Pro-inflammatory GPR75 and anti-apoptotic phospholipase signaling pathways contribute to the ameliorating effect of soluble epoxide hydrolase inhibition on chronic experimental autoimmune encephalomyelitis in mice.

Soluble epoxide hydrolase (sEH) inhibition has currently emerged as a therapeutic target in the treatment of various neuroinflammatory neurodegenerative diseases, including multiple sclerosis. Previously, we reported that treatment of mice with a sEH-selective inhibitor, 1-(1-propanoylpiperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl]urea; TPPU), ameliorated chronic experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein 35-55 peptide immunization followed by injection of pertussis toxin to mice via regulating pro-inflammatory and anti-inflammatory pathways in the central nervous system. This study tested the hypothesis that the pro-inflammatory G protein-coupled receptor (GPR) 75 and anti-apoptotic phospholipase C (PLC) signaling pathways also contribute to the ameliorating effect of TPPU on chronic EAE. Brains and spinal cords of phosphate-buffered saline-, dimethyl sulfoxide-, or TPPU (3 mg/kg)-treated mice were used for the measurement of sEH, GPR75, Gaq/11, activator protein (AP)-1, PLC ß4, phosphoinositide 3-kinase (PI3K) p85a, Akt1, mitogen-activated protein kinase kinase (MEK) 1/2, extracellular signal-regulated kinase (ERK) 1/2, cyclic adenosine monophosphate-response element-binding protein (CREB) 1, B-cell lymphoma (Bcl)-2, semaphorin (SEMA) 3A, and myelin proteolipid protein (PLP) expression and/or activity by using the immunoblotting method. Expression of sEH, GPR75, Gaq/11, c-jun, phosphorylated c-Jun, and SEMA3A was lower, while PLCß4, phosphorylated PI3K p85a, phosphorylated Akt1, phosphorylated MEK1/2, phosphorylated ERK1/2, phosphorylated CREB1, Bcl-2, and myelin PLP expression was higher in the tissues of TPPU (3 mg/kg)-treated mice as compared with the EAE and vehicle control groups. Inhibition of sEH by TPPU ameliorates chronic EAE through suppressing pro-inflammatory GPR75/Gaq/11/AP-1 pathway and reducing expression of the remyelination inhibitor, SEMA3A, as well as increasing anti-apoptotic PLC/PI3K/Akt1/MEK1/2/ERK1/2/CREB1/Bcl-2 pathway activity and myelin PLP expression.

NLRX1 ligand, docosahexaenoic acid, ameliorates LPS-induced inflammatory hyperalgesia by decreasing TRAF6/IKK/IkB-a/NF-kB signaling pathway activity.

The nucleotide-binding oligomerization domain-like receptor X1 (NLRX1) has been associated with various anti-inflammatory mechanisms. We investigated whether the NLRX1 ligand docosahexaenoic acid (DHA) ameliorates lipopolysaccharide (LPS)-induced inflammatory hyperalgesia by interacting with tumor necrosis factor receptor-associated factor 6 (TRAF6)/inhibitor of kB (IkB) kinase (IKK)/IkB-a/nuclear factor-κB (NF-κB) signaling pathway in the central nervous system. Reaction time to thermal stimuli within 30 seconds was measured in male mice injected with saline, lipopolysaccharide (LPS), and/or DHA after 6 hours using the hot plate test. Co-immunoprecipitation and immunoblotting studies were performed to determine the activation of the TRAF6/IKK/IkB-a/NF-kB pathway in the brains and spinal cords of animals. Latency to the thermal stimulus was reduced by 30% in LPS-injected endotoxemic mice compared with saline-injected mice. Treatment with DHA significantly improved latency compared with endotoxemic mice. In the brain and spinal cord of LPS-injected mice, treatment with DHA also prevented the increase in the expression and/or activity of (1) IKKa/IKKß, IKKg, and K63 U in the NLRX1-immunoprecipitated tissues, (2) IKKa/IKKß, K63 U, and K48 U in the IKKg-immunoprecipitated tissues, and (3) IkB-α, NF-kB p65, and interleukin-1ß associated with decreased IkB-α expression. These findings suggest that inhibition of IKK/IkB-a/NF-kB signaling by dissociation of NLRX1 from TRAF6 in response to LPS treatment contributes to the protective effect of DHA against inflammatory hyperalgesia.

