Augmented CCL5/CCR5 signaling in brown adipose tissue inhibits adaptive thermogenesis and worsens insulin resistance in obesity.

Chemokine (C-C motif) ligand 5 (CCL5) and CCR5, one of its receptors have been reported to be highly expressed in white adipose tissue (WAT) and are associated with the progression of inflammation and the development of insulin resistance in obese humans and mice. However, the role of CCL5/CCR5 signaling in obesity-associated dysregulation of energy metabolism remains unclear. Here, we demonstrate that global CCL5/CCR5 double knockout (DKO) mice have higher cold stress-induced energy expenditure and thermogenic function in brown adipose tissue (BAT) than wildtype (WT) mice. DKO mice have higher cold stress-induced energy expenditure and thermogenic function in BAT than WT mice. KEGG pathway analysis indicated that deletion of CCL5/CCR5 further facilitated the cold-induced expression of genes related to oxidative phosphorylation (OxPhos) and lipid metabolic pathways. In primary brown adipocytes of DKO mice, the augmentation of CL-316243-stimulated thermogenic and lipolysis responses was reversed by co-treatment with AMPKα1 and α2 short interfering RNA (siRNA). Overexpression of BAT CCL5/CCR5 genes by local lentivirus injection in WT mice suppressed cold stress-induced lipolytic processes and thermogenic activities. In contrast, knockdown of BAT CCL5/CCR5 signaling further up-regulated AMPK phosphorylation as well as thermogenic and lipolysis responses to chronic adrenergic stimuli and subsequently decreased level of body weight gain. Chronic knockdown of BAT CCL5/CCR5 signaling improved high-fat diet (HFD)-induced insulin resistance in WT mice. It is suggested that obesity-induced augmentation of adipose tissue (AT) CCL5/CCR5 signaling could, at least in part, suppress energy expenditure and adaptive thermogenesis by inhibiting AMPK-mediated lipolysis and oxidative metabolism in thermogenic AT to exacerbate the development of obesity and insulin resistance.

Rapid screening for mutations associated with malignant hyperthermia using high-resolution melting curve analysis.

OBJECTIVES: The diagnosis of malignant hyperthermia (MH) is based on clinical signs or laboratory testing. The gold standard laboratory test is the in vitro contracture test, although it is invasive, expensive, and only performed at specialized centers. Genetic diagnosis is another option, although direct mutation screening is a laborious task. Therefore, we evaluated whether high-resolution melting (HRM) curve analysis could be used as a rapid screening tool to target MH-associated mutations. MATERIALS AND METHODS: The feasibility of HRM analysis was evaluated using plasmids that were constructed by cloning wild-type or mutated versions of the ryanodine receptor 1 (RYR1) gene into the pCR2.1 plasmid. We obtained engineered plasmids and patient DNA extracted from blood samples with known wild-type or mutated sequences that are associated with MH. Amplicon lengths were kept relatively short (<250 bp) to improve discrimination between the engineered and patient plasmids. Real-time polymerase chain reaction (PCR) cycling and HRM analysis of the engineered plasmids and patient DNA were performed using the LightCycler 480 System (Roche). RESULTS: The HRM results were clearly different from those obtained using real-time PCR. Furthermore, the HRM analysis provided sufficient resolution to identify two single-nucleotide variants in the tested RYR1 exons. CONCLUSION: We conclude that HRM analysis can provide high resolution for identifying single-nucleotide variants in RYR1, which might be useful for predicting the risk of MH in the preanesthesia setting.

Importance of PLC-Dependent PI3K/AKT and AMPK Signaling in RANTES/CCR5 Mediated Macrophage Chemotaxis.

