| Background:Psoriasis is a genetic, chronic, and immune-mediated inflammatory skin disorder, the main pathological characteristic histological features of which are hyperproliferation and abnormal differentiation of keratinocytes, dilatation and elongation of capillaries, infiltration of neutrophils and lymphocytes. At present, more and more researches show that psoriasis is a systemic disease which is associated with autoimmune disease, metabolic syndrome, cardiovascular disease, cancer, mental illness and so on. There gradually form the point of view that psoriasis affect overall health. Although the pathogenesis of psoriasis remains unclear, the imbalance of immune and epidermal keratinocyte hyper-proliferation are involved in the pathogenesis, especially closely related with cytokines and inflammatory mediators. Psoriasis is now believed to be a mixed Thl, Th17 and Th22 cell-mediated autoimmune disease because the activated T lymphocytes release numerous cytokines to stimulate the proliferation of keratinocytes and recruit neutrophils. The cytokines-activated keratinocytes can also express a broad array of cytokines, chemokines and membrane molecules in return that induce the recruitment and activated of T lymphocytes in the skin, thus forming cytokine network to induce and aggravate the pathological changes of psoriasis.The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway, a canonical pathway used to transmit signals from extracellular receptors to the nucleus, has attracted more and more attention. The combination of numous cytokines with corresponding receptors of target cells exerts a variety of biological effects by the signal from the cell membrane to the nucleus cytoplasm. The JAK-STAT signaling pathway is an important ways to lymphocyte activation. As the important components of JAK/STAT signaling pathway, STAT1 and STAT3 play an important role in maintaining the normal proliferation and differentiation of cells under normal circumstances. Our previous studies have shown that the expression of STAT1 and STAT3 in of psoriatic lesions were significantly increased. Activation of the STAT1 signaling pathway is initiated upon IFN-y which has long been considered the central cytokine involved in psoriasis pathogenesis, then the activated STAT1 combined into promoter region of activation sites and up-regulted the expression of IFN-y. In addition, IFN-y and IL-17 can promotes the expression of cytokeratin 17 (K17) through the JAK/STAT1 pathway. K17 is a kind of psoriasis associated cytokeratin, it’s only expressed in the lesions of psoriasis rather than in normal cells. K17 could lead to the abnormal activation effect of T lymphocytes. The vicious spiral of "IFN-γ- K17- T lymphocytes" induce and aggravate inflammatory reaction in psoriasis. JAK/STAT3 signaling pathway can be activated by IL-6, which together with IL-17, IL-23 to constitutes complex cytokine regulation network. Some previous studies had demonstrated that transgenic mice with keratinocytes expressing a constitutively active Stat3 (K5.Stat3C mice) develop a skin phenotype either spontaneously, or in response to wounding, that closely resembles psoriasis, meanwhile, the level of Stat3 in human psoriatic lesions, particularly in the nuclei of keratinocytes was increased. The mechanism of STAT1 and STAT3 in the hyper-proliferation keratinocytes in psoriasis patientes need for further research.The SOCS family, another family of proteins in the JAK/STAT pathway, are consisted of some relatively conservative composition SH2 domain in the N zone, SH2 zone and the C-terminus. The SOCS proteins are functionally related by their ability to negatively regulate cytokine signaling. The expression of intracellular SOCS is extremely low, but it can rising quickly with the stimulation of cytokines. the SOCS 1 and SOCS3 act in a classical negative-feedback loop to cytokine signal transduction manners. SOCS1 has been shown to associate with JAKs to inhibit their activity and SOCS3 appear to inhibit signaling by interacting with activated cytokine receptors. There is no one-to-one correspondence between SOCS and cytokines, SOCS1 can be induced by INF-y, IL-2 and IL-6. SOCS3 can be induced by IL-2, IL-6, IL-12 and TNF-a. SOCS1 and SOCS3 are the strongest STAT inhibitor in SOCS protein family by far. SOCS1 plays important role in maintaining immune homeostasis, delelation of the SOCS1 gene lead to a catastrophic monocytic inflammation because of excessive INF-y and interleukin signaling, it may cause severe inflammation of heart, liver, kidney, skin and other organs. SOCS3 not only affect the magnitude of the signaling response, but also the quality of IL-6 signaling response. SOCS1 and SOCS3 mRNA are not highly expressed in unstimulated cells, but are encoded rapidly in cytokines treated cells with an activated-STAT-dependent manner. The expression of SOCS1 