Weeks In Mice Research Articles

SGLT1/2 inhibition improves glycemic control and multi-organ protection in type 1 diabetes

Sodium glucose cotransporters (SGLTs) are transport proteins that are expressed throughout the body. Inhibition of SGLTs is a relatively novel therapeutic strategy to improve glycemic control and has been shown to promote cardiorenal benefits. Dual SGLT1/2 inhibitors (SGLT1/2i) such as sotagliflozin target both SGLT1 and 2 proteins. Sotagliflozin or vehicle was administered to diabetic Akimba mice for 8weeks at a dose of 25mg/kg/day. Urine glucose levels, water consumption, and body weight were measured weekly. Serum, kidney, pancreas, and brain tissue were harvested under terminal anesthesia. Tissues were assessed using immunohistochemistry or ELISA techniques. Treatment with sotagliflozin promoted multiple metabolic benefits in diabetic Akimba mice resulting in decreased blood glucose and improved polydipsia. Sotagliflozin also prevented mortalities associated with diabetes. Our data suggests that there is the possibility that combined SGLT1/2i may be superior to SGLT2i in controlling glucose homeostasis and provides protection of multiple organs affected by diabetes.

Effects of perfluorododecanoic acid on testicular function in mice.

Perfluorodecanoic acid (PFDoA) is a widely distributed environmental pollutant that can affect the functions of many organs. However, systematic evaluations of the effects of PFDoA on testicular functions are lacking. The aim of this study was to investigate the effects of PFDoA on mouse testicular functions, including spermatogenesis, testosterone synthesis, and stem Leydig cells (SLCs) in the interstitial tissue of the testis. PFDoA (0, 2, 5, 10mg/kg/d) was administered via gavage to 2-month-old mice for 4weeks. Serum hormone levels and sperm quality were assayed. Furthermore, to investigate the mechanisms by which PFDoA affects testosterone synthesis and spermatogenesis in vivo, the expression of StAR and P450scc in testicular tissue was measured by immunofluorescence staining and quantitative real-time PCR. In addition, the levels of SLC markers, including nestin and CD51, were studied. PFDoA decreased the luteinizing hormone concentration and sperm quality. Although the difference was not statistically significant, mean testosterone levels showed a downward trend. The expression of StAR, P450scc, CD51, and nestin was also suppressed in the PFDoA-treated groups compared with the control group. Our study suggested that PFDoA exposure can decrease testosterone biosynthesis, and even reduce the number of SLCs. These results indicated that PFDoA suppressed the main functions of testis, and further researches are required to identify strategies for preventing or reducing the effect of PFDoA on testicular function.

Phosphoproteomics analysis of diabetic cardiomyopathy in aging-accelerated mice and effects of D-pinitol.

The molecular mechanisms of diabetic cardiomyopathy (DCM) development and D-pinitol (DP) in its treatment remain unclear. The present study is to explore the underlying mechanism of DCM in an elderly diabetic mouse model and to seek the protective targets of DP by phosphoproteomics. We used streptozotocin to induce diabetes in SAMP8 and DP (150mg/kg/day) intragastrically administrated to diabetic mice for 8weeks. The heart tissues were harvested for label-free phosphoproteomic analysis from diabetic mice. Some differentially regulated phosphorylation sites were confirmed by parallel reaction monitoring. Our results showed that 612 phosphorylation sites on 454 proteins had their phosphorylation levels significantly changed in the heart of untreated diabetic mice (DM). Of these phosphorylation sites, 216 phosphorylation sites on 182 proteins were normalized after DP treatment. We analyzed the functional signaling pathways in the heart of DP treated diabetic mice (DMT), including glucagon signaling pathway, insulin signaling pathway, mitophagy, apoptosis, and longevity regulating pathway. Two consensus motifs identified were targeted by Src and epidermal growth factor receptor between DMT and DM groups. Our study might help to better understand the mechanism of DCM, provide novel targets for estimating the protective effects of DP.

Hyaluronic acid biomaterial for human tissue-engineered skin substitutes: Preclinical comparative in vivo study of wound healing.

