Therapy Targets Of Curcumin On Proliferation Of HaCaT Cells And Role Of Curcumin On TPA Induced Psoriasis Model

Psoriasis vulgaris is a chronic skin disease clinically characterized by recurrentepisodes of red and scaly skin plaques that are sharply demarcated from adjacent normalskin. Although the lesions often appear in areas susceptible to epidermal trauma such aselbows and knees, all parts of the body may be affected in the late stage. Since the lesionsoften combined with itch and appear striking and recurrent, it reduced the life qulity andloaded a big burden on financial and psychological to patients. Since the pathogenesis ofpsoriasis has not been fully unstood so far, it was seemed that as a chornic inflammtorydisease, immunocytes take an important role for a long time. It was accepted that psoriasiswas the result of combined action of innate immuocyte, activated T cells and functionallydisregulated keratinocytes, with IL-23/IL-17/IL-22axis takes the pivotal role in thepathogenesis. Currently treatment for psoriasis vulgaris was foucsed onimmunosuppressive agents (eg: Cyclosporine, methotrexate, tacrolimus, acitretin), topicalagents(vitamin D,steroids) or UVB phototherapy. Biological therapies are now routinelyused when traditional systemic agents fail, or not tolerated, or unsuitable owing tocomorbid conditions. The current biological agents approved by the U.S. Food and DrugAdministration are alefacept, infliximab, etanercept, adalimumab, golimumab, andustekinumab. Targeted therapy made them clinical efficiency. However, the high expenseand relapse after withdrawal forced us to look for more safely, high effiency and economicdrugs targeting for psoriasis.Curcumin is the yellow powder extract from the Zingiberaceae tumetric. More andmore attentions were paid due to its anti-inflammtory, anti-oxidation, anti-tumor and highsecurity. It was used widly used for cancer therapy including Cron’s disease, breast cancer,multiple myeloma because its limited water solubility and oral bioavailability. Two clinicalstudies have been initiated for treating psoriasis. However, the low oral bioavailabilitycurbed its useage. Studies on structure and formulation modification to increase watersolubility and bioavailability of curcumin were the main force.The aim of this study was to use a topical formulation of curcumin and investigate theeffects and mechanism of curcumin on K14-VEGFtransgene mice model of psoriasis.There were three parts in this study:Part1: Curcumin inhibited proliferation in Interleukin-22treated HaCaT cells IL-22was significantly elevated in lesions of psoriasis. And the main effect was toinduce proliferation and inhibit differentiation of keratinocytes and finally made epidermalhyperplasia, up-regulate proteins of S100A7, S100A8in KC to increase anti-beactirea ofpeidermal. Besides, IL-22also induced KC to secrete IL-20, which strengthened effects ofIL-22. There were four groups in this study: control group, curcumin treatment group, IL-22treatment group and IL-22plus curcumin treatment group.First, growth of HaCaT was invested under the regulation of IL-22(50ng/ml and100ng/ml) and curcumin. After stimulated HaCaT cells with IL-22for2h, curcumin was usedto stimulate afterwards. The OD value of living cells was recorded with CCK-8kit every6h to record the growth of cells under each stimulation. Curcumin inhibited proliferation ofHaCaT cells with the induction of IL-22or not.Second, the mechanism of curcumin restrained IL-22induced proliferation of HaCaTwas invested. Since the main effects of IL-22on KC was mediated by JAK-STAT3pathway, level of p-STAT3was measured under different treatment with western-blotanalysis. Activation of STAT3was supressed by curcumin.Next, since cyclins have close relationship with growth of cells, real-time PCR andwestern-blot was used to detect expression of cyclin D1and cyclin E in different group.Results showed that curcumin restrained expression of cyclin D1and cyclin E. Expressionsof both proteins had no significant variation under stimmulation of IL-22compared withexpression of control.Finally, expressions of IL-20were investigated with real-time PCR and ELISA undereach induction. Curcumin and IL-22had no significant influence on mRNA of IL-20whilecurcumin upregulated protein of IL-20combined with IL-22.Conclusion: curcumin exhibited a significant anti-proliferation effect on HaCaT cells nomatter with the stimulation of IL-22or not.. Curcumin inhibited IL-22induced activationof STAT3. In addition, curcumin down-regulated expression of cyclin D1and cyclin E,which may blocked cell cycle progression from IL-22induced cells. However, curcumininduced the production of IL-20when combined with IL-22.Part2: To study the expression of IL-22in keratinocytes under the induction of IL-17Aand IL-17F individually or they combined with IFN-γ、TNF-α、IL-6respectively.Expression of IL-17A and IL-17F secreted from dermal γδT cell was elevated underinduction of IL-23in another our study with the therapy of curcumin onimiquimod-induced psoriasis in mice. Expression of IL-22secreted from the same cells was also elevated significantly. However, the fold of increasement of IL-22was ten timesand even