Pathogenicity and host cytokines response of EqHV-8 infection in C57BL/6J mice.

Equid herpesvirus type 8 (EqHV-8) is known to cause abortion, respiratory signs, and viral encephalitis in equines. EqHV-8 has been reported to cause serious economic losses in large-scale donkey farms in China. However, little is known about the viral replication and immune reaction in the brains and lungs of EqHV-8-induced C57BL/6J mice. We determined the pathogenicity and immune status in a mice model. The C57BL/6J mice were infected with the EqHV-8 donkey/Shandong/10/2021 strain, and the clinical signs and body weights were evaluated every day. In addition, viremia, virus loads, and the expression of pro-inflammatory cytokines in mice brains and lungs were assessed at 1, 3, 5, and 7 days post infection (dpi). Our results demonstrated that mice in the EqHV-8 infected group displayed body weight loss, dyspnea signs, and viremia. The expression of interleukin (IL)-1ß, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-6 mRNA was increased in the brains and lungs of EqHV-8-infected mice than that in control group at 5 dpi and 7 dpi, and IL-12a expression was increased at 7 dpi. These data indicated that EqHV-8 elicited a strong cytokines response, caused neurogenic disease and respiratory signs in C57BL/6J mice, thus revealing the pathogenicity of EqHV-8.

NREM sleep loss increases neurofilament light chain levels in APP/PS1 and C57BL/6 J mice.

PURPOSE: Sleep disturbances exacerbate the progression of Alzheimer's disease (AD), but disturbances of non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep may have different effects. Neurofilament light chain (NfL), an axon-specific protein, is an indicator of the severity of neuronal apoptosis. To investigate whether or not NREM or REM sleep is crucial to neuronal survival, we examined the effects of induced NREM or REM sleep loss on NfL levels in APP/PS1 mice, a model of AD, and their wild-type (WT) C57BL/6 J littermates. METHODS: At 6 months of age, WT mice and AD mice were equally divided into six groups, namely, the WT-normal sleep (S), WT-total sleep deprivation (TSD), WT-REM deprivation (RD), AD-S, AD-TSD and AD-RD groups, according to the type of sleep intervention applied. All mice underwent 6 days of sleep intervention. Cerebrospinal fluid (CSF) and plasma NfL levels were measured at baseline and on days 2, 4 and 6, and spatial memory was assessed in the Morris water maze (MWM) test. RESULTS: Among the 18 WT and 18 AD mice, CSF and plasma NfL levels were higher in AD-TSD mice than in AD-S or AD-RD mice, while no significant difference was observed between the latter two groups. In AD-TSD mice, CSF and plasma NfL levels increased with the duration of sleep deprivation. A similar pattern of results was observed for the WT groups. CONCLUSIONS: NREM sleep loss may increase CSF and plasma NfL levels in both WT and AD mice.

A Comparative Study of Skin Changes in Different Species of Mice in Chronic Photoaging Models.

This study aimed to design a novel mouse model of chronic photoaging. We used three different species of mice (C57BL/6J, ICR, and KM) to create a chronic photoaging model of the skin. The irradiation time was gradually increased for 40 consecutive days. The skins of the mice were removed on day 41 and subjected to staining to observe them for morphological changes. Immunohistochemistry was used to detect tumor necrosis factor-α (TNF-α) and p53 expression; superoxide dismutase (SOD) and malondialdehyde (MDA) were measured as well. Compared with C57BL/J mice, which showed hyperpigmentation, the irradiated skin of ICR and KM mice showed more obvious skin thickening and photoaging changes of the collagen and elastic fibers. KM mice had higher levels of inflammation, oxidative stress, and senescent cells. Compared with the 5-month-old KM mice, the photoaging changes of the 9-month-old KM mice were more pronounced, the SOD values were lower, and the MDA values were higher. In summary, KM mice have higher levels of abnormal elastic fibers, inflammation, cellular senescence, and oxidative stress than ICR mice, and are more suitable for studies related to chronic skin photoaging. C57BL/6J mice were found to be suitable for studies related to skin pigmentation due to photoaging.

A carcinogenicity study of diphenylarsinic acid in C57BL/6J mice in drinking water for 78 weeks.