There is not an ideal biomaterial for tissue-engineered skin substitutes (TESSs), and most of the studies or existing therapies use xenogeneic origin natural biomaterials or biosynthetic scaffolds. To analyse clinical, histological integration and homeostasis parameters of a human TESS manufactured with fibrin-hyaluronic acid biomaterial (HA-Skin), grafted in immunodeficient mice for 8weeks, and compared with the gold standard treatment (Autograft), a human TESS manufactured with fibrin-agarose biomaterial (AG-Skin) and secondary wound healing dressings. Human TESSs and autografts were implanted into BALB/c mice after surgical excision. Secondary wound healing approach was achieved with biosynthetic collagen wound dressing (Biobrane® ) and fibrin-hyaluronic acid or fibrin-agarose biomaterial without cells (Total N=44). Clinical integration and homeostasis parameters were evaluated every two weeks for two months. Histological and immunohistochemical analyses were performed four and eight weeks after grafting. HA-Skin, AG-Skin and Autograft groups showed a proper clinical integration and epithelization eight weeks later. Scar evaluation revealed better results for Autograft and HA-Skin. Homeostasis analysis indicated similar values of transepidermal water loss and elasticity between HA-Skin (6.42±0.75g/h/m2 , 0.42±0.08AU), Autograft (6.91±1.28g/h/m2 , 0.40±0.08AU) and healthy mouse skin (6.40±0.43g/h/m2 , 0.35±0.03AU). Histological results showed that human TESSs and autografts presented better skin structuration and higher expression of cytokeratins. This study suggests that human TESS based on fibrin-hyaluronic acid biomaterial could be suitable for clinical application in the treatment of several dermatological pathologies (wound healing).

Dietary Selenium Deficiency Facilitated Reduced Stomatin and Phosphatidylserine Externalization, Increasing Erythrocyte Osmotic Fragility in Mice.

Selenium (Se) is an essential trace element that maintains normal physiological functions in organisms. Since the discovery of glutathione peroxidase (GSH-PX), public interest in selenoproteins has gradually increased. Based on previous studies, dietary Se maintains erythrocyte homeostasis through selenoprotein-induced mediation of redox reactions. Furthermore, both the surface phosphatidylserine (PS) and intramembrane stomatin contents can be used as indicators of erythrocyte osmotic fragility. This study focused on the mechanism by which dietary Se deficiency increases erythrocyte osmotic fragility. We fed Se-deficient grain to mice for 8weeks to establish a Se deficiency model in mice. We measured Se levels in the blood as well as the activities of antioxidant enzymes associated with selenoproteins in a Se-deficient environment. We used Western blotting, routine blood analysis, and other methods to detect red blood cell oxidative stress levels, membrane stomatin levels, and PS externalization. Fresh blood was collected to test erythrocyte osmotic fragility. The results showed that antioxidant enzyme activity was affected by dietary Se deficiency. Oxidative stress increased lipid peroxidation and the ROS content in the blood of the mice. Under such conditions, decreased PS exposure and stomatin content in the erythrocyte membrane eventually affected the structure of the erythrocyte membrane and increased erythrocyte osmotic fragility.

Activity-Dependent Neurodegeneration and Neuroplasticity of Auditory Neurons Following Conductive Hearing Loss in Adult Mice.

We examined the functional and structural changes of auditory neurons (ANs) in adult mice after conductive hearing loss (CHL). Earplugs (EPs) were bilaterally inserted in male 8-week-old mice for 4weeks [EP(+) group] and subsequently removed for 4weeks [EP(+/-) group]. We examined the control mice [EP(-) group] with no EPs inserted at 12weeks. The auditory brainstem response (ABR) was measured to determine the cochlear function before and after EP insertion, after EP removal, and at 4weeks following EP removal. We examined the cochleae for hair cell (HC) and spiral ganglion neuron survival, synaptic and neural properties, and AN myelination. There was a significant elevation of the ABR threshold across all tested frequencies after EP insertion. After removing the occlusion, these threshold shifts were fully recovered. Compared with the EP(-) mice, the EP(+) mice showed a significant decrease in the ABR peak 1 amplitude and a significantly prolonged latency at all tested frequencies. There was no significant effect of auditory deprivation on the survival of HCs and ANs. Conversely, auditory deprivation caused significant damage to the synapses and myelin and a significant decrease in the AN size. Although functional changes in the ABR amplitude and latency did not fully recover in the EP(+/-) mice, almost all anatomical changes were fully recovered in the EP(+/-) mice; however, cochlear synapses only showed partial recovery. These results suggest that auditory activities are required to maintain peripheral auditory synapses and myelination in adults. The auditory deprivation model allows for assessment of the mechanisms of synaptopathy and demyelination in the auditory periphery, and synaptic and myelin regeneration in sensorineural hearing loss.

Mealworm larvae (Tenebrio molitor L.) exuviae as a novel prebiotic material for BALB/c mouse gut microbiota.

