Inhibition of the hyaluronan oligosaccharides inflammatory response: reduction of adenosine 2A receptor activation by EPAC and PKA

The aim of this study was to investigate the involvement of exchange proteins directly activated by cyclic adenosine (ADO) monophosphate (EPAC) in 4‐mer hyaluronan (HA) oligosaccharide‐induced inflammatory response in mouse normal synovial fibroblasts (NSF). Treatment of NSF with 4‐mer HA increased Toll‐like receptor‐4, TNF‐alpha and IL‐1beta mRNA expression and of the related proteins, as well as nuclear factor kappaB (NF‐kB) activation. Addition to NSF, previously stimulated with 4‐mer HA oligosaccharides, of ADO significantly reduced NF‐kB activation, TNF‐alpha and IL‐1beta expression. The pre‐treatment of NSF with cyclic ADO monophosphate and/or PKA and/or EPAC‐specific inhibitors significantly inhibited the anti‐inflammatory effect exerted by ADO. In particular, the EPAC inhibitor reduced the ADO effect to a major extent than the PKA inhibitor. These results mean that both PKA and EPAC pathways are involved in ADO‐induced NF‐kB inhibition although EPAC seems to be more involved than PKA. Copyright © 2014 John Wiley & Sons, Ltd.     

Glycosaminoglycans modulate inflammation and apoptosis in LPS‐treated chondrocytes

Previous studies reported that hyaluronic acid (HA), chondroitin sulphate (CS) and heparan sulphate (HS) were able to reduce the inflammatory process in a variety of cell types after lypopolysaccharide (LPS) stimulation. The aim of this study was to investigate the anti‐inflammatory effect of glycosaminoglycans (GAGs) in mouse articular chondrocytes stimulated with LPS. Chondrocyte treatment with LPS (50 µg/ml) generated high levels of TNF‐α, IL‐1β, IL‐6, IFN‐γ, MMP‐1, MMP‐13, iNOS gene expression and their related proteins, increased NO concentrations (evaluated in terms of nitrites formation), NF‐κB activation and IkBα degradation as well as apoptosis evaluated by the increase in caspase‐3 expression and the amount of its related protein. The treatment of chondrocytes using two different doses (0.5 and 1.0 mg/ml) of HA, chondroitin‐4‐sulphate (C4S), chondroitin‐6‐sulphate (C6S), HS, keratan sulphate (KS) and dermatan sulphate (DS) produced a number of effects. HA exerted a very small anti‐inflammatory and anti‐apoptotic effect while it significantly reduced NO levels, although the effect on iNOS expression and activity was extremely slight. C4S and C6S reduced inflammation mediators and the apoptotic process. C6S failed to decrease NO production, although iNOS expression and activity were significantly reduced. HS, like C4S, was able to reduce all the effects stimulated by LPS treatment. KS and DS produced no reduction in any of the parameters considered. These results give further support to the hypothesis that GAGs actively participate in the regulation of inflammatory and apoptotic processes. J. Cell. Biochem. 106: 83–92, 2009. © 2008 Wiley‐Liss, Inc.     

Cistanches alleviates sevoflurane‐induced cognitive dysfunction by regulating PPAR‐γ‐dependent antioxidant and anti‐inflammatory in rats

