mRNA Expression Of Pro-inflammatory Markers Research Articles

Abstract P302: EFFECTS OF FINERENONE ON MYOCARDIAL INJURY AND SODIUM ACCUMULATION IN ALDOSTERONE-INFUSED RATS

The aim of this study was to examine the effects of finerenone, a nonsteroidal mineralocorticoid receptor (MR) antagonist, on myocardial injury and sodium accumulation in rats subjected to aldosterone and salt treatment. Finerenone was administered orally at a dose of 10 mg/kg body weight, while aldosterone was given subcutaneously at a rate of 0.75 μg/hr and salt was provided in drinking water at a concentration of 1% NaCl. Aldosterone-infused rats experienced mortality within 30 days, while finerenone-treated rats did not. Echocardiography and gene expression analyses were conducted to evaluate cardiac remodeling and function in rats exposed to salt/aldosterone loading for 4 weeks. The results showed adverse cardiac remodeling and cardiac dysfunction with preserved ejection fraction in these rats. However, finerenone treatment completely prevented cardiac dysfunction and improved cardiac remodeling. Sodium accumulation in the left ventricular tissues was markedly elevated in salt/aldosterone-loaded rats, but significantly reduced in rats treated with finerenone. Furthermore, finerenone treatment significantly reduced gene expression of F4/80, a macrophage marker. In vitro studies with RAW 264.7 cells showed that sodium treatment increased mRNA expression of pro-inflammatory markers and upregulated NFAT5, a transcription factor that regulates genes involved in osmotic stress, while reducing the expression of anti-inflammatory markers. Finerenone treatment suppressed the elevation of pro-inflammatory markers and NFAT5, but did not affect anti-inflammatory gene levels. These findings suggest that finerenone protects against aldosterone-induced myocardial injury by suppressing sodium accumulation and subsequent inflammation, as well as adverse cardiovascular remodeling.

Abstract 12359: Thioridazine Suppresses Endothelial Inflammation and Atherogenesis Through Inhibition of Yap-jnk Signaling Axis

Introduction: Atherosclerosis is a critical public health issue as cardiovascular disease has been on the rise globally for decades. Our previous study found that YAP plays a critical role in endothelial inflammation and atherosclerosis, indicating that YAP is a promising therapeutic target against atherosclerotic vascular diseases. Therefore, we established the drug screening platform, aiming to seek new YAP inhibitors through repurposing FDA-approved drugs. Methods and Results: Drug screening was initially performed by luciferase reporter gene assay. We found that the anti-psychotic drug thioridazine can significantly inhibit the YAP/TAZ activity in human endothelial cells. Thioridazine downregulated the mRNA levels of YAP/TAZ target genes (ANKRD1, CTGF, CYR61) in endothelial cells. Thioridazine also increased the phosphorylation of YAP and inhibited the nuclear translocation of YAP in endothelial cells. Thioridazine suppressed endothelial inflammation, as demonstrated by downregulation of JNK phosphorylation, activity of JNK effector AP-1 as well as mRNA expression of pro-inflammatory markers including JNK target genes. Moreover, we observed that oral administration of thioridazine alleviated the formation of atherosclerotic plaques in ApoE -/- mice on a western diet. Conclusion: Overall, the FDA-approved clinical drug, thioridazine suppresses endothelial inflammation and atherogenesis through inhibition of YAP-JNK signaling pathway. As a novel YAP inhibitor, thioridazine might need further investigation and development for potential use of treating atherosclerotic vascular disease.

Orlistat Mitigates Oxidative Stress-Linked Myocardial Damage via NF-κβ- and Caspase-Dependent Activities in Obese Rats.

