Combination of conjugated linoleic acid with fish oil prevents age-associated bone marrow adiposity in C57Bl/6J mice

The inverse relationship between fat in bone marrow and bone mass in the skeleton of aging subjects is well known. However, there is no precise therapy for the treatment of bone marrow adiposity. We investigated the ability of conjugated linoleic acid (CLA) and fish oil (FO), alone or in combination, to modulate bone loss using 12 months old C57Bl/6J mice fed 10% corn oil diet as control or supplemented with 0.5% CLA or 5% FO or 0.5% CLA+5% FO for 6 months. We found, CLA-fed mice exhibited reduced body weight, body fat mass (BFM) and enhanced hind leg lean mass (HLLM) and bone mineral density (BMD) in different regions measured by dual energy x-ray absorptiometry (DXA); however, associated with fatty liver and increased insulin resistance; whereas, FO fed mice exhibited enhanced BMD, improved insulin sensitivity, with no changes in BFM and HLLM. Interestingly, CLA+FO fed mice exhibited reduced body weight, BFM, peroxisome proliferator-activated receptor gamma and cathepsin K expression in bone marrow with enhanced BMD and HLLM. Moreover, CLA+FO supplementation reduced liver hypertrophy and improved insulin sensitivity with remarkable attenuation of bone marrow adiposity, inflammation and oxidative stress in aging mice. Therefore, CLA with FO combination might be a novel dietary supplement to reduce fat mass and improve BMD.     

Induction of antitumor activities in spleen cells from mice and rats activated with lipopolysaccharide immobilized on beads

Summary Lipopolysaccharides (LPS) were coupled to polystyrene beads in order to apply the LPS without toxicity. The antitumor activity of the LPS-immobilizing beads was studied in experiments in vitro and in vivo. In vitro studies showed that spleen cells from C3H/HeN mice stimulated by beads immobilizing LPS fromEscherichia coli produced cytolytic activity as strong as that of lymphokine-activated killer (LAK) cells. Spleen cells from Sprague-Dawley rats stimulated by beads immobilizing LPS fromSalmonella minnesota produced cytolytic activity stronger than that of LAK cells. However, spleen cells stimulated by beads immobilizing each component of the LPS separately could not induce cytolysis. Contact stimulation, even for a brief period, sufficed for cytolytic activity, and was enhanced by culture for 48–72 h. Through in vivo studies, the suppression of tumor growth and a prolongation of the survival time were observed in tumorbearing mice injected with spleen cells activated by beads immobilizing LPS fromE.coli, and in mice injected with LAK cells. The effect of the activated spleen cells was stronger than that of the LAK cells. In rats bearing metastatic tumors, spleen cells activated by beads immobilizing LPS fromS.minnesota suppressed lung metastases more strongly than did LAK cells. These findings indicate that LPS immobilized by beads induced killer cells more strongly than interleukin-2. Ex vivo immunomodulation with LPS-immobilizing beads can be applied usefully as an anticancer treatment.     

Loss of smelling is an early marker of aging and is associated with inflammation and DNA damage in C57BL/6J mice

Olfactory dysfunction is a prevalent symptom and an early marker of age-related neurodegenerative diseases in humans, including Alzheimer's and Parkinson's Diseases. However, as olfactory dysfunction is also a common symptom of normal aging, it is important to identify associated behavioral and mechanistic changes that underlie olfactory dysfunction in nonpathological aging. In the present study, we systematically investigated age-related behavioral changes in four specific domains of olfaction and the molecular basis in C57BL/6J mice. Our results showed that selective loss of odor discrimination was the earliest smelling behavioral change with aging, followed by a decline in odor sensitivity and detection while odor habituation remained in old mice. Compared to behavioral changes related with cognitive and motor functions, smelling loss was among the earliest biomarkers of aging. During aging, metabolites related with oxidative stress, osmolytes, and infection became dysregulated in the olfactory bulb, and G protein coupled receptor-related signaling was significantly down regulated in olfactory bulbs of aged mice. Poly ADP-ribosylation levels, protein expression of DNA damage markers, and inflammation increased significantly in the olfactory bulb of older mice. Lower NAD⁺ levels were also detected. Supplementation of NAD⁺ through NR in water improved longevity and partially enhanced olfaction in aged mice. Our studies provide mechanistic and biological insights into the olfaction decline during aging and highlight the role of NAD⁺ for preserving smelling function and general health.     