This study aimed to investigate the protective effects and underlying mechanisms of cistanche on sevoflurane‐induced aged cognitive dysfunction rat model. Aged (24 months) male SD rats were randomly assigned to four groups: control group, sevoflurane group, control + cistanche and sevoflurane + cistanche group. Subsequently, inflammatory cytokine levels were measured by ELISA, and the cognitive dysfunction of rats was evaluated by water maze test, open‐field test and the fear conditioning test. Three days following anaesthesia, the rats were killed and hippocampus was harvested for the analysis of relative biomolecules. The oxidative stress level was indicated as nitrite and MDA concentration, along with the SOD and CAT activity. Finally, PPAR‐γ antagonist was used to explore the mechanism of cistanche in vivo. The results showed that after inhaling the sevoflurane, 24‐ but not 3‐month‐old male SD rats developed obvious cognitive impairments in the behaviour test 3 days after anaesthesia. Intraperitoneal injection of cistanche at the dose of 50 mg/kg for 3 consecutive days before anaesthesia alleviated the sevoflurane‐induced elevation of neuroinflammation levels and significantly attenuated the hippocampus‐dependent memory impairments in 24‐month‐old rats. Cistanche also reduced the oxidative stress by decreasing nitrite and MDA while increasing the SOD and CAT activity. Moreover, such treatment also inhibited the activation of microglia. In addition, we demonstrated that PPAR‐γ inhibition conversely alleviated cistanche‐induced protective effect. Taken together, we demonstrated that cistanche can exert antioxidant, anti‐inflammatory, anti‐apoptosis and anti‐activation of microglia effects on the development of sevoflurane‐induced cognitive dysfunction by activating PPAR‐γ signalling.     

Anti‐inflammatory effects of doxepin hydrochloride against LPS‐induced C6‐glioma cell inflammatory reaction by PI3K‐mediated Akt signaling

Recent studies have shown that tricyclic antidepressants (TCAs) may have anti‐inflammatory and anticonvulsant effects in addition to its antidepressant effects. So far, the nonantidepressant effects of TCAs and their molecular pharmacological mechanisms remain completely unclear. Chronic inflammation in the brain parenchyma may be related to the pathogenesis and progression of various neurodegenerative diseases. As a common antidepressant and anti‐insomnia drug, doxepin also may be a potential anti‐inflammatory and anticonvulsant drug, so the study on the anti‐inflammatory protective effect of doxepin and its molecular mechanism has become a very important issue in pharmacology and clinical medicine. Further elucidating the anti‐inflammatory and neuroprotective effects of doxepin and its molecular mechanism may provide the important theoretical and clinical basis for the prevention and treatment of neurodegenerative disease. This study was designed to understand the glio‐protective mechanism of doxepin against the inflammatory damage induced by lipopolysaccharide (LPS) exposure in C6‐glioma cells. We found the treatment of C6‐glioma cells with LPS results in deleterious effects, including the augmentation of inflammatory cytokine levels (tumor necrosis factor‐α, interleukin‐1β), and suppresses the Akt phosphorylation. Furthermore, our outcomes demonstrated that doxepin was able to suppress these effects induced by LPS, through activation of the phosphatidylinositol‐3‐kinase‐mediated protein kinase B (Akt) pathway. To sum up, these results highlight the potential role of doxepin against neuroinflammatory‐related disease in the brain.     

Anti-inflammatory activity of superoxide dismutase conjugated with sodium hyaluronate

Superoxide dismutase (SOD) from bovine erythrocytes was conjugated with sodium hyaluronate (HA) with a mean molecular weight of 106 to have greater anti-inflammatory activity in vivo. Amino groups of SOD were coupled with carboxyl groups in the hyaluronate molecule using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The HA-SOD conjugate was composed of 1.5 mol of SOD molecule per 1 mol of hyaluronate on the average, and retained 70% of the activity of unmodified SOD. The conjugate was essentially non-immunogenic in mice, and exhibited much higher anti-inflammatory activities than HA or SOD in models of inflammatory diseases such as ischemic oedema of the foot-pad in mice, carrageenin-induced pleurisy and adjuvant arthritis in rats. This revised version was published online in November 2006 with corrections to the Cover Date.     

Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes

Hyaluronan (HA) action depends upon its molecular size. Low molecular weight HA elicits pro-inflammatory responses by modulating the toll-like receptor-4 (TLR-4) or by activating the nuclear factor kappa B (NF-kB). In contrast, high molecular weight HA manifests an anti-inflammatory effect via CD receptors and by inhibiting NF-kB activation. Lipopolysaccharide (LPS) –mediated activation of TLR-4 complex induces the myeloid differentiation primary-response protein (MyD88) and the tumor necrosis factor receptor-associated factor-6 (TRAF-6), and ends with the liberation of NF-kB/Rel family members. The aim of this study was to investigate the influence of HA at different MWs (low, medium, high) on TLR-4 modulation in LPS-induced inflammatory response in mouse chondrocyte cultures.     

