左旋紫草素抗炎作用与其抑制蛋白酶体活性关系的研究
摘要
背景与目的:
炎症的本质即为机体免疫系统对外来异物或自身破碎组织细胞进行清除的过程。适度的炎症反应对机体具有保护作用,但当机体遭受过度或失控性的炎症反应时,则可引起宿主自身组织结构和生理功能的广泛损害,导致系统性炎症反应综合症(Systemic inflammatory response syndrome, SIRS)的发生,最终引起脓毒症及多器官功能障碍综合症(Multiple organ dysfunction syndrome, MODS),甚至死亡。
过度炎症性反应主要是细胞分泌的炎症介质之间相互作用导致炎症信号“瀑布式”放大的效应。大量细胞因子释放入血,TNF-α、ROS、NO及COX-2的产生等将引起SIRS,过度SIRS造成远离组织器官的损伤,继发MODS、脓毒血症。而近几年应用的针对单一炎症介质抑制或拮抗的措施并不能较好地阻断炎症信号“网络”介导的整个炎症反应演进过程。
NF-κB是一种核转录因子,现被认为在炎症性疾病的发病中起关键性调节作用。在正常细胞内,NF-κB和它的抑制性蛋白I-κBα相结合,以非活性的形式存在于胞浆中。当细胞受到细菌、病毒、LPS、TNF-α等外源性刺激后,I-κBα即发生磷酸化,进而泛素化,泛素化后的I-κBα即可被26S proteasome识别并降解;至此,活化的NF-κB便移位到细胞核中,结合到靶基因的转录起始区域,激活细胞因子、粘附分子和COX-2等的表达。26S proteasome是细胞内最主要的非溶酶体蛋白酶复合物,其通过细胞周期时限特异性的生物学机制精确的调控细胞内70-90%的蛋白降解,NF-κB的抑制因子I-κBα及许多与细胞周期循环和凋亡相关的重要因子、转录因子等多经泛素-蛋白酶体途径(UPS)降解。
左旋紫草素是中药紫草的主要活性成分,据文献报道,其具有抗炎、抗肿瘤等作用。本研究组运用建立的蛋白酶体抑制剂筛选平台,证明左旋紫草素可能是一种新的蛋白酶体抑制剂,其抗肿瘤作用与蛋白酶体功能抑制有关。但对其抗炎及其他作用与蛋白酶体抑制效应的关系尚未明确。本实验通过观察左旋紫草素的抗炎作用与其蛋白酶体活性抑制效应之间的关系,探讨其抗炎作用与抑制蛋白酶体活性的机制;同时复制了三种动物模型探讨其对急性炎症抗炎和脓毒症小鼠存活率的影响,为左旋紫草素用于炎症相关性疾病的防治提供实验依据。
方法
1.无菌收集培养SD大鼠腹腔巨噬细胞,分别加入左旋紫草素预处理及脂多糖(LPS,1μg/ml)刺激。实验组予以不同浓度左旋紫草素预处理加LPS刺激、阳性对照组予以脂多糖刺激。
2.应用ELISA方法检测各组细胞培养上清液中炎症介质TNF-α的含量,观察左旋紫草素的抗炎效应。
3.应用细胞免疫荧光方法检测转录因子NF-κB各组细胞内核转位情况,观察左旋紫草素对各组细胞NF-κB活性的抑制效应。
4.应用Western blot方法检测左旋紫草素对各组腹腔巨噬细胞泛素化蛋白(Ub-prs)和NF-κB抑制蛋白I-κBα的表达水平,观察转录因子NF-κB活性抑制与蛋白酶体活性抑制的关系。
5.应用流式细胞仪,AnnexinV和PI双染法检测分析由左旋紫草素诱导的小鼠巨噬细胞系Ana/1细胞凋亡情况。
6.分别采用二甲苯致小鼠耳廓肿胀、醋酸致小鼠腹腔毛细血管通透性增高的方法,探讨左旋紫草素对急性炎症的抗炎作用。
7.复制由LPS(35mg/kg)诱导的小鼠脓毒症模型,观察左旋紫草素对小鼠脓毒症存活率的影响。
结果
1.左旋紫草素抑制巨噬细胞释放炎症介质TNF-α,且呈浓度依赖。结果显示,正常大鼠腹腔巨噬细胞在体外RPMI1640培养基中表达少量的细胞因子TNF-α;LPS刺激后,TNF-α表达较空白对照组明显增加(P<0.05);用左旋紫草素预处理后,其细胞培养上清液中TNF-α的分泌量与阳性组相比明显下降(P<0.05),且呈浓度依赖,并于4h时抑制效果更明显。
2.左旋紫草素抑制转录因子NF-κB核转位,且呈浓度依赖。结果显示,LPS阳性对照组细胞核内NF-κBp65的表达明显增强;左旋紫草素预处理的腹腔巨噬细胞,NF-κBp65的核内表达与LPS组相比有明显的下降,且表达量与药物浓度负相关。
3.左旋紫草素对腹腔巨噬细胞Ub-prs和I-κBα蛋白表达水平的影响。结果表明,反映UPS系统功能的内源性蛋白Ub-prs、I-κBα的表达量在LPS阳性组有明显的下降,而左旋紫草素处理组与阳性组相比有明显的升高,且有剂量依赖关系。
4.左旋紫草素诱导小鼠巨噬细胞系Ana/1细胞凋亡,且呈浓度依赖。结果显示,左旋紫草素对Ana/1细胞在低剂量时即有促凋亡作用,在剂量4μmol/L时凋亡率达到66.5%。
5.左旋紫草素抑制二甲苯所致小鼠耳廓肿胀及减轻醋酸所致小鼠腹腔毛细血管的通透性。结果显示,左旋紫草素各剂量组均能明显抑制二甲苯所致小鼠耳廓肿胀程度及减轻醋酸所致小鼠腹腔毛细血管的通透性(P<0.05),且呈剂量依赖性。
6.左旋紫草素增加了由LPS诱导的小鼠脓毒症的存活率。结果显示,各左旋紫草素处理组均能明显增加由LPS诱导的小鼠脓毒症存活率(P<0.05),且以2mg/kg剂量组效果最好。
结论
1.左旋紫草素能够下调由LPS刺激活化的腹腔巨噬细胞中炎症因子TNF-α的表达释放。
2.左旋紫草素对LPS激活的巨噬细胞蛋白酶体活性具有抑制作用。
3.左旋紫草素能抑制LPS激活的巨噬细胞NF-κB途径的活化。
4.左旋紫草素对炎症动物模型有抗炎作用。
Introduction:
Inflammation is a process by which the body’s immune system protect us from infection and foreign substances such as bacteria and viruses. When inflammation occurs normally, it is basically a protective mechanism. Sometimes, however, excessive inflammatory response causes damage to the body’s tissues, and finally sepsis and multiple organ dysfunction syndrome(MODS).
