Amelioration of Endotoxin-Induced Uveitis in Rabbit by Topical Administration of Tacrolimus Proglycosome Nano-Vesicles

This work was aimed to improve the efficacy of tacrolimus in the treatment of endotoxin-induced uveitis (EIU) using propylene glycol modified lipid vesicles termed as proglycosome nano-vesicles (PNVs). PNVs were prepared by modified film hydration method. Experimental uveitis in rabbit eye was induced by an intravitreal injection of 20 μL of the endotoxin solution containing 100 ng of lipopolysaccharide endotoxin. In vivo efficacy of PNVs was determined by studying clinical symptoms of uveitis using slit lamp examination and by quantitatively measuring levels of tumor necrosis factor-alpha, interleukin-6, leukocytes and total proteins in aqueous humor, 24 h after intravitreal injection of endotoxin. Comparison was made with healthy, untreated and tacrolimus solution treated eyes. PNVs developed were nano-sized, deformable and showed sustained release of tacrolimus over period of 12 h. In vivo results indicated statistically significant difference between the effects of PNVs in the treatment of EIU compared to tacrolimus. PNV treatment not only subsides clinical symptoms of uveitis but also prevented breakdown of blood aqueous barrier. Tacrolimus loaded PNVs are potential new topical treatment for uveitis.     

静脉麻醉药对脂多糖诱导的神经星形胶质细胞增殖的影响及其机制

目的 检测静脉麻醉药对脂多糖诱导的神经星形胶质细胞的影响及其机制。 方法 利用MTT实验、集落形成实验和Annexin V-FITC/PI双染色法检测静脉麻醉药对脂多糖作用后的神经星形胶质细胞增殖和凋亡的影响。Western blotting技术检测相关机制。 结果 静脉麻醉药可以逆转脂多糖诱导的神经星形胶质细胞增殖。经麻醉药处理后的神经星形胶质细胞p-Akt和p-mTOR蛋白表达下降，Caspase-3和Caspase-9表达水平升高。结论 静脉麻醉药可以通过抑制mTOR通路逆转脂多糖诱导的神经星形胶质细胞增殖。     

Enterohemorrhagic Escherichia coli O157 outer membrane vesicles induce interleukin 8 production in human intestinal epithelial cells by signaling via Toll-like receptors TLR4 and TLR5 and activation of the nuclear factor NF-κB

Proinflammatory cytokines play important roles in the pathogenesis of diseases caused by enterohemorrhagic Escherichia coli (EHEC) O157, but the spectrum of bacterial components involved in the proinflammatory responses is not fully understood. Here, we investigated the abilities of outer membrane vesicles (OMVs), nanoparticles released by EHEC O157 during growth, to induce production of proinflammatory cytokines in human intestinal epithelial cells. OMVs from both EHEC O157:H7 and sorbitol-fermenting (SF) EHEC O157:H⁻ induced production of interleukin-8 (IL-8) in Caco-2, HCT-8, and HT-29 intestinal epithelial cell lines. H7 flagellin was the key IL-8-inducing component of EHEC O157:H7 OMVs, whereas cytolethal distending toxin V and O157 lipopolysaccharide (LPS) largely contributed to IL-8 production elicited by flagellin-lacking OMVs from SF EHEC O157:H⁻. The H7 flagellin-mediated signaling via Toll-like receptor (TLR) 5, and O157 LPS-mediated signaling via TLR4/MD-2 complex, which were followed by activation of the nuclear factor NF-κB were major pathways underlying IL-8 production induced by EHEC O157 OMVs. The proinflammatory and immunomodulatory capacities of EHEC O157 OMVs have pathogenetic implications and support the OMVs as suitable vaccine candidates.     

脂多糖受体簇和大电导Ca2+活K+通道在脂多糖信号识别中的作用

背景 细菌脂多糖(lipopolysaccharide,LPS)可激活细胞合成和释放多种细胞因子,导致全身炎症反应.LPS识别及跨膜信号转导是引起细胞效应的关键,成为近年的研究热点.目的 对新近提出的“LPS受体簇”理论和大电导Ca2+激活K+通道(MaxiK)在LPS信号识别中的作用研究进展进行综述.内容 LPS与CD14结合后,不同的信号分子在脂质筏内聚集,LPS被释放到脂质双分子层,并与由多种受体分子组成的受体簇相互作用.根据不同的细胞类型和细菌刺激,形成了不同的LPS受体簇.MaxiK通道在LPS诱导的巨噬细胞信号转导过程的早期即被激活.并且以IκB-α/NF-κB为中心的促炎症反应依赖MaxiK的功能.趋向 需要进一步研究来阐明LPS受体簇在细胞膜特定区域内形成的确切分子机制,以及组成受体簇的几种蛋白分子在刺激识别和信号转导过程中的作用.     