慢性PM2.5暴露对C57BL/6J小鼠肺组织炎症和NLRP3炎性小体活性的影响

目的 研究慢性PM2.5暴露对小鼠肺炎症和NLRP3炎性小体活性的影响,为防治PM2.5所致肺损伤提供新靶点。方法 雄性C57BL/6J小鼠通过不同剂量气管滴注法进行PM2.5染毒,剂量为2,10mg/(kg·bw),对照组小鼠滴注生理盐水。小鼠连续滴注20次,每3d染毒1次后,取血和肺组织。三组小鼠进行血细胞计数;用免疫荧光染色法检测肺组织巨噬细胞水平;用试剂盒测定肺组织中白细胞介素(interleukin,IL)-1β,IL-18水平及caspase-1活性;用实时定量PCR法检测肺组织NLRP3炎性小体相关mRNA表达水平。结果 两个剂量PM2.5染毒均能明显降低单核细胞百分比(P<0.01),增加中性粒白细胞百分比(P<0.01);导致肺炎症发生;增加肺组织caspase-1活性(P<0.01)及NLRP3和ASC的mRNA表达(P<0.01)。与对照组相比,两个剂量组小鼠肺组织IL-1β和IL-18水平均显著增高(P<0.01)。结论 慢性PM2.5暴露可能通过激活肺组织NLRP3炎性小体导致肺炎症发生。     

Experimental and finite element analysis of strains induced by axial tibial compression in young-adult and old female C57Bl/6 mice

Axial compression of the mouse tibia is used to study strain-adaptive bone (re)modeling. In some studies, comparisons between mice of different ages are of interest. We characterized the tibial deformation and force–strain relationships in female C57Bl/6 mice at 5-, 12- and 22-months age. A three-gauge experimental method was used to determine the strain distribution at the mid-diaphysis, while specimen-specific finite element analysis was used to examine strain distribution along the tibial length. The peak strains in the tibial mid-diaphyseal cross-section are compressive and occur at the postero-lateral apex. The magnitudes of these peak compressive strains are 1.5 to 2 times those on the opposite, antero-medial face (a site often used for strain gauge placement). For example, −10 N force applied to a 5-months old mouse engenders a peak compressive strain of −2800 µε and a tensile strain on the antero-medial face of +1450 µε. The orientation of the neutral axis at the mid-diaphysis did not differ with age (p=0.46), indicating a similar deformation mode in young and old tibiae. On the other hand, from 5- to 22-months there is a 25% reduction in cortical thickness and moment of inertia (p<0.05), resulting in significantly greater tibial strain magnitudes in older mice for equivalent applied force (p<0.05). We conclude that comparisons of tibial loading responses in young-adult and old C57Bl/6 tibiae are facilitated by similar deformation pattern across ages, but that modest adjustment of force levels is required to engender matching peak strains.     

Brain Diurnal Levels of Adenosine 3', 5' Cyclic Monophasphate in C57 Bl/6 and Balb/C Mice

Adenosine 3', 5'-cyclic monophosphate (cAMP) was measured in whole brain of two inbred strains of mice (BALB/C and C57 BL/6) submitted to a lighting schedule consisting of 12 hr light (0700-1900) and 12 hr darkness (1900-0700). Different mean levels of cAMP were found in each strain. Furthermore, statistical analysis of diurnal brain cAMP fluctuations showed different nycthemeral rhythms in both strains. BALB/C was mainly characterized by the presence of very significant 0600 and 0800 harmonics and C57 BL/6 by an orthophase around 1700 hr.

Because both strains were studied under the same experimental conditions of light, temperature and food availability, these factors cannot account for the observed differences, which were thus tentatively interpreted in terms of genetic regulatory processes.     