Activation of GPR75 Signaling Pathway Contributes to the Effect of a 20-HETE Mimetic, 5,14-HEDGE, to Prevent Hypotensive and Tachycardic Responses to Lipopolysaccharide in a Rat Model of Septic Shock.

ABSTRACT: The orphan receptor, G protein-coupled receptor (GPR) 75, which has been shown to mediate various effects of 20-hydroxyeicosatetraenoic acid (20-HETE), is considered as a therapeutic target in the treatment of cardiovascular diseases in which changes in the production of 20-HETE play a key role in their pathogenesis. Our previous studies showed that 20-HETE mimetic, N -(20-hydroxyeicosa-5[Z],14[Z]-dienoyl)glycine (5,14-HEDGE), protects against vascular hyporeactivity, hypotension, tachycardia, and arterial inflammation induced by lipopolysaccharide (LPS) in rats. This study tested the hypothesis that the GPR75 signaling pathway mediates these effects of 5,14-HEDGE in response to systemic exposure to LPS. Mean arterial pressure reduced by 33 mm Hg, and heart rate increased by 102 beats/min at 4 hours following LPS injection. Coimmunoprecipitation studies demonstrated that (1) the dissociation of GPR75/Gα q/11 and GPR kinase interactor 1 (GIT1)/protein kinase C (PKC) α, the association of GPR75/GIT1, large conductance voltage and calcium-activated potassium subunit ß (MaxiKß)/PKCα, MaxiKß/proto-oncogene tyrosine-protein kinase (c-Src), and epidermal growth factor receptor (EGFR)/c-Src, MaxiKß, and EGFR tyrosine phosphorylation were decreased, and (2) the association of GIT1/c-Src was increased in the arterial tissues of rats treated with LPS. The LPS-induced changes were prevented by 5,14-HEDGE. N -[20-Hydroxyeicosa-6( Z ),15( Z )-dienoyl]glycine, a 20-HETE antagonist, reversed the effects of 5,14-HEDGE in the arterial tissues of LPS-treated rats. Thus, similar to 20-HETE, by binding to GPR75 and activating the Gα q/11 /PKCα/MaxiKß, GIT1/PKCα/MaxiKß, GIT1/c-Src/MaxiKß, and GIT1/c-Src/EGFR signaling pathways, 5,14-HEDGE may exert its protective effects against LPS-induced hypotension and tachycardia associated with vascular hyporeactivity and arterial inflammation.

mTOR inhibition as a possible pharmacological target in the management of systemic inflammatory response and associated neuroinflammation by lipopolysaccharide challenge in rats.

Neuroinflammation plays a critical role during sepsis triggered by microglial activation. Mammalian target of rapamycin (mTOR) has gained attraction in neuroinflammation, however, the mechanism remains unclear. Our goal was to assess the effects of mTOR inhibition by rapamycin on inflammation, microglial activation, oxidative stress, and apoptosis associated with the changes in the inhibitor-κB (IκB)-α/nuclear factor-κB (NF-κB)/hypoxia-inducible factor-1α (HIF-1α) pathway activity following a systemic challenge with lipopolysaccharide (LPS). Rats received saline (10 mL/kg), LPS (10 mg/kg), and (or) rapamycin (1 mg/kg) intraperitoneally. Inhibition of mTOR by rapamycin blocked phosphorylated form of ribosomal protein S6, NF-κB p65 activity by increasing degradation of IκB-α in parallel with HIF-1α expression increased by LPS in the kidney, heart, lung, and brain tissues. Rapamycin attenuated the increment in the expression of tumor necrosis factor-α and interleukin-1ß, the inducible nitric oxide synthase, gp91phox, and p47phox in addition to nitrite levels elicited by LPS in tissues or sera. Concomitantly, rapamycin treatment reduced microglial activation, brain expression of caspase-3, and Bcl-2-associated X protein while it increased expression of B cell lymphoma 2 induced by LPS. Overall, this study supports the hypothesis that mTOR contributes to the detrimental effect of LPS-induced systemic inflammatory response associated with neuroinflammation via IκB-α/NF-κB/HIF-1α signaling pathway.