Men and women differ in inflammatory and neuroendocrine responses to endotoxin but not in the severity of sickness symptoms

Impaired mood and increased anxiety represent core symptoms of sickness behavior that are thought to be mediated by pro-inflammatory cytokines. Moreover, excessive inflammation seems to be implicated in the development of mood/affective disorders. Although women are known to mount stronger pro-inflammatory responses during infections and are at higher risk to develop depressive and anxiety disorders compared to men, experimental studies on sex differences in sickness symptoms are scarce. Thus, the present study aimed at comparing physiological and psychological responses to endotoxin administration between men and women. Twenty-eight healthy volunteers (14 men, 14 women) were intravenously injected with a low dose (0.4 ng/kg) of lipopolysaccharide (LPS) and plasma concentrations of cytokines and neuroendocrine factors as well as negative state emotions were measured before and until six hours after LPS administration. Women exhibited a more profound pro-inflammatory response with significantly higher increases in tumor necrosis factor (TNF)-α and interleukin (IL)-6. In contrast, the LPS-induced increase in anti-inflammatory IL-10 was significantly higher in men. The cytokine alterations were accompanied by changes in neuroendocrine factors known to be involved in inflammation regulation. Endotoxin injection induced a significant increase in noradrenaline, without evidence for sex differences. The LPS-induced increase in cortisol was significantly higher in woman, whereas changes in dehydroepiandrosterone were largely comparable. LPS administration also increased secretion of prolactin, but only in women. Despite these profound sex differences in inflammatory and neuroendocrine responses, men and women did not differ in endotoxin-induced alterations in mood and state anxiety or non-specific sickness symptoms. This suggests that compensatory mechanisms exist that counteract the more pronounced inflammatory response in women, preventing an exaggerated sickness response. Disturbance of these compensatory mechanisms by environmental factors such as stress may promote the development of affective disorders in women.     

Cognitive deficits develop 1 month after diffuse brain injury and are exaggerated by microglia-associated reactivity to peripheral immune challenge

Traumatic brain injury (TBI) elicits immediate neuroinflammatory events that contribute to acute cognitive, motor, and affective disturbance. Despite resolution of these acute complications, significant neuropsychiatric and cognitive issues can develop and progress after TBI. We and others have provided novel evidence that these complications are potentiated by repeated injuries, immune challenges and stressors. A key component to this may be increased sensitization or priming of glia after TBI. Therefore, our objectives were to determine the degree to which cognitive deterioration occurred after diffuse TBI (moderate midline fluid percussion injury) and ascertain if glial reactivity induced by an acute immune challenge potentiated cognitive decline 30 days post injury (dpi). In post-recovery assessments, hippocampal-dependent learning and memory recall were normal 7 dpi, but anterograde learning was impaired by 30 dpi. Examination of mRNA and morphological profiles of glia 30 dpi indicated a low but persistent level of inflammation with elevated expression of GFAP and IL-1β in astrocytes and MHCII and IL-1β in microglia. Moreover, an acute immune challenge 30 dpi robustly interrupted memory consolidation specifically in TBI mice. These deficits were associated with exaggerated microglia-mediated inflammation with amplified (IL-1β, CCL2, TNFα) and prolonged (TNFα) cytokine/chemokine expression, and a marked reactive morphological profile of microglia in the CA3 of the hippocampus. Collectively, these data indicate that microglia remain sensitized 30 dpi after moderate TBI and a secondary inflammatory challenge elicits robust microglial reactivity that augments cognitive decline.Statement of SignificanceTraumatic brain injury (TBI) is a major risk factor in development of neuropsychiatric problems long after injury, negatively affecting quality of life. Mounting evidence indicates that inflammatory processes worsen with time after a brain injury and are likely mediated by glia. Here, we show that primed microglia and astrocytes developed in mice 1 month following moderate diffuse TBI, coinciding with cognitive deficits that were not initially evident after injury. Additionally, TBI-induced glial priming may adversely affect the ability of glia to appropriately respond to immune challenges, which occur regularly across the lifespan. Indeed, we show that an acute immune challenge augmented microglial reactivity and cognitive deficits. This idea may provide new avenues of clinical assessments and treatments following TBI.     