Excessive inflammatory response is mainly a complex network of activated inflammatory mediators. Recreuitment and activation of various inflammatory cells cause a release of various proinflammatory mediators such as TNF-α、IL-1、IL-6 and other chemokines and anti-inflammatory factors such as IL-10. An imbalance in the pathway results in a sustained or exaggerated systemic inflammatory response syndrome(SIRS), followed by widespread tissue damage and death from MODS and septicaemia. Single cytokine antagonist therapy has not yet proven to be of clinical benefit on excessive inflammatory diseases.
Nuclear factor-κB(NF-κB) is a pluripotent transcription factor which plays an important role in regulating the host inflammatory, immune and anti-apoptotic responses. Under basal conditions, NF-κB is sequestered in an inactive form in the cytoplasm through an association with the inhibitory protein I-κBα. When stimulated by inflammatory stress such as TNF-α、IL-1 or lipopolysaccharide(LPS), I-κBαis phosphorylated, ubipuitylated, and subsequently degraded by the 26S proteasome. The degradation of I-κBαallows NF-κB to translocate into the nucleus to direct the transcription of target genes encoding cytokines, chemokines, anti-apoptotic proteins and adhension molecules required for neutrophil adhension and migration. Therefore, inhibition of the UPS may represent a novel therapeutic strategy for acute and chronic inflammatory diseases.
Chinese medicine shikonin has a beneficial effect on treating inflammatory diseases, which has proved by the many year’s clinical application. It has proved that shikonin may be a novel proteasome inhibitor and its anti-tumor function may be closely associated with inhibition of the proteasome activity. But the association of its anti-inflammatory effects with proteasome inhibition is not clear. In our study, the aim was to observe the anti-inflammatory effects of shikonin on LPS-induced peritoneal macrophages and the association of its anti-inflammatory effects with proteasome inhibition to provide experimental evidences for treating systemic inflammatory response diseases and looking for more fitable drug-effecting targets.
Methods:
1. Peritoneal macrophages(PM) were obtained by lavage using PBS. Macrophages were randomly divided into these groups: control group,received RPMI media; LPS group,received LPS 1μg/ml; shikonin groups,received different doses of shikonin 1.5h before stimulation with LPS 1μg/ml.
2. TNF-αproduction in cell cultured supernatants was determined by ELISA kit.
3. NF-κB translocation from the cytoplasm to the nucleus were detected by immunocyto-staining method.
4. The proteasome substrate proteins Ub, I-κBαwere detected by western blot assay.
5. Cell apoptosis analysis was carried out by AnnexinV-FITC/PI staining and Fluorescence-activated cell sorting (FACS).
6. The animal models of mouse’s auricle swelling induced by xylene , the increase of capillary permeability in mice induced by acetic acid were used to determine the acute anti-inflammatory effects of shikonin.
7. Effects of shikonin on mouse survival in LPS(35mg/kg)-induced murine septic models.
Results:
1. Shikonin inhibited the expression of TNF-αin primary macrophage in a dose-dependent manner.
LPS induced a dramatic increase in the concentration of TNF-αin cell cultured supernatants. Pretreatment with shikonin attenuated the effect of LPS in dose-dependent manner. Furthermore, it was found that shikonin significantly inhibited TNF-αproduction at 4h(P<0.05).
2. Shikonin inhibited the expression of NF-κB in the nuclei in a dose-dependent manner.
The nuclear NF-κB/p65 protein level in the LPS stimulated PM markedly increased when compared to the control. Pretreatment of macrophages with shikonin significantly reduced the LPS induced expression of NF-κB/p65 in a dose-dependent manner.
3. Shikonin accumulated I-κBαand ubiquitinated proteins in primary macrophages.
It was shown that the endogenous proteasome substrates: both Ub and I-κBαwere increased in a concentration-dependent manner.
4. Shikonin induced apoptosis of Ana/1 cells in a dose-dependent manner.
It was found that Ana/1 cells could be induced to apoptosis by Shikonin in a dose-dependent manner and the apoptotic rate reached as high as 66.5% at 4μmol/L.
5. Shikonin inhibited mouse’s auricle swelling induced by xylene and the increase of capillary permeability in mice induced by acetic acid.
It was found that different doses of shikonin have obvious inhibitory action on mouse’s auricle swelling induced by xylene and the increase of capillary permeability in mice induced by acetic acid (P<0.05)
6. Shikonin increased the survival rates of LPS-induced sepsis in mice.
The results demonstrated that the survival rates of mice challenged with LPS were significantly higher in groups pretreated with shikonin than that in controls (p<0.05).
Conclusions:
1. Shikonin can inhibit the expression of TNF-αprotein in LPS-induced peritoneal macrophages.
2. Shikonin can inhibit proteasome activity of LPS-induced peritoneal macrophages.
3. Shikonin can inhibit NF-κB activity of LPS-induced peritoneal macrophages.
4. Shikonin have remarkable anti-inflammatory effects in the animal models and increase the survival rates of LPS-induced sepsis.
