姜黄素影响HaCaT细胞增殖的靶点及对TPA诱导的银屑病小鼠模型的作用研究
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摘要
寻常型银屑病是一种常见的慢性皮肤病，临床表现为与周围皮肤界限分明的红色隆起，表面常带有白色小半鞘翅。因其发病部位常分布在全身各处且伴随剧烈瘙痒，严重影响外观和患者的生活质量。银屑病又极易反复发作，因此给患者带来极大的生理、心理和经济负担。尽管银屑病在我国的发病率较欧美地区低，但总体患病人数较多。长期以来人们认为银屑病是一种免疫细胞介导的慢性炎症性疾病，但银屑病具体的发病机制还不甚清楚，目前主要认为是以IL-23、IL-17A、IL-17F、IL-22、TNF-α、IFN-γ、IL-1β等IL-23/IL-17/IL-22细胞因子轴为中心、固有免疫细胞和活化的T细胞参与、功能失调的角质形成细胞共同作用的结果。目前针对寻常型银屑病的治疗主要集中免疫抑制剂（如：环孢菌素、甲氨蝶呤、阿维A、他克莫司等）、激素治疗（丙酸倍他米松、维A酸类、丙酸氯倍他索等）、窄谱光照射疗法等，由于其副作用及不良反应因而限制了其应用。针对银屑病发病机制中较关键的炎症因子infliximab、etanercept、adalimumab等生物制剂由于其靶向性更强等取得了很好的临床疗效，但由于其价格昂贵且停药后易复发等缺点，迫使人们继续寻找新的安全、有效、经济的抗银屑病药物。
姜黄素是从姜科植物姜黄中提取的黄色粉末，由于其抗炎、抗氧化、抗肿瘤等广泛的药理活性及安全性等越来越受到人们的关注。但由于其水溶性较差，口服生物利用度很低等缺点，目前主要用于克罗恩氏病、乳腺癌等的研究。很早也有关于姜黄素用于银屑病的临床研究，可以有效改善银屑病患者皮损症状，但该实验样本量小且缺乏有效的对照等缺点，主要是由于姜黄素口服生物利用度较低。但为姜黄素皮肤局部给药治疗银屑病提供了理论支持。目前人们对姜黄素研究的热点是将其结构改造后增加水溶性进而对其药效学进行考察，或制成新剂型以改善其生物利用度。
本课题的研究目的是利用姜黄素凝胶，通过经皮给药的方式考察姜黄素对银屑病小鼠模型的作用及其机制。
本课题主要进行了以下研究内容：第一部分：姜黄素对IL‐22诱导下角质形成细胞增殖的影响及机制研究
银屑病时IL-22大量增加，主要作用为刺激角质过度增殖并抑制其分化，最终引起表皮增厚；另一方面还可以刺激角质形成细胞(KC)产生炎症因子IL-20，和IL-22发挥协同作用，加重皮肤炎症状况；同时上调抗菌蛋白S100A7、S100A8、S100A9等使银屑病皮肤抗菌性增强。
1、本部分研究首先考察姜黄素对IL-22诱导永生化的人角质形成细胞(HaCaT)生长状况的影响，分别利用20ng/ml和100ng/ml的IL-22刺激HaCaT细胞2h后，再利用姜黄素刺激。每隔6h通过CCK-8测活细胞吸光度，记录各处理组细胞吸光度从而记录细胞生长曲线。无论IL-22是否存在，姜黄素均可以抑制HaCaT的增殖。
2、探讨了姜黄素抑制细胞增殖的机制：IL-22与KC上的受体结合后，主要通过激活JAK-STAT3通路发挥作用。本研究利用western-blot检测了不同处理组STAT3的磷酸化水平，发现姜黄素不仅强烈抑制了IL-22诱导的STAT3磷酸化，而且几乎完全抑制了正常细胞内的磷酸化。
3、周期蛋白是调控细胞增殖重要的内部因素。本研究利用real-time PCR检测了姜黄素作用2h和6h后周期蛋白cyclin D1和cyclin E在各处理组中的表达。此外还利用western-blot检测了姜黄素作用24h后不同处理组中cyclin D1和cyclin E的变化。结果显示，与空白组相比，IL-22对周期蛋白的影响不显著，但姜黄素在核酸和蛋白水平上均显著抑制了IL-22诱导后周期蛋白cyclin D1和cyclin E的表达。