Regulation of activin receptor-interacting protein 2 expression in mouse hepatoma Hepal-6 cells and its relationship with collagen type Ⅳ

AIM: To investigate the regulation of activin receptor-interacting protein 2 (ARIP2) expression and its possible relationships with collagen type Ⅳ (collagen Ⅳ) in mouse hepatoma cell line Hepal-6 cells.METHODS: The ARIP2 mRNA expression kinetics in Hepal-6 cells was detected by RT-PCR, and its regulation factors were analyzed by treatment with signal transduction activators such as phorbol 12-myristate 13-acetate (PMA), forskolin and A23187. After pcDNA3-ARIP2 was transfected into Hepal-6 cells, the effects of ARIP2 overexpression on activin type Ⅱ receptor (ActRII)and collagen Ⅳ expression were evaluated.RESULTS: The expression levels of ARIP2 mRNA in Hapel-6 cells were elevated in time-dependent manner 12 h after treatment with activin A and endotoxin LPS, but not changed evidently in the early stage of stimulation (2 or 4 h). TheARIP2 mRNA expression was increased after stimulated with signal transduction activators such as PMA and forskolin in Hepal-6 cells, whereas decreased after treatment with A23187 (25.3% ± 5.7% vS 48.1% ± 3.6%, P ＜ 0.01). ARIP2 overexpression could remarkably suppress the expression of ActRIIA mRNA in dose-dependent manner, but has no effect on ActRIIB in Hepal-6 cells induced by activin A. Furthermore, we have found that overexpression of ARIP2 could inhibit collagen Ⅳ mRNA and protein expressions induced by activin A in Hapel-6 cells.CONCLUSION: These findings suggest that ARIP2 expression can be influenced by various factors. ARIP2 may participate in the negative feedback regulation of signal transduction in the late stage by affecting the expression of ActRIIA and play an important role in regulation of development of liver fibrosis induced by activin.     

内毒素血症时肝窦内皮细胞脂多糖受体CD14蛋白表达的观察

目的观察内毒素血症时肝窦内皮细胞(LSECs)中CD14蛋白合成和CD14 基因的表达,以及CD14蛋白在内毒素介导LSECs激活中的作用. 方法经尾静脉注入脂多糖(LPS,E coli O111B4)5mg/kg,建立大鼠内毒素血症动物模型,分别于术后0(对照组)、3、6、12、24h活杀取材.用兔抗鼠CD14抗体和异硫氢酸荧光素(FITC)标记的羊抗兔IgG对LSECs进行孵育后,流式细胞仪测定LSECs的平均荧光强度(MFI)及FITC阳性细胞数;用原位杂交法测定LSEC中CD14 mRNA的表达.用原位胶原酶灌注法分离大鼠LSECs,用不同浓度LPS(0、0.01、1、10、100μg/ml)刺激LSECs.并用CD14抗体阻断LSECs的 CD14蛋白后,再用不同浓度LPS(0、0.01、1、10、100μg/ml)刺激LSECs.测定LPS介导LSECs肿瘤坏死因子(TNF)-α及白细胞介素-6(IL-6)分泌及CD14抗体对LSECs细胞因子分泌的影响. 结果内毒素血症大鼠3、6、12和24h 时LSECs的MFI明显增加;FITC阳性细胞数也明显增多,分别为54.32%、65.83%、85.61%和45.65%,与对照组的4.45%比较差异有非常显著意义(P＜0.01).原位杂交显示,内毒素血症大鼠LSECs中CD14 mRNA的表达明显增强,而对照组CD14 mRMA无阳性表达.LPS组TNF-α的含量(pg/ml)分别为54.49±6.02、84.65±10.16、206.54±23.55、349.87±39.47和365.76±40.31;CD14阻断组TNF-α的含量(pg/ml)分别为55.93±6.95、63.32±7.81、85.34±9.72、112.75±13.54、198.66±21.54;两组间比较差异有非常显著意义(P＜0.01).LPS组IL-6的含量(pg/ml)分别为103.34±12.52、187.39±20.31、243.87±27.83、289.51±30.15、298.53±31.94;CD14阻断组IL-6的含量(pg/ml)分别为104.37±11.49、125.02±13.58、164.59±19.47、183.47±20.17、221.76±26.43;两组间比较差异有非常显著意义(P＜0.01). 结论内毒素血症时LSECs能合成CD14蛋白及表达CD14基因;抗CD14抗体对LPS诱导LSECs TNF-α和IL-6的分泌有抑制作用;CD14蛋白的表达在内毒素介导LSECs激活中可能起重要作用.