Normal Control Mice Research Articles

Effect of Probiotics on Pharmacokinetics of Orally Administered Acetaminophen in Mice.

Orally administered probiotics change gut microbiota composition and enzyme activities. Thus, coadministration of probiotics with drugs may lead to changes in the pharmacokinetic parameters of the drugs. In this study, we investigated the pharmacokinetics of acetaminophen in mice treated with probiotics. Oral administration of probiotics changed the gut microbiota composition in the mice. Of these probiotics, Lactobacillus reuteri K8 increased the numbers of clostridia, bifidobacteria, and enterococci, and Lactobacillus rhamnosus K9 decreased the number of bifidobacteria, determined by culturing in selective media. Next, we performed a pharmacokinetic study of acetaminophen in mice orally treated with K8 and K9 for 3 days. Treatment with K8 reduced the area under the curve (AUC) of orally administered acetaminophen to 68.4% compared with normal control mice, whereas K9 did not affect the AUC of acetaminophen. Oral administration to mice of K8, which degraded acetaminophen, increased the degradation of acetaminophen by gut microbiota, whereas K9 treatment did not affect it. Treatment with K8 increased the number of L. reuteri adhered in the upper small intestine, whereas the number of L. rhamnosus was not affected by treatment with K8 or K9. K8 increased the number of cyanobacteria, whereas K9 increased the number of deferribacteres. These results suggest that the intake of probiotics may make the absorption of orally administered drugs fluctuate through the disturbance of gut microbiota-mediated drug metabolism and that the subsequent impact on microbiota metabolism could result in altered systemic concentrations of the intact drug.

Arthritic role of Porphyromonas gingivalis in collagen-induced arthritis mice.

Epidemiological studies show an association between rheumatoid arthritis (RA) and periodontal disease. Porphyromonas gingivalis (P.gingivalis) is a well-known pathogen in periodontitis. This study investigated the pathogenic effects of P.gingivalis on autoimmune arthritis in vivo. Collagen-induced arthritis (CIA) mice were intraperitoneally injected with W83 and 2561 strains of P.gingivalis. Infection with P.gingivalis exacerbated arthritis score in CIA mice. Synovial inflammation and bone destruction in CIA mice infected with P.gingivalis were more severe than in uninfected CIA mice. Both W83 and 2561 strains were more pro-arthritic after arthritis symptom was fully activated. Interestingly, only W83 strain was arthritogenic before autoimmune reaction initiated. Citrullination was detected in synovial tissue of CIA mice and CIA mice inoculated with P.gingivalis, but not in normal control mice. The citrullinated area was greater in P.gingivalis-infected CIA mice than in non-infected CIA mice. This study showed that P.gingivalis exacerbated disease in a mouse model of autoimmune arthritis and increased the expression of citrullinated antigens in the synovium. The arthritogenic effects of P.gingivalis were at least in part, dependent upon the bacterial strain with or without fimbriae expression, route and time of infection. P.gingivalis-mediated citrullination may explain the possible link between periodontal disease and RA.

Smooth Muscle-Selective Nuclear Factor-κB Inhibition Reduces Phosphate-Induced Arterial Medial Calcification in Mice With Chronic Kidney Disease.

BackgroundHyperphosphatemia is a major factor promoting the formation of arterial medial calcification in chronic kidney disease (CKD). However, arterial medial calcification begins to occur during the early stages of CKD, when hyperphosphatemia is not yet apparent. It is predicted that other factors also play a role. The aim of the present study was to determine the role of pro‐inflammatory nuclear factor‐κB (NF‐κB) signaling in smooth muscle cells (SMCs) for phosphate‐induced arterial medial calcification in CKD mice.Methods and ResultsWe first sought to establish a novel mouse model of CKD with arterial medial calcification. CKD was induced in DBA/2 mice by feeding them a low concentration of adenine, and these mice were fed a normal or high‐phosphorus diet. Severe calcification was seen in CKD mice fed the high‐phosphorus diet, while it was undetectable in CKD mice fed the normal phosphorus diet or control mice fed the high‐phosphorus diet. Arterial medial calcification was accompanied by phenotypic switching of SMCs into osteogenic cells. Interestingly, NF‐κB inhibitors, tempol and triptolide, both reduced arterial medial calcification in CKD mice fed the high‐phosphorus diet. Moreover, formation of arterial medial calcification, as well as SMC phenotypic switching, was also markedly attenuated in transgenic mice, in which the NF‐κB activity was inhibited selectively in SMCs. Mechanistic studies revealed that Krüppel‐like factor 4 was involved in NF‐κB‐induced SMC phenotypic switching and calcification.ConclusionsResults of the present studies suggest that the NF‐κB signaling in SMCs plays an important role in high phosphate‐induced arterial medial calcification in CKD.

Effects of Pu-erh ripened tea on hyperuricemic mice studied by serum metabolomics.

