Expression Of Several Inflammatory Mediators Research Articles

The effect of antibody-mediated PCSK9 inhibitor on bare-metal stent implantation outcome

Abstract Introduction Percutaneous coronary intervention (PCI) is the gold standard for the management of acute coronary syndrome, and has been benefited from advances in pharmacotherapy and device innovation. Nevertheless, in-stent restenosis and stent thrombosis remain serious complications following PCI procedure with stent implantation. Proprotein convertase subtilisin kexin type 9 (PCSK9) is an enzyme known for its role in plasma cholesterol homeostasis. Efficiently reducing plasma cholesterol, PCSK9 inhibition recently became a pivotal treatment strategy for hypercholesterolemia. However, the effect of PCSK9 inhibition on stent implantation outcomes remains uninvestigated. Methods Carotid artery bare-metal stent (BMS) implantation was performed on C57Bl/6 mice, which then a parallel assessment of various inflammatory mediators was performed. To inhibit PCSK9, alirocumab was administered weekly to antiplatelet-treated mice started before stent implantation. Six weeks after the stent implantation procedure, animals were harvested and histomorphometric analysis was performed on stented arteries. In vitro cell migration and cellular senescence were also assessed on alirocumab-treated primary human aortic endothelial (EC) and vascular smooth muscle cells (VSMC). Results Compared to sham intervention, stent implantation was associated with increased expression of several inflammatory mediators, including PCSK9. Interestingly, the increase in PCSK9 level was confirmed in the stented vascular tissue, but not in plasma (Fig 1A). Following alirocumab treatment, PCSK9 inhibition results in an increased intimal hyperplasia in the stented vascular segment of alirocumab-treated animals compared to controls (Fig 1B). In vitro, alirocumab promoted migration and inhibited the onset of senescence in primary human VSMC, while blunted the migration and increased the senescence of EC. Conclusion The results of this study demonstrated that antibody-based PCSK9 inhibition worsen bare-metal stent implantation outcome in mice. This observed effect is mediated, in part, by the differential effect on VSMC and EC senescence, thus promoting in-stent intimal hyperplasia while blunting vascular healing. The herein-reported data warrant additional investigations concerning the use of PCSK9 inhibitors in patients undergoing PCI with stent implantation.

Antibody-mediated PCSK9 neutralization worsens outcome after bare-metal stent implantation in mice

AimsDespite advances in pharmacotherapy and device innovation, in-stent restenosis (ISR) and stent thrombosis (ST) remain serious complications following percutaneous coronary intervention (PCI) procedure with stent implantation. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme involved in plasma cholesterol homeostasis and recently emerged as a therapeutic target for hypercholesterolemia. Antibody-based PCSK9 inhibition is increasingly used in different subsets of patients, including those undergoing PCI. However, whether PCSK9 inhibition affects outcome after stent implantation remains unknown. Methods and results12 to 14 weeks old C57Bl/6 mice underwent carotid artery bare-metal stent implantation. Compared to sham intervention, stent implantation was associated with increased expression of several inflammatory mediators, including PCSK9. The increase in PCSK9 protein expression was confirmed in the stented vascular tissue, but not in plasma. To inhibit PCSK9, alirocumab was administered weekly to mice before stent implantation. After 6 weeks, histological examination revealed increased intimal hyperplasia in the stented segment of alirocumab-treated animals compared to controls. In vitro, alirocumab promoted migration and inhibited the onset of senescence in primary human vascular smooth muscle cells (VSMC). Conversely, it blunted the migration and increased the senescence of endothelial cells (EC). ConclusionAntibody-based PCSK9 inhibition promotes in-stent intimal hyperplasia and blunts vascular healing by increasing VSMC migration, while reducing that of EC. This effect is likely mediated, at least in part, by a differential effect on VSMC and EC senescence. The herein-reported data warrant additional investigations concerning the use of PCSK9 inhibitors in patients undergoing PCI with stent implantation.

