Selective brain perfusion improves the neurological outcomes after extracorporeal cardiopulmonary resuscitation in a rat model.

BACKGROUND: The major concern in patients who have suffered from cardiac arrest (CA) and undergone successful extracorporeal cardiopulmonary resuscitation (E-CPR) is poor neurological outcomes. In this study, we aimed to introduce a rat model of selective brain perfusion (SBP) during E-CPR to improve the neurological outcome after CA. METHODS: The rats underwent 7 min of untreated asphyxial CA and then were resuscitated with E-CPR for 30 min. The right external jugular vein and right femoral artery were separately cannulated to the E-CPR outflow and inflow. The right common carotid artery was cannulated from the proximal to the distal side for SBP. Subsequently, rats were removed from E-CPR, wounds were closed, and 90 min of intensive care were provided. Neurological deficit scores were tested after 4 h of recovery when the rats were mechanical ventilation-free. S100 calcium-binding protein B (S100B) and glial fibrillary acidic protein (GFAP) were detected through immunohistochemistry (IHC) of brain tissue. RESULTS: The rats that received SBP while resuscitated by E-CPR showed markedly better neurological performances after 4-h recovery than those resuscitated by E-CPR only. The IHC staining of GFAP and S100B in the hippocampus was low in the rats receiving SBP during E-CPR, but only GFAP showed significant differences. CONCLUSIONS: We successfully developed a novel and reproducible rat model of SBP while resuscitated by E-CPR to ameliorate the neurological performances after CA. This achievement might have opportunities for studying how to improve the neurological outcome in the clinical condition.

Folic Acid Ameliorates Renal Injury in Experimental Obstructive Nephropathy: Role of Glycine N-Methyltransferase.

Folic acid exerts both anti-inflammatory and antifibrotic effects. Glycine N-methyltransferase (GNMT), the major folic acid-binding protein in the liver, is a crucial enzyme that regulates the cellular methylation process by maintaining S-adenosylmethionine levels. However, as yet neither the therapeutic effects of folic acid in renal fibrosis nor whether GNMT is involved in these folic acid-associated mechanisms has been investigated. First, the expression of GNMT was examined in human kidneys with or without obstructive nephropathy. Later, wild-type and GNMT knockout (GNMT-/-) mice were subjected to unilateral ureteral obstruction (UUO) and then treated with either folic acid or vehicle for 14 days. Renal tubular injury, inflammation, fibrosis, and autophagy were evaluated by histological analysis and Western blotting. We observed increased expression of GNMT in humans with obstructive nephropathy. Furthermore, UUO significantly increased the expression of GNMT in mice; in addition, it caused renal injury as well as the development of both hydronephrosis and tubular injury. These were all alleviated by folic acid treatment. In contrast, GNMT-/- mice exhibited exacerbated UUO-induced renal injury, but the protective effect of folic acid was not observed in GNMT-/- mice. We propose a novel role for folic acid in the treatment of renal fibrosis, which indicates that GNMT may be a therapeutic target.

Genetic Deletion of HLJ1 Does Not Affect Blood Coagulation in Mice.

HLJ1 (also called DNAJB4) is a member of the DNAJ/Hsp40 family and plays an important role in regulating protein folding and activity. However, there is little information about the role of HLJ1 in the regulation of physiological function. In this study, we investigated the role of HLJ1 in blood coagulation using wild-type C57BL/6 mice and HLJ1-null (HLJ1-/-) mice. Western blot analysis and immunohistochemistry were used to assess the expression and distribution of HLJ1 protein, respectively. The tail bleeding assay was applied to assess the bleeding time and blood loss. A coagulation test was used for measuring the activity of extrinsic, intrinsic and common coagulation pathways. Thromboelastography was used to measure the coagulation parameters in the progression of blood clot formation. The results showed that HLJ1 was detectable in plasma and bone marrow. The distribution of HLJ1 was co-localized with CD41, the marker of platelets and megakaryocytes. However, genetic deletion of HLJ1 did not alter blood loss and the activity of extrinsic and intrinsic coagulation pathways, as well as blood clot formation, compared to wild-type mice. Collectively, these findings suggest that, although HLJ1 appears in megakaryocytes and platelets, it may not play a role in the function of blood coagulation under normal physiological conditions.

Role of TRPA1 in Tissue Damage and Kidney Disease.