Oxidative stress contributes to major complications of obesity. This study intended to identify whether orlistat could mitigate myocardial damage in obese animal models. The tested rats were divided into two groups and fed either with normal chow (n = 6 per group) or with a high-fat diet (HFD) for 6 weeks to induce obesity (n = 12 per group). Obese rats were further subjected to treatment either with distilled water (OB group) or orlistat 10 mg/kg/day (OB + OR group). Key indices of oxidative stress, inflammation, and apoptosis were assessed using an immunohistochemical-based technique and real-time PCR. The OB group showed significant increases of oxidative stress markers (TBARs and PCO), with significant decreases of anti-oxidant markers (Nrf2, SOD, CAT, and GPx). Furthermore, mRNA expression of pro-inflammatory markers (TNF-α and NF-κβ) and pro-apoptosis markers (Bax, Caspase-3, Caspase-8, and Caspase-9) were significantly upregulated in the OB group. Obese rats developed pathological changes of myocardial damages as evidenced by the presence of myocardial hypertrophy and inflammatory cells infiltration. Orlistat dampened the progression of myocardial damage in obese rats by ameliorating the oxidative stress, and by inhibiting NF-κβ pathway and caspase-dependent cell apoptosis. Our study proposed that orlistat could potentially mitigate oxidative stress-linked myocardial damage by mitigating inflammation and apoptosis, thus rationalizing its medical usage.

Curcumin-loaded niosomes downregulate mRNA expression of pro-inflammatory markers involved in asthma: an in vitro study.

Aim: In this study, curcumin was encapsulated in niosomes (Nio-Curc) to increase its effectiveness for the treatment of asthma. Materials & methods: The formulation underwent various physicochemical characterization experiments, an in vitro release study, molecular simulations and was evaluated for in vitro anti-inflammatory activity. Results: Results showed that Nio-Curc had a mean particle size of 284.93±14.27nm, zeta potential of -46.93 and encapsulation efficacy of 99.62%, which demonstrates optimized physicochemical characteristics. Curcumin release in vitro could be sustained for up to 24h. Additionally, Nio-Curc effectively reduced mRNA transcript expression of pro-inflammatory markers; IL-6, IL-8, IL-1β and TNF-α in immortalized human airway basal cell line (BCi-NS1.1). Conclusion: In this study, we have demonstrated that Nio-Curc mitigated the mRNA expression of pro-inflammatory markers in an in vitro study, which could be applied to treatment of asthma with further studies.

479-P: Inhibition of the NLRP3 Inflammasome by MCC950 Accelerates Renal Injury in Diabetes Model

Background: Previous studies suggest that NLRP3 inflammasome activation could have pathological relevance in diabetic kidney disease (DKD). A recent study demonstrated that NLRP3 inhibition in a preventional approach in db/db mice provided reno-protection, potentially via a ROS dependent mechanism. We aimed to examine the therapeutical potential of the NLRP3 inflammasome inhibitor, MCC950 in a more clinically relevant interventional approach in a model of DKD and its impact on renal pathology. Methods: Diabetes was induced by streptozotocin in ApoE-/- mice. A subgroup of control and diabetic mice was treated with MCC950 (5mg/kg/3 times/week). Treatment was commenced after 5 or 9 weeks of diabetes and continued for another 5 or 9 weeks for the assessment of renal gene expression as well its impact on albuminuria and renal morphology, respectively. Results: Diabetes-induced mesangial expansion was further increased in MCC950 treated diabetic mice when compared to untreated diabetic group (18.9±0.6% vs. 16.3±0.7%, respectively). MCC950 treated diabetic mice showed a trend of increased albuminuria when compared to untreated diabetic mice (43±9 vs. 31±5 ug/24hrs, respectively). Gene expression of markers of fibrosis (fibronectin, collagen IV, α-smooth muscle actin and PCNA) and pro-oxidant enzymes (Nox2 and Nox4) were upregulated in diabetic mice compared to nondiabetic controls. These changes were further upregulated in MCC950-treated diabetic mice when compared to untreated diabetic mice. In addition, mRNA expression of pro-inflammatory markers (MCP-1 and IL-6) were increased in untreated diabetic animals as compared to controls and MCC950 treatment did not show additional upregulation of these parameters. Conclusions: The present study indicates that inhibition of the NLRP3 inflammasome by MCC950 after establishment of disease, may not have a reno-protective effect. Our data rather shows an adverse effect of this drug at least in this model of DKD. Disclosure J.A. Østergaard: None. J.C. Jha: None. A. Sharma: None. A. Dai: None. M.E. Cooper: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Mundipharma International. Research Support; Self; Boehringer Ingelheim International GmbH, Novo Nordisk A/S. Speaker’s Bureau; Self; AstraZeneca, Merck Sharp & Dohme Corp., Novartis AG, Sanofi-Aventis, Servier. J.B. de Haan: Research Support; Self; Bayer Inc., Reata Pharmaceuticals. K. Jandeleit-Dahm: None.