4、考察了姜黄素对IL-22诱导的IL-20影响：利用real-time PCR对姜黄素作用6h后对IL-20mRNA的表达做出检测；同时利用ELISA对姜黄素作用24h后IL-20的量进行了考察。结果显示，与空白组相比，姜黄素和IL-22在核酸水平对IL-22的影响不显著，但姜黄素在蛋白水平上调了IL-22诱导下IL-20的表达。
结论：姜黄素可以抑制IL-22诱导下HaCaT细胞的增殖，其作用可能表现为一方面通过抑制STAT3磷酸化，另一方面可能是通过抑制PI3K通路，下调了周期蛋白Cyclin D1和Cyclin E的表达。第二部分：通过IL-17F、IL-17A单用或联合IFN-γ、TNF-α、IL-6刺激角质形成细胞，研究其对IL-22表达的诱导作用
在本课题组另一部分实验——姜黄素对咪喹莫特诱导的银屑病小鼠的治疗作用及其机制时发现，咪喹莫特刺激小鼠时，在IL-23的诱导下，来源于真皮δγT细胞的IL-17A、IL-17F表达上调，同样主要来源于δγT细胞的IL-22表达明显上调，且表达量是IL-17A和IL-17F的数十倍甚至百倍。因此，推测IL-17A或IL-17F是否刺激了其它的细胞（如KC）产生IL-22，进而将炎症反应级联放大？由于此模型中INF-γ、TNF-α、IL-6也高表达，是否存在IL-17A/IL-17F在INF-γ、TNF-α、IL-6的协调作用下刺激KC产生了大量的IL-22的可能？为此本课题进行了如下实验：
1、利用real-time PCR考察IL-17A或IL-17F单独刺激KC时，是否引起IL-22基因表达上调。结果显示，IL-17A和IL-17F单独作用时，IL-22的表达和空白组相比无差异，均没有IL-22基因的扩增。
2、利用real-time PCR考察IL-17A或IL-17F联合INF-γ、TNF-α、IL-6刺激KC时，是否能够引起IL-22基因表达上调。结果显示，以上刺激因素均不能引起IL-22目的基因的扩增，不能产生IL-22。
3、利用ELISA分别对IL-17A、IL-17F联合INF-γ、TNF-α、IL-6作用24h后的KC细胞上清进行收集，检测是否有IL-22的产生。结果显示，与空白组相比，两种条件刺激下IL-22的蛋白水平和空白组基本无差异，且量很少，可以认为IL-17A、IL-17F联合INF-γ、TNF-α、IL-6作用于KC时，不能引起IL-22的表达。
结论：IL-17A或IL-17F单独或联合INF-γ、TNF-α、IL-6刺激KC不能产生IL-22。第三部分：姜黄素对TPA诱导的K14-VEGF银屑病小鼠模型的影响及作用机制研究
本部分研究首先比较了野生型和6-8周转基因小鼠生理基线的差异：利用数码相机记录小鼠耳部皮肤形态学；数字测厚仪测量并记录野生型和转基因小鼠的耳厚；通过HE染色对两种小鼠耳部皮肤组织学特征进行比较；采用real-time PCR对两种小鼠耳部皮肤中IL-23/IL17/IL-22作用轴上的一系列细胞因子进行了考察，此外，还利用western-blot和免疫组化对γδT细胞在两种小鼠耳部皮肤中的分布、及真皮中的表达进行了考察。
与野生型小鼠相比，转基因小鼠耳部皮肤自发产生红肿、血管明显、增厚。HE染色发现转基因小鼠由于表皮基底层轻微增殖而增厚、皮下组织增生，同时伴有部分炎症细胞浸润和水肿。Real-time PCR考察结果显示，除IL-23外，转基因小鼠和野生型小鼠在其它细胞因子（如：IL-17A、IL-17F、IL-22、IL-21、TNF、IL-6、VEGF等）水平上无差异，但western-blot结果表明，转基因小鼠VEGF表达明显增强，真皮中γδT细胞特异性标记物CCR6无明显差异。免疫组化结果显示，γδT细胞在两种小鼠耳部皮肤中的分布及表达无差异。