The objective of this study was to evaluate the effect of yellow mealworm (Tenebrio molitor L.) exuviae (ME) given as a prebiotic in 20% of the diet fed to BALB/c mice. Analysis of the ME revealed that it was mostly composed of crude protein (52.94%), crude fiber (10.70%), and moisture (10.54%). When ME was fed to mice for 8weeks, the number of intestinal lactic acid bacteria increased, reaching similar numbers (4.50 ± 0.80CFU/mL) to those (4.70 ± 0.80CFU/mL) of the control group not fed ME. Microbiome analysis showed that 8weeks feeding of ME promoted the growth of Bifidobacteriaceae and Lactobacillaceae compared to the POS group, indicating the positive effects of feeding 20% ME on the intestinal microbiota of mice. These results suggest that ME can be considered as a dietary prebiotics to improve human gut microbial population, but further application study to human is necessary.

Intracisternal delivery of PEG-coated gold nanoparticles results in high brain penetrance and long-lasting stability

BackgroundThe increasing use of gold nanoparticles (AuNPs) in the field of neuroscience instilled hope for their rapid translation to the clinical practice. AuNPs can be engineered to carry therapeutics or diagnostics in the diseased brain, possibly providing greater cell specificity and low toxicity. Although there is a general enthusiasm for these tools, we are in early stages of their development. Overall, their brain penetrance, stability and cell specificity are critical issues that must be addressed to drive AuNPs to the clinic.ResultsWe studied the kinetic, distribution and stability of PEG-coated AuNPs in mice receiving a single injection into the cisterna magna of the 4th ventricle. AuNPs were conjugated with the fluorescent tag Cy5.5 (Cy5.5-AuNPs) to track their in vivo distribution. Fluorescence levels from such particles were detected in mice for weeks. In situ analysis of brains by immunofluorescence and electron microscopy revealed that Cy5.5-AuNPs penetrated the brain parenchyma, spreading in the CNS parenchyma beneath the 4th ventricle. Cy5.5-AuNPs were preferentially found in neurons, although a subset of resting microglia also entrapped these particles.ConclusionsOur results suggest that the ICM route for delivering gold particles allows the targeting of neurons. This approach might be pursued to carry therapeutics or diagnostics inside a diseased brain with a surgical procedure that is largely used in gene therapy approaches. Furthermore, this approach could be used for radiotherapy, enhancing the agent’s efficacy to kill brain cancer cells.

The severity of lung injury and metabolic disorders induced by ambient PM2.5 exposure is associated with cumulative dose

Lots of epidemiological and experimental studies have found that ambient fine particulate matter (PM2.5) exposure is associated with the development of cardiopulmonary diseases, obesity and diabetes. This study focused on the effects of cumulative PM2.5 exposure on pulmonary and systemic inflammation and insulin resistance. Thirty-two 6-week-old male Balb/c mice were randomly divided into four groups (FA, PM, WEEK and DAY groups) and were continuously or intermittently exposed to concentrated PM2.5 or filtered air (FA) for four weeks using Shanghai Meteorological and Environmental Animal Exposure System (“Shanghai-METAS”). The levels of IL-6 and TNF-α in serum, bronchoalveolar lavage fluid (BALF), lung tissues and white adipose tissue (WAT) were measured. Meanwhile, the expression of NF-κB and phosphor-NF-κB in lung tissue was detected by Western blot. Glucose tolerance and insulin resistance were also determined at the end of exposure. The results found that the mice in PM group displayed moderate inflammatory cell infiltration in lung, whereas the mice in WEEK and DAY groups displayed slight inflammatory cell infiltration in lung. Compared with the mice in FA group, the mRNA expressions of IL-6 and TNF-α in lung tissue and WAT significantly increased in the mice of PM group. Importantly, IL-6 and TNF-α mRNA expressions in PM group were higher than those in WEEK and DAY groups. The protein expression of phospho-NF-κB in lung tissue showed that PM group showed the activation of NF-κB, which was higher than that in the WEEK and DAY groups. Meanwhile, the mice in PM group showed more severe glucose tolerance and insulin resistance than that in the WEEK and DAY groups. The results suggested that the reduction of PM2.5 cumulative exposure may alleviate pulmonary and adipose inflammation, insulin resistance and glucose tolerance impairment. The results provided a clue that the interruption of ambient PM2.5 exposures by systems such as indoor air purification could be of benefit to people’s health.