A New Rat Model of Pouchitis After Proctocolectomy and Ileal Pouch-Anal Anastomosis Using 2,4,6-Trinitrobenzene Sulfonic Acid

BackgroundPouchitis is a common complication after ileal pouch-anal anastomosis (IPAA) in patients with ulcerative colitis. However, an ideal model remains lacking. Therefore, we aimed to establish an appropriate model resembling human pouchitis. MethodsSprague-Dawley rats were randomly assigned to five groups: TNBS group, DSS group, NS group (following IPAA procedure, administrated with TNBS enema, DSS orally, normal saline enema, respectively), NI group (underwent IPAA), and sham group (underwent switch abdominal surgery). General status, weight change, hematochezia, and fecal scores were recorded. Fecal microbiota were counted under a microscope and analyzed by 16S rRNA gene high-throughput sequencing. Specimens of ileal pouch and small intestine (proximal, mid, distal) were collected to evaluate myeloperoxidase and occludin expression by immunohistochemistry and mRNA expression of pro-inflammatory markers by PCR. ResultsGeneral status, hematochezia, fecal score, and increased mRNA expression of interleukin-6 and TNF-α in the TNBS group were similar to those in the DSS group, whereas the TNBS-induced model displayed a more stable weight change and more serious dysbacteriosis, not only was fecal bacterial diversity reduced, the dominant microbiota was altered. Histopathology scores of the distal small intestine in the TNBS group were lower compared with those in the DSS group (P < 0.05). A significant difference in myeloperoxidase and occludin expression in the small intestine was also detected between the TNBS and DSS groups. ConclusionsOur model mimicked the characteristics of human pouchitis and avoided potential side effects in the small intestine, and thus could be employed for further research.

Lignin-derived low-molecular-weight oxidized lignophenol stimulates AMP-activated protein kinase and suppresses renal inflammation and interstitial fibrosis in high fat diet-fed mice

Excess weight and obesity increase the risk of developing major risk factors for chronic kidney disease. Lignin comprises 20%–30% of the global plant biomass; however, it is not well utilized because of its resistance to chemical and biological degradation. We investigated whether low-molecular-weight oxidized lignophenol (LOLP), a lignin derivative, could alter inflammation and fibrosis in the kidneys of a high-fat diet (HFD)-fed mice. Male mice were divided into three treatment groups: HFD; HFD +0.3% LOLP; and HFD +0.6% LOLP. The control mice (Cont) were fed a low-fat diet. Macrophage kinetics, the degree of fibrosis, the extent of phosphorylation of AMP-activated protein kinase (AMPK), and mRNA expression of proinflammatory mediators in the kidneys were examined. The number of macrophages, the percentage of fibrotic area, and the mRNA expression of proinflammatory markers, TNF-α and Ccl2, and a marker of fibrosis, TGF-β, were significantly higher in the kidneys of mice in the HFD group than those in the Cont group. Conversely, treatment with 0.6% LOLP for 8 weeks significantly suppressed the degree of macrophage infiltration, interstitial fibrotic area, and the increased mRNA expression of proinflammatory and fibrosis markers induced by HFD. In conclusion, LOLP suppressed macrophage infiltration and the increase in fibrotic area, and upregulated AMPK phosphorylation in the kidneys of HFD-fed mice; thus, it may ameliorate HFD-induced kidney injury.

