Upregulation Of Tumor Necrosis Factor Alpha Research Articles

Interleukin-1 Beta and Tumor Necrosis Factor Alpha Upregulation and Nuclear Factor Kappa B Activation in Skeletal Muscle from a Mouse Model of Chronic/Progressive Parkinson Disease.

BackgroundSkeletal muscle atrophy has been reported in patients with Parkinson disease (PD). The purpose of this study was to examine the potential implication of interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and nuclear factor kappa B (NF kappa B) in skeletal muscle atrophy following PD induction.Material/MethodsChronic Parkinsonism was induced in 10 albino mice by MPTP/probenecid treatment, while 10 other albino mice remained without treatment and were subsequently used as controls. Gastrocnemius muscles were examined for the expression of IL-1β and TNF-α, as well as the nuclear localization of NF kappa B, indicative of its activation, using immunohistochemistry in the 2 different groups.ResultsIL-1β and TNF-α expression and NF kappa B nuclear localization were significantly higher in the PD skeletal muscle compared with those in the controls (P value <0.01).ConclusionsThe present data are indicative of an association of PD with IL-1β and TNF-α upregulation and NF kappa B activation in gastrocnemius muscles, potentially promoting the atrophy frequently observed in PD.

Reawakening of dormant estrogen-dependent human breast cancer cells by bone marrow stroma secretory senescence

BackgroundDormant estrogen receptor positive (ER+) breast cancer micrometastases in the bone marrow survive adjuvant chemotherapy and recur stochastically for more than 20 years. We hypothesized that inflammatory cytokines produced by stromal injury can re-awaken dormant breast cancer cells.MethodsWe used an established in vitro dormancy model of Michigan Cancer Foundation-7 (MCF-7) breast cancer cells incubated at clonogenic density on fibronectin-coated plates to determine the effects of inflammatory cytokines on reactivation of dormant ER+ breast cancer cells. We measured induction of a mesenchymal phenotype, motility and the capacity to re-enter dormancy. We induced secretory senescence in murine stromal monolayers by oxidation, hypoxia and estrogen deprivation with hydrogen peroxide (H2O2), carbonyl-cyanide m-chlorophenylhydrazzone (CCCP) and Fulvestrant (ICI 182780), respectively, and determined the effects on growth of co-cultivated breast cancer cells.ResultsExogenous recombinant human (rh) interleukin (IL)-6, IL-8 or transforming growth factor β1 (TGFβ1) induced regrowth of dormant MCF-7 cells on fibronectin-coated plates. Dormant cells had decreased expression of E-cadherin and estrogen receptor α (ERα) and increased expression of N-cadherin and SNAI2 (SLUG). Cytokine or TGFβ1 treatment of dormant clones induced formation of growing clones, a mesenchymal appearance, increased motility and an impaired capacity to re-enter dormancy. Stromal injury induced secretion of IL-6, IL-8, upregulated tumor necrosis factor alpha (TNFα), activated TGFβ and stimulated the growth of co-cultivated MCF-7 cells. MCF-7 cells induced secretion of IL-6 and IL-8 by stroma in co-culture.ConclusionsDormant ER+ breast cancer cells have activated epithelial mesenchymal transition (EMT) gene expression programs and downregulated ERα but maintain a dormant epithelial phenotype. Stromal inflammation reactivates these cells, induces growth and a mesenchymal phenotype. Reactivated, growing cells have an impaired ability to re-enter dormancy. In turn, breast cancer cells co-cultured with stroma induce secretion of IL-6 and IL-8 by the stroma, creating a positive feedback loop.

A comparative study: Difference in omega-6/omega-3 balance and saturated fat in diets for Atlantic salmon (Salmo salar) affect immune-, fat metabolism-, oxidative and apoptotic-gene expression, and eicosanoid secretion in head kidney leukocytes