结论：由于周龄较小，K14-VEGF转基因小鼠VEGF表达明显增强，但由于缺乏IL-1β的联合刺激，真皮中的γδT细胞并未增殖活化，因此IL-6、TNF、IL-17等还未高表达，和野生型小鼠相比，6-8周转基因小鼠只出现了轻微的表皮增厚，并未有显著的组织学变化，与以往报导的研究一致。
在对野生型和转基因小鼠比较的基础上，本课题考察了姜黄素对TPA诱导的K14-VEGF银屑病小鼠模型的影响及作用机制研究。此部分实验分为5组：转基因小鼠空白对照组、TPA模型诱导组、姜黄素单独作用组、姜黄素治疗组、丙酸氯倍他索治疗组。各组按以下内容进行：
1、在实验的第0、2、4、6、8、10、12天测量、记录小鼠耳厚，并对各处理组小鼠耳部皮肤进行炎症评分。在实验进行的第12天利用数码相机记录不同处理组间小鼠耳部皮肤的形态学差异。通过HE染色对不同处理组的小鼠耳部皮肤组织学变化进行分析。结果显示，TPA作用于K14-VEGF转基因小鼠耳部皮肤后，诱导其产生了红肿、增厚、鳞屑、血管明显等银屑病样症状，姜黄素则逆转了上述银屑病样症状。HE染色表明，TPA诱导转基因小鼠产生表皮增厚、水肿、大量炎症细胞浸润等，姜黄素和丙酸氯倍他索减轻了以上症状。
2、利用免疫组化和western-blot考察不同处理因素对小鼠耳部皮肤中δγT细胞的影响，主要采用标记CCR6记录各处理因素对真皮中δγT的影响；利用western-blot检测δγTCR和RORγ来考察δγT整体水平的变化。免疫组化和western-blot结果表明，TPA促进了δγT细胞的增殖，但真皮中的CCR6+δγT细胞增殖并不显著，表皮中的δγT大量增加，姜黄素和丙酸氯倍他索对TPA诱导的表皮δγT增殖无显著影响。
3、利用real-time PCR对各处理组小鼠耳部皮肤中的炎症因子IL-23、IL-1β、IL-17A、 IL-17F、IL-22、TNF-α、IL-6、IL-21、VEGF等进行考察，探明姜黄素的作用机制。结果表明TPA并不通过激活IL-23/IL-17/IL-22作用轴诱导转基因小鼠产生银屑病样特征，姜黄素也不通过此通路发挥作用,但姜黄素不能抑制VEGF的高表达。
4、为进一步探明TPA诱导K14-VEGF转基因小鼠产生银屑病的机制及姜黄素对其治疗机制，利用real-time PCR对各处理组小鼠耳部皮肤中炎症因子IL-12、IL-27、IFN-γ、IL-2、CXCL9、CXCL10的变化进行了考察。结果表明，TPA诱导下Th1型上游炎症因子IL-27及下游炎症因子IFN-γ、及相应趋化因子CXCL9、CXCL10高表达，而姜黄素和丙酸氯倍他索则显著下调了TPA诱导的以上炎症因子的上调。
结论：TPA可以诱导短周龄K14-VEGF转基因小鼠出现银屑病样特征，但这种作用并不依赖于δγT的增殖和激活，此外，IL-23/IL-17/IL-22作用轴上一系列细胞因子并不参与此炎症过程。TPA诱导K14-VEGF转基因小鼠出现银屑病样症状是TPA作用于皮肤后，通过激活IL-27/Th1/IFN-γ通路诱导小鼠产生了长期的迟发型超敏反应，产生大量的IFN-γ、CXCL9、CXCL10等Th1型炎症因子，与VEGF慢性过表达造成的血管炎性反应相互作用，造成了银屑病样症状。姜黄素有效减轻银屑病样症状可能是通过灭活IL-27诱导下Th1的活化和TPA诱导下蛋白激酶C的活化从而发挥作用，在对抗TPA作用于K14/VEGF转基因小鼠时可以进一步诱导VEGF高表达，但一方面可以通过阻断VEGF与受体结合后激活的下游信号通路，另一方面通过负反馈机制下调了VEGFR的表达，从而阻断VEGF的作用。
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 mice
Differences 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.
