基于p38MAPK信号转导通路探讨土茯苓提取物对角质形成细胞增殖及OPN和VEGF表达的影响

Psoriasis is a common，chronic，inflammatory disease，which affected severely the patient's physical and mental health. The cause and pathogenesis of psoriasis is still unknown. The disease is characterized by keratinocyte hyperproliferation, vascular hyperplasia and infiltration of T lymphocytes，neutrophils in affected skin. Vascular endothelial growth factor(VEGF) is a crucial regulator of angiogenesis and vascular permeability in both physiological and pathological conditions such as tumor growth and chronic inflammation. VEGF is expressed and secreted by epidermal keratinocytes in normal human skin. Keratinocytes overexpress VEGF in clinically involved and uninvolved skin of patients with chronic plaque psoriasis. Osteopontin(OPN) is produced by dendritic cells, other antigen-presenting cells and keracinocytes. OPN is clearly elevated in psoriasis lesions. Many previous studies have demonstrated that VEGF and OPN might play an important role in the pathogenesis of psoriasis. Qingre Liangxue method is an effective method and Smilax glabra Roxb is one of the most importment Chinese traditional herbal medicines. Astilbin consists of the most components. However, the mechanism of Astilbin on psoriasis remains unclear and need further investigation. The mitogen-activated protein kinase（MAPK）pathways are the best characterized of intracellular protein kinase cascades. MAPK plays a role in signal transduction associated with cell proliferation, differentiation and the production of cytokines. There are three well-characterized MAPK subfamilies in mammalian cells:extra cellular-signal-regulated protein kinase 1/2(ERK1/2), the p38 MAPK and the c-Jun N-terminal kinase（JNK）. A strong link has been established between the p38 pathway and inflammation. However, little is known about the exact role of Astilbin in HaCaT cells. We hypothesized that Astilbin may modulate VEGF and OPN expression. We examined whether this effect functioned via the MAPKs signal transduction pathway,particularly p38 MAPK. .Based on this, we easked if Astilbin is able to induce the proliferation and VEGF and OPN production in epidermal keratinocytes, and investigated the role of MAPK signal pathway in the proliferation and VEGF and OPN production in epidermal keratinocytes. This research successfully will be further provided theoretical basis for Smilax glabra Roxb in treating psoriasis.

银屑病是一种常见的慢性炎症性疾病，严重影响患者的生活质量。清热凉血法是治疗银屑病的有效方法，其中土茯苓是主要药物之一。中药单体研究是目前研究的热点，也是中医药现代化的重要路径之一。p38MAPK信号转导通路在银屑病发病中起重要作用。本研究在大量前期研究基础上，以挖掘清热凉血中药土茯苓治疗银屑病的科学内涵为目标，提出土茯苓提取单体物-落新妇苷抑制HaCaT增殖可能是通过p38MAPK途径发挥作用的科学假说；以与银屑病发病密切相关的血管增生和角质细胞增殖分化为主线，以体外培养的人表皮角质形成细胞系HaCaT细胞分泌的VEGF和OPN为切入点，通过分子生物学技术结合生物信息学的研究方法，观察p38 MAPK信号转导通路中，落新妇苷对HaCaT细胞增殖、VEGF和OPN表达的影响，以期从分子、蛋白、基因组学层面揭示清热凉血中药治疗银屑病的科学内涵和微观作用机制，进而应用于临床诊断、治疗和药物开发。

本研究以与银屑病发病密切相关的血管增生和角质细胞增殖分化为主线，以清热利湿凉血中药为干预手段，通过体外实验，观察清热利湿中药对HaCaT细胞增殖、凋亡及对HaCaT细胞系分泌VEGF、OPN 、IL-6、IL-8和IL-10表达的影响；结果显示：（1）清热利湿中药土茯苓提取物在一定浓度范围内能抑制HaCaT细胞增殖，并能促进HaCaT细胞早期凋亡；（2）清热利湿中药土茯苓提取物能抑制p38MAPK、NF-κB p65、OPN、VEGF mRNA及NF-κB p65、OPN、VEGF蛋白的表达。（3）中药清热利湿饮对HaCaT细胞周期有一定的影响。其作用后S期细胞明显增加，MTX将HaCaT细胞阻滞于G1期，而清热利湿饮提取液则将其阻滞于S期。（4）清热利湿饮对经TNF-α诱导活化的HaCaT细胞产生的炎性细胞因子IL-6mRNA和IL-8mRNA的表达有下调作用；清热利湿饮对HaCaT细胞分泌IL-8和IL-10的影响清热利湿饮对经TNF-α诱导活化的HaCaT细胞可降低IL-8的分泌，但对IL-10的影响不明显。结论：清热利湿中药土茯苓提取物能抑制HaCaT细胞增殖、诱导细胞凋亡，其机制可能与制抑细胞NF-κB p65、OPN、VEGF mRNA表达有关；清热利湿饮对HaCaT细胞增殖有明显的抑制作用，且在一定浓度范围内其抑制率呈明显的时间和剂量依赖性；作用于HaCaT细胞后均可诱导其凋亡；干扰HaCaT细胞周期，将细胞阻滞于S期；下调IL-6mRNA、IL-8mRNA的表达，IL-10mRNA的表达随药物浓度的升高而增强；可降低IL-8的分泌，但对IL-10的影响不明显。提示中药清热利湿饮治疗银屑病的机制可能与此有关。