TRPA1, a nonselective cation channel, is expressed in sensory afferent that innervates peripheral targets. Neuronal TRPA1 can promote tissue repair, remove harmful stimuli and induce protective responses via the release of neuropeptides after the activation of the channel by chemical, exogenous, or endogenous irritants in the injured tissue. However, chronic inflammation after repeated noxious stimuli may result in the development of several diseases. In addition to sensory neurons, TRPA1, activated by inflammatory agents from some non-neuronal cells in the injured area or disease, might promote or protect disease progression. Therefore, TRPA1 works as a molecular sentinel of tissue damage or as an inflammation gatekeeper. Most kidney damage cases are associated with inflammation. In this review, we summarised the role of TRPA1 in neurogenic or non-neurogenic inflammation and in kidney disease, especially the non-neuronal TRPA1. In in vivo animal studies, TRPA1 prevented sepsis-induced or Ang-II-induced and ischemia-reperfusion renal injury by maintaining mitochondrial haemostasis or via the downregulation of macrophage-mediated inflammation, respectively. Renal tubular epithelial TRPA1 acts as an oxidative stress sensor to mediate hypoxia-reoxygenation injury in vitro and ischaemia-reperfusion-induced kidney injury in vivo through MAPKs/NF-kB signalling. Acute kidney injury (AKI) patients with high renal tubular TRPA1 expression had low complete renal function recovery. In renal disease, TPRA1 plays different roles in different cell types accordingly. These findings depict the important role of TRPA1 and warrant further investigation.

Renal Tubular Epithelial TRPA1 Acts as An Oxidative Stress Sensor to Mediate Ischemia-Reperfusion-Induced Kidney Injury through MAPKs/NF-κB Signaling.

Oxidative stress and inflammation play important roles in the pathophysiology of acute kidney injury (AKI). Transient receptor potential ankyrin 1 (TRPA1) is a Ca2+-permeable ion channel that is sensitive to reactive oxygen species (ROS). The role of TRPA1 in AKI remains unclear. In this study, we used human and animal studies to assess the role of renal TRPA1 in AKI and to explore the regulatory mechanism of renal TRPA1 in inflammation via in vitro experiments. TRPA1 expression increased in the renal tubular epithelia of patients with AKI. The severity of tubular injury correlated well with tubular TRPA1 or 8-hydroxy-2'-deoxyguanosine expression. In an animal model, renal ischemia-reperfusion injury (IR) increased tubular TRPA1 expression in wild-type (WT) mice. Trpa1-/- mice displayed less IR-induced tubular injury, oxidative stress, inflammation, and dysfunction in kidneys compared with WT mice. In the in vitro model, TRPA1 expression increased in renal tubular cells under hypoxia-reoxygenation injury (H/R) conditions. We demonstrated that H/R evoked a ROS-dependent TRPA1 activation, which elevated intracellular Ca2+ level, increased NADPH oxidase activity, activated MAPK/NF-κB signaling, and increased IL-8. Renal tubular TRPA1 may serve as an oxidative stress sensor and a crucial regulator in the activation of signaling pathways and promote the subsequent transcriptional regulation of IL-8. These actions might be evident in mice with IR or patients with AKI.

Endothelial Nitric Oxide Mediates the Anti-Atherosclerotic Action of Torenia concolor Lindley var. Formosama Yamazaki.

Torenia concolor Lindley var. formosama Yamazaki ethanolic extract (TCEE) is reported to have anti-inflammatory and anti-obesity properties. However, the effects of TCEE and its underlying mechanisms in the activation of endothelial nitric oxide synthase (eNOS) have not yet been investigated. Increasing the endothelium-derived nitric oxide (NO) production has been known to be beneficial against the development of cardiovascular diseases. In this study, we investigated the effect of TCEE on eNOS activation and NO-related endothelial function and inflammation by using an in vitro system. In endothelial cells (ECs), TCEE increased NO production in a concentration-dependent manner without affecting the expression of eNOS. In addition, TCEE increased the phosphorylation of eNOS at serine 635 residue (Ser635) and Ser1179, Akt at Ser473, calmodulin kinase II (CaMKII) at threonine residue 286 (Thr286), and AMP-activated protein kinase (AMPK) at Thr172. Moreover, TCEE-induced NO production, and EC proliferation, migration, and tube formation were diminished by pretreatment with LY294002 (an Akt inhibitor), KN62 (a CaMKII inhibitor), and compound C (an AMPK inhibitor). Additionally, TCEE attenuated the tumor necrosis factor-α-induced inflammatory response as evidenced by the expression of adhesion molecules in ECs and monocyte adhesion onto ECs. These inflammatory effects of TCEE were abolished by L-NG-nitroarginine methyl ester (an NOS inhibitor). Moreover, chronic treatment with TCEE attenuated hyperlipidemia, systemic and aortic inflammatory response, and the atherosclerotic lesions in apolipoprotein E-deficient mice. Collectively, our findings suggest that TCEE may confer protection from atherosclerosis by preventing endothelial dysfunction.