Deficient E-cadherin adhesion in C57BL/6J-Min/+ mice is associated with increased tyrosine kinase activity and RhoA-dependent actomyosin contractility

The Min/+ mouse is a model for APC-dependent colorectal cancer (CRC). We showed that tumorigenesis in this animal was associated with decreased E-cadherin adhesion and increased epidermal growth factor receptor (Egfr) activity in the non-tumor intestinal mucosa. Here, we tested whether these abnormalities correlated with changes in the actin cytoskeleton due to increased Rho-ROCK signaling. We treated Apc+/+ (WT) littermate small intestine with EGTA, an inhibitor of E-cadherin, and with LPA, an RhoA activator; both induced effects on adhesion and kinase activity that mimicked the Min/+ phenotype. GTP-bound Rho was increased in Min/+ enterocytes relative to WT. Since RhoA activity is associated with actomyosin contractility, markers of this signaling cascade were assessed including phosphorylated myosin light chain (MLC), cofilin, Pyk2, Src, and MAPK kinases. The increased actomyosin contractility characterizing Min/+ intestinal tissue was suppressed by the ROCK inhibitor, Y27632, but was inducible in the WT by EGTA or LPA. Finally, ultrastructural imaging revealed changes consistent with actomyosin contractility in Min/+ enterocytes. Thus, the positive regulation of E-cadherin adhesion provided by Apc+ in vivo allows proper negative regulation of Egfr, Src, Pyk2, and MAPK, as well as RhoA activities.     

C1q/TNF-related protein-3 (CTRP-3) attenuates lipopolysaccharide (LPS)-induced systemic inflammation and adipose tissue Erk-1/-2 phosphorylation in mice in vivo

Background

The C1q/TNF-related proteins comprise a growing family of adiponectin paralogous proteins. CTRP-3 represents a novel adipokine with strong expression in adipose tissue and was shown to inhibit chemokine and cytokine release in adipocytes and monocytes in vitro. The aim of the study was to gain the proof of principle that CTRP-3 is a potent anti-inflammatory adipokine in vivo.

Methods

C57BL/6N mice were treated intraperitoneally (i.p.) with bacterial lipopolysaccharide (LPS) for 2 h. The effects of a 30 min pre-treatment with CTRP-3 i.p. or intravenously (i.v.) on systemic and on epididymal, perirenal and subcutaneous adipose tissue inflammation was analyzed via real-time RT-PCR, ELISA and Western blot analysis.

Results

LPS (1 μg i.p.) significantly increased serum IL-6 and MIP-2 levels as well as epididymal adipose tissue expression of IL-6 and MIP-2 in mice, whereas CTRP-3 (10 μg i.p.) alone or PBS (i.p.) had no effect. Pre-treatment of mice by CTRP-3 i.p. prior to LPS application significantly attenuated LPS-induced cytokine levels but had no effect on adipose tissue cytokine mRNA expression. In contrast to i.p. application of CTRP-3, systemic i.v. application was not sufficient to inhibit LPS-induced cytokine levels or mRNA tissue expression. CTRP-3 given i.p. significantly attenuated LPS-induced phosphorylation of Erk-1/-2 in inguinal adipose tissue.

Conclusion

The present study shows the proof of principle that the novel adipokine CTRP-3 is a potent inhibitor of LPS-induced systemic inflammation and LPS-induced signaling in adipose tissue in vivo.     

Bordetella pertussis respiratory infection in C57B1/6 and Balb/c mice: Pathophysiology and immune responses

A virulent clone of Bordetella pertussis, injected intranasally into C57B1/6 or Balb/c mice, induced a respiratory tract infection that mimicked the infectious process of whooping cough. The density of the inoculum influenced the kinetics of in vivo bacterial growth, as well as the associated leucocytosis, which were of equivalent intensity in both strains of mice. Convalescing mice became resistant to re-infection, but not to the effects of the leucocytosis-promoting factor of the pertussis toxin. Prominent immune response, associated with acquired resistance, was a delayed type hypersensitivity (DTH) reaction, the intensity of which depended upon the genotype of the mice when the eliciting antigen contained the pertussis toxin in a biologically active form. Intranasal infection of congenic mice may represent an improved quantitative test for reproducible measurement of virulence and immunogenicity of B. pertussis.     