Omega‐3‐rich Isochrysis sp. biomass enhances brain docosahexaenoic acid levels and improves serum lipid profile and antioxidant status in Wistar rats

Isochrysis sp. is a marine microalga, rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The potential use of its biomass as an alternative source of polyunsaturated fatty acids (PUFAs) has not been studied in animal models. Male albino Wistar rats were divided into three groups and treated for 28 days. The rats were fed with (1) standard chow (control group), (2) microalgal biomass rich in EPA and DHA along with standard chow (microalga group), and (3) fish oil that contains equivalent amounts of EPA and DHA along with standard chow (fish oil group). After intervention, biochemical indices, histopathological indices, relative mRNA expression of PUFA genes, antioxidant genes, inflammatory markers, and the fatty acid profile of major tissues were studied. Animals treated with microalgal biomass showed significantly increased serum HDL levels (P < 0.05) and reduced oxidative stress markers with a concomitant decrease in urea and creatinine levels. Oral supplementation of microalgal biomass did not show any toxicity or damage in any major organs. The mRNA expression of PUFA genes was significantly downregulated (P < 0.05) and antioxidant genes were upregulated. Furthermore, the mRNA expression of pro-inflammatory markers was significantly downregulated (P < 0.05) and anti-inflammatory markers were upregulated. Oral supplementation of microalgal biomass improved DHA status in brain and liver. The present study demonstrated that Isochrysis sp. can be used as a safe, alternative food supplement for ω-3 fatty acids. © 2019 Society of Chemical Industry.

SUN-306 ESTABLISHING EQUIVALENT DIABETES IN MALE AND FEMALE NOS3 DEFICIENT MICE RESULTS IN SIMILAR ONSET OF DIABETIC KIDNEY DISEASE

Streptozotocin (STZ)-induced diabetes in male mice lacking endothelial nitric oxide synthase (Nos3-/-) is a widely used model for examining the development of diabetic kidney disease (DKD). Female mice are partially protected from developing STZ-induced diabetes due to their abundance of 17β-estradiol and lack of testosterone, and are not normally used for studies of DKD. In this study, we sought to identify whether male and female Nos3-/- mice with equivalent diabetes develop comparable renal injury, facilitating the potential to use both sexes in studies of DKD. Male and female Nos3-/- mice were made diabetic with 5 or 6 intraperitoneal injections of STZ (55mg/kg/day), respectively. Groups of male and female mice with equivalent diabetes at week 3 after STZ were assessed for diabetic kidney injury (albuminuria, podocyte loss, inflammation, renal function impairment, glomerulosclerosis, tubular injury) at week 8. STZ-treated male and female Nos3-/- mice maintained similar elevated blood glucose levels between weeks 3 and 8 and had equivalent levels of glycated hemoglobin (HbA1c) and hypertension. At week 8, Urine albumin/creatinine levels were increased 8-fold in both diabetic males and females compared to non-diabetic, and were accompanied by a similar (17-20%) loss of podocytes. In diabetic males and females, plasma cystatin-C levels were increased by 33% at week 8, demonstrating similar renal function impairment. Glomerular area of collagen IV immunostaining was also similarly increased by 30% in both genders, signifying equal development of glomerulosclerosis. In addition, kidney mRNA expression of proinflammatory markers (CD68, TNF-α, CCL2), profibrotic markers (TGF-β, Collagen I, Collagen IV, Fibronectin) and KIM-1 were equally elevated in males and females, indicating similar renal inflammation, renal fibrosis and tubular injury. Our study shows that equivalent diabetes induces a similar onset of DKD in male and female Nos3-/- mice, demonstrating it is possible to include males and females together in studies of DKD.

The α/β-hydrolase domain 6 inhibitor WWL70 decreases endotoxin-induced lung inflammation in mice, potential contribution of 2-arachidonoylglycerol, and lysoglycerophospholipids.

