论文部分内容阅读
研究背景树突状细胞(DCs)是体内功能最强大的抗原递呈细胞,具有独特的激活初始T淋巴细胞的功能,是启动和调控特异性免疫应答的中心环节。近年来的大量基础和临床研究证实,动脉粥样硬化(As)是一种慢性炎症和免疫性疾病,DCs直接或间接参与As的发生发展。以DCs为作用靶点可能是干预As的有效方法。丹参酚酸B(Sal B)是自中药丹参中提取出的水溶性单体,是丹参的重要药理活性成份,化学结构明确,性质稳定。以往的研究证实,Sal B对心、脑、肝、肾等多个器官具有重要的保护作用。近期的研究还发现,Sal B具有抗炎症、抗免疫反应的作用,能够影响As的发生发展,但具体作用机制和靶点还不明确。目的从免疫炎症的角度探讨Sal B对氧化低密度脂蛋白(ox-LDL)诱导的人单核细胞源DCs免疫功能成熟的影响,进一步研究其作用机制,为临床As的防治提供新靶点和新思路。方法培养人单核细胞源DCs,Sal B预处理后,再与ox-LDL共孵育。流式细胞术检测DCs表面分子(CD40、CD1a、CD86和HLA-DR)的表达,ELISA法检测细胞培养上清液细胞因子(IL-12和TNF-α)的浓度,Western blot法检测PPARγ和MAPKs的蛋白表达,RT-PCR法检测PPARγ的基因表达,Luciferase报告系统检测PPARγ的DNA结合活性。结果与ox-LDL组相比,Sal B预处理组DCs的表面分子CD40、CD1a、CD86和HLA-DR的表达明显下降(P<0.05),细胞因子IL-12和TNF-α的浓度明显降低(P<0.01),证明Sal B预处理明显抑制ox-LDL诱导的人单核细胞源DCs的免疫功能成熟。与ox-LDL组相比,Sal B预处理组DCs的MAPKs的蛋白表达明显下调,主要是P38蛋白表达明显降低(P<0.05),表明Sal B预处理明显抑制ox-LDL诱导的人单核细胞源DCs的MAPKs蛋白表达。进一步采用siRNA技术使PPARγ基因沉默后,Sal B预处理抑制ox-LDL诱导的人单核细胞源DCs表面分子、细胞因子和MAPKs蛋白表达的作用被明显翻转,提示Sal B通过影响PPARγ抑制DCs的免疫功能成熟。接下来采用Luciferase技术证实Sal B可以特异的上调PPARγ的DNA结合活性,是转录后的水平,而不是mRNA水平的改变。结论 Sal B通过激活PPARγ抑制ox-LDL诱导的人单核细胞源DCs免疫功能成熟,这可能是Sal B抑制As的一个机制。
Background Dendritic cells (DCs) are the most potent antigen-presenting cells in the body. They have the unique function of activating initial T lymphocytes and are central to the initiation and regulation of specific immune responses. In recent years, a large number of basic and clinical studies have confirmed that atherosclerosis (As) is a chronic inflammation and immune diseases, DCs directly or indirectly involved in the occurrence and development of As. Using DCs as a target may be an effective way to interfere with As. Salvia miltiorrhiza B (Sal B) is a water-soluble monomer extracted from Salvia miltiorrhiza. It is an important pharmacologically active ingredient of Salvia miltiorrhiza. Its chemical structure is clear and its properties are stable. Previous studies confirmed that Sal B has important protective effects on many organs such as heart, brain, liver and kidney. Recent studies also found that Sal B has anti-inflammatory and anti-immune effects and can affect the occurrence and development of As, but the specific mechanism of action and target are not clear. Objective To investigate the effect of Sal B on the immune function of human monocyte-derived DCs induced by ox-LDL from the perspective of immune inflammation and further study its mechanism of action to provide a new target for prevention and treatment of clinical As New ideas. Methods Human monocyte-derived DCs were cultured and pretreated with Sal B before co-incubation with ox-LDL. The expression of CD40, CD1a, CD86 and HLA-DR on DCs were detected by flow cytometry. The concentrations of cytokines (IL-12 and TNF-α) in supernatants were determined by ELISA. MAPKs protein expression, RT-PCR method to detect PPARγ gene expression, Luciferase reporter system to detect PPARγ DNA binding activity. Results Compared with ox-LDL group, the expression of CD40, CD1a, CD86 and HLA-DR of DCs in Sal B pretreatment group was significantly decreased (P <0.05), and the concentrations of cytokines IL-12 and TNF-α were significantly decreased (P <0.01), Sal B pretreatment significantly inhibited ox-LDL-induced human monocyte-derived DCs immune function maturation. Compared with the ox-LDL group, the protein expression of MAPKs in DCs of Sal B pretreatment group was significantly downregulated, mainly P38 protein expression was significantly decreased (P <0.05), indicating that Sal B pretreatment significantly inhibited ox-LDL-induced human mononuclear MAPKs protein expression in cell-derived DCs. After further silencing PPARγ gene by siRNA, the effect of Sal B pretreatment on ox-LDL-induced human monocyte-derived DCs surface molecules, cytokines and MAPKs protein expression was reversed significantly, suggesting that Sal B can inhibit the proliferation of DCs induced by PPARγ Immune function is mature. Followed by Luciferase technology confirmed that Sal B can specifically upregulate PPARγ DNA binding activity, is the level of post-transcription, rather than changes in mRNA levels. Conclusion Sal B inhibits ox-LDL-induced immune function of human monocyte-derived DCs by activating PPARγ, which may be a mechanism by which Sal B inhibits As.