Modulation of the endocannabinoid system by focal brain ischemia in the rat is involved in neuroprotection afforded by 17beta-estradiol

Endogenous levels of the endocannabinoid anandamide, and the activities of the synthesizing and hydrolyzing enzymes, i.e. N-acylphosphatidylethanolamine-hydrolyzing phospholipase D and fatty acid amide hydrolase, respectively, were determined in the cortex and the striatum of rats subjected to transient middle cerebral artery occlusion. Anandamide content was markedly increased ( approximately 3-fold over controls; P < 0.01) in the ischemic striatum after 2 h of middle cerebral artery occlusion, but not in the cortex, and this elevation was paralleled by increased activity of N-acylphosphatidylethanolamine-hydrolyzing phospholipase D ( approximately 1.7-fold; P < 0.01), and reduced activity ( approximately 0.6-fold; P < 0.01) and expression ( approximately 0.7-fold; P < 0.05) of fatty acid amide hydrolase. These effects of middle cerebral artery occlusion were further potentiated by 1 h of reperfusion, whereas anandamide binding to type 1 cannabinoid and type 1 vanilloid receptors was not affected significantly by the ischemic insult. Additionally, the cannabinoid type 1 receptor antagonist SR141716, but not the receptor agonist R-(+)-WIN55,212-2, significantly reduced (33%; P < 0.05) cerebral infarct volume detected 22 h after the beginning of reperfusion. A neuroprotective intraperitoneal dose of 17beta-estradiol (0.20 mg x kg(-1)) that reduced infarct size by 43% also minimized the effect of brain ischemia on the endocannabinoid system, in an estrogen receptor-dependent manner. In conclusion, we show that the endocannabinoid system is implicated in the pathophysiology of transient middle cerebral artery occlusion-induced brain damage, and that neuroprotection afforded by estrogen is coincident with a re-establishment of anandamide levels in the ischemic striatum through a mechanism that needs to be investigated further.     

p38 MAPK反义寡聚脱氧核苷酸阻断肢体缺血预处理诱导的脑缺血耐受

目的:观察p38MAPK反义寡聚脱氧核苷酸(As-ODN)对肢体缺血预处理(LIP)诱导的脑缺血耐受的影响。方法:48只永久凝闭双侧椎动脉的Wistar大鼠分为8组(n=6):sham组、LIP组、脑缺血损伤组、LIP+脑缺血损伤组、双蒸水+LIP+脑缺血损伤组、p38MAPKAs-ODN组和p38MAPKAs-ODN+LIP+脑缺血损伤组,p38MAPKAs-ODN的剂量又分为5nmol/5μl和10nmol/5μl。所有动物均在sham手术后或末次全脑缺血/再灌注后7天断头取脑,硫堇染色观察海马CA1区锥体神经元迟发性死亡情况。结果:sham组和LIP组均未见延迟性神经元死亡(DND)。与sham、LIP组相比,脑缺血损伤组出现了明显的DND,表现为组织学分级(HG)升高和锥体神经元密度(ND)下降(P0.05)。LIP可显著抑制脑缺血损伤引起的DND。与LIP+脑缺血损伤组相比,p38MAPKAs-ODN+LIP+脑缺血损伤组出现了显著的DND,表现为HG升高、ND降低(P0.05),且此种变化与p38MAPKAs-ODN的注射剂量呈明显正相关。结论:p38MAPKAs-ODN可阻断LIP诱导的脑缺血耐受,进一步证实了p38MAPK表达上调参与了LIP诱导的脑缺血耐受。     

JNK-interacting protein 3 associates with Toll-like receptor 4 and is involved in LPS-mediated JNK activation

Lipopolysaccharide (LPS) is recognized by Toll-like receptor (TLR) 4 and activates NF-kappaB and a set of MAP kinases. Here we have investigated proteins associated with the cytoplasmic domain of mouse TLR4 by yeast two-hybrid screening and identified JNK-interacting protein 3 (JIP3), a scaffold protein for JNK, as a TLR4-associated protein. In mammalian cells, JIP3, through its N-terminal region, constitutively associates with TLR4. The association is specific to JIP3, as the two other JIPs, JIP1 and JIP2, failed to bind TLR4. In HEK 293 cells exogenously expressing TLR4, MD2 and CD14, co-expression of JIP3 significantly increased the complex formation of TLR4-JNK and LPS-mediated JNK activation. In contrast, expression of C-terminally truncated forms of JIP3 impaired LPS-induced JNK activation in a mouse macrophage cell line, RAW264.7. Moreover, RNA interference of JIP3 inhibited LPS-mediated JNK activation. In RAW264.7 cells, JIP3 associates MEKK-1, but not with TAK-1. Finally, JIP3 also associates with TLR2 and TLR9, but not with TLR1 or TLR6. Altogether, our data indicate the involvement of JIP3 in JNK activation in downstream signals of some TLRs.     