The aim of this study was to compare how different dietary vegetable oil n-6/n-3 ratios affect gene responses involved in inflammation, signaling pathways, fatty acid synthesis and oxidation, oxidation and apoptosis as well as eicosanoid production in salmon head kidney tissues and isolated head kidney leukocytes. Salmon smolts (200 g) were fed four different diets where the main lipid components were palm oil (n-6/n-3 ratio = 0.7), rapeseed oil (n-6/n-3 ratio = 0.9), and soybean oil (n-6/n-3 ratio = 2.4) and a high soybean oil diet with an n-6/n-3 ratio = 4. Both head kidney tissue and leukocytes isolated from head kidneys were sampled from the four diets, but from different fish. Leukocytes isolated from the head kidneys were seeded into culture wells and added lipopolysaccharide (LPS) to induce inflammatory responses. Controls without LPS were included. Head kidney leukocytes and the tissues should have the same phenotype reflecting the different diets. Interleukin 1β (IL-1β) transcription was elevated in head kidney tissue and especially in LPS treated leukocytes isolated from soybean oil (n-6/n-3 = 2.4) fed salmon, which confirmed the suitability of the in vitro model in this experiment. Leukocytes, treated with LPS, and isolated from salmon fed the soybean oil diet (n-6/n-3 = 2.4) also upregulated tumor necrosis factor alpha (tnf-α), cyclooxygenase (cox2), prostaglandin D and E synthase (ptgds, ptges), fatty acyl synthase (fas), 5 and 6 desaturases (5des, 6 des) and a fatty acid translocase protein (cd36) when compared to the other diets. The results suggest that diets with a specific n-6/n-3 ratio influence the transcription of pro-inflammatory genes and may be cross-linked to transcription of selected fatty acid metabolism genes. Salmon fed the palm oil diet (n-6/n-3 = 0.7) showed a lower expression of inflammatory genes. Instead, peroxisome proliferator activated receptor β1 (pparβ1), acyl coenzyme A (aco), apoptosis regulator (bax) and superoxide dismutase (sod) were upregulated in leukocytes in vitro, while head kidney tissue transcription of a dendritic marker (cd83) was lower than measured in tissues from fish fed the other diets. The concentration of LTB4 (10–20 ng/mL) were relatively constant in leukocyte supernatants, all diets. Head kidney leukocytes from soybean oil (n-6/n-3 = 2.4) fed fish produced LPS induced PGE2 (mean 0.5 ng/mL) while leukocytes isolated from palm oil diet (n-6/n-3 = 0.7) secreted very high amounts of LTB5 (50–70 ng/mL). In addition, equal amounts of LPS induced PGE2 and PGE3 (mean 0, 5 ng/mL) were produced, indicating that the n-6/n-3 ratio of this saturated fatty acid may have a specific impact on eicosanoid production in the head kidney of salmon.

The role of P2X7R/ERK signaling in dorsal root ganglia satellite glial cells in the development of chronic postsurgical pain induced by skin/muscle incision and retraction (SMIR)

The mechanisms of chronic postsurgical pain remain to be elucidated. We reported here that skin/muscle incision and retraction (SMIR), a rat model of postsurgical pain, phosphorylated the extracellular regulated protein kinases (ERK) signaling components c-Raf, MEK (ERK kinase) and ERK1/2 in lumbar 3 dorsal root ganglion (L3 DRG) in rats. Intrathecal injection of ERK specific inhibitor SCH772984 suppressed the mechanical allodynia induced by SMIR. Furthermore, SMIR upregulated tumor necrosis factor alpha (TNFα) in L3 DRG, which could be inhibited by SCH772984. Intrathecal injection of TNF antagonist Etanercept could also inhibit the mechanical allodynia and the increased ERK phosphorylation in L3 DRG induced by SMIR. In addition, immunofluorescent data showed that P2X7R was located exclusively in GFAP labeled satellite glial cells and was highly colocalized with p-ERK1/2 following SMIR. Pretreatment with P2X7R antagonist Brilliant Blue G (BBG) could also block the mechanical allodynia, inhibited the phosphorylation of c-Raf, MEK, ERK1/2, and decrease the expression of TNF-α. Finally, intrathecal injection of BzATP produced mechanical allodynia and induced ERK phosphorylation in satellite glial cells in L3 DRG. Thus, P2X7R activation in satellite glial cells in L3 DRG, leading to a positive feedback between ERK pathway activation and TNF-α production, is suggested to be involved in the induction of chronic postsurgical pain following SMIR.

Effect of Shensong Yangxin on the Progression of Paroxysmal Atrial Fibrillation is Correlated with Regulation of Autonomic Nerve Activity.

