| Objective:To investigate the effects of low-dose 5-fluorouracil(5-FU)on Th2 response and airway hyperresponsiveness(AHR)in ovalbumin(OVA)-induced mouse models of asthma.Methods:1.The effects of intraperitoneal administration of low-dose 5-FU before immunization on Th2 response and AHR in OVA-induced asthmatic mice.Fifteen female BALB/c mice that are wild type(WT)were divided into normal control group,phosphate-buffered saline(PBS)intervention asthma group and pre-sensitization low-dose 5-FU intervention asthma group in a random manner.The mice in PBS intervention asthma group and pre-sensitization low-dose 5-FU intervention asthma group were sensitized with 20μg of OVA absorbed in 2 mg of alum in 200μL of PBS through intraperitoneal injection on day 0 and 14.This was followed by intranasal OVA challenges(100μg in 50μl of PBS)on day 21,22,and 23.The mice in normal control group were similarly immunized and challenged with an identical volume of PBS.The mice in pre-sensitization low-dose 5-FU intervention asthma group received a single intraperitoneal injection of low-dose 5-FU(50mg/kg)one day before OVA immunization on day 0.The mice in PBS intervention asthma group and normal control group received identical volume of PBS.Mice were sacrificed 24 h after the final OVA challenge on day 24 to further analyze.Lung sections were stained by hematoxylin and eosin(HE)staining to assess airway inflammation and by periodic acid-Schiff(PAS)staining to evaluate goblet cell hyperplasia using light microscopy.Goblet cell hyperplasia was determined by counting the number of PAS-positive goblet cells in the epithelium of the central airway with a digital image.The circumferences of the airways at the basement membrane were measured using Image J software.Goblet cell hyperplasia were expressed as the number of PAS-positive cells per unit length(mm)of the basement membrane.Cells in the bronchoalveolar lavage fluid(BALF)were obtained to stain with May-Grunwalsd Giemsa for total cell counting and differential cell counting.Measurement of IL-4,IL-5,IL-13,and IFN-γin the BALF was performed using an ELISA kit according to the manufacturer’s protocols.The concentrations of OVA-specific Ig E in the serum were determined using ELISA kits.AHR was measured by invasive assessment of airway resistance.2.The effects of intraperitoneal administration of low-dose 5-FU before challenge on Th2 response and AHR in OVA-induced mouse models of asthma.Fifteen female BALB/c mice that are WT were divided into normal control group,PBS intervention asthma group and pre-challenge low-dose 5-FU intervention asthma group in a random manner.The mice in PBS intervention asthma group and pre-challenge low-dose 5-FU intervention asthma group were sensitized with 20μg of OVA absorbed in 2 mg of alum in 200μL of PBS through intraperitoneal injection on day 0and 14.This was followed by intranasal OVA challenges(100μg in 50μl of PBS)on day 21,22,and 23.The mice in normal control group were similarly immunized and challenged with an identical volume of PBS.The mice in pre-challenge low-dose 5-FU intervention asthma group received a single intraperitoneal injection of low-dose 5-FU(50mg/kg)one day before OVA challenge on day 21.The mice in PBS intervention asthma group and normal control group received identical volume of PBS.Mice were sacrificed 24 h after the final OVA challenge on day 24 to further analyze.Lung sections were stained by HE staining to assess airway inflammation and by PAS staining to evaluate goblet cell hyperplasia using light microscopy.Goblet cell hyperplasia was determined by counting the number of PAS-positive goblet cells in the epithelium of the central airway with a digital image.The circumferences of the airways at the basement membrane were measured using Image J software.Goblet cell hyperplasia were expressed as the number of PAS-positive cells per unit length(mm)of the basement membrane.Cells in the BALF were obtained to stain with May-Grunwalsd Giemsa for total cell counting and differential cell counting.Measurement of IL-4,IL-5,IL-13,and IFN-γin the BALF was performed using an ELISA kit according to the manufacturer’s protocols.The concentrations of OVA-specific Ig E in the serum were determined using ELISA kits.AHR was measured by invasive assessment of airway resistance.3.The effects of intraperitoneal administration of low-dose 5-FU after challenge on Th2response and AHR in OVA-induced mouse models of asthma.Fifteen female WT BALB/c mice were randomly divided into 3 groups.Mice weresensitized to OVA on day 0 and 14,and challenged on days 21-23 with daily intranasal OVA.On day 24,mice received a single intraperitoneal injection of low-dose 5-FU(50mg/kg)or identical volume of PBS.Subsequently,mice were challenged for another three times on days 25-27 by daily intranasal OVA.The mice in normal control group were treated with identical volume of PBS.Mice were sacrificed 24 h after the final OVA challenge on day 28 to further analyze.Lung sections were stained by HE staining to assess airway inflammation and by PAS staining to evaluate goblet cell hyperplasia using light microscopy.Goblet cell hyperplasia was determined by counting the number of PAS-positive goblet cells in the epithelium of the central airway with a digital image.The circumferences of the airways at the basement membrane