| Psoriasis is a common skin disease that is characterized by chronic relapsing inflammatory reaction. In its pathological manifestations, excessive proliferation and abnormal differentiation of epidermal keratinocytes (keratinocytes, KC) causes hyperplasia, dyskeratosis, acanthosis, and the change of stratum corneum in granular layer[1]. This shows that the pathogenesis of psoriasis is closely related to its signal pathway and abnormal KC cell-signal transduction is one of the important pathogenesis of psoriasis[2]. MAPK signal pathway can lead NF-κB and active the activating protein, and then activate the inflammatory factors in the genetic transcription. Along with the development and amplification of the inflammation, the NF-κB and MAPK signaling pathway may have a very important effect in pathogenesis of psoriasis[3].Human beta-defensin-2(HBD-2) was obtained from skin lesions of psoriasis patients by separation purity. Researchers found that psoriasis patients had skin lesions such as erythema, papules, scales, scratches, and even scabs, but ra rely skin infection. Research of HBD-2is related with psoriasis, but the sp ecific mechanism still needs to be investigated continuously. Traditional Ch inese Medicine treatment has significant effect in psoriasis, but the exact mechanism has not been found yet. In the molecular biology perspective of pa thogenesis of psoriasis, the study of HBD-2can help us find the mechanism o f Chinese Medicine by providing new train of thoughts for treatment of disea se.Cytokine such as IL-1βand TNF-α, which are released after the activa tion of Keratinocytes (KCs), are strong inducers of HBD-2. IL-1β and TNF-α are regulated by NF-κB and MAPK signal pathway while inducing HBD-2. The s ignaling pathway of NF-κB and MAPK is activated in epidermis of psoriasis, especially in the link of hyperplasia of keratinocytes and excessive secret ion of its proinflammatory cytokines. MAPK signal pathway can also turn the initial CD4+T cells into Thl or Th2cells, which leads to different immune r esponses. JNK1can induce CD4+T cell to Th2, and JNK2to Thl, while promotin g Thl cytokines. P38can be differentiated to Thl and promote differentiatio n of Th2.Quyin granule, which has clearing heat and cooling blood detoxification effect, was made by Professor Bai Yanping herself. It has shown its effectiveness through animal experiments, but the specific mechanism of action has not been completely explained.Objective:This study is to reveal the changes of the p38, JNK, NF-κB type psoriasis in-vitro model after taking Quyin granule; to observe the effect of Quyin granule on in-vitro culture Keratinocytes NF-κB and MAPK pahtway level, as well as the mechanism of HBD-2; to elucidating the correlation between NF-KB&MAPK signaling pathway and HBD-2by studying intervention of Quyin granule to psoriasis; to provide objective experimental data of controlling psoriatic HBD-2by therapeutic effect of Quyin granule effecting NF-KB and MAPK signaling pathway.Methods:This study uses advanced foreign technology production of psoriasis in-vitro epidermal reconstruction model. Moreover, through observing forms of tissue and validating if the model is successful or not by HE staining and immunohistochemistry method, can provide a new model for the study of psoriasis in-vitro. After the experiment of in-vitro psoriasis epidermal reconstruction model, it is possible to obtain the pathological and molecular biological characteristic skin substitute of psoriasis morphology, which could provide further study of morphology, molecular pathology and pharmacology.Selected HaCaT cells were put in Transwell culture dish to establish psoriatic in-vitro epidermal reconstruction model; Quyin granule control group (A retinoic acid) and negative control (normal saline) medicated serum were prepared. Immunohistochemistry and real-time fluorescence quantitative PCR method were used to investigate the expression of HBD-2in psoriasis. A mixture of TNF-α, IL-1α, IL-6, IL-22was added into the cytology in-vitro model to induce high development of HBD-2with NF-κB and MAPK signaling pathway activation; at the cellular level, by applying immunecytochemistry and using real-time fluorescent quantitative PCR, ELISA method to observe the HBD-2influences on HaCat cells and NF-κB&MAPK signal pathway in the of human epidermal keratinocyte in-vitro.Quyin granule, by using the Western blot method, is told to affect the phosphorylation of HBD-2, P-I κ Bα/IκBα, P-P38/P38, P-JNK1/JNK1and P-JN, and could modulate the NF-κB&MAPK signal pathway.Results:(1) Psoriasis in-vitro skin reconstruction model was successful ly established by using HaCaT cells in transwell vessel. Through HE staining and immunohistochemistry method it verified its good stability and reproduc ibility, which provides a pathological and molecular biological characterist ic of psoriasis skin substitute for the in-vitro study of psoriasis morpholo gy. Furthermore, it provides a new tool for basic research methods and offer s a certain economic value by screening studies of effective prescription fo r the treatment of psoriasis.(2) Studies of immunohistochemistry, qPCR, and Elisa found out the Quyin granule can reduce the HBD-2indexes of culture supernatant and cytology in-vitro model of psoriasis.The result of positive control group (A retinoic acid) was significant.(3) In Western blot method it was seen that Quyin granule can significantly reduce P-I KBa, P-P38, P-JNK1and P-JNK2protein activation. This leads to inhibition of NF-κB, P38MAPK and JNKMAPK signal pathway, which blocks the IL-1a, TNF-a, IL-6and IL-22activation of HBD-2.Conclusion:Quyin granule can significantly lower the index of cytology i n-vitro psoriasis reconstruction model and reduce the development of HBD-2in culture supernatant; the effectiveness of positive control group was greater t han that of the negative control group. Quyin granule inhibits JNK2and MAPK signaling pathway and restrains the activation of HBD-2by blocking IL-1α, T NF-a, IL-6, IL-22, and therby brings cure to psoriasis. |
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