| Background Psoriasis is a kind of common chronic inflammatory skin disease with increasing incidence year by year. The histopathological changes of psoriasis include hyperplasia of the epidermis (acanthosis), abnomal differentiation of keratinocyte (KC), dilation and growth of dermal papilla capillaries, and infiltration of leukocytes into both the dermis and epidermis. Recent studies have shed light on the immunologic mechanism of psoriasis. Based on the studies of the characteristics of T cells in the skin lesions of psoriasis patients and their function of inducing cytokines, researchers found that a new regulatory T cell subset that is, Th17cells secrete cytokines of Interleukin-17A(IL-17A), IL-17F and IL-22, which play important roles in the pathogenesis of psoriasis. Of note, the expression of IL-22in skin lesions and plasma of psoriasis patients is increased significantly. IL-22is known to not only block the terminal differentiation of KC but also induce KC migration and the expression of proinflammatory genes. However, the cellular and molecular mechanism of KC effect mediated by IL-22is not well understood so far. Grb2-associated binder1/2(Gab1/2) are a kind of adaptor proteins which are involved in signal transduction of membrane surface receptor tyrosine kinase and non-receptor tyrosine kinase. Phosphorylated Gabl regulates the epidermal growth and differentiation induced by epidermal growth factor (EGF) through interaction with Src homology2domain-containing tyrosine phosphatase(Shp-2), but it is not known whether Gabl and Gab2, which are highly expressed in KC, paly roles in proliferation, migration and differention of KC induced by IL-22.Psoriasis belongs to categories of "baibi","snake lice","loose skin tinea" in Traditional Chinese Medicine (TCM).Clearing heat and detoxification, cooling blood and invigorating circulation are the important TCM therapeutic principles for the disease. Zicao tastes sweet and salty with cold properties, and has effects of cooling blood, invigorating circulation, detoxification, as well as promoting eruptions. Shikonin is one major effective components of Zicao with a wide range of bioactivities. It has high medicine values including anti-tumor, antibacterial, antiviral, sedation, heat-clearing and detoxifying, immunoregulation, healing wounds. Investigating the apoptosis mechanism of tumor cell induced by shikonin revealed that it inhibits the proliferation of tumor cells by regulating the activity of MAPK pathway and reducing the phosphorylation of Erk, Jnk, and Stat3. Though the effect of shikonin treatment for psoriasis is well demonstrated, the underlying mechanism remains largely unknown.We speculate that shikonin exerts it effect on the proliferation, differentiation and migration of KC by antagonizing IL-22-induced phosphorylation of Gab1/2on KC, which resulting in the down-regulation of the activation of Erkl/2/MAPK singnal pathway.ObjectivesPart one:To investigate the effect of shikonin on cell biological behaviour of HaCaT mediated by IL-221. Effect of shikonin on proliferation and migration of HaCaT cell stimulated by IL-22;2. Effect of shikonin on expression of proinflammatory genes S100A7, S100A8, S100A9mRNA stimulated by IL-22.Part two:The mechanisms study of the effect of shikonin on IL-22-mediated HaCaT biological behaviour.1. Detect the expression of proteins in Erkl/2/MAPK pathway in HaCaT stimulated by IL-22;2. Explore the expression of proteins in Erkl/2/MAPK pathway in HaCaT infected with adenovirus expressing Gab1and Gab2mutants (Shp2-binding defective);3. Explore the expression of proteins in Erk1/2/MAPK pathway in HaCaT with adeno virus expressing siRNA targeting Gab1and Gab2;4. Study on whether both Gabl and Gab2play roles in cell proliferation, migration and differentiation of HaCaT induced by IL-22;5. Study on the phosphorylation of Gabl, Gab2and Erkl/2stimulated by IL-22and the intervention of shikonin.Methods1. Detection of cell proliferation by WST-1assay2. Detection of cell migration with Transwell assay3. Detection of the expression of mRN A by Real-Time PCR4. Detection of the expression of proteins by Western blot assay5. Deterction of the interactions between specific proteins by immunoprecipitation6. Construction of recombinant adenovirus (Shp2-binding defective Gabl and Gabl, siRNA knockouted Gab1and Gab2, siRNA knockouted Gab1+Gab2)7. Statistical analysis with SPSS17.0software (difference has statistical significance when P<0.05)Results1. Shikonin inhibited the growth of HaCaT cells in a dose-dependent manner. It should be noted that low concentration of shikonin (0.1and0.25ug/ml) didn’t affect the cell activity and had no damage and toxicity to HaCaT cells.2. Shikonin treatment decreased HaCaT cells proliferation and migration mediated by IL-22.. The experiment about the proliferation and migration of HaCaT inhibited by shikonin is performed as following groups:blank control (without shikonin and IL-22), incubate with IL-22for24h, incubate with IL-22and shikonin for24h, incubate with IL-22for48h, and incubate with IL-22and shikonin for48h. The concentration of shikonin and IL-22were0.25ug/ml and100ng/ml, respectively. After adding IL-22for24h and48h, the proliferation and migration of HaCaT were all increased (P<0.05), however these effects were inhibited by adding the shikonin (P<0.05). 