The phosphatase activity of soluble epoxide hydrolase regulates ATP-binding cassette transporter-A1-dependent cholesterol efflux.

The contribution of soluble epoxide hydrolase (sEH) to atherosclerosis has been well defined. However, less is understood about the role of sEH and its underlying mechanism in the cholesterol metabolism of macrophages. The expression of sEH protein was increased in atherosclerotic aortas of apolipoprotein E-deficient mice, primarily in macrophage foam cells. Oxidized low-density lipoprotein (oxLDL) increased sEH expression in macrophages. Genetic deletion of sEH (sEH-/- ) in macrophages markedly exacerbated oxLDL-induced lipid accumulation and decreased the expression of ATP-binding cassette transporters-A1 (ABCA1) and apolipoprotein AI-dependent cholesterol efflux following oxLDL treatment. The down-regulation of ABCA1 in sEH-/- macrophages was due to an increase in the turnover rate of ABCA1 protein but not in mRNA transcription. Inhibition of phosphatase activity, but not hydrolase activity, of sEH decreased ABCA1 expression and cholesterol efflux following oxLDL challenge, which resulted in increased cholesterol accumulation. Additionally, oxLDL increased the phosphatase activity, promoted the sEH-ABCA1 complex formation and decreased the phosphorylated level of ABCA1 at threonine residues. Overexpression of phosphatase domain of sEH abrogated the oxLDL-induced ABCA1 phosphorylation and further increased ABCA1 expression and cholesterol efflux, leading to the attenuation of oxLDL-induced cholesterol accumulation. Our findings suggest that the phosphatase domain of sEH plays a crucial role in the cholesterol metabolism of macrophages.

Genetic deletion of soluble epoxide hydrolase delays the progression of Alzheimer's disease.

BACKGROUND: Soluble epoxide hydrolase (sEH) is a bifunctional enzyme with COOH-terminal hydrolase and NH2-terminal lipid phosphatase activities. It is expressed in various cell types in the brain and is involved in the pathogenesis of inflammatory and neurodegenerative diseases. Alzheimer's disease (AD) is a progressive neuroinflammatory and neurodegenerative disease. However, the pathological significance of sEH and underlying molecular mechanism in AD remain unclear. METHODS: To examine the role of sEH in pathogenesis of AD, we used wild-type (WT) mice, soluble epoxide hydrolase deficient (sEH-/-) and two mouse models of AD, including amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic (APP/PS1 Tg) and APP/PS1 Tg/sEH-/- mice. Western blotting analysis and immunohistochemistry assay were performed to evaluate the protein expression. Locomotion, nesting building ability, Y-maze, and Morris water maze tests were conducted to study mouse behavior. The levels of interleukin (IL)-1ß, IL-4, IL-6, and IL-10 and the activities of NF-κB and nuclear factor of activated T cells (NFAT) were measured by commercial assay kits. The quantitative protein level profiling in the brain lysate was analyzed using LC-MS/MS approaches. RESULTS: We demonstrated that the level of sEH was increased in the brain and predominantly appeared in hippocampal astrocytes of APP/PS1 Tg mice. Genetic ablation of sEH in APP/PS1 Tg mice delayed the progression of AD as evidenced by the alleviation in behavior outcomes and Aß plaque deposition. In addition, loss of the function of sEH in APP/PS1 Tg mice increased astrogliosis and the production of astrocyte-derived anti-inflammatory cytokines including IL-1ß, IL-4, and IL-10, as well as the activity of NF-kB and NFAT. Moreover, analysis of gene ontology in the AD brain revealed that important signaling pathways and processes related to AD pathogenesis such as translational regulation, oxidative stress, cytoskeleton reorganization, and small GTPase signal transduction were altered in APP/PS1 Tg/sEH-/- mice compared with APP/PS1 Tg mice. CONCLUSION: Our results suggest that sEH is a crucial regulator in the progression of AD and might be a potential therapeutic target for the treatment of AD.