Diphenylarsinic acid (DPAA), a neurotoxic organic arsenical, is present in groundwater and soil in some regions of Japan owing to illegal dumping. The present study evaluated the potential carcinogenicity of DPAA, including investigating whether bile duct hyperplasia in the liver that was observed in a chronic study on 52 week mouse, develops into a tumor when administered to mice in their drinking water for 78 weeks. DPAA was administered to 4 groups of male and female C57BL/6J mice at concentrations of 0, 6.25, 12.5, and 25 ppm in drinking water for 78 weeks. A significant decrease in the survival rate was found for females in the 25 ppm DPAA group. Body weights of males in the 25 ppm and females in the 12.5 and 25 ppm DPAA groups were significantly lower than those of the controls. Histopathological evaluation of neoplasms in all tissues showed no significant increase in tumor incidence in any organ or tissue of 6.25, 12.5, or 25 ppm DPAA-treated male or female mice. In conclusion, the present study demonstrated that DPAA is not carcinogenic to male or female C57BL/6J mice. Taken together with the fact that the toxic effect of DPAA is predominantly restricted to the central nervous system in humans, and the finding that DPAA was not carcinogenic in a previous 104-week rat carcinogenicity study, our results suggest that DPAA is unlikely to be carcinogenic in humans.

Development of tolerance upon repeated administration with the GABAB receptor positive allosteric modulator, COR659, on alcohol drinking in rodents.

Background: Recent work has demonstrated that acute administration of the novel positive allosteric modulator of the GABAB receptor, COR659, reduces several alcohol-related behaviors in rodents.Objective: To assess whether COR659 continues to lessen alcohol intake after repeated administration, a fundamental feature of drugs with therapeutic potential.Methods: Male C57BL/6J mice (n = 40) were exposed to daily 2-hour drinking sessions (20% (v/v) alcohol) under the 1-bottle "drinking in the dark" protocol and male Sardinian alcohol-preferring rats (n = 40) were exposed to daily 1-hour drinking sessions under the 2-bottle "alcohol (10%, v/v) vs water" choice regimen. COR659 (0, 10, 20, and 40 mg/kg in the mouse experiment; 0, 5, 10, and 20 mg/kg in the rat experiment) was administered intraperitoneally before 7 consecutive drinking sessions.Results: Alcohol intake in vehicle-treated mice and rats averaged 2.5-3.0 and 1.5-1.6 g/kg/session, respectively, indicative of high basal levels. In both experiments, treatment with COR659 resulted in an initial, dose-related suppression of alcohol intake (up to 70-80% compared to vehicle treatment; P < .0005 and P < .0001 in mouse and rat experiments, respectively). The magnitude of the reducing effect of COR659 on alcohol drinking diminished progressively, until vanishing over the subsequent 2-4 drinking sessions.Conclusion: COR659 effectively reduced alcohol intake in two different rodent models of excessive alcohol drinking. However, tolerance to the anti-alcohol effects of COR659 developed rapidly. If theoretically transposed to humans, these data would represent a possible limitation to the clinical use of COR659.

Cross-Omics Analysis of Fenugreek Supplementation Reveals Beneficial Effects Are Caused by Gut Microbiome Changes Not Mammalian Host Physiology.

Herbal remedies are increasing in popularity as treatments for metabolic conditions such as obesity and Type 2 Diabetes. One potential therapeutic option is fenugreek seeds (Trigonella foenum-graecum), which have been used for treating high cholesterol and Type 2 diabetes. A proposed mechanism for these benefits is through alterations in the microbiome, which impact mammalian host metabolic function. This study used untargeted metabolomics to investigate the fenugreek-induced alterations in the intestinal, liver, and serum profiles of mice fed either a 60% high-fat or low-fat control diet each with or without fenugreek supplementation (2% w/w) for 14 weeks. Metagenomic analyses of intestinal contents found significant alterations in the relative composition of the gut microbiome resulting from fenugreek supplementation. Specifically, Verrucomicrobia, a phylum containing beneficial bacteria which are correlated with health benefits, increased in relative abundance with fenugreek. Metabolomics partial least squares discriminant analysis revealed substantial fenugreek-induced changes in the large intestines. However, it was observed that while the magnitude of changes was less, significant modifications were present in the liver tissues resulting from fenugreek supplementation. Further analyses revealed metabolic processes affected by fenugreek and showed broad ranging impacts in multiple pathways, including carnitine biosynthesis, cholesterol and bile acid metabolism, and arginine biosynthesis. These pathways may play important roles in the beneficial effects of fenugreek.

