Effect Of Conjugated Linoleic Acid On2,4-Dinitrofluorobenzene-Induced Atopic Dermatitis In Mice

BackgroundAtopic dermatitis,also known as atopic eczema,is a genetic predisposition,chronic relapsing inflammatory skin disease characterized by intense pruritus.AD substantially reduces quality of life,mainly due to intense pruritus that leads to sleep loss.Furthermore,in a process referred to as the“atopic march”,many patients with AD develop asthma and/or food allergies later in life,which can be life-threatening.The pathogenesis of AD involves a complex inflammatory process and is not fully understood,but is believed to be involved in genetic and environmental factors.contributing to skin barrier dysfunction and immune dysregulation in AD.Skin barrier dysfunction and immune dysregulation changes of local cytokines are closely related to the onset and development of AD.Conjugated linoleic acid?CLA?has exhibited potent immunomodulatory and anti-inflammatory properties that are demonstrated in a wide range of inflammatory disorders including inflammatory bowel disease,diabetes,atherosclerosis and allergic diseases.ObjectiveTo study the therapeutic effect and its possible mechanism of conjugated linoleic acid?CLA?in 2,4-dinitrofluorobenzene-induced AD mice model.Methods1.After one week acclimation,mice were randomly divided into 4 experimental groups with 8 mice each:?1?Normal?NOR?group;?2?AD group;?3?AD+CLA?100mg/kg;0.1 m L/10g?group;?4?AD+DEX?0.1 mg/kg;0.1 m L/10g?group.Dexamethasone?DEX?was used as a positive control in the present study and dissolved in the ethanol/distilled water vehicle?v/v;1/99?prior to use.The dorsal flank and ear pinnas of mice were sensitized and challenged with 1%or 0.5%DNFB for four weeks in order to establish AD model.The mice were were fed respectively with vehicle,CLA and DEX by gavage once a day from the first day of modeling.During the experiment,the mortality,body weight and food consumption were carefully monitored.Simultaneously,we dynamically observed and recorded the changes of skin lesions,scratching behaviors and ear thickness of each mice.2.On day 29,mice were sacrificed and their blood samples and skin specimens of all the mice were collected.Total and differential leukocyte counts in mice blood were calculated by blood routine examination.Levels of Ig E,IL-4,IFN-?,PGE₂ and LTB₄in serum or skin lesions of the mice in each group were determined by ELISA.The DNFB-damaged skins of mice were made into paraffin sections and stained by hematoxylin-eosin?HE?and toluidine blue to observe its pathological changes and the infiltration of mast cells in the dermis.Immunohistochemical analysis was performed to detect the protein expression of CD4,NF-?B p65 and TNF-?in damaged skins.3.Total protein were extracted from the skin tissues and the relative expression of i NOS,COX-2,5-LOX,TLR4 and My D88 proteins were detected by using western blotting.Results1.Scratching behaviors,dermatitis scores and ear swelling were significantly increased in the AD group compared with that in the NOR group?p<0.05 or p<0.01?.Oral administration with 100 mg/kg CLA remarkably inhibited scratching behaviors,dermatitis scores and ear swelling.Pathological changes,including epidermal hyperplasia and infiltration of inflammatory cells and mast cells in the dermis were significantly relieved in the CLA group?p<0.05 or p<0.01?.2.The total white blood cells,monocytes and neutrophils in the AD model group were significantly elevated as compared to the NOR group?p<0.05 or p<0.01?,which were characteristics of allergic diseases.Compared with the AD group,total and differential leukocyte counts were significantly reduced in the CLA group?p<0.05 or p<0.01?.DEX,as a positive control drug,showed much more remarkable reductions?p<0.01?.Elisa results showed that Serum Ig E was significantly higher in mice than in the normal group.100 mg/kg CLA dramatically reduced levels of Ig E,IL-4 and IFN-?in mouse serum or.The expression of CD4 which are closely related to the production of IL-4 and IFN-?was decreased in the CLA group compared with the AD model group.3.Sensitization and repeated topical application of DNFB significantly elevated PGE₂ and LTB₄ release in serum or lesional skin of AD mice as compared to NOR group,which phenomenon was reversed by 100 mg/kg doses of CLA?p<0.05 or p<0.01?.Western blotting studies have identified that CLA down-regulated COX-2 and 5-LOX expression,exhibiting similar trends of CLA on modulation of PGE₂ and LTB₄production in AD-like skin lesions.Meanwhile,the expression levels of NF-?B p65,TNF-?,TLR4 and MYD88,closely related to inflammation process were remarkably down-regulated following CLA or DEX treatment.ConclusionCollectively,the results obtained in this study suggest that CLA,as the conventional dietary,can alleviate AD-like symptoms in mice,including dorsal skin thickness,mast cell infiltration and release of Ig E,supporting the clinical potential of dietary supplementation with CLA as an alternative therapeutic strategy in AD patients.CLA exert favorable protective effects against DNFB-induced AD,which might be partially mediated by by modulating the COX-2/5-LOX and TLR4/My D88/NF-?B signaling pathways.