Lung inflammation plays a crucial role in the pathogenesis of many respiratory diseases that are in need of new therapeutic strategies. Previously, we showed that inhibition of α/β-hydrolase domain 6 (ABHD6) decreased macrophage activation and exerted anti-inflammatory effects. Therefore, we thought to assess the effects of ABHD6 inhibition in a mouse model of acute lung injury (ALI) induced by intratracheal administration of lipopolysaccharides. ABHD6 inhibition with N-methyl-N-{[3-(4-pyridinyl)phenyl]methyl}-carbamic acid 4'-(aminocarbonyl)(1,1'-biphenyl)-4-yl ester (WWL70) decreases most of the hallmarks of ALI, including neutrophil infiltration, cytokine secretion, and protein extravasation. mRNA expression of proinflammatory markers in the cells recovered in the bronchoalveolar lavage was also decreased. Interestingly, ABHD6 inhibition was more efficient than monoacylglycerol lipase inhibition by 4-nitrophenyl-4-[dibenzo(d)(14)dioxol-5-yl(hydroxy)methyl]piperidine-1-carboxylate. We also studied ABHD6 inhibition on primary alveolar macrophages and neutrophils to explore their potential implication in the effects of ABHD6 inhibition in vivo. Moreover, we quantified by high-performance liquid chromatography-mass spectrometry the levels of reported substrates of ABHD6 [i.e., 2-arachidonoylglycerol (2-AG) and lysophospholipids]. The potential implication of these lipid mediators in the effects of WWL70 was further investigated on primary alveolar macrophages. Taken together, these data support ABHD6 inhibition as an interesting anti-inflammatory strategy in acute lung inflammation and assess the possible contribution of 2-AG and lysophospholipids in the observed effects.-Bottemanne, P., Paquot, A., Ameraoui, H., Alhouayek, M., Muccioli, G. G. The α/β-hydrolase domain 6 inhibitor WWL70 decreases endotoxin-induced lung inflammation in mice, potential contribution of 2-arachidonoylglycerol, and lysoglycerophospholipids.

Long-term treatment with the P2X7 receptor antagonist Brilliant Blue G reduces liver inflammation in a humanized mouse model of graft-versus-host disease

Allogeneic haematopoietic stem cell transplantation (HSCT) is a frequent curative therapy for numerous haematological malignancies. However, HSCT is limited by the occurrence of graft-versus-host disease (GVHD), with current therapies restricted to general immunosuppression. Activation of the P2X7 receptor by extracellular adenosine triphosphate (ATP) causes inflammation and tissue damage in GVHD. Short-term pharmacological blockade of P2X7 has been shown to reduce clinical disease and/or reduce inflammatory markers in allogeneic and humanized mouse models of GVHD. The current study demonstrates that long-term P2X7 blockade by intra-peritoneal injection of Brilliant Blue G (BBG) thrice weekly for up to 10 weeks did not impact human (h) peripheral blood mononuclear cell (PBMC) engraftment, predominantly T cells, in blood at 3 weeks post-hPBMC injection or in spleens at end-point in humanized mice. Histological analysis demonstrated long-term BBG treatment reduced leukocyte infiltration in the livers of humanized mice. Immunohistochemical analysis demonstrated that BBG treatment reduced liver apoptosis. Long-term BBG treatment did not alter clinical disease, mRNA expression of pro-inflammatory markers in tissues or serum human interferon (IFN)-γ concentrations. Therefore, this study demonstrates that P2X7 activation plays a role in GVHD pathogenesis in the livers of humanized mice, supporting a role for this receptor in GVHD development in HSCT recipients.

Gold nanoparticles as cell regulators: beneficial effects of gold nanoparticles on the metabolic profile of mice with pre-existing obesity

BackgroundWe have previously shown that intraperitoneal injection of gold nanoparticles (AuNPs, 20–30 nm) into mice, decreases high-fat diet (HFD) induced weight gain and glucose intolerance, via suppression of inflammatory responses in both fat and liver tissues. This study investigates whether AuNPs provide similar benefit to mice with pre-existing obesity. Male C57BL/6 mice were fed a HFD for 15 weeks. AuNPs (OB-EAu 0.0785 μg/g/day, OB-LAu 0.785 μg/g/day, OB-HAu7.85 μg/g/day, ip) were administered to subgroups of HFD-fed mice over the last 5 weeks. Control group was fed standard chow and administered vehicle injection.ResultsOnly the OB-LAu group demonstrated significant weight loss (12%), while all AuNP treated groups showed improved glycaemic control and reduced blood lipid levels. In the fat tissue, mRNA expression of pro-inflammatory markers were unchanged following AuNP treatment, while glucose and lipid metabolic markers were improved in OB-LAu and OB-HAu mice. In the liver, AuNP treatment downregulated inflammatory markers and improved lipid metabolic markers, with marked effects in OB-EAu and OB-LAu groups.ConclusionsAuNP treatment can improve glucose and fat metabolism in mice with long-term obesity, however weight loss was only observed in a single specific dose regime. AuNP therapy is a promising new technology for managing metabolic disorders in the obese.

