The Effect Of Calcipotriol On JAK/STAT Signal Transduction Pathway Of Psoriasis

Background:Psoriasis is a common chronic inflammatory proliferative disease, stubborn disease, easy relapse, and can cause systemic damage,of which etiopathogenisis and pathogenesis have not been completely known yet. And do not have special treatment, there is no effective method to cure, serious influence patient’s quality of life, even survival cycle, is one of the focus on the disease at home and abroad.The main pathology of psoriasis is abnormal proliferation of keratinocytes, parakeratosis, angiogenesis and inflammatory cells infiltration. Research shows that the disorder of immune balance and (or)myelodysplastic epidermal keratinocytes is closely related with the pathogenesis of psoriasis, Which cytokines on keratinocyte proliferation and differentiation, pathology and cell kinetics have a role. At present, it has been demonstrated many believe that the abnormal activation of T cells and T cells-keratinocytes plays a very important role in the pathogenesis of psoriasis.In recent years JAK/STAT pathway relations with psoriasis by more and more attention, a lot of research work show that the JAK/STAT pathway is the most important cytokines information transfer conditions of a channel.The secretion of avariety of cytokines by activated T cells, and in combination with the corresponding receptors on the cell surface. The extracellular signals to the nucleus by JAK/STAT pathway regulate keratinocyte cell and other cells such as proliferation and differentiation. Thus play an important role in the immune process of psoriasis. However, the exact roles of STAT mechanisms are not fully understood.The majority of research results at home and abroad show that activated T cells to produce a series of cytokines in psoriatic lesions. These cytokines with the keratinocytes cell surface receptor combination, may cause changes in expression of Keratin patterns in keratinocytes. And the study found that normal skin does not express keratin17(Keratin17K17), but high expression in psoriatic lesions. Accordingly, K17is considered to be one of the markers of psoriasis. Cytokines IFN-y can activate STAT1, induction above the basal layer of the epidermis of keratinocytes express keratin K17. Some studies also show that expression of IL-17A in the keratinocyte cells by STAT1and STAT3pathway dose dependency raised K17expression. At present most studies have shown that high expression of STAT3protein in the cytoplasm and nucleus of the lower epidermis cell in psoriatic skin lesions of patients. Also significantly increased its expression in the blood and with the severity of the disease was positively correlated.Meanwhile, studies have shown that abnormal activation of STAT3can promote cell differentiation and proliferation, inhibition of apoptosis, eventually leading to abnormal cell proliferation and (or) malignant transformation, which can lead to many diseases.Prompted STAT3plays an important role in the pathogenesis of psoriasis.Therefore, STAT1and STAT3in keratinocytes form abnormal cell proliferation in patients with psoriasis, whether there is a high expression and synergistic result in the pathogenesis of psoriasis worthy of further study.Recently, it has been clear that there are at least three different types of inhibitory protein is associated with negative regulation of cytokine signal transduction,SOCS family members are a negative regulator of most cytokine signal transduction pathways involved in many immunological pathogenesis of inflammatory disease.And, the role of SOCS1and SOCS3of SOCS family in the pathogenesis of psoriasis are more and more attention. Study found that SOCS1is negative feedback regulation of IFN-γ signal activity factor, SOCS3can inhibit the inflammation caused by IL-2.Whereas IFN-, IL-2for keratinocytes is the most powerful pro-inflammatory cytokines,IFN-γ and IL-2through the induction of JAK-STAT signal transduction pathway, formation,The keratinocyte cell immune inflammatory reaction.Although most studies have confirmed the JAK/STAT’s and its negative regulator factor important role on the pathogenesis of psoriasis, however, However, its specific mechanism is not fully clear, the need for further in-depth study.Calcipotriol is1,25(OH)2D3analogs, recent clinical studies found that the efficacy of1,25(OH)2D3and its analogs to treat psoriasis.The experiment study found,strong affinity with calcitriol receptor. And calcipotriol reduce skin psoriasis patients with IL-6content and distribution, as well as the activation of epidermal T lymphocytes, thus to correct the detected abnormal proliferation and differentiation of psoriasis.Meanwhile, it is also found that the anti-proliferative effect of the CPT ssociated with phosphorylation of pRB,The dephosphorylated pRB and UE2F after transcription factor binding can reduce a variety of cell cycle regulatory proteins and positive expression of transcription factors (such as of CyclinDl, CDK4, C-Myc) expression. At the same time, CPT may promote increased synthesis of TGF-β1, TGF-β1and inhibition of CDK4expression.The confluence of these findings, JAK/STAT pathway in the pathogenesis of psoriasis and Calcipotriol relationship with the JAK/STAT pathways is is worth in-depth study. Whether can put forward Calcipotriol is a direct role in the JAK/STAT signal transduction pathway, so as to achieve the purpose of the treatment of psoriasis?In order to solve the above problems, this research object for the study of human keratinocytes HaCaT cell line.Using RNA interference technology, the design of the specificity of siRNA against STAT1/STAT3then silence sequence was transfected into HaCaT cells, respectively silence STAT1/STAT3gene. To detection the silence effect of STAT1/STAT3by Q-PCR and Western-blot. Filter out the best silent sequence. To further investigate the pathogenesis of