TAK1信号通路在高糖诱导骨髓来源巨噬细胞激活中的作用

发布时间：2018-01-07 23:10

本文关键词：TAK1信号通路在高糖诱导骨髓来源巨噬细胞激活中的作用　出处：《安徽医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：背景与目的患糖尿病人数逐年增加,糖尿病肾病(DN)的发病率也随之升高。目前普遍认为糖尿病肾病是一种慢性低度炎症性疾病,巨噬细胞在DN发生发展中起重要作用,而静止的巨噬细胞对肾脏无损伤作用,只有活化的巨噬细胞才能发挥促炎性损伤及促纤维化作用。然而巨噬细胞活化的分子机制目前尚不清楚。转化生长因子β激活激酶1(TGF-βactivated kinase-1,TAK1)属于丝裂原活化蛋白激酶激酶激酶(mitogen activated protein kinase kinase kinase,MAP3K)家族成员,是MAPKs和核因子-κB(nuclear factor-kappa B,NF-κB)通路共同上游信号分子,能介导多条炎症通路激活,发挥促炎效应。然而在高糖条件下TAK1信号通路是否介导骨髓来源巨噬细胞(bone marrow derived macrophages,BMDM)活化尚未见报道。本研究通过观察高浓度葡萄糖刺激及不同浓度TAK1特异性抑制剂5Z-7-oxozeaenol作用下,细胞上清液中肿瘤坏死因子(TNF)-α、白介素(IL)-1β、单核细胞趋化因子(MCP)-1分泌情况及磷酸化(p)-TAK1、TAK1结合蛋白(TAK1binding protein,TAB1)、p-JNK、NF-κB p65蛋白表达变化,探讨高糖刺激巨噬细胞活化及炎症因子分泌增加是否由TAK1信号通路部分介导。方法1.小鼠骨髓来源巨噬细胞(BMDM)的分离、培养。2.应用流式细胞术鉴定BMDM纯度。3.采用CCK-8法检测不同浓度5Z-7-oxozeaenol(10-1000nmol/L)的细胞毒性。4.应用Transwell小室构建巨噬细胞和系膜细胞共培养模型。5.实验分组:按培养方式分为巨噬细胞和系膜细胞共培养、巨噬细胞单培养2种,每种培养方式按培养条件分7组:(1)抑制剂对照组(OZ300);(2)渗透压对照组(mannitol control,MC);(3)正常对照组(normal control,NC);(4)高糖组(high glucose,HG);(5)高糖+30n M抑制剂组(HG+OZ30);(6)高糖+100n M抑制剂组(HG+OZ100);(7)高糖+300n M抑制剂组(HG+OZ300)。6.采用免疫荧光双染法检测M1巨噬细胞表型标志诱导型一氧化氮合酶(i NOS)的表达。7.采用ELISA法检测单培养和共培养模型中各组细胞上清液TNF-α、IL-1β、MCP-1含量变化。8.应用Western印迹技术检测高糖刺激不同时间点单培养巨噬细胞p-TAK1、TAB1、p-JNK、NF-κB p65蛋白表达变化及不同浓度5Z-7-oxozeaenol抑制作用。9.通过激光共聚焦技术检测TAK1抑制剂对高糖刺激巨噬细胞NF-κB p65核转移的影响。结果1.经流式细胞术检测BMDM纯度达99.36%。2.本实验观察范围内5Z-7-oxozeaenol浓度(30-300nmol/L)对高糖诱导的BMDM活性均无影响(P均㧐0.05),给予1000nmol/L的5Z-7-oxozeaenol干预可使BMDM活性降低(P㩳0.05)。3.高糖组巨噬细胞表型标志i NOS荧光强度明显高于对照组,抑制剂干预组i NOS荧光强度较高糖组明显减弱。4.高糖刺激下,单培养和共培养细胞上清液中TNF-α、IL-1β、MCP-1含量均较对照组升高(P均0.05),巨噬细胞与系膜细胞共培养模型组上清液中TNF-α、IL-1β、MCP-1含量较BMDM单培养组升高(P0.05)。与高糖组比较,TAK1抑制剂呈浓度依赖性降低各组上清液中炎症因子含量(P0.05)。5.在BMDM单培养条件下,与对照组比较,高糖刺激巨噬细胞p-TAK1、TAB1、p-JNK、NF-κB p65蛋白表达水平均上调(P均0.05),抑制剂组5Z-7-oxozeaenol呈浓度依赖性降低巨噬细胞p-TAK1、TAB1、p-JNK、NF-κB p65蛋白表达量(P0.05)。6.通过激光共聚焦显微镜观察到高糖刺激能诱导BMDM胞浆中NF-κB p65亚基入核,300nmol/L的5Z-7-oxozeaenol干预可明显抑制NF-κB p65核转移。结论1.高糖刺激可上调i NOS表达,诱导骨髓来源巨噬细胞(BMDM)向M1型巨噬细胞活化。2.TAK1可能通过激活下游JNK和NF-κB信号通路,从而促进巨噬细胞向促炎表型极化。3.高糖刺激可诱导BMDM分泌TNF-α、IL-1β和MCP-1,而巨噬细胞与系膜细胞共培养能进一步促进这些炎症因子的分泌,加重炎症反应。4.TAK1特异性抑制剂5Z-7-oxozeaenol能抑制TAK1/JNK和TAK1/NF-κB通路激活,降低高糖条件下M1型巨噬细胞活化,并减少炎症因子TNF-α、IL-1β和趋化因子MCP-1的产生,从而减轻炎症反应。
[Abstract]:Background and objective: diabetes has increased the number of diabetic nephropathy (DN), the incidence rate is also increased. It is generally believed that the diabetic nephropathy is a chronic inflammation disease, macrophages play an important role in the pathogenesis of DN, but still no damage to kidney macrophages, activated macrophages can only exert pro-inflammatory damage and profibrotic effect. However, the molecular mechanism of macrophage activation is unclear. Transforming growth factor activated kinase 1 (TGF- beta activated kinase-1, TAK1) belong to the mitogen activated protein kinase (mitogen activated protein kinase kinase kinase, MAP3K) family members, MAPKs and nuclear factor kappa B (nuclear factor-kappa B NF-, K B) pathways upstream signal molecule mediated by multiple inflammatory pathways activation play a proinflammatory effect. However, in high glucose conditions TAK1 signaling pathway Is mediated by bone marrow macrophages (bone marrow derived macrophages, BMDM) activation has not been reported. This study through the observation of high glucose stimulation and the effects of different concentrations of TAK1 specific inhibitor 5Z-7-oxozeaenol and tumor necrosis factor in the supernatant (TNF) - alpha, interleukin (IL) -1 beta, monocyte chemotactic factor (MCP) -1 secretion and phosphorylation of -TAK1 (P), TAK1 binding protein (TAK1binding, p-JNK, protein, TAB1) expression of NF- kappa B p65 protein, on glucose stimulated macrophage activation and inflammatory cytokines secretion is mediated in part by TAK1 signaling pathway. Methods 1. mouse bone marrow-derived macrophages (BMDM) separation.2. training, flow cytometry was used to