Anti-inflammatory properties of raw honey and its clinical applications in daily practice

Background: Honey is a natural product; and in terms of phytochemical composition, it contains a high number of flavonoids and polyphenols such as quercetin, kaempferol, chrysin, and apigenin. Flavonoids found in honey mitigate the inflammatory processes, thereby exhibiting the anti-inflammatory potential of honey. Besides their anti-oxidant activities, flavonoids have the ability to inhibit pro-inflammatory enzymes such as LOX, COX, iNOS, and pro-inflammatory mediators, including cytokines, nitric oxide, and chemokines. Furthermore, flavonoids modulate transcriptional factors such as NF-?B, thereby controlling the expression of several inflammatory mediators.Methods: Clinical uses of natural raw honey have been studied at our Umm Ghuwailina Health Center, Doha, Qatar, with daily application under occlusion with promising results, in various patients with multiple conditions, including eczema, seborrheic dermatitis, and psoriasis. A layer of honey was applied to the lesion site, covered with a transparent securement dressing, and washed off for 7 consecutive days for patients with atopic dermatitis and psoriasis. The Three Item Severity (TIS) score, which includes erythema, edema/papulation, and excoriation on a scale from 0 to 3, was used for atopic dermatitis. For psoriasis, the primary outcome measure was the intensity component of the validated Psoriasis Area and Severity Index (PASI) and the Visual Analogue Score (VAS). Participants who were allergic to honey or receiving any corticosteroid within the last one month were excluded.Results: Twelve patients with atopic dermatitis had a significant improvement from a TIS score of 6 to 1 noticed in the honey-treated lesion 1 week later. With regard to psoriatic lesions, 6 patients were studied. There was a significant overall improvement in their psoriatic lesions, reaching a PASI score of 80 even one month after stopping the topical honey application. The vast majority of patients showed a significant improvement and no side effects.Conclusion: Raw natural honey might be a promising efficacious and cost-effective alternative to advanced anti-inflammatory products. However, well-designed double-blind placebo-controlled clinical trials are needed to confirm the benefits of honey from different botanical sources.

Plantaricin NC8 \u03b1\u03b2 prevents Staphylococcus aureus-mediated cytotoxicity and inflammatory responses of human keratinocytes

Multidrug resistance bacteria constitue an increasing global health problem and the development of novel therapeutic strategies to face this challenge is urgent. Antimicrobial peptides have been proven as potent agents against pathogenic bacteria shown by promising in vitro results. The aim of this study was to characterize the antimicrobial effects of PLNC8 αβ on cell signaling pathways and inflammatory responses of human keratinocytes infected with S. aureus. PLNC8 αβ did not affect the viability of human keratinocytes but upregulated several cytokines (IL-1β, IL-6, CXCL8), MMPs (MMP1, MMP2, MMP9, MMP10) and growth factors (VEGF and PDGF-AA), which are essential in cell regeneration. S. aureus induced the expression of several inflammatory mediators at the gene and protein level and PLNC8 αβ was able to significantly suppress these effects. Intracellular signaling events involved primarily c-Jun via JNK, c-Fos and NFκB, suggesting their essential role in the initiation of inflammatory responses in human keratinocytes. PLNC8 αβ was shown to modulate early keratinocyte responses, without affecting their viability. The peptides have high selectivity towards S. aureus and were efficient at eliminating the bacteria and counteracting their inflammatory and cytotoxic effects, alone and in combination with low concentrations of gentamicin. We propose that PLNC8 αβ may be developed to combat infections caused by Staphylococcus spp.

Protective effect of Alpha-Linolenic acid on Lipopolysaccharide-Induced Orchitis in mice.

