脂多糖上调人牙髓细胞B细胞淋巴瘤-2蛋白及其相关X蛋白的表达

目的：探讨脂多糖（LPS）作用于人牙髓细胞后,B细胞淋巴瘤-2（Bc1-2）蛋白、Bcl-2相关X蛋白（Bax）在正常人牙髓和炎症牙髓中的表达,探讨其在牙髓炎症过程中的作用机制。方法在体外用不同质量浓度LPS刺激人牙髓细胞后,采用免疫细胞化学染色方法在不同的时间点检测Bcl-2、Bax的表达,用图像分析系统Simple?PCI?version?5.1分析。结果正常人牙髓细胞中Bcl-2、Bax表达量不高,而在牙髓炎症过程中两者均增强,但是随着LPS质量浓度的增加,Bcl-2的表达量开始下降,而Bax的表达则持续增强。结论内毒素能使人牙髓细胞膜上的Bcl-2、Bax表达增强,但Bax的表达强于Bcl-2,内毒素可能通过两者介导的信号传导途径引起细胞凋亡。     

褪黑素对血管内皮细胞损伤的保护作用及机制

目的研究褪黑素对脂多糖引起的人脐静脉内皮细胞损伤的保护作用,以及对其机制探讨。方法在体外建立脂多糖血管内皮细胞损伤模型,添加不同浓度的褪黑素（低剂量组100μmol／L,高剂量组500μmol／L）,采用MTT法观察褪黑素对血管内皮细胞存活率的影响;用流式细胞仪检测细胞凋亡率,细胞免疫荧光技术检测细胞色素C的释放;用酶联免疫吸附试验（ELISA）法测定细胞裂解液中超氧化物歧化酶的浓度;用划痕修复实验检测血管内皮细胞的迁移功能。结果脂多糖损伤后,血管内皮细胞存活率减少约50％,凋亡细胞百分比增加35．7％,细胞色素c大量释放到细胞质中,细胞裂解液中超氧化物歧化酶浓度下降约50％,且血管内皮细胞的迁移能力受损。加入褪黑素后,可明显减轻脂多糖对细胞的损伤作用,恢复细胞存活率至70％-80％,减少细胞凋亡率至15．1％-20．6％,保护细胞的迁移能力,且呈剂量依赖性,高剂量组的效果明显好于低剂量组,各指标差异有统计学意义（P〈0．05）。结论褪黑素可保护和修复脂多糖引起的血管内皮细胞损伤,其作用途径可能与抑制细胞凋亡．减轻氧化损伤以及保护迁移功能有关。     

Spironolactone inhibits production of proinflammatory mediators in response to lipopolysaccharide via inactivation of nuclear factor-κB

The effect of spironolactone (SPIR) on lipopolysaccharide (LPS)-induced production of proinflammatory mediators was examined using RAW 264.7 macrophage-like cells and mouse peritoneal macrophages. SPIR significantly inhibited LPS-induced production of nitric oxide (NO), tumor necrosis factor-α and prostaglandin E2. The inhibition was not mediated by cell death. SPIR reduced the expression of an inducible NO synthase mRNA in response to LPS. SPIR significantly inhibited phosphorylation of p65 nuclear factor (NF)-κB in response to LPS. Furthermore, SPIR inhibited phosphorylation of IκB kinase (IKK) as an upstream molecule of NF-κB in response to LPS. LPS did not induce the production of aldosterone in RAW 264.7 cells. Taken together, SPIR is suggested to inhibit LPS-induced proinflammatory mediators via inactivation of IKK/NF-κB in LPS signaling.     

The kiwi fruit peptide kissper displays anti‐inflammatory and anti‐oxidant effects in in‐vitro and ex‐vivo human intestinal models