DDAH-2 alleviates contrast medium iopromide-induced acute kidney injury through nitric oxide synthase.

BACKGROUND: Contrast medium-induced acute kidney injury (CI-AKI) is one of the most common causes of hospital-acquired acute renal failure. However, the pathogenesis of CI-AKI remains unclear. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor that is largely metabolised by dimethylarginine dimethylaminohydroxylase (DDAH) in humans. Two isoforms of DDAH exist, namely, DDAH-1 and DDAH-2. In the present study, we examined whether the DDAH-2/ADMA/NOS pathway is involved in the pathogenesis of CI-AKI. METHODS AND RESULTS: Exposure to the contrast medium iopromide led to increase in creatinine and blood urea nitrogen (BUN) levels, accumulation of ADMA, increase in reactive oxygen species (ROS) generation, and an inflammatory response in mice kidney tissue. The injection of adenovirus-harbouring DDAH-2 lowered renal ADMA levels and had a reno-protective effect against contrast-medium injury by decreasing cell apoptosis, ROS, and fibrosis. By contrast, contrast medium-induced renal injury was exacerbated in heterozygous DDAH-2 knockout mice. In the in vitro study, overexpression of DDAH-2 increased the levels of nitrite and intracellular cGMP, while the DDAH-2 knockdown induced the opposite effect. These findings were also observed in the in vivo sample. CONCLUSIONS: Our findings provide the first evidence that the DDAH-2/ADMA/NOS pathway is involved in the pathogenesis of CI-AKI and that the protective effect of DDAH-2 probably arises from the modulation of NOS activity, oxidative stress, and the inflammatory process.

Atypical Antipsychotic Drug Olanzapine Deregulates Hepatic Lipid Metabolism and Aortic Inflammation and Aggravates Atherosclerosis.

BACKGROUND/AIMS: Olanzapine, an atypical antipsychotic drug, has therapeutic effects for schizophrenia. However, clinical reports indicate that patients taking atypical antipsychotic drugs are at high risk of metabolic syndrome with unclear mechanisms. We investigated the effect of olanzapine on atherosclerosis and the mechanisms in apolipoprotein E-null (apoE-/-) mice. METHODS: ApoE-/- mice were used as in vivo models. Western blot analysis was used to evaluate protein expression. Conventional assay kits were applied to assess the levels of cholesterol, triglycerides, free cholesterol, cholesteryl ester, fatty acids, glycerol, and cytokines. RESULTS: Daily treatment with olanzapine (3 mg/kg body weight) for four weeks increased mean arterial blood pressure and the whitening of brown adipose tissue in mice. In addition, olanzapine impaired aortic cholesterol homeostasis and exacerbated hyperlipidemia and aortic inflammation, which accelerated atherosclerosis in mice. Moreover, lipid accumulation in liver, particularly total cholesterol, free cholesterol, fatty acids, and glycerol, was increased with olanzapine treatment in apoE-/- mice by upregulating the expression of de novo lipid synthesis-related proteins and downregulating that of cholesterol clearance- or very low-density lipoprotein secretion-related proteins. CONCLUSION: Olanzapine may exacerbate atherosclerosis by deregulating hepatic lipid metabolism and worsening hyperlipidemia and aortic inflammation.

Role of transient receptor potential ankyrin 1 channels in Alzheimer's disease.