Previous studies have demonstrated that polyunsaturated fatty acids (PUFAs) have anti-inflammatory effects. One specific PUFA, alpha-linolenic acid (ALA), shows both anti-inflammatory and antioxidant properties. In the testes, inflammatory mediators are known to increase when orchitis is induced by bacterial lipopolysaccharide (LPS). This study aimed to determine whether the anti-inflammatory properties of ALA could have a protective effect against LPS-induced orchitis in mice. The mice were divided into untreated control, orchitis and ALA-treated orchitis groups. Orchitis was induced by intraperitoneal injection of LPS. The ALA-treated group was administered ALA by gavage three days before intraperitoneal LPS injection. Cyclooxygenase-2 (COX-2), cytosolic phospholipase A2 (cPLA2), inducible nitric oxide synthase (iNOS) enzymes and nuclear factor kappa-B (NF-κB) in the testes, as well as serum interleukin 6 (IL-6) and tumour necrosis factor-alpha (TNF-α), were analysed using enzyme-linked immunosorbent assay (ELISA) tests. LPS administration increased the expression of several inflammatory mediators, including IL-6, TNF-α and NF-κB, as well as the COX-2, cPLA2 and iNOS enzymes. ALA administration significantly prevented the LPS-induced increases in these inflammatory mediators and enzymes (p<.05). The anti-inflammatory and antioxidant effects of ALA may make it a useful candidate for the treatment of orchitis caused by bacterial LPS.

Inflammatory cardiac fibroblast phenotype underlies chronic alcohol-induced cardiac atrophy and dysfunction

AimsThe purpose of this study was to investigate mechanisms of chronic alcohol-induced cardiac remodeling and dysfunction. We also sought to determine the role of cardiac fibroblasts, which play a dynamic role in cardiac remodeling, in mediating these effects. Main methodsAdult male Wistar rats were exposed to ethanol (EtOH) vapor inhalation for 16 weeks. Echocardiography was performed to assess terminal cardiac structure and function. Cardiac fibroblasts were isolated from the left ventricle (LV) for both ex vivo and in vitro analysis. Cultured H9C2 cells were also exposed to conditioned media from alcohol-exposed cardiac fibroblasts. Gene expression in whole LV tissue, isolated cardiac fibroblasts, or cultured H9C2 cells was determined by real-time PCR, and protein expression was determined by Western blot. Key findingsEtOH led to LV wall thinning and impaired systolic function, and decreased contractile protein mRNA levels. EtOH increased LV inflammatory markers, JNK and Akt activation, and decreased mTOR expression. EtOH induced myofibroblast activation as assessed by flow cytometry, and increased LV collagen III expression. EtOH increased expression of several inflammatory mediators in cardiac fibroblasts both ex vivo and in vitro. Administration of conditioned media from EtOH-treated fibroblasts decreased contractile protein mRNA levels and impaired Akt and mTOR signaling in differentiated H9C2 cardiomyocytes. SignificanceOur results indicate that EtOH-induced cardiac atrophy and dysfunction is associated with activation of inflammatory pathways. Furthermore, EtOH may induce a pro-inflammatory cardiac fibroblast phenotype, leading to aberrant fibroblast-myocyte cross-talk. Thus, EtOH may promote cardiac muscle wasting in part by activation of pro-inflammatory fibroblasts.

Effect of sodium salicylate on COX-2 expression in neonatal rat cardiomyocytes

Salicylates, one of the oldest medicinal compounds known to humans, have been reported to show anti-inflammatory effects via cyclooxygenase (COX) inhibition. However, the pathophysiological role of COX-2 in the heart is conflicting, and the role of sodium salicylate in the regulation of cardiac inflammation has not yet been elucidated. We aimed to investigate the effect of salicylate on COX-2 expression and its associated prostaglandin production using cultured neonatal rat cardiomyocytes. The cells were incubated in the presence or absence of sodium salicylate (8 mM). Treatment with sodium salicylate significantly increased COX-2 expression at both mRNA and protein levels, induced prostaglandin D2 release, and increased TNF-α mRNA expression in cardiomyocytes. In addition, salicylate treatment induced cardiomyocyte hypertrophy. Taken together, we demonstrated that salicylate induced COX-2 expression, which in turn resulted in the regulation of expression of several inflammatory mediators in cardiomyocytes.