Soluble epoxide hydrolase inhibitor trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea prevents hyperalgesia through regulating NLRC4 inflammasome-related pro-inflammatory and anti-inflammatory signaling pathways in the lipopolysaccharide-induced pain mouse model.

Epoxyeicosatrienoic acids (EETs) have anti-inflammatory effects and soluble epoxide hydrolase (sEH) inhibition might be a useful therapeutic approach to manage inflammatory disorders. The purpose of the study was to investigate whether nucleotide-binding and oligomerization domain-like receptor (NLR) C4 inflammasome-related pro-inflammatory and anti-inflammatory signaling pathways in the central nervous system (CNS) participates in the effect of trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea (TPPU), a potent sEH inhibitor, to prevent hyperalgesia in the LPS-induced pain mouse model. The latency of pain within 30 s was measured by the hot plate test in male mice injected with saline, lipopolysaccharide (LPS) (10 mg/kg), and/or TPPU (0.3, 0.5, or 1 mg/kg) after 6 h. Hyperalgesia induced by LPS was associated with decreased 14,15-dihydroxyeicosatrienoic acid and interleukin (IL)-1ß levels and enhanced expression of NLRC4, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), caspase-1 p20, IL-1ß, and caspase-11 p20 in the brains and spinal cords of the animals. Besides the increased expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX) subunits (gp91phox and p47phox ) and nitrotyrosine, a decrease in NLRC3, inducible nitric oxide synthase (iNOS), and neuronal NOS (nNOS) expression was also observed in the tissues of LPS-treated mice. TPPU at 0.5 mg/kg dose prevented the changes induced by LPS. Likely, decreased activity of pro-inflammatory NLRC4/ASC/pro-caspase-1 and caspase-11 inflammasomes and NOX in addition to enhanced levels of anti-inflammatory EETs and expression of NLRC3, iNOS, and nNOS in the CNS of mice participates in the protective effect of TPPU against LPS-induced hyperalgesia.

Suppression of TLR4/MyD88/TAK1/NF-κB/COX-2 Signaling Pathway in the Central Nervous System by Bexarotene, a Selective RXR Agonist, Prevents Hyperalgesia in the Lipopolysaccharide-Induced Pain Mouse Model.

A selective RXR agonist, bexarotene, has been shown to have anti-inflammatory, anti-nociceptive, and neuroprotective effects in several models of numerous neurological diseases characterized by systemic inflammation. The mechanisms underlying these effects remains unknown. To elucidate these mechanisms, we investigated whether the TLR4/MyD88/TAK1/NF-κB/COX-2 signaling pathway in the CNS mediates the effect of bexarotene to prevent hyperalgesia in the LPS-induced inflammatory pain mouse model. The reaction time to thermal stimuli within 30 s was evaluated by the hot plate test in male mice treated with saline, LPS (10 mg/kg), DMSO, and/or bexarotene (0.1, 1, 3, or 10 mg/kg) after 6 h. The latency to the thermal stimulus (18.11 ± 1.36 s) in the LPS-treated mice was significantly decreased by 30% compared with saline-treated mice (25.84 ± 1.99 s). Treatment with bexarotene only at a dose of 10 mg/kg showed a significant increase in the latency by 22.49 ± 1.00 s compared with LPS-treated mice. Bexarotene also prevented the reduction in RXRα protein expression associated with a rise in the expression of TLR4, MyD88, phosphorylated TAK1, NF-κB p65, phosphorylated NF-κB p65, COX-2, and IL-1ß proteins, in addition to COX-2 activity and levels of PGE2 and IL-1ß in the brains and spinal cords of the LPS-treated animals. Likely, decreased activity of TLR4/MyD88/TAK1/NF-κB/COX-2 signaling pathway in addition to increased pro-inflammatory cytokine formation in the CNS of mice participates in the protective effect of bexarotene against hyperalgesia induced by LPS.

