The Effect Of Inflammatory Reaction On HaCaT Cells Induced By DINP

ObjectiveIn order to explore the relationship and mechanism between DINP and atopic dermatitis,build in vitro model of cell's exposure to investigate the effect of DINP on the inflammatory response of human epidermal cells(HaCaT)which has been exposed to allergen(DNCB).Methods1.HaCaT cells culture.Cells was cultured by DMEM containing 10% FBS and 1%Double antibody of penicillin and streptomycin at a temperature of 37?,5%CO2.2.Detect the cell viability of HaCaT cells after the exposure to different concentrations of DINP and DNCB by MTT kit.3.Elisa method was used to detect the effect on the level of MPO production in HaCaT cells by DINP.4.Determine the content of IL-8 in HaCaT cells which caused by DINP and DNCB,used Elisa kit.5.Detect the level of mRNA expressions of IL-6,IL-8,IFN-?,CYP1A1,ICAM-1 and S1008 A in HaCaT cells induced by DINP and DNCB,used RT-PCR kit.6.Explore the regulatory effect of DINP on ERK signaling pathway in HaCaT cells by Western Blot.Results1.When the HaCaT cells was treated by DNCB in concentration 20?M 24 h,the survival rate was 88.84±5.54%,compared with the control group,the difference was statistically significant(t=2.92,P<0.05).When exposed to 200?M DINP for 24 hours,the survival rate of HaCaT cells was 86.54±6.33%,compared with the control group,the difference was statistically significant(t=3.87,P<0.01).2.When treat HaCaT cells by DINP30?M,at 30 min,60min,MPO content was signific-antly higher than that of the control group(P<0.01;P<0.001).3.When treat HaCaT cells by DINP and DNCB at 0.5h?1h?3h?6h,IL-8 concentration was significantly higher than the control group,and both group used alone(P <0.05).4.DINP can coordinate DNCB increase the IL-6,IL-8,CYP1A1,ICAM-1,IFN-?and S1008 A mRNA expression,the difference was statistically significant(t=3.87,P<0.05).5.DINP increased P-ERK1/2 expression to induced ERK signal pathway by(P<0.05).Conclusions1.DINP has obvious toxicity and inflammation on skin keratinocytes in vitro,and it has synergistic effect on inflammatory induced by DNCB.2.DINP may aggravated AD through promoting the expression and secretion of inflammatory related proteins,adhesion molecules,and dermatitis related proteins in epidermal cells,DINP can activate the ERK signaling pathway.