Regulated upon activation, normal T cell expressed, and secreted (RANTES), also known as chemokine ligand 5 (CCL5), has been reported to facilitate macrophage migration, which plays a crucial role in tissue inflammation. The aim of this study is to investigate the characteristics and underlying mechanism of RANTES on macrophage chemotaxis under physiological and pathological conditions. The study was conducted on macrophage RAW264.7 cell and bone marrow-derived macrophages (BMDM) isolated from CCL receptor 5 (CCR5) knockout mice. The macrophage migration and glucose uptake was assessed in time and dose dependent manners. Moreover, reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were used to characterize mRNA and protein level related to the underlying mechanism. The present result showed that the maraviroc, a selective CCR5 inhibitor, dose-dependently suppressed RANTES-induced rapid increases in glucose uptake and cell migration in RAW264.7 cells. Similar effects were observed in the BMDM isolated from CCR5 knockout mice compared with wild type control. RANTES treatment promptly enhanced membrane glucose transporter 1 (GLUT1) expression, glucose uptake as well as phosphorylation of AKT on Thr308, Ser473 within min and has prolonged effect on phosphorylation of AMP-activated protein kinase (AMPK) on Thr172, which were abrogated by maraviroc, CCR5 siRNA or phospholipase C (PLC) inhibitor in RAW264.7 cells. Inhibition of PI3K and AMPK by LY294002 and Compound C significantly suppress RANTES-stimulated macrophage glucose uptake and migration, respectively. RANTES has biphasic effect on activating PLC signaling including prompt action on PI3K/AKT phosphorylation and prolong action on AMPK phosphorylation via CCR5 which leads to increased GLUT1-mediated glucose uptake and macrophage migration under physiopathological states.

IL-17 deficiency attenuates acetaminophen-induced hepatotoxicity in mice.

Acetaminophen (APAP) overdose results in the production of reactive oxygen species (ROS), hepatocyte necrosis, and cell death, and leads to acute liver failure. Interleukin-17 (IL-17), a pro-inflammatory cytokine, plays a key role in the recruitment of neutrophils into sites of inflammation and subsequent damage after liver ischemia-reperfusion injury. In this study, we employed IL-17 knockout (KO) mice to investigate the role of IL-17 in APAP-induced hepatotoxicity. IL-17 wide type (WT) and IL-17 KO mice received an intraperitoneal injection of APAP (300 mg/kg). After 16 h of treatment, the hepatic injury, inflammatory mediators, immune cell infiltration, and western blotting were examined and analyzed. The serum alanine transferase (ALT) enzyme levels and hepatic myeloperoxidase (MPO) activity were significantly elevated 16 h after APAP treatment in the WT mice. IL-17 deficiency significantly attenuates APAP-induced liver injury, MPO activity, pro-inflammatory cytokines (tumor necrosis factor-α, IL-6 and interferon-γ) levels and inflammatory cell (neutrophils, macrophage) infiltration in the liver. Moreover, phosphorylated extracellular signal-regulated kinase (ERK) was significantly decreased at 16 h after APAP treatment in the IL-17 KO mice compared with the IL-17 WT mice. Our data suggests that IL-17 plays a pivotal role in APAP-induced hepatotoxicity through modulation of inflammatory response, and perhaps in part through the ERK signaling pathway. Blockade of IL-17 could be a potential therapeutic target for APAP-induced hepatotoxicity.

C-C Chemokine Ligand-5 is critical for facilitating macrophage infiltration in the early phase of liver ischemia/reperfusion injury.

CCL5/RANTES, a chemoattractant for myeloid cells, is induced by hepatic ischemia/reperfusion injury (IRI). The roles of CCL5 in hepatic IRI were carried out by means of CCL5 immunodepletion, antagonistic competition by Met-CCL5, and treatment with recombinant murine CCL5 (rmCCL5). Depletion or inhibition of CCL5 reduced severity of hepatic IRI, whereas rmCCL5 treatment aggravated liver IRI as manifested in elevated serum alanine aminotransferase (ALT) and tissue myeloperoxidase (MPO) levels. Moreover, IRI severity was reduced in CCL5-knockout (CCL5-KO) mice versus wildtype (WT) mice, with drops in serum ALT level, intrahepatic MPO activity, and histological pathology. Bone marrow transplantion (BMT) studies show that myeloid cells and tissue cells are both required for CCL5-aggravated hepatic IRI. The profile of liver-infiltrating leukocyte subsets after hepatic reperfusion identified CD11b+ cells as the only compartment significantly reduced in CCL5-KO mice versus WT controls at early reperfusion phase. The role of CCL5 recruiting CD11b+ cells in early reperfusion was validated by in vitro transwell migration assay of murine primary macrophages (broadly characterized by their CD11b expression) in response to liver lysates after early reperfusion. Taken together, our results demonstrate a sequence of early events elicited by CCL5 chemoattracting macrophage that result in inflammatory aggravation of hepatic IRI.

