NF-κB抑制剂PDTC对急性白血病细胞HL-60增殖与凋亡的影响

目的:探讨核因子κB(NF-κB)抑制剂吡咯烷二硫基甲酸盐(PDTC)对急性白血病细胞HL-60增殖与凋亡的影响。方法:用PDTC 0、25、50、100μmol/L处理HL-60细胞24、48、72 h,CCK-8实验检测细胞增殖抑制率,Hoechst染色检测细胞凋亡,流式细胞术检测细胞周期,Western blot检测细胞中B淋巴细胞瘤-2(BCL-2)、BCL-2相关X蛋白(BAX)、细胞周期蛋白D1(cyclinD1)、活化型含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved caspase 3)、cleaved caspase 8及NF-κB信号通路相关蛋白表达。结果:25、50、100μmol/L PDTC处理HL-60细胞24、48、72 h后,同一时间点下随着PDTC浓度升高,细胞增殖抑制率越高(r=0.924,P<0.01),在同一PDTC浓度下,随着时间推移,细胞增殖抑制率增高(r=0.952,P<0.01)。在25、50、100μmol/L PDTC处理HL-60细胞48 h后,与对照组比较,PDTC能显著提高HL-60细胞凋亡率,并使细胞周期阻滞在G1期(P<0.01);PDTC能显著下调HL-60细胞BCL-2,cyclinD1、p-NF-κB p65表达(P<0.05),上调BAX、cleaved-caspase 3、cleaved-caspase 8表达(P<0.01);50、100μmol/L PDTC能显著下调HL-60细胞p-NF-κB抑制蛋白(p-IκBα)表达(P<0.01)。结论:PDTC能显著抑制急性白血病细胞HL-60的增殖,并诱导细胞凋亡。

PDTC对高血压大鼠继发左心室肥厚及心力衰竭的作用

目的评价核因子(NF)-κB抑制剂吡咯烷二硫基甲酸盐(PDTC)对高血压-心室肥厚-充血性心力衰竭大鼠心脏NF-κB活性和血清中各炎症递质的抑制作用。方法 42只雄性Dahl盐敏感大鼠建立高血压-心肌肥厚-心力衰竭动物模型,按随机数字表法分为A组(正常饲料喂养组)、B组(高盐饲料喂养组)、C组(早期NF-κB抑制组)、D组(高血压期NF-κB抑制组)、E组(心肌肥厚期12周NF-κB抑制组)、F组(心肌肥厚期16周NF-κB抑制组)、G组(心力衰竭期NF-κB抑制组),每组6只。各组分别按特定时间段予以8%高盐饲养及PDTC 100 mg/(kg·d)腹腔内注射,观察不同时长的PDCT给药期对血压、左心室舒张末期室间隔厚度(IVSD)、左室舒张末期后壁厚度(LVPWD)、左室舒张末期内径(LVEDD)、左室收缩末期内径(LVESD)、左室射血分数(LVEF)、心肺质量/体质量比值、NF-κB活性、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6、单核细胞趋化蛋白(MCP)-1、C反应蛋白(CRP)的影响。结果早期给予PDTC可抑制NF-κB和促炎症细胞因子水平,大鼠心脏结构改变减轻且心脏功能仅呈轻度降低,给药时间越长,对心脏的保护作用越大,可有效地控制血压,在一定程度上阻断未来可能发生的左室肥厚与左室衰竭。当高血压持续、心肌肥厚形成并伴有心力衰竭的情况下,PDTC用药效果作用有限。结论 PDTC对大鼠高血压、心室肥厚及充血性心力衰竭的发生发展具有预防和治疗作用,早期应用对维持大鼠心脏的正常心功能作用更加明显。

高糖对INS-1细胞胰岛素基因表达的影响及PDTC的保护作用

目的研究高糖毒性对INS-1细胞胰岛素基因及其转录因子PDX-1和Maf A表达的影响,及NF-κB抑制剂吡咯烷二硫基甲酸盐(PDTC)的保护作用,探讨"糖毒性"的生化机制。方法分别以4.0 mmol/L葡萄糖(NG组)、16.7mmol/L葡萄糖(HG组)及16.7 mmol/L葡萄糖+50μmol/L PDTC(HG+P组)培养INS-1细胞,48 h后,离心收集细胞,提取总RNA,以real-ti me PCR法检测胰岛素基因及转录因子PDX-1和Maf A的mRNA水平。结果HG组与NG组比较,胰岛素mRNA的表达下降了64.9%(P<0.05);PDX-1 mRNA的表达下降了82.3%(P<0.01);Maf A mRNA的表达下降了80.9%(P<0.01)。HG+P组与HG组相比,胰岛素mRNA的表达则提高了6.67倍(P<0.05);PDX-1mRNA的表达提高了7.52倍(P<0.05);Maf A mRNA的表达提高了5.70倍(P<0.01);而与NG组相比,胰岛素、PDX-1和Maf A mRNA的表达差异均无统计学意义(均P>0.05)。结论"糖毒性"可以通过激活INS-1细胞内的NF-κB途径,使胰岛素转录因子PDX-1和Maf A的表达下降,而导致胰岛素基因表达下降,抑制NF-κB可起到保护作用。