were measured using Image J software.Goblet cell hyperplasia were expressed as the number of PAS-positive cells per unit length(mm)of the basement membrane.Cells in the BALF were obtained to stain with May-Grunwalsd Giemsa for total cell counting and differential cell counting.Measurement of IL-4,IL-5,IL-13,and IFN-γin the BALF was performed using an ELISA kit according to the manufacturer’s protocols.The concentrations of OVA-specific Ig E in the serum were determined using ELISA kits.AHR was measured by invasive assessment of airway resistance.4.The effects of intraperitoneal administration of low-dose 5-FU on the quantity of lung inflammatory DCs(inf DCs).Fifteen female BALB/c mice that were WT were divided into normal control group,PBS intervention asthma group and low-dose 5-FU intervention asthma group in a random manner.The mice in PBS intervention asthma group and low-dose 5-FU intervention asthma group were sensitized with 20μg of OVA absorbed in 2 mg of alum in 200μL of PBS through intraperitoneal injection on day 0 and 14.This was followed by intranasal OVA challenges(100μg in 50μl of PBS)on day 21,22,and 23.The mice in normal control group were similarly immunized and challenged with an identical volume of PBS.The mice in low-dose 5-FU intervention asthma group received a single intraperitoneal injection of low-dose 5-FU(50mg/kg)one day before OVA challenge on day 21.The mice in PBS intervention asthma group and normal control group received identical volume of PBS.Mice were sacrificed 24 h after the final OVA challenge on day 24 to further analyze.The effect of low-dose 5-FU on the quantity of lung inf DCs was evaluated via flow cytometry.Result:1.The effects of intraperitoneal administration of low-dose 5-FU before immunization on Th2 response and AHR in OVA-induced mouse models of asthma.No inflammatory cell infiltration was observed in lung tissue of normal control group by HE staining,while there was infiltration of a large number of inflammatory cells around the airway and blood vessels in the lung tissue of the mice in the PBS intervention asthma group by HE staining.The inflammatory cell infiltration in the lung tissue of OVA-induced asthmatic mice was alleviated by low-dose 5-FU treatment before sensitization compared with that of mice in the asthma group.PAS~+cells were not found in the normal control group,and the number of PAS~+cells in pre-sensitization low-dose 5-FU intervention asthma group were less than PBS intervention asthma group(P<0.01).The frequency of total cells and the eosinophils macrophages lymphocytes and neutrophils frequency in the BALF,the concentration of cytokines,such as IL-4,IL-5,and IL-13 in the BALF,as well as the amount of OVA-specific Ig E in the serum of the asthmatic mice induced by ovalbumin with low-dose 5-FU treatment before OVA sensitization were markedly decreased,compared with OVA-induced asthmatic mice treated with PBS(P<0.01).In addition,the RL of mice in pre-sensitization low-dose 5-FU intervention asthma group was lower than PBS intervention asthma group(P<0.01),by contrast,no difference in Cdyn was shown these two groups(P>0.05).2.The effects of intraperitoneal administration of low-dose 5-FU before challenge on Th2 response and AHR in OVA-induced mouse models of asthma.low-dose 5-FU treatment before OVA challenge significantly reduced the airways inflammatory cell infiltration,PAS-positive cells(goblet cells),and the frequency of total cells and the eosinophils macrophages lymphocytes and neutrophils frequency in the BALF,and the concentrations of IL-4,IL-5,and IL-13 in the BALF,as well as the levels of OVA-specific Ig E in the OVA-induced asthmatic mice,compared with PBS treatment(P<0.01).In addition,decreased RL to Mch was observed in the asthmatic mice induced by OVA with low-dose 5-FU treatment before OVA challenge,compared with PBS treatment(P<0.01).By contrast,no difference in Cdyn was shown these two groups(P>0.05).3.The effects of intraperitoneal administration of low-dose 5-FU after challenge on Th2response and AHR in OVA-induced mouse models of asthma.low-dose 5-FU treatment after OVA challenge on day 24 significantly reduced theairways inflammatory cell infiltration,goblet cells(PAS-positive cells),and the frequency of total cells and the eosinophils macrophages lymphocytes and neutrophils frequency in the BALF,and the concentrations of IL-4,IL-5,and IL-13 in the BALF,as well as the levels of OVA-specific Ig E in the OVA-induced asthmatic mice,compared with PBS treatment(P<0.01).In addition,decreased RL to Mch was observed in the asthmatic mice induced by OVA with low-dose 5-FU treatment after OVA challenge,compared with PBS treatment(P<0.01).By contrast,no difference in Cdyn was shown these two groups(P>0.05).4.The effects of intraperitoneal administration of low-dose 5-FU on the quantity of lung inf DCs.The number of lung inf DCs from low-dose 5-FU treatment group was lower than PBS intervention asthma group(P<0.01),but higher than normal control group(P<0.01).Conclusion:5-FU can alleviate Th2 inflammatory response and AHR in OVA-induced asthmatic mice at sensitization stage or challenge stage or acute attack stage.The inhibition of 5-FU on asthmatic inflammation may be related to the reduction of inf DCs in asthmatic mice induced by 5-FU. |
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