姜黄素促HaCaT细胞凋亡机理及对小鼠银屑病模型治疗研究

发布时间：2018-02-22 05:34

本文关键词： 姜黄素 HaCaT细胞氮酮凝胶银屑病小鼠 gamma/delta T细胞细胞因子　出处：《第二军医大学》2013年博士论文　论文类型：学位论文

【摘要】：姜黄素是从姜科植物姜黄(Curcuma longa)中提取的一种酚类化合物,是植物界很稀少的具有二酮的色素。姜黄素能够通过抑制多种丝氨酸/苏氨酸蛋白激酶的磷酸化,如蛋白激酶A、蛋白激酶C、精蛋白激酶、磷酸化酶激酶、自身磷酸化活性蛋白激酶以及pp60c-src酪氨酸激酶等,影响细胞的信号转导,表现出多样化的生物学作用,包括影响转录因子、酶、细胞因子和生长因子等,从而展现多重药理活性,如促进凋亡,抗增生,抗氧化,抗炎,抗肿瘤和抗血管增生等作用。姜黄素不溶于水,可应用的静脉给药方法至今未见报道。国内外目前已有的临床研究资料都是以口服给药为途径。但是姜黄素的生物利用度之低却另人失望。在一项姜黄素Ⅰ期临床研究中,口服3.6g/天的剂量也仅仅能够产生11.1±0.6nmol/L的血浆峰浓度,而低于此剂量则未检测到姜黄素。鉴于以上原因,虽然众多关于姜黄素的体内外研究已明确其药理作用,但是它极低的生物利用度严重制约了对其应用研究的脚步。银屑病是临床常见的皮肤病,发病率占世界人口的0.1%-3%。因该病顽固难治,仍被列为当今世界皮肤科领域的重要研究课题,是全世界皮肤科重点防治疾病之一。虽然银屑病的发病机理目前尚未完全明了,但越来越多的证据已经证明IL-23→IL-17→IL-22作用链以及其他细胞因子在疾病的发展中起了至关重要的作用。银屑病皮损区皮肤在基因与蛋白水平均可检测到IL-23, IL-21，IL-22和IL-17的高表达,并且IL-23可在小鼠皮肤诱发与人类银屑病类似的病变特征,如红斑,表皮增生(棘皮征),角化不全和白细胞浸润,同时真皮层的炎症与棘皮征被认为与细胞因子IL-22有关,此过程还需趋化因子受体CCR6的参与。IL-23诱导的皮肤炎症主要通过IL-17及其下游相关细胞因子实现的。除了上述提及的细胞因子以外,尚有TNF-α, IL-lα, IL-1β, IL-6, IL-8, VEGF, TGF-β等参与银屑病的病理生理过程。银屑病参与的细胞因子众多,牵涉到的信号转导更是十分庞杂。现在已知的涉及到NF-κB激活的细胞因子有IL-17A, IL-17F, TNF-α, IL-6等。姜黄素多靶点的作用机制与银屑病复杂的信号转导网络有诸多的叠加之处,如抑制NF-κB激活、抑制STAT3磷酸化等。考虑到姜黄素在银屑病治疗中潜在的应用价值,着眼于银屑病病变部位的浅表特点,本研究通过验证姜黄素对其靶细胞——KC细胞的药理作用基础上,通过局部给药的方式(规避姜黄素口服给药难以在银屑病靶细胞产生有效浓度的难题),通过小鼠银屑病动物模型评估姜黄素局部给药的治疗效果,为后续的应用研究打下坚实的实验基础。我们的研究发现：TRAIL及其受体在姜黄素诱导的HaCaT细胞凋亡中起重要作用,此作用包括TRAIL-R1和TRAIL-R2表达的上调以及凋亡抑制蛋白BcL-XL, IAP-1,IAP-2表达的抑制。以上证据说明TRAIL及其受体通路在姜黄素诱导细胞凋亡的作用中占有重要地位。此外姜黄素还能够抑制TNF-α诱导的NF-κB的激活,并减少炎症因子IL-6和IL-8的分泌。这无疑使其具备了理想的,对抗银屑病中TNF-α作用的潜力。借鉴以往的研究基础,在原有配方的基础上,引入了另外的透皮促进剂：氮酮,并适当调整了组方的比例,成功配制出了符合实验研究需要、稳定、能够强效促透的姜黄素凝胶制剂。以此制剂为基础,我们验证评估了姜黄素对咪喹莫特诱导的银屑病小鼠模型的实验治疗效果,结果令人振奋。咪喹莫特小鼠银屑病模型最早于2009年见诸报道,是迄今为止最能够最大程度模拟银屑病发病机理的模型。我们发现姜黄素可以抑制咪喹莫特导致的皮肤增厚和炎症反应；显著抑制咪喹莫特诱导的银屑病模型小鼠耳廓皮肤的IL-17A, IL-17F, IL-22, IL-1β, TNF-α和IL-6的mRNA水平的上升。并且还发现,姜黄素可能会抑制咪喹莫特诱导的实验小鼠真皮丫δ T细胞的增殖,而这一细胞被认为在银屑病的发病机制中占有重要地位。本研究在细胞和实验动物水平上系统的探讨了姜黄素对于银屑病治疗的机理和效果评估,并为实现这一研究目标对姜黄素的外用凝胶制剂进行了研究和改进。首次在银屑病动物模型上观察研究了姜黄素对IL-23/IL-17A/IL-22细胞因子轴的影响,并和类固醇激素的作用进行了对比,为该化合物未来的开发方向奠定了重要的理论和实验基础。
[Abstract]:Curcumin is from Zingiberaceae turmeric (Curcuma longa) is a kind of phenolic compounds extracted from plants, is very rare with two ketone pigment. Curcumin can inhibit a variety of the serine / threonine protein kinase phosphorylation, such as protein kinase A, protein kinase C, egg white kinase and phosphorylase kinase itself. The phosphorylation activity of protein kinase pp60c-src and tyrosine kinase, signal transduction of cells, showing diverse biological effects, including the effects of transcription factors, enzymes, cytokines and growth factors, so as to show a variety of pharmacological activities, such as promoting apoptosis, anti proliferative, antioxidant, anti-inflammatory, anti-tumor and anti angiogenesis effect.
Curcumin is not soluble in water, can be used for intravenous administration method has not been reported at home and abroad. The current clinical research data are based on the existing oral administration way. But the low bioavailability of curcumin was disappointing. In a phase I clinical study of curcumin, peak plasma concentration of oral 3.6g/ days dose only can produce 11.1 + 0.6nmol/L, and lower than the dose not detected by curcumin. In view of the above reasons, although a number of curcumin in vitro and in vivo studies have confirmed its pharmacological effects, but its low bioavailability seriously restrict the application of the pace.
Psoriasis is a common skin disease in clinic, the incidence rate of 0.1%-3%. of the world's population with the disease is difficult to cure, still listed as an important research topic in the world in the field of Department of Dermatology, Department of dermatology is one of the world's focus on disease prevention and control. Although the pathogenesis of psoriasis is not yet entirely clear, but more and more evidences have shown that IL-23, IL-17 IL-22, chain and other cytokines play a crucial role in the development of the disease. The skin lesions of patients with psoriasis in gene and