hundred of times higher than that of IL-17A and/or IL-17F. Since IL-22andIL-20belonged to the same family and IL-20can be produced by KC under the inductionof IL-22, thus we infer that maybe IL-22could also be produced by KC under induction ofIL-17F or IL-17A or IL-17A/IL-17F combined with INF-γ、TNF-α、IL-6. Becauseexpressions of INF-γ、TNF-α、IL-6were also elevated in psoriasis lesions. So thefollowing researches were performed.Frist, real-time PCR was carried out to test whether IL-22could be expressed underinduction of IL-17A or IL-17F respectively. Melting curves and cycling curves showedthat there were no expressions of IL-22under such irritation.Second, real-time PCR was carried out to test whether IL-22could be expressed underinduction of IL-17A or IL-17F combined with INF-γ、TNF-α、IL-6. Melting curves andcycling curves also showed that there were no expression of IL-22under such irritations.Last, ELISA was used to test the concentration of IL-22in the supernatant of cellculter sysytem. IL-17A/IL-17F combined with INF-γ、TNF-α、IL-6was used to stimulateHaCaT cells. Results showed that expression of IL-22was quite low in all treatmentgroups and there was no difference in concentration of IL-22between the control groupand treatment groups.Conclusion: IL-22could not be produced by KC neither under induction ofIL-17A/IL-17F singly nor under induction of IL-17A/IL-17F combined with INF-γ, TNF-α,IL-6.Part3: Therapy of curcumin on TPA-induced psoriasis-like response in transgenicK14-VEGF miceDifferences of physiological baseline between wild-type mice and k14-VEGFtrangene mice were compared in the first part of this study: ear skin morphology andhistological features in both types of mice were recorded using digital camera and H&Estaining respectively. Expressions of cytokines in the IL-23/IL-17/IL-22axis (such asIL-23, IL-17A, IL-17F, IL-22, IL-21, TNF, VEGF, IL-6) in both types were tested usingreal-time PCR. Besides, expression and distribution of γδT in the ear skin in both types ofmice were recorded by western-blot and immunohistochemisty.Results showed that ear skin of transgenic mice displayed slight erythema, clear bloodvessel and incresed thickness. Analysis of H&E-stained section from the transgenic miceskin showed increased epidermal thickeness in ear skin. Such acanthosis was partly caused by hyperproliferation of keratinocytes. However, edema was also occurred in the ear skinof K14-VEGF trangene mice which partly result in ear thicken. Results from real-timePCR showed that there were no differences between the wild type mice and transgenicmice in IL-23/IL-17/IL-22axis cytokines. Results from immunohistochemisty showed thatthere were no differences between wild-tpye and transgenic mice in expression of dermalγδTcell.Based on comparations the difference between wild-type and K14-VEGF mice,mechanism and effects of curcumin on TPA induced psoriasis in K14-VEGF transgenemice was performed. There were five groups in this part of study: control group, TPAtreatment group, curcumin treatment group, curcumin and TPA treatment group, clobetasolpropionate and TPA treatment group. The studies were as follows:First, skin of ear thickness and inflammtory scores were recordrd at day0,2,4,6,8,10,12in each group. Morphology and H&E were used to record modification of ear skinin histology. Psoriasis-like symptoms were foud in skin of TPA treated mice, curcumin andclobetasol propionate reduced the syptoms.Second, Results from immunohistochemisty and western-blot showed that quantity ofIL-17-producing δγT cells was unchanged while δγT cells from epidermal significantlyincreased under induction of TPA. And curcumin could not relieve the enhancement ofepidermal δγT cells.Third, mRNAs of cytokines in the IL-23/IL-17/IL-22axis were recorded usingreal-time PCR in each treatment group. Expressions of cytokines from this axis were notchanged after TPA and curcumin treatment. However, expression of VEGF induced byTPA was upregulated by curcumin.Last, to clear the pathogenesis of TPA induced psoriasis-like syptoms in K14-VEGFtransgene mice, skin biospy collected at9d after each treatment were used for real-timePCR to test expression of cytokines in IL-12/IL-27/Th1/IFN-γ axis. Results showed thatexpression of IL-27, IFN-γ, CXCL9and CXCL10were significantly elevated after TPAinduction while expressions of these cytokine were potentially reduced after curcumintreatment.Conclusion: TPA induced psoriasis-like sypmtoms in K14-VEGF mice were neithermediated by IL-23/IL-17/IL-22axis nor by proliferated γδT cells. It was likely adelayd-type hypersensitivity reaction which Th1cells took the pivotal role. And curcuminalleviated inflammatory reactions mainly by inhibited expression of IFN-γ and IFN-γ induced CXCL9and CXCL10. What’s more, curcumin up-regulated the expression ofVEGF when used to anti-TPA-induced inflammation in K14-VEGF transgenic mice.Maybe curcumin inhibited the signaling pathway after VEGF combined with VEGFreceptor on the one side. On the other side, receptors of VEGF may decrease afterexpression of VEGF increased as a negative feedback. Anyhow, curcumin could efficientlycurbed TPA induced psoriasis-like symptoms in K14-VEGF trangene mice.