Background:Shensong Yangxin (SSYX), a traditional Chinese herbal medicine, has long been used clinically to treat arrhythmias in China. However, the mechanism of SSYX on atrial fibrillation (AF) is unknown. In this study, we tested the hypothesis that the effect of SSYX on the progression of paroxysmal AF is correlated with the regulation of autonomic nerve activity.Methods:Eighteen mongrel dogs were randomly divided into control group (n = 6), pacing group (n = 6), and pacing + SSYX group (n = 6). The control group was implanted with pacemakers without pacing; the pacing group was implanted with pacemakers with long-term intermittent atrial pacing; the pacing + SSYX group underwent long-term intermittent atrial pacing and SSYX oral administration.Results:Compared to the pacing group, the parameters of heart rate variability were lower after 8 weeks in the pacing + SSYX group (low-frequency [LF] component: 20.85 ± 3.14 vs. 15.3 ± 1.89 ms2, P = 0.004; LF component/high-frequency component: 1.34 ± 0.33 vs. 0.77 ± 0.15, P < 0.001). The atrial effective refractory period (AERP) was shorter and the dispersion of the AERP was higher after 8 weeks in the pacing group, while the changes were suppressed by SSYX intake. The dogs in the pacing group had more episodes and longer durations of AF than that in the pacing + SSYX group. SSYX markedly inhibited the increase in sympathetic nerves and upregulation of tumor necrosis factor-alpha and interleukin-6 expression in the pacing + SSYX group. Furthermore, SSYX suppressed the decrease of acetylcholine and α7 nicotinic acetylcholine receptor protein induced by long-term intermittent atrial pacing.Conclusions:SSYX substantially prevents atrial electrical remodeling and the progression of AF. These effects of SSYX may have association with regulating the imbalance of autonomic nerve activity and the cholinergic anti-inflammatory pathway.

The levels of neuropeptide S in the brain of asthmatic mice with anxiety and the relationship with the inflammatory mediators

Objective: To investigate the levels of neuropeptide S in the brain of asthmatic mice with anxiety and the effects of inflammatory mediatores on changes of neuropeptide S in in vitro experiments. Methods: According to the random number table method, 40 BALB/C mice were randomly divided into 4 groups: the control group, the asthma group, the anxiety group and the asthma and anxiety group. The relative expressions of neuropeptide S mRNA in the brain tissue of each group were detected by quantitative real-time polymerase chain reaction(QRT-PCR). Rat cortex neurons obtained by primary culture were divided into 4 groups: the PBS control group, the interleukin-1 beta group, the interleukin-6 group and the tumor necrosis factor-alpha group. After stimulation with inflammatory cytokines the mRNA expressions of neuropeptide S were measured by QRT-PCR and neuropeptide S levels in the cell culture supernatants were measured by emzyme linked immunosorbent assay(ELISA). Results: The relative expressions of neuropeptide S mRNA were decreased in the anxiety group(0.87±0.05) and the asthma and anxiety group(0.79±0.03)compared with the control group(1.00±0.05)and the asthma group(0.96±0.06), most notably in the asthma and anxiety group (all P<0.05). Compared to the PBS control group[(1.00±0.06), (50.6±1. 8)ng/L] and the interleukin-1 beta group[(0.94±0.08), (49.5±1.0)ng/L], the levels of neuropeptide S mRNA and neuropeptide S were decreased in the interleukin-6 group[(0.88±0.07), (45.4±1.2)ng/L] and the tumor necrosis factor-alpha group[(0.86±0.07), (46.0±1.0)ng/L](all P<0.05). There were no significant differences between the interleukin-1 beta group and the PBS control group(all P>0.05). Conclusions: Up-regulated interleukin-6 and tumor necrosis factor-alpha in asthma can inhibit the secretion of neuropeptide S in neuronal cells. The decline of brain neuropeptide S, which has anti-anxiety effect, may lead to the occurrence of anxiety, which may be a potential mechanism of comorbidity of asthma and anxiety.

PKCδ knockout mice are protected from para-methoxymethamphetamine-induced mitochondrial stress and associated neurotoxicity in the striatum of mice

Para-methoxymethamphetamine (PMMA) is a para-ring-substituted amphetamine derivative sold worldwide as an illegal psychotropic drug. Although PMMA use has been reported to lead to severe intoxication and even death, little is known about the mechanism(s) by which PMMA exerts its neurotoxic effects. Here we found that PMMA treatment resulted in phosphorylation of protein kinase Cδ (PKCδ) and subsequent mitochondrial translocation of cleaved PKCδ. PMMA-induced oxidative stress was more pronounced in mitochondria than in the cytosol. Moreover, treatment with PMMA consistently resulted in significant reductions in mitochondrial membrane potential, mitochondrial complex I activity, and mitochondrial Mn superoxide dismutase-immunoreactivity. In contrast, PMMA treatment led to a significant increase in intramitochondrial Ca2+ level. Treatment with PMMA also significantly increased ionized calcium binding adaptor molecule 1 (Iba-1)-labeled microglial activation and upregulated tumor necrosis factor alpha (TNF-α) gene expression. PKCδ knockout attenuated these mitochondrial effects and dampened the neurotoxic effects of PMMA. Importantly, TNF-α knockout mice were significantly protected from PMMA-induced increases in phospho-PKCδ expression, mitochondrial translocation of cleaved PKCδ, and Iba-1-labeled microgliosis. Both rottlerin, a pharmacological inhibitor of PKCδ, and etanercept, a pharmacological inhibitor of TNF-α, significantly protected against PMMA-mediated induction of apoptosis, as assessed by terminal deoxynucleotidyl transferase dUDP nick end labeling (TUNEL) assays. In addition, PKCδ knockout and TNF-α knockout both resulted in decreased PMMA-mediated induction of dopaminergic loss. Therefore, our results suggest that PKCδ mediates PMMA-induced neurotoxicity by facilitating oxidative stress (mitochondria > cytosol), mitochondrial dysfunction, microglial activation, and pro-apoptotic signaling. Our results also indicate that PMMA-induced PKCδ activation requires the proinflammatory cytokine TNF-α.