Comparative analysis of clinics,pathologies and immune responses in BALB/c and C57BL/6 mice infected with Streptobacillus moniliformis

Streptobacillus (S.) moniliformis is a rat-associated zoonotic pathogen that occasionally causes disease in other species. We investigated the working hypothesis that intranasal infection might lead to different immune responses in C57BL/6 and BALB/c mice associated with distinct pathologies. This study confirmed with 75% mortality the known high susceptibility of C57BL/6 mice to Streptobacillus moniliformis infection in comparison to BALB/c mice which did not develop signs of disease. Main pathologies in C57BL/6 mice were purulent to necrotizing lymphadenitis and pneumonia. Significant seroconversion was recorded in surviving mice of both strains. Differentiation of IgG-subclasses revealed mean ratios of IgG2b to IgG1 below 0.5 in sera of all mice prior to infection and of BALB/c mice post infection. In contrast, C57BL/6 mice had a mean IgG2b/IgG1 ratio of 2.5 post infection indicating a Th1 immune response in C57BL/6 versus a Th2 response in BALB/c mice. Evaluation of different sentinel systems revealed that cultural and serological investigations of these animals might not be sufficient to detect infection. In summary, an intranasal S. moniliformis infection model in C57BL/6 mice leading to purulent to necrotizing inflammations in the lung, the lymph nodes and other organs associated with a Th1 immune response is described.     

Macrophage cholesterol efflux to free apoprotein A-I in C3H and C57BL/6 mice

Cholesterol efflux from peritoneal macrophages of mice C57BL/6 susceptible and C3H resistant to atherosclerosis was compared, using apoprotein A-I as acceptor. The elicited macrophages were labeled with 3H-cholesterol and cholesterol enriched by incubation for 24 h with acetylated LDL. After incubation for 6 or 24 h, 3H-cholesterol efflux to free apoA-I (10 microg/ml) was significantly higher with macrophages derived from C3H mice compared to C57BL/6 mice. The cells were also pretreated with 0.3-0.45 mM cyclic AMP, 10 microM 9-cis-retinoic acid or 10 microM 22(R)-hydroxycholesterol, RXR and LXR ligands. Treatment with cyclic AMP, RXR, or LXR ligands, resulted in enhancement of 3H-cholesterol efflux in both strains. Under all conditions, 3H-cholesterol efflux was significantly higher in C3H compared to C57BL/6 macrophages. In conclusion, the higher cholesterol efflux from C3H macrophages could contribute toward the resistance of this strain to diet-induced atherosclerosis despite hypercholesterolemia.     

The ACE inhibitors enalapril and captopril modulate cytokine responses in Balb/c and C57Bl/6 normal mice and increase CD4CD103CD25 splenic T cell numbers

Increasing evidence implies beneficial effects of angiotensin-converting enzyme (ACE) inhibitors beyond those of their original indications to control hypertension. One of the most attractive non-hemodynamic properties of ACE inhibitors is their ability to regulate cytokine production. The mechanism(s) underlying the role of ACE inhibitors on cytokine synthesis are not well understood but they have traditionally been attributed to the inhibition of angiotensin (Ang) II formation. In fact, it has been extensively demonstrated that ACE inhibitors decrease Ang II-induced production of proinflammatory cytokines and chemokines. However, it is not well described if inhibition of endogenous Ang II generation by ACE inhibitors modulates systemic cytokine production in mice. To verify that, in this work, we investigated the effects of treatment with the ACE inhibitors enalapril and captopril on cytokine synthesis in C57Bl/6 and Balb/c mice. Our results show that enalapril up regulates IL-10 produced by splenocytes from Balb/c and C57Bl/6 mice and captopril increased it only in Balb/c mice. Furthermore, CD4⁺CD103⁺ presented increased IL-10 production after enalapril treatment. Enalapril as well as captopril short-term treatment enhanced IL-2 synthesis in Balb/c mice. Besides, enhanced IL-2 and IL-10 levels correlates with increased CD4⁺CD103⁺CD25^negative T cells numbers in spleens from enalapril-treated mice.     