核因子κB抑制剂PDTC对颈椎病大鼠椎间盘炎症反应的抑制作用

目的探讨核因子κB(NF-κB)抑制剂吡咯烷二硫基甲酸盐(PDTC)对颈椎病大鼠椎间盘炎症反应的抑制作用。方法构建动静力失衡性颈椎病大鼠模型,将造模成功大鼠分为模型组,PDTC低、中、高剂量组,另外设立假手术组(仅切开颈部皮肤),每组大鼠10只,PDTC低、中、高剂量组大鼠每天分别腹腔注射50 mg/kg、100 mg/kg、200 mg/kg PDTC,假手术组及模型组腹腔注射等量生理盐水,连续28 d。酶联免疫吸附试验(ELISA)检测各组大鼠椎间盘组织中肿瘤坏死因子-α(TNF-α),白介素-1β(IL-1β)及IL-6含量,Realtime PCR检测TNF-α,IL-1β及IL-6 mRNA表达,Western blot检测各组大鼠椎间盘组织中NF-κB p65、NF-κB抑制蛋白(IκBα)表达。结果与假手术组比较,模型组中大鼠椎间盘组织中TNF-α,IL-1β及IL-6 mRNA及蛋白含量提高(P <0. 05),p-NF-κB p65、p-IκBα表达量上调(P <0. 05)。与模型组比较,PDTC低、中、高剂量组大鼠椎间盘组织中TNF-α,IL-1β及IL-6 mRNA及蛋白含量降低(P <0. 05),p-NF-κB p65、p-IκBα表达量下调(P <0. 05),并呈剂量依赖性。结论 PDTC能显著地抑制颈椎病大鼠椎间盘炎症反应,并其作用呈剂量依赖性。

PDTC对心衰大鼠心肌细胞炎症通路的影响

目的观察NF-κB抑制剂吡咯烷二硫基甲酸盐(PDTC)对大鼠心衰模型心脏核因子-κB活性的干预及炎症递质的影响。方法雄性Dahl盐敏感大鼠建立高血压-心肌肥厚-心力衰竭动物模型42只,随机分为A组(喂养正常饲料组)、B组(喂养高盐饲料组)、C组(早期加用PDTC组)、D组(高血压期加用PDTC组)、E组(心肌肥厚期12周后加用PDTC组)、F组(心肌肥厚期16周加用PDTC组)、G组(心力衰竭期加用PDTC组)共7组(n=6)。各组分别按特定时间段予以8%高盐饲养及PDTC 100 mg/kg/d腹腔内注射,统一观察不同时长的PDCT给药期对NF-κB活性、TNF-α、IL-6、MCP-1、CRP的影响。结果早期给予PDTC能更好的降低心衰大鼠心肌的炎症反应。结论 PDTC能够抑制心衰大鼠心肌的炎症反应,早期应用作用更加明显。

Activation of nuclear factor-kappa B and effects of pyrrolidine dithiocarbamate on TNBS-induced rat colitis

AIM: To explore the changes of nuclear factor-kappa B (NF-κB) DNA-binding activity, the expression of intercellular adhesion molecule-1(ICAM-1) regulated by IMF-κB at various times and to evaluate the effects of pyrrolidine dithiocarbamate (PDTC) on trinitrobenzene sulfonic acid (TNBS)-induced rat colitis. METHODS: TNBS of 0.6 mL was mixed with ethanol of 0.3 mL solution and instilled into the lumen of the rat colon. The rat models were divided into 6 groups, which were killed at 24 h, 3, 7,14, and 21 d after enema. Colonic inflammation and damage were assessed by macroscopical and histological criteria. Activity of NF-κB DNA-binding was analyzed by electrophoresis mobility shift assays (EMSA). Expression of ICAM-1 was detected by in situ hybridization (ISH) and immunohistochemistry (IH). Then various doses of PDTC were injected into rat abdomen 30 min before enema with TNBS/ethanol as pretreatment. The rats were killed 4 h after enema and the colonic inflammation, myeloperoxidase (MPO) activity, malondialdehyde (MDA) level, and DNA-binding activity of NF-κB were assessed. Finally, PDTC was injected intraperitoneally after colitis was induced. Changes of morphology were assayed. RESULTS: During the first week, hyperemia, hemorrhage, edema and ulceration of the colonic mucosa appeared with predominant infiltration of leukocytes. Neutrophils, macrophages, lymphocytes infiltrated in mucosa and submucosa 14 d later. Fibroblasts and granuloma-like structures were also obviously seen. The binding activity of NF-κB began to increase at 24 h time point and reached a peak at 14 d, then decreased but still was higher than control group at 21 d (P<0.01). Levels of ICAM-1 mRNA and protein significantly elevated at 24 h and the peak was at 21 d. Pretreatment with PDTC could attenuate the development of inflammation but not by reducing NF-KB activity. This attenuation of inflammation had a positive relationship with the dose of PDTC. PDTC at the dose of 100 mg/kg had no therapeutic effect after colitis was induced. CONCLUSION: NF-κB activation is an important event that may be involved in acute and chronic inflammation development and may contribute to self-protection against early inflammation damage. NF-κB also regulates ICAM-1 expression during colonic inflammation. Pretreatment of PDTC may attenuate the inflammation development. But PDTC has no therapeutic effect after the colitis is induced.