Assessment of Bone Microstructure by Micro CT in C57BL/6J Mice for Sex-Specific Differentiation.

It remains uncertain which skeletal sites and parameters should be analyzed in rodent studies evaluating bone health and disease. In this cross-sectional mouse study using micro-computed tomography (µCT), we explored: (1) which microstructural parameters can be used to discriminate female from male bones and (2) whether it is meaningful to evaluate more than one bone site. Microstructural parameters of the trabecular and/or cortical compartments of the femur, tibia, thoracic and lumbar vertebral bodies, and skull were evaluated by µCT in 10 female and 10 male six-month-old C57BL/6J mice. The trabecular number (TbN) was significantly higher, while the trabecular separation (TbSp) was significantly lower in male compared to female mice at all skeletal sites assessed. Overall, bone volume/tissue volume (BV/TV) was also significantly higher in male vs. female mice (except for the thoracic spine, which did not differ by sex). Most parameters of the cortical bone microstructure did not differ between male and female mice. BV/TV, TbN, and TbSp at the femur, and TbN and TbSp at the tibia and lumbar spine could fully (100%) discriminate female from male bones. Cortical thickness (CtTh) at the femur was the best parameter to detect sex differences in the cortical compartment (AUC = 0.914). In 6-month-old C57BL/6J mice, BV/TV, TbN, and TbSp can be used to distinguish male from female bones. Whenever it is not possible to assess multiple bone sites, we propose to evaluate the bone microstructure of the femur for detecting potential sex differences.

Toll-like receptor 2 and 4 antagonism for the treatment of experimental autoimmune encephalomyelitis (EAE)-related pain.

Neuropathic pain is a major symptom of multiple sclerosis (MS) with up to 92% of patients reporting bodily pain, and 85% reporting pain severe enough to cause functional disability. None of the available therapeutics target MS pain. Toll-like receptors 2 and 4 (TLR2/TLR4) have emerged as targets for treating a wide array of autoimmune disorders, including MS, as well as having demonstrated success at suppressing pain in diverse animal models. The current series of studies tested systemic TLR2/TLR4 antagonists in males and females in a low-dose Myelin oligodendrocyte glycoprotein (MOG) experimental autoimmune encephalomyelitis (EAE) model, with reduced motor dysfunction to allow unconfounded testing of allodynia through 50+ days post-MOG. The data demonstrated that blocking TLR2/TLR4 suppressed EAE-related pain, equally in males and females; upregulation of dorsal spinal cord proinflammatory gene expression for TLR2, TLR4, NLRP3, interleukin-1ß, IkBα, TNF-α and interleukin-17; and upregulation of dorsal spinal cord expression of glial immunoreactivity markers. In support of these results, intrathecal interleukin-1 receptor antagonist reversed EAE-induced allodynia, both early and late after EAE induction. In contrast, blocking TLR2/TLR4 did not suppress EAE-induced motor disturbances induced by a higher MOG dose. These data suggest that blocking TLR2/TLR4 prevents the production of proinflammatory factors involved in low dose EAE pathology. Moreover, in this EAE model, TLR2/TLR4 antagonists were highly effective in reducing pain, whereas motor impairment, as seen in high dose MOG EAE, is not affected.

Prolonged-release pirfenidone prevents obesity-induced cardiac steatosis and fibrosis in a mouse NASH model.