Preserved glucose tolerance in high-fat-fed C57BL/6 mice transplanted with PPARgamma-/-, PPARdelta-/-, PPARgammadelta-/-, or LXRalphabeta-/- bone marrow

Macrophage lipid metabolism and inflammatory responses are both regulated by the nuclear receptors PPAR and LXR. Emerging links between inflammation and metabolic disease progression suggest that PPAR and LXR signaling may alter macrophage function and thereby impact systemic metabolism. In this study, the function of macrophage PPAR and LXR in Th1-biased C57BL/6 mice was tested using a bone marrow transplantation approach with PPARgamma(-/-), PPARdelta(-/-), PPARgammadelta(-/-), and LXRalphabeta(-/-) cells. Despite their inhibitory effects on inflammatory gene expression, loss of PPARs or LXRs in macrophages did not exert major effects on obesity or glucose tolerance induced by a high-fat diet. Treatment with rosiglitazone effectively improved glucose tolerance in mice lacking macrophage PPARgamma, suggesting that cell types other than macrophages are the primary mediators of the anti-diabetic effects of PPARgamma agonists in our model system. C57BL/6 macrophages lacking PPARs or LXRs exhibited normal expression of most alternative activation gene markers, indicating that macrophage alternative activation is not absolutely dependent on these receptors in the C57BL/6 background under the conditions used here. These studies suggest that genetic background may be an important modifier of nuclear receptor effects in macrophages. Our results do not exclude a contribution of macrophage PPAR and LXR expression to systemic metabolism in certain contexts, but these factors do not appear to be dominant contributors to glucose tolerance in a high-fat-fed Th1-biased bone marrow transplant model.     

Regulation of adipose tissue inflammation and systemic metabolism in murine obesity by polymer implants loaded with lentiviral vectors encoding human interleukin-4

Dysfunctional adipose tissue plays a central role in the pathogenesis of the obesity-related metabolic disease, including type 2 diabetes. Targeting adipose tissue using biopolymer implants is a novel therapeutic approach for metabolic disease. We transplanted porous poly(lactide-co-glycolide) (PLG) implants coated with human interleukin-4 (hIL-4)-expressing lentivirus into epididymal white adipose tissue (eWAT) of mice fed a high-fat diet. Tissue and systemic inflammation and metabolism were studied with flow cytometry, immunohistochemistry, quantitative real-time polymerase chain reaction, adipose tissue histology, and in vivo glucose tolerance testing at 2 and 10 weeks of a high-fat diet. PLG implants carrying hIL-4-expressing lentivirus implanted into epididymal white adipose tissue of mice-regulated adipose tissue inflammation, including increased CD3⁺CD4⁺ T-cell frequency, increased eWAT adipocyte hypertrophy, and decreased FASN and ATGL expression, along with reduced fasting blood glucose levels. These effects were observed in early obesity but were not maintained in established obesity. Local delivery of bioimplants loaded with cytokine-expressing lentivirus vectors to adipose tissue influences tissue inflammation and systemic metabolism in early obesity. Further study will be required to show more durable metabolic effects. These data demonstrate that polymer biomaterials implanted into adipose tissue have the potential to modulate local tissue and systemic inflammation and metabolism.     

Short amyloid-beta immunogens show strong immunogenicity and avoid stimulating pro-inflammatory pathways in bone marrow-derived dendritic cells from C57BL/6J mice in vitro

Amyloid beta peptide 1-15 (Aβ1-15) and its derivatives have attracted the attention of the scientific community as candidate vaccines for Alzheimer's disease (AD) immunotherapy. Recent studies suggested that Aβ1-42 modulated the immune system by inducing pro-inflammatory dendritic cells (DCs) with reduced antigen-presenting function. However, it remains elusive how Aβ1-15 impacts DCs function. We therefore investigated the modulation by short Aβ peptides of DCs from C57Bl/6J mice. Two new immunogens, a tandem repeat of two-lysine-linked Aβ1-15 sequences with or without an addition of a RGD motif, were tested. Chemotaxis, endocytosis, antigen presenting function and producing cytokines were measured. Both peptides increased migration/endocytosis of immature DCs and MHC II molecule expression/alloreactive T cell activation in TNF-α-matured DCs. In addition, they exhibited decreased production of Th1/Th2 cytokines and pro-inflammatory cytokines. Overall, the two peptides demonstrated strong immunogenicity but did not stimulate pro-inflammatory pathways. These results support the use of short Aβ immunogens in AD immunotherapy.     

