Regulation Mechanism Of Substance P And The Intervention Of Drugs In Cutaneous Allergy Reaction

The cutaneous allergy reaction is an anaphylaxis which has a high incidence rate. The anaphylaxis often emerges in skin when the body is stimulated by special sensibiligen. It is especially serious for the soldiers who work in border defense or on island. In the fields of dermatology, more and more researchers increasingly pay more attention on the role of the network of immune-neuro-endocrine in dermatic diseases. The nervous system effects the function of cutaneous cells through the neurotransmitters, regulating the physiologic and physiopathologic processes of skin. The specific neurokinin receptor antagonists may play therapeutic role by intervening in the physiologic processes through regulating the receptor-ligand binding and the signal transduction. It has become a focus to explore the new targets or the new active compounds involved in the cutaneous allergy reaction .The neuropeptide substance P (SP) is a member of tachykinin peptide family and widely distributed in the central and peripheral nervous system. SP is accepted to be the major contributor to skin allergic and inflammatory reaction by preferentially activating a specific receptor, the SP receptor (SPR) (also designated NK-1 receptor for neurokinin 1 receptor). The aspect about skin immune system (SIS) mediated by keratinocytes, the principle epidermal cells, and the dermal fibroblasts has attracted the attention of both immunologists and dermatopathologists. A fact that the mechanism of the SP and NK-1 receptor in the cutaneous allergy reaction has received only little notice so far in the world of classical immunology and no specific neurotransmitters antagonists has been on the market until now. So it is very important and necessary to explore the role of SP and NK-1 receptor in the cutaneous allergy reaction and try to find out the new active component for the therapy of skin hypersensitiveness and inflammation.In this study, an animal model of IgE-dependent triphasic cutaneous reaction caused by DNFB in the ears of BALB/c mice was established to investigate the expression of SP in mice ear skin and the effect of Cetirizine Hydrochloride on it. The expression of NK-1 receptor protein in HaCaT cells and fibroblasts was examined by immunohistochemistry, and themRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The expression and regulation characteristics of NK-1 receptor were measured by flow cytometry and Western blotting treated with various stimuli and drugs .The effect of SP and Cetirizine on the secretion of IL-10, IFN-γ and MCP-1 in HaCaT cells and fibroblasts were also been invesgated. The potentiality of Matrine and Schizonepetae Oil extracted from traditional Chinese medicine herbs Sophora flavescens Ait. and Schizonepeta tenuifolia Briq. on the regulation processes of SP was been explored at last.Part I: The establishment of IgE-dependent triphasic cutaneous reaction caused by DNFB in the ears of BALB/c mice1. An animal model of IgE-dependent triphasic cutaneous reaction caused by DNFB in the ears of BALB/c mice was established. BALB/c mice were passively sensitized by intravenous infection of anti-DNP IgE monoclonal antibody 24 h before challenge. Skin reaction was elicited by applying DNFB to both sides of each ear of sensitized mice. The mice exhibited a triphasic cutaneous reaction with an immediate-phase response (IPR) at 1 h, a late-phase response (LPR) at 24 h and a very late-phase response (vLPR) at 7 days after challenge with DNFB. The ear swelling was 15.8±2.6 μm, 22.1±3.2 μrn, 52.3±7.8 μm, respectively. The results were confirmed by skin histological examinations.2. The concentrations of IgE in mice serum were assayed by ELISA. The level of serum IgE changed with the development of allergy compared with the control. In IPR, the concentrations of IgE increased from 18.50±2.07 pg · mL^-1 to 24.42±5.92 pg · mL^-1, which changed from 17.91 ±4.72 pg·mL^-1 to 32.53 ±6.70 pg·mL^-1 in LPR. The increasing of IgE is the major factor of IPR and LPR. The serum IgE decreased to 22.14 ±2.56 pg· mL^-1, close to the normal amounts in vLPR, which indicated that IgE may be not the domain mediator of vLPR.Part II The expression of SP in triphasic cutaneous reaction and the regulation of Cetirizine1. The mice were treated with Cetirizine (1 mg · kg^-1, 10 mg·kg^-1, ig) 1 h before injection of anti-DNP IgE monoclonal antibody and once a day after chanlleged with DNFB. Cetirizine was