Activation of NPFFR2 leads to hyperalgesia through the spinal inflammatory mediator CGRP in mice.

Neuropeptide FF (NPFF) is recognized as an opioid modulating peptide that regulates morphine-induced analgesia. The aim of this study was to delineate the role of NPFFR2 in pain transmission. We found the expression levels of NPFF and NPFFR2 were increased in the lumbar dorsal horn of animals with CFA- and carrageenan-induced inflammation and both NPFFR2 over-expressing transgenic (NPFFR2-Tg) and NPFFR2 agonist-treated mice displayed hyperalgesia. BOLD signals from functional MRI showed that NPFFR2-Tg mice exhibited increased activation of pain-related brain regions after painful stimulation when compared to WT mice. Inflammatory mediators within the spinal cord, calcitonin gene-related peptide (CGRP) and substance P (SP), were up-regulated in NPFFR2-Tg and chronic NPFFR2 agonist-treated mice. In DRG cultures, treatment with an NPFFR2 agonist induced the expression and release of CGRP, an action which was blocked by NPFFR2 siRNA. Furthermore, treatment with a CGRP antagonist ameliorated the pain hyperalgesia in NPFFR2-Tg mice, returning the pain threshold to a control level. However, treatment with a SP antagonist reduced the pain responses in both WT and NPFFR2-Tg mice and did not suppress pain hypersensitivity in NPFFR2-Tg mice. Together, these results demonstrate that NPFFR2 activation modulates pain transmission by up-regulating the pain mediator CGRP, leading to hyperalgesia.

ERK Signaling Pathway Plays a Key Role in Baicalin Protection Against Acetaminophen-Induced Liver Injury.

Acetaminophen (APAP) overdose causes hepatocytes necrosis and acute liver failure. Baicalin (BA), a major flavonoid of Scutellariae radix, has potent hepatoprotective properties in traditional medicine. In the present study, we investigated the protective effects of BA on a APAP-induced liver injury in a mouse model. The mice received an intraperitoneal hepatotoxic dose of APAP (300[Formula: see text]mg/kg) and after 30[Formula: see text]min, were treated with BA at concentrations of 0, 15, 30, or 60[Formula: see text]mg/kg. After 16[Formula: see text]h of treatment, the mice were sacrificed for further analysis. APAP administration significantly elevated the serum alanine transferase (ALT) enzyme levels and hepatic myeloperoxidase (MPO) activity when compared with control animals. Baicalin treatment significantly attenuated the elevation of liver ALT levels, as well as hepatic MPO activity in a dose- dependent manner (15-60[Formula: see text]mg/kg) in APAP-treated mice. The strongest beneficial effects of BA were seen at a dose of 30[Formula: see text]mg/kg. BA treatment at 30[Formula: see text]mg/kg after APAP overdose reduced elevated hepatic cytokine (TNF-[Formula: see text] and IL-6) levels, and macrophage recruitment around the area of hepatotoxicity in immunohistochemical staining. Significantly, BA treatment can also decrease hepatic phosphorylated extracellular signal-regulated kinase (ERK) expression, which is induced by APAP overdose. Our data suggests that baicalin treatment can effectively attenuate APAP-induced liver injury by down-regulating the ERK signaling pathway and its downstream effectors of inflammatory responses. These results support that baicalin is a potential hepatoprotective agent.

Baicalin Attenuates IL-17-Mediated Acetaminophen-Induced Liver Injury in a Mouse Model.