Ergothioneine and melatonin attenuate oxidative stress and protect against learning and memory deficits in C57BL/6J mice treated with D-galactose

Abstract

Male C57BL/6J mice treated with D-galactose (DG) were used to examine the effects of ergothioneine (EGT), melatonin (MEL), or their combination (EGT+MEL) on learning and memory abilities. The mice were divided into five groups and injected subcutaneously with DG (0.3 mL of 1% DG/mouse) except for group 1 (normal controls). Group 3 was orally supplemented with EGT [0.5 mg/kg body weight (bw)], group 4 with MEL (10 mg/kg bw, p.o.), and group 5 with EGT+MEL. EGT and MEL were provided daily for 88 days, while DG was provided between days 7 to 56. Active avoidance task and Morris water-maze task were used to evaluate learning and memory abilities. DG treatment markedly increased escape latency and decreased the number of avoidance in the active avoidance test, whereas EGT and MEL alone significantly improved the performance. DG also impaired the learning and memory abilities in the water-maze task, and EGT and MEL alone also significantly improved the performance. EGT+MEL produced the strongest effects in both tasks. EGT and MEL alone markedly decreased β-amyloid protein accumulation in the hippocampus and significantly inhibited lipid peroxidation and maintained glutathione/glutathione disulfide ratio and superoxide dismutase activity in brain tissues of DG-treated mice. MEL alone completely prevented the rise in brain acetylcholine esterase activity induced by DG, whereas EGT and EGT+MEL were only partially effective. Overall, EGT, MEL, and, in particular, the combination of EGT and MEL effectively protect against learning and memory deficits in C57BL/6J mice treated with DG, possibly through attenuation of oxidative damage.     

The distinct effects of a butanol fraction of <Emphasis Type="Italic">Bidens pilosa</Emphasis> plant extract on the development of Th1-mediated diabetes and Th2-mediated airway inflammation in mice

Bidens pilosa is claimed to be useful for immune or anti-inflammatory disorders; however, little scientific evidence has been published concerning its function. In this paper, immune disease mouse models were used to study the function of a butanol fraction of B.pilosa. We demonstrated treatment with the butanol fraction of B.pilosa ameliorated Th1 cell-mediated autoimmune diabetes in nonobese diabetic (NOD) mice but caused deterioration of Th2 cell-mediated airway inflammation induced by ovalbumin (OVA) in BALB/c mice. We next showed that Th2 cytokines (IL-4 and/or IL-5) increased but Th1 cytokine (IFN-) decreased following injections with the butanol fraction of B.pilosa in both mouse strains. Accordingly, Th2 cytokine-regulated IgE production in mouse serum increased following treatment with this fraction. Finally, we found that the butanol fraction of B.pilosa inhibited Th1 cell differentiation but promoted Th2 cell differentiation. Taken together, the butanol fraction of B.pilosa has a dichotomous effect on helper T cell-mediated immune disorders, plausibly via modulation of T cell differentiation.     

Effect of Cheonggukjang supplementation upon hepatic acyl-CoA synthase, carnitine palmitoyltransferase I, acyl-CoA oxidase and uncoupling protein 2 mRNA levels in C57BL/6J mice fed with high fat diet

This study investigated the effect of Cheonggukjang on mRNA levels of hepatic acyl-CoA synthase (ACS), carnitine palmitoyltransferase I (CPT-I), acyl-CoA oxidase (ACO) and uncoupling protein 2 (UCP2), and on serum lipid profiles in C57BL/6J mice. Thirty male C57BL/6J mice were divided into three groups; normal diet (ND), high fat diet (HD) and high fat diet with 40% Cheonggukjang (HDC). Energy intake was significantly higher in the HDC group than in the ND and HD groups. The HDC group normalized in weight gain, epididymal and back fat (g/100 g) accumulation which are increased by high fat diet. Serum concentrations of triglyceride and total cholesterol in the HDC were significantly lower than those in the HD group. These results were confirmed by hepatic mRNA expression of enzymes and protein (ACS, CPT-1, ACO, UCP2) which is related with lipid metabolism by RT-PCR. Hepatic CPT-I, ACO and UCP2 mRNA expression was increased by Cheonggukjang supplementation. We demonstrated that Cheonggukjang supplement leads to increased mRNA expressions of enzymes and protein involved in fatty acid oxidation in liver, reduced accumulation of body fat and improvement of serum lipids in high fat diet fed mice.