BACKGROUND: Transient receptor potential ankyrin 1 (TRPA1) channel plays an important role in pain and inflammation. However, little is known about the significance of the TRPA1 channel in the pathophysiology of Alzheimer's disease (AD). METHODS: Wild-type (WT), TRPA1(-/-), amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic (APP/PS1 Tg) mice, the mouse model of AD, and APP/PS1 Tg/TRPA1(-/-) mice were used to examine the role of TRPA1 in pathogenesis of AD. Western blot was used for protein expression; immunohistochemistry was used for histological examination. The mouse behaviors were evaluated by locomotion, nesting building, Y-maze and Morris water maze tests; levels of interleukin (IL)-1ß, IL-4, IL-6 and IL-10 and the activities of protein phosphatase 2B (PP2B), NF-κB and nuclear factor of activated T cells (NFAT) were measured by conventional assay kits; Fluo-8 NW calcium (Ca(2+)) assay kit was used for the measurement of intracellular Ca(2+) level in primary astrocytes and HEK293 cells. RESULTS: The protein expression of TRPA1 channels was higher in brains, mainly astrocytes of the hippocampus, from APP/PS1 Tg mice than WT mice. Ablation of TRPA1-channel function in APP/PS1 Tg mice alleviated behavioral dysfunction, Aß plaque deposition and pro-inflammatory cytokine production but increased astrogliosis in brain lesions. TRPA1 channels were activated and Ca(2+) influx was elicited in both astrocytes and TRPA1-transfected HEK293 cells treated with fibrilized Aß1-42; these were abrogated by pharmacological inhibition of TRPA1 channel activity, disruption of TRPA1 channel function or removal of extracellular Ca(2+). Inhibition of TRPA1 channel activity exacerbated Aß1-42-induced astrogliosis but inhibited Aß1-42-increased PP2B activation, the production of pro-inflammatory cytokines and activities of transcriptional factors NF-κB and NFAT in astrocytes and in APP/PS1 Tg mice. Pharmacological inhibition of PP2B activity diminished the fibrilized Aß1-42-induced production of pro-inflammatory cytokines, activation of NF-κB and NFAT and astrogliosis in astrocytes. CONCLUSIONS: TRPA1 - Ca(2+) - PP2B signaling may play a crucial role in regulating astrocyte-derived inflammation and pathogenesis of AD.

Glycine N-methyltransferase deficiency in female mice impairs insulin signaling and promotes gluconeogenesis by modulating the PI3K/Akt pathway in the liver.

BACKGROUND: Glycine N-methyltransferase (GNMT) is abundantly expressed in the normal liver but is down-regulated in liver cancer tissues. GNMT knockout (Gnmt-/-) mice can spontaneously develop chronic hepatitis, fatty liver, and liver cancer. We previously demonstrated that hepatic GNMT is decreased in high-fat-diet-induced type 2 diabetes mellitus, but its contribution to metabolic syndrome is unclear. Here we show that GNMT modulates key aspects of metabolic syndrome in mice. METHODS: Eleven-week-old Gnmt-/- and wild-type (WT) mice with a C57BL/6 genetic background were used in this study. The metabolic defects of GNMT deficiency were measured by glucose and insulin tolerance tests, lipid homeostasis, gluconeogenesis, and insulin signaling. RESULTS: Gnmt-/- mice, especially females, exhibited glucose intolerance and insulin resistance. However, their body fat and lean mass, food and water intakes, and energy expenditure did not differ from those of WT mice. In addition, glucose-stimulated insulin secretion and insulin-stimulated glucagon secretion were normal in the serum and pancreatic islets of Gnmt-/- mice. Importantly, we found that GNMT deficiency increased lipogenesis and triglycerides in the liver. The elevated triglycerides disrupted the ability of insulin to induce Akt and S6 ribosomal protein phosphorylation, and then triggered insulin resistance and gluconeogenesis in female Gnmt-/- mice. CONCLUSIONS: Our data indicate that hepatic GNMT regulates lipid and glucose homeostasis, and provide insight into the development of insulin resistance through modulating the PI3K/Akt pathway.

Di-(2-ethylhexyl) phthalate accelerates atherosclerosis in apolipoprotein E-deficient mice.

Di-(2-ethylhexyl) phthalate (DEHP) is associated with atherosclerosis-related cardiovascular disease complications, but we lack direct evidence of its unfavorable effect on atherogenesis. In this study, we aimed to clarify in vivo and in vitro the contribution of DEHP to the development of atherosclerosis and its underlying mechanisms. Apolipoprotein E-deficient (apoE(-/-)) mice chronically treated with DEHP for 4 weeks showed exacerbated hyperlipidemia, systemic inflammation, and atherosclerosis. In addition, DEHP promoted low-density lipoprotein (LDL) oxidation, which led to inflammation in endothelial cells as evidenced by increased protein expression of pro-inflammatory mediators. Furthermore, chronic DEHP treatment increased hepatic cholesterol accumulation by downregulating the protein expression of key regulators in cholesterol clearance including LDL receptor, cholesterol 7α-hydrolase, ATP-binding cassette transporter G5 and G8, and liver X receptor α. Moreover, the adiposity and inflammation of white adipose tissues were promoted in DEHP-treated apoE(-/-) mice. In conclusion, DEHP may disturb cholesterol homeostasis and deregulate the inflammatory response, thus leading to accelerated atherosclerosis.