BACKGROUND: IL-17 has been shown to be involved in liver inflammatory disorders in both mice and humans. Baicalin (BA), a major compound extracted from traditional herb medicine (Scutellariae radix), has potent hepatoprotective properties. Previous study showed that BA inhibits IL-17-mediated lymphocyte adhesion and downregulates joint inflammation. The aim of this study is to investigate the role of IL-17 in the hepatoprotective effects of BA in an acetaminophen (APAP)-induced liver injury mouse model. METHODS: Eight weeks male C57BL/6 (B6) mice were used for this study. Mice received intraperitoneal hepatotoxic injection of APAP (300 mg/kg) and after 30 min of injection, the mice were treated with BA at a concentration of 30 mg/kg. After 16 h of treatment, mice were killed. Blood samples and liver tissues were harvested for analysis of liver injury parameters. RESULTS: APAP overdose significantly increased the serum alanine transferase (ALT) levels, hepatic activities of myeloperoxidase (MPO), expression of cytokines (TNF-α, IL-6, and IL-17), and malondialdehyde (MDA) activity when compared with the control animals. BA treatment after APAP administration significantly attenuated the elevation of these parameters in APAP-induced liver injury mice. Furthermore, BA treatment could also decrease hepatic IL-17-producing γδT cells recruitment, which was induced after APAP overdose. CONCLUSION: Our data suggested that baicalin treatment could effectively decrease APAP-induced liver injury in part through attenuation of hepatic IL-17 expression. These results indicate that baicalin is a potential hepatoprotective agent.

tPA-MMP-9 Axis Plays a Pivotal Role in Mobilization of Endothelial Progenitor Cells from Bone Marrow to Circulation and Ischemic Region for Angiogenesis.

We examined the role of tissue plasminogen activator- (tPA-) matrix metalloproteinase- (MMP-) 9 in mobilizing endothelial progenitor cells (EPCs) from bone marrow to circulation and critical limb ischemia (CLI) region. Male C57BL/6J mice having been irradiated were categorized into wild-type mice (WT) receiving WT bone marrow cell (BMC) transfusion (group 1), WT mice receiving MMP-9 knockout (MMP-9(-/-)) BMC (group 2), MMP-9(-/-) receiving MMP-9(-/-) BMC (group 3), and MMP-9(-/-) receiving WT BMC (group 4), each of which was subdivided into sham control (SC), CLI, SC-tPA, and CLI-tPA. In groups 1 and 4, by post-CLI 18 h and day 14, circulating EPC (C-kit+/CD31+, Sca-1+/KDR+) levels were highest in CLI-tPA subgroup. In groups 2 and 3, EPC levels did not differ among all subgroups. The EPC levels in bone marrow were higher in groups 2 and 3 than those in groups 1 and 4. By day 14, in animals with CLI, expression levels of proangiogenic factors (CXCR4, SDF-1α, and VEGF) showed similar trends as circulating EPC levels. Moreover, the number of infiltrated neutrophils and macrophages in quadriceps was higher in groups 1 and 4 than groups in 2 and 3. In conclusion, tPA-MMP-9 axis plays a crucial role in EPC mobilization and angiogenesis in experimental CLI.

Chronic activation of NPFFR2 stimulates the stress-related depressive behaviors through HPA axis modulation.