PURPOSE: Obesity is associated with systemic insulin resistance and cardiac hypertrophy with fibrosis. Peroxisome proliferator-activated receptors (PPARs) regulate carbohydrate and lipid metabolism, improving insulin sensitivity, triglyceride levels, inflammation, and oxidative stress. We previously demonstrated that prolonged-release pirfenidone (PR-PFD) is an agonistic ligand for Pparα with anti-inflammatory and anti-fibrotic effects, and might be a promising drug for cardiac diseases-treatment. Here, we investigated the effects of PR-PFD in ventricular tissue of mice with nonalcoholic steatohepatitis (NASH) and obesity induced by high-fat/high-carbohydrate (HFHC) diet. METHODS: Five male C57BL/6 J mice were fed with normal diet (ND) and ten with HFHC diet for 16 weeks; at 8 weeks of feeding, five mice with HFHC diet were administered PR-PFD (350 mg/kg/day) mixed with HFHC diet. RESULT: Systemic insulin resistance, heart weight/body weight ratio, myocardial steatosis with inflammatory foci, hypertrophy, and fibrosis were prevented by PR-PFD. In addition, HFHC mice showed significantly increased desmin, Tgfß1, Timp1, collagen I (Col I), collagen III (Col III), TNF-α, and Nrf2 mRNA levels, including α-SMA, NF-kB, Nrf2, troponin I, Acox1, Cpt1A, and Lxrα protein levels compared with the ND ventricular tissues. Mechanistically, HFHC mice with PR-PFD treatment significantly decreased these genes overexpressed by HFHC diet. Furthermore, PR-PFD overexpressed the Pgc1a mRNA levels and Pparα, Pparγ, Acox1, and Cpt1A protein levels. CONCLUSIONS: The results suggest that PR-PFD could be a promising drug for the prevention and treatment of cardiac steatosis and fibrosis induced by obesity.

Lotus leaf flavonoids induce apoptosis of human lung cancer A549 cells through the ROS/p38 MAPK pathway.

BACKGROUND: Leaves of the natural plant lotus are used in traditional Chinese medicine and tea production. They are rich in flavonoids. METHODS: In this study, lotus leaf flavonoids (LLF) were applied to human lung cancer A549 cells and human small cell lung cancer cells H446 in vitro to verify the effect of LLF on apoptosis in these cells through the ROS/p38 MAPK pathway. RESULTS: LLF had no toxic effect on normal cells at concentrations up to 500 µg/mL, but could significantly inhibit the proliferation of A549 cells and H446 cells. Flow cytometry showed that LLF could induce growth in A549 cells. We also found that LLF could increase ROS and MDA levels, and decrease SOD activity in A549 cells. Furthermore, qRT-PCR and western blot analyses showed that LLF could upregulate the expression of p38 MAPK (p-p38 MAPK), caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9 and Bax and downregulate the expression of Cu/Zn SOD, CAT, Nrf2, NQO1, HO-1, and Bcl-2 in A549 cells. Results of HPLC showed that LLF mainly contain five active substances: kaempferitrin, hyperoside, astragalin, phloridzin, and quercetin. The apoptosis-inducing effect of LLF on A549 cells came from these naturally active compounds. CONCLUSIONS: We have shown in this study that LLF is a bioactive substance that can induce apoptosis in A549 cells in vitro, and merits further research and development.

Impact of C57BL/6J and SV-129 Mouse Strain Differences on Ischemia-Induced Postnatal Angiogenesis and the Associated Leukocyte Infiltration in a Murine Hindlimb Model of Ischemia.

Strain-related differences in arteriogenesis in inbred mouse strains have already been studied excessively. However, these analyses missed evaluating the mouse strain-related differences in ischemia-induced angiogenic capacities. With the present study, we wanted to shed light on the different angiogenic potentials and the associated leukocyte infiltration of C57BL/6J and SV-129 mice to facilitate the comparison of angiogenesis-related analyses between these strains. For the induction of angiogenesis, we ligated the femoral artery in 8-12-week-old male C57BL/6J and SV-129 mice and performed (immuno-) histological analyses on the ischemic gastrocnemius muscles collected 24 h or 7 days after ligation. As evidenced by hematoxylin and eosin staining, C57BL/6J mice showed reduced tissue damage but displayed an increased capillary-to-muscle fiber ratio and an elevated number of proliferating capillaries (CD31+/BrdU+ cells) compared to SV-129 mice, thus showing improved angiogenesis. Regarding the associated leukocyte infiltration, we found increased numbers of neutrophils (MPO+ cells), NETs (MPO+/CitH3+/DAPI+), and macrophages (CD68+ cells) in SV-129 mice, whereas macrophage polarization (MRC1- vs. MRC1+) and total leukocyte infiltration (CD45+ cells) did not differ between the mouse strains. In summary, we show increased ischemia-induced angiogenic capacities in C57BL/6J mice compared to SV-129 mice, with the latter showing aggravated tissue damage, inflammation, and impaired angiogenesis.

