局部麻醉剂通过丝裂原活化蛋白激酶途径诱导人甲状腺癌细胞凋亡

目的探讨局部麻醉剂对人甲状腺癌细胞凋亡的影响及其作用机制。方法MTT法检测人甲状腺癌细胞经0 mM、1 mM、2 mM、4 mM、8 mM和16 mM的利多卡因及0 mM、0.2 mM、0.4 mM、0.8 mM、1.6 mM和3.2 mM的布比卡因分别干预24 h和48 h后的细胞活力流式细胞仪检测经4 mM和8 mM的利多卡因和0.8 mM和1.6 mM的布比卡因干预48 h后癌细胞的凋亡及线粒体膜电位;Western Blot检测凋亡相关因子及丝裂原活化蛋白激酶(MAPK)途径相关蛋白的表达;分别采用SP600125(JNK抑制剂)、PD98059(MEK抑制剂)、SB203580(p38 MAPK抑制剂)联合利多卡因或布比卡因对细胞进行处理,Western Blot检测凋亡相关蛋白的表达。结果利多卡因和布比卡因干预24 h和48 h均可呈剂量依赖式抑制人甲状腺癌细胞的生长。利多卡因和布比卡因干预48 h可降低癌细胞线粒体膜电位,增加Caspase-3和Bax的表达,降低Bcl-2的表达激活p38和JNK,抑制ERK的活性。MAPK信号通路抑制剂可降低利多卡因和布比卡因诱导Caspase-3和Bax的表达,增加Bcl-2的表达。结论局部麻醉剂利多卡因和布比卡因可诱导人甲状腺癌细胞生存抑制和凋亡,其机制与MAPK通路激活有关。     

地塞米松诱导成骨细胞凋亡的蛋白激酶C途径

背景：地塞米松以增加细胞周期时间的方法提高细胞凋亡来减少成骨细胞和骨细胞的数量。蛋白激酶C是细胞内信号传导通路的一种,与细胞凋亡有关已有相关文献报道。目的：探讨地塞米松诱导成骨细胞凋亡在蛋白激酶C细胞内信号转导途径方向的机制。方法：取胎鼠骨髓间充质干细胞进行成骨诱导,分为塞米松模型组、佛波醇组和星型胞菌素组。地塞米松模型组加入1×10-6 mol/L地塞米松,佛波醇组加入1×10-6 mol/L地塞米松和1×10-7 mol/L佛波醇,星型胞菌素组加入1×10-6 mol/L地塞米松和1×10-7结果与结论：地塞米松可明显的诱导凋亡,加入佛波醇后凋亡明显增加,加入星形孢菌素后凋亡明显减少。加入地塞米松后胞浆的蛋白激酶C明显减少,包膜明显增加。加入地塞米松不同时间,胞浆和胞膜的蛋白激酶C变化在30 min最明显。说明地塞米松诱导成骨细胞凋亡的机制与蛋白激酶C有关,地塞米松为蛋白激酶C激动剂。细胞受到刺激后,胞浆的蛋白激酶C转移至包膜,胞浆中的蛋白激酶C量减少,包膜中的增加。mol/L星型胞菌素,观察不同干预组细胞的增殖或抑制,并对细胞膜及细胞质中蛋白激酶C含量的变化进行检测。     

Human recombinant C5a enhances lipopolysaccharide-induced synthesis of interleukin-6 by human monocytes

The effect of human recombinant C5a (hrC5a) on the synthesis of interleukin-6 (IL-6) was studied in human monocytes. Monocytes incubated in the absence of hrC5a and of bacterial lipopolysaccharide (LPS) produced only low amounts (less than 100 U/2 x 10(6) cells/16 h) of IL-6 activity. LPS in concentrations from 10 pg ml-1 to 10 ng ml-1 greatly stimulated the synthesis of IL-6 to about 50.000 U/10(6) cells/16 h. When hrC5a was added to the monocyte media maximal IL-6 synthesis was reached at lower LPS concentrations, i.e. at 0.1 ng ml-1 LPS in the presence of 100 ng ml-1 hrC5a. Maximal IL-6 production was not significantly enhanced by hrC5a. Metabolic labelling with [35S]-methionine followed by immunoprecipitation of IL-6 showed that the increased IL-6 activity in the medium of hrC5a treated monocytes was due to a stimulation of the de novo synthesis of IL-6. Increased amounts of IL-6 mRNA were found in monocytes treated with LPS and hrC5a compared with monocytes stimulated only with LPS. HrC5a prolonged the elevation of IL-6 mRNA levels after stimulation of monocytes with LPS. HrC5a thus enhanced the LPS-induced synthesis of IL-6 by human monocytes.     