炎症的本质即为机体免疫系统对外来异物或自身破碎组织细胞进行清除的过程。适度的炎症反应对机体具有保护作用,但当机体遭受过度或失控性的炎症反应时,则可引起宿主自身组织结构和生理功能的广泛损害,导致系统性炎症反应综合症(Systemic inflammatory response syndrome, SIRS)的发生,最终引起脓毒症及多器官功能障碍综合症(Multiple organ dysfunction syndrome, MODS),甚至死亡。
过度炎症性反应主要是细胞分泌的炎症介质之间相互作用导致炎症信号“瀑布式”放大的效应。大量细胞因子释放入血,TNF-α、ROS、NO及COX-2的产生等将引起SIRS,过度SIRS造成远离组织器官的损伤,继发MODS、脓毒血症。而近几年应用的针对单一炎症介质抑制或拮抗的措施并不能较好地阻断炎症信号“网络”介导的整个炎症反应演进过程。
NF-κB是一种核转录因子,现被认为在炎症性疾病的发病中起关键性调节作用。在正常细胞内,NF-κB和它的抑制性蛋白I-κBα相结合,以非活性的形式存在于胞浆中。当细胞受到细菌、病毒、LPS、TNF-α等外源性刺激后,I-κBα即发生磷酸化,进而泛素化,泛素化后的I-κBα即可被26S proteasome识别并降解;至此,活化的NF-κB便移位到细胞核中,结合到靶基因的转录起始区域,激活细胞因子、粘附分子和COX-2等的表达。26S proteasome是细胞内最主要的非溶酶体蛋白酶复合物,其通过细胞周期时限特异性的生物学机制精确的调控细胞内70-90%的蛋白降解,NF-κB的抑制因子I-κBα及许多与细胞周期循环和凋亡相关的重要因子、转录因子等多经泛素-蛋白酶体途径(UPS)降解。
左旋紫草素是中药紫草的主要活性成分,据文献报道,其具有抗炎、抗肿瘤等作用。本研究组运用建立的蛋白酶体抑制剂筛选平台,证明左旋紫草素可能是一种新的蛋白酶体抑制剂,其抗肿瘤作用与蛋白酶体功能抑制有关。但对其抗炎及其他作用与蛋白酶体抑制效应的关系尚未明确。本实验通过观察左旋紫草素的抗炎作用与其蛋白酶体活性抑制效应之间的关系,探讨其抗炎作用与抑制蛋白酶体活性的机制;同时复制了三种动物模型探讨其对急性炎症抗炎和脓毒症小鼠存活率的影响,为左旋紫草素用于炎症相关性疾病的防治提供实验依据。
方法
1.无菌收集培养SD大鼠腹腔巨噬细胞,分别加入左旋紫草素预处理及脂多糖(LPS,1μg/ml)刺激。实验组予以不同浓度左旋紫草素预处理加LPS刺激、阳性对照组予以脂多糖刺激。
2.应用ELISA方法检测各组细胞培养上清液中炎症介质TNF-α的含量,观察左旋紫草素的抗炎效应。
3.应用细胞免疫荧光方法检测转录因子NF-κB各组细胞内核转位情况,观察左旋紫草素对各组细胞NF-κB活性的抑制效应。
4.应用Western blot方法检测左旋紫草素对各组腹腔巨噬细胞泛素化蛋白(Ub-prs)和NF-κB抑制蛋白I-κBα的表达水平,观察转录因子NF-κB活性抑制与蛋白酶体活性抑制的关系。
5.应用流式细胞仪,AnnexinV和PI双染法检测分析由左旋紫草素诱导的小鼠巨噬细胞系Ana/1细胞凋亡情况。
6.分别采用二甲苯致小鼠耳廓肿胀、醋酸致小鼠腹腔毛细血管通透性增高的方法,探讨左旋紫草素对急性炎症的抗炎作用。
7.复制由LPS(35mg/kg)诱导的小鼠脓毒症模型,观察左旋紫草素对小鼠脓毒症存活率的影响。
结果
1.左旋紫草素抑制巨噬细胞释放炎症介质TNF-α,且呈浓度依赖。结果显示,正常大鼠腹腔巨噬细胞在体外RPMI1640培养基中表达少量的细胞因子TNF-α;LPS刺激后,TNF-α表达较空白对照组明显增加(P<0.05);用左旋紫草素预处理后,其细胞培养上清液中TNF-α的分泌量与阳性组相比明显下降(P<0.05),且呈浓度依赖,并于4h时抑制效果更明显。
2.左旋紫草素抑制转录因子NF-κB核转位,且呈浓度依赖。结果显示,LPS阳性对照组细胞核内NF-κBp65的表达明显增强;左旋紫草素预处理的腹腔巨噬细胞,NF-κBp65的核内表达与LPS组相比有明显的下降,且表达量与药物浓度负相关。
3.左旋紫草素对腹腔巨噬细胞Ub-prs和I-κBα蛋白表达水平的影响。结果表明,反映UPS系统功能的内源性蛋白Ub-prs、I-κBα的表达量在LPS阳性组有明显的下降,而左旋紫草素处理组与阳性组相比有明显的升高,且有剂量依赖关系。
4.左旋紫草素诱导小鼠巨噬细胞系Ana/1细胞凋亡,且呈浓度依赖。结果显示,左旋紫草素对Ana/1细胞在低剂量时即有促凋亡作用,在剂量4μmol/L时凋亡率达到66.5%。
5.左旋紫草素抑制二甲苯所致小鼠耳廓肿胀及减轻醋酸所致小鼠腹腔毛细血管的通透性。结果显示,左旋紫草素各剂量组均能明显抑制二甲苯所致小鼠耳廓肿胀程度及减轻醋酸所致小鼠腹腔毛细血管的通透性(P<0.05),且呈剂量依赖性。
6.左旋紫草素增加了由LPS诱导的小鼠脓毒症的存活率。结果显示,各左旋紫草素处理组均能明显增加由LPS诱导的小鼠脓毒症存活率(P<0.05),且以2mg/kg剂量组效果最好。
结论
1.左旋紫草素能够下调由LPS刺激活化的腹腔巨噬细胞中炎症因子TNF-α的表达释放。
2.左旋紫草素对LPS激活的巨噬细胞蛋白酶体活性具有抑制作用。
3.左旋紫草素能抑制LPS激活的巨噬细胞NF-κB途径的活化。
4.左旋紫草素对炎症动物模型有抗炎作用。
Introduction:
Inflammation is a process by which the body’s immune system protect us from infection and foreign substances such as bacteria and viruses. When inflammation occurs normally, it is basically a protective mechanism. Sometimes, however, excessive inflammatory response causes damage to the body’s tissues, and finally sepsis and multiple organ dysfunction syndrome(MODS).