Potential use of C-phycocyanin in non-alcoholic fatty liver disease.

C-phycocyanin (C-PC) is a kind of photosynthetically assisted pigment, which is ubiquitous in cyanobacteria cells. We investigated the effect of C-PC on non-alcoholic fatty liver disease (NAFLD) and its mechanism. Through oil red O staining, TC/TG detection, liver SOD/MDA detection and liver H&E staining, we found that C-PC could significantly reduce the lipid accumulation in the steatosis L02 cells and the liver of non-alcoholic steatohepatitis (NASH) mice, and improve the antioxidant capacity of liver. The results of Western Blotting showed that C-PC upregulated the expression of AMPK phosphorylation and downregulated SREBP-1c and its target genes ACC and FAS expression levels. Furthermore, C-PC also upregulated the expression of transcription factor PPARα, which was regulated by AMPK, and its target genes CPT1 level. In addition, C-PC could promote AMPK phosphorylation in hepatocytes while increasing the phosphorylation level of ACC in vivo and in vitro. Besides, C-PC could also improve the liver inflammatory infiltration by upregulated the expression of PPARγ and downregulated the expression of CD36, IL6 and TNFα. These results indicate that C-PC may improve hepatic lipid accumulation and inflammation in the non-alcoholic fatty liver mice by activating AMPK pathway of hepatocytes. The finding provides important help for the research and development of C-PC in the nutraceuticals and therapeutics of NAFLD.

Intake of Watermelon or Its Byproducts Alters Glucose Metabolism, the Microbiome, and Hepatic Proinflammatory Metabolites in High-Fat-Fed Male C57BL/6 J Mice.

BACKGROUND: Watermelon intake has demonstrated effects on blood pressure regulation along with other health benefits. OBJECTIVE: We hypothesized that intake of whole watermelon and products made from watermelon rind (WR) and watermelon skin (WS) would remediate metabolic complications in C57BL/6 J male mice fed a diet modeling a Western-style diet. METHODS: Ten-week-old male C57BL/6 J mice were provided either a low-fat (LF) diet [10% fat (by energy), 8% sucrose (by energy) and no added cholesterol], a high-fat (HF) diet [45% fat (by energy), 20% kcal sucrose (by energy), and 1% (w/w) cholesterol], or an HF diet plus WS, WR, or watermelon flesh (WF) for 10 wk. Dried WF was provided at 8% of total energy (equivalent to 2 servings/d) and watermelon skin and rind were added at 2.25% (w/w, dry weight of additives) of diet. Animals were provided experimental diets ad libitum. Body weights, food intake, and glucose tolerance were determined. Serum insulin, inflammatory markers, microbiome, and the relative hepatic concentrations of 709 biochemicals were measured postmortem. RESULTS: The final body weight of the LF control group was significantly lower than that of the HF-fed control group (32.8 ± 0.9 g compared with 43.0 ± 1.7 g, P ≤ 0.05). Mice in treatment groups fed HF supplemented with watermelon products had final body weights similar to those of the HF-fed control mice. Serum insulin concentrations were reduced by ∼40% in mice fed an HF diet with WR supplementation compared with mice fed an HF diet alone (P ≤ 0.05). Depending on the individual species or group, microbiome populations changed significantly. Supplementation with WF resulted in a return to the basal hepatic concentrations of monohydroxy fatty acids and eicosanoids observed in LF-fed mice (P ≤ 0.05). CONCLUSIONS: In obese male mice, supplementation with each of the watermelon products to an HF diet improved fasting blood glucose, circulating serum insulin concentrations, and changes in hepatic metabolite accumulation. At a modest level of supplementation to an HF diet, fiber-rich additives made from WR and WS further improved glucose metabolism and energy efficiency and shifted the microbiome composition.