Abstract 267: Activation of a Novel Estrogen Receptor Ameliorates Experimental Pulmonary Hypertension in Male Rats

Introduction: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling that leads to uncompensated RV failure, and premature death. Preclinical studies have demonstrated that activation of the G protein-coupled estrogen receptor (GPER) is cardioprotective in male rats, and that a selective agonist G1, elicits vascular relaxation in rats of either sex. This work investigated the effects of G1 in male rats with monocrotaline (MCT)-induced PAH. Methods and Results: Male Wistar rats received a single intraperitoneal injection of MCT (60 mg/kg) for PAH induction. Experimental groups were: control, MCT + vehicle, and MCT + G1 (400 μg/kg/day s.c.). Animals were treated with vehicle or G1 for 14 days after the onset of disease (n = 5 per group). Treadmill test and transthoracic echocardiography were performed to access exercise capacity and cardiac function, respectively. RV systolic pressure (RVSP) and mean arterial pressure (MAP) were measured. Time to exhaustion in the treadmill (s) was reduced from 1042.0 ± 66.4 to 188.0 ± 53.1 (MCT + vehicle) and recovered to 809.4 ± 61.5 after G1 treatment. Pulmonary acceleration time (PAT) (ms) was reduced from 44.7 ± 1.4 to 24.7 ± 1.2 in MCT + vehicle group and restored to 41.8 ± 1.0 in MCT + G1 group. RVSP (mmHg) was increased from 24.6 ± 0.6 to 41.1 ± 1.4 (MCT + vehicle) and was reduced to 27.5 ± 0.7 (MCT + G1). MAP (mmHg) was reduced from 100.9 ± 1.1 to 77.1 ± 2.4 (MCT + vehicle), indicating HF induced by the RV dysfunction, and G1 normalized it to 92.4 ± 1.4. G1 decreased pulmonary vascular remodeling and normalized endothelial nitric oxide enzyme levels in lungs from PAH rats. PLB/SERCA2a ratio increased, as a sign of contractility dysfunction, in PAH rats and tumor necrosis factor alpha upregulation was significantly correlated with these Ca 2+ handling proteins impairment. Activation of GPER beneficially reduced PLB/SERCA2a ratio and TNF-α levels in RV tissue of MCT-induced PAH rats. Conclusions: G1 was effective to reverse PAH-induced RV dysfunction and exercise intolerance in male rats, a finding that may have important implications for ongoing clinical evaluation of new drugs for the treatment of the disease.

Upregulation of tumor necrosis factor-alpha in nucleus accumbens attenuates morphine-induced rewarding in a neuropathic pain model

Treatment of neuropathic pain with opioid analgesics remains controversial and a major concern is the risk of addiction. Here, we investigated this issue with spared nerve injury (SNI) model of neuropathic pain in rats and mice. SNI prevented conditioned place preference (CPP) induced by low dose (3.5mg/kg) of morphine (MOR), which was effective for anti-allodynia, but not by high dose (⩾5.0mg/kg) of MOR. Tumor necrosis factor-alpha (TNF-α) was upregulated in nucleus accumbens (NAcc) following SNI. The inhibitory effect of SNI on MOR-induced CPP was blocked by either genetic deletion of TNF receptor 1 (TNFR1) or microinjection of anti-TNF-α into the NAcc and was mimicked by intra-NAcc injection of TNF-α in sham rats. Furthermore, SNI reduced dopamine (DA) level and upregulated dopamine transporter (DAT) in the NAcc, but did not affect total tyrosine hydroxylase (TH) or phospho-TH (p-TH), a rate-limiting enzyme of catecholamine biosynthesis, in ventral tegmental area (VTA). Accordingly, the increase in DA reuptake but not decrease in its synthesis may lead to the reduction of DA level. Finally, the upregulation of DAT in the NAcc of SNI animals was again blocked by either genetic deletion of TNFR1 or NAcc injection of anti-TNF-α, and was mimicked by NAcc injection of TNF-α in sham animals. Thus, our data provided novel evidence that upregulation of TNF-α in NAcc may attenuate MOR-induced rewarding by upregulation of DAT in NAcc under neuropathic pain condition.