Excessive inflammatory response is mainly a complex network of activated inflammatory mediators. Recreuitment and activation of various inflammatory cells cause a release of various proinflammatory mediators such as TNF-α、IL-1、IL-6 and other chemokines and anti-inflammatory factors such as IL-10. An imbalance in the pathway results in a sustained or exaggerated systemic inflammatory response syndrome(SIRS), followed by widespread tissue damage and death from MODS and septicaemia. Single cytokine antagonist therapy has not yet proven to be of clinical benefit on excessive inflammatory diseases.
Nuclear factor-κB(NF-κB) is a pluripotent transcription factor which plays an important role in regulating the host inflammatory, immune and anti-apoptotic responses. Under basal conditions, NF-κB is sequestered in an inactive form in the cytoplasm through an association with the inhibitory protein I-κBα. When stimulated by inflammatory stress such as TNF-α、IL-1 or lipopolysaccharide(LPS), I-κBαis phosphorylated, ubipuitylated, and subsequently degraded by the 26S proteasome. The degradation of I-κBαallows NF-κB to translocate into the nucleus to direct the transcription of target genes encoding cytokines, chemokines, anti-apoptotic proteins and adhension molecules required for neutrophil adhension and migration. Therefore, inhibition of the UPS may represent a novel therapeutic strategy for acute and chronic inflammatory diseases.
Chinese medicine shikonin has a beneficial effect on treating inflammatory diseases, which has proved by the many year’s clinical application. It has proved that shikonin may be a novel proteasome inhibitor and its anti-tumor function may be closely associated with inhibition of the proteasome activity. But the association of its anti-inflammatory effects with proteasome inhibition is not clear. In our study, the aim was to observe the anti-inflammatory effects of shikonin on LPS-induced peritoneal macrophages and the association of its anti-inflammatory effects with proteasome inhibition to provide experimental evidences for treating systemic inflammatory response diseases and looking for more fitable drug-effecting targets.
Methods:
1. Peritoneal macrophages(PM) were obtained by lavage using PBS. Macrophages were randomly divided into these groups: control group,received RPMI media; LPS group,received LPS 1μg/ml; shikonin groups,received different doses of shikonin 1.5h before stimulation with LPS 1μg/ml.
2. TNF-αproduction in cell cultured supernatants was determined by ELISA kit.
3. NF-κB translocation from the cytoplasm to the nucleus were detected by immunocyto-staining method.
4. The proteasome substrate proteins Ub, I-κBαwere detected by western blot assay.
5. Cell apoptosis analysis was carried out by AnnexinV-FITC/PI staining and Fluorescence-activated cell sorting (FACS).
6. The animal models of mouse’s auricle swelling induced by xylene , the increase of capillary permeability in mice induced by acetic acid were used to determine the acute anti-inflammatory effects of shikonin.
7. Effects of shikonin on mouse survival in LPS(35mg/kg)-induced murine septic models.
Results:
1. Shikonin inhibited the expression of TNF-αin primary macrophage in a dose-dependent manner.
LPS induced a dramatic increase in the concentration of TNF-αin cell cultured supernatants. Pretreatment with shikonin attenuated the effect of LPS in dose-dependent manner. Furthermore, it was found that shikonin significantly inhibited TNF-αproduction at 4h(P<0.05).
2. Shikonin inhibited the expression of NF-κB in the nuclei in a dose-dependent manner.
The nuclear NF-κB/p65 protein level in the LPS stimulated PM markedly increased when compared to the control. Pretreatment of macrophages with shikonin significantly reduced the LPS induced expression of NF-κB/p65 in a dose-dependent manner.
3. Shikonin accumulated I-κBαand ubiquitinated proteins in primary macrophages.
It was shown that the endogenous proteasome substrates: both Ub and I-κBαwere increased in a concentration-dependent manner.
4. Shikonin induced apoptosis of Ana/1 cells in a dose-dependent manner.
It was found that Ana/1 cells could be induced to apoptosis by Shikonin in a dose-dependent manner and the apoptotic rate reached as high as 66.5% at 4μmol/L.
5. Shikonin inhibited mouse’s auricle swelling induced by xylene and the increase of capillary permeability in mice induced by acetic acid.
It was found that different doses of shikonin have obvious inhibitory action on mouse’s auricle swelling induced by xylene and the increase of capillary permeability in mice induced by acetic acid (P<0.05)
6. Shikonin increased the survival rates of LPS-induced sepsis in mice.
The results demonstrated that the survival rates of mice challenged with LPS were significantly higher in groups pretreated with shikonin than that in controls (p<0.05).
Conclusions:
1. Shikonin can inhibit the expression of TNF-αprotein in LPS-induced peritoneal macrophages.
2. Shikonin can inhibit proteasome activity of LPS-induced peritoneal macrophages.
3. Shikonin can inhibit NF-κB activity of LPS-induced peritoneal macrophages.
4. Shikonin have remarkable anti-inflammatory effects in the animal models and increase the survival rates of LPS-induced sepsis.
引文
1. Abraham E,et al. Why immunomodulatory therapries have not worked in sepsis. Intensive Care Med, 1999,25(6):556-566.
2. Baue AE ,et al. Multiple organ failure,multiple organ dysfunction syndrome and systemic inflammatory response syndrome:Why no magic bullets? Arch Surg.1997,132(7):703-707.
3. Zeni F,et al. Anti-inflammatory therapies to treat sepsis and septic shock: a reassessment. Crit Care Med,1997,25(7):1115-1124.
4. Sigal LH ,et al. Basic science for the clinician: NF– kappaB-function, activation, control, and consequences. J Clin Rheumatol, 2006, 12(4): 207-211.
5.Chung KC, et al . Novel biphasic effect of pyrrolidine dithiocarbamate on neuronal cell viability is mediated by the differential regulation of intracellular zinc and copper ion levels , NF - kappaB ,and MAP kinases. J Neurosci Res , 2000 , 59 : 117 - 125.