P091 Inflammation in relation to mucosal barrier function in chronic colitis: a study in time

The pathogenesis of inflammatory bowel diseases (IBD) is multifactorial. Midst in the complex interaction between genetic, environmental and immunological factors is the intestinal barrier. IBD patients have been observed to have increased intestinal permeability which can partially explain the exaggerated immune response towards luminal antigens. Inflammatory mediators perpetuate this barrier defect by causing further damage to the intestinal barrier. Our aim is to investigate the interaction between permeability and inflammation in a chronic colitis mouse model. Colitis was induced in immunodeficient SCID mice by the adoptive transfer of naive T cells, isolated from BALB/c mice. Mice were sacrificed at fixed time points to study disease progression resulting in five groups: control mice (Week 0); colitis mice in week 1, 2, 4 and 6 of the experimental protocol (Week 1; Week 2; Week 4, Week 6; all n = 10). FITC-dextran was used to assess intestinal permeability, next to the messenger RNA of the tight junction proteins Claudin-1 and Occludin. After sacrifice, colonic inflammation was assessed by macro- and microscopic scoring, myeloperoxidase (MPO) activity and cytometric bead array (CBA) for TNF- α and IL-1β . Messenger RNA of transcription factors that regulate T helper (Th) cell differentiation including T-bet (Th1), GATA-3 (Th2), ROR- γt (Th17) were quantified using RT-qPCR technique. Mucosal inflammation appeared gradually in the colon of the diseased mice. Macroscopically there was a significant increase starting from Week 2 on, gradually increasing until Week 6. The same was true for the MPO activity. Microscopically, however, the first significant signs of inflammation were observed at Week 1, which then also gradually increased with the highest score at Week 6. The colonic inflammation was mainly Th1/Th2 driven, since the transcription factors T-bet and GATA-3 were significantly upregulated from respectively Week 2 and Week 4 on. TNF-α and IL-1β were significantly elevated starting from Week 1. Intestinal permeability was significantly elevated at Week 1 and remained elevated at Week 2, 4 and 6. Data are presented as mean±SEM and analysed by one-way ANOVA with multiple comparisons SNK posthoc testing. *<0.05 vs. WEEK 0 # <0.05 vs. WEEK 1. Our results show a gradual increase of intestinal inflammation over time which, for most parameters, started at Week 2. However, we observed an unexpectedly early increase in intestinal permeability at Week 1. This shows that the mucosal barrier function is of great importance in disease onset and could be an interesting therapeutic target or biomarker predicting flares.

Fibroblast growth factor 21 increases hepatic oxidative capacity but not physical activity or energy expenditure in hepatic peroxisome proliferator-activated receptor γ coactivator-1α-deficient mice.

What is the central question of this study? Does a reduction in hepatic peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which has been observed in an insulin-resistant obese state, impair the ability of fibroblast growth factor 21 (FGF21) to modulate metabolism? What is the main finding and its importance? A deficit in hepatic PGC-1α does not compromise the ability of FGF21 to increase hepatic fatty acid oxidation; however, the effects of FGF21 to regulate whole-body metabolism (i.e. total and resting energy expenditure), as well as ambulatory activity, were altered when hepatic PGC-1α was reduced. Fibroblast growth factor 21 (FGF21) treatment drives metabolic improvements, including increased metabolic flux and reduced hepatic steatosis, but the mechanisms responsible for these effects remain to be elucidated fully. We tested whether a targeted reduction in hepatic peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which has been shown to occur with obesity, had a negative impact on the metabolic effects of FGF21. We infused FGF21 (1mgkg-1 day-1 ) or saline in chow-fed wild-type (WT) and liver-specific PGC-1α heterozygous (LPGC-1α) mice for 4weeks. Administration of FGF21 lowered serum insulin and cholesterol (P≤0.05) and tended to lower free fatty acids (P=0.057). The LPGC-1α mice exhibited reduced complete hepatic fatty acid oxidation (FAO; LPGC-1α, 1788±165nmolg-1 h-1 compared with WT, 2572±437nmolg-1 h-1 ; P<0.001), which was normalized by FGF21 treatment (2788±519nmolg-1 h-1 ; P<0.001). FGF21 also increased hepatic incomplete FAO by 12% in both groups and extramitochondrial FAO by 89 and 56% in WT and LPGC-1α mice, respectfully (P=0.001), and lowered hepatic triacylglycerol by 30-40% (P<0.001). Chronic treatment with FGF21 lowered body weight and fat mass (P<0.05), while increasing food consumption (P<0.05), total energy expenditure [7.3±0.60 versus 6.6±0.39 kcal(12h)-1 in WT mice; P=0.009] and resting energy expenditure [5.4±0.89 versus 4.6±0.21kcal(12h)-1 in WT mice; P=0.005]. Interestingly, FGF21 only increased ambulatory activity in the WT mice (P=0.03), without a concomitant increase in non-resting energy expenditure. In conclusion, although reduced hepatic PGC-1α expression was not necessary for FGF21 to increase FAO, it does appear to mediate FGF21-induced changes in total and resting energy expenditure and ambulatory activity in lean mice.

