The effects of imiquimod on an animal model of asthma

Objective To study the mechanism of imiquimod on asthma animals.Methods(1) 40 mice and 48 rats were divided into 4 groups:control,asthma,dexamethasone and imiquimod groups. The asthma model was established.The mice and rats in the imiquimod group were exposed to an aerosol of 0.15% imiquimod.Lung inflammation and airway responsiveness were measured 24 h after the last ovalbumin(OVA)challenge.The expression of Interleukin-4(IL-4),interferon γ(IFN-γ),eotaxin, macrophage-derived chemokine(MDC),thymus and activation-regulated ehemokine(TARC),T-bet, GATA-3,STAT6 mRNA in the lung were determind by reverse transcription polymerase chain reaction(RT- PCR).The levels of eotaxin,MDC,and TARC in sara were tested by enzymelinked immunosorbentassy (ELISA).The expression of T-bet,GATA-3 and STAT6 proteins in the lung were measured by Western blot.(2)Parabronchial lymphnodes(PBLN)were isolated and cultured.The PBLN cells were divided into blank control,positive control,dexamethasone and drug groups(1-3 subgroups),cultured for different hours,and the expressions of IL-4 and IFN-γ in supernatants were determined by ELISA,The mRNA expressions of the eytokines in cells weredetected by RT-PCR.(3)Flow cytometry was used to detect intracellular IL-4 and IFN-γ production in spleen T lymphocytes.(4)CD_4~+ T cell of spleen pellets were subject to assessment of T-bet and GATA-3 protein and mRNA expression respectively.Results The expiration resistance was determined before and after injection of acetylcholine chloride(20-160 μg/ml), and expiration resistances of the asthmatic group(6.26±0.85),(11.55±3.09),(28.74±5.94), (3710.83±197.49)cm H_2O·ml~(-1)·s~(-1),were significantly elevated compared with those of the control group(1.34±0.16),(3.47±0.49),(9.29±1.27),(25.22±5.44)cm H_2O·ml~(-1)·s~(-1),D= 88.98,56.00,45.00,108.00,all P0.01).The numbers of eosinophils and lymphoeytes,the thicknesses of WA/Pi and ASM/Pi in the asthmatic group[(26.0±1.6)/mm~2,(45.2±3.2)/mm~2,12.0±1.4, 6.7±0.6]were all significantly higher than those of the imiquimod group[(12.4±2.9)/mm~2,(24.2± 3.7)/mm~2,9.2±0.6,4.0±0.5,D or q=193.00,16.92,185.50,7.66,all P0.01].In the imiquimod group,the mRNA and protein expressions of T-bet(0.48±0.08,0.48±0.17)were significantly increased compared with those of the asthmatic group(0.08±0.12,0.18±0.06,D= 120.96,177.98,all P0.01),the mRNA and protein expressions of GATA-3 in the imiquimod group were both significantly decreased compared with those of the asthmatic group(D=166.96,310.97,all P 0.01).In the control group,only low concentrations of IFN-γ[(22±5,31±5)pg/ml]were detected in PBLN cell cultures.After 24 or 48 h stimulation,the concentrations of IFN-γ in drug 2 subgroup[(149± 31),(154±28)pg/ml]and drug 3 subgroup[(166±30),(158±31)pg/ml]were increased significantly;Levels of IL-4[druug 2 subgroup:(23±5),(39±11)pg/ml,drug 3 subgroup:(43±13), (56±12)pg/ml]were increased slowly compared with those in the OVA group(drug 2 subgroup 24 h IL-4, D=9.90;drug 3 subgroup 24 h IL-4,D=8.79,drug 2 subgroup 48 h IL-4,D=8.80,drug 3 subgroup 48 h IL-4,D=8.10,drug 2 subgroup 24 h IFN-γ,q=4.80,drug 3 subgroup 24 h IFN-γ,q=6.40,drug 2 subgroup 48 h IFN-γ,q=3.95,drug 3 subgroup 48 h IFN-γ,q=4.31,all P0.05).After imiquimod treatment,the mRNA and protein levels of T-bet in imiquimod group CD_4~+ T cells were increased significantly compared with those in OVA group,and the mRNA and protein levels of GATA-3 were decreased significantly in CD_4~+ T cells of imiquimod group compared with those in OVA group.The eotaxin, MDC and TARC levels of serum in asthma group[(593±41)pg/ml,(170±20)pg/ml,(221±25) pg/ml]were significant different from those in control group[(288±66)pg/ml,(100±33)pg/ml, (84±49)pg/ml],(eotaxin:q=12.20,MDC:q=8.00,TARC:q=10.50,all P0.01).MDC and TARC levels of serum in imiquimod group[(84±13)pg/ml,(163±35)pg/ml]decreased as compared with those in asthma group(MDC:q=9.80,TARC:q=4.50,all P0.01)and MDC levels in imiquimod group were no different with normal group(q=1.80,P0.05).eotaxin levels of serum in imiquimod group[(501±76)pg/ml]increased as compared with those from normal group(q=8.50,P0.01), and decreased as compared with those from asthma group(q=3.70,P0.05).(4)The expression of eoaxin,MDC,TARC and STAT_6 on the bronchial epithelium in imiquimod group was decreased as compared with asthma group,but increased as compared with normal group.The eotaxin,MDC and TARC mRNA expression of the lung in asthma group(0.85±0.11,0.96±0.10,0.94±0.28)had significant differences from those in the control group(0.45±0.08,0.39±0.09,0.24±0.08,eotaxin:q=3.00,MDC:q= 15.40,TARC:q=5.90,all P0.01)and those in imiquimod group(0.65±0.17,0.66±0.12,0.66± 0.34,eotaxin:q=1.50,MDC:q=8.10,TARC:q=2.40,all P0.05).Conclusion These findings suggested that imiquimod can inhibit the airway inflammation of asthma animals by reducing GATA-3 mRNA and protein expression and increasing T-bet,STAT_6 mRNA and protein expression.