6.Sen R , et al. Multiple nuclear factors interact with the immunol globulin enhancer sequences. Cell , 1986 , 46 : 705 - 716.
7.Siebenlist U , et al. Structure ,regulation and function of NF-κB. Annu Rev Cell Biol , 1994 , 10 : 405 - 455.
8.Baldwin AS Jr , et al. Series introduction : the transcription factor NF– kappaB and human disease. J Clin Invest , 2001 , 107 : 3 - 6.
9. Zandy AJ, et al. Proteolytic mechanisms underlying mitochondrial degradation in the ocular lens. Znvest Ophthalmol Vis Sci, 2007,21(2): 245-249.
10. Williams A J, et al. Neuroprotection with the proteasome inhibitor MLN519 in focal ischemic brain injury: relation to nuclear factor KappaB(NF–kappaB), inflammatory gene expression, and leukocyte infiltration. Neurochem Int,2006,49(2):106-112.
11. Peng Z, et al. Evidence for a physical association of the COP9 signalosome, the proteasome, and specific SCF E3 ligases in vivo. Curr Biol. 2003, 8(13): 504–505.
12. Ciechanover, et al. The ubiquitin proteolytic system: From a vague idea, through basicmechanisms, and onto human diseases and drug targeting Neurology. Inclusion-body myositis: Clinical and pathologic aspects, and basic research potentially relevant to treatment. 2006, 66(2): 7-19.
13. Osterlund MT, et al. The role of COP1 in repression of Arabidopsis photomorphogenic development. Trends Cell Biol. 1999, 9(3):113-118.
14. Schwechheimer C, et al. Multiple ubiquitin ligase-mediated processes require COP9 signalosome and AXR1 function.Plant Cell. 2002, 14(10): 2553 -2563.
15. Naumann, M, et al. COP9 signalosome-directed c-Jun activation/ stabiliza- tion is in- dependent of JNK. J.Biol.Chem.1999, 274: 35297–35300.
16. Ho MS, et al. F-box proteins: the key to protein degradation J Biomed Sci, 2006, 13(2):181-91.
17. Pan ZQ, et al. Nedd8 on cullin: building an expressway to protein destruction. Onco- gene, 2004, 23: 1985–1997.
18. Musikacharoen T, et al. NF-κB and STATs play important roles in the regulation of mouse Toll-like receptor gene expression. Immunol. 2001,166:4516-4520.
19. Driessler F, et al. Molecular mechanisms of interleukin-10-mediated inhibition of NF–kappaB activity: a role for p50. Clin. Exp. Immunol. 2004,135:64-73.
20. Dijsselbloem N, et al. A critical role for p53 in the control of NF–kappaB-dependent gene expression in TLR4-stimulated dendritic ceel exposed to genistein. J Immunol. 2007,15,178(8):5048-5057.
21. Ulloa L, et al. Ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation. Proc Natl Acad Sci USA. 2002,99:12351-6.
22. Ciechanover A, et al. The ubiquitin-mediated proteolytic pathway: mode of action and clinical implication[J]. J Cell Biochem,Suppl 2000,34:40-51.
23. Rock KL, et al. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC classⅠmolecules. Cell. 1994,78:761-771.
24. Chenggong Z, et al. Regulation of murine cardiac 20S proteasomes role of associatingpartners. Circ Res,2006,99(7):372-380.
25.Niranjan Rajapakse, et al. Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-stimulated RAW264.7 Cells by carboxybutyrylated glucosamine takes place via down-regulation of mitogen-activated protein kinase-mediated nuclear factor-κB signaling. Immunology. 123:348-357.
26. Eilliott PJ, et al. Proteasome inhibition: a new anti-inflammatory strategy[J]. J Mol Med, 2003,81(4):235-245.
27.戴翠莲等.蛋白酶体抑制剂MG-132下调炎症因子表达抑制大鼠心肌再灌注损伤第三军医大学学报2007, 29(2):129-133.
28. Sinn DI, et al. Proteasomal inhibition in intracerebral hemorrhage: neuroprotective and anti-inflammatory effects of bortezomib. Neurosci Res. 2007,58:12-18.
29. Meiners S, et al. Suppression of cardiomyocyte hypertrophy by inhibition of the ubiquitin-proteasome system. Hypertension, 2008,51(2):302-308.
30. Zhou H, Wertz I, O`Rourke K, et al. Bcl10 activates the NF-kappa B pathway through ubiquitination of NEMO. Nature. 2004, 127(6970): 167-171
31.尹会男柴家科姚咏明等.泛素一蛋白酶体途径对烫伤脓毒症大鼠肠道炎症反应与屏障功能的作用研究。中国危重病急救医学,2006,18(11):649-652
32.尹会男柴家科姚咏明等.蛋白酶体抑制剂在烫伤脓毒症大鼠肺脏炎症反应中的作用。中华急诊医学杂志,2006,15(10):886-889
33. Jing Shen, et al. Key inflammatory signaling pathways are regulated by the proteasome. Shock. 2006,25(5):472-84.
34. Kim CS, et al. Capsaicin exhibits anti-inflammatory property by inhibiting IκBa degradation in LPS-stimulated peritoneal macrophages. Cell Signal. 2003,15:299-306.
35. Yang H, et al. Shikonin exerts antitumor activity via proteasome inhibition and cell death induction in vitro and in vivo. IntJ Cancer. 2009,124(10):2450-9.
36. Span LF, et al. The dynamic process of apoptosis analyzed by flow cytometry using Annexin-V/propidium iodide and a modified in situ end labeling technique[J]. Cytometry,2002,47(1):24-31.
37. Budihardjo I, et al. Biochemical pathways of caspase activation during apoptosis[J]. Annu Rev Cell Dev Biol. 1999,15:269-90.
38. Hengartner MO, et al. The biochemistry of apoptosis[J]. Nature, 2000, 407 (6805): 770-6.
39. Saikumar P, et al. Apoptosis: definition, mechanisms,and relevance to disease[J]. Am J Med,1999,107(5):489-506.
40. Rao RV, et al. Coupling endoplasmic reticulum stress to the cell death program. Mechnism of caspase activation[J]. J Biol Chem,2001, 276(36): 33869-33874.
