Activation mechanism of NF-kB and development of therapeutic strategy through its regulation.

NF-kB 的激活机制以及通过其调节制定治疗策略。

• 批准号: 11671061
• 负责人: KASHIHARA Naoki
• 金额: $ 2.18万
• 依托单位: Kawasaki Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671061/
• 关键词: NF-KB; Transcription factor; Cytokine; Glucocorticoid; Renal diseases

NF-kB plays an important role in cellular response to injury, such as mesangial cell (MC) proliferation or infiltration of inflammatory cells, in glomerulonephritis through activation of a number of cytokines, growth factors and adhesion molecules. We attempted to modulate NF-kB activity by using several reagents, such as glucocorticoid, anti-oxidant (PDTC). We also verified feasibility of oligonucleotide (ODN) which contains the consensus NF-kB binding site (decoy DNA) in an attempt to inhibit NF-kB activation. We evaluated inhibitory effect of these reagents on mesangial cell proliferation in vitro and therapeutic effects in the rat anti-Thyl.l nephritis model as well. First, the effect of these reagents on MC proliferation was assessed in vitro. The nuclear extracts were prepared from the cells treated with these reagents. Electrophoretic mobility-shift assay (EMSA) was performed to confirm the effect on NF-kB DNA binding activity. Secondly, these three reagents were used in vivo. T … More he rat anti-Thyl.l model was induced by injection of monoclonal anti-Thyl.l antibody (oX-7). Renal biopsy was performed at day 8.The total cell number and the number of proliferating nuclear cell antigen (PCNA)-positive cells per glomeralar cross section were determined. Glomerular mRNA was extracted and the mRNA expressions of IL-1, TNF-a, MCP-1 and ICAM-1 of which gene expression was regulated by NF-kB, were measured by 'reverse transcriptase polymerase chain reaction (RT-PCR) method. NF-kB decoy inhibited the MC growth in a dose dependent manner in vitro. Ten mM of decoy inhibited the MC growth by 75%. EMSA revealed that this inhibitory effect was mediated by decreased NF-kB binding activity. Glucocorticoid, PDTC and NF-kB decoy suppressed MC proliferation in the Thy1.1 glomerulonephritis model. The number of glomeralar cell decreased by 25%. Decreased mRNA expressions of IL-1, TNF-a, MCP-1 and ICAM-1 were recognized in the experimental group. Thus, inhibition of mesangial cell proliferation was possibly resulted from suppressed expression of these cytokines at least in part.These results indicate that the reagents which modulate NF-kB function would be a novel therapeutic agent for inflammatory glomeralar diseases. Less

NF-kB通过激活多种细胞因子、生长因子和粘附分子，在肾小球肾炎中对损伤的细胞反应中发挥重要作用，如系膜细胞（MC）增殖或炎症细胞浸润。我们试图通过使用几种试剂，如糖皮质激素，抗氧化剂（PDTC）来调节NF-kB活性。我们还验证了含有NF-kB结合位点（诱饵DNA）的寡核苷酸（ODN）抑制NF-kB激活的可行性。我们在体外研究了这些试剂对肾小球系膜细胞增殖的抑制作用和对大鼠抗thyl的治疗作用。L型肾炎模型。首先，在体外评估这些试剂对mcc增殖的影响。用这些试剂处理的细胞制备核提取物。电泳迁移位移试验（EMSA）证实了对NF-kB DNA结合活性的影响。其次，这三种试剂在体内使用。更多的是老鼠反泰尔。单克隆抗thyl注射诱导l模型。l抗体（oX-7）。第8天进行肾活检。测定肾小球横切面细胞总数和增殖核细胞抗原（PCNA）阳性细胞数。提取肾小球mRNA，采用逆转录聚合酶链反应（RT-PCR）法检测NF-kB调控基因表达的IL-1、TNF-a、MCP-1和ICAM-1 mRNA表达量。NF-kB诱骗剂对体外mcc生长有剂量依赖性。10 mM诱捕剂可抑制mc75 %的生长。EMSA显示，这种抑制作用是由NF-kB结合活性降低介导的。糖皮质激素、PDTC和NF-kB诱饵抑制了Thy1.1型肾小球肾炎模型中MC的增殖。肾小球细胞数量减少25%。实验组IL-1、TNF-a、MCP-1、ICAM-1 mRNA表达降低。因此，系膜细胞增殖的抑制可能至少部分是由于这些细胞因子的表达受到抑制。这些结果表明，调节NF-kB功能的试剂将成为炎症性肾小球疾病的新型治疗剂。少

项目成果

柏原直樹: "Mechanisms for induction of apoptosis and glomerular disease"Nephrol Dial Transplant. 14. 770-775 (1999)

Naoki Kashihara：“诱导细胞凋亡和肾小球疾病的机制”Nephrol Dial Transplant 14. 770-775 (1999)。

柏原直樹: "糸球体硬化とアポトーシス"興和医報. 42. 19-23 (1999)

Naoki Kashihara：“肾小球硬化和细胞凋亡”Kowa Medical Journal 42. 19-23 (1999)。

Okamoto K, Kashihara N, Yamasaki Y, Kanao K, Maeshima Y, Sekikawa T, Sugiyama H, Murakami T, and Makino H: "Calqesmon isofonn associated with phenotypic modulation of mesangial cells."Exp Nephron. 8. 20-27 (2000)

Okamoto K、Kashihara N、Yamasaki Y、Kanao K、Maeshima Y、Sekikawa T、Sugiyama H、Murakami T 和 Makino H：“Calqesmon isofonn 与系膜细胞的表型调节相关。”Exp Nephron。

Jyo O Y, Sasaki T, Kawakarni Y, Nohno T, Itoh N, Osawa G, and Kashihara N:: "Expression of the fibroblast growth factor receptor 1-4 genes in glomeruli in anti-Thyl. I mesangial proliferative glomerulonephritis."Virchows Archiv. 435. 501-508 (1999)

Jyo O Y、Sasaki T、Kawakarni Y、Nohno T、Itoh N、Osawa G 和 Kashihara N：：“抗 Thyl 肾小球中成纤维细胞生长因子受体 1-4 基因的表达。I 系膜增生性肾小球肾炎。”Virchows Archive

Jyo OY: "Expression of the fibroblast growth factor receptor 1-4 genes in glomeruli in anti-Thy1.1 mesangial proliferative glomerulonephritis"Virchows Arch. 435. 501-508 (1999)

Jyo OY：“抗 Thy1.1 系膜增生性肾小球肾炎中肾小球中成纤维细胞生长因子受体 1-4 基因的表达”Virchows Arch。