Hyaluronan differently modulates TLR-4 and the inflammatory response in mouse chondrocytes

Hyaluronic acid (HA) may exert different action depending on its degree of polymerization. Small HA fragments induce proinflammatory responses, while highly polymerized HA exerts a protective effect in inflammatory pathologies such as rheumatoid arthritis. In both cases the toll-like receptor 4 (TLR-4) seems to be involved in the modulation of the inflammation process. The aim of this study was to investigate the influence of short HA oligosaccharides (HA 4-mers) and high molecular weight HA (HMWHA) in the inflammatory response in normal mouse chondrocytes. Messenger RNA and related protein levels were measured for TLR-4, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and interleukin-18 (IL-18) in cells with and without the addition of HA. NF-kB activation was also evaluated. 4-mer HA treatment produced a significant up-regulation of all parameters considered while HMWHA did not exert any activity in untreated cells although it was able to reduce the effects of 4- mers HA significantly. Specific TLR-4 small interference RNA (siRNA) was used to confirm TLR-4 as the target of HA action. This study suggests that HA may modulate proinflammatory cytokines via its different degree of polymerization and inflammatory action may be modulated as a result of the interaction between HA and TLR-4.     

Proteomic analysis of the anti‐inflammatory action of minocycline

Minocycline possesses anti‐inflammatory properties independently of its antibiotic activity although the underlying molecular mechanisms are unclear. Lipopolysaccharide (LPS)‐induced cytokines and pro‐inflammatory protein expression are reduced by minocycline in cultured macrophages. Here, we tested a range of clinically important tetracycline compounds (oxytetracycline, doxycycline, minocycline and tigecycline) and showed that they all inhibited LPS‐induced nitric oxide production. We made the novel finding that tigecycline inhibited LPS‐induced nitric oxide production to a greater extent than the other tetracycline compounds tested. To identify potential targets for minocycline, we assessed alterations in the macrophage proteome induced by LPS in the presence or absence of a minocycline pre‐treatment using 2‐DE and nanoLC‐MS. We found a number of proteins, mainly involved in cellular metabolism (ATP synthase β‐subunit and aldose reductase) or stress response (heat shock proteins), which were altered in expression in response to LPS, some of which were restored, at least in part, by minocycline. This is the first study to document proteomic changes induced by minocycline. The observation that minocycline inhibits some, but not all, of the LPS‐induced proteomic changes shows that minocycline specifically affects some signalling pathways and does not completely inhibit macrophage activation.     

Concentration dependent dual effect of thrombin in endothelial cells via Par‐1 and Pi3 Kinase

Disruption of endothelial barrier is a critical pathophysiological factor in inflammation. Thrombin exerts a variety of cellular effects including inflammation and apoptosis through activation of the protease activated receptors (PARs). The activation of PAR‐1 by thrombin is known to have a bimodal effect in endothelial cell permeability with a low concentration (pM levels) eliciting a barrier protective and a high concentration (nM levels) eliciting a barrier disruptive response. It is not known whether this PAR‐1‐dependent activity of thrombin is a unique phenomenon specific for the in vitro assay or it is part of a general anti‐inflammatory effect of low concentrations of thrombin that may have a physiological relevance. Here, we report that low concentrations of thrombin or of PAR‐1 agonist peptide induced significant anti‐inflammatory activities. However, relatively high concentration of thrombin or of PAR‐1 agonist peptide showed pro‐inflammatory activities. By using function‐blocking anti‐PAR‐1 antibodies and PI3 kinase inhibitor, we show that the direct anti‐inflammatory effects of low concentrations of thrombin are dependent on the activation of PAR‐1 and PI3 kinase. These results suggest a role for cross communication between PAR‐1 activation and PI3 kinase pathway in mediating the cytoprotective effects of low concentrations of thrombin in the cytokine‐stimulated endothelial cells. J. Cell. Physiol. 219: 744–751, 2009. © 2009 Wiley‐Liss, Inc.     