Pleiotropic Roles of Nrf2 as Regulators of Chondrocyte Apoptosis, Oxidative Stress, Inflammatory Response and Catabolic and Anabolic Pathways in Osteoarthritis

Nrf2, a redox regulated transcription factor, has recently been shown to play a role in cartilage integrity but the mechanism remains largely unknown. Osteoarthritis (OA) is a multifactorial disease in which focal degradation of cartilage occurs. Here, we investigated the regulation of oxidative stress, inflammatory response, apoptosis, catabolic and anabolic factors by Nrf2 in IL-1β-stimulated human OA chondrocytes. Ectopic expression of Nrf2 in OA chondrocytes suppressed the generation of ROS while Nrf2 knockdown significantly increased the ROS levels in IL-1β-stimulated OA chondrocytes. Further, Nrf2 activation significantly inhibited the IL-1β-induced activation of extrinsic and intrinsic apoptotic pathways as determined by inhibition of DNA fragmentation, activation of Caspase-3,-8,-9, cleavage of PARP, release of cytochrome-c, suppression of mitochondrial dysfunction, suppression of pro-apoptotic proteins Bax, Bad and Bid expression and enhanced expression of anti-apoptotic proteins Bcl2, Bcl-xl and Mcl-1 in OA chondrocytes. To examine the role of Nrf2 in IL-1β-induced inflammatory response, we determined the mRNA expression and protein levels of several cytokines and chemokines using PCR array and multiplex immunoassay. Our data demonstrate that Nrf2 activation inhibited the IL-1β-mediated expression of several inflammatory mediators and Gene Ontology analysis revealed significant enrichment of MAPK signaling, TNF signaling, NF-κβ signaling and cytokine receptor signaling pathways. Additionally, Nrf2 activation inhibited the expression matrix degrading proteases-MMP-13, 3, 9, and ADAMTS-4 and enhanced the expression of OA related anabolic factors-COL2A1, ACAN, FRZB and GDF5 and SOX-5, 8 and 9 in IL-1β-stimulated OA chondrocytes. Importantly, we also discovered that Nrf2 mediated suppression of catabolic factors was dependent on ERK1/2 activation by Nrf2. Our data demonstrate that Nrf2 regulates multiple pathways involved in chondrocyte apoptosis, inflammation and matrix degradation via activation of ERK1/2/ELK1-P70S6K-P90RSK axis. Our findings collectively support the development of therapies targeting Nrf2 for the management of OA.

Gabapentin Attenuates Ocular Inflammation: In vitro and In vivo Studies.

To investigate the effects of gabapentin, a structural analog of γ-amino butyric acid (GABA), on the inflammatory response of lipopolysaccharide (LPS)-stimulated rabbit corneal cells (SIRC) and on endotoxin-induced uveitis (EIU) in rabbits. We investigated the LPS-induced expression of several inflammatory mediators, such as TNF-α, IL-1β, cPLA2, COX-2, and PGE2 in the SIRC cells with or without gabapentin treatment. Gabapentin treatment significantly (p < 0.05) attenuated cytokines production, cPLA2 activation, COX-2 expression, and PGE2 levels in SIRC. EIU was induced by an intraocular injection of 0.1 μg of LPS in albino rabbit eye. After 7 and 24 h from LPS injection clinical signs of ocular inflammation were examined by slit lamp with or without topical treatment of 0.5% gabapentin. Tears, aqueous, cornea, conjunctiva, and iris-ciliary body were collected and inflammatory biomarkers assessed. Topical treatment with gabapentin significantly (p < 0.05) reduced clinical signs and biomarkers of inflammation compared with the LPS group both at 7 and 24 h. In conclusion, the results generated in the present study suggest that ophthalmic formulation based on gabapentin may be useful in the treatment of inflammatory conditions associated to ocular pain such as uveitis, and that clinical studies to evaluate this possibility may be warranted.