Literature reports describe kiwi fruit as a food with significant effects on human health, including anti‐oxidant and anti‐inflammatory activity. Fresh fruit or raw kiwi fruit extracts have been used so far to investigate these effects, but the molecule(s) responsible for these health‐promoting activities have not yet been identified. Kissper is a kiwi fruit peptide displaying pore‐forming activity in synthetic lipid bilayers, the composition of which is similar to that found in intestinal cells. The objective of this study was to investigate the kissper influence on intestinal inflammation using cultured cells and ex‐vivo tissues from healthy subjects and Crohn's disease (CD) patients. The anti‐oxidant and anti‐inflammatory properties of kissper were tested on Caco‐2 cells and on the colonic mucosa from 23 patients with CD, by challenging with the lipopolysaccharide from Escherichia coli (EC‐LPS) and monitoring the appropriate markers by Western blot and immunofluorescence. EC‐LPS challenge determined an increase in the intracellular concentration of calcium and reactive oxygen species (ROS). The peptide kissper was highly effective in preventing the increase of LPS‐induced ROS levels in both the Caco‐2 cells and CD colonic mucosa. Moreover, it controls the calcium increase, p65‐nuclear factor (NF)‐kB induction and transglutaminase 2 (TG2) activation inflammatory response in Caco‐2 cells and CD colonic mucosa. Kissper efficiently counteracts the oxidative stress and inflammatory response in valuable model systems consisting of intestinal cells and CD colonic mucosa. This study reports the first evidence supporting a possible correlation between some beneficial effects of kiwi fruit and a specific protein molecule rather than generic nutrients.     

LPS对乳腺癌MDA-MB-231细胞上皮间质转化的诱导作用及其对β-catenin表达的影响

目的：探讨脂多糖（LPS）对人乳腺癌MDA-MB-231细胞中上皮间质转化（EMT）标志物及β-连环素（β-catenin）表达的影响,阐明其可能机制。方法：人乳腺癌MDA-MB-231细胞分为对照组和不同浓度（5、10、20和40 mg·L^-1）LPS组,倒置显微镜观察各组MDA-MB-231细胞的形态表现,免疫荧光实验检测各组MDA-MB-231细胞中β-catenin表达及定位,实时荧光定量PCR（RT-qPCR）法和Western blotting法检测各组MDA-MB-231细胞中EMT标志物E-钙黏蛋白（E-cadherin）、波形蛋白（Vimentin）和β-catenin mRNA及蛋白表达水平。结果：对照组MDA-MB-231细胞形态表现为上皮细胞表型,不同浓度LPS组MDA-MB-231细胞形态表现为间质细胞表型。免疫荧光实验,β-catenin表达定位主要在细胞核。与对照组比较,不同浓度LPS组细胞中Vimentin和β-catenin mRNA及蛋白表达水平明显升高（P<0.05或P<0.01）,以20 mg·L^-1LPS组升高最为明显;与对照组比较,不同浓度LPS组细胞中E-cadherin mRNA和蛋白表达水平明显降低（P<0.05或P<0.01）,以20 mg·L^-1 LPS组降低最为明显。结论：LPS通过下调E-cadherin表达、上调Vimentin表达促进乳腺癌MDA-MB-231细胞发生EMT、侵袭和转移,其作用机制可能与Wnt/β-catenin信号通路有关。     

Significance of platelet and monocyte activation for therapeutic immunoglobulin-induced thromboembolism

Objectives

Thromboembolic events (TEE) in patients receiving infusions of intravenous immunoglobulin (IVIG) products have recently been associated with contaminating factor XIa. We studied whether platelet and monocyte activation could also be involved.

Methods

Twenty IVIG samples from five manufacturers were tested for the induction of visible whole blood clot formation. A selection of TEE-associated and not associated lots was further analyzed for effects on thromboelastometry, platelet activation and adhesion, as well as monocyte tissue factor surface expression. Pure factor XIa was included for comparison. Western blotting was applied to analyze anti-CD154-reactive proteins in IVIG.

Results

In whole blood, IVIG enhanced macroscopic clotting additively with factor XIa. In monocytes, all IVIG products induced the FcγRII-dependent tissue factor expression to a similar extent, which was not affected by addition of factor XIa. Testing platelet aggregation, IVIG strengthened the ADP and TRAP-6-elicited response. Furthermore, IVIG increased platelet-monocyte adhesion and annexin V binding to platelet microvesicles, and promoted platelet adhesion to IVIG-coated surfaces. The strongest effects were observed with TEE-associated lots. CD154-related proteins were detected in all IVIG products. CD154-related high molecular weight complexes were particularly found in the TEE-associated IVIG. In platelet aggregation, recombinant soluble CD154 enhanced aggregate formation and stability.

Conclusion

Our data demonstrate that IVIG modulate platelet and monocyte activation and can thereby affect the hemostatic balance. These effects are either additive to or independent from factor XIa. CD154-related proteins are assumed to be involved in these interactions, the mechanism of which needs to be elucidated in further studies.     