Unloading-Induced Cortical Bone Loss is Exacerbated by Low-Dose Irradiation During a Simulated Deep Space Exploration Mission.

Spaceflight-induced bone losses have been reliably reproduced in Hind-Limb-Unloading (HLU) rodent models. However, a considerable knowledge gap exists regarding the effects of low-dose radiation and microgravity together. Ten-week-old male C57BL/6J mice, randomly allocated to Control (CONT), Hind-Limb Unloading (HLU), and Hind-Limb Unloading plus Irradiation (HLUIR), were acclimatized at 28 °C, close to thermoneutral temperature, for 28 days prior to the 14-day HLU protocol. HLUIR mice received a 25 mGy dose of X-ray irradiation, simulating 14 days of exposure to the deep space radiation environment, on day 7 of the HLU protocol. Trabecular bone mass was similarly reduced in HLU and HLUIR mice when compared to CONT, with losses driven by osteoclastic bone resorption rather than changes to osteoblastic bone formation. Femoral cortical thickness was reduced only in the HLUIR mice (102 µm, 97.5-107) as compared to CONT (108.5 µm, 102.5-120.5). Bone surface area was also reduced only in the HLUIR group, with no difference between HLU and CONT. Cortical losses were driven by osteoclastic resorption on the posterior endosteal surface of the distal femoral diaphysis, with no increase in the numbers of dead osteocytes. In conclusion, we show that low-dose radiation exposure negatively influences bone physiology beyond that induced by microgravity alone.

Discordant susceptibility of inbred C57BL/6 versus outbred CD1 mice to experimental fungal sepsis.

Individual susceptibility differences to fungal infection following invasive and/or immunosuppressive medical interventions are an important clinical issue. In order to explore immune response-related factors that may be linked to fungal infection susceptibility, we have compared the response of inbred C57BL/6J and outbred CD1 mouse strains to different experimental models of fungal sepsis. The challenge of animals with the zymosan-induced generalised inflammation model revealed poorer survival rates in C57BL/6J, consistent with lower Th1 cytokine interferon (IFN)-γ serum levels, compared with CD1 mice. Likewise, ex vivo exposure of C57BL/6J splenocytes to zymosan but also bacterial lipopolisaccharide or lipoteichoic acid, resulted in lower IFN-γ secretion compared with CD1 mice. C57BL/6J susceptibility could be reverted by rescue infusion of relative low IFN-γ doses (0.2 µg/kg) either alone or in combination with the ß-glucan-binding CD5 protein (0.7 mg/kg) leading to improved post zymosan-induced generalised inflammation survival. Similarly, low survival rates to systemic Candida albicans infection (2.86 × 104  CFU/gr) were ameliorated by low-dose IFN-γ infusion in C57BL/6J but not CD1 mice. Our results highlight the importance of strain choice in experimental fungal infection models and provide a susceptibility rationale for more specific antifungal immunotherapy designs.

[Establishment of mouse lung cancer model induced by benzo[a]pyrene dynamic inhalation exposure].

OBJECTIVE: To establish a mouse lung cancer model induced by benzo[a]pyrene(B[a]P) dynamic inhalation exposure. METHODS: A total of 96 C57 BL/6 J mice weighing 18 to 20 g were randomly divided into the control group(n=48)and the experimental group(n=48), male and female in half. The experimental group was treated with 10. 0 µg/m~3 BaP for 13 weeks or 25 weeks(6 h per day and 5 days per week) by dynamic inhalation exposure, while the control group was given dimethyl sulfoxide(DMSO). The 13 weeks or 25 weeks after B[a]P exposure, 24 mice were sacrificed and their lung tissues dissected and observed for tumor formation. During B[a]P exposure, the gas in the poisoning cabinet was collected by the active carbon tube method regularly, and the concentration of B[a]P in the poisoning cabinet was analyzed by gas chromatography mass spectrometer, and the(7 R, 8 S)-dihydroxy-(9 S, 10 R)-epoxy-7, 8, 9, 10-tetrahydrobenzo [a] pyrene(BPDE)-DNA adduct content in the whole blood of mice was determined by ELISA. RESULTS: Concentration of B[a]P in the dynamic inhalation cabinet was stable at(12. 794±0. 518)µg/m~3. There was no significant difference in the BPDE-DNA content in the experimental objects between 13 weeks and 25 weeks after B[a]P exposure(P>0. 05). After 25 weeks of B[a]P exposure, the lung cancer formation rate in the experimental group was significantly higher than that in the control group(P<0. 05), the tumor formation rate of female mice was up to 100%. CONCLUSION: The mice lung cancer model induced by B[a]P dynamic inhalation exposure was established successfully.