A Synergistic Vascular Effect of Airborne Particulate Matter and Nickel in a Mouse Model

Both epidemiological and empirical studies have indicated that nickel (Ni) may play an important role in PM2.5 exposure-induced adverse cardiovascular effects. However, the underlying mechanism remains unclear. In the present study, we exposed mice to concentrated ambient PM2.5 (CAP), Ni, or coexposure to both CAP + Ni in a specially designed whole-body exposure system for a duration of 3 months and investigated their effects on vascular function, oxidative stress, and vascular inflammation. CAP + Ni exposure induced greater endothelial dysfunction compared with CAP or Ni alone. Ni exposure decreased endothelial nitric oxide synthase (eNOS) dimers in the aorta, which was potentiated by coexposure with CAP. CAP alone did not reduce NOS dimers but was more effective than Ni in decreasing phosphorylation of eNOS (S1177) and Akt (T308). Ni had minimal effects on the expression of vascular inflammatory genes but synergized with CAP in marked upregulation of tumor necrosis factor-alpha and monocyte chemotactic protein-1. The effects of Ni on NOS monomer formation in endothelial cells were redox dependent as evidenced by attenuation of effects by Tiron in cultured endothelial cells. Ni synergized with lipopolysaccharide, another bioactive component of CAP in reducing eNOS dimerization in cultured endothelial cells. Ni exposure induces endothelial dysfunction through oxidative stress-dependent inhibition of eNOS dimerization. Its interaction with other components of CAP may significantly contribute to the adverse cardiovascular effects of CAP exposure.

Protection of MPTP-induced neuroinflammation and neurodegeneration by Pycnogenol

Oxidative stress and inflammation play a crucial role in Parkinson’s disease (PD) pathogenesis and may represent a target for treatment. Current PD drugs provide only symptomatic relief and have limitations in terms of adverse effects and inability to prevent neurodegeneration. Flavonoids have been suggested to exert human health benefits by its anti-oxidant and anti-inflammatory properties. Therefore, in the present study, using 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine (MPTP)-induced mouse model of Parkinsonism, we investigated the neuroprotective potential of bioflavonoid compound Pycnogenol® (PYC), an extract of Pinus maritime bark. MPTP injected mice developed significantly severe oxidative stress and impaired motor coordination at day 1 and day 7 postinjection. This was associated with significantly increased inflammatory responses of astrocyte and microglia as assessed by ionized calcium binding adaptor molecule 1 (Iba 1) and glial fibrillary acidic protein (GFAP) immunohistochemistry, and nuclear transcription factor-κB (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in the striata by Western blot. Additionally, there was significant upregulation of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) expression in the striata of MPTP injected mice compared to saline controls. The MPTP-induced neuroinflammation, neurodegeneration and behavioral impairments were markedly repudiated by treatment with PYC. These results suggest that PYC protects dopaminergic neurons from MPTP-induced toxicity in the mouse model of PD. Thus, the present finding of PYC-induced adaptation to oxidative stress and inflammation could suggest a novel avenue for clinical intervention in neurodegenerative diseases including PD.

Mitochondrial DNA Maintenance Is Regulated in Human Hepatoma Cells by Glycogen Synthase Kinase 3β and p53 in Response to Tumor Necrosis Factor α