Non-redundant role of the chemokine receptor CX3CR1 in the anti-inflammatory function of gut macrophages

Mucosal immunity at the intestinal level is constantly challenged by the presence of external food and microbial antigens and must be kept under strict control to avoid the rise of aberrant inflammation. Among cells of the innate immunity, macrophages expressing the chemokine receptor CX3CR1 are strategically located near the gut epithelial barrier. These cells contribute to the maintenance of homeostasis by producing the anti-inflammatory cytokine IL-10; however, their role in the control of full blown inflammation and tissue injury is controversial. In this study we investigated mice proficient or deficient for the expression of the CX3CR1 receptor in a model of dextran sulphate sodium (DSS) induced acute colitis. We found that KO mice (CX3CR1GFP/GFP) had a more severe disease compared to WT mice (CX3CR1GFP/+), both in terms of histological examination of colonic tissues and leukocyte infiltration, with an expansion of macrophages and CD4-Th17 lymphocytes. The expression of several inflammatory mediators (IL-1β, IL-6, IFNγ, iNOS) was also significantly upregulated in KO mice, despite higher IL-10 production. Overall, our study demonstrates that macrophages expressing a functional CX3CR1 receptor have an important and non-redundant role in controlling the abnormal intestinal inflammation that may lead to tissue damage.

Anti-inflammatory and Anti-tumor Activities of Parthenolide: An Update

Parthenolide (PTL), the secondary metabolite of feverfew plant (Tanaceum parthenium), has been used in various medical purposes globally. Inflammation represents a physiological response to injury and helps to restore tissue homeostasis. Inflammation and cancer both are associated with genotoxicity, invasion, metastasis, and abnormal tissue repair mechanisms. PTL inhibit major cellular inflammatory and proliferation pathways like NFκB, STAT3, and MAPK along with the activity and expression of several inflammatory mediators including COX. NFκB pathway plays a key role in controlling cell cycle progression and apoptosis together with metastasis and cancer of various types. Elevated NFκB, Wnt/β-catenin pathways are crucial factors of tumorogenesis. PTL inhibits NFκB and Wnt/β-catenin pathways, and thereby promotes apoptosis and suppresses cell proliferation. Experimental data showed that PTL protects normal cells from apoptosis; whereas in cancer cells it induces apoptotic cell death. Hence, parthenolide could be useful in controlling inflammatory diseases alone or together with tumorogenesis due to its evident anticancer potency and anti-inflammatory nature.

YCG063 inhibits Pseudomonas aeruginosa LPS-induced inflammation in human retinal pigment epithelial cells through the TLR2-mediated AKT/NF-κB pathway and ROS-independent pathways.

YCG063 is known as an inhibitor of reactive oxygen species (ROS); however, its intracellular mechanisms of action remain poorly understood. In the present study, we investigated the effects of YCG063 on the inflammatory response of Pseudomonas aeruginosa lipopolysaccharide (PA-LPS)‑stimulated human retinal pigment epithelial cells (RPE cells). Human adult RPE cells (ARPE‑19) were stimulated with PA-LPS. We then investigated the LPS-induced expression of several inflammatory mediators, such as interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and intracellular adhesion molecule-1 (ICAM-1) in the ARPE-19 cells. We performed an enzyme-linked immunosorbent assay (ELISA), western blot analysis, electrophoretic mobility shift assay (EMSA) and fluorescence-activated cell sorting (FACS) to elucidate the mechanisms involved in the anti-inflammatory effects of YCG063 in the PA-LPS-stimulated cells. The results revealed that treatment with YCG063 significantly inhibited the levels of IL-6, IL-8, MCP-1 and ICAM-1 in the PA-LPS-stimulated ARPE-19 cells. YCG063 also markedly inhibited the phosphorylation of AKT in the PA‑LPS-stimulated cells. In addition, the activation of nuclear factor-κB (NF-κB) was also attenuated folllowing treatment with YCG063. ROS were not generated in the PA-LPS-stimulated cells. In conclusion, our data indicate that YCG063 may prove to be a potential protective agent against inflammation, possibly through the downregulation of Toll‑like receptor 2 (TLR2) and the AKT-dependent NF-κB activation pathway in PA-LPS-stimulated ARPE-19 cells. Furthermore, this anti-inflammatory activity occurred through ROS-independent signaling pathways.

Coffee and liver health.