41. Safranek, et al. Modulation of inflammatory response in sepsis by proteasome inhibition. 2006, Oct;87(5):369-72.
1.Pickart CM, Eddins MJ. Ubiquitin: structures, functions, mechanisms[J]. Biochim BiophysActa, 2004, 1695 (1-3) : 55 - 72.
2.Herrmann J, CiechanoverA, Lerman LO, et al. The ubiquitin -proteasome system in cardiovascular diseases: a hypothesis extended[J]. Cardiovasc Res, 2004, 61 (1) : 11 - 21.
3.Patterson C. Search and destroy: the role of protein quality control in maintaining cardiac function [J]. J Mol Cell Cardiol, 2006, 40(4) : 438 - 441.
4.Raasi S, Wolf DH. Ubiquitin receptors and ERAD: A network of pathways to the proteasome [J]. Sem in Cell Dev Biol, 2007, 18(6) : 78 - 91.
5.Fricke B, Heink S, Steffen J, et al. The proteasome maturation protein POMP facilitates major steps of 20S proteasome formation at the endoplasmic reticulum[J]. EMBO Rep, 2007, 8 (12) : 1170 -5.
6.Meiners S, Ludwig A, Stangl V, et al. Proteasome inhibitors: Poisons and remedies[J]. Med Res Rev, 2008, 28 (2) : 309 - 27.
7.MarquesA J, Glanemann C, Ramos P C, et al. The C terminal extension of the beta 7 subunit and activator complexes stabilize nascent 20s proteasomes and promote their maturation [J]. J Biol Chem , 2007, 282 (48) : 34869 - 76.
8.王岚,孙圣刚,曹学兵,等.格尔德霉素通过诱导热休克蛋白反应抑制DA能神经元蛋白水解应激的研究[J].中国药理学通报, 2007, 23 (8) : 1025 - 9.
9.Wang L, Sun SG, Cao XB, et al. Geldanamycin activates a heat shock response and inhibits proteolytic stress in dopaminergic neurons[J]. Chin Pharmacol Bull, 2007, 23 (8) : 1025 - 9.
10.Kim H T, Kim K P, Lledias F, et al. Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize non-degradable forked ubiquitin chains containing all possible isopeptide linkages [J]. J Biol Chem , 2007. 282( 24 ) :17375 - 86.
11.Kwon Y T, Kashina A S, Varshavsky A. Alternative splicing results in differential expression, activity, and localization of the two forms of arginyl tRNA protein transferase, a component of the N-end rule pathway[J]. Mol Cell Biol, 1999. 19 (1) : 182– 93.
12.Aaron C, Schwartz AL.Ubiquitin-mediated degradation of cellular proteins in health and disease. Hepatology, 2002,35(1):3-6.
13.Sun Z, Andersson R. NF-kappaB activation and inhibition. Shock, 2002, 18: 99-106.
14. Jesenberger V, Jentsch S. Deadly encounter: ubiquition meets apoptosis. Nat Rev Mol Cell Biol, 2002, 3(2): 112-21.
15.Simon R,Struckmann K,Schraml P. Amplification pattern of 12q13q15 genes(MDM2 CDK4, CLI) in urinary bladder cancer[J]. Oncogene, 2002, 21(16): 2476-2483.
16.Bloom J,Pagano M. Deregulated degradation of the cdk inhibitor p27 and malignant transformation[J]. Semin Cancer Biol, 2003, 13(1):41-47.
17 . Viglietto C. Fusee A. Understanding p27 Kipl deregulation in cancer: Down-regulation or mislocalisation[J]. Cell Circle, 2002, l(6): 394-400.
18.Gstaiger M,Jordan R,Lim M,et a1. Skp2 is oncogenic and overexpressed in human cancer[J]. Pmc Natl Acad Sci USA. 200l, 98(9): 5043-5048.
19.Pugh CW,Ratcliffe PJ. The von Hippela-Lindau tumor suppressor, hypoxia-inducible factor-l(HIF-1) degradation and cancer pathogenesis[J]. Semin Cancer Biol, 2003,13(1): 83-89.
20.Lorrick KL,Jensen JP,Fang S. et al. RING fingers mediate ubiquitin—conjugating enzyme(E2)-dependent ubiquitination[J]. Proc Natl Acad Sci USA, l999, 96(20): ll364-ll369.
21.Mani A ,Gelmann EP. The ubiquitin-proteasome Pathway and its role in cancer[J]. Clin Oncol, 2005, 23(21): 4776-4789.
22.Williams A J, et al. Neuroprotection with the proteasome inhibitor MLN519 in focal ischemic brain injury: relation to nuclear factor KappaB(NF–kappaB), inflammatorygene expression, and leukocyte infiltration. Neurochem Int,2006,49(2):106-112.
23.Qureshi N, et al. Key inflammatory signaling pathways are regulated by the proteasome. Shock. 2006,25(5):472-84.
24.Qureshi N, et al. The proteasome: a central regulator of inflammation and macrophage function. Immunol Res. 2005;31:243-260.
25.Ward CL,Omura S,Kopito RR. Degradation of CFTR by the ubiquitin-proteasome pathway[J]. Cell,1995, 83(1): 121-127.
26.Kitada T,Asakawa S,Hmtori N.et a1. Mutations in the Parkin gene cause autosomal recessive juvenile parkinsonism[J]. Nature, 1998, 392(6676): 60l5—608.
27.Muyang L,Delin C,Ariel S, et a1. Deubiquitination of p53 by HAUSP is all important pathway for p53 stabili action[J]. Nature, 2002, 416 (6881) : 648– 653.
28.GiovanniD,Angela H, Frank M C, et al. Infetion Imunity, 2000, 68 ( 6) : 3097 -3102.
2. Baue AE ,et al. Multiple organ failure,multiple organ dysfunction syndrome and systemic inflammatory response syndrome:Why no magic bullets? Arch Surg.1997,132(7):703-707.
3. Zeni F,et al. Anti-inflammatory therapies to treat sepsis and septic shock: a reassessment. Crit Care Med,1997,25(7):1115-1124.