Pharmacological inhibition of soluble epoxide hydrolase attenuates chronic experimental autoimmune encephalomyelitis by modulating inflammatory and anti-inflammatory pathways in an inflammasome-dependent and -independent manner.

We aimed to determine the effect of soluble epoxide hydrolase (sEH) inhibition on chronic experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), associated with changes in inflammasome-dependent and -independent inflammatory and anti-inflammatory pathways in the CNS of mice. C57BL/6 mice were used to induce chronic EAE by using an injection of MOG35-55 peptide/PT. Animals were observed daily and scored for EAE signs for 25 days after immunization. Following the induction of EAE, the scores were increased after 9 days and reached peak value as determined by ≥ 2 or ≤ 3 with 8% mortality rate on day 17. On day 17, mice were administered daily PBS, DMSO, or TPPU (a potent sEH inhibitor) (1, 3, or 10 mg/kg) until the end of the study. TPPU only at 3 mg/kg dose decreased the AUC values calculated from EAE scores obtained during the disease compared to EAE and vehicle control groups. On day 25, TPPU also caused an increase in the PPARα/ß/γ and NLRC3 proteins and a decrease in the proteins of TLR4, MyD88, NF-κB p65, p-NF-κB p65, iNOS/nNOS, COX-2, NLRC4, ASC, caspase-1 p20, IL-1ß, caspase-11 p20, NOX subunits (gp91phox and p47phox), and nitrotyrosine in addition to 14,15-DHET and IL-1ß levels compared to EAE and vehicle control groups. Our findings suggest that pharmacological inhibition of sEH attenuates chronic EAE likely because of enhanced levels of anti-inflammatory EETs in addition to PPARα/ß/γ and NLRC3 expression associated with suppressed inflammatory TLR4/MyD88/NF-κB signalling pathway, NLRC4/ASC/pro-caspase-1 inflammasome, caspase-11 inflammasome, and NOX activity that are responsible for inflammatory mediator formation in the CNS of mice.

Eicosanoids derived from cytochrome P450 pathway of arachidonic acid and inflammatory shock.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Septic shock, the most common form of vasodilatory shock, is a subset of sepsis in which circulatory and cellular/metabolic abnormalities are severe enough to increase mortality. Inflammatory shock constitutes the hallmark of sepsis, but also a final common pathway of any form of severe long-term tissue hypoperfusion. The pathogenesis of inflammatory shock seems to be due to circulating substances released by pathogens (e.g., bacterial endotoxins) and host immuno-inflammatory responses (e.g., changes in the production of histamine, bradykinin, serotonin, nitric oxide [NO], reactive nitrogen and oxygen species, and arachidonic acid [AA]-derived eicosanoids mainly through NO synthase, cyclooxygenase, and cytochrome P450 [CYP] pathways, and proinflammatory cytokine formation). Therefore, refractory hypotension to vasoconstrictors with end-organ hypoperfusion is a life threatening feature of inflammatory shock. This review summarizes the current knowledge regarding the role of eicosanoids derived from CYP pathway of AA in animal models of inflammatory shock syndromes with an emphasis on septic shock in addition to potential therapeutic strategies targeting specific CYP isoforms responsible for proinflammatory/anti-inflammatory mediator production.