Upregulation of microRNA-351 exerts protective effects during sepsis by ameliorating skeletal muscle wasting through the Tead-4-mediated blockade of the Hippo signaling pathway.

Sepsis-induced skeletal muscle wasting may lead to various severe clinical consequences. Understanding molecular mechanisms of the regulation of the loss of skeletal muscle mass in septic patients remains a significant clinical challenge. The current study was conducted to establish septic mice models to explore the relationship between microRNA (miR)-351 and the transcription element apical (TEA) domain transcription factor (Tead)-4 gene and to investigate its effects on the skeletal muscle through mediating the Hippo signaling pathway in mice with acute sepsis. A total of 60 mice were collected to establish mouse models of acute sepsis. The positive expression rate of Tead-4 and the apoptotic index (AI) were measured. A dual-luciferase reporter gene assay was conducted to verify the targeting relationship between miR-351 and Tead-4. Furthermore, the muscle fiber diameter (MFD) and area (MFA) and the content of 3-methylhistidine (3-MH) and tyrosine (Tyr) were assessed. The expression levels of miR-351, p38-MAPK, Yes-associated protein, Tead-4, B-cell lymphoma X protein (Bax), and Caspase-3 were determined with quantitative RT-PCR and Western blot analysis. Finally, cell viability, apoptosis, and levels of inflammatory factors, including IL-1ß, IL-6, IGF-1, TNF-α, and monocyte chemoattractant protein-1 were detected by 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and ELISA. Initially, Tead-4 protein expression was higher in skeletal muscle tissues of mice with acute sepsis. Tead-4 was identified to negatively regulate miR-351. Upregulation of miR-351 increased MFA and MFD, muscle weight water content, Bcl-2 expression levels, and cell viability. Up-regulation of miR-351 reduced AI; 3-MH and Tyr content; positive expression of Tead-4 protein; the expression levels of p38-MAPK, Yap, Tead-4, Bax, and Caspase-3; apoptosis; and inflammatory responses. The current study demonstrated that up-regulation of miR-351 inhibits the degradation of skeletal muscle protein and the atrophy of skeletal muscle in mice with acute sepsis by targeting Tead-4 through suppression of the Hippo signaling pathway. Thus, miR-351 overexpression may be a future therapeutic strategy for acute sepsis.-Zhang, L.-N., Tian, H., Zhou, X.-L., Tian, S.-C., Zhang, X.-H., Wu, T.-J. Upregulation of microRNA-351 exerts protective effects during sepsis by ameliorating skeletal muscle wasting through the Tead-4-mediated blockade of the Hippo signaling pathway.

Housing conditions modulate spontaneous physical activity, feeding behavior, aerobic running capacity and adiposity in C57BL/6J mice.