Standardized extract of Zingiber zerumbet suppresses LPS-induced pro-inflammatory responses through NF-κB, MAPK and PI3K-Akt signaling pathways in U937 macrophages

BackgroundZingiber zerumbet rhizome has been used as spices and in traditional medicine to heal various immune-inflammatory related ailments. Although the plant was reported to have potent anti-inflammatory and immunosuppressive properties by several studies, the molecular mechanisms underlying the effects have not been well justified. PurposeThe study was carried out to investigate the molecular mechanisms underlying the anti-inflammatory properties of the standardized 80% ethanol extract of Z. zerumbet through its effect on mitogen-activated protein kinase (MyD88)-dependent nuclear factor-kappa B (NF-кB), mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/Akt (PI3K-Akt) signaling pathways in lipopolysaccharide (LPS)-induced U937 human macrophages. MethodsStandardization of the 80% ethanol extract of Z. zerumbet was performed by using a validated reversed-phase HPLC method, while LC-MS/MS was used to profile the secondary metabolites. The release of pro-inflammatory markers, tumor necrosis factor (TNF)-α, interleukin (IL)-1β and prostaglandin E2 (PGE2) was evaluated by enzyme-linked immunosorbent assay (ELISA), while the Western blot technique was executed to elucidate the expression of mediators linked to MyD88-dependent respective signaling pathways. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay was carried out to quantify the relative gene expression of cyclooxygenase (COX)-2 and pro-inflammatory mediators at the transcriptional level. ResultsThe quantitative and qualitative analyses of Z. zerumbet extract showed the presence of several compounds including the major chemical marker zerumbone. Z. zerumbet extract suppressed the release of pro-inflammatory mediators, COX-2 protein expression and downregulated the mRNA expression of pro-inflammatory markers. Z. zerumbet-treatment also blocked NF-κB activation by preventing the phosphorylation of IKKα/β and NF-κB (p65) as well as the phosphorylation and degradation of IκBα. Z. zerumbet extract concentration-dependently inhibited the phosphorylation of respective MAPKs (JNK, ERK, and p38) as well as Akt. Correspondingly, Z. zerumbet extract suppressed the upstream signaling adaptor molecules, TLR4 and MyD88 prerequisite for the NF-κB, MAPKs, and PI3K-Akt activation. ConclusionThe findings suggest that Z. zerumbet has impressive role in suppressing inflammation and related immune disorders by inhibition of various pro-inflammatory markers through the imperative MyD88-dependent NF-κB, MAPKs, and PI3K-Akt activation.

Alternative microglial activation is associated with cessation of progressive dopamine neuron loss in mice systemically administered lipopolysaccharide

Inflammation arising from central and/or peripheral sources contributes to the pathogenesis of multiple neurodegenerative diseases including Parkinson's disease (PD). Emerging data suggest that differential activation of glia could lead to the pathogenesis and progression of PD. Here, we sought to determine the relationship between lipopolysaccharide (LPS) treatment, loss of dopaminergic neurons and differential activation of glia. Using a model of repeated injections with LPS (1mg/kg, i.p. for 4days), we found that LPS induced a 34% loss of dopamine neurons in the substantia nigra 19days after initiation of treatment, but no further cell loss was observed at 36days. LPS induced a strong pro-inflammatory response with increased mRNA expression of pro-inflammatory markers, including tumor necrosis factor-α (4.8-fold), inducible nitric oxide synthase (2.0-fold), interleukin-1 beta (8.9-fold), interleukin-6 (10.7-fold), and robust glial activation were observed at 1day after final dose of LPS. These pro-inflammatory genes were then reduced at 19days after treatment, when there was a rise in the anti-inflammatory genes Ym1 (1.8-fold) and arginase-1 (2.6-fold). Additionally, 36days after the last LPS injection there was a significant increase in interleukin-10 (2.1-fold) expression. The qPCR data results were supported by protein data, including cytokine measurements, western blotting, and immunofluorescence in brain microglia. Taken together, these data demonstrate that progressive neurodegeneration in the substantia nigra following LPS is likely arrested by microglia shifting to an anti-inflammatory phenotype. Thus, strategies to promote resolution of neuroinflammation may be a promising avenue to slow the progressive loss of dopamine neurons in PD.

