| 姚书东.TJ-5抑制ubch5c介导NFMO泛素化调控NF-κB通路对MHD体内微炎症的影响[J].浙江中西医结合杂志,2020,30(1): |
| TJ-5抑制ubch5c介导NFMO泛素化调控NF-κB通路对MHD体内微炎症的影响 |
| Influence of tj-5 on microinflammation in MHD by inhibiting ubch5c mediated NFMO ubiquitinization regulating NF- kappa B pathway |
| 投稿时间：2019-06-13 修订日期：2019-12-02 |
| DOI： |
| 中文关键词: 维持性血液透析，泛素化，NF-κB通路，微炎症，倍半萜内酯 |
| 英文关键词:Maintenance hemodialysis with ubiquitination, NF- kappab pathway, Microinflammation |
| 基金项目:基金课题：湖州市科学技术局任务项目；项目编号（2018GY35） |
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| 中文摘要: |
| 目的：通过观察倍半萜内酯化合物TJ-5调控 ubch5c介导NFMO泛素化对维持性血液透析( maintenance hemodialysis,MHD)患者体内微炎症的影响,探讨其通过调控NF-κB通路抑制MHD患者体内炎症因子表达的机制。方法：抽取MHD患者和正常人外周血。流式检测两组患者外周血中Th17/Treg比例；分别分离SLE患者和正常人外周血单一核细胞(PBMC),T细胞分选试剂盒分离纯化得到CD 4+T细胞,并进行鉴定。分离得到的外周血CD 4+T细胞使用不同浓度TJ-5处理,MHD患者分为：空白组(0μM/mL TJ-5处理)、高低浓度组(10、5μM /mL TJ-5处理)以及正常组(正常人)。LISA 法检测MHD患者外周血CD 4+T细胞在TJ-5处理后TNF-a、IL- 1、IL-6及IL-10及因子的表达情况；Western Blot法检测SLE患者和正常人外周血CD 4+T细胞P65、I-κB蛋白及其磷酸化；采用镍柱亲和纯化获得ubch5c,通过体外泛素化实验检测泛素连接酶ubch5c活性变化情况；免疫共沉淀法检测NEMO线性泛素化情况。结果：MHD患者外周血中Th17/Treg比例显著高于正常人(P<0.05)；TJ-5处理后,CD 4+T细胞促炎性因子TNF-a、IL- 1、IL-6明显下调,抗炎性因子IL-10明显上调(P<0.05)；P65、I-κB蛋白及其磷酸化显著降低(P<0.05)；ubch5c活性受到抑制(P<0.05)；NEMO线性泛素化减弱(P<0.05)。结论：倍半萜内酯化合物TJ-5拮抗CD 4+T细胞ubch5c活性,抑制NEMO线性泛素化调控NF-κB通路改善MHD体内微炎症。 |
| 英文摘要: |
| Objective: Ubch5c mediated by observing the TJ - 5 regulation NFMO ubiquitin change of maintenance hemodialysis (maintenance hemodialysis, MHD) patients with micro inflammation in the body, the influence of its through regulating the nf-kappa B pathway inhibition mechanism of MHD patients inflammation factor expression.Methods: Peripheral blood was extracted from MHD patients and normal people.The Th17/Treg ratio in peripheral blood of the two groups was detected by flow cytometry.Peripheral blood mononuclear cells (PBMC) were isolated from SLE patients and normal people, and CD 4+T cells were isolated and purified with T cell sorting kit, and identified.The isolated peripheral blood CD 4+T cells were treated with different concentrations of tj-5. MHD patients were divided into the blank group (0 micron /mL tj-5 treatment), the high and low concentration group (10 micron /mL tj-5 treatment) and the normal group (normal). The expression of TNF- a, il-1, il-6, il-10 and factors in CD 4+T cells in peripheral blood of MHD patients after tj-5 treatment was detected by LISA method.Western blotting was performed to detect P65, i-kappab protein and phosphorylation of CD 4+T cells in peripheral blood of SLE patients and normal patients.Ubch5c was purified by nickel column affinity, and the activity of ubiquitin ligase ubch5c was detected by in vitro ubiquitination experiment.Detection of NEMO linear ubiquitination by immunocoprecipitation.Result：Th17/Treg ratio in peripheral blood of MHD patients was significantly higher than that of normal patients (P<0.05).After tj-5 treatment, the pro-inflammatory factors TNF- a, il-1 and il-6 of CD 4+T cells were significantly down-regulated, while the anti-inflammatory factors il-10 were significantly up-regulated (P<0.05).P65 and i-kappab protein and their phosphorylation were significantly decreased (P<0.05).Ubch5c activity was inhibited (P<0.05).Decreased NEMO linear ubiquitination (P<0.05).Conclusion: Tj-5 antagonized ubch5c activity of CD 4+T cells, inhibited NEMO linear ubiquitinization regulating NF- kappa B pathway and improved microinflammation in MHD. |
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