and SOCS3, to a certain extent, reflects the activation of STAT.Acitretin, as one of the second generation of retinoids, is a time-honored treatment for psoriasis, either in monotherapy or in combination. Compared with other systemic therapies, acitretin has a much shorter half-life and poor lipophilic, what’s more, acitretin hardly affects the immune system, all of those advantages lay foundations of its unique position. Previous clinical trials showed that acitretin is especially useful in the treatment of pulstular psoriasis, erythrodemic psoriasis and chronic plaque psoriasis, it can promote the differentiation of lymphocytes and mononuclear cells, activate macrophages and epidermal cells Hans Lange. Some authors found that acitretin not only modulate the proliferatin of epidermal keratinocytes, but also reduce INF-γ, IL-17, IL-18, IL-23 and VEGF level in psoriasis patients. In a word, there may exits some relationship between acitretin and the epidermal keratinocytes of psoriasis, but the mechanisms are not fully clear.In recent years, the relationship between JAK/STAT signal transduction pathway and psoriasis have received more and more attention. Although most studies confirm the important role of JAK/STAT and its negative regulatory factors in the pathogenesis of psoriasis, but its mechanism is not entirely clear, so the detailed study of JAK/STAT signal transduction process and control links will help to understand the pathogenesis of many diseases, and may provide some theory basis for the corresponding pharmacological effect of drugs. The JAK/STAT signal transduction pathway in the pathogenesis of psoriasis mechanism and the relationship between acitretin with JAK/STAT pathway is worth depth research. We supposed that acitretin could directly effect on JAK/STAT signal transduction pathway and achieve the purpose of treatment. In order to proved the aboved hypothesis, we choose human keratinocyte cell line HaCaT as the research object, specific interference RNA targeting for STAT1 and STAT3 (siRNA) were designed and transfected into HaCaT cells respectively. Then, Filter out the best silent sequence by Q-PCR and Western-blot methods. We then selected the best silent sequence to explore the pathogenesis of psoriasis.Objective:.1. To establish the STAT1 and STAT3 low expression experimental prototype by using RNA interference technolog and to investigate the biological function and gene regulation mechanism of STAT1, STAT3, SOCS1 and SOCS3 in HaCaT cells ofpsorisis.2. To explore the effect of acitretin on JAK/STAT pathway, thus may contribute to confirm acitretin target spots and mechanism for the treatment of psoriasis.Methods:1. An immortalized human keratinocytes cell line, HaCaT cells, was and cultured in DEMN supplemented with 100U/ml streptomycin and 10% heat-inactivated fetal bovine serum. HaCaT cells were kept at 37℃ and 5% CO2 in humidified atmosphere.2. HaCaT cells were treated with acitretin at concentrations of 0.1 uM, 1uM, 5uM for 12h,24h,48h and 72h. The groups treated without acitretin intervention was set up as control. The proliferation activity of HaCaT cells was measured by MTS after the cells were collected. Then, Filter out the best inhibitory concentrate of acitretin and observed the dose-dependent manner.3. After HaCaT cells treated with the appropriate concentration, the level of STAT1, p-STAT1, STAT3, p-STAT3, SOCS1 and SOCS3 expression in cells was mesured by Q-PCR and Western blot.4. Construction and transfection of three pairs of siRNA-STAT1 and siRNA-STAT3.① The gene sequences of STAT1 mRNA and STAT3 mRNA were searched from genetic databases (such as www.ncbi.nlm.nih.gov), and homology analysis with BLAST software. ② In accordance with the siRNA design principle, three pairs of siRNA-STAT1 and siRNA-STAT3 sequences were designed after choosing the whole gene sequence of STAT1 and STAT3mRNA as template. ③ Three pairs of siRNA-STAT1 and siRNA-STAT3 were synthesized according to the design sequence. The negative control group was established. ④Three pairs of siRNA targeting STAT1 and STAT3 were transfected into HaCaT cells respectively by Lipo-2000.5. The silencing effects:The silencing effects were verified by Q-PCR and Western-blot in terms of STAT1, STAT3 mRNA and protein level. We screened si-RNA sequences with best silence effection.6. The chosen si-RNA sequences were subsequently transfected into the HaCaT cells again, we utilize MTS, Q-PCR and Western-blot to detect the role of STAT1 and STAT3, as well as the expression of SOCS and SOCS3.7. Drug intervention:The chosen STAT1-siRNA and STAT3-siRNA were transfected to HaCaT cells which were treated with or without 5mol/L acitretin for 48h.The expression of STAT1, p-STAT1, STAT3, p-STAT3, SOCS1 and SOCS3 were measured by Q-PCR and Western-Blot.8. Dates were expressed as mean±SD of three independent experiments. Comparisons