| ObjectiveTo establish mice model of atopic dermatitis by using DNCB after the induction and provocation phase.To study the effect and mechanism of DINP on mice AD model by DNCB.MethodsThe mice model of atopic dermatitis could be established by repeated sensitization and stimulation of DNCB.The ear thickness was measured with a digital caliper,and the ear thickness difference was calculated.HE staining was used to observe the changes of skin structure and inflammatory cell infiltration in mice.Observing the number of mouse skin lesions and mast cells degranulation by the toluidine blue staining.The expression of TSLP in mouse skin lesions was measured by immunohistochemistry.Enzyme linked immunosorbent assay was used to measure the levels of IgE,IL-4,IFN-?and IL-31 in serum of mice.Results1.Comparison of mice weight:Weight before the experiment was 13.598±0.928 g,the difference between the five groups was not statistically significant(F=0.041,P=0.997).Weight after the experiment were 19.638±1.003g in control group,19.450±0.845g in model group,19.550±0.996g in 4 mg/kg/day DINP+DNCB group,19.400±0.953g in 40mg/kg/day DINP+DNCB group,19.088±0.849g in 400 mg/kg/day DINP+DNCB group.The difference between the five groups were not statistically significant(F=0.405,P=0.804).DINP exposure had no significant effect on weight.2.Comparison of ear changes:The control group had no change.The right ears are red in DNCB group and 4mg/kg/day DINP+DNCB group.The right ears appear dry,scaling,pulp callus obviously and hair loss in 40 mg/kg/day DINP+DNCB and 400mg/kg/day DINP+DNCB group.3.Comparison of ear thickness difference:The differences between control group(0.001±0.025 mm),DNCB group(171±0.038mm),4 mg/kg/day DINP+DNCB group(0.203±0.035 mm),40 mg/kg/day DINP+DNCB group(0.300±0.056mm)and 400mg/kg/day DINP+DNCB group(0.363±0.048mm)was statistically significant(F=87.633,P(27)0.001).It can be concluded that the five groups were not the same.The results of multiple comparison showed that DINP exposure resulted in an increase in ear thickness difference,which was dose dependent.4.Comparison of spleen index:The differences between control group(3.660±0.256mg/10g),DNCB group(3.916±0.483mg/10g),4 mg/kg/day DINP+DNCB group(3.971±0.353 mg/10g),40 mg/kg/day DINP+DNCB group(4.337±0.482 mg/10g)and 400mg/kg/day DINP+DNCB group(4.387±0.435mg/10g)was statistically significant(F=4.401,P=0.005).It can be concluded that the five groups were not the same.The results of multiple comparison showed that 400 mg/kg/day DINP exposure resulted in an increase in spleen index,There was no significant effect on spleen index in 4 and 40mg/kg/day DINP exposure.5.Comparison of HE staining:Control group has no obvious change.DNCB group shows a small amount of inflammatory cell infiltration.With the increase of DINP exposure,inflammatory cell infiltration increasing.6.Comparison of toluidine blue staining:The number of mast cell and degranulation:control group(10.417±2.742,2.375±0.667),DNCB group(16.875±3.673,3.458±0.689),4(16.167±4.612,2.458±0.796),40(25.792±4.102,4.667±0.777)and 400 mg/kg/day DINP+DNCB group(35.417±5.418,6.917±1.257).In mast cell counts,the differences between the five groups was statistically significant(F=43.316,P(27)0.001).It can be concluded that the total number of mast cell of five groups were not the same.The results of Multiple comparison showed that 40 and 400 mg/kg/day DINP exposure resulted in an increase,but there was no significant effect in 4 mg/kg/day DINP exposure.In degranulation counts,the differences between the five groups was statistically significant(F=38.044,P(27)0.001).It can be concluded that the degranulation mast cell of five groups were not the same.The results of Multiple comparison showed that 4 mg/kg/day DINP exposure resulted in an decrease,but 40 and 400 mg/kg/day DINP exposure resulted in an increase.7.Comparison of TSLP integrated opticdensity:The differences between control group(280.292±86.57),DNCB group(1515.625±364.113),4(1129.500±135.550),40(5042.542±810.476),400 mg/kg/day DINP+DNCB group(5974.417±751.653)was statistically significant(?~2=35.488,P(27)0.001).It can be concluded that the five groups were not the same.The results of Multiple comparison showed that 4 mg/kg/day DINP exposure resulted in an decrease,but 40 and 400 mg/kg/day DINP exposure resulted in an increase.8.Comparison of serum IgE:The differences between the control group(2.455±0.228U/ml),DNCB group(3.592±0.375 U/ml),4(3.500±0.364 U/ml),40(4.160±0.390 U/ml),400 mg/kg/day DINP+DNCB group(4.412±0.423 U/ml)are statistically significant(F=34.876,P(27)0.001).It can be concluded that the five groups were not the same.The results of multiple comparison showed that 4 mg/kg/day DINP exposure resulted in obvious change,but 40 and 400 mg/kg/day DINP exposure resulted in an increase.9.Comparison of serum IL-4:The differences between the control group(7.363±0.690 ng/L),DNCB group(8.243±0.940 ng/L),4(8.334±0.859 ng/L),40(9.081±0.869 ng/L),400mg/kg/day DINP+DNCB group(9.429±0.841 ng/L)was statistically significant(F=7.266,P(27)0.001).It can be concluded that the five groups were not the same.The results of Multiple comparison showed that 4,40 and 400 mg/kg/day DINP exposure resulted in an increase.10.Comparison of serum IFN-?:The difference between the control group(183.134±8.969 pg/ml),DNCB group(175.481±13.383 pg/ml),4(175.153±13.039 pg/ml),40(170.051±12.180 pg/ml)and 400 mg/kg/day DINP+DNCB group(167.892±12.632pg/ml)was no statistically significant(F=1.888,P=0.134).It can not be concluded that the serum IFN-?levels of five groups was different.11.Comparison of serum IFN-?/IL-4:The differences between control group(24.970±1.305),DNCB group(21.421±1.692),4(21.175±2.327),40(18.846±1.951),400mg/kg/day DINP+DNCB group(17.885±1.560)are statistically significant(?~2=26.045,P=0.002).The results of multiple comparison showed that 4 mg/kg/day DINP exposure resulted in an increase,but not significant.40 and 400 mg/kg/day DINP exposure resulted in a significant increase.12.Comparison of serum IL-31:The differences between control group(8.737±0.571ng/L),DNCB group(9.236±0.857 ng/L),4(9.509±0.648 ng/L),40(10.880±1.243 ng/L),400 mg/kg/day DINP+DNCB group(11.891±0.919 ng/L)was statistically significant(F=17.455,P(27)0.001).It can be concluded that the five groups were not the same.The results of multiple comparison showed that 4,40 and 400 mg/kg/day DINP exposure resulted in an significant increase.Conclusions1.The mice model of atopic dermatitis could be established by repeated sensitization and stimulation of DNCB.2.Exposure to 40 mg/kg/day and 400mg/kg/day of DINP aggravate dermatitis in mice by DNCB induced.3.Th1/Th2 imbalance is an important immunological mechanism of DINP aggravated DNCB induced dermatitis in mice. |
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