4. Sigal LH ,et al. Basic science for the clinician: NF– kappaB-function, activation, control, and consequences. J Clin Rheumatol, 2006, 12(4): 207-211.
5.Chung KC, et al . Novel biphasic effect of pyrrolidine dithiocarbamate on neuronal cell viability is mediated by the differential regulation of intracellular zinc and copper ion levels , NF - kappaB ,and MAP kinases. J Neurosci Res , 2000 , 59 : 117 - 125.
6.Sen R , et al. Multiple nuclear factors interact with the immunol globulin enhancer sequences. Cell , 1986 , 46 : 705 - 716.
7.Siebenlist U , et al. Structure ,regulation and function of NF-κB. Annu Rev Cell Biol , 1994 , 10 : 405 - 455.
8.Baldwin AS Jr , et al. Series introduction : the transcription factor NF– kappaB and human disease. J Clin Invest , 2001 , 107 : 3 - 6.
9. Zandy AJ, et al. Proteolytic mechanisms underlying mitochondrial degradation in the ocular lens. Znvest Ophthalmol Vis Sci, 2007,21(2): 245-249.
10. Williams A J, et al. Neuroprotection with the proteasome inhibitor MLN519 in focal ischemic brain injury: relation to nuclear factor KappaB(NF–kappaB), inflammatory gene expression, and leukocyte infiltration. Neurochem Int,2006,49(2):106-112.
11. Peng Z, et al. Evidence for a physical association of the COP9 signalosome, the proteasome, and specific SCF E3 ligases in vivo. Curr Biol. 2003, 8(13): 504–505.
12. Ciechanover, et al. The ubiquitin proteolytic system: From a vague idea, through basicmechanisms, and onto human diseases and drug targeting Neurology. Inclusion-body myositis: Clinical and pathologic aspects, and basic research potentially relevant to treatment. 2006, 66(2): 7-19.
13. Osterlund MT, et al. The role of COP1 in repression of Arabidopsis photomorphogenic development. Trends Cell Biol. 1999, 9(3):113-118.
14. Schwechheimer C, et al. Multiple ubiquitin ligase-mediated processes require COP9 signalosome and AXR1 function.Plant Cell. 2002, 14(10): 2553 -2563.
15. Naumann, M, et al. COP9 signalosome-directed c-Jun activation/ stabiliza- tion is in- dependent of JNK. J.Biol.Chem.1999, 274: 35297–35300.
16. Ho MS, et al. F-box proteins: the key to protein degradation J Biomed Sci, 2006, 13(2):181-91.
17. Pan ZQ, et al. Nedd8 on cullin: building an expressway to protein destruction. Onco- gene, 2004, 23: 1985–1997.
18. Musikacharoen T, et al. NF-κB and STATs play important roles in the regulation of mouse Toll-like receptor gene expression. Immunol. 2001,166:4516-4520.
19. Driessler F, et al. Molecular mechanisms of interleukin-10-mediated inhibition of NF–kappaB activity: a role for p50. Clin. Exp. Immunol. 2004,135:64-73.
20. Dijsselbloem N, et al. A critical role for p53 in the control of NF–kappaB-dependent gene expression in TLR4-stimulated dendritic ceel exposed to genistein. J Immunol. 2007,15,178(8):5048-5057.
21. Ulloa L, et al. Ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation. Proc Natl Acad Sci USA. 2002,99:12351-6.
22. Ciechanover A, et al. The ubiquitin-mediated proteolytic pathway: mode of action and clinical implication[J]. J Cell Biochem,Suppl 2000,34:40-51.
23. Rock KL, et al. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC classⅠmolecules. Cell. 1994,78:761-771.
24. Chenggong Z, et al. Regulation of murine cardiac 20S proteasomes role of associatingpartners. Circ Res,2006,99(7):372-380.
25.Niranjan Rajapakse, et al. Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-stimulated RAW264.7 Cells by carboxybutyrylated glucosamine takes place via down-regulation of mitogen-activated protein kinase-mediated nuclear factor-κB signaling. Immunology. 123:348-357.
26. Eilliott PJ, et al. Proteasome inhibition: a new anti-inflammatory strategy[J]. J Mol Med, 2003,81(4):235-245.
27.戴翠莲等.蛋白酶体抑制剂MG-132下调炎症因子表达抑制大鼠心肌再灌注损伤第三军医大学学报2007, 29(2):129-133.
28. Sinn DI, et al. Proteasomal inhibition in intracerebral hemorrhage: neuroprotective and anti-inflammatory effects of bortezomib. Neurosci Res. 2007,58:12-18.
29. Meiners S, et al. Suppression of cardiomyocyte hypertrophy by inhibition of the ubiquitin-proteasome system. Hypertension, 2008,51(2):302-308.
30. Zhou H, Wertz I, O`Rourke K, et al. Bcl10 activates the NF-kappa B pathway through ubiquitination of NEMO. Nature. 2004, 127(6970): 167-171
31.尹会男柴家科姚咏明等.泛素一蛋白酶体途径对烫伤脓毒症大鼠肠道炎症反应与屏障功能的作用研究。中国危重病急救医学,2006,18(11):649-652
32.尹会男柴家科姚咏明等.蛋白酶体抑制剂在烫伤脓毒症大鼠肺脏炎症反应中的作用。中华急诊医学杂志,2006,15(10):886-889
33. Jing Shen, et al. Key inflammatory signaling pathways are regulated by the proteasome. Shock. 2006,25(5):472-84.
34. Kim CS, et al. Capsaicin exhibits anti-inflammatory property by inhibiting IκBa degradation in LPS-stimulated peritoneal macrophages. Cell Signal. 2003,15:299-306.
35. Yang H, et al. Shikonin exerts antitumor activity via proteasome inhibition and cell death induction in vitro and in vivo. IntJ Cancer. 2009,124(10):2450-9.
36. Span LF, et al. The dynamic process of apoptosis analyzed by flow cytometry using Annexin-V/propidium iodide and a modified in situ end labeling technique[J]. Cytometry,2002,47(1):24-31.
37. Budihardjo I, et al. Biochemical pathways of caspase activation during apoptosis[J]. Annu Rev Cell Dev Biol. 1999,15:269-90.