protein level were detected in IL-23, IL-21, high expression of IL-22 and IL-17, and IL-23 in the pathological characteristics, and human induced psoriasis like mouse skin such as erythema, epidermal hyperplasia (spine skin syndrome), parakeratosis and leukocyte infiltration and inflammation and dermal acanthosis syndrome were found and cytokines related to IL-22, this process also need to chemokine receptor CCR6 Skin inflammation induced by.IL-23 is mainly achieved by IL-17 and its downstream related cytokines. Besides the above mentioned cytokines, there are TNF-, IL-l, IL-1, IL-6, IL-8, VEGF and TGF- beta participate in the pathophysiology process of psoriasis.
Cytokines in psoriasis in many signal transduction involved is very complex. Now known to involve NF- cell factor kappa B activation of IL-17A, IL-17F, TNF- alpha, IL-6. Signal transduction mechanism and psoriasis curcumin targets have many complex superimposed place, such as inhibition of NF- k the activation of B, inhibit the phosphorylation of STAT3. Considering the potential application value of curcumin in the treatment of psoriasis, superficial characteristics focus on psoriasis lesion, this study verified by curcumin in KC cells and the target cells of pharmacological basis, through local administration way (oral administration is difficult to avoid the problem of curcumin in the target cell of psoriasis produce effective concentration), topical therapy through mice psoriasis animal model for experimental evaluation of curcumin, a solid foundation for the application of subsequent lay.
Our study found that: TRAIL and its receptors play an important role in HaCaT cell apoptosis induced by curcumin in this role, including the expression of TRAIL-R1 and upregulation of TRAIL-R2 and inhibitor of apoptosis protein BcL-XL, IAP-1, inhibit the expression of IAP-2. The above evidence plays an important role of TRAIL and its receptor pathway in curcumin induced apoptosis. The activation of the addition of curcumin TNF- can also inhibit NF- induced by alpha kappa B, and reduce the secretion of inflammatory cytokines IL-6 and IL-8. This undoubtedly makes it have the ideal effect against psoriasis TNF- alpha potential. Basis of previous studies, based on the original formula, the penetration enhancers azone, and another. Appropriate adjustment of the prescription ratio was prepared successfully with the experimental research, stability, can be powerful penetration. This gel preparation of curcumin based formulations, we verify the evaluation Evaluate the experimental therapeutic effect of curcumin on psoriasis animal model induced by imiquimod, exciting results. Imiquimod psoriasis model mice. The earliest reported in 2009, is by far the most to the greatest extent simulating the pathogenesis of psoriasis model. We found that skin thickening and inflammatory reaction of curcumin can inhibit the cause of imiquimod; significant inhibition of imiquimod induced psoriasis model mice auricle skin IL-17A, IL-17F, IL-22, IL-1 beta, TNF- alpha and IL-6 increased the level of mRNA. It was also found that curcumin may inhibit imiquimod induced mice dermal Ya delta proliferation of T cells, and the cells are thought to play an important role in the pathogenesis of psoriasis.
In this study, experimental animal cells and on the level of the system is discussed for the mechanism and effect of curcumin in the treatment of psoriasis and evaluation, research and improvement for the realization of the goal of topical gel preparation of curcumin was carried out. For the first time in animal models of psoriasis were studied on the effects of curcumin on IL-23/IL-17A/IL-22 cell factor axis, and comparison of the effects of steroid hormones, laid an important theoretical and experimental basis for the development direction of the compound in the future.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R758.63;R-332

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