Maternal exposure to di-(2-ethylhexyl) phthalate exposure deregulates blood pressure, adiposity, cholesterol metabolism and social interaction in mouse offspring.

Long-term exposure to di-(2-ethylhexyl) phthalate (DEHP) is highly associated with carcinogenicity, fetotoxicity, psychological disorders and metabolic diseases, but the detrimental effects and mechanisms are not fully understood. We investigated the effect of exposing mouse mothers to DEHP, and the underlying mechanism, on blood pressure, obesity and cholesterol metabolism as well as psychological and learning behaviors in offspring. Tail-cuff plethysmography was used for blood pressure measurement; Western blot used was for phosphorylation and expression of protein; hematoxylin and eosin staining, Nissl staining and Golgi staining were used for histological examination. The serum levels of cholesterol, triglycerides and glucose were measured by blood biochemical analysis. Hepatic cholesterol and triglyceride levels were assessed by colorimetric assay kits. Offspring behaviors were evaluated by open-field activity, elevated plus maze, social preference test and Morris water maze. Maternal DEHP exposure deregulated the phosphorylation of endothelial nitric oxide synthase and upregulated angiotensin type 1 receptor in offspring, which led to increased blood pressure. It led to obesity in offspring by increasing the size of adipocytes in white adipose tissue and number of adipocytes in brown adipose tissue. It increased the serum level of cholesterol in offspring by decreasing the hepatic capacity for cholesterol clearance. The impaired social interaction ability induced by maternal DEHP exposure might be due to abnormal neuronal development. Collectively, our findings provide new evidence that maternal exposure to DEHP has a lasting effect on the physiological functions of the vascular system, adipose tissue and nerve system in offspring.

Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation.

In chronic liver diseases, regardless of their etiology, the development of fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. Hepatic stellate cells (HSCs) are the main profibrogenic cells that promote the pathogenesis of liver fibrosis, and so it is important to identify the molecules that regulate HSCs activation and liver fibrosis. Niemann-Pick type C2 (NPC2) protein plays an important role in the regulation of intracellular cholesterol homeostasis by directly binding with free cholesterol. However, the roles of NPC2 in HSCs activation and liver fibrosis have not been explored in detail. Since a high-cholesterol diet exacerbates liver fibrosis progression in both rodents and humans, we propose that the expression of NPC2 affects free cholesterol metabolism and regulates HSCs activation. In this study, we found that NPC2 is decreased in both thioacetamide- and carbon tetrachloride-induced liver fibrosis tissues. In addition, NPC2 is expressed in quiescent HSCs, but its activation status is down-regulated. Knockdown of NPC2 in HSC-T6 cells resulted in marked increases in transforming growth factor-ß1 (TGF-ß1)-induced collagen type 1 α1 (Col1a1), α-smooth muscle actin (α-SMA) expression, and Smad2 phosphorylation. In contrast, NPC2 overexpression decreased TGF-ß1-induced HSCs activation. We further demonstrated that NPC2 deficiency significantly increased the accumulation of free cholesterol in HSCs, increasing Col1a1 and α-SMA expression and activating Smad2, and leading to sensitization of HSCs to TGF-ß1 activation. In contrast, overexpression of NPC2 decreased U18666A-induced free cholesterol accumulation and inhibited the subsequent HSCs activation. In conclusion, our study has demonstrated that NPC2 plays an important role in HSCs activation by regulating the accumulation of free cholesterol. NPC2 overexpression may thus represent a new treatment strategy for liver fibrosis.

Regulation of Cigarette Smoke Induction of IL-8 in Macrophages by AMP-activated Protein Kinase Signaling.