NF-κB activation mediates LPS-or zymosan-induced hypotension and inflammation reversed by BAY61-3606, a selective Syk inhibitor, in rat models of septic and non-septic shock.

We have previously demonstrated that the activation of the spleen tyrosine kinase (Syk)/inhibitory-κB (IκB)-α/nuclear factor-κB (NF-κB) p65 signalling pathway contributes to hypotension and inflammatory response in a rat models of zymosan (ZYM)-induced non-septic shock. The purpose of this study was to further examine the possible mechanism underlying the effect of inhibition of Syk by BAY61-3606 via NF-κB activity at the level of nuclear translocation regarding the production of vasodilator and proinflammatory mediators in lipopolysaccharide (LPS) (septic)- and ZYM (non-septic)-induced shock. Administration of LPS (10 mg/kg, ip) or ZYM (500 mg/kg, ip) to male Wistar rats decreased mean arterial pressure and increased heart rate that was associated with an increase in the activities of cyclooxygenase and nitric oxide synthase, tumour necrosis factor-α, and interleukin-8 levels, and NF-κB activation and nuclear translocation in sera and/or cardiovascular and renal tissues. BAY61-3606 (3 mg/kg, ip), the selective Syk inhibitor, given 1 hour after LPS- or ZYM injection reversed all the above-mentioned effects. These results suggest that Syk contributes to the LPS- or ZYM-induced hypotension and inflammation associated with transactivation of NF-κB in septic and non-septic shock.

Inhibition of NLRP3 Inflammasome Prevents LPS-Induced Inflammatory Hyperalgesia in Mice: Contribution of NF-κB, Caspase-1/11, ASC, NOX, and NOS Isoforms.

The nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3), an intracellular signaling molecule that senses many environmental- and pathogen/host-derived factors, has been implicated in the pathogenesis of several diseases associated with inflammation. It has been suggested that NLRP3 inflammasome inhibitors may have a therapeutic potential in the treatment of NLRP3-related inflammatory diseases. The aim of this study was to determine whether inhibition of NLRP3 inflammasome prevents inflammatory hyperalgesia induced by lipopolysaccharide (LPS) in mice as well as changes in expression/activity of nuclear factor κB (NF-κB), caspase-1/11, nicotinamide adenine dinucleotide phosphate oxidase (NOX), and endothelial/neuronal/inducible nitric oxide synthase (eNOS/nNOS/iNOS) that may regulate NLRP3/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/pro-caspase-1 inflammasome formation and activity by using a selective NLRP3 inflammasome inhibitor, MCC950. Male mice received saline (10 ml/kg; i.p.), LPS (10 mg/kg; i.p.), and/or MCC950 (3 mg/kg; i.p.). Reaction time to thermal stimuli within 1 min was evaluated after 6 h. The mice were killed and the brains, hearts, and lungs were collected for measurement of NF-κB, caspase-1, caspase-11, NLRP3, ASC, NOX subunits (gp91phox; NOX2), and p47phox; NOXO2), nitrotyrosine, eNOS, nNOS, iNOS, and ß-actin protein expression, NOS activity, and interleukin (IL)-1ß levels. LPS-induced hyperalgesia was associated with a decrease in eNOS, nNOS, and iNOS protein expression and activity as well as an increase in expression of NF-κB p65, caspase-1 p20, caspase-11 p20, NLRP3, ASC, gp91phox, p47phox, and nitrotyrosine proteins in addition to elevated IL-1ß levels. The LPS-induced changes were prevented by MCC950. The results suggest that inhibition of NLRP3/ASC/pro-caspase-1 inflammasome formation and activity prevents inflammatory hyperalgesia induced by LPS in mice as well as changes in NF-κB, caspase-11, NOX2, NOXO2, and eNOS/nNOS/iNOS expression/activity.

Quality of life, clinical effectiveness, and satisfaction in patients with beta thalassemia major and sickle cell anemia receiving deferasirox chelation therapy.