Toll样受体4在吗啡应用和耐受中的作用

Toll样受体4(Toll-like receptor 4) 是主要表达于小胶质细胞表面开启哺乳动物先天免疫反应的重要受体.近年研究表明,TLR4参与疼痛及炎症的形成.TLR4 能够被吗啡激活,其结果导致小胶质细胞活化,细胞因子合成和释放增加,从而提高疼痛感受细胞的兴奋性,减小或抵消吗啡的镇痛作用,即形成吗啡耐受.抑制TLR4可以增加吗啡的镇痛作用,减缓吗啡耐受的形成.TLR4与经典阿片受体之间存在立体选择特异性差异,(-)和(+)吗啡均能使之激活.吗啡-TLR4-胶质细胞作用链的研究为治疗吗啡耐受产生提供新的路径.     

Alteration of Toll-like receptor 4 activation by 4-hydroxy-2-nonenal mediated by the suppression of receptor homodimerization

Toll-like receptors (TLRs) detect invading microbial pathogens and initiate immune responses as part of host defense mechanisms. They also respond to host-derived substances released from injured cells and tissues to ensure wound healing and tissue homeostasis. Dysregulation of TLRs increases the risk of chronic inflammatory diseases and immune disorders. Inflammatory events are often accompanied by oxidative stress, which generates lipid peroxidation products such as 4-hydroxy-2-nonenal (4-HNE). Therefore, we investigated if 4-HNE affects TLR activation. We found that 4-HNE blocked LPS (a TLR4 agonist)-induced activation of NFκB and IRF3 as well as expression of IFNβ, IP-10, RANTES, and TNFα. To investigate the mechanism of inhibition by 4-HNE, we examined its effects on TLR4 dimerization, one of the initial steps in TLR4 activation. 4-HNE suppressed both ligand-induced and ligand-independent receptor dimerization. The thiol donors, DTT and NAC, prevented the inhibitory effects of 4-HNE on TLR4 dimerization, and LC–MS/MS analysis showed that 4-HNE formed adducts with cysteine residues of synthetic peptides derived from TLR4. These observations suggest that the reactivity of 4-HNE with sulfhydryl moieties is implicated in the inhibition of TLR4 activation. Furthermore, inhibition of TLR4 activation by 4-HNE resulted in down-regulation of the phagocytic activity of macrophages. Collectively, these results demonstrate that 4-HNE blocks TLR4-mediated macrophage activation, gene expression, and phagocytic functions, at least partly by suppressing receptor dimerization. They further suggest that 4-HNE influences innate immune responses at sites of infection and inflammation by inhibiting TLR4 activation.     

Toll-like receptor 4 is involved in inflammatory and joint destructive pathways in collagen-induced arthritis in DBA1J mice

In rheumatoid arthritis, a significant proportion of cytokine and chemokine synthesis is attributed to innate immune mechanisms. TLR4 is a prominent innate receptor since several endogenous ligands known to activate the innate immune system bind to it and may thereby promote joint inflammation. We generated TLR4 deficient DBA1J mice by backcrossing the TLR4 mutation present in C3H/HeJ strain onto the DBA1J strain and investigated the course of collagen-induced arthritis in TLR4 deficient mice in comparison to wild type littermates. The incidence of collagen- induced arthritis was significantly lower in TLR4 deficient compared to wild type mice (59 percent vs. 100 percent). The severity of arthritis was reduced in the TLR4 deficient mice compared to wild type littermates (mean maximum score 2,54 vs. 6,25). Mice deficient for TLR4 were virtually protected from cartilage destruction, and infiltration of inflammatory cells was reduced compared to wt mice. In parallel to the decreased clinical severity, lower anti-CCP antibody concentrations and lower IL-17 concentrations were found in the TLR4 deficient mice. The study further supports the role of TLR4 in the propagation of joint inflammation and destruction. Moreover, since deficiency in TLR4 led to decreased IL-17 and anti-CCP antibody production, the results indicate a link between TLR4 stimulation and the adaptive autoimmune response. This mechanism might be relevant in human rheumatoid arthritis, possibly in response to activating endogenous ligands in the affected joints.     