姜黄素是从姜科植物姜黄中提取的黄色粉末，由于其抗炎、抗氧化、抗肿瘤等广泛的药理活性及安全性等越来越受到人们的关注。但由于其水溶性较差，口服生物利用度很低等缺点，目前主要用于克罗恩氏病、乳腺癌等的研究。很早也有关于姜黄素用于银屑病的临床研究，可以有效改善银屑病患者皮损症状，但该实验样本量小且缺乏有效的对照等缺点，主要是由于姜黄素口服生物利用度较低。但为姜黄素皮肤局部给药治疗银屑病提供了理论支持。目前人们对姜黄素研究的热点是将其结构改造后增加水溶性进而对其药效学进行考察，或制成新剂型以改善其生物利用度。
本课题的研究目的是利用姜黄素凝胶，通过经皮给药的方式考察姜黄素对银屑病小鼠模型的作用及其机制。
本课题主要进行了以下研究内容：第一部分：姜黄素对IL‐22诱导下角质形成细胞增殖的影响及机制研究
银屑病时IL-22大量增加，主要作用为刺激角质过度增殖并抑制其分化，最终引起表皮增厚；另一方面还可以刺激角质形成细胞(KC)产生炎症因子IL-20，和IL-22发挥协同作用，加重皮肤炎症状况；同时上调抗菌蛋白S100A7、S100A8、S100A9等使银屑病皮肤抗菌性增强。
1、本部分研究首先考察姜黄素对IL-22诱导永生化的人角质形成细胞(HaCaT)生长状况的影响，分别利用20ng/ml和100ng/ml的IL-22刺激HaCaT细胞2h后，再利用姜黄素刺激。每隔6h通过CCK-8测活细胞吸光度，记录各处理组细胞吸光度从而记录细胞生长曲线。无论IL-22是否存在，姜黄素均可以抑制HaCaT的增殖。
2、探讨了姜黄素抑制细胞增殖的机制：IL-22与KC上的受体结合后，主要通过激活JAK-STAT3通路发挥作用。本研究利用western-blot检测了不同处理组STAT3的磷酸化水平，发现姜黄素不仅强烈抑制了IL-22诱导的STAT3磷酸化，而且几乎完全抑制了正常细胞内的磷酸化。
3、周期蛋白是调控细胞增殖重要的内部因素。本研究利用real-time PCR检测了姜黄素作用2h和6h后周期蛋白cyclin D1和cyclin E在各处理组中的表达。此外还利用western-blot检测了姜黄素作用24h后不同处理组中cyclin D1和cyclin E的变化。结果显示，与空白组相比，IL-22对周期蛋白的影响不显著，但姜黄素在核酸和蛋白水平上均显著抑制了IL-22诱导后周期蛋白cyclin D1和cyclin E的表达。
4、考察了姜黄素对IL-22诱导的IL-20影响：利用real-time PCR对姜黄素作用6h后对IL-20mRNA的表达做出检测；同时利用ELISA对姜黄素作用24h后IL-20的量进行了考察。结果显示，与空白组相比，姜黄素和IL-22在核酸水平对IL-22的影响不显著，但姜黄素在蛋白水平上调了IL-22诱导下IL-20的表达。
结论：姜黄素可以抑制IL-22诱导下HaCaT细胞的增殖，其作用可能表现为一方面通过抑制STAT3磷酸化，另一方面可能是通过抑制PI3K通路，下调了周期蛋白Cyclin D1和Cyclin E的表达。第二部分：通过IL-17F、IL-17A单用或联合IFN-γ、TNF-α、IL-6刺激角质形成细胞，研究其对IL-22表达的诱导作用