Suppression of Heme Oxygenase-1 by Prostaglandin E2-Protein Kinase A-A-Kinase Anchoring Protein Signaling Is Central for Augmented Cyclooxygenase-2 Expression in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Purpose

Prostaglandin (PG) E₂ is an immunomodulatory lipid mediator generated mainly via the cyclooxygenase-2 (COX-2) pathway from arachidonic acid at sites of infection and inflammation. A positive feedback loop of PGE₂ on COX-2 expression is critical for homeostasis during toll-like receptor (TLR)-mediated inflammatory processes. The mechanism of PGE₂-regulated COX-2 expression remains poorly understood. The low-molecular-weight stress protein heme oxygenase-1 (HO-1) contributes to the anti-inflammatory, anti-oxidant and anti-apoptotic response against environmental stress.

Methods

We explored the involvement of HO-1 on PGE₂ regulation of LPS-induced COX-2 expression in RAW 264.7 macrophages.

Results

LPS-induced COX-2 expression in RAW 264.7 macrophages was enhanced by exogenous PGE₂ or cyclic AMP (cAMP) analogue and was suppressed by a COX inhibitor (indomethacin), a protein kinase A (PKA) inhibitor (KT5720), and A kinase anchoring protein (AKAP) disruptors (Ht31 and RIAD). This result suggests that the stimulatory effects of endogenous and exogenous PGE₂ on COX-2 expression are mediated by a cAMP-PKA-AKAP-dependent pathway. The induction of HO-1 was observed in LPS-stimulated RAW 264.7 macrophages. This induction was suppressed by exogenous PGE₂ and enhanced by blockage of the endogenous PGE₂ effect by the PKA inhibitor or AKAP disruptors. In addition, HO-1 induction by the HO activator copper protoporphyrin suppressed LPS-induced COX-2 expression, which was restored by the addition of exogenous PGE₂. The induction of HO-1 inhibited LPS-induced NF-κB p-65 nuclear expression and translocation.

Conclusions

AKAP plays an important role in PGE₂ regulation of COX-2 expression, and the suppression of HO-1 by PGE₂-cAMP-PKA-AKAP signaling helps potentiate the LPS-induced COX-2 expression through a positive feedback loop in RAW 264.7 macrophages.     

TNF-α及LPS刺激下牙周膜干细胞IκB磷酸化及其下游基因的表达

目的:研究TNF-α及LPS刺激下牙周膜干细胞IκB磷酸化及其下游基因的表达.方法:体外分离、培养人牙周膜干细胞,分别在10 ng/mL TNF-α和500 ng/mL Ecoli.LPS刺激下进行培养,并于刺激培养即时(0 h)、5、30 min和1、2h,采用Western Blot法检测IκBα总蛋白和磷酸化IκBα蛋白的表达变化;Real-time PCR检测IL-6、IL-8 mRNA的表达水平.结果:牙周膜干细胞在10 ng/mL TNFα或500 ng/mL Ecoli.LPS刺激下培养0～2h不同时间后,IκBα总蛋白随刺激时间逐渐降低,1h时最低;磷酸化IκBα随刺激时间逐渐升高,2h时最高;NFκB的下游基因IL-6及IL-8mRNA表达水平均随刺激时间逐渐升高,2h时升高最明显(P＜0.05).结论:炎症细胞因子TNF-α或LPS均可促进牙周膜干细胞中IκBα蛋白的磷酸化,并上调IL-6、IL-8 mRNA的表达;提示NFκB信号通路的活化可能是导致炎症状态下牙周膜干细胞功能损害的重要因素.     

依布硒啉对急性肺损伤大鼠氧化应激的影响

目的 观察依布硒啉对内毒素性急性肺损伤的保护作用.方法 健康雄性SD大鼠,随机分成6组:正常对照组、模型组、依布硒啉高剂量组、中剂量组、低剂量组和地塞米松对照组.通过尾静脉注射LPS(5 mg/kg)建立模型,治疗组于造模前30 min,大鼠腹腔注射依布硒啉(7.5 mg/kg、15 mg/kg、30 mg/kg),对照组和模型组分别注入等量溶剂.造模后6h,麻醉放血处死动物,并取肺组织,测定肺湿质量/干质量.采用硫代巴比妥酸和黄嘌呤氧化法分别检测肺组织中超氧化物歧化酶SOD活性和丙二醛MDA及髓过氧化物酶(MPO)含量.光镜下观察肺组织病理学改变.结果 依布硒啉治疗组与模型组相比,肺湿质量/干质量降低,肺组织SOD的活性明显增高,MDA和MPO含量显著降低(P＜0.05),病理改善明显.结论 依布硒啉对内毒素性急性肺损伤有一定保护作用,其机制可能与提高抗氧化能力有关.