Effect Of Silencing STAT3 On The STAT3-VEGF Pathway In Th17 Cells Of Psoriatic And The Role Of Tacrolimus On It

Background:Psoriasis is a common chronic inflammatory disease,of which etiopathogenisis and pathogenesis have not been completely known yet.The main pathology of psoriasis are abnormal proliferation of keratinocytes,parakeratosis,angiogenesis and inflammatory cells infiltration.The pathogenesis of psoriasis is profusely investigated in the world focusing on abnormal proliferation of keratinocytes and disorder of immunologic system.However,immune disorder is more inclined to study relate in the main to the following area:Thl/Th2 imbalance.Recently,a new type of CD4+effect T cells Th17 cells different from Th1 cells and Th2 may play more important role in the pathogenesis of psoriasis.The pathogenesis of psoriasis is complicated involved in a variety of inflammatory cells,cytokines and growth factors and so on[1].In fact,maintaining the normal function of the basic biology mechanism is the transmission of information between cells,and the signal of defect can lead to a variety of abnormal hyperplastic diseases（such as tumor）[2].Thus,our research would concentrate on signal transduction to find the key mechanisms of psoriasis and give consideration to the other factors in order to fight with psoriasis in an effective and comprehensive way.It has been verified[3,4]that the abnormal activation of STAT3 can promote the differentiation and proliferation of cells,also restrain cell apoptosis.Ultimately,those changes should induce cells to proliferate abnormally and malignant transformation,both of which could result in diseases.Recently,the activated STAT3 which acts on the promoter of VEGF directly has been reported to up-regulate VEGF expression.Most of studies showed that STAT3-VEGF signal pathway was activated more frequently closely relating to the proliferation and metastasis of tumor cells.As a result,blocking the activation of the STAT3-VEGF pathway may become a targeted ablate therapy in cancer.Thus,we wonder if the strong expression of STAT3 and VEGF cause psoriasis?Do both of them work as the STAT3-VEGF pathways in cancer?It is reported that STAT3 protein highly expressed in the cell cytoplasm and middle of the nucleus in the lesions of patient with psoriasis,as well as in the serum.There was a significantly positive correlation between the levels of STAT3 and psoriasis[7,8].In short,STAT3 plays an important role in psoriasis.As we all know,VEGF is the most important effective media in angiogenesis.The levels of VEGF also increased in the lesions and serum of psoriasis.In addition,the abnormal proliferation of subcutaneous vascular and the increased vascular permeability in the patient with psoriasis could lead to lesions edema and the infiltration of inflammatory cells and aggravate the development of psoriasis.Therefore,the recurrent bouts of psoriasis were relevant to the proliferation of microvascular endothelial cells in the psoriasis lesions.Although most researches suggest that STAT3 and VEGF play an important role in psoriasis,many matters still need to be studied because of its complex mechanism.As we mentioned before,Th17 cells play an important role in the pathogenesis of psoriasis and provide a new idea to further study the pathogenesis and treatment of psoriasis.Visibly,STAT3,VEGF and Th17 all take part in the occurrence of psoriasis.Thus we suppose that there is a signal pathway stimulated by some factor and its activation cause some changes in Th17（such as the proliferation and dysfunction）to induce psoriasis.It is discovered that the development and function of Th17 was different from Th1,Th2 cells.Th17 cells express IL-17,IL-21,IL-22 highly,but also produce IL-6 and TNF-α etc[9].And the development of Th17 which was controlled by STAT3 needed the initial activation of IL-6,TGF-β and the maintenance of IL-23.At present,studies show that[10,11,31]STAT3 and VEGF were all involved in the differentiation and development of Th17 in other diseases,and influenced Th17 to excrete effect factors.Therefore,how are STAT3 and VEGF connected to Th17?Dose STAT3-VEGF pathway play a role in the proliferation and function of Th17?Those look worthy of further research.In order to discuss the correlation of STAT3 and VEGF with Th17,Th17 cells would be isolated from peripheral blood in the patient with psoriasis and culture in vitro.Three target sequences in STAT3 gene were designed and transfected into the Th17 by Lipofectamine2000.Then the expression of STAT3 and VEGF were compared to the best siRNA sequence for the follow-up experiments by fluorescence quantitative PCR and Western blot.As we all know,tacrolimus is a widely used T-cell-targeted immunosuppression drug known as a calcineurin inhibitor in organ transplantation.In recent years,topical tacrolimus therapy is effective in the patients with psoriasis,with only mild adverse effect.