3. Shikonin down-regulated the expression of S100A7, S100A8and S100A9induced by IL-22. We detected the effect of shikonin on the expression of S100A7, S100A8and S100A9mRNA induced by IL-22by Real-Time PCR. The results showed that2-△△Ct values of S100A7and S100A8induced by IL-22were all more than2, while adding shikonin, the2-△△Ct values were less than1, that indicates shikonin can down-regulate the mRNA expression of S100A7, S100A8induced by IL-22in HaCaT, but has no influence on the expression of S100A9.4. IL-22induced the activation of Jakl/Tyk2/Stat and MAPK pathway. HaCaT cells were serum starved for16-18h, and then stimuilated with or without IL-22(100ng/ml) for10min,20min,30min,60min and120min.and total cells lysates were analyzed by Western blot, The results of Western blot showed that Stat3Tyr705, Stat Ser727, Jakl and Tyk2in Jakl/Tyk2/Stat pathway and Erkl/2and p38in MAPK pathway were phosphorylated after10min stimulation by IL-22. The activation of Erkl/2, the proliferation and migration of HaCa T cells induced by IL-22were suppressed when the HaCaT cells were pretreated with Mek inhibitor PD98059(10μM) for1h. However, Loricrin, the marker protein of the differentiation of KC, was still not detected at72h after the stimulation of IL-22, while Loricrin expression of HaCaT was increased significantly when PD98059was added before IL-22stimulation.5. IL-22induced the phosphorylation of Gabl and Gab2, and then activated Erkl/2. After the stimulation of serum-starved HaCaT cells with IL-22for10min, co-immunoprecipitation and Western blot assays showed that the phosphorylation of Gabl and Gab2increased markedly and the binding with Shp2was also enhanced. The activation of Erkl/2and Stat3Tyr795were tested after the HaCaT cells were infected by recombinant adenovirus AdGab1EF, AdGab2F, AdGab1WT, AdGab2WT. The results showed that there was a marked decrease in the phosphorylation of Erkl/2in groups of (AdGablEF) HaCaT and (AdGab2F) HaCaT, while the phosphorylation of Stat3Tyr795in groups of (AdGab2F) HaCaT,(AdGab1WT) HaCaT and (AdGab2WT) HaCaT had no change. Besides, the activations of Erkl/2and Stat3Tyr795in (AdGablsiRNA) HaCaT,(AdGab2siRNA) HaCaT and (AdGablsiRNA+AdGab2siRNA) HaCaT were also tested. The results showed that the phosphorylation of Erkl/2all decreased in the three groups, but that of Stat3Tyr795still had no markedly changes.6. The influence of Gab1and Gab2on the proliferation, migration and differentiation of HaCaT induced by IL-22is related with Shp2. The cell proliferation rate and migration were detected after the stimulation of(AdGab1WT) HaCaT,(AdGab2WT) HaCaT,(AdGab1FF) HaCaT and (AdGab2F) HaCaT by IL-22for24h and48h. The results showed that the proliferation rate and migration cells of (AdGablFF) HaCaT and (AdGab2F) HaCaT all reduced (P<0.01). Similar results were obtained in (AdGab1siRNA) HaCaT、(AdGab2siRNA) HaCaT and (AdGab1siRNA+AdGab2siRNA) HaCaT. Compaired with the (AdU6Vector) HaCaT, cells proliferation rate decreased in the experimental groups within24h and48h(P<0.05and P<0.01, respectively);migration cells also decreaed(both P<0.05);Stimulating adenovirus-infected HaCaT with IL-22for72h, we observed that the expression of Loricin in mutant and silent taget gene groups were higher than that in wild type groups or the control virus.7. Shikonin inhibited IL-22-induced phosphorylation of Gab1, Gab2and Erkl/2on KC. The levels of phosphorylation of Gabl, Gab2and Erkl/2in shikonin-treated HaCaT cells upon IL-22stimulation were lower than in the groups without shikonin.Conclusions1. Shikonin inhibited the proliferation and migration of HaCaT cells and reduced the expression of S100A7and S100A8mRNA in HaCaT cells induced by IL-22.2. IL-22induced the activation of Jakl/Tyk2/Stat and MAPK pathways in HaCaT cells and mediated the cell proliferation, migration and differentiation.3. Gabl and Gab2participated jointly in IL-22mediated keratinocyte proliferation, migration and differentiation mainly via activation of Erk/MAPK pathway.4. Shikonin inhibited the phosphorylation of Gabl, Gab2and Erkl/2induced by IL-22, suggesting that the mechanism of shikonin treatment for psoriasis may be related with the inhibition of activation of Gabl, Gab2and futhur down-regulated the activation of Erkl/2/MAPK pathway. |
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