Coffee is one of the most widely used beverages in the world. It includes a wide array of components that can have potential implications for health. Several epidemiological studies associate coffee consumption with a reduced incidence of various chronic diseases such as diabetes, cardiovascular diseases, and neurodegenerative diseases. Over the past 20 years, an increasing number of epidemiological and experimental studies have demonstrated the positive effects of coffee on chronic liver diseases. Coffee consumption has been inversely associated with the activity of liver enzymes in subjects at risk, including heavy drinkers. Coffee favours an improvement in hepatic steatosis and fibrosis, and a reduction in cirrhosis and the risk of hepatocellular carcinoma. The mechanisms of action through which it exerts its beneficial effects are not fully understood. Experimental studies show that coffee consumption reduces fat accumulation and collagen deposition in the liver and promotes antioxidant capacity through an increase in glutathione as well as modulation of the gene and protein expression of several inflammatory mediators. Animal and in vitro studies indicate that cafestol and kahweol, 2 diterpens, can operate by modulating multiple enzymes involved in the detoxification process of carcinogens causing hepatocellular carcinoma. It is unclear whether the benefits are significant enough to "treat" patients with chronic liver disease. While we await clarification, moderate daily unsweetened coffee use is a reasonable adjuvant to therapy for these patients.

IL-1β Induces MMP-9-Dependent Brain Astrocytic Migration via Transactivation of PDGF Receptor/NADPH Oxidase 2-Derived Reactive Oxygen Species Signals.

Matrix metalloproteinase-9 (MMP-9) plays a crucial role in pathological processes of brain inflammation, injury, and neurodegeneration. Moreover, cytokines such as interleukin-1β (IL-1β) induce expression of several inflammatory mediators in brain astrocytes, which may be important for brain inflammatory disorders. Recent studies have implicated that increased oxidative stress may contribute to the brain injury and inflammation. However, whether IL-1β-induced MMP-9 expression mediated through oxidative stress remains unclear. Therefore, we investigated the role of redox signals in IL-1β-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells). Herein, we first demonstrated that reactive oxygen species (ROS) play a crucial role in ILβ-induced MMP-9 expression by zymography, real-time PCR, and ROS staining in cultured RBA-1 cells. Next, IL-1β-induced MMP-9 expression is mediated through a c-Src-mediated transactivation of PDGFR/PI3K/Akt cascade linking to p47(phox)/NADPH oxidase 2 (Nox2)/ROS signaling pathway. Nox2-dependent ROS generation led to activation of MAPKs and the downstream transcription factors NF-κB and AP-1 (i.e., ATF2), which enhanced MMP-9 promoter activity, and thereby turned on transcription of MMP-9 gene. Functionally, IL-1β-induced MMP-9 expression promoted astrocytic migration. These results demonstrated that in RBA-1 cells, activation of NF-κB and AP-1 (ATF2) by the c-Src/PDGFR/PI3K/Akt-mediated Nox2/ROS/MAPKs signals is required for upregulation of MMP-9 and cell migration enhanced by IL-1β.

Chronic antidepressant treatments resulted in altered expression of genes involved in inflammation in the rat hypothalamus

To gain insight into the possible immune targets of antidepressant, we evaluated the expression of several inflammatory mediators in the hypothalamus of rats chronically (28 days) treated with the serotonin selective reuptake inhibitor fluoxetine (5mg/kg, i.p.) or the tricyclic compound imipramine (15mg/kg, i.p.). We focused our attention on the hypothalamus as it plays a key role in determining many of the somatic symptoms experienced by depressed patients. This brain region, critical also for expression of motivated behaviours, participates in the control of the hypothalamic-pituitary-adrenal axis activity and in stress response as well as coordinates physiological functions such as sleep and food intake that have been found altered in a high percentage of depressed patients. Notably, hypothalamus is a key structure for brain cytokine expression and function as it integrates signals from the neuro, immune, endocrine systems. By means of quantitative Real Time PCR experiments we demonstrated that a chronic treatment with either fluoxetine or imipramine resulted in a reduction of IL-6 and IFN-γ mRNAs and increased IL-4 mRNA expression in the rat hypothalamus. Moreover, we demonstrated that hypothalamic expression of members of IL-18 system was differentially affected by chronic antidepressant treatments. Chronically administered fluoxetine decreased IL-8 and CX3CL1 hypothalamic expression, while a chronic treatment with imipramine decreased p11 mRNA. Our data suggest that a shift in the balance of the inflammation toward an anti-inflammatory state in the hypothalamus may represent a common mechanism of action of both the chronic treatments with fluoxetine and imipramine.

