic Acid Schiff（PAS）staining were used to observe the changes of lung histopathology and airway mucus level.The concentr ations of inflammatory cytokines IL-4,IL-5 and IL-13 in serum and I g E and BALF were detected by enzyme-linked immunosorbent assay（E LISA）.The expressions of SIRT1 and GATA-3 in mouse lung tissue w ere detected by immunohistochemistry and Western blot.Results:1.Behavioral observation:In the sensitization stage,there was no obvious abnormality in the behavior of mice in each group;in the atomization stimulation stage,the NC group began to show increased activity 3 minutes after the start of atomization,but soon returned to normal;after multiple atomization stimulation,there was no significant change in hair luster,diet and weight.OVA group of mice scratching the ear,sneezing,and then appear breathing rate increased,irritability,upright,upright hair performance,and mouse hair glossiness,diet and weight decreased significantly;OVA+VD₃group of mice scratching the chin,bowed upright fewer times,after multiple stimulation of hair gloss,diet and weight slightly decreased.2.Pathological changes of lung tissue:HE staining results showed that the structure of alveoli in NC group was clear,the size of alveoli was uniform,the epithelial structure of airway mucosa was complete,the cilia were arranged neatly,there was no inflammatory cell infiltration around trachea and blood vessels,and few eosinophils were found.A large number of inflammatory cells were infiltrated around the airway and blood vessels of mice in the OVA group,including significant increase of eosinophils,narrowing of bronchial lumen,shedding of epithelial cells in part of the bronchus and other changes.A small amount of inflammatory cell infiltration was observed around the airway and blood vessels in the OVA+VD₃ group,and eosinophils were significantly reduced compared with the OVA group.The bronchial lumen was more regular and the epithelium was more complete.PAS staining results showed that the bronchial epithelial goblet cells and airway mucus secretion were significantly increased in the OVA group compared with the NC group,and the secretion of airway goblet cells and mucus was significantly decreased in the OVA+VD₃group compared with the OVA group,but still higher than that in the NC group.3.Serum Ig E,IL-4,IL-5 and IL-13 levels in BALF:Compared w ith NC group,Serum Ig E（187.23±17.06ng/m L）,cytokine IL-4（264.41±24.20pg/ml）,IL-5（20.25±3.55pg/ml）and IL-13（78.44±13.79ng/L）in BALF were significantly increased in OVA group.The difference was statisti cally significant（P<0.05）.Compared with the OVA group,Ig E（122.91±7.46ng/m L）,IL-4（211.99±16.94pg/m L）,IL-5（13.60±1.98pg/m L）and IL-13（62.58±10.36ng/L）levels in the OVA+VD3 group were significantly decreased.The difference was statistically significant（P<0.05）.4.Expression of SIRT1 and GATA-3 protein in lung tissue:Immu nohistochemistry showed that the positive expressions of SIRT1 and G ATA-3 were yellow-brown or brown,mainly distributed in the nuclei o f inflammatory cells in bronchi and around blood vessels,lung tissue and lung interstitium.Compared with NC group,the expression range of SIRT1 decreased in OVA group,and the expression range of GATA-3 increased in OVA group.Compared with the OVA group,the expres sion range of SIRT1 in the OVA+VD₃ group was enhanced,while the expression range of GATA-3 was decreased.The average optical densit y value（average OD value）was used to represent the expression amo unt of detected protein.The average OD of SIRT1 protein in OVA gro up（0.058±0.019）was significantly lower than that in NC group（0.117±0.017）.The average OD value of SIRT1 protein in OVA+VD₃ group（0.089±0.010）was significantly higher than that in OVA group,and the differ ences were statistically significant（P<0.05）.The average OD value of GATA-3 protein in OVA group（0.111±0.016）was significantly highe r than that in NC group（0.063±0.011）,average OD value of GATA-3protein in OVA+VD₃ group（0.094±0.010）was significantly lower than that in OVA group,the differences were statistically significant（P<0.05）.Subsequently,Western blot was used to detect the expression leve ls of SIRT1 and GATA-3 proteins.The results showed that compared with NC group,the expression level of SIRT1 protein in lung tissue o f mice in OVA group was significantly decreased,while the expression level of GATA-3 protein was significantly increased,with statistical s ignificance（P<0.05）.The expression level of SIRT1 in OVA+VD₃ gr oup was higher than that in OVA group,and the expression level of GATA-3 was lower than that in OVA group,with statistical significanc e（P<0.05）.5.Correlation analysis:There was a negative correlation between SIRT1 and GATA-3 expression in lung tissue（r=-0.7219,P<0.0001）.SI RT1 expression in lung tissue was negatively correlated with serum Ig E（r=-0.8320,P<0.0001）,IL-4（r=-0.8319,P<0.0001）,IL-5（r=-0.6236,P=0.0011）and IL-13（r=-0.6026,P=0.0018）concentrations in B ALF（r=-0.8320,P<0.0001）.The expression level of GATA-3 in lun g tissue was positively correlated with the concentrations of Ig E（r=0.8319,P<0.0001）in serum,IL-4（r=0.7709,P<0.0001）,IL-5（r=0.5586,P=0.0046）and IL-13（r=0.5197,P=0.0093）in BALF（r=0.8319,P<0.0001）.Conclusion:1.The expression of SIRT1 was decreased and the expression of GATA-3was up-regulated in the lung tissue of asthmatic mice.SIRT1 and GATA-3 were involved in the formation of airway inflammation in asthmatic mice.2.1α,25-dihydroxy vitamin D₃ can reduce the levels of Ig E in serum,IL-4,IL-5,IL-13 in BALF of asthmatic mice,and relieve airway inflammation of asthmatic mice.3.1α,25-dihydroxy vitamin D₃ may improve airway inflammation in asthmatic mice by up-regulating the expression of SIRT1 in lung tissue,inhibiting the expression of GATA-3,and then inhibiting inflammatory factors（IL-4,IL-5,IL-13）.