The complement component C5a induces the expression of plasminogen activator inhibitor-1 in human macrophages via NF-κB activation

BACKGROUND: Atherosclerosis is considered to be a chronic inflammatory disorder. Activation of the complement cascade is a major aspect of chronic inflammatory diseases. Complement components were identified in atherosclerotic plaques, and a correlation between adverse events and C5a plasma levels was found. These findings support the notion that complement activation contributes to development and progression of atherosclerotic lesions. OBJECTIVES: We investigated whether complement components C3a and C5a regulate plasminogen activator inhibitor (PAI-1) in human macrophages. METHODS: Human monocyte-derived macrophages (MDM) and human plaque macrophages were cultured and incubated with the complement component C5a. RESULTS: C5a increased PAI-1 up to 11-fold in human MDM and up to 2.7-fold in human plaque macrophages. These results were confirmed at the mRNA level using real time-polymerase chain reaction. Pertussis toxin or anti-C5aR/CD88 antibody completely abolished the effect of recombinant human C5a on PAI-1 production, suggesting a role of the C5a receptor. Experiments with antitumor necrosis factor (TNF)-alpha antibodies and tiron showed that the effect of C5a was not mediated by TNF-alpha or oxidative burst. Furthermore C5a induced NF-kappaB binding to the cis element in human macrophages and the C5a-induced increase in PAI-1 was completely abolished by an NF-kappaB inhibitor. CONCLUSIONS: We conclude that C5a upregulates PAI-1 in macrophages via NF-kappaB activation. We hypothesize that - if operative in vivo- this effect could favor thrombus development and thrombus stabilization in the lesion area. On the other hand one could speculate that C5a-induced upregulation of PAI-1 in plaque macrophages could act as a defense mechanism against plaque destabilization and rupture.     

Rho kinase inhibition by fasudil suppresses lipopolysaccharide-induced apoptosis of rat pulmonary microvascular endothelial cells via JNK and p38 MAPK pathway

Apoptosis of microvascular endothelial cells plays a crucial role in the progression of various lung diseases and triggers microcirculatory disorder and organ dysfunction. LPS, an outer membrane component of Gram-negative bacteria, is one of the major virulence factors for lung diseases. Recent studies have shown that the Rho/Rho kinase (ROCK) pathway plays an important role in the regulation of apoptosis, inflammatory cell migration and chemokine production in various cell types and animal models. We therefore undertake this study to investigate the inhibitory effect of fasudil, a potent and selective inhibitor of ROCK, on LPS-induced apoptosis of rat pulmonary microvascular endothelial cells (PMVECs). The results suggested that fasudil effectively prevented LPS-induced injury of rat PMVECs, as determined by MTT assay, LDH activity assay, apoptosis and western blot analysis of apoptosis-related proteins Bcl-2 and Bax. Furthermore, the mechanisms underlying the protective effect were evaluated. We found that LPS-induced MYPT-1 phosphorylation was markedly suppressed by fasudil. Moreover, fasudil pretreatment obviously inhibited the activation of JNK and p38 MAPKs induced by LPS, whereas that of ERK1/2 was not affected by fasudil. In addition, inhibiting the JNK and p38 pathways by SP600125 and SB203580 respectively attenuated the LPS-induced apoptosis and regulated the expression of apoptosis-related proteins Bcl-2 and Bax. Taken together, these results demonstrate that fasudil exerts an anti-apoptotic effect in rat PMVECs, which is mediated by the inhibition of Rho/ROCK and its downstream JNK and p38 MAPKs.     

