Effect Of BCG-PSN On Mice Model Of Atopic Dermatitis

PrefaceAtopic dermatitis (AD) is a common skin disease mediated by IgE and usually accompanied with allergic rhinitis, asthma and other allergic disease-related. Clinical manifestations of AD patients vary with age and are usually divided into three stages. In infancy eczema-like lesions usually appear on the cheek and scalp. Scratching often occurs in a few weeks followed by erosion scab. In childhood, the lesions appear on the creases, items back as well as the extensor surfaces of limbs. In adolescence and adulthood, moss-like lesions usually affect the limbs, flexor side of the head and neck. There will be varying degrees of itching everyday and this lead to lack of sleep which would seriously affect the patients' quality of life at each stage. AD are becoming more common in infants and children and sometimes protracting illness into adulthood. AD does seriously impact on quality of life. The severity of the disease and the recurrence rate is also increasing and these catch the researchers' attention. What a pity, the exact pathogenesis is not fully understood so far and the therapeutic strategy could not satisfy the patients anticipation. Therefore, the Study of the pathogenesis of AD and the search for therapeutic drugs is very necessary.According to the cell sub-type theory, human T helper cells can be divided into Th1 and Th2 according to their function. Th1/Th2 cells perform their immuno-modulatory function by secreting different cytokines. Th1 cells produce IL-2, IFN-γand these cytokines promote macrophages and NK cells proliferation and participate in bacterial or viral infection. Th2 cells hold great importance in the hypersensitisation and could secrete IL-4, IL-5, IL-6, IL-10 and IL-13. The cytokines mainly are involved IgE-mediated typeⅠallergy characterized by eosinophil accumulation. A balance of cytolines production by Th1 and Th2 cells guanrantees the normal immunity in the physiological circumstances.More and more researches have shown a Th2-type cytokines domination in skin lesions and blood from the AD patients,suggestting that Th1/Th2 imbalance would plays a crucial role in the pathogenesis. Cellular immune response to Th2 subsets in cell-mediated immune response drift, The increased IL-4, IL-5, IL-6, IL-10 and IL-13 levels and decreased IFN-γand IL-12 provided a cytokine miroenviroment for Th2 polarization which meant a depressed Thl proliferation meanwhile. IL-4 is produced by activated T cells and could promote B lymphocytes differentiation and proliferation. IL-4 is a major determinant for Th2 differentiation and promoting the synthesis of IgE in B cells. Recent studies have found that IL-12 is a determinant to Thl cells polarization and can promote Th1 differentiation and proliferation, controlling of cell-mediated immunity, promoting T cells and natural killer cell developing and proliferation, and stimulating those cells to produce IFN-γ. IFN-y is a Thl type cytokines and was confirmed that IFN-γcan inhibit IL-4 mediated IgE production in healthy persons and AD patients. Researches have showed a decreased level in AD skin lesions and peripheral blood and an increased expression as long as the improvement of clinical symptoms of AD, indicating that regulating the Thl/Th2 balance between cytokines suppress Th2 response advantages the treatment of AD has important significance.BCG-polysaccharide nucleic acid (BCG-PSN) worked as an immunomodulatoyr agent extracted from BCG polysaccharide with the hot phenol method. Studies have shown that therapeutic application of BCG-PSN could prevent allergic asthma, chronic urticaria and other allergic diseases and has achieved satisfactory results. It reported that BCG-PSN could enable the normal mouse spleen cells to produce IFN-y and enhance ConA spleen cells secreting IFN-y and IL-2 in vitro. Our research group has found that BCG-PSN pre-AD patients could promote IL-12 secretion in PBMC, rectify Thl/Th2 imbalance. So far, although BCG-PSN for the treatment of atopic dermatitis has a significant effect, but information on their treatment of atopic dermatitis system pre-clinical studies and research related to mechanism of action is incomplete. The experiment with NC/Nga mice model with AD was considered as the object, the BCG-PSN on DC2.4 cells in mice to reveal the BCG-PSN treatment of atopic dermatitis of the possible mechanism. Objective To explore the establishment of AD model by DNCB sensitization on Nc/Nga mice and to study the therapeutic effect of BCG-PSN on the animal model of atopic dermatitis Nc/Nga mice.Methods 20 Nc/Nga mice were randomly divided into 4 groups (n =5), control group:stimulated with acetone and intraperitoneal injection of 100μl saline; model group:stimulated with DNCB and intraperitoneal injection of saline 100μl; BCG-PSN group:stimulated with DNCB and intraperitoneal injection of 0.5mg/kg of BCG/PSN; DXM group: stimulated with DNCB and intraperitoneal injection of 0.1mg/kg DXM. The scratching behavior and ear thickness are counted once a week. vernier caliper is used to measure the ear thickness of mice. The mice skin inflammation is observed and for histological analysis. ELISA assay was used to dectecct serum IgE and IL-4, IL-10, IL-12, IFN-y levels. Real-time quantitative RT-PCR was used to detect the IL-4, IL-10, IL-12, IFN-y expression from spleen cells.Results Compared with the control mice, mice from all the other experimental group appeared, erythema, exudation, skin