Multiple Factors from Bradykinin-Challenged Astrocytes Contribute to the Neuronal Apoptosis: Involvement of Astroglial ROS, MMP-9, and HO-1/CO System

Bradykinin (BK) has been shown to induce the expression of several inflammatory mediators, including reactive oxygen species (ROS) and matrix metalloproteinases (MMPs), in brain astrocytes. These mediators may contribute to neuronal dysfunction and death in various neurological disorders. However, the effects of multiple inflammatory mediators released from BK-challenged astrocytes on neuronal cells remain unclear. Here, we found that multiple factors were released from brain astrocytes (RBA-1) exposed to BK in the conditioned culture media (BK-CM), including ROS, MMP-9, and heme oxygenase-1 (HO-1)/carbon monoxide (CO), leading to neuronal cell (SK-N-SH) death. Exposure of SK-N-SH cells to BK-CM or H2O2 reduced cell viability and induced cell apoptosis which were attenuated by N-acetyl cysteine, indicating a role of ROS in these responses. The effect of BK-CM on cell viability and cell apoptosis was also reversed by immunoprecipitation of BK-CM with anti-MMP-9 antibody (MMP-9-IP-CM) or MMP2/9 inhibitor, suggesting the involvement of MMP-9 in BK-CM-mediated responses. Astroglial HO-1/CO in BK-CM induced cell apoptosis and reduced cell viability which was reversed by hemoglobin. Consistently, the involvement of CO in these cellular responses was revealed by incubation with a CO donor CO-RM2 which was reversed by hemoglobin. The role of HO-1 in BK-CM-induced responses was confirmed by overexpression of HO-1 in SK-N-SH infected with Adv-HO-1. BK-CM-induced cell apoptosis was due to the activation of caspase-3 and cleavage of PARP. Together, we demonstrate that BK-induced several neurotoxic factors, including ROS, MMP-9, and CO released from astrocytes, may induce neuronal death through a caspase-3-dependent apoptotic pathway.

Mast Cells Are an Essential Component of Human Radiation Proctitis and Contribute to Experimental Colorectal Damage in Mice

Radiation proctitis is characterized by mucosal inflammation followed by adverse chronic tissue remodeling and is associated with substantial morbidity and mortality. Mast cell hyperplasia has been associated with diseases characterized by pathological tissue remodeling and fibrosis. Rectal tissue from patients treated with radiotherapy shows mast cell hyperplasia and activation, suggesting that these cells play a role in the development of radiation-induced sequelae. To investigate the role of mast cells in radiation damage, experimental radiation proctitis was induced in a mast cell-deficient (W(sh)/W(sh)) mouse model. The colon and rectum of W(sh)/W(sh) and wild-type mice were exposed to 27-Gy single-dose irradiation and studied after 2 and 14 weeks. Irradiated rodent rectum showed mast cell hyperplasia. W(sh)/W(sh) mice developed less acute and chronic rectal radiation damage than their control littermates. Tissue protection was associated with increased tissue neutrophil influx and expression of several inflammatory mediators immediately after radiation exposure. It was further demonstrated that mast cell chymase, tryptase, and histamine could change human muscularis propria smooth muscle cells into a migrating/proliferating and proinflammatory phenotype. These data show that mast cells have deleterious effects on both acute and chronic radiation proctitis, possibly by limiting acute tissue neutrophil influx and by favoring phenotypic orientation of smooth muscle cells, thus making them active participants in the radiation-induced inflammatory process and dystrophy of the rectal wall.