A gold bullet to treat obesity related metabolic disorders

Objectives: Obesity is associated with chronic low grade inflammation. Adipose resident macrophages (ATMs) in obese individuals are known to produce increased levels of proinflammatory cytokines. We have previously shown that intraperitoneal (IP) injection of 20—30 nm gold nanoparticles (AuNPs) in lean mice can induce significant fat loss along with reduced adiposetissue-derived, tumour necrosis factor (TNF) mRNA expression without a concomitant change in ATM number. This study aimed to investigate whether AuNPs also reduce fat mass and metabolic disorders in mice fed a high-fat diet (HFD). Method: Male C57BL/6 mice (6 weeks) were fed a HFD with/without daily IP injection of AuNPs (LAu 0.785 g/g/d, HAu 7.85 g/g/d) for 9 weeks. Control group was fed standard chow with vehicle injection. IP-glucose tolerance test (IP-GTT) was performed a week before the endpoint. mRNA expression of pro-inflammatory cytokines and fat metabolic markers were determined in retroperitoneal fat and liver using real-time PCR. Results: At the end point, body weight was similar between AuNP-treated and non-treated HFD-fed mice, but adiposity and liver weight were reduced by AuNP treatment. Blood glucose levels during IPGTT were significantly lower in AuNP-treated mice compared to the non-treated HFD-fed group. In addition, AuNP treatment improved plasma free fatty acid, triglyceride and cholesterol levels in HFD-fed mice. In the fat tissue, mRNA expression of pro-inflammatory markers was significantly reduced by AuNP treatment, including TNF . In the liver, AuNP treatment downregulated inflammatory markers and improved lipid metabolic markers. Interestingly, liver mRNA expression of glucose transporter-4 was upregulated by AuNP treatment. Conclusion: AuNPs treatment in HFD-fed mice prevented their development of glucose intolerance with improved fat metabolism and reduced inflammation in both the fat and liver. Our results therefore support the potential development of AuNPs as a new therapeutic strategy for obesity related metabolic disorders.

The association between obesity and excessive daytime sleepiness in Australian workers

Objectives: Obesity is associated with chronic low grade inflammation. Adipose resident macrophages (ATMs) in obese individuals are known to produce increased levels of proinflammatory cytokines. We have previously shown that intraperitoneal (IP) injection of 20—30 nm gold nanoparticles (AuNPs) in lean mice can induce significant fat loss along with reduced adiposetissue-derived, tumour necrosis factor (TNF) mRNA expression without a concomitant change in ATM number. This study aimed to investigate whether AuNPs also reduce fat mass and metabolic disorders in mice fed a high-fat diet (HFD). Method: Male C57BL/6 mice (6 weeks) were fed a HFD with/without daily IP injection of AuNPs (LAu 0.785 g/g/d, HAu 7.85 g/g/d) for 9 weeks. Control group was fed standard chow with vehicle injection. IP-glucose tolerance test (IP-GTT) was performed a week before the endpoint. mRNA expression of pro-inflammatory cytokines and fat metabolic markers were determined in retroperitoneal fat and liver using real-time PCR. Results: At the end point, body weight was similar between AuNP-treated and non-treated HFD-fed mice, but adiposity and liver weight were reduced by AuNP treatment. Blood glucose levels during IPGTT were significantly lower in AuNP-treated mice compared to the non-treated HFD-fed group. In addition, AuNP treatment improved plasma free fatty acid, triglyceride and cholesterol levels in HFD-fed mice. In the fat tissue, mRNA expression of pro-inflammatory markers was significantly reduced by AuNP treatment, including TNF . In the liver, AuNP treatment downregulated inflammatory markers and improved lipid metabolic markers. Interestingly, liver mRNA expression of glucose transporter-4 was upregulated by AuNP treatment. Conclusion: AuNPs treatment in HFD-fed mice prevented their development of glucose intolerance with improved fat metabolism and reduced inflammation in both the fat and liver. Our results therefore support the potential development of AuNPs as a new therapeutic strategy for obesity related metabolic disorders.