丁香苷抗炎镇痛作用及部分机制研究

研究丁香苷抗炎镇痛作用及部分机制。以阿司匹林作阳性对照药,观察丁香苷对二甲苯致小鼠耳廓肿胀、醋酸致小鼠毛细血管通透性增加、角叉菜胶致大鼠足趾肿胀、棉球致大鼠肉芽肿的抗炎作用;对小鼠热板试验、醋酸扭体试验的镇痛作用;同时测定角叉菜胶致大鼠炎足炎性渗出物中的PGE2、MDA和血清中的NO、SOD,初步探讨丁香苷抗炎镇痛的部分机制。结果表明,丁香苷对急慢性炎症反应有明显抑制作用,能明显降低角叉菜胶致炎足炎性渗出物中PGE2、MDA和血清中NO含量,明显增加血清中SOD的活性。因此,丁香苷具有较强的抗炎镇痛作用,其机制可能与抑制PGE2、NO等炎症介质生成、增强自由基清除能力有关。     

Fucoxanthin Inhibits the Inflammation Response in Paw Edema Model through Suppressing MAPKs,Akt, and NFκB

Undaria pinnatifida is a well‐known traditional Korean food with a variety of biological activities. Carrageenan (carr) is commonly used to induce paw edema in animal models. This study was designed to elucidate the processes underlying the anti‐inflammatory effect of fucoxanthin isolated from the sporophyll of U. pinnatifida in carr‐induced paw edema in ICR mice. Fucoxanthin significantly decreased carr‐induced increased nitric oxide levels in the plasma of mice with carr‐induced paw edema. Fucoxanthin protected catalase (CAT) and superoxide dismutase (SOD) activity against disruption in mice with carr‐induced paw edema. In addition, fucoxanthin repressed carr‐induced activation of inducible nitric oxide synthase, cyclooxygenase‐2, and nuclear factor kappa B, as well as carr‐induced phosphorylation of mitogen‐activated protein kinase, extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase, p38, and protein kinase B/Akt. These results suggest that fucoxanthin may have therapeutic potential as a treatment for patients with inflammatory diseases.     

Specific calcineurin targeting in macrophages confers resistance to inflammation via MKP‐1 and p38

Macrophages contribute to tissue homeostasis and influence inflammatory responses by modulating their phenotype in response to the local environment. Understanding the molecular mechanisms governing this plasticity would open new avenues for the treatment for inflammatory disorders. We show that deletion of calcineurin (CN) or its inhibition with LxVP peptide in macrophages induces an anti‐inflammatory population that confers resistance to arthritis and contact hypersensitivity. Transfer of CN‐targeted macrophages or direct injection of LxVP‐encoding lentivirus has anti‐inflammatory effects in these models. Specific CN targeting in macrophages induces p38 MAPK activity by downregulating MKP‐1 expression. However, pharmacological CN inhibition with cyclosporin A (CsA) or FK506 did not reproduce these effects and failed to induce p38 activity. The CN‐inhibitory peptide VIVIT also failed to reproduce the effects of LxVP. p38 inhibition prevented the anti‐inflammatory phenotype of CN‐targeted macrophages, and mice with defective p38‐activation were resistant to the anti‐inflammatory effect of LxVP. Our results identify a key role for CN and p38 in the modulation of macrophage phenotype and suggest an alternative treatment for inflammation based on redirecting macrophages toward an anti‐inflammatory status.     