among groups were carried out using One-Way ANOVA. In all analyses, P value<0.05 was taken to indicate statistical significance.Results:1. Human epidermal keratinocytes were treated with 0.1 uM, 1uM,5uM acitretin for 12,24,48 and 72 hours. The cellular proliferation was determined by measuring MTS assay. Compared with the blank control group, HaCaT cells proliferation was inhibited by acitretin with a time-dependent and dose-dependent manner. Acitretin with 5uM concentration could significantly inhibit cellular proliferation after 24 hours later. Therefore, acitretin with 5uM was optimal for the subsequent experiments. The expression of STAT1, p-STAT1, STAT3, p-STAT3, SOCS1 and SOCS3 were down-regulated in HaCaT cells which were treated with 5uM acitretin.2. Transfection efficiency was detected by immunofluorescence under fluorescence microscopy. STAT1-siRNA and STAT3-siRNA were introduced into HaCaT cells at 25,50, 100nMol/L independently. The potential of siRNA for inhibition of STAT1 and STAT3 were measured by Q-PCR and Western-blot. STAT1 and STAT3 mRNA were down-regulated by STAT1-siRNA and STAT3-siRNA respectively compared with the control group(p<0.05). As for the protein levels, STAT1 and STAT3 protein expression in the treated groups were lower than the control group. Therefore,50nMol/L siRNA-STAT1-003 and 50nMol/L siRNA-STAT3-001 were optimal for the subsequent experiments.3. HaCaT cells transfected with 50nmol/L STAT1-siRNA and STAT3-siRNA were treated with or without 5umol/L acitretin. Eight groups containing cells group (A), lipofectamine-2000 group (B), NC-siRNA group (C), acitretin group (D), STAT1-siRNA group (E), STAT3-siRNA group (F), STAT1-siRNA+acitretin group (G), STAT3-siRNA +acitretin group (H) were set up. HaCaT cells proliferation existed significant differences in eight groups (P<0.05), however, there were no significant differences between A, B and C groups (P>0.05) after incubation for 12, 24,48,72h. The cell proliferation of groups D, E, F, G, and H were inhibited compared with group A at each time (P<0.05). What’s more, there were no significant differences between E and F groups, as well as G and H groups (P>0.05), the inhibition ratio of G and H groups were higher than E and F groups. The value of OD in descending order were G, H< E, F<D<A, B and C. The comparison of eight groups indicated that acitretin, STAT1-siRNA and STAT3-siRNA alone or combination with each other could inhibit HaCaT cells proliferation, the inhibitory ability of acitretin was weaker than STAT1-siRNA and STAT3-siRNA.4. The STAT1, STAT3, SOCS1 and SOCS3 mRNA level were significant differences between the eight groups (P<0.05). However, all of them didn’t exit significant differences in group A, B and C, which indicated little influence of lipo-2000 and NC-siRNA on STAT and SOCS expression. The expression of STAT1 mRNA was inhibited in D, E, G and H groups. The level of STAT3 mRNA also decreased in D, F, G and H groups. As for the expression of SOCS1 and SOCS3 mRNA, the mRNA levels in D, E, F, G, and H group were lower than A, B, and C group. Those results shows that STAT1-siRNA could inhibit the expression of STAT1 and SOCS1 mRNA, but STAT1-siRNA may not down-regulated the STAT3 mRNA, and STAT3-siRNA could inhibit the expression of STAT3 and SOCS3 mRNA but didn’t down-regulated the STAT1 mRNA, however, acitretin could down-regulated STAT1, STAT3, SOCS1 and SOCS3 mRNA. The content of STAT1 protein was decreased in acitretin or STAT1-siRNA treated groups, and the same trend of STAT3 protein in acitretin or STAT3-siRNA treated groups. As for the SOCS1 and SOCS3 protein, both of which in acitretin, STAT1-siRNA or STAT3-siRNA treated groups was less than in blank, lipo-2000 or NC-siRNA groups. These results suggested that STAT1-siRNA could inhibit the expression of STAT1, SOCS1 and SOCS3, STAT3-siRNA could inhibit the expression of STAT3, SOCS1 and SOCS3. What’s more, acitretin could inhibit the expression of STAT1, STAT3, SOCSl and SOCS3.Conclusion:1. Acitretin could inhibit the proliferation of HaCaT cells as well as the expression of STAT1, STAT3, SOCS1 and SOCS3. Silencing the STAT1 or STAT3 gene induced the down-regulation of SOCS1 and SOCS3. Therefore, we speculated that acitretin might modulate HaCaT cells proliferation in psoriasis by decreasing the expressionof STAT- and STAT3-dependent mechanism2. Our research helps to deepen the understanding the roles of JAK/STAT signal transduction pathway and its negative regulatory factors SOCS1 and SOCS3. The JAK/STAT pathway is a potential key point of the pathogenesis of psoriasis, What’s more, acitretin could inhibit this signals by down-regulated the expression STAT1 and STAT3. This study also provide a theoretical basis for some new research and development of antipsoriasis drug in clinical application. |
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