To evaluate effects of Pu-erh ripened tea in hyperuricemic mice, a mouse hyperuricemia model was developed by oral administration of potassium oxonate for 7 d. Serum metabolomics, based on gas chromatography-mass spectrometry, was used to generate metabolic profiles from normal control, hyperuricemic and allopurinol-treated hyperuricemic mice, as well as hyperuricemic mice given Pu-erh ripened tea at three doses. Pu-erh ripened tea significantly lowered serum uric acid levels. Twelve potential biomarkers associated with hyperuricemia were identified. Pu-erh ripened tea and allopurinol differed in their metabolic effects in the hyperuricemic mice. Levels of glutamic acid, indolelactate, L-allothreonine, nicotinoylglycine, isoleucine, l-cysteine and glycocyamine, all involved in amino acid metabolism, were significantly changed in hyperuricemic mice treated Pu-erh ripened tea. Thus, modulating amino acid metabolism might be the primary mechanism of anti-hyperuricemia by Pu-erh ripened tea.

Anxiolytic-like effect of Bifidobacterium adolescentis IM38 in mice with or without immobilisation stress.

To better understand the role of gut microbiota in the anxiety, we isolated bifidobacteria and lactobacilli from the human faecal microbiota, investigated their inhibitory effects on the expression of interleukin (IL)-6 and tumour necrosis factor (TNF)-α in lipopolysaccharide-stimulated macrophages, and examined the anxiolytic-like effect of Bifidobacterium adolescentis IM38 in mice treated with or without immobilisation stress using the elevated plus maze (EPM) task. Oral administration of IM38 at a dose of 1×109 cfu/mouse showed a significant anxiolytic-like effect both in mice exposed to immobilisation stress and in control mice using the EPM test (P<0.05). Moreover, IM38 treatment significantly increased the amount of time spent on open arms and open arm entries. The anxiolytic-like effect of IM38 was comparable to that of buspirone (1 mg/kg). Moreover, this anxiolytic-like effect was blocked by treatment with flumazenil (3 mg/kg, i.p.), a benzodiazepine receptor antagonist, but was not affected by treatment with bicuculine or WAY-100635. IM38 treatment also reduced the blood levels of corticosterone and IL-6 in mice with or without immobilisation stress, whereas this effect was abolished by treatment with flumazenil. IM38 treatment also reduced the blood TNF-α level in mice subjected to immobilisation stress but not in normal control mice. Treatment with flumazenil also significantly increased TNF-α and IL-6 levels in immobilisation stress-free mice treated with IM38. These findings suggest that IM38 may attenuate anxiety through modulation of the benzodiazepine site on the GABAA receptor and modulate stress-related cytokine expression.

ROS-induced Oxidative Injury involved in Pathogenesis of Fungal Keratitis via p38 MAPK Activation

This study was to explore the mechanism by which reactive oxygen species (ROS)-induced oxidative stress involved in the pathogenesis of fungal keratitis using an in vivo experimental keratitis mouse model and an in vitro culture model of human corneal epithelial cells (HCECs). Compared to normal control mice and HCECs, ROS production was markedly increased in fungal corneas and HCECs exposed to Candida albicans, accompanied by p38 mitogen-activated protein kinases (MAPK) activation. Increased products of oxidative markers, malondialdehyde (MDA), 4–hydroxynonenal (HNE), mitochondria DNA 8-OHdG and aconitase-2 were observed in fungal infected corneas and HCECs. Fungal infection also increased the mRNA expression and protein production of heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2), with suppressed levels of antioxidant enzymes, superoxide dismutase-1 (SOD1), glutathione peroxidase-1 (GPx1) and peroxiredoxin-4 (PRDX4). Interestingly, the levels of ROS, oxidative markers and oxygenases were significantly reduced by co-cultured p38 inhibitor SB203580. Furthermore, SB203580 restored the levels of antioxidant enzymes suppressed by fungus. Our findings demonstrated for the first time that ROS-induced oxidative injury is involved in pathogenesis of fungal keratitis via p38 MAPK pathway, suggesting the novel therapeutic targets for the potential treatment of fungal keratitis.

Restoring diabetes-induced autophagic flux arrest in ischemic/reperfused heart by ADIPOR (adiponectin receptor) activation involves both AMPK-dependent and AMPK-independent signaling

