摘要
目的观察倍半萜内酯化合物TJ-5调控泛素结合酶(ubch5c)介导NF-κB必须调节蛋白(NEMO)泛素化对维持性血液透析(MHD)患者体内微炎症的影响。方法选取2018年2月-2019年2月浙江中医药大学附属湖州中医院收治的MHD患者60例,同期选取体检中心健康体检者20名。抽取MHD患者和健康者外周血,分离单一核细胞(PBMC)及CD4+T细胞。分离纯化得到的外周血CD4+T细胞使用不同浓度TJ-5处理,MHD患者血样本分离纯化得到的CD4+T细胞分为空白组(0μM/mL TJ-5处理)和高、低浓度组(10、5μM/mL TJ-5处理),每组20份,健康对照组(健康者不予处理)20份。ELISA法检测MHD患者外周血CD4+T细胞经TJ-5处理后肿瘤坏死因子-α(TNF-α)、白细胞介素1β(IL-1β)、白介素-6(IL-6)及白介素-10(IL-10)表达情况;Western Blot法检测MHD患者和健康志愿者外周血CD4+T细胞核转录因子(P65)、I-κB(NF-κB抑制蛋白)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Th17/Treg比例显著高于健康志愿者[(1.57±0.25)%比(0.93±0.11)%,P<0.05];与空白组比较,TJ-5处理后,低、高浓度组CD4+T细胞促炎性因子TNF-α、IL-1β和IL-6明显下调[TNF-α:(5.53±0.37)ng/L、(2.27±0.26)ng/L比(8.11±1.05)ng/L,P均<0.05;IL-1β:(1.59±0.30)pg/L、(0.84±0.22)pg/L比(2.75±0.46)pg/L,P均<0.05;IL-6:(30.94±6.47)pg/L、(12.81±5.51)pg/L比(41.26±7.25)pg/L,P均<0.05];抗炎性因子IL-10明显上调[(15.82±1.01)pg/L、(20.47±1.16)pg/L比(7.34±1.71),P均<0.05];与空白组比较,核转录因子(P65)、磷酸化p65(p-P65)水平显著下调[P65:(0.72±0.12)、(0.40±0.10)比(1.19±0.14),P均<0.05;p-P65:(0.27±0.15)、(0.18±0.11)比(0.32±0.11),P均<0.05];核因子κB(NF-κB)抑制蛋白(I-κB)显著上调[(0.40±0.14)、(0.56±0.12)比(0.27±0.08),P均<0.05;磷酸化I-κB(p-I-κB)显著下调[(0.53±0.17)、(0.33±0.08)比(0.76±0.14),P均<0.05];ubch5c活性抑制率增高[(11.18±2.11)、(26.80±2.68)比(0.00±0.00),P均<0.05];NEMO线性泛素化减弱[(0.573±0.036)、(0.234±0.037)比(0.833±0.079),P均<0.05]。结论倍半萜内酯化合物TJ-5能拮抗CD4+T细胞ubch5c活性,抑制NEMO线性泛素化调控NF-κB通路及炎症因子表达,改善MHD患者微炎症。
Objective To observe the effect of sesquiterpene lactone compound TJ-5 on ubiquitin-binding enzyme(ubch5 c)-mediated NF-κB essential modulator(NEMO) ubiquitination on microinflammation in patients with maintenance hemodialysis(MHD). Methods From February 2018 to February 2019, 60 cases of MHD patients admitted to Huzhou Hospital of Traditional Chinese Medicine, Zhejiang Chinese Medical University, were recruited.Twenty healthy individuals from the physical examination center were selected as controls. Peripheral blood was collected from MHD patients and healthy individuals, and peripheral blood mononuclear cells(PBMCs) and CD4+ T cells were isolated. CD4+ T cells were treated with different concentrations of TJ-5. CD4+ T cells obtained from highly purified blood samples of MHD patients were divided into blank group(0μM/mL TJ-5), and high(10μM/mL TJ-5) and low concentration group(5μM/mL TJ-5), 20 copies of each group. The healthy control group was also divided into 20 copies without any treatment. The expressions of tumor necrosis factor-α (TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6) and interleukin-10(IL-10) were detected by ELISA. Nuclear transcription factor P65,NF-κB inhibitor(I-κB) and its phosphorylation were tested by Western blot. Ubch5 c was obtained by affinity purification with a nickel column, and the ubiquitin ligase ubch5 c activity was detected by in vitro ubiquitination experiments;immunocoprecipitation was used to detect the linear ubiquitination of NEMO. Results The ratio of Th17/Treg in MHD patients was significantly higher than that of healthy volunteers[(1.57±0.25)% vs(0.93±0.11)%,P<0.05]. Compared to the blank group, proinflammatory cytokines, TNF-α, IL-1β and IL-6 of CD4+ T cells in the low and high concentration groups down-regulated significantly, i.e., TNF-α: [(5.53 ±0.37) and(2.27 ±0.26) vs(8.11±1.05)ng/L, P<0.05, respectively], IL-1β[(1.59±0.30) and(0.84±0.22) vs(2.75±0.46)pg/L, P<0.05, respectively], IL-6: [(30.94±6.47) and(12.81±5.51) vs(41.26±7.25)pg/L, P<0.05, respectively], While the anti-inflammatory factors IL-10 up-regulated significantly, [(15.82±1.01) and(20.47±1.16) vs(7.34±1.71)pg/L, P<0.05, respectively]. Also, P65 and phosphorylated P65(p-P65) down-regulated significantly, P65: [(0.72±0.12) and(0.40±0.10) vs(1.19±0.14), P<0.05, respectively], p-P65: [(0.27±0.15) and(0.18±0.11) vs(0.32 ±0.11), P<0.05, respectively];NF-κB up-regulated significantly, [(0.40±0.14) and(0.56±0.12) vs(0.27±0.08), P<0.05, respectively];phosphorylated I-κB(p-I-κB) down-regulated significantly, [(0.53±0.17) and(0.33±0.08) vs(0.76±0.14), P<0.05,respectively];The inhibition rate of ubch5 c activity increased, [(11.18±2.11) and(26.80±2.68) vs(0.00±0.00), P<0.05, respectively];NEMO linear ubiquitination weakened, [(0.573±0.036) and(0.234±0.037) vs(0.833±0.079),P<0.05, respectively]. Conclusion Sesquiterpene lactone compound TJ-5 can antagonize ubch5 c activity of CD4+ T cells and inhibit NEMO linear ubiquitination regulating NF-κB pathway and inflammatory factor expression, improves microinflammation in patients with MHD.
作者
姚书东
吴佳
罗文荣
YAO Shudong;WU Jia;LUO Wenrong(Department of Nephrology,Huzhou Hospital of Traditional Chinese Medicine,Zhejiang Chinese Medical University,Huzhou,Zhejiang province,313000,China)
出处
《浙江中西医结合杂志》
2020年第1期5-9,I0002,共6页
Zhejiang Journal of Integrated Traditional Chinese and Western Medicine
基金
浙江省湖州市公益性技术应用研究(一般)项目(No.2018GY35)
关键词
维持性血液透析
微炎症
泛素化
NF-ΚB通路
倍半萜内酯
Maintenance hemodialysis
Microinflammation
Ubiquitination
NF-κB pathway
Sesquiterpene lactone