Imoxin attenuates high fructose‐induced oxidative stress and apoptosis in renal epithelial cells via downregulation of protein kinase R pathway

Double‐stranded RNA (dsRNA)‐activated protein kinase R (PKR), a ubiquitously expressed serine/threonine kinase, is a key inducer of inflammation, insulin resistance, and glucose homeostasis in obesity. Recent studies have demonstrated that PKR can respond to metabolic stress in mice as well as in humans. However, the underlying molecular mechanism is not fully understood. The aim of this study was to examine the effect of high fructose (HF) in cultured renal tubular epithelial cells (NRK‐52E) derived from rat kidney and to investigate whether inhibition of PKR could prevent any deleterious effects of HF in these cells. PKR expression was determined by immunofluorescence staining and Western blotting. Oxidative damage and apoptosis were measured by flow cytometry. HF‐treated renal cells developed a significant increase in PKR expression. A significant increase in reactive oxygen species generation and apoptosis was also observed in HF‐treated cultured renal epithelial cells. All these effects of HF were attenuated by a selective PKR inhibitor, imoxin (C16). In conclusion, our study demonstrates PKR induces oxidative stress and apoptosis, is a significant contributor involved in vascular complications and is a possible mediator of HF‐induced hypertension. Inhibition of PKR pathway can be used as a therapeutic strategy for the treatment of cardiovascular and metabolic disorders.     

丙型肝炎病毒NS5a区血清型特异性多肽的选择及应用

目的:从HCV非结构5a区(NS5a)选出基因型特异性多肽,并应用酶免疫法(EIA)进行HCV血清分型。方法:根据HCV NS5a高免疫源区(2182～2343)的氨基酸序列,设计并合成305个特异性多肽,应用已知基因1～6型的抗-HCV阳性血清,通过EIA法检测每个多肽的抗原性,并用基因型特异性多肽进行血清分型,同时将其与序列分析法基因分型结果进行比较。结果:不同基因型间NS5a区氨基酸的同源性较低,相同基因型不同区段氨基酸的同源性也很低。对305个特异性多肽抗原性检测结果表明,主要线性抗原区位于氨基酸残基R3、R7和R9区。18个来自保守区的多肽可与不同基因型抗-HCV阳性血清反应;12个多肽具基因型特异性。血清基因分型结果与基因分型的结果高度一致。结论:HCV NS5a高免疫源区存在主要的线性抗原;来自高度保守区的多肽抗原无基因型特异性,可用于HCV抗体检测;基因型特异性多肽可用于HCV基因分型,特别适用于HCV RNA阴性患者。     

C5a反义肽对脓毒症小鼠心肌中性粒细胞浸润和P－选择素表达的影响

目的：探讨C5a反义肽对脓毒症小鼠心肌中性粒细胞（PMN）浸润的影响。方法盲肠结扎穿孔法（CLP）制备小鼠脓毒症模型,随机分为健康对照组、假手术组、脓毒症组和C5a反义肽治疗组四组。分别在2、4、8、12 h时间点观察心肌组织的病理改变,测心肌组织髓过氧化物酶（ MPO）活性,采用实时定量PCR和免疫印迹法分别检测心肌组织P－选择素mRNA和蛋白表达水平。结果 CLP造模4 h后,脓毒症组心肌病理可见心肌纤维结构疏松,局灶性肌溶解,胞质淡染,间质水肿,伴充血,细胞核肿胀等改变,C5a反义肽治疗组病变程度均较造模组轻;脓毒症组和C5a反义肽治疗组心肌组织MPO活性较对照组明显增高（P＜0．05）,C5a反义肽治疗组低于脓毒症组（P＜0.05）;P－选择素mRNA和蛋白表达量均明显增高（P＜0．05）,且随时间的增加而明显增加（P＜0．05）。与脓毒症组比较,C5a反义肽治疗组各时间点P－选择素mRNA和蛋白表达量明显下降（ P＜0．05）。结论 C5a反义肽可减少脓毒症小鼠心肌组织PMN浸润,C5a反义肽可能是通过减少P－选择素表达来减少PMN浸润的。