Inhibition of inflammation and oxidative stress by an imidazopyridine derivative X22 prevents heart injury from obesity

Inflammation and oxidative stress plays an important role in the development of obesity‐related complications and cardiovascular disease. Benzimidazole and imidazopyridine compounds are a class of compounds with a variety of activities, including anti‐inflammatory, antioxidant and anti‐cancer. X22 is an imidazopyridine derivative we synthesized and evaluated previously for anti‐inflammatory activity in lipopolysaccharide‐stimulated macrophages. However, its ability to alleviate obesity‐induced heart injury via its anti‐inflammatory actions was unclear. This study was designed to evaluate the cardioprotective effects of X22 using cell culture studies and a high‐fat diet rat model. We observed that palmitic acid treatment in cardiac‐derived H9c2 cells induced a significant increase in reactive oxygen species, inflammation, apoptosis, fibrosis and hypertrophy. All of these changes were inhibited by treatment with X22. Furthermore, oral administration of X22 suppressed high‐fat diet‐induced oxidative stress, inflammation, apoptosis, hypertrophy and fibrosis in rat heart tissues and decreased serum lipid concentration. We also found that the anti‐inflammatory and anti‐oxidative actions of X22 were associated with Nrf2 activation and nuclear factor‐kappaB (NF‐κB) inhibition, respectively, both in vitro and in vivo. The results of this study indicate that X22 may be a promising cardioprotective agent and that Nrf2 and NF‐κB may be important therapeutic targets for obesity‐related complications.     

磁性纳米粒介导沉默PI3Kγ基因调控肿瘤相关巨噬细胞抗小鼠肺癌细胞效应的研究

为了探讨超顺磁性Fe3O4纳米粒子(superparamagnetic iron oxide nanoparticles,SPIONs)介导的磷脂酰肌醇3激酶γ(phosphatidylinositol 3 kinaseγ,PI3Kγ)抑制表达调控的肿瘤相关巨噬细胞(tumor-associated macrophages,TAM)对小鼠Lewis肺癌细胞(Lewis lung carcinoma,LLC)增殖和凋亡的影响,该研究构建了能启动巨噬细胞(macrophage,MФ)特异性表达PI3Kγ催化亚基p100 siRNA的pSilencer-EGFP-SP-p110质粒,通过SPIONs负载成磁性纳米质粒复合物(SPIONs-DNA),在强磁作用下转染MФ,通过普鲁士蓝染色法检测SPIONs-DNA在细胞内的分布,Real-time PCR和Western blot检测细胞PI3Kγp110亚基的表达水平。建立M1、M2型MФ模型,将SPIONs-DNA在强磁作用下转染M2型MФ,通过Real-time PCR和Western blot鉴定细胞表型,明确M2型MФ转化为M1型的强度。采用Transwell系统建立SPIONs-DNA转染的M2型MФ与小鼠LLC细胞的共培养模型,通过锥虫蓝染色法检测LLC细胞的活细胞数并绘制细胞生长曲线,CCK-8法检测LLC细胞增殖情况,硝酸还原酶法检测共培养液上清中NO含量,流式细胞术检测LLC细胞凋亡情况。结果显示,制备的SPIONs-DNA在强磁作用下成功转染MФ并大量分布在细胞胞核周围,SPIONs-DNA转染组细胞PI3Kγp110 mRNA和蛋白表达水平显著低于空白细胞对照组(P<0.05)。建立的M1型MФ高表达iNOS(P<0.001),M2型MФ高表达ARG-1(P<0.001)。M2型MФ转染SPIONs-DNA后细胞iNOS mRNA和蛋白的表达显著增加(P<0.001),ARG-1 mRNA和蛋白的表达显著降低(P<0.01)。在共培养组中,SPIONs-DNA转染的M2型MФ组能大量分泌NO,LLC细胞生长和增殖能力显著降低(P<0.05),凋亡率显著增高(P<0.01)。结果表明,磁性纳米粒负载pSilencer-EGFP-SP-p110重组质粒能够特异性靶向抑制巨噬细胞PI3Kγp110的表达,诱导M2型MФ转化为M1型;其转染的M2型MФ可显著抑制LLC细胞的生长和增值,促进细胞凋亡,这与其大量分泌NO有关。该磁性纳米质粒复合物可诱导TAM发挥抗肿瘤作用,为研究开发有效的抗肺癌基因治疗措施奠定基础。     

Rutin ameliorates copper sulfate‐induced brain damage via antioxidative and anti‐inflammatory activities in rats