GUB06-046, a novel secretin/glucagon-like peptide 1 co-agonist, decreases food intake, improves glycemic control, and preserves beta cell mass in diabetic mice.

Bariatric surgery is currently the most effective treatment of obesity, which has spurred an interest in developing pharmaceutical mimetics. It is thought that the marked body weight-lowering effects of bariatric surgery involve stimulated secretion of appetite-regulating gut hormones, including glucagon-like peptide 1. We here report that intestinal expression of secretin is markedly upregulated in a rat model of Roux-en-Y gastric bypass, suggesting an additional role of secretin in the beneficial metabolic effects of Roux-en-Y gastric bypass. We therefore developed novel secretin-based peptide co-agonists and identified a lead compound, GUB06-046, that exhibited potent agonism of both the secretin receptor and glucagon-like peptide 1 receptor. Semi-acute administration of GUB06-046 to lean mice significantly decreased cumulative food intake and improved glucose tolerance. Chronic administration of GUB06-046 to diabetic db/db mice for 8weeks improved glycemic control, as indicated by a 39% decrease in fasting blood glucose and 1.6% reduction of plasma HbA1c levels. Stereological analysis of db/db mice pancreata revealed a 78% increase in beta-cell mass after GUB06-046 treatment, with no impact on exocrine pancreas mass or pancreatic duct epithelial mass. The data demonstrate beneficial effects of GUB06-046 on appetite regulation, glucose homeostasis, and beta-cell mass in db/db mice, without proliferative effects on the exocrine pancreas and the pancreatic duct epithelium. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Ectopic models for endochondral ossification: comparing pellet and alginate bead culture methods.

Key aspects of native endochondral bone development and fracture healing can be mimicked in mesenchymal stem cells (MSCs) through standard in vitro chondrogenic induction. Exploiting this phenomenon has recently emerged as an attractive technique to engineer bone tissue, however, relatively little is known about the best conditions for doing so. The objective of the present study was to compare the bone-forming capacity and angiogenic induction of hypertrophic cell constructs containing human adipose-derived stem cells (hASCs) primed for chondrogenesis in two different culture systems: high-density pellets and alginate bead hydrogels. The hASC constructs were subjected to 4weeks of identical chondrogenic induction in vitro, encapsulated in an agarose carrier, and then implanted subcutaneously in immune-compromised mice for 8weeks to evaluate their endochondral potential. At the time of implantation, both pellets and beads expressed aggrecan and type II collagen, as well as alkaline phosphatase (ALP) and type X collagen. Interestingly, ASCs in pellets formed a matrix containing higher glycosaminoglycan and collagen contents than that in beads, and ALP activity per cell was higher in pellets. However, after 8weeks in vivo, pellets and beads induced an equivalent volume of mineralized tissue and a comparable level of vascularization. Although osteocalcin and osteopontin-positive osteogenic tissue and new vascular growth was found within both types of constructs, all appeared to be better distributed throughout the hydrogel beads. The results of this ectopic model indicate that hydrogel culture may be an attractive alternative to cell pellets for bone tissue engineering via the endochondral pathway. Copyright © 2016 John Wiley & Sons, Ltd.

Red raspberry (Rubus idaeus L.) intake decreases oxidative stress in obese diabetic (db/db) mice.

Red raspberry fruit intake was investigated on obese diabetic (db/db) mice for 8weeks. Animals fed isocaloric diets (5.3% freeze-dried raspberry, or control) were assessed for obesity-diabetes-disease risk biomarkers. Results showed that raspberry intake improved antioxidant status and lessened plasma interleukin (IL)-6 (0.3-fold of control, p<0.1); most likely through enhancing glutathione peroxidase (GPx) activity in liver (4.3-fold of control), and in blood (2.1-fold of control). Other disease-risk biomarkers were similar between groups (p>0.05). Plasma levels of total cholesterol (T-CHL), low density lipoprotein-cholesterol (LDL-CHL), and resistin were higher in the raspberry group. Overall, the enhanced detoxifying cell defenses exerted by raspberry intake might be due to its polyphenolics and fibre. This study demonstrates in vivo that raspberry intake, at a dose that can be achieved by human consumption, might protect against diabetes-induced oxidative stress.