There is evidence of reduced adiposity in rodents living in a large cages (LC) as compared to animals housed in small cages (SC). Because spontaneous physical activity (SPA) provides an important portion of the total daily energy expenditure, an increase of SPA in rodents kept in LC could explain their reduced body fat accumulation. The relationship between SPA and components of physical fitness (i.e. aerobic and anaerobic fitness and body leanness) has not been previously determined. We examined the effects of eight weeks of LC exposure on SPA, body composition, feeding behavior, as well as aerobic and anaerobic running capacity in adult C57BL/6J mice. Male mice were housed in cages of two different sizes for 8 weeks: a small (SC, n = 10) and large (LC n = 10) cages with 1320 cm2 and 4800 cm2 floor space, respectively. SPA was measured gravimetrically, and food and water intake were recorded daily. Mice had critical velocity (CV) and anaerobic running capacity (ARC) evaluated at the beginning, middle course (4th week) and at the end of study (8th week). Despite non-significant differences in each week LC-mice were more active than SC-mice by considering all SPA values obtained in the entire period of 8 weeks. The difference in SPA over the whole day was mainly due to light phase activity, but also due to activity at dark period (from 6 pm to 9 pm and from 5 am to 6 am). LC-mice also exhibited higher food and water intake over the entire 8-wk period. LC-mice had lower content of fat mass (% of the eviscerated carcass) than SC-mice (SC: 8.4 ±â€¯0.4 vs LC: 6.3 ±â€¯0.3, p < 0.05). LC-mice also exhibited reduced epididymal fat pads (% of body mass) compared to SC-mice (SC: 1.3 ±â€¯0.1 vs LC: 0.9 ±â€¯0.1, p < 0.05) and retroperitoneal fat pads (SC: 0.4 ±â€¯0.05 vs LC: 0.2 ±â€¯0.02, p < 0.05). The LC-group showed significantly higher critical velocity than SC-group at the fourth week (SC: 14.9 ±â€¯0.6 m·min-1 vs LC: 18.0 ±â€¯0.3 m·min-1, p < 0.05) and eighth week (SC: 17.1 ±â€¯0.5 m·min-1 vs LC: 18.8 ±â€¯0.6 m·min-1, p < 0.05). Our findings demonstrate that eight weeks of LC housing increases SPA of C57BL/6J mice, and this may lead to reduced fat accumulation as well as higher aerobic fitness. Importantly, our study implies that SC limits SPA, possibly generating experimental artifacts in long-term rodent studies.

Induction of tolerogenic properties by Anisakis larval antigens on murine dendritic cells.

AIMS: The objective of this work is to investigate whether Anisakis simplex larval antigens present immunomodulatory properties by the induction of tolerogenic dendritic cells (DCs) from two strains of mice (BALB/c and C57BL/6J). METHODS AND RESULTS: We used mouse bone marrow-derived DCs. We determined their antigen-presenting ability by expression of membrane markers (MHC I and MHC II, CD80, CD86) and intracellular expression levels of IL-10 and IL-12 cytokines. We also analysed whether stimulation with A simplex larval antigens is enhanced by the co-administration of the TLR4 and TLR9 agonists [LPS E coli 026B6 and CpG (ODN1826), respectively]. Two differential types of responses were found in the two mouse strains studied: the BALB/c strain showed an acute and inflammatory response, whereas the C57BL/6J mice developed a more discrete and resistant response. This suggests the coexistence of two opposing responses generated by A simplex larval antigens and confirms that the host genetic basis plays a role in the development of a Th2 or Treg response. CONCLUSION: The study of the mechanisms by which Anisakis manipulates the immune response through anti-inflammatory molecules is of interest not only for the direct application on the development of anthelmintic strategies, but also for the development of new anti-inflammatory products.

Donepezil Attenuates Obesity-Associated Oxidative Stress and Central Inflammation and Improves Memory Deficit in Mice Fed a High-Fat Diet.

BACKGROUND/AIMS: Obesity is associated with chronic inflammation and cognitive decline, and is considered a major risk factor for neurodegeneration. Meanwhile, neuroinflammation is important in the pathogenesis and progression of neurodegenerative diseases. METHODS: In this study, we tested the hypothesis that donepezil would attenuate central inflammation and oxidative damage and improve memory deficit in high-fat diet (HFD)-fed mice. After 16 weeks on a HFD, C57BL/6J mice were given either donepezil (3 mg/kg, i.p.) or saline for 4 weeks in parallel to a control diet (CD) group. Thereafter, the step-through test was used to assess learning and memory function. RESULTS: In the brain of HFD-fed mice, levels of the proinflammatory cytokines interleukin 16 and tumor necrosis factor α were reduced by donepezil treatment. Similarly, HFD-induced protein levels of advanced glycation end-products and oxidative stress in the brain were significantly decreased by donepezil treatment. CONCLUSION: Our results indicate that donepezil may reverse obesity-related central inflammation and oxidative damage and improve memory deficit in HFD-fed mice.