During chronic liver inflammation, up-regulated Tumor Necrosis Factor alpha (TNF-α) targets hepatocytes and induces abnormal reactive oxygen species (ROS) production responsible for mitochondrial DNA (mtDNA) alterations. The serine/threonine Glycogen Synthase Kinase 3 beta (GSK3β) plays a pivotal role during inflammation but its involvement in the maintenance of mtDNA remains unknown. The aim of this study was to investigate its involvement in TNF-α induced mtDNA depletion and its interrelationship with p53 a protein known to maintain mtDNA copy numbers. Using quantitative polymerase chain reaction (qPCR) we found that at 30 min in human hepatoma HepG2 cells TNF-α induced 0.55±0.10 mtDNA lesions per 10 Kb and a 52.4±2.8% decrease in mtDNA content dependent on TNF-R1 receptor and ROS production. Both lesions and depletion returned to baseline from 1 to 6 h after TNF-α exposure. Luminol-amplified chemiluminescence (LAC) was used to measure the rapid (10 min) and transient TNF-α induced increase in ROS production (168±15%). A transient 8-oxo-dG level of 1.4±0.3 ng/mg DNA and repair of abasic sites were also measured by ELISA assays. Translocation of p53 to mitochondria was observed by Western Blot and co-immunoprecipitations showed that TNF-α induced p53 binding to GSK3β and mitochondrial transcription factor A (TFAM). In addition, mitochondrial D-loop immunoprecipitation (mtDIP) revealed that TNF-α induced p53 binding to the regulatory D-loop region of mtDNA. The knockdown of p53 by siRNAs, inhibition by the phosphoSer15p53 antibody or transfection of human mutant active GSK3βS9A pcDNA3 plasmid inhibited recovery of mtDNA content while blockade of GSK3β activity by SB216763 inhibitor or knockdown by siRNAs suppressed mtDNA depletion. This study is the first to report the involvement of GSK3β in TNF-α induced mtDNA depletion. We suggest that p53 binding to GSK3β, TFAM and D-loop could induce recovery of mtDNA content through mtDNA repair.

Ginsenoside Rg&lt;sub&gt;3&lt;/sub&gt; Attenuates Microglia Activation Following Systemic Lipopolysaccharide Treatment in Mice

Neuroinflammation, characterized by activation of microglia and expression of major inflammatory mediators, contributes to neuronal damage in addition to acute and chronic central nervous system (CNS) disease progression. The present study investigated the immune modulatory effects of ginsenoside Rg3, a principle active ingredient in Panax ginseng, on pro-inflammatory cytokines and microglia activation in brain tissue induced by systemic lipopolysaccharide (LPS) treatment in C57BL/6 mice. Systemic LPS treatment induces immediate microglia activation in the brain. Based on this information, ginsenoside Rg3 was treated orally with 10, 20, and 30 mg/kg 1 h prior to the LPS (3 mg/kg, intraperitoneally (i.p.)) injection. Ginsenoside Rg3 at 20 and 30 mg/kg oral doses significantly attenuated up-regulation of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and IL-6 mRNA in brain tissue at 4 h after LPS injection. Morphological activation of microglia and Iba1 protein expression by systemic LPS injection were reduced with ginsenoside Rg3 (30 mg/kg) treatment. In addition, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in brain tissue were also attenuated with oral treatment of ginsenoside Rg3 at 30 mg/kg. These results indicate that ginsenoside Rg3 plays a modulatory role in neuroinflammation. This study shows that ginsenoside Rg3 attenuates microglia activation using an in vivo animal model.

A peptidic fraction from milk fermented with Lactobacillus helveticus protects mice against Salmonella infection

A peptidic fraction, isolated from milk fermented with Lactobacillus helveticus, was assessed for its ability to bolster the immunity in mice against an oral Salmonella enteritidis challenge. Following four days of feeding with the fraction or saline, mice were challenged with a single Salmonella dose. Mice fed at 0.02 μg day −1 (PO2 group) had higher general health score and survival rate than mice in the control group and in those fed at 0.01 μg day −1. The Salmonella burden in the spleen and liver of mice in the control group was higher than that of mice in the PO2 group. The active fraction up-regulated tumour necrosis factor-alpha (TNF-α) production but had no effect on interferon gamma (IFN-γ) secretion. Pathological studies showed that the fraction was non toxic at a dose of 0.2 μg day −1. Antivirulent and antimicrobial properties of the active fraction were also studied to understand the mechanisms associated with the in vivo effects observed.

Acetylcholine inhibits hypoxia‐induced tumor necrosis factor‐α production via regulation of MAPKs phosphorylation in cardiomyocytes