Excessive exposure to Copper (Cu) may result in Cu toxicity and adversely affect health outcomes. We investigated the protective role of rutin on Cu‐induced brain damage. Experimental rats were treated as follows: group I: control; group II: Cu‐sulfate: 200 mg/kg; group III: Cu‐sulfate, and rutin 100 mg/kg; and group IV: rutin 100 mg/kg, for 7 weeks. Cu only treatment significantly decreased body weight gain, while rutin cotreatment reversed this decrease. Cu treatment increased malondialdehyde, nitric oxide level, and myeloperoxidase activity and decreased superoxide dismutase and catalase activities in rat brain. Immunohistochemistry showed that COX‐2, iNOS, and Bcl‐2 proteins were strongly expressed, while Bax was mildly expressed in the brain of Cu‐treated rats. Furthermore, brain histology revealed degenerated neurons, and perforated laminae of cerebral cortex in the Cu‐only treated rats. Interestingly, coadministration of Cu and rutin reduced the observed histological alteration, improved inflammatory and antioxidant biomarkers, thereby protecting against Cu‐induced brain damage via antioxidative and anti‐inflammatory mechanisms.     

Myricetin suppresses lipoteichoic acid‐induced interleukin‐1β and cyclooxygenase‐2 expression in human gingival fibroblasts

Periodontitis is an inflammatory disease affecting the connective tissue and supporting bone surrounding the teeth. In periodontitis, human gingival fibroblasts (HGFs) synthesize IL‐1β, causing a progressive inflammatory response. Flavones demonstrate a variety of biological activity: among others, they possess anti‐inflammatory properties. Myricetin is a flavone with a strong anti‐inflammatory activity. The objective of this study was to evaluate the effect of the flavonoid myricetin on HGFs under inflammatory conditions induced by lipoteichoic acid (LTA). the effect of myricetin on HGFs was assessed by measuring cell viability, signaling pathways and IL‐1β expression and synthesis. It was found that, over time, myricetin did not affect cell viability. However, it inhibited activation of p38 and extracellular‐signal‐regulated kinase‐1/2 in LTA‐treated HGFs and also blocked IκB degradation and cyclooxygenase‐2 and prostaglandin E2 synthesis and expression. These findings suggest that myricetin has therapeutic effects in the form of controlling LTA‐induced inflammatory responses.     

Anti‐arthritis activity of cationic materials

Cationic materials exhibit remarkable anti‐inflammatory activity in experimental arthritis models. Our aim was to confirm this character of cationic materials and investigate its possible mechanism. Adjuvant‐induced arthritis (AIA) models were used to test cationic materials for their anti‐inflammatory activity. Cationic dextran (C‐dextran) with different cationic degrees was used to investigate the influence of the cationic elements of materials on their anti‐inflammatory ability. Peritoneal macrophages and spleen cells were used to test the expression of cytokines stimulated by cationic materials. Interferon (IFN)‐γ receptor‐deficient mice and macrophage‐depleted rats were used to examine the possible mechanisms of the anti‐inflammatory activity of cationic materials. In AIA models, different cationic materials shared similar anti‐inflammatory characters. The anti‐inflammatory activity of C‐dextran increased with as the cationic degree increased. Cationic materials stimulated interleukin (IL)‐12 expression in peritoneal macrophages, and strong stimulation of IFN‐γ secretion was subsequently observed in spleen cells. In vivo experiments revealed that circulating IL‐12 and IFN‐γ were enhanced by the cationic materials. Using IFN‐γ receptor knockout mice and macrophage‐depleted rats, we found that IFN‐γ and macrophages played key roles in the anti‐inflammatory activity of the materials towards cells. We also found that neutrophil infiltration at inflammatory sites was reduced when AIA animals were treated with C‐dextran. We propose that cationic signals act through an unknown receptor on macrophages to induce IL‐12 secretion, and that IL‐12 promotes the expression of IFN‐γ by natural killer cells (or T cells). The resulting elevated systemic levels of IFN‐γ inhibit arthritis development by preventing neutrophil recruitment to inflammatory sites.