在本课题组另一部分实验——姜黄素对咪喹莫特诱导的银屑病小鼠的治疗作用及其机制时发现，咪喹莫特刺激小鼠时，在IL-23的诱导下，来源于真皮δγT细胞的IL-17A、IL-17F表达上调，同样主要来源于δγT细胞的IL-22表达明显上调，且表达量是IL-17A和IL-17F的数十倍甚至百倍。因此，推测IL-17A或IL-17F是否刺激了其它的细胞（如KC）产生IL-22，进而将炎症反应级联放大？由于此模型中INF-γ、TNF-α、IL-6也高表达，是否存在IL-17A/IL-17F在INF-γ、TNF-α、IL-6的协调作用下刺激KC产生了大量的IL-22的可能？为此本课题进行了如下实验：
1、利用real-time PCR考察IL-17A或IL-17F单独刺激KC时，是否引起IL-22基因表达上调。结果显示，IL-17A和IL-17F单独作用时，IL-22的表达和空白组相比无差异，均没有IL-22基因的扩增。
2、利用real-time PCR考察IL-17A或IL-17F联合INF-γ、TNF-α、IL-6刺激KC时，是否能够引起IL-22基因表达上调。结果显示，以上刺激因素均不能引起IL-22目的基因的扩增，不能产生IL-22。
3、利用ELISA分别对IL-17A、IL-17F联合INF-γ、TNF-α、IL-6作用24h后的KC细胞上清进行收集，检测是否有IL-22的产生。结果显示，与空白组相比，两种条件刺激下IL-22的蛋白水平和空白组基本无差异，且量很少，可以认为IL-17A、IL-17F联合INF-γ、TNF-α、IL-6作用于KC时，不能引起IL-22的表达。
结论：IL-17A或IL-17F单独或联合INF-γ、TNF-α、IL-6刺激KC不能产生IL-22。第三部分：姜黄素对TPA诱导的K14-VEGF银屑病小鼠模型的影响及作用机制研究
本部分研究首先比较了野生型和6-8周转基因小鼠生理基线的差异：利用数码相机记录小鼠耳部皮肤形态学；数字测厚仪测量并记录野生型和转基因小鼠的耳厚；通过HE染色对两种小鼠耳部皮肤组织学特征进行比较；采用real-time PCR对两种小鼠耳部皮肤中IL-23/IL17/IL-22作用轴上的一系列细胞因子进行了考察，此外，还利用western-blot和免疫组化对γδT细胞在两种小鼠耳部皮肤中的分布、及真皮中的表达进行了考察。
与野生型小鼠相比，转基因小鼠耳部皮肤自发产生红肿、血管明显、增厚。HE染色发现转基因小鼠由于表皮基底层轻微增殖而增厚、皮下组织增生，同时伴有部分炎症细胞浸润和水肿。Real-time PCR考察结果显示，除IL-23外，转基因小鼠和野生型小鼠在其它细胞因子（如：IL-17A、IL-17F、IL-22、IL-21、TNF、IL-6、VEGF等）水平上无差异，但western-blot结果表明，转基因小鼠VEGF表达明显增强，真皮中γδT细胞特异性标记物CCR6无明显差异。免疫组化结果显示，γδT细胞在两种小鼠耳部皮肤中的分布及表达无差异。
结论：由于周龄较小，K14-VEGF转基因小鼠VEGF表达明显增强，但由于缺乏IL-1β的联合刺激，真皮中的γδT细胞并未增殖活化，因此IL-6、TNF、IL-17等还未高表达，和野生型小鼠相比，6-8周转基因小鼠只出现了轻微的表皮增厚，并未有显著的组织学变化，与以往报导的研究一致。
在对野生型和转基因小鼠比较的基础上，本课题考察了姜黄素对TPA诱导的K14-VEGF银屑病小鼠模型的影响及作用机制研究。此部分实验分为5组：转基因小鼠空白对照组、TPA模型诱导组、姜黄素单独作用组、姜黄素治疗组、丙酸氯倍他索治疗组。各组按以下内容进行：
