【作者】 伍丹；

【导师】 朱峰； 段秋红；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2016， 硕士

【摘要】 目的:探讨红景天苷抑制日光性皮炎的分子机制。方法:(1)通过免疫组织化学技术(IHC)分析临床日光性皮炎标本中与日光紫外线(SUV)相关MAPK通路因子p-JNKs,p-p38和炎症因子Ki67,CD45,COX-2的表达。(2)选取适宜的SUV照射剂量(不引起细胞凋亡),对JB6 Cl41(小鼠上皮细胞)和Ha Ca T(人角质形成细胞)进行处理,用western blot检测p-p38,p-JNKs和COX-2的表达。(3)在Ha Ca T细胞中采用基因沉默技术使COX-2表达下调,观察MAPK通路因子p-p38,p-JNKs的变化。(4)采用同源建模和分子对接方法预测COX-2的抑制剂,以及相互作用的位点。(5)采用体外药物蛋白结合技术(in vitro pull-down assay),检测红景天苷能否与COX-2结合。(6)MTS方法测定细胞存活率,观察红景天苷对细胞是否有毒性。(7)选取不同浓度的红景天苷和适宜浓度不同时间段预处理JB6 Cl41和Ha Ca T细胞,SUV照射后,收集细胞培养上清,酶联免疫吸附实验(ELISA)测定红景天苷是否抑制COX-2活性,即下游PGE2的产生以及炎症因子IL-6和TNF-α水平;提取细胞总蛋白,采用western blot分析红景天苷对细胞MAPK通路因子及COX-2的作用。(8)建立小鼠日光性皮炎模型,在小鼠背部皮肤处涂抹红景天苷或者丙酮,然后进行SUV照射,取皮肤组织进行H-E染色观察皮肤炎症的病理变化,以及免疫组化技术来分析皮肤组织中COX-2,p-p38,p-JNKs的表达变化。结果:(1)COX-2,p-JNKs,p-p38,Ki67和CD45在日光性皮炎中表达上调。(2)SUV诱导Ha Ca T和JB6 Cl41细胞中COX-2,p-p38和p-JNK的表达增加。(3)在Ha Ca T细胞中沉默COX-2后,SUV诱导的p38或JNKs的磷酸化水平下降。(4)红景天苷在体外与COX-2直接结合且对Ha Ca T和JB6 Cl41细胞无毒性。(5)红景天苷抑制COX-2的活性,减轻SUV辐射引起的Ha Ca T和JB6Cl41细胞炎症反应。(6)红景天苷预处理过的小鼠皮肤经SUV照射后,皮肤组织表层明显增厚,炎症细胞减少,COX-2,p-p38和p-JNKs的表达均有减少。结论:红景天苷可靶向COX-2抑制日光性皮炎。红景天苷作为潜在的COX-2抑制剂,有助于改善皮肤光损伤和治疗光敏性皮肤疾病。为开发天然安全的中药防护剂和临床日光性皮肤炎症的治疗提供了科学依据。更多还原

【Abstract】 Objective: To investigate the mechanism of salidroside suppressing solar ultraviolet-induced skin inflammation.Methods:(1) The expression of COX-2, p-p38, p-JNKs, Ki67 and CD45 were detected by immunohistochemistry(IHC) assay in human solar dermatitis.(2)Appropriate doses of solar UV were selected to irradiate Ha Ca T and JB6 Cl41 cells,the expression of p-p38, p-JNKs and COX-2 were detected by western blot.(3) The gene silencing technology was performed to knock down COX-2 expression in Ha Ca T cells, and test the level of p-p38 or p-JNKs.(4) In vitro pull-down assay was performed to show whether salidroside can directly bind with COX-2. The cell viability was measured by MTS assay to observe the cytotoxicity of salidroside to cells.(5) The levels of COX-2, p-p38 or p-JNKs in salidroside pre-treated Ha Ca T and JB6 Cl41 cells were detected by western blot after SUV irradiation. The production of PGE2 and inflammatory factors IL-6 and TNF-α were detected by enzyme-linked immunosorbent assay(ELISA).(6) The skin inflammation model of mice was established, the mice were pre-treated with salidroside or acetone and then irradiated by SUV, H-E staining in skin tissures showed the pathological changes of skin inflammation, the levels of COX-2, phosphorylated of p38 or JNKs were detected by IHC.Results:(1) The expression of COX-2, p-p38, p-JNKs, Ki67 and CD45 are increased in human solar dermatitis.(2) SUV irradiation induces the expression of COX-2, the phosphorylation of p38 or JNKs in Ha Ca T and JB6 Cl41 cells.(3) Knocking down COX-2 inhibits SUV-induced phosphorylation of p38 or JNKs in Ha Ca T cells.(4) Salidroside can directly bind with COX-2 and has no toxicity to Ha Ca T and JB6 Cl41 cells.(5) Salidroside inhibits the activity of COX-2 and decreases the inflammatory process induced by SUV irradiation ex vivo.(6) Salidroside inhibits inflammation induced by SUV irradiation in vivo.Conclusions: In brief, our data provided the evidences for the protective role of salidroside against SUV-induced inflammation by targeting COX-2, salidroside might be a promising drug for the treatment of SUV-induced skin inflammation.更多还原