Hepatoprotective effect of engineered silver nanoparticles coated bioactive compounds against diethylnitrosamine induced hepatocarcinogenesis in experimental mice

Nanoparticle based drug delivery can rapidly improves the therapeutic potential of anti-cancer agents. The present study focused to evaluate the hepatoprotective activity of silver nanoparticles (AgNPs) synthesized using aqueous extracts of Andrographis paniculata leaves (ApAgNPs) and Semecarpus anacardium nuts (SaAgNPs) against diethylnitrosamine (DEN) induced liver cancer in mice model. The physico-chemical properties of synthesized AgNPs were characterized by Fourier transform infrared (FTIR) spectroscopy, Transmission Electron Microscopy (TEM), Selected Area Electron Diffraction (SAED), X-ray Diffraction (XRD), Energy Dispersive X-ray (EDX) spectrum, Zeta potential and Dynamic Light Scattering (DLS) analysis. The surface plasmon resonance (SPR) absorption spectrum revealed a strong peak at 420nm for both SaAgNPs and ApAgNPs. FTIR results exhibited the presence of possible functional groups in the synthesized AgNPs. TEM analysis determined the hexagonal, and spherical shape of the synthesized silver nanoparticles. The XRD and SAED pattern confirmed the crystalline nature and crystalline size of the AgNPs. EDX result clearly showed strong silver signals in the range between 2 and 4keV. Zeta potential measurements indicated a sharp peak at −3.93 and −13.8mV for ApAgNPs and SaAgNPs, respectively. DLS measurement expressed the particle size distribution was 70 and 60nm for ApAgNPs and SaAgNPs, respectively. DEN (20mg/kg b.wt.) was subjected to induce liver cancer in mice for 8weeks and treated with biosynthesized silver nanoparticles. Interestingly, ApAgNPs and SaAgNPs treated DEN induced animal groups show a decreased level of aspartate amino transferase (AST), alanine amino transferase (ALT), serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) activity and elevated level of catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST) and superoxide dismutase (SOD) activity over untreated DEN control animals group. Histopathological investigation reveals decreased fat accumulation, appearance of binucleated cells in nanoparticle treated animals and showed mere normal cells induced by DEN. Argyrophilic nucleolar organiser region (AgNORs) had a significant decrease in number of acidic proteins and mast cells assay showed decrease of metachromatic cells in nanoparticles treated animal groups over control. Present results strongly suggest that biomolecule coated silver nanoparticles exposure showed potential hepatoprotective effect against DEN induced liver cancer and could be used as an effective anticancer nanodrug.

Naringenin interferes with the anti-diabetic actions of pioglitazone via pharmacodynamic interactions.

Pioglitazone is a peroxisome proliferator-activated receptor gamma (PPARγ) full agonist and useful for the treatment of type 2 diabetes mellitus. Naringenin is a citrus flavonoid with anti-inflammatory actions, which has been shown to prevent obesity-related diseases and to activate PPARγ. The aim of this study was to investigate whether dietary naringenin affects the actions of pioglitazone. We administered naringenin (100mg/kg) and pioglitazone (10mg/kg) to Tsumura Suzuki Obese Diabetes (TSOD) mice for 4weeks and then conducted an oral glucose tolerance test. We found that oral administration of naringenin attenuated the hypoglycemic action of pioglitazone in TSOD mice. However, pioglitazone and naringenin did not affect fasting blood glucose levels, epididymal fat pad weight and body weight changes in this administration period. Pioglitazone suppressed expression of obesity-related adipokines such as tissue inhibitor of metalloproteinases-1 in adipose tissue of TSOD mice, but this effect was attenuated by naringenin. However, naringenin did not affect the pharmacokinetics of pioglitazone after single or repeated administration. Naringenin exhibited weak partial agonist activity in time-resolved fluorescence resonance energy transfer assay, but naringenin interfered with pioglitazone agonism, consistent with partial agonism. Our results suggest that it is advisable to avoid administering a combination of naringenin and pioglitazone.