ABSTRACTMacroautophagy/autophagy is increasingly recognized as an important regulator of myocardial ischemia-reperfusion (MI-R) injury. However, whether and how diabetes may alter autophagy in response to MI-R remains unknown. Deficiency of ADIPOQ, a cardioprotective molecule, markedly increases MI-R injury. However, the role of diabetic hypoadiponectinemia in cardiac autophagy alteration after MI-R is unclear. Utilizing normal control (NC), high-fat-diet-induced diabetes, and Adipoq knockout (adipoq−/−) mice, we demonstrated that autophagosome formation was modestly inhibited and autophagosome clearance was markedly impaired in the diabetic heart subjected to MI-R. adipoq−/− largely reproduced the phenotypic alterations observed in the ischemic-reperfused diabetic heart. Treatment of diabetic and adipoq−/− mice with AdipoRon, a novel ADIPOR (adiponectin receptor) agonist, stimulated autophagosome formation, markedly increased autophagosome clearance, reduced infarct size, and improved cardiac function (P < 0.01 vs vehicle). Mechanistically, AdipoRon caused significant phosphorylation of AMPK-BECN1 (Ser93/Thr119)-class III PtdIns3K (Ser164) and enhanced lysosome protein LAMP2 expression both in vivo and in isolated adult cardiomyocytes. Pharmacological AMPK inhibition or genetic Prkaa2 mutation abolished AdipoRon-induced BECN1 (Ser93/Thr119)-PtdIns3K (Ser164) phosphorylation and AdipoRon-stimulated autophagosome formation. However, AdipoRon-induced LAMP2 expression, AdipoRon-stimulated autophagosome clearance, and AdipoRon-suppressed superoxide generation were not affected by AMPK inhibition. Treatment with MnTMPyP (a superoxide scavenger) increased LAMP2 expression and stimulated autophagosome clearance in simulated ischemic-reperfused cardiomyocytes. However, no additive effect between AdipoRon and MnTMPyP was observed. Collectively, these results demonstrate that hypoadiponectinemia impairs autophagic flux, contributing to enhanced MI-R injury in the diabetic state. ADIPOR activation restores AMPK-mediated autophagosome formation and antioxidant-mediated autophagosome clearance, representing a novel intervention effective against MI-R injury in diabetic conditions.

Longevity is impacted by growth hormone action during early postnatal period.

Life-long lack of growth hormone (GH) action can produce remarkable extension of longevity in mice. Here we report that GH treatment limited to a few weeks during development influences the lifespan of long-lived Ames dwarf and normal littermate control mice in a genotype and sex-specific manner. Studies in a separate cohort of Ames dwarf mice show that this short period of the GH exposure during early development produces persistent phenotypic, metabolic and molecular changes that are evident in late adult life. These effects may represent mechanisms responsible for reduced longevity of dwarf mice exposed to GH treatment early in life. Our data suggest that developmental programming of aging importantly contributes to (and perhaps explains) the well documented developmental origins of adult disease.

The hypoglycemic effects of Viola tianshanica Maxim

Objective To explore the hypoglycemic effects of Viola tianshanica Maxim. Methods Extraction conditions by orthogonal design research, in total flavonoids as index by reflux extraction for Viola tianshanica Maxim extracted.Established mouse model of diabetes with streptozotocin(STZ), reused Viola tianshanica Maxim extract for hypoglycemic experiment.The mice were divided into high dose group, middle dosage group, low dose group, blank control group, positive control group and normal mice group, 15 mice in each group.The mice blood glucose and body weight of the different groups were observed. Results Viola tianshanica Maxim in a high concentration had significantly hypoglycemic effect in diabetic mice, average blood glucose level decreased from (18.4±5.8)mmol/L to (11.6±3.4)mmol/L, and the concentration of the extract could reduce the level of blood glucose in diabetic mice, average blood glucose level decreased from (24.8±5.6)mmol/L to (18.5±4.6)mmol/L, the differences were statistically significant(all P<0.05). Conclusion Viola tianshanica Maxim crude extract has a certain hypoglycemic effect. Key words: Violaceae; Flavones; Hypoglycins

The protective effect of Ginsenoside Rg1 on aging mouse pancreas damage induced by D-galactose.

The protective effect and mechanism of Ginsenoside Rg1 on aging mouse pancreas damaged by D-galactose (D-gal)-induced was studied. Two-month-old male C57BL/6J mice were randomly divided into three groups of 10 mice per group. The D-gal group of mice received hypodermic injection of D-gal (120 mg/kg/day) for 42 days; the Rg1+D-gal group of mice receiving D-gal + intraperitoneal injection Rg1 (40 mg/kg/day) for 27 days from the 16th day of D-gal replication; and the naïve group that constituted the normal control mice receiving the same dose of saline instead of the drug. The related indicators were tested on the second day after modeling and administration. Fasting blood glucose (FBG), oral glucose tolerance test (OGTT) and fasting insulin level were measured by taking peripheral blood. Samples of pancreas were weighed and visceral index was calculated. Paraffin sections were prepared. H&E staining sections were produced to observe pancreatic tissue morphology. Immunohistochemical staining was used to observe advanced glycation end products (AGEs) and integral optical density (IOD) of stained positive tissue in pancreas. Ultrathin slices were used to observe ultrastructural change of pancreatic tissue. Frozen sections were prepared to test the relative optical density of positive cells that were stained by senescence-associated β-galactosidase (SA-β-gal) in pancreatic tissue. Superoxide dismutase (SOD), malonaldehyde (MDA) and total antioxidant capacity (T-AOC) were detected by preparing pancreas tissue homogenates. Compared with the control group, Rg1+D-gal mice had significantly decreased pancreatic wet weight and visceral index and significantly lower FBG; OGTT for 30 and 120 min. There was no significant difference of the blood sugar level between the groups. The area under the curve and the number and size of the nucleated cells within islet were markedly reduced. In addition, SA-β-gal-positive particles in pancreas tissue intracytoplasmic cells significantly decreased and relative optical density also reduced. The IOD of AGEs in pancreas tissue and MDA content decreased. SOD and T-AOC activity significantly increased. Ginsenoside Rg1 can be effective antagonistic structure and function of the pancreas injