Effects of tacrolimus on the expression of nuclear factor-κB and glucocorticoid receptor by HaCaT cells in vitro

Objective To investigate the effect of tacrolimus on the expression of nuclear factor-κB (NF-κB) in HaCaT cells stimulated by tumor necrosis factor-α(TNF-α),and on the expression of glucocorticoid receptor (GR)α and β in untreated HaCaT cells in vitro.Methods Cultured Ha CaT cells were treated with TNF-α(10μg/L) only,combination of TNF-α(10μg/L) and various concentrations (10-8mol/L, 10-7mol/L,10-6moL/L) of tacrolimus or tacrolimus of different concentrations only.After additional 12-,24-, 36- or 48-hour cnlture, Westem blot and immunofluorescenee-confocal laser scanning microscopy were used to detect the expressions of NF-κB,GRα and GRβ in HaCaT cells.Those untreated HaCaT cells served as the control.Results The relative protein expression level of NF-κB was increased in HaCaT cells after treatment with TNF-α for 24 and 48 hours zompared with untreated ceils (0.73±0.0316 and 0.8925±0.0171 vs 0.4988±0.03506,both P<0.05);however,the increase in NF-κB expression was inhibited by the combination treatment with tacrolimus,and the relative expression level of NF-κB protein was 0.6825±0.0263.0.6200±0.0163 and 0.5575±0.0299 in HaCaT cells treated with TNF-α plus tacrolimus of 10-8mol/L 10-7mol/L and 10-6mol/L,respectively;the difference was significant etween TNF-α-treated cells and those dealt with the combination of NF-α and tacrolimus of 10-7 or 10-6 mol/L (both P<0.05).No significant difference was observed in the expression of NF-κB by HaCaT cells between different time oints treated with tacrolimus of 10-8,10-7 or 10-6 mol/L.Also,there was no zignificant difference in the expression of GRα or GRβ between untreated HaCaT cells and those treated with tacrolimus of 10-8, 10-7 or 10-6 mol/L at any time point.Conclusions Tacrolimus ould inhibit the expression of NF-κB by TNF-α-stimulated HaCaT cells,but does not affect the expression of GRα or GRβ,in untreated HaCaT cells.     

他克莫司对HaCaT细胞核因子-κB和糖皮质激素受体表达的影响

目的 探讨他克莫司对TNF-α刺激的HacaT细胞核因子-κB(NF-κB)表达的影响,以及对HaCaT细胞糖皮质激素受体(GR)α和β表达的影响.方法 采用Western印迹和免疫荧光-激光共聚焦显微镜技术观察:①以TNF-α(10μg/L)单独或TNF-α(10μg/L)与他克莫司(10-8mol/L、10-7mol/L、10-6mol/L)同时作用HaCaT细胞,在24h和48h分别观察胞核NF-κB的表达;②以他克莫司(10-8mol/L、10-7mol/L、10-6mol/L)作用HaCaT细胞,在12h、24h和36h分别观察GRα和GRβ的表达.结果 未处理组HaCaT细胞的胞核有NF-κB(p65)蛋白的表达,灰度值比值为0.4988±0.03506;TNF-α作用24h和48h后,HaCaT细胞胞核NF-κB(p65)表达随时间延长而增强,灰度值比值分别为0.73±0.0316和0.8925±0.0171,与未处理组相比较.差异均有统计学意义(P<0.05).TNF-α与10-8 mol/L、10-7 mol/L、10-6mol/L他克莫司同时作用组HaCaT细胞胞核NF-κB表达灰度值比值分别为0.6825±0.0263、0.6200±0.0163和0.5575±0.0299,NF-κB表达随他克莫司浓度增加而减弱,后两组与TNF-α单独作用组相比,差异均有统计学意义(P<0.05).各浓度他克莫司作用24h和48h的NF-κB表达差异均无统计学意义(P>0.05).与未处理组相比,不同浓度他克莫司作用HaCaT细胞后,各时相点GRα和GRβ的表达差异均无统计学意义(P>0.05).结论 他克莫司能以浓度依赖方式抑制TNF-α刺激的角质形成细胞胞核NF-κB的表达,不影响角质形成细胞GRα和GRβ的表达.     

磷酸化NF-κB亚基P65介导化学性缺氧诱导的HaCaT细胞炎症损伤

目的 探讨核因子-κB(NF-κB)亚基P65磷酸化是否参与化学性缺氧模拟剂氯化钴(CoCl2)诱导的永生化人皮肤角质形成细胞(HaCaT)毒性作用及炎症反应.方法 用2 mmol/L CoCl2处理HaCaT细胞,建立化学性缺氧诱导HaCaT细胞损伤的体外模型.RNA干扰法下调NF-κB亚基P65的表达.细胞计数试剂盒-8比色法检测细胞存活率;ELISA法检测培养基中IL-6和IL-8的含量;Western印迹法检测总量P65及磷酸化P65的蛋白表达.结果 CoCl2处理HaCaT细胞0～4 h,可促进NF-κB亚基P65的磷酸化,在0.5 h时P65亚基开始磷酸化,1.5 h时P65亚基的磷酸化水平达到高峰,约为对照组的6.6倍,而4 h基本恢复到正常水平.CoCl2处理HaCaT细胞0～6 h,可时间依赖性地降低细胞活力,2、4和6 h的细胞存活率与对照组比较,P值分别＜0.05、＜0.01及＜0.01.CoCl2处理6 h,还引起IL-6和IL-8的释放显著增加.RNA干扰法下调P65的表达后,CoCl2处理引起的HaCaT细胞毒性作用被明显减弱,即使细胞存活率升高了11%左右,下调P65表达还明显抑制了CoCl2处理引起的IL-6和Il-8释放增多.结论 磷酸化NF-κB亚基P65介导CoCl2诱导的HaCaT细胞毒性及炎症反应.

Abstract：

Objective To explore whether the phosphorylation of NF-κB P65 subunit is involved in the cytotoxicity to and inflammation in an immortal human keratinocyte cell line HaCaT during cobalt chloride (CoCl2-induced chemical hypoxia. Methods HaCaT cells were treated with CoCl2 of 2 mmol/L to set up a chemical hypoxia-induced cell model of injury. Then, RNA interference was used to down-regulate the expression of P65 in CoCl2-induced HaCaT cells. After additional culture, cell viability was tested by cell counting kit8 (CCK-8), the levels of interleukin 6 (IL-6) and interleukin 8 (IL-8) were detected by ELISA kits, phosphorylated and total P65 protein was measured by Western blot. Results The exposure of HaCaT cells to 2 mmol/L CoCl2 for 0 to 4 hours enhanced the phosphorylation of P65, which began at 0.5 hour, peaked at 1.5 hours, and restored to the normal level at 4 hours, and the level of P65 phosphorylation was about 6.6 times that in the untreated control group. The CoCl2 of 2 mmol/L decreased the cell viability of HaCaT cells in a time dependent manner, and a significant difference was observed in the viability of HaCaT cells between CoCl2-treated and untreated HaCaT cells at 2, 4, and 6 hours (P ＜ 0.05, 0.01, 0.01 ). The release of IL-6 and IL-8 from HaCaT cells was also promoted by CoCl2 treatment. The knockdown of P65 expression with siRNA markedly suppressed the CoCl2-induced cytotoxicity to and increase in the release of IL-6 and IL-8 from HaCaT cells,despite of an increment in cell viability by about 11%. Conclusion The phosphorylated P65 subunit mediates CoCl2-induced cytotoxicity and inflammatory injury to HaCaT cells.     

他克莫司联合罗格列酮对TNF-α诱导的HaCaT细胞LL37及NF-κB表达的影响

目的:确定他克莫司与罗格列酮单独及联合应用对肿瘤坏死因子-α(TNF-α)诱导的HaCaT细胞HL37及核因子- κB(NF-κB)表达的影响.方法:利用TNF-α诱导体外培养的HaCaT细胞处于炎性状态,在不同浓度的他克莫司与罗格列酮单独及联合作用下,采用RT- PCR法检测LL37的表达情况,采用免疫细胞化学(SABC)法检测NF-κB的表达情况.结果:(1)TNF-α诱导的HaCaT细胞中LL37及NF- κB的表达显著高于对照组(P＜0.05).(2)他克莫司、罗格列酮单独及联合应用均可显著抑制TNF-α诱导的HaCaT细胞LL37及NF- κB的表达(P＜0.05);二者联合应用的作用效果均较单独用药组更强(p＜0.05).结论:他克莫司和罗格列酮联合应用能增强其对TNF-α诱导的LL37及NF-κB表达的抑制作用.     

他克莫司联合罗格列酮对TNF-α诱导的HaCaT细胞LL37及NF—κB表达的影响

目的：确定他克莫司与罗格列酮单独及联合应用对肿瘤坏死因子-α（TNF—α）诱导的HaCaT细胞LL37及核因子-κB（NF-κB）表达的影响。方法：利用TNF—α诱导体外培养的HaCaT细胞处于炎性状态,在不同浓度的他克莫司与罗格列酮单独及联合作用下,采用RT—PCR法检测LL37的表达情况,采用免疫细胞化学（SABC）法检测NF—κB的表达情况。结果：（1）TNF-α诱导的HaCaT细胞中LL37及NF—κB的表达显著高于对照组（P〈0．05）。（2）他克莫司、罗格列酮单独及联合应用均可显著抑制TNF-α诱导的HaCaT细胞LL37及NF—κB的表达（P〈0．05）;二者联合应用的作用效果均较单独用药组更强（P〈0．05）。结论：他克莫司和罗格列酮联合应用能增强其对TNF-α诱导的LL37及NF—κB表达的抑制作用。     

核因子-κB反义寡核苷酸抑制紫外线诱导的HaCaT细胞反义分泌白介素6

目的 探讨核因子-κB(NF-κB)反义寡核苷酸转染体外培养的HaCaT细胞反义株HaCaT后,对紫外线(UV)诱导的HaCaT细胞反义分泌炎症因子白介素6(IL-6)的抑制作用。方法 蛋白质印迹方法测定不同剂量中波紫外线(UVB)辐射HaCaT细胞反义后NF-κBp65的变化;逆转录-聚合酶链反应测定NF-κBp65反义寡核苷酸转染后p65m RNA表达的变化;酶联免疫吸附测定法测定NF-κBp65反义寡核苷酸转染后紫外线辐射的HaCaT细胞反义分泌IL-6水平。结果 10、20、30mJ/cm² UVB辐照HaCaT细胞反义后均可显著促进NF-κBp65表达(P<0.05),不同浓度的NF-κBp65反义寡核苷酸转染后对30mJ/cm² UVB诱导的p65mRNA表达和IL-6分泌均有显著抑制作用(P<0.05)。结论 脂质体介导的NF-κBp65反义寡核苷酸转染HaCaT细胞反义,可以抑制UV辐射诱导的HaCaT细胞反义产生IL-6,表明UV诱导HaCaT细胞反义产生IL-6可能是通过UV激活NF-κBp65的表达产生的。     

卡马拉素对HaCaT细胞体外增殖及对白细胞介素17C、CCL20、NF-κB表达的影响

目的 探讨卡马拉素对HaCaT细胞体外增殖及对白细胞介素17C(IL-17C)、趋化因子CCL20、NF-κB表达的影响.方法 用不同浓度卡马拉素组(0.5、1.0、2.0、4.0 μmol/L)、含与4.0 μmol/L卡马拉素等体积二甲基亚砜的RPMI 1640培养液(溶媒组)、RPMI 1640培养液(对照组)分别作用于HaCaT细胞.采用CCK8法检测卡马拉素作用于HaCaT细胞24、48、72 h对细胞体外增殖的影响;RT-PCR法检测卡马拉素对HaCaT细胞IL-17C和CCL20 mRNA表达的影响;Western印迹法检测卡马拉素对HaCaT细胞IL-17C、CCL20和NF-κB蛋白表达的影响.统计学处理采用重复测量的方差分析、单因素方差分析和Pearson相关分析.结果 各浓度组卡马拉素对HaCaT细胞增殖的抑制作用有随时间变化的趋势(F=126.936,P＜ 0.05),药物作用时间越长,抑制作用越强;HaCaT细胞增殖的抑制率也随卡马拉素浓度的增加而升高(F=838.308,P＜ 0.05);不同浓度卡马拉素组对HaCaT细胞体外增殖的抑制作用随时间变化的趋势不同,药物浓度与时间存在交互作用(F=15.961,P＜ 0.05).不同浓度卡马拉素作用于HaCaT细胞48 h后,IL-17C mRNA及其蛋白、CCL20 mRNA及其蛋白、NF-κB蛋白表达量均随卡马拉素浓度的增加而不断降低,差异均有统计学意义(F值分别为206.041、233.887、143.883、162.431、577.915,均P＜0.05).结论 卡马拉素可抑制HaCaT细胞的体外增殖,并可在mRNA和蛋白水平下调IL-17C、CCL20的表达,而两者表达量的降低可能与NF-κB表达下调有关.     

狼疮肾炎患者外周血单个核细胞NF-κB与糖皮质激素受体mRNA的表达

目的：检测狼疮肾炎患者单个核细胞核因子-κB（NF一κB）与糖皮质激素受体mRNA（GR-mRNA）的表达水平。方法：免疫组织化学方法及逆转录酶链反应检测狼疮肾炎患者和正常对照组外周血单个核细胞NF-κB与糖皮质激素受体mRNA的表达。结果：与对照组相比,狼疮肾炎患者外周血单个核细胞GRmRNA表达水平降低,NF-κB表达水平升高（P〈0．01）;激素治疗前,激素敏感组、激素依赖组及激素抵抗组GRmRNA表达水平均降低（P〈0．01）,NF—κB表达水平升高（P〈0．01）。激素敏感组与激素抵抗组GRmRNA表达水平有统计学差异（P〈0．01）。结论：NF—κB与GRmRNA的异常表达可能与狼疮。肾炎病情及糖皮质激素疗效不同有关。