Neuropeptide FF (NPFF) is a morphine-modulating peptide that regulates the analgesic effect of opioids, and also controls food consumption and cardiovascular function through its interaction with two cognate receptors, NPFFR1 and NPFFR2. In the present study, we explore a novel modulatory role for NPFF-NPFFR2 in stress-related depressive behaviors. In a mouse model of chronic mild stress (CMS)-induced depression, the expression of NPFF significantly increased in the hypothalamus, hippocampus, medial prefrontal cortex (mPFC) and amygdala. In addition, transgenic (Tg) mice over-expressing NPFFR2 displayed clear depression and anxiety-like behaviors with hyperactivity in the hypothalamic-pituitary-adrenal (HPA) axis, reduced expression of glucocorticoid receptor (GR) and neurogenesis in the hippocampus. Furthermore, acute treatment of NPFFR2 agonists in wild-type (WT) mice enhanced the activity of the HPA axis, and chronic administration resulted in depressive and anxiety-like behaviors. Chronic stimulation of NPFFR2 also decreased the expression of hippocampal GR and led to persistent activation of the HPA axis. Strikingly, bilateral intra-paraventricular nucleus (PVN) injection of NPFFR2 shRNA predominately inhibits the depressive-like behavior in CMS-exposed mice. Antidepressants, fluoxetine and ketamine, effectively relieved the depressive behaviors of NPFFR2-Tg mice. We speculate that persistent NPFFR2 activation, in particular in the hypothalamus, up-regulates the HPA axis and results in long-lasting increases in circulating corticosterone (CORT), consequently damaging hippocampal function. This novel role of NPFFR2 in regulating the HPA axis and hippocampal function provides a new avenue for combating depression and anxiety-like disorder.

Preactivated and Disaggregated Shape-Changed Platelets Protected Against Acute Respiratory Distress Syndrome Complicated by Sepsis Through Inflammation Suppression.

BACKGROUND: This study tested the hypothesis that preactivated and disaggregated shape-changed platelet (PreD-SCP) therapy attenuates lung injury from acute respiratory distress syndrome (ARDS) induced by 100% oxygen inhalation and complicated by sepsis through peritoneal administration of 1.5 mg/kg lipopolysaccharide (LPS). METHODS: Adult male Sprague-Dawley rats, weighing 325 to 350 g, were randomized into group 1 (normal controls [NC]), group 2 (NC + PreD-SCP [3.0 × 10, intravenous administration]), group 3 (ARDS-LPS), and group 4 (ARDS-LPS + PreD-SCP), and sacrificed by 72 h after ARDS induction. RESULTS: The lung injury score was significantly higher in group 3 than that in other groups, and significantly higher in group 4 than that in groups 1 and 2, whereas the numbers of alveolar sacs and oxygen saturation (%) showed a reversed pattern compared with that of lung injury score among the four groups (all P < 0.0001) without significant difference between groups 1 and 2. The expressions of proinflammatory cells (CD11+, CD14+, CD68+) and proteins (tumor necrosis factor [TNF]-α, nuclear factor [NF]-κB, interleukin [IL]-1ßß, matrix metalloproteinase [MMP]-9, inducible nitric oxide synthase, intercellular adhesion molecule-1) exhibited a pattern identical to the lung injury score. Circulating levels of white blood cell, IL-6, TNF-α, myeloperoxidase and CCL5, and pulmonary protein expressions of oxidative stress (NOX-1/NOX-2, oxidized protein), apoptotic (Bax, cleaved caspase 3/poly (ADP-ribose) polymerase), fibrotic (Smad3, transforming growth factor [TGF]-ß), and DNA damage (γ-H2AX) biomarkers showed an identical pattern, whereas protein expressions of antifibrotic (Smad1/5, bone morphogenetic protein [BMP]-2) and anti-inflammatory (Bcl-2) biomarkers demonstrated an opposite pattern compared with the proinflammatory indices among the four groups (all P < 0.001). CONCLUSIONS: PreD-SCP therapy effectively improved lung injury in ARDS complicated by sepsis.

RANTES mediates kidney ischemia reperfusion injury through a possible role of HIF-1α and LncRNA PRINS.

RANTES (Regulated on activation, normal T-cell expressed and secreted), recruits circulating leukocytes and augments inflammatory responses in many clinical conditions. Inflammatory responses in ischemia-reperfusion injury (IRI) significantly affect the unfavorable outcomes of acute kidney injury (AKI), and that infiltrating immune cells are important mediators of AKI. However, the significance of RANTES in AKI and whether hypoxia-induced LncRNAs are involved in the regulatory process of AKI are not known. Here we show that, in the kidney IRI mice model, significant RANTES expression was observed in renal tubular cells of wild type mice. RANTES deficient (RANTES(-/-)) mice showed better renal function by reducing the acute tubular necrosis, serum creatinine levels, infiltration of inflammatory cells and cytokine expressions compared to wild type. In vitro, we found that RANTES expression was regulated by NF-κB. Further, renal tubular cells showed deregulated LncRNA expression under hypoxia. Among HIF-1α dependent LncRNAs, PRINS (Psoriasis susceptibility-related RNA Gene Induced by Stress) was significantly up regulated in hypoxic conditions and had specific interaction with RANTES as confirmed through reporter assay. These observations show first evidence for RANTES produced by renal tubular cells act as a key chemokine in AKI and HIF-1α regulated LncRNA-PRINS might be involved in RANTES production.

Resveratrol exerts anti-obesity effects in high-fat diet obese mice and displays differential dosage effects on cytotoxicity, differentiation, and lipolysis in 3T3-L1 cells.

Studies on resveratrol in a wide range of concentrations on obese mice and adipose cells are necessary to comprehend its range of diverse and contradictory effects. In this study, we examined the anti-obesity effects of resveratrol on high-fat diet (HFD)-induced obese mice at dosages ranging from 1 to 30 mg/kg treatment for 10 wk. We also evaluated the effects of resveratrol on cytotoxicity, proliferation, adipogenic differentiation, and lipolysis of 3T3-L1 cells at concentrations ranging from 0.03 to 100 µM. In HFD obese mice, resveratrol treatment for 10 wk without decreased calories intake significantly attenuated HFD-induced weight gain in a dose-dependent manner. Resveratrol treatment also protected against HFD-induced lipid deposition in adipose tissues and liver. In cultured 3T3-L1 preadipocytes, high dosage (10 to 100 µM) resveratrol treatment produced cytotoxicity in both preadipocytes and mature adipocytes. In contrast, low concentration resveratrol treatment (1 to 10 µM) significantly inhibited the capacity of 3T3-L1 cells differentiated into mature adipocytes. Low dose resveratrol treatment also downregulated peroxisome proliferator-activated receptor gamma (PPARγ) and perilipin protein expressions in differentiated adipocytes. Additionally, tumor necrosis factor alpha (TNFα)-induced lipolysis was inhibited by low concentration resveratrol treatment in mature adipocytes. At concentrations of 10-100 µM, resveratrol exerted cytotoxicity. In contrast, at concentrations of 1-10 µM resveratrol inhibited adipogenic differentiation in preadipocytes and suppressed lipolysis in mature adipocytes. Our results suggest that resveratrol possessed anti-obesity effects by induction of cytotoxicity at high dosage and that it influences preadipocyte differentiation and mature adipocyte lipolysis at low concentration.

Tiny tweaks, big changes: An alternative strategy to empower ethical culture of human research in anesthesia (A Taiwan Acta Anesthesiologica Taiwanica-Ethics Review Task Force Report).

For this guidance article, the Ethics Review Task Force (ERTF) of the Journal reviewed and discussed the ethics issues related to publication of human research in the field of anesthesia. ERTF first introduced international ethics principles and minimal requirements of reporting of ethics practices, followed by discussing the universal problems of publication ethics. ERTF then compared the accountability and methodology of several medical journals in assuring authors' ethics compliance. Using the Taiwan Institutional Review Board system as an example, ERTF expressed the importance of institutional review board registration and accreditation to assure human participant protection. ERTF presented four major human research misconducts in the field of anesthesia in recent years. ERTF finally proposed a flow-chart to guide journal peer reviewers and editors in ethics review during the editorial process in publishing. Examples of template languages applied in the Ethics statement section in the manuscript are expected to strengthen the ethics compliance of the authors and to set an ethical culture for all the stakeholders involved in human research.

Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment.