1、在实验的第0、2、4、6、8、10、12天测量、记录小鼠耳厚，并对各处理组小鼠耳部皮肤进行炎症评分。在实验进行的第12天利用数码相机记录不同处理组间小鼠耳部皮肤的形态学差异。通过HE染色对不同处理组的小鼠耳部皮肤组织学变化进行分析。结果显示，TPA作用于K14-VEGF转基因小鼠耳部皮肤后，诱导其产生了红肿、增厚、鳞屑、血管明显等银屑病样症状，姜黄素则逆转了上述银屑病样症状。HE染色表明，TPA诱导转基因小鼠产生表皮增厚、水肿、大量炎症细胞浸润等，姜黄素和丙酸氯倍他索减轻了以上症状。
2、利用免疫组化和western-blot考察不同处理因素对小鼠耳部皮肤中δγT细胞的影响，主要采用标记CCR6记录各处理因素对真皮中δγT的影响；利用western-blot检测δγTCR和RORγ来考察δγT整体水平的变化。免疫组化和western-blot结果表明，TPA促进了δγT细胞的增殖，但真皮中的CCR6+δγT细胞增殖并不显著，表皮中的δγT大量增加，姜黄素和丙酸氯倍他索对TPA诱导的表皮δγT增殖无显著影响。
3、利用real-time PCR对各处理组小鼠耳部皮肤中的炎症因子IL-23、IL-1β、IL-17A、 IL-17F、IL-22、TNF-α、IL-6、IL-21、VEGF等进行考察，探明姜黄素的作用机制。结果表明TPA并不通过激活IL-23/IL-17/IL-22作用轴诱导转基因小鼠产生银屑病样特征，姜黄素也不通过此通路发挥作用,但姜黄素不能抑制VEGF的高表达。
4、为进一步探明TPA诱导K14-VEGF转基因小鼠产生银屑病的机制及姜黄素对其治疗机制，利用real-time PCR对各处理组小鼠耳部皮肤中炎症因子IL-12、IL-27、IFN-γ、IL-2、CXCL9、CXCL10的变化进行了考察。结果表明，TPA诱导下Th1型上游炎症因子IL-27及下游炎症因子IFN-γ、及相应趋化因子CXCL9、CXCL10高表达，而姜黄素和丙酸氯倍他索则显著下调了TPA诱导的以上炎症因子的上调。
结论：TPA可以诱导短周龄K14-VEGF转基因小鼠出现银屑病样特征，但这种作用并不依赖于δγT的增殖和激活，此外，IL-23/IL-17/IL-22作用轴上一系列细胞因子并不参与此炎症过程。TPA诱导K14-VEGF转基因小鼠出现银屑病样症状是TPA作用于皮肤后，通过激活IL-27/Th1/IFN-γ通路诱导小鼠产生了长期的迟发型超敏反应，产生大量的IFN-γ、CXCL9、CXCL10等Th1型炎症因子，与VEGF慢性过表达造成的血管炎性反应相互作用，造成了银屑病样症状。姜黄素有效减轻银屑病样症状可能是通过灭活IL-27诱导下Th1的活化和TPA诱导下蛋白激酶C的活化从而发挥作用，在对抗TPA作用于K14/VEGF转基因小鼠时可以进一步诱导VEGF高表达，但一方面可以通过阻断VEGF与受体结合后激活的下游信号通路，另一方面通过负反馈机制下调了VEGFR的表达，从而阻断VEGF的作用。
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 mice
Differences 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.
引文
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