Thymocyte depletion during acute Trypanosoma cruzi infection in C57BL/6 mice is partly reverted by lipopolysaccharide pretreatment

Infection with Trypanosoma cruzi in C57BL/6 mice leads to a progressive fatal disease accompanied by thymocyte depletion, which is not related with a higher parasite burden but with increased serum levels of tumour necrosis factor alpha (TNF- alpha). Because this situation may result from an excessive inflammatory syndrome, mice were now given anti-TNF-alpha mAbs throughout their acute infection, or subjected to a LPS desensitization protocol before parasite challenge. Treatment with anti-TNF-alpha mAbs failed to ameliorate thymocyte depletion but shortened survival time and increased parasite load. Pretreatment with LPS (desensitization followed by a sublethal LPS dose) prolonged survival time with a trend to reduce parasitemias and TNF-alpha serum concentrations. Given that pentoxifylline (PTx) interferes with in vitro LPS tolerance, experiments by administering PTx in combination with the tolerance-inducing LPS doses were also performed. Such schedule significantly reduced mortality, TNF-alpha and IL-6 serum concentrations, and CD4+ CD8+ thymocyte loss. LPS pretreatment allowed a better infection control and protected from the accompanying tissue damage.     

Induction of murine macrophage TNF-alpha synthesis by Mycobacterium avium is modulated through complement-dependent interaction via complement receptors 3 and 4 in relation to M. avium glycopeptidolipid

We studied whether complement receptor (CR) mediated Mycobacterium avium interaction modulated macrophage TNF-alpha expression. Compared to control conditions, infections performed with C3-depletion yielded significantly higher TNF-alpha levels. Blockage of the CR4 iC3b site yielded increases in TNF-alpha for all morphotypic variants of a virulent serovar-8 strain (smooth transparent (SmT), smooth opaque (SmO), serovar-specific glycopeptidolipid (ssGPL) deficient knockout mutant) whereas CR3 blockage increased TNF-alpha only for SmT and ssGPL-deficient strains. Thus, complement-mediated binding of M. avium to CR3 and CR4 was shown to modulate TNF-alpha expression. The differential activation of morphotypic and isogenic variants of a single strain provides an excellent model system to delineate signaling pathways.     

A dual negative regulation model of Toll-like receptor 4 signaling for endotoxin preconditioning in human endotoxemia

We discuss a model illustrating how the outcome of repeated endotoxin administration experiments can emerge as a natural consequence of the tightly regulated signaling pathways and also highlight the importance of a dual negative feedback regulation including PI3K/Akt and IRAK-M (IRAK3). We identify the relative time scales of the onset and the magnitude of the stimulus as key determinants of outcome in repeated administration experiments. The results of our simulations involve potentiated response, tolerance, and protective tolerance. Moreover, the knockout of negative regulators shows that IRAK-M is a necessary and sufficient factor for generation of endotoxin tolerance (ET). The effects of the knockout of IRAK-M gene or administration of PI3K inhibitor do yield predictions that have been verified experimentally. Finally, the pretreatment with PI3K inhibitor reveals the interaction between these two negative regulations.     

Gene expressions of Toll-like receptor 2, but not Toll-like receptor 4, is induced by LPS and inflammatory cytokines in mouse macrophages

Toll-like receptors (TLRs) are a family of mammalian homologues of Drosophila Toll and play important roles in host defense. Two of the TLRs, TLR2 and TLR4, mediate the responsiveness to LPS. Here the gene expression of TLR2 and TLR4 was analyzed in mouse macrophages. Mouse splenic macrophages responded to an intraperitoneal injection or in vitro treatment of LPS by increased gene expression of TLR2, but not TLR4. Treatment of a mouse macrophage cell line with LPS, synthetic lipid A, IL-2, IL-15, IL-1beta, IFN-gamma, or TNF-alpha significantly increased TLR2 mRNA expression, whereas TLR4 mRNA expression remained constant. TLR2 mRNA increase in response to synthetic lipid A was severely impaired in splenic macrophages isolated from TLR4-mutated C3H/HeJ mice, suggesting that TLR4 plays an essential role in the process. Specific inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase and p38 kinase did not significantly inhibit TLR2 mRNA up-regulation by LPS. In contrast, LPS-mediated TLR2 mRNA induction was abrogated by pretreatment with a high concentration of curcumin, suggesting that NF-kappaB activation may be essential for the process. Taken together, our results indicate that TLR2, in contrast to TLR4, can be induced in macrophages in response to bacterial infections and may accelerate the innate immunity against pathogens.     