identify BMDM purity of.3. with different concentration of 5Z-7-oxozeaenol was detected by CCK-8 (10-1000nmol/L) on the cytotoxicity of.4. application of Transwell small chamber construction and mesangial cells were macrophages Training model of.5. experimental groups: according to the training mode is divided into co cultured macrophages and macrophages cultured mesangial cells, single 2 kinds, each kind of culture by way of culture conditions were divided into 7 groups: control group (1) inhibitor (OZ300); (2) osmotic pressure control group (mannitol control, MC); (3) normal the control group (normal, control, NC); (4) high glucose group (high glucose, HG); (5) high glucose +30n M inhibitor group (HG+OZ30); (6) high glucose +100n M inhibitor group (HG+OZ100); (7) high glucose +300n M inhibitor group (HG+OZ300) immunofluorescence double staining of M1 macrophages phenotype of inducible nitric oxide synthase by.6. (I NOS) on the expression of.7. was detected by ELISA single and co cultured supernatant of cells in each group of TNF- alpha, IL-1 beta model, the change of the content of MCP-1.8. using Western blot of high glucose at different time points of single cultured macrophage p-TAK1, TAB1, p-JNK, and expression of NF- K B p65 protein Effects of different concentrations of 5Z-7-oxozeaenol inhibited.9. by confocal laser technology to detect TAK1 inhibitors on glucose stimulated macrophage NF- kappa B p65 nuclear transfer. Results 1. the purity of BMDM cells was detected by flow cytometry as 99.36%.2. we observed the concentration range of 5Z-7-oxozeaenol (30-300nmol/L) had no effect on high glucose induced BMDM activity (P? 0.05) 5Z-7-oxozeaenol, give intervention 1000nmol/L can make the activity of BMDM decreased (P? 0.05).3. high glucose group macrophage phenotype marker I NOS fluorescence intensity was significantly higher than the control group, I inhibitor group, NOS fluorescence intensity high sugar group significantly reduced.4. stimulated by high glucose, single and co cultured in the supernatant of TNF- alpha, IL-1 beta, MCP-1 the content were higher than those in control group (P 0.05), TNF- alpha, model group was co cultured with macrophages and mesangial cells of IL-1 beta, MCP-1 content is BMDM single culture group (P0.05) and high rise. Sugar group, TAK1 inhibitor showed a concentration dependent decrease inflammatory cytokine content in supernatant were measured by.5. BMDM (P0.05) in single culture conditions, compared with the control group, high glucose stimulated p-TAK1, TAB1, p-JNK, expression of NF- kappa B p65 protein levels were up-regulated (P 0.05), 5Z-7-oxozeaenol inhibitor group in a concentration dependent manner. Reduce TAB1, macrophage p-TAK1, p-JNK expression of NF- kappa B protein p65 (P0.05).6. by laser confocal microscopy to high glucose induced BMDM cytoplasmic NF- kappa B subunit p65 in the nucleus, 300nmol/L 5Z-7-oxozeaenol intervention can significantly inhibit the NF- kappa B p65 nuclear translocation. Conclusion high glucose stimulated expression of 1. upregulation of I NOS induced bone marrow-derived macrophages (BMDM) activation to M1 macrophages.2.TAK1 may activate downstream JNK and NF- B signaling pathway, which promotes macrophage to proinflammatory phenotype polarization.3. high glucose can induce the secretion of BMDM TNF- alpha, IL-1 beta and MCP-1, and mesangial cells were co cultured with macrophages can further promote the secretion of these cytokines, aggravate the inflammatory response of.4.TAK1 specific inhibitor 5Z-7-oxozeaenol can inhibit TAK1/JNK and TAK1/NF- K B activation, reduce the activation of M1 macrophages under high glucose condition, and reduce the inflammatory factor TNF- alpha, IL-1 beta and chemotaxis factor MCP-1, in order to reduce inflammation.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.1

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