Recent findings have reported that up-regulation of tumor necrosis factor-alpha (TNF-α) induced by myocardial hypoxia aggravates cardiomyocyte injury. Acetylcholine (ACh), the principle vagal neurotransmitter, protects cardiomyocytes against hypoxia by inhibiting apoptosis. However, it is still unclear whether ACh regulates TNF-α production in cardiomyocytes after hypoxia. The concentration of extracellular TNF-α was increased in a time-dependent manner during hypoxia. Furthermore, ACh treatment also inhibited hypoxia-induced TNF-α mRNA and protein expression, caspase-3 activation, cell death and the production of reactive oxygen species (ROS) in cardiomyocytes. ACh treatment prevented the hypoxia-induced increase in p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation, and increased extracellular signal-regulated kinase (ERK) phosphorylation. Co-treatment with atropine, a non-selective muscarinic acetylcholine receptor antagonist, or methoctramine, a selective type-2 muscarinic acetylcholine (M(2) ) receptor antagonist, abrogated the effects of ACh treatment in hypoxic cardiomyocytes. Co-treatment with hexamethonium, a non-selective nicotinic receptor antagonist, and methyllycaconitine, a selective alpha7-nicotinic acetylcholine receptor antagonist, had no effect on ACh-treated hypoxic cardiomyocytes. In conclusion, these results demonstrate that ACh activates the M(2) receptor, leading to regulation of MAPKs phosphorylation and, subsequently, down-regulation of TNF-α production. We have identified a novel pathway by which ACh mediates cardioprotection against hypoxic injury in cardiomyocytes.

Inhibition of NF-kappaB prevents mechanical allodynia induced by spinal ventral root transection and suppresses the re-expression of Nav1.3 in DRG neurons in vivo and in vitro

Activation of nucleus factor-kappaB (NF-κB) in the dorsal root ganglia (DRG) is critical for development of neuropathic pain. The underlying mechanisms, however, are largely unknown. In the present work we tested if the activation of NF-κB is required for re-expression of Nav1.3, which is important for development of neuropathic pain, in uninjured DRG neurons. We found that intrathecal injection of pyrrolidine dithiocarbamate (PDTC), a NF-κB inhibitor, completely blocked the mechanical allodynia induced by L5 ventral root transection (L5-VRT), when applied 30 min before or 8 h after operation, but at 7 d after L5-VRT the same manipulation had no effect on established allodynia. Pre-treatment with PDTC also prevented the re-expression of Nav1.3 induced by L5-VRT. As our previous work has shown that up-regulation of tumor necrosis factor-alpha (TNF-α) in DRG is responsible for the re-expression of Nav1.3 in uninjured DRG neurons following L5 ventral root injury, we investigated whether activation of NF-κB is essential for the up-regulation of Nav1.3 by TNF-α. Results showed that application of rat recombinant TNF-α (rrTNF) into the cultured normal adult rat DRG neurons increased the immunoreactive (IR) of Nav1.3 localized mainly around the cell membrane and pre-treatment with PDTC blocked the change dose-dependently. The data suggested that injury to ventral root might lead to neuropathic pain and the re-expression of Nav1.3 in primary sensory neurons by activation of NF-κB.

Differential regulation of TNF receptors by vagal nerve stimulation protects heart against acute ischemic injury

Vagal nerve stimulation (VS) has been reported to improve the survival after both acute and chronic myocardial infarction through the release of neurotransmitter ACh. However, the precise mechanism behind its beneficial effect is still unknown. In this study, we demonstrate the upregulation of tumor necrosis factor-alpha (TNF-α) and its cell survival TNF receptor-2 (TNFR2) as the mechanism behind VS induced myocardial protection.We investigated the effects of efferent VS on myocardial ischemic injury with in vivo and in vitro mouse models. In in vivo hearts VS significantly increased the expression of TNF-α both at the messenger and protein level after 3-hours of myocardial ischemia. In the in vitro studies ACh treatment before hypoxia, induced a significant upregulation of TNF-α compared to the untreated cardiomyocytes. Immunofluorescence analysis confirmed the synthesis of TNF-α by cardiomyocytes both in vivo and in vitro. VS also significantly reduced the myocardial infarct size (23.9±5.7% vs. 56±1.9%) and activated the cell survival Akt cascade system. Further, ACh upregulated the cell survival TNFR2 expression, while downregulating the cell destructive TNF receptor 1 (TNFR1) expression. These results were confirmed using the TNF receptors deficient mice, where the VS mediated protection was lost both in vivo and in vitro in TNFR2 (TNFR2-/-) and TNF receptors double knock out (TNFR1-/-2-/-) mice.VS and ACh protects the heart against acute ischemia or hypoxic injury by differentially regulating the TNF receptor subtypes.