exfoliation, crusting, bilateral ear swelling, bleeding, crusting and other manifestations. Histopathology showed hyperkeratosis, dermal epidermal thickening, obvious infiltration of a large number of lymphocytes, plasma cells and eosinophil in model mice. All the pathological changes in BCG-PSN mice appeared a little slighter. Compared with the control mice, the scratching behavior in model mice increased significantly. Plasma IgE and IL-4 and IL-10 of Model mice were also significantly increased. The spleen IL-4 and IL-10 mRNA expression is increased, with obvious difference. Application of BCG-PSN may lighten skin lesions and the inflammation degree and reduced erythema, exudation, crusting in the DNCB sensitized Nc/Nga mice. Pathology shows only a slight hyperkeratosis, acanthosis cytoplasmic vacuolization, dermal light layer of inflammatory cell infiltration in BCG-PSN group and reduced skin lesions when compared with model mice. BCG-PSN and DXM treated mince showed an enhanced IL-12 and IFN-y concentration and a decreased plasma IgE levels. The differences between the BCG-PSN and DXM treated mice did not hold obvious significance.Conclusion The AD mice model was successfully established by DNCB sensitization in Nc/Nga mice; BCG-PSN treatment could inhibite the skin lesion, probably due to the reduction of IgE levels and increase expression of IL-12 and INF-y. Objective To study the influnce of BCG-PSN with concentration gradient to the cytokines secretion the expression of costimulatory molecules in mouse DC2.4 cells.Methods The DC2.4 cells were cocultured with5μg/ml,20μg/ml, 50μg/ml of BCG-PSN and were observed with inverted microscope. Flow cytometry was used to detect major histocompatibility complex class molecules MHC-Ⅱand costimulatory molecules CD83, CD86 expression, ELISA assay was used to detect IL-12, IL-10 levels in supernatant. Real-time quantitative RT-PCR was used to determinate IL-10 and IL-12 p40 mRNA expression. DC2.4 cells were preconditioned with anti-TLR2, TLR4, TLR9 antibodies before BCG-PSN co-incubation. ELISA assay was used to detect IL-12 of cell culture supernatant levels and Real-time quantitative RT-PCR was used to determinate IL-12 p40 mRNA expression. DC2.4 cells were preconditioned with PDTC for 1 hour, expression of CD83 and MHC-Ⅱwere dectected by cytoflow metry and IL-12 secretion was determined by ELISA assay.Results The increased volume and thickened synapses were observed when DC2.4 was co-cultivated with BCG-PSN, and the changes appeared more and more obvious along with the increased BCG-PSN concentration. An enhanced expression of CD83, MHC-Ⅱ molecule were obtained in 5μg/ml,20μg/ml and 50μg/ml BCG-PSN50 group when compared with the control group (P<0.05). CD86 expression in all the group were increased, without difference among them (P>0.05). IL-12 in DC2.4 cell culture supernatant increased gradually in BCG-PSN group along with the BCG-PSN concentration increase when compared with the control group (P<0.05). The expression of IL-12p40 mRNA in BCG-PSN treated DC2.4 increased, with a dose dependant pattern. There is no significant difference in IL-12 secretion and IL-12 p40 mRNA expression after anti-TLR2, anti-TLR4 and anti-TLR9 antibody preconditioning when compared with the control group. The IL-12 secreation and IL-12 p40 mRNA expression in DC2.4 cells were obvious retarded after PDTC preconditoning.Conclusion BCG-PSN could promote the CD83 and MHC-Ⅱexpression, IL-12 secretion on DC cells. BCG-PSN could be adopted by TLR4 receptors followed the activation of NF-κB, which, consenquently promote the IL-12 transcription on DC cells. Objective To study the preventive role of multiple injections of BCG-PSN at early stage on atopic dermatitis-like lesions in the Nc/Nga mice with and to explore its possible mechanism.Methods Newborn Nc/Nga mice were randomly divided into 3 groups:model group, low dose BCG-PSN group and high dose BCG-PSN.20μg/ml and 100μg/ml BCG-PSN were injected subcutaneously at 1,8,15,22 days after birth inow dose BCG-PSN group and high dose BCG-PSN group. The model mice were given the same amount of saline instead. After 49 days,1% DNCB was used to sensitize the mice. At day 56,63 amd 70,0.1% DNCB was used to stimulate the mice. At day 57,64 and 71, dermatitis score, pictures, orbital vein blood, and scratch behavior were observed. At day 72, the mice were killed and specimens, skin lesion and sperum were collected. ELISA was used to detect the concentration of IgE in plasma and IL-4 and IL-12 concentrations in spleen mononuclear cell culture supernatants. Immunohistochemistry was used to detect the IFN-y in lesions, HE staining was used to observe Histopathological changes.Results Repeated injection of BCG-PSN at early stage could significantly reduce the skin lesions of the Nc/Nga mice, reduce dermatitis score, and significantly reduce the cratching behavior in adult Nc/Nga mice sensitized by DNCB. There is no difference of scratching frequency between the different doses of BCG-PSN. BCG-PSN treatment could reduce the plasma levels of IgE,with a dose-dependent pattern. 0.5mg/kg BCG-PSN treatment could increase the secretion of IFN-γin skin lesions and reduces the IL-4 level and increase IL-12 concentration in spleen mononuclear cell culture supernatant.Conclusion The early injection of BCG-PSN could improve the skin lession in Nc/Nga mice probably due to the promotion of secretion of IFN-y and IL-12, reduction of IL-4 and IgE levels.