OBJECTIVES: There is a need to remove excess iron with iron chelation therapy (ICT) to avoid the serious clinical sequelae associated with iron overload in patients with beta thalassemia major (BTM) and sickle cell anemia (SCA). Due to the effects of the diseases and their treatments, ICT is still a major reason for unsatisfactory compliance. The aim of this single-center observational study was to evaluate the quality of life, clinical effectiveness, and satisfaction in pediatric and adult patients with BTM and SCA receiving deferasirox (DFX) chelation therapy. METHODS: In this study, 37 pediatric and 35 adult patients with BTM or SCA receiving DFX for at least 6 months participated. Upon receipt of Informed Consent Form, Case Report Form, Demographic Data Collection Form, Child Health Questionnaire-Parent Form, Life Quality Survey Short Form-36, and ICT Satisfaction Survey were used to obtain data for the effectiveness of ICT and parameters that may affect compliance to treatment and life quality of the participants. RESULTS: As a main index for the effectiveness of DFX chelation therapy, serum ferritin levels were higher than the normal values in the patients receiving DFX. The increased ferritin levels were also associated with hematological and biochemical abnormalities. Our findings regarding quality of life and satisfaction with DFX chelation therapy indicated that the patients with BTM or SCA had lower scores. Overall, problems with treatment regimen and side effects appeared to be common causes of poor compliance to DFX chelation therapy. CONCLUSIONS: Our findings suggest that health care providers should be aware of the importance of monitoring iron load with timely initiation of DFX chelation therapy and ongoing adjustments to chelation regimens and/or transfusion methods to decrease hospitalizations and improve compliance to ICT of the patients with BTM and SCA.

Contribution of PPARα/ß/γ, AP-1, importin-α3, and RXRα to the protective effect of 5,14-HEDGE, a 20-HETE mimetic, against hypotension, tachycardia, and inflammation in a rat model of septic shock.

OBJECTIVES: We have previously demonstrated that downregulation of the MyD88/TAK1-dependent signaling pathway associated with increased CYP4A1 expression and 20-HETE formation participates in the protective effect of N-(20-hydroxyeicosa-5[Z],14[Z]-dienoyl)glycine (5,14-HEDGE), a 20-HETE mimetic, against vascular hyporeactivity, hypotension, tachycardia, inflammation, and mortality in a rodent model of septic shock. The aim of this study was to determine whether increased renal and cardiovascular expression of PPARα/ß/γ and RXRα associated with decreased expression and/or activity of AP-1 and importin-α3 participates in the protective effect of 5,14-HEDGE in response to systemic administration of lipopolysaccharide (LPS). METHODS: Conscious male Wistar rats received saline (4 ml/kg) or LPS (10 mg/kg) at time 0. Blood pressure and heart rate were measured using a tail-cuff device. Separate groups of LPS-treated rats were given 5,14-HEDGE (30 mg/kg) 1 h after injection of saline or LPS. The rats were killed 4 h after saline or LPS administration and the kidney, heart, thoracic aorta, and superior mesenteric artery were collected for measurement of protein expression. RESULTS: Blood pressure fell by 33 mmHg and heart rate rose by 72 beats/min at 4 h after LPS administration. In LPS-treated rats, tissue protein expressions of cytosolic/nuclear PPARα/ß/γ and nuclear RXRα, in addition to nuclear translocation of PPARα/ß/γ proteins, were decreased, while cytosolic/nuclear AP-1 subunit c-jun/phosphorylated c-jun and importin-α3 protein expression as well as their nuclear translocation were increased. The LPS-induced changes were prevented by 5,14-HEDGE. CONCLUSIONS: The results suggest that an increase in the expression of PPARα/ß/γ and RXRα as well as a decrease in AP-1 and importin-α3 expression/activity participates in the protective effect of 5,14-HEDGE against hypotension, tachycardia, and inflammation during endotoxemia and thus have a beneficial effect in septic shock treatment.