shown to significantly inhibit the ear swellings induced by DNFB in IPR and LPR. Theear swelling decreased from 17.3 ±4.8 μm to 5.2±2.4 μm (10 mg · kg^-1) and 7.2 ± 3.1 μm (1 mg · kg^-1) (P<0.01) in IPR., which changed from 22.7±4.7 μm to 17.4±4.0 μm (P<0.05) treated by Cetirizine (10 mg · kg^-1) in LPR. But Cetirizine has no obvious effect on ear swelling in vLPR.2. The mice ears were removed for immunohistochemical staining of SP at definited time after challenge. The results show that the expression of SP was very thin in nomal mice ears. The obvious positive expression can be seen in epidermis and dermis of mice ears after chanllenged. The expression became stronger with the process of allergy.3. The contents of SP in skin of mice ears were measured by radioimmunoassay (RIA). Cetirizine can inhibit the expression of SP in triphasic cutaneous reaction. The content of SP in mice ears decreased to 4.3 ± 1.0 pg·mg^-1 from 5.8 ± 1.6 pg · mg^-1(P<0.05) in IPR. In LPR and vLPR, the content of SP were 4.5 ± 1.9 pg·mg^-1 and 4.0 ±0.5 pg · mg^-1 compared of 6.5 ±2.4 pg · mg^-1(P<0.05) and 7.7±2.3 pg · mg^-1(P<0.01).The results above indicated that SP is involved in the pathogenesis of allergic dermatitis. Cetirizine can inhibit the expression of SP in IgE-dependent triphasic cutaneous reaction. It might be part of the mechanisms of anti-anaphylaxis of Cetirizine.Part III The expression characteristics of Substance P receptor in human keratinocyte and fibroblasts and the regulation of drugs1. HaCaT cells, a human keratinocyte cell line, and fibroblasts were cultured. The expression of NK-IR protein was examined by immunohistochemistry and the mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). NK-1 receptor positive expression was observed both in HaCaT cells and fibroblasts and the mRNA levels in HaCaT cells was more than that of fibroblasts, which concordance with the results of flow cytometry.2. The expression and regulation of NK-IR were measured by flow cytometry and Western blotting in HaCaT cells and fibroblasts treated with SP, LPS, IFN-γ and drugs. The results showed that the expression of NK-IR was up-regulated 39.3% and 33.1%(P<0.01) stimulated by SP and IFN-γ in HaCaT cells , respectively. The expression was up-regulated 47.0% (P<0.01) and 30.9% stimulated by SP and IFN-γ in fibroblasts. LPS can up-regulated theexpression of NK-IR in HaCaT cells while down-regulated the expression in fibroblasts. Spantide I , a pan-specific neurokinin receptor antagonist, and Cetirizine can degrad the expression of NK-1 receptor stimulated by SP .The results above indicated that the keratinocytes and fibroblasts were involved in the regulation of skin immune and the NK-1 receptor may play an important role in the patho-genesis of cutaneous allergic inflammation.Part IV The regulation of SP on the secretion of cytokines and chemotatic factors in cutaneous cells and the effect of Cetirizine1. The time course and dose-response relationship of SP on the secretion of IFN-γ, IL-10 in HaCaT cells and fibroblasts were investigated by ELISA. SP can induce the production of IFN-γ, IL-10 in two type cutaneous cells. Cetirizine (10^-6M and 10^-4M) can increase the secretion of IFN-γ, but no significant deviation compared with the control (P>0.05) .Cetirizine has no significant effect on the secretion of IL-10.2. The secretion of MCP-1 in HaCaT cells and fibroblasts simulated by SP were asssyed by ELISA. The production of MCP-1 increased to 361.40± 19.02pg · mL^-1, 2.17 times as much as the control (P<0.01) in HaCaT cells .Cetirizine (10^-6, 10^-5, 10^-4M) can degrade the secretion of MCP-1 incubation with SP. The concentrations were 183.70 ±21.83, 160.26 ±31.32, 211.73 ± 16.78 pg · mL^-1, respectively. The production of MCP-1 increased to 559.90± 22.74pg · mL^-1, 1.55 times as much as the control (P<0.01) in fibroblasts stimulated by SP. Cetirizine (10^-6 , 10^-5, 10^-4M) can degrad the secretion of MCP-1 incubation with SP. The concentrations were 428.40± 18.68, 422.93± 10.82, 413.40 ± 36.45 pg · mL^-1, respectively.The results above showed that Cetirizine has no significant effect on the secretion of IL-10, IFN-γ, while inhibited the secretion of MCP-1, which may be a new effective path of Cetirizine.Part V The effect of Martine and Schizonepetae Oil on the regulation of SP in cutaneous cells1 .The Matrine and Schizonepetae Oil were extracted from traditional Chinese medicine herbs Sophora flavescens Ait. and Schizonepeta tenuifolia Briq. The effect of Mat and Sch on the...