Enoxaparin Improves the Course of Dextran Sodium Sulfate-Induced Colitis in Syndecan-1-Deficient Mice

Syndecan-1 (Sdc1) plays a major role in wound healing and modulates inflammatory responses. Sdc1 expression is reduced in lesions of patients with ulcerative colitis. The aim of this study was to investigate the role of Sdc1 in murine dextran sodium sulfate (DSS)-induced colitis. DSS colitis was induced in Sdc1-deficient (knockout (KO)) and wild-type mice by oral administration of 3% DSS. KO mice exhibited a significantly increased lethality as compared with wild-type controls (61 versus 5%, P < 0.05). Impaired mucosal healing and prolonged recruitment of inflammatory cells in KO mice were accompanied by significant up-regulation of tumor necrosis factor-alpha, CC chemokine ligand 3/macrophage inflammatory protein-1alpha, and vascular cell adhesion molecule-1, as determined by histological correlation between 0 and 15 days after colitis induction, TaqMan low-density array analysis, and quantitative real-time PCR. Treatment from days 7 through 14 with enoxaparin, a functional analogue of the Sdc1 heparan sulfate chains, significantly reduced lethality of KO mice due to DSS-induced colitis, which was correlated with improved mucosal healing. In vitro, Sdc1-deficient polymorphonuclear cells displayed increased adhesion to endothelial cells and intercellular adhesion molecule-1, and enoxaparin reverted adhesion to wild-type levels. Small interfering RNA-mediated knockdown of Sdc1 expression resulted in reduced basic fibroblast growth factor-mediated mitogen-activated protein kinase signaling and reduced Caco-2 cell proliferation. We conclude that Sdc1 has a protective effect during experimental colitis. The modification of missing Sdc1 function by heparin analogues may emerge as a promising anti-inflammatory approach.

Co–Cr–Mo alloy particles induce tumor necrosis factor alpha production in MLO-Y4 osteocytes: A role for osteocytes in particle-induced inflammation

Wear debris-induced osteolysis is purportedly the limiting problem affecting the long term results of joint arthroplasty. Pathogenic effects of wear debris in peri-implant cells such as macrophages, osteoblasts and osteoclasts have been well studied. In contrast, the effects of wear debris on osteocytes, which make up over 90% of all bone cells, remain unknown. We hypothesized that metal implant debris can induce the pro-inflammatory response in osteocytes. This study demonstrated the effects of cobalt–chromium–molybdenum alloy (Co–Cr–Mo) particles on a well-characterized MLO-Y4 osteocyte cell line. Co–Cr–Mo alloy particle treatment significantly ( p < 0.05) up-regulated tumor necrosis factor alpha (TNFα) gene expression after 3 and 6 h and TNFα protein production after 24 h, but down-regulated interleukin-6 (IL-6) gene expression after 6 h. Co–Cr–Mo alloy particle treatment also induced osteocyte apoptosis after 24 h. This apoptotic effect was partially (40%) dependent on TNFα. Therefore, our results suggest that osteocytes play a role in particle-induced inflammation and bone resorption following total joint arthroplasty by inducing pro-inflammatory cytokines and inducing osteocyte apoptosis.

Minocycline reduces intracerebral hemorrhage-induced brain injury

Objectives: Microglial activation and thrombin formation contribute to brain injury after intracerebral hemorrhage. Tumor necrosis factor-alpha and interleukin-1beta are two major pro-inflammatory cytokines. The present study investigated if thrombin stimulates tumor necrosis factor-alpha and interleukin-1beta secretion in vitro and if microglial inhibition reduces intracerebral hemorrhage-induced brain injury in vivo.Methods: There were two parts in this study. In the first part, cultured rat microglial cells were treated with vehicle, thrombin (10 U/ml) or thrombin plus minocycline (1 or 10 μM), an inhibitor of microglia activation. Levels of tumor necrosis factor-alpha and interleukin-1beta in culture medium were measured by enzyme-linked immunosorbent assay 24 hours after thrombin treatment. In the second part, rats had an intracerebral injection of 100 μl autologous whole blood. Rats received minocycline or vehicle treatment. Brain edema was measured at day 3 and brain atrophy was determined at day 28 after intracerebral hemorrhage.Results: Thrombin receptors were expressed in cultured microglia cells, and tumor necrosis factor-alpha and interleukin-1beta levels in the culture medium were increased after thrombin treatment. Minocycline reduced thrombin-induced up-regulation of tumor necrosis factor-alpha and interleukin-1beta. In vivo, minocycline reduced perihematomal brain edema, neurological deficits and brain atrophy.Discussion: Thrombin stimulates microglia to release the pro-inflammatory cytokines, tumor necrosis factor-alpha and interleukin-1beta, and microglial inhibition with minocycline reduces brain injury after intracerebral hemorrhage, suggesting a critical role of microglia activation in intracerebral hemorrhage-related brain injury.