Anti-inflammatory action of exendin-4 in human islets is enhanced by phosphodiesterase inhibitors: potential therapeutic benefits in diabetic patients

Exendin-4, a glucagon-like peptide-1 (GLP-1) analogue, is reported to have modest anti-inflammatory effects in addition to that of improving beta cell survival. We therefore sought to determine whether exendin-4 decreases expression of the gene encoding chemokine (C-X-C motif) ligand (CXCL)10, which plays a role in initiating insulitis in type 1 diabetes. The expression of CXCL10 in human islets was determined at the mRNA level by real-time RT-PCR analysis and at the protein level by western blotting. The level of CXCL10 in culture medium was measured by ELISA. Pathway-specific gene expression profiling was carried out to determine the expression of a panel of genes encoding chemokines and cytokines in human islets exposed to cytokines. IFN-γ induced expression of CXCL10 through activation of signal transducer and activator of transcription-1 (STAT-1). A combination of cytokines (IL-1β, TNF-α and IFN-γ) showed strong synergy in the induction of numerous chemokines and cytokines through nuclear factor kappa B and STAT-1. Exendin-4 suppressed basal expression of several inflammatory mediators. In combination with phosphodiesterase inhibitors, exendin-4 also decreased IFN-γ-induced CXCL10 expression in human islets and in MIN6 cells (a mouse beta cell line), and its secretion into the culture medium. Exendin-4 action was mimicked by forskolin, an activator of adenylyl cyclase, and by dibutyryl cyclic AMP. Protein kinase A was not involved in mediating exendin-4 action on CXCL10. The mechanism of exendin-4's anti-inflammatory action involved decreases in STAT-1 levels. These findings suggest that the GLP-1-cyclic AMP pathway decreases islet inflammation in addition to its known effects on beta cell survival.

Timing of Expression of Inflammatory Mediators in Skeletal Muscles from Mice Acutely Infected with the RA Strain of Trypanosoma cruzi

Objective: Chagas’ disease is caused by persistent Trypanosoma cruzi infection in muscle cells that ultimately results in chronic inflammation and tissue destruction. The goal of this study was to determine the expression of different chemokines and their receptors, as well as proinflammatory cytokines and inducible nitric oxide synthase, in muscles from mice acutely infected with T. cruzi. Methods: Histological, semiquantitative reverse transcriptase polymerase chain reaction and immunohistochemical studies were performed on skeletal muscle and myocardium of BALB/c mice infected with T. cruzi, RA strain. Results: Early induction of muscular mRNA expression for CCL5/CCR5 and CXCL9/CXCR3, as well as for iNOS, IFN-γ, TNF-α and MIF, was demonstrated accompanied by progressive increases in parasitism and leukocyte recruitment. Protein overexpression for MIF and CCL5/CCR5 was also verified in the infected muscles. Conclusions: In muscles from acutely T. cruzi RA-infected mice, upregulated gene expression of proinflammatory chemokines, chemokine receptors, cytokines and iNOS is associated with the severity of parasite burden and myopathic alterations. Compared to the heart, striated muscles displayed differential timing of expression of several inflammatory mediators throughout acute infection. Our findings suggest that enhanced early production of these factors could contribute to T. cruzi-dependent inflammatory damage to skeletal muscles.

A novel therapy for acute hepatitis utilizing dehydroepiandrosterone in the murine model of hepatitis

Dehydroepiandrosterone (DHEA), one of the major androgens secreted by the adrenal cortex, has been shown to have potential immunoreguratory properties. In this study, we examined the effect of DHEA in a mouse model of hepatitis. Mice were treated with DHEA and injected with concanavalin A (Con A) or lipopolysaccharide (LPS)/D-galactosamine (GalN). Cytokine expression was measured by quantitative RT-PCR and ELISA. Apoptosis was detected by the TUNEL method and by DNA fragmentation analysis. In the DHEA-treated mice, the serum levels of ALT and expression of inflammatory mediators were significantly decreased. The number of apoptotic cells was also much lower than that observed in control, untreated mouse liver tissue. There were fewer tumor necrosis factor-α (TNF-α)-induced apoptotic cells in H4IIE hepatoma cells treated with DHEA than in non-treated cells. DHEA decreased the expression levels of mRNA transcripts encoding TNF-α and iNOS. These results suggest that DHEA can reduce T-cell-mediated injury in the liver as manifest by inhibition of the expression of several inflammatory mediators and hepatocyte apoptosis. DHEA should, thus, be considered as a novel candidate for the therapy of liver injury.