High concentrations of adenosine in tumor microenvironments inhibit antitumor cytotoxic lymphocyte responses. Although T cells express inhibitory adenosine A2A receptors (A2AR) that suppress their activation and inhibit immune killing of tumors, a role for myeloid cell A2ARs in suppressing the immune response to tumors has yet to be investigated. In this study, we show that the growth of transplanted syngeneic B16F10 melanoma or Lewis lung carcinoma cells is slowed in Adora2a(f/f)-LysMCre(+/-) mice, which selectively lack myeloid A2ARs. Reduced melanoma growth is associated with significant increases in MHCII and IL12 expression in tumor-associated macrophages and with >90% reductions in IL10 expression in tumor-associated macrophages, dendritic cells (DC), and Ly6C(+) or Ly6G(+) myeloid-derived suppressor cells (MDSC). Myeloid deletion of A2ARs significantly increases CD44 expression on tumor-associated T cells and natural killer (NK) cells. Depletion of CD8(+) T cells or NK cells in tumor-bearing mice indicates that both cell types initially contribute to slowing melanoma growth in mice lacking myeloid A2A receptors, but tumor suppression mediated by CD8(+) T cells is more persistent. Myeloid-selective A2AR deletion significantly reduces lung metastasis of melanomas that express luciferase (for in vivo tracking) and ovalbumin (as a model antigen). Reduced metastasis is associated with increased numbers and activation of NK cells and antigen-specific CD8(+) T cells in lung infiltrates. Overall, the findings indicate that myeloid cell A2ARs have direct myelosuppressive effects that indirectly contribute to the suppression of T cells and NK cells in primary and metastatic tumor microenvironments. The results indicate that tumor-associated myeloid cells, including macrophages, DCs, and MDSCs all express immunosuppressive A2ARs that are potential targets of adenosine receptor blockers to enhance immune killing of tumors.

Insulin renders diabetic rats resistant to acute ischemic stroke by arresting nitric oxide reaction with superoxide to form peroxynitrite.

BACKGROUND: The functions of free radicals on the effects of insulin that result in protection against cerebral ischemic insult in diabetes remain undefined. This present study aims to explain the contradiction among nitric oxide (NO)/superoxide/peroxynitrite of insulin in amelioration of focal cerebral ischemia-reperfusion (FC I/R) injury in streptozotocin (STZ)-diabetic rats and to delineate the underlying mechanisms. Long-Evans male rats were divided into three groups (age-matched controls, diabetic, and diabetic treated with insulin) with or without being subjected to FC I/R injury. RESULTS: Hyperglycemia exacerbated microvascular functions, increased cerebral NO production, and aggravated FC I/R-induced cerebral infarction and neurological deficits. Parallel with hypoglycemic effects, insulin improved microvascular functions and attenuated FC I/R injury in STZ-diabetic rats. Diabetes decreased the efficacy of NO and superoxide production, but NO and superoxide easily formed peroxynitrite in diabetic rats after FC I/R injury. Insulin treatment significantly rescued the phenomenon. CONCLUSIONS: These results suggest that insulin renders diabetic rats resistant to acute ischemic stroke by arresting NO reaction with superoxide to form peroxynitrite.

Hippocampal transcriptional dysregulation after renal ischemia and reperfusion.

Neurological complications contribute largely to the morbidity and mortality in patients with acute renal failure. In order to study pathophysiological complications of renal failure, a murine model of renal ischemia/reperfusion-induced acute kidney injury (AKI) was generated by 60min bilateral ischemia, and followed by 2h or 24h reperfusion (B-60'IRI). Compared to the sham-operated mice, B-60'IRI mice exhibited a significant inflammatory injury to remote brain. We found that serum and brain levels of KC, G-CSF and MCP-1 were significantly increased in B-60'IRI mice after 2h and 24h reperfusion when compared with sham-operated mice. Moreover, B-60'IRI mice exhibited increased numbers of activated microglial cells in the brain, and severe blood-brain barrier (BBB) permeability when compared with the control sham mice. The technology of cDNA microarray and quantitated RT-PCR are used to identify hippocampal genes whose expression is altered in response to AKI in B-60' IRI mice. The initiation of transcriptional abnormality was indicated by the finding that B-60' IRI mice exhibited upregulated mRNA levels of genes involved in inflammation, cell signaling, extracellular matrix and cell-cycle regulation and downregulated mRNA levels of genes involved in transporters, G protein-coupled receptor signaling, cell survival and chaperone. Our data suggest that renal IR contributes to a complicated hippocampal gene irregulation in inflammation and physiological homeostasis.