Short communication: Effects of trans fatty acids on markers of inflammation in bovine mammary epithelial cells

Trans fatty acids (tFA) contribute to inflammation. The objective was to investigate the effects of tFA on mRNA expression of proinflammatory markers in cultured bovine mammary epithelial cells (MAC-T cell line). Bovine mammary epithelial cells were grown in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum. Cells were then subcultured in a medium lacking fetal bovine serum, to which incremental concentrations (up to 90μM) of elaidic acid (trans-9 C18:1) or linoleidic acid (trans-9, trans-12 C18:2) were added. Bovine serum albumin (fatty acid-free) solutions were added and cells were collected at specific time points over 48h. Then, RNA was extracted and converted to complementary DNA for quantitative real-time PCR analysis of proinflammatory gene expression. Presence of elaidic acid caused increases in mRNA expression of interleukin (IL)-1β (3.4-fold; dose-independently over a 6-h period) and intercellular adhesion molecule (ICAM)-1 (up to 1.4-fold) relative to that for cells treated with no tFA, whereas expression of IL-6 and IL-8 was reduced 0.75- and 0.85-fold, respectively. Presence of linoleidic acid reduced mRNA expression of IL-6 and IL-8 relative to that for control (0.95- and 0.87-fold, respectively). Trans mono- and dienoic fatty acids upregulated mRNA expression of IL-1β and ICAM-1, whereas expression of IL-6 and IL-8 was downregulated in MAC-T cells. Because these genes are ultimately involved in inflammation, elaidic or linoleidic acid, either directly fed or formed in the rumen during biohydrogenation, may alter the risk for mastitis in vivo.

1-(2′,4′-dichlorophenyl)-6-methyl-N-cyclohexylamine-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide, a novel CB2 agonist, alleviates neuropathic pain through functional microglial changes in mice

Neuropathic pain is a devastating neurological disease that seriously affects quality of life in patients. The mechanisms leading to the development and maintenance of neuropathic pain are still poorly understood. However, recent evidence points towards a role of spinal microglia in the modulation of neuronal mechanisms. In this context, cannabinoids are thought to modulate synaptic plasticity as well as glial functions. Here, we have investigated the effect of chronic treatment with a selective agonist of cannabinoid type 2 receptor (CB2), 1-(2′,4′-dichlorophenyl)-6-methyl-N-cyclohexylamine-1,4-dihydroindeno[1,2-c]pyrazole-3 carboxamide (NESS400), on pain thresholds in the spared nerve injury (SNI) model in the mouse and on the distribution and activation of spinal microglia. Repeated treatment with NESS400 (4 mg/kg) significantly alleviated neuropathic mechanical allodynia and thermal hyperalgesia. In the dorsal horn (L4–L6) of neuropathic mice microglia activation (quantification of the length of microglial processes) and astrocytosis were associated with CB2 receptor over-expression on both cell types. Treatment with NESS400 significantly reduced the number of hypertrophic microglia while leaving microglial cell number unaffected and reduced astrogliosis. Moreover, prolonged administration of NESS400 reduced mRNA expression of pro-inflammatory markers and enhanced anti-inflammatory marker gene expression in dorsal horn extracts. In conclusion, we show that selective CB2 receptor stimulation prevents thermal hyperalgesia, alleviates mechanical allodynia and facilitates the proliferation of anti-inflammatory microglial phenotype in the ipsilateral dorsal horn of the spinal cord in SNI mice.