The Effect Of G31P, CXCR1/2Antagonist, On Lesional Skin Of Epidermolysis Bullosa Acquisita

Objective: Epidermolysis bullosa acquisita (EBA) in the majority of patientsinvolving the skin and mucosal surfaces, especially in the mouth, nose, pharynx, larynxmucosa and conjunctiva. It is subepidermal disease with clinical performance of blisters,erosions and scars associated with autoimmunity to type VII collagen and neutrophilaccumulation at derm–epidermal junctions (DEJ). Histopathological examination oflesional epidermis show the separation of the dermal-epidermal, which is IgGantibodies, complement C3linear deposition in the basement membrane of thedermal-epidermal. Mouse model of epidermolysis bullosa acquisita (EBA) around eyes,lips, limbs, trunk, and tail of mice, it is subepidermal disease with clinical performanceof blisters, erosions and scars. After successfully established mouse model ofepidermolysis bullosa acquisita (EBA),respectively, different groups of the EBA modelinject G31P or NS at predetermined time point. By comparing difference between NSgroup and G31P group, we observe that if CXCR1/2antagonist is able to inhibit typeVII collagen antibody damaging in skin. It is certain significance in research thepathogenesis of EBA and remising clinical symptoms of EBA and its treatment.Methods: G31P preparation: take G31P powder1mg,adding into10ml NS,formulated into a working solution concentration10g/100l,each mouse was injected10g/20g(100l). Animal groups:20Balb/c mice are divided into four groups: G31Pfemale group, G31P male group, NS female group, NS male group, five mice in eachgroup. Animal model: the GST-mCol7c and Freund’s complete adjuvant (FCA) wereinjected in rabbits by passive immunization, and purified IgG antibodies obtained from these rabbits serum, then inject IgG antibodies to Balb/c mice by active immunization.G31P were injected on alternative day in G31P male and female groups on from1stdayof drug NS male and female groups were injected with NS with same time period ofG31P.On2,4,7day of experiment, all four groups were injected P-IgG. Detectionmethods: On5thand9thday of experiment, peripheral blood of mice obtained from tail,tail skin were used for FACS (Gr-1), IF/IHC. Degree of skin damage in mice werescored on every day of experiment. On12thday of experiment, mice were dissected, andcollect peripheral blood to detect cytokines by FACS (CBA). One part of mice earpreserved in formalin for HE staining, another part freeze at-80℃formyeloperoxidase (MPO) and immunofluorescence (IF) also store damaged skin forHE staining and spleen for FACS (Gr-1, T/B).Results:1.The observations of G31P group and NS group show that the performance ofNS group appeared earlier than G31P group, which is with clinical performance ofblisters, erosions and scars around ears，eyes, tail of mice.The NS group is more seriousand obvious than the G31P group；2. Skin damage in G31P male and female groups were less than control NSgroups of EBA mice model, P <0.05, statistically significant;3.HE staining of tail skin, eye lids, ears of G31P and NS groups compared,separation at dermal–epidermal junction (DEJ), was more serious and more intensiveinflammatory cells mainly neutrophils were observed;4. Tail skin, eye lids, ears of G31P and NS groups was observed by directimmunofluorescence detection of IgG and C3, both groups show separate and lineardeposition of IgG and C3at dermal-epidermal junction, but NS group showed greaterfluorescence intensity, P>0.05, not statistically significant;5. Detection of Gr-1in peripheral blood by flow sorting technique in all fourgroups of mice, results were significantly higher in NS groups than G31P groups on9th day, P <0.05, statistically significant;6. Gr-1, CD3, CD9in spleen were detected by flow cytometry in both groups, result between two groups were not significantly different.Conclusion:1. G31P can significantly reduce the symptoms of skin damage on EBA modelmice;2. G31P can reduce the infiltration of inflammatory cells under the epidermisand relieve the separation of the dermis and epidermis on skin lesion site on EBA modelmice;3. G31P can reduce the number of neutrophils in the blood, but can not affectthe number of neutrophils、B cells and T cells in spleen on EBA model mice;4. G31P do not significantly effect skin damage caused linear deposition of IgGand C3under the epidermis on EBA model mice.