38. Hengartner MO, et al. The biochemistry of apoptosis[J]. Nature, 2000, 407 (6805): 770-6.
39. Saikumar P, et al. Apoptosis: definition, mechanisms,and relevance to disease[J]. Am J Med,1999,107(5):489-506.
40. Rao RV, et al. Coupling endoplasmic reticulum stress to the cell death program. Mechnism of caspase activation[J]. J Biol Chem,2001, 276(36): 33869-33874.
41. Safranek, et al. Modulation of inflammatory response in sepsis by proteasome inhibition. 2006, Oct;87(5):369-72.
1.Pickart CM, Eddins MJ. Ubiquitin: structures, functions, mechanisms[J]. Biochim BiophysActa, 2004, 1695 (1-3) : 55 - 72.
2.Herrmann J, CiechanoverA, Lerman LO, et al. The ubiquitin -proteasome system in cardiovascular diseases: a hypothesis extended[J]. Cardiovasc Res, 2004, 61 (1) : 11 - 21.
3.Patterson C. Search and destroy: the role of protein quality control in maintaining cardiac function [J]. J Mol Cell Cardiol, 2006, 40(4) : 438 - 441.
4.Raasi S, Wolf DH. Ubiquitin receptors and ERAD: A network of pathways to the proteasome [J]. Sem in Cell Dev Biol, 2007, 18(6) : 78 - 91.
5.Fricke B, Heink S, Steffen J, et al. The proteasome maturation protein POMP facilitates major steps of 20S proteasome formation at the endoplasmic reticulum[J]. EMBO Rep, 2007, 8 (12) : 1170 -5.
6.Meiners S, Ludwig A, Stangl V, et al. Proteasome inhibitors: Poisons and remedies[J]. Med Res Rev, 2008, 28 (2) : 309 - 27.
7.MarquesA J, Glanemann C, Ramos P C, et al. The C terminal extension of the beta 7 subunit and activator complexes stabilize nascent 20s proteasomes and promote their maturation [J]. J Biol Chem , 2007, 282 (48) : 34869 - 76.
8.王岚,孙圣刚,曹学兵,等.格尔德霉素通过诱导热休克蛋白反应抑制DA能神经元蛋白水解应激的研究[J].中国药理学通报, 2007, 23 (8) : 1025 - 9.
9.Wang L, Sun SG, Cao XB, et al. Geldanamycin activates a heat shock response and inhibits proteolytic stress in dopaminergic neurons[J]. Chin Pharmacol Bull, 2007, 23 (8) : 1025 - 9.
10.Kim H T, Kim K P, Lledias F, et al. Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize non-degradable forked ubiquitin chains containing all possible isopeptide linkages [J]. J Biol Chem , 2007. 282( 24 ) :17375 - 86.
11.Kwon Y T, Kashina A S, Varshavsky A. Alternative splicing results in differential expression, activity, and localization of the two forms of arginyl tRNA protein transferase, a component of the N-end rule pathway[J]. Mol Cell Biol, 1999. 19 (1) : 182– 93.
12.Aaron C, Schwartz AL.Ubiquitin-mediated degradation of cellular proteins in health and disease. Hepatology, 2002,35(1):3-6.
13.Sun Z, Andersson R. NF-kappaB activation and inhibition. Shock, 2002, 18: 99-106.
14. Jesenberger V, Jentsch S. Deadly encounter: ubiquition meets apoptosis. Nat Rev Mol Cell Biol, 2002, 3(2): 112-21.
15.Simon R,Struckmann K,Schraml P. Amplification pattern of 12q13q15 genes(MDM2 CDK4, CLI) in urinary bladder cancer[J]. Oncogene, 2002, 21(16): 2476-2483.
16.Bloom J,Pagano M. Deregulated degradation of the cdk inhibitor p27 and malignant transformation[J]. Semin Cancer Biol, 2003, 13(1):41-47.
17 . Viglietto C. Fusee A. Understanding p27 Kipl deregulation in cancer: Down-regulation or mislocalisation[J]. Cell Circle, 2002, l(6): 394-400.
18.Gstaiger M,Jordan R,Lim M,et a1. Skp2 is oncogenic and overexpressed in human cancer[J]. Pmc Natl Acad Sci USA. 200l, 98(9): 5043-5048.
19.Pugh CW,Ratcliffe PJ. The von Hippela-Lindau tumor suppressor, hypoxia-inducible factor-l(HIF-1) degradation and cancer pathogenesis[J]. Semin Cancer Biol, 2003,13(1): 83-89.
20.Lorrick KL,Jensen JP,Fang S. et al. RING fingers mediate ubiquitin—conjugating enzyme(E2)-dependent ubiquitination[J]. Proc Natl Acad Sci USA, l999, 96(20): ll364-ll369.
21.Mani A ,Gelmann EP. The ubiquitin-proteasome Pathway and its role in cancer[J]. Clin Oncol, 2005, 23(21): 4776-4789.
22.Williams A J, et al. Neuroprotection with the proteasome inhibitor MLN519 in focal ischemic brain injury: relation to nuclear factor KappaB(NF–kappaB), inflammatorygene expression, and leukocyte infiltration. Neurochem Int,2006,49(2):106-112.
23.Qureshi N, et al. Key inflammatory signaling pathways are regulated by the proteasome. Shock. 2006,25(5):472-84.
24.Qureshi N, et al. The proteasome: a central regulator of inflammation and macrophage function. Immunol Res. 2005;31:243-260.
25.Ward CL,Omura S,Kopito RR. Degradation of CFTR by the ubiquitin-proteasome pathway[J]. Cell,1995, 83(1): 121-127.
26.Kitada T,Asakawa S,Hmtori N.et a1. Mutations in the Parkin gene cause autosomal recessive juvenile parkinsonism[J]. Nature, 1998, 392(6676): 60l5—608.
27.Muyang L,Delin C,Ariel S, et a1. Deubiquitination of p53 by HAUSP is all important pathway for p53 stabili action[J]. Nature, 2002, 416 (6881) : 648– 653.
28.GiovanniD,Angela H, Frank M C, et al. Infetion Imunity, 2000, 68 ( 6) : 3097 -3102.