[33,34].However,its pharmacologic effects have not been fully elucidated.In transplantation,it was demonstrated that Tacrolimus could inhibit the differentiation and proliferation of Th17 cells and expression of IL-17 messenger RNA in order to reduce deeper inflammation.Low concentration of tacrolimus could prevent the original T cells from differentiating to Th1,Th2 and Th17 cells and reduce the production of IFN-γ,IL-4 and IL-17.In addition,tacrolimus still could inhibit the STAT3 phosphorylation,down-regulate the expression of VEGF expression.Consequently,we speculate that tacrolimus may restrain the differentiation of Th17 cells and the secretion of cytokines through affecting the STAT3-VEGF signaling pathway in psoriasis.Our previous studies have indicated that the expression of IL-22,IL-23 in Th17 cells showed the decreasing trend after application of tacrolimus and attained the lowest in 50 ng/ml;And the expression of STAT3 and VEG mRNA also descented,consistent in 50 ng/ml.Based on these results,we utilized the best siRNA sequence to inhibit STAT3 expression in Th17 cells incubated with 50ng/ml tacrolimus for 24h.Then the changes of STAT3 and VEGF were detected in order to further study whether tacrolimus showed treatment effect to psoriasis through adjusting the STAT3-VEGF pathway in Th17 cells.This study would play a significant role in knowing active site of tacrolimus and provide the theory basis in the study of the potential drug target on psoriasis related cell signal transduction pathway.Objective:Using RNAi-mediated gene silencing of STAT3 in the Th17 to observe the expression of STAT3 and VEGF mRNA as well as levels of STAT3 and VEGF protein and screen the best siRNA-STAT3 sequence.Th17 cells silenced with the optimal sequence were incubated with 50ng/ml tacrolimus for 24h.And then the mRNA and protein expressions of STAT3 and VEGF were detected.Methods:1.20ml blood sample from a patient with psoriasis were collected.And Th17 cells separated by flow cytometry were cultured in vitro.2.SiRNA-STAT3 construction and transfection:①STAT3mRNA human gene sequences were searched from CNKI（such as www.ncbi.nlm.nih.gov）using BLAST homology analysis software.②using 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-STAT3-001,siRNA-STAT3-002,siRNA-STAT3-003and negative control groups was established at the same time.④siRNA sequences were transfected into Th17cells by Lipo2000.3.Detection effects:the efficiency of transfection were estimated by detecting the expression of STAT3 and VEGF in order to screen the best siRNA sequence.4.Drug intervention:Th17 cells silenced with the optimal sequence were incubated with 50ng/ml tacrolimus for 24h.The expression of STAT3,VEGFmRNA were detected by RT-PCR and STAT3,VEGF protein expression detected by Western-Blot.Results:1.Our results showed that three different target siRNA-STAT3 sequences could inhibit the expression STAT3 and VEGF.Because of working best,the siRNA-STAT3-003 sequence was appointed to the follow-up experiments.2.The STAT3 and VEGF expression in tacrolimus group and Si-STAT3-003 group were all inhibited with no obvious difference.However,the expression of STAT3 and VEGF in tacrolimus combined with si-STAT3-003 group obviously decreased than that of other groups.Conclusion:1.This experiment showed that siRNA could effectively inhibit the expression of STAT3 mRNA and protein as well as levels of VEGF.The STAT3-VEGF pathway resided in Th17 and its activation may influence the proliferation and function of Th17.It was speculated that the STAT3-VEGF pathway was a critical point in psoriasis.STAT3 as a target for the treatment of psoriasis is feasible.2.The STAT3 and VEGF expression in tacrolimus group and Si-STAT3-003 group were all inhibited with no obvious difference suggested that tacrolimus may act through inhibiting the activation of STAT3-VEGF pathway in psoriasis.The expression of STAT3 and VEGF decreased significantly in tacrolimus combined with siRNA-STAT3-003 group than that of other groups.This indicated that tacrolimus may also inhibited the expression of STAT3 or VEGF through other pathways.This study would have significant roles in further understanding the effect of Th17 in the pathogenesis of psoriasis and the active site of tacrolimus.This study would also provide the theory basis in the study of the potential drug target on psoriasis related cell signal transduction pathway.Moreover,the preparation of Lipo2000-Si-STAT3-003 which could markedly decrease the expression of STAT3,VEGF mRNA and protein may be an important biological agent to treat psoriasis.Those look worthy of further research.