Lack of interleukin-17 leads to a modulated micro-environment and amelioration of mechanical hypersensitivity after peripheral nerve injury in mice.

Interleukin-17 (IL-17) is involved in a wide range of inflammatory disorders and in recruitment of inflammatory cells to injury sites. A recent study of IL-17 knock-out mice revealed that IL-17 contributes to neuroinflammation and neuropathic pain after peripheral nerve injury. Surprisingly, little is known of micro-environment modulation by IL-17 in injured sites and in pathologically related neuroinflammation and chronic neuropathic pain. Therefore, we investigated nociceptive sensitization, immune cell infiltration, myeloperoxidase (MPO) activity, and expression of multiple cytokines and opioid peptides in damaged nerves of wild-type (IL-17(+/+)) and IL-17 knock-out (IL-17(-/-)) mice after partial sciatic nerve ligation. Our results demonstrated that the IL-17(-/-) mice had less behavioral hypersensitivity after partial sciatic nerve ligation, and inflammatory cell infiltration and pro-inflammatory cytokine (tumor necrosis factor-α, IL-6, and interferon-γ) levels in damaged nerves were significantly decreased, with the levels of anti-inflammatory cytokines IL-10 and IL-13, and expressions of enkephalin, ß-endorphin, and dynorphin were also decreased compared to those in wild-type control mice. In conclusion, we provided evidence that IL-17 modulates the micro-environment at the level of the peripheral injured nerve site and regulates progression of behavioral hypersensitivity in a murine chronic neuropathic pain model. The attenuated behavioral hypersensitivity in IL-17(-/-) mice could be a result of decreased inflammatory cell infiltration to the injured site, resulting in modulation of the pro- and anti-inflammatory cytokine milieu within the injured nerve. Therefore, IL-17 may be a critical component for neuropathic pain pathogenesis and a novel target for therapeutic intervention for this and other chronic pain states.

The essential role of endothelial nitric oxide synthase activation in insulin-mediated neuroprotection against ischemic stroke in diabetes.

BACKGROUND: Stroke patients with diabetes have a higher mortality rate, worse neurologic outcome, and more severe disability than those without diabetes. Results from clinical trials comparing the outcomes of stroke seen with intensive glycemic control in diabetic individuals are conflicting. Therefore, the present study was aimed to identify the key factor involved in the neuroprotective action of insulin beyond its hypoglycemic effects in streptozotocin-diabetic rats with ischemic stroke. METHODS: Long-Evans male rats were divided into three groups (control, diabetes, and diabetes treated with insulin) and subjected to focal cerebral ischemia-reperfusion (FC I/R) injury. RESULTS: Hyperglycemia aggravated FC I/R injuries with an increase in cerebral infarction and neurologic deficits, inhibition of glucose uptake and membrane-trafficking activity of glucose transporter 1, and reduction of Akt and endothelial nitric oxide synthase (eNOS) phosphorylation in the cerebrum. Insulin treatment alleviated hyperglycemia and the symptoms of diabetes in streptozotocin-diabetic rats. Insulin administration also significantly decreased cerebral infarction and neurologic deficits and increased phosphorylation of Akt and eNOS protein in the cerebrum of FC I/R-injured diabetic rats. However, the glucose uptake and membrane trafficking activity of glucose transporter 1 in the cerebrum were not restored by insulin treatment. Coadministration of the eNOS inhibitor, N-iminoethyl-L-ornithine, with insulin abrogated beneficial effects of insulin on cerebral infarct volume and neurologic deficits in FC I/R-injured diabetic rats without affecting the hypoglycemic action of insulin. CONCLUSIONS: These results suggest that eNOS activation is required for the neuroprotection of insulin against ischemic stroke in patients with diabetes.