Inhaled cigarette smoke (CS) causes persistent lung inflammation in smokers. Interleukin 8 (IL-8) released from macrophages is a key chemokine during initiation and progression of CS-induced lung inflammation, yet its regulation is largely unknown. AMP-activated protein kinase (AMPK), a crucial energy homeostasis regulator, may modulate inflammation. Here we report that CS extract (CSE) increased the level of intracellular reactive oxygen species (ROS), activating AMPK, mitogen-activated protein kinases (MAPKs), and NF-κB, as well as inducing IL-8, in human macrophages. N-acetyl-cysteine (ROS scavenger) or hexamethonium [nicotinic acetylcholine receptor (nAChR) antagonist] attenuated the CSE-induced increase in intracellular ROS, activation of AMPK and NF-κB, as well as IL-8 induction, which suggests that nAChRs and ROS are important. Prevention of AMPK activation by compound C or AMPK siRNA reduced CSE-induced IL-8 production, confirming the role of AMPK. Compound C or AMPK siRNA also inhibited the activation of MAPKs and NF-κB by CSE, which suggests that these molecules are downstream of AMPK. Additionally, exposure of human macrophages to nicotine activated AMPK and induced IL-8 and that these effects were lessened by hexamethonium or compound C, implying that nicotine in CS may be causative. Furthermore, chronic CS exposure in mice promoted AMPK phosphorylation and expression of MIP-2 (an IL-8 homolog) in infiltrated macrophages and in lung tissues, as well as induced lung inflammation, all of which were reduced by compound C treatment. Thus, we identified a novel nAChRs-dependent, ROS-sensitive, AMPK/MAPKs/NF-κB signaling pathway, which seems to be important to CS-induced macrophage IL-8 production and possibly to lung inflammation.

Soluble epoxide hydrolase inhibitor enhances synaptic neurotransmission and plasticity in mouse prefrontal cortex.

BACKGROUND: The soluble epoxide hydrolase (sEH) is an important enzyme chiefly involved in the metabolism of fatty acid signaling molecules termed epoxyeicosatrienoic acids (EETs). sEH inhibition (sEHI) has proven to be protective against experimental cerebral ischemia, and it is emerging as a therapeutic target for prevention and treatment of ischemic stroke. However, the role of sEH on synaptic function in the central nervous system is still largely unknown. This study aimed to test whether sEH C-terminal epoxide hydrolase inhibitor, 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) affects basal synaptic transmission and synaptic plasticity in the prefrontal cortex area (PFC). Whole cell and extracellular recording examined the miniature excitatory postsynaptic currents (mEPSCs) and field excitatory postsynaptic potentials (fEPSPs); Western Blotting determined the protein levels of glutamate receptors and ERK phosphorylation in acute medial PFC slices. RESULTS: Application of the sEH C-terminal epoxide hydrolase inhibitor, AUDA significantly increased the amplitude of mEPSCs and fEPSPs in prefrontal cortex neurons, while additionally enhancing long term potentiation (LTP). Western Blotting demonstrated that AUDA treatment increased the expression of the N-methyl-D-aspartate receptor (NMDA) subunits NR1, NR2A, NR2B; the α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR1, GluR2, and ERK phosphorylation. CONCLUSIONS: Inhibition of sEH induced an enhancement of PFC neuronal synaptic neurotransmission. This enhancement of synaptic neurotransmission is associated with an enhanced postsynaptic glutamatergic receptor and postsynaptic glutamatergic receptor mediated synaptic LTP. LTP is enhanced via ERK phosphorylation resulting from the delivery of glutamate receptors into the PFC by post-synapse by treatment with AUDA. These findings provide a possible link between synaptic function and memory processes.

Soluble epoxide hydrolase activity regulates inflammatory responses and seizure generation in two mouse models of temporal lobe epilepsy.