psoriasis and calcipotriol treatment of psoriasis mechanism.Objective:1. To investigate the function of STAT1/STAT3and SOCS1/SOCS3in Psorisis, the down regulatation the STAT1/STAT3expression in HaCaT cell line by using RNA interference technology was established, and we can study the biological function and mechanism in Psorisis.2. To investigate the effect of calcipotriol in JAK/STAT pathway, thus confirming calcipotriol targets and mechanism for the treatment of psoriasis.Methods:1. HaCaT cells were cultured in Dulbecco’s Modified Eagle’s Minimal Essential Medium (DMEM) with10%fetal bovine serum(FBS)under a humidified atmosphere containing5%CO2at37℃.2. Calcipotriol treated HaCaT cells at concentrations of0.1nM,1nM,10nM,100nM,1uM,10uM,and in Oh,12h,24h,48h,72h after the cells were collected, Without applying the intervention HaCaT cells as control group. Theproliferation of HaCaT cells was evaluated by MTS after Calcipotriol. Then, Filter out the best inhibitory concentrate of Calcipotriol. Observed the dose-dependent manner.3. The best inhibitory concentrationThe level of STAT1、STAT3、P-STAT1、 P-STAT1、SOCS1、SOCS3expression was evaluated by Q-PCR,Western blot after the best inhibitory concentration of Calcipotriol tread. 4. siRNA-STAT1/siRNA-STAT3construction and transfection:①STAT1mRNA、STAT3mRNA human gene sequences were searched from CNKI (such as www.ncbi.nlm.nih.gov) using BLAST homology analysis software.②using STAT1mRNA、STAT3mRNA good will of the entire gene sequence as a template, three sections of the expression of STAT3-specific siRNA sequences were designed in accordance with the principle of siRNA design.③According to the design of the sequence, three double-stranded siRNAs were synthesized by a chemical method, such as siRNA-STAT1-001/siRNA-STAT3-001、siRNA-STAT1-002/siRNA-STAT3-002、siRNA-STAT1-003/siRNA-STAT3-003and negative control groups was established at the same time.④siRNA sequences were transfected into HaCaT cells by Lipo2000.5. Detection effects:the efficiency of transfection were estimated by detecting the expression of STAT1、STAT3in order to screen the best siRNA sequence. we used multiple methods such as Q-PCR, MTS and Western-blot.6. To study the proliferation of HaCaT cells and the gene expression of JAK/STAT passway after transfection, we used multiple methods such as Q-PCR, MTS and Western-blot.7. Drug intervention:STAT1/STAT3silenced with the optimal sequence were incubated with luM Calcipotriol for24h.The expression of STAT1mRNA,STAT3mRNA were detected by RT-PCR and STAT1,STAT3protein expression detected by Western-Blot.8. The statistical analysis was performed with the software SPSS13.0, and the significance was set at P value≤0.05.Results:1. After Calcipotriol treated HaCaT cells at different concentrations, The optimum OD values detected by MTS, and the inhibitory concentration of calcipotriol was lOnM. The expression of STAT1、STAT3、P-STAT1、P-STAT1、 SOCS1and SOCS3in gene and protein levels were decreased.2. Different concentrations of the siRNA-STAT1/siRNA-STAT3transfected into HaCaT cells.After transfection observed a large number of green fluorescent for24h in fluorescence microscope.the STAT1/STAT3silenced effect were detected by RT-PCR and Western-Blot. we confirmed that STAT1/STAT3with downregulated expressed of was HaCaT cells obtained. the best silent sequence were siRNA-STAT1-003-50um and siRNA-STAT3-001-50um.3. The cells were divided into eight groups after the siRNA-STAT1/siRNA-STAT3successfully transfected into HaCaT cells, A.cell group;B.lipo2000group;C.NCsiRNA group; D.lOnM Calcipotriol group; E.STAT1siRNA group;F.STAT3siRNA group; G.STAT1siRNA+luM Calcipotriol group; H. STAT3siRNA+luM Calcipotriol. The STAT1, STAT3, P-STAT1, P-STAT1, SOCS1and SOCS3expression of the gene and protein level was no significant difference in A, B, and Cgroups (P>0.05).And the D, E, F, G and H expression of relevant factor in the gene and protein level was decreased. Moreover, the expression of STAT1/p-STAT1were no significant difference between E and G, the expression of STAT3/p-STAT3were no significant difference between F and H.4. The optimum OD values of HaCaT cells detected by MTS in8groups. The levels of HaCaT cells were decreased. in D、E、F、G and H groups.Conclusion:1. After HaCaT cells were cultured with calcipotriol, the expression of STAT1, STAT3, SOCS1and SOCS3in gene and protein level were decreased. And HaCaT cell proliferation was inhibited.This experiment showed that siRNA could effectively inhibit the expression of STAT1, STAT3, SOCS1and SOCS3level in mRNA and protein as well as calcipotriol group. Calcipotriol may be prompted by JAK/STAT signaling pathway may affect HaCaT cell differentiation, proliferation. It was speculated that the JAK/STAT pathway was a critical point in psoriasis. STAT1/STAT3as a target for the treatment of psoriasis is feasible.2. The STAT1、STAT3、P-STAT1、P-STAT1、SOCS1and SOCS3expression in siRNA-STAT1-003-50um group、siRNA-STAT1-001-50um group and calcipotriol group were all inhibited with no obvious difference suggested that calcipotriol may act through inhibiting the activation of JAK/STAT pathway in psoriasis. And the expression of STAT1/p-STAT1were no significant difference between E and G, the expression of STAT3/p-STAT3were no significant difference between F and H. The G.This indicated that calcipotriol may also inhibit the expression of STAT1and STAT3through other pathways. Prompted the JAK/STAT signaling pathway is regulated by a variety of factors, needs further study.3. This experiment will help to in-depth understanding of the the JAK/STAT signal transduction pathway and its suppressor of cytokine signaling (SOCS1, SOCS3) role in the pathogenesis of psoriasis and calcipotriol its related signaling pathways,This study would also provide the theory basis in the study of the potential drug target on psoriasis related cell signal transduction pathway.