Inhibition of homodimerization of toll-like receptor 4 by 6-shogaol

Toll-like receptors (TLRs) play a critical role in sensing microbial components and inducing innate immune and inflammatory responses by recognizing invading microbial pathogens. Lipopolysaccharide-induced dimerization of TLR4 is required for the activation of downstream signaling pathways including nuclear factor-kappa B (NF-κB). Therefore, TLR4 dimerization may be an early regulatory event in activating ligand-induced signaling pathways and induction of subsequent immune responses. Here, we report biochemical evidence that 6-shogaol, the most bioactive component of ginger, inhibits lipopolysaccharide-induced dimerization of TLR4 resulting in the inhibition of NF-κB activation and the expression of cyclooxygenase-2. Furthermore, we demonstrate that 6-shogaol can directly inhibit TLR-mediated signaling pathways at the receptor level. These results suggest that 6-shogaol can modulate TLR-mediated inflammatory responses, which may influence the risk of chronic inflammatory diseases.     

Activation of Toll-like receptor 4 is associated with insulin resistance in adipocytes

Chronic inflammation is closely associated with metabolic disorders such as obesity and type 2 diabetes, however, the underlying mechanism is unclear. Toll-like receptors (TLRs) play a key role in innate immune response as well as inflammatory signals. Here, we observed that mRNA level of TLR4 was induced during adipocyte differentiation and remarkably enhanced in fat tissues of obese db/db mice. In addition, activation of TLR4 with either LPS or free fatty acids stimulated NFkappaB signaling and expression of inflammatory cytokine genes, such as TNFalpha and IL-6 in 3T3-L1 adipocytes. Furthermore, we discovered that TLR4 activation in 3T3-L1 adipocytes provoked insulin resistance. Taken together, these results suggest that activation of TLR4 in adipocyte might be implicated in the onset of insulin resistance in obesity and type 2 diabetes.     

间歇性低压低氧预处理对脑缺血大鼠海马p-p38 MAPK表达的影响

目的:本文旨在观察间歇性低压低氧(IH)预处理诱导脑缺血耐受过程中,大鼠海马CA1区磷酸化p38MAPK(p-p38 MAPK)的表达以及表达p-p38 MAPK的星形胶质细胞数量。方法:将30只健康雄性Wistar大鼠随机分为6组(n=5):假手术(sham)0 min组、IH+sham 0 min组、sham 7 d组、IH+sham 7 d组、损伤性缺血(Is)7 d组、IH+Is 7 d组。通过硫堇染色对各组大鼠海马CA1区锥体神经元进行神经病理学评价;免疫组织化学染色观察pp38 MAPK的表达;免疫荧光双标法观察表达p-p38 MAPK的星形胶质细胞数量。结果:IH预处理可以诱导脑缺血耐受,同时引起大鼠海马CA1区p-p38 MAPK的表达明显增加,且上调星形胶质细胞中p-p38 MAPK的表达。结论:低压低氧预处理促大鼠海马CA1区锥体神经元和星形胶质细胞中p-p38MAPK上调可能是IH预处理保护脑的一个途经。     

Toll-like receptor 2 is required for LPS-induced Toll-like receptor 4 signaling and inhibition of ion transport in renal thick ascending limb

Previously we demonstrated that basolateral LPS inhibits HCO(3)(-) absorption in the renal medullary thick ascending limb (MTAL) through TLR4-dependent ERK activation. Here we report that the response of the MTAL to basolateral LPS requires TLR2 in addition to TLR4. The basolateral addition of LPS (ultrapure Escherichia coli K12) decreased HCO(3)(-) absorption in isolated, perfused MTALs from wild-type mice but had no effect in MTALs from TLR2(-/-) mice. In contrast, inhibition of HCO(3)(-) absorption by lumen LPS was preserved in TLR2(-/-) MTALs, indicating that TLR2 is involved specifically in mediating the basolateral LPS response. LPS also did not increase ERK phosphorylation in MTALs from TLR2(-/-) mice. TLR2 deficiency had no effect on expression of TLR4, MD-2, or MyD88. However, LPS-induced recruitment of MyD88 to the basolateral membrane was impaired in TLR2(-/-) MTALs. Inhibition of HCO(3)(-) absorption by LPS did not require CD14. Co-immunoprecipitation studies demonstrated an association between TLR4 and TLR2. Inhibition of HCO(3)(-) absorption by TLR2-specific ligands was preserved in MTALs from TLR4(-/-) mice. These results indicate that the effect of basolateral LPS to inhibit HCO(3)(-) absorption in the MTAL through MyD88-dependent ERK activation depends on a novel interaction between TLR4 and TLR2. TLR2 plays a dual role in the induction of intracellular signals that impair MTAL function, both through cooperation with TLR4 to mediate ERK signaling by LPS and through a TLR4-independent signaling pathway activated by Gram-positive bacterial ligands. Regulation of TLR2 expression and its interaction with TLR4 may provide new mechanisms for controlling and therapeutic targeting of TLR4-mediated LPS responses.