Neuroinflammation is known to be involved in epileptogenesis with unclear mechanisms. Inhibition of soluble epoxide hydrolase (sEH) seems to offer anti-inflammatory protection to ischemic brain injury in rodents. Thus, it is hypothesized that sEH inhibition might also affect the neuroinflammatory responses caused by epileptic seizures. In the present study, we investigated the involvement of sEH in neuroinflammation, seizure generation and subsequent epileptogenesis using two mouse models of temporal lobe epilepsy. Experimental epileptic seizures were induced by either pilocarpine or electrical amygdala kindling in both wild-type (WT) C57BL/6 mice and sEH knockout (sEH KO) mice. The sEH expression in the hippocampus was detected by immunohistochemistry and Western blot analysis. The effects of the sEH hydrolase inhibitors, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) and N-[1-(1-oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy) phenyl)-urea (TPPU), and of the genetic deletion of sEH on seizure-induced neuroinflammatory responses and the development of epilepsy were evaluated. In the hippocampus of WT mice, sEH was mainly expressed in astrocytes (GFAP(+)), neurons (NeuN(+)) and scattered microglia (Iba-1(+)) in the regions of CA1, CA3 and dentate gyrus. Expression of sEH was significantly increased on day 7, 14, 21 and 28 after pilocarpine-induced status epilepticus (SE). Administration with sEH inhibitors attenuated the SE-induced up-regulation of interleukin-1ß (IL-1ß) and interleukin-6 (IL-6), the degradation of EETs, as well as IκB phosphorylation. Following treatment with AUDA, the frequency and duration of spontaneous motor seizures in the pilocarpine-SE mice were decreased and the seizure-induction threshold of the fully kindled mice was increased. Up-regulation of hippocampal IL-1ß and IL-6 was found in both WT and sEH KO mice after successful induction of SE. Notably, sEH KO mice were more susceptible to seizures than WT mice. Seizure related neuroinflammation and ictogenesis were attenuated by pharmacological inhibition of sEH enzymatic activity but not by sEH genetic deletion. Therefore, sEH may play an important role in the generation of epilepsy. Furthermore, the effectiveness of AUDA in terms of anti-inflammatory and anti-ictogenesis properties suggests that it may have clinical therapeutic implication for epilepsy in the future, particularly when treating temporal lobe epilepsy.

Lung Epithelial TRPA1 Transduces the Extracellular ROS into Transcriptional Regulation of Lung Inflammation Induced by Cigarette Smoke: The Role of Influxed Ca²âº.

The mechanism underlying the inflammatory role of TRPA1 in lung epithelial cells (LECs) remains unclear. Here, we show that cigarette smoke extract (CSE) sequentially induced several events in LECs. The Ca(2+) influx was prevented by decreasing extracellular reactive oxygen species (ROS) with the scavenger N-acetyl-cysteine, removing extracellular Ca(2+) with the chelator EGTA, or treating with the TRPA1 antagonist HC030031. NADPH oxidase activation was abolished by its inhibitor apocynin, EGTA, or HC030031. The increased intracellular ROS was halted by apocynin, N-acetyl-cysteine, or HC030031. The activation of the MAPKs/NF-κB signaling was suppressed by EGTA, N-acetyl-cysteine, or HC030031. IL-8 induction was inhibited by HC030031 or TRPA1 siRNA. Additionally, chronic cigarette smoke (CS) exposure in wild-type mice induced TRPA1 expression in LECs and lung tissues. In CS-exposure trpa1 (-/-) mice, the increased BALF level of ROS was similar to that of CS-exposure wild-type mice; yet lung inflammation was lessened. Thus, in LECs, CSE may initially increase extracellular ROS, which activate TRPA1 leading to an increase in Ca(2+) influx. The increased intracellular Ca(2+) contributes to activation of NADPH oxidase, resulting in increased intracellular ROS, which activate the MAPKs/NF-κB signaling leading to IL-8 induction. This mechanism may possibly be at work in mice chronically exposed to CS.

ß Common Receptor Mediates Erythropoietin-Conferred Protection on OxLDL-Induced Lipid Accumulation and Inflammation in Macrophages.

Erythropoietin (EPO), the key factor for erythropoiesis, also protects macrophage foam cells from lipid accumulation, yet the definitive mechanisms are not fully understood. ß common receptor (ßCR) plays a crucial role in the nonhematopoietic effects of EPO. In the current study, we investigated the role of ßCR in EPO-mediated protection in macrophages against oxidized low-density lipoprotein- (oxLDL-) induced deregulation of lipid metabolism and inflammation. Here, we show that ßCR expression was mainly in foamy macrophages of atherosclerotic aortas from apolipoprotein E-deficient mice. Results of confocal microscopy and immunoprecipitation analyses revealed that ßCR was colocalized and interacted with EPO receptor (EPOR) in macrophages. Inhibition of ßCR activation by neutralizing antibody or small interfering RNA (siRNA) abolished the EPO-conferred protection in oxLDL-induced lipid accumulation. Furthermore, EPO-promoted cholesterol efflux and upregulation of ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 were prevented by pretreatment with ßCR neutralizing antibody or ßCR siRNA. Additionally, blockage of ßCR abrogated the EPO-conferred anti-inflammatory action on oxLDL-induced production of macrophage inflammatory protein-2. Collectively, our findings suggest that ßCR may play an important role in the beneficial effects of EPO against oxLDL-elicited dysfunction of macrophage foam cells.