Preparation of bioactive β-tricalcium phosphate microspheres as bone graft substitute materials

In this study, β-tricalcium phosphate (Ca3PO4, β-TCP) microspheres with different diameters were fabricated via a solid-in-oil-in-water (S/O/W) emulsion method. After soaking in simulated body fluid (SBF), the fabricated β-TCP microspheres were fully covered with a new bone-like apatite layer; subsequent analysis suggested that the microspheres have excellent bioactivity properties, specifically in inducing apatite deposition. The calcium release profiles of the microspheres were tested in pH7.4 Tris-HCl buffer, and results demonstrated that the Ca2+ continually released from microspheres during the two-week test period. We then co-cultured bone marrow stem cells (BMSCs) in vitro with β-TCP microspheres, and performed SEM and confocal microscope analyses to find that β-TCP microspheres efficiently promoted BMSC attachment and bone-related gene expression. The co-cultured BMSCs and microspheres were successfully implanted subcutaneously into nude mice for 8weeks. The H&E neo-tissue staining results showed that abundant new bone-like structures had formed between the β-TCP microspheres, implying that β-TCP microspheres used as a cell carrier and bone graft substitute material show highly promising potential application for irregular-shaped bone defect regeneration.

Social isolation mediated anxiety like behavior is associated with enhanced expression and regulation of BDNF in the female mouse brain

Adverse early life experience is prominent risk factors for numerous psychiatric illnesses, including mood and anxiety disorders. It imposes serious long-term costs on the individual as well as health and social systems. Hence, developing therapies that prevent the long-term consequences of early life stress is of utmost importance, and necessitates a better understanding of the mechanisms by which early life stress triggers long-lasting alterations in gene expression and behavior. Post-weaning isolation rearing of rodents models the behavioral consequences of adverse early life experiences in humans and it is reported to cause anxiety like behavior which is more common in case of females. Therefore, in the present study, we have studied the impact of social isolation of young female mice for 8weeks on the anxiety like behavior and the underlying molecular mechanism. Elevated plus maze and open field test revealed that social isolation caused anxiety like behavior. BDNF, a well-known molecule implicated in the anxiety like behavior, was up-regulated both at the message and protein level in cerebral cortex by social isolation. CREB-1 and CBP, which play a crucial role in BDNF transcription, were up-regulated at mRNA level in cerebral cortex by social isolation. HDAC-2, which negatively regulates BDNF expression, was down-regulated at mRNA and protein level in cerebral cortex by social isolation. Furthermore, BDNF acts in concert with Limk-1, miRNA-132 and miRNA-134 for the regulation of structural and morphological plasticity. Social isolation resulted in up-regulation of Limk-1 mRNA and miRNA-132 expression in the cerebral cortex. MiRNA-134, which inhibits the translation of Limk-1, was decreased in cerebral cortex by social isolation. Taken together, our study suggests that social isolation mediated anxiety like behavior is associated with up-regulation of BDNF expression and concomitant increase in the expression of CBP, CREB-1, Limk-1 and miRNA-132, and decrease in the expression of HDAC-2 and miRNA-134 in the cerebral cortex.

Functional Studies of Ex Vivo Expanded Hematopoietic Stem Cells in Mice and Monkeys

Hematopoietic stem cell (HSC) transplantation has been widely applied for the treatment of malignant blood diseases. However, obtaining sufficient HLA-matched stem/progenitors for cell transplantation is an obstacle for clinical applications.We reported here that an optimal cytokine cocktail in a modified IMDM basal medium was developed that contained stem cell factor, Flt-3 ligand, thrombopoietin, interleukin 3, G-CSF and GM-CSF. Up to 7.3 folds of expanded CD34+ cells with 66.3% CD34+ of whole cells were obtained after 4 days' culture from human umbilical cord blood. Colony-forming unit (CFU) assays showed that expanded CD34+ cells retained the same renewal ability as the pre-expanded counterparts. To test the repopulating ability of the expanded CD34+ in vivo, sixteen NOD/SCID mice were divided to four groups and injected with saline (group 1), 0.4 million pre-expanded CD34+ cells (group 2), 0.4 million 4-day expanded CD34+ cells (group 3), and 2.9 million expanded CD34+ cells (group 4), respectively. Multi-lineage differentiations in the peripheral blood were assessed by flow cytometry with antibodies against a panel of human cell surface markers. In week 3, human CD34+ cells were decreased below 1% in groups 2 and 3, and 1.717%±0.65% in group 4. Whereas, human CD45+ was increased up to 3.831%±1.54%, 3.108%±1.18% and 10.408%±3.27% for groups 2, 3 and 4, respectively. The other human CD41+, CD71+ and CD15+ were also increased in groups 2-4. No expression of any human cell lineage markers was detected in group 1, indicating that expanded human CD34+ cells possessed the repopulating viability of HSCs in vivo. Furthermore, in week 12, the human CD34+ cells were re-isolated from the bone morrow of the mice (one mouse from each group). The isolated human CD34+ cells were again transfused into new NOD/SCID mice for the secondary transplantation. In week 6, human CD45+, CD15+ and CD19+ were observed from the bone morrow cells of sacrificed mice. On the other hand, human CD45+, CD15+ and CD19+ were also detectable in bone morrow cells for all remaining mice in week 24, suggesting that the expanded CD34+ cells could be successfully engrafted into mice in a long term.In addition, the cytokine cocktail was further evaluated for its safety and efficacy in primates. The CD34+ cells were isolated from the peripheral blood of cynomolgus monkeys and expanded for about 8 folds were obtained on day 9. Harvested CD34+ cells were transducted with the gene of green fluorescent protein (GFP). These cynomolgus monkeys (n=11) were administered with cyclophosphamide via intravenous injection at a dose of 50 mg/kg/day for two days. The myelo-suppressed monkeys were randomly divided into three groups as follows: a control group treated with saline (n=3), a group with autologous CD34- cells (n=3), and a group treated with GFP-labeled, expanded autologous CD34+ cells (n=5), respectively. After autologous transplantation, routine blood tests and flow cytometry analysis were performed to determine the proportion of GFP+ cells in the peripheral blood.The flow cytometry analysis revealed that the white blood cells (WBC), neutrophil (NEU) and platelets (PLT) in peripheral blood of cynomolgus monkeys were completely recovered to the normal levels on days 12, 11 and 10 post autologous transplantation of expended CD34+ cells, respectively. For the control groups, WBC, NEU and PLT returned to the normal on days 22, 22 and 12 for the saline treatment and on days 20, 20 and 12 for the CD34- group, respectively. Similarly, the lymphocytes of cynomolgus monkeys were recovered completely on day 20 post autologous CD34+ cell transplantation compared with the saline control (day 25) and the CD34- group (day 22). On day 30 after the autologous transplantation, the GFP+ ratio in CD45+ populations was around 2% in the peripheral blood. GFP+ cells (ranging from 1.8% to 4.1%) were also detected in bone morrow of cynomolgus monkeys. All primates transplanted with the expanded autologous CD34+ cells have survived for 18 months without any noticeable abnormalities.In conclusion, our results indicate that expanded CD34+ cells can be safely and efficiently used for repopulating stem cell compartment in mice and primates, underscoring the potential applications in the clinic. Furthermore, the results from successful autologous transplantation of cynomolgus CD34+ cells strongly suggest a possible application for personalized treatment of blood diseases. DisclosuresQin:Biopharmagen. corp: Employment. Ren:Biopharmagen corp: Employment.

Characterization of Humoral Responses Induced by an H7N9 Influenza Virus-Like Particle Vaccine in BALB/C Mice.

In April 2013, human infections with a novel avian influenza (H7N9) virus emerged in China. It has caused serious concerns for public health throughout the world. However, there is presently no effective treatment, and an A (H7N9) H7 subtype influenza vaccine is not available. Vaccination with virus-like particles (VLPs) has showed considerable promise for many other subtype influenza viruses. To produce H7N9 VLPs, full length, unmodified hemagglutinin (HA), neuraminidase (NA), and matrix1 (M1) genes from the A/Wuxi/1/2013(H7N9) were cloned into a pCDNA5.1 FRT vector. By co-transfection, VLPs containing HA, NA, and M1 were secreted by 293T cells. VLPs were purified by ultracentrifugation and injected into mice by the intramuscular route. In animal experiments, humoral and cellular immunoresponse were all triggered by H7N9 VLPs. High levels of specific antibodies and the isotypes of IgG were detected by ELISA. Anamnestic cellular immune responses were examined by detecting specific cytotoxic T cell for IFN-γ production in ELISPOT assay. The hemagglutination-inhibition (HAI) against the homologous virus was more than 1:64, and cross-reactive HAI titers against the heterologous virus (H1N1 and H3N2) were more than 1:16. Moreover, VLPs immunized mice showed a rapid increase of neutralizing antibodies, with neutralizing antibody titers more than 1:8, which increased four-fold against PBS immunized mice in week four. By week six, the mice had high neutralization ability against the given strain and held a potent homologous virus neutralizing capacity. Thus, VLPs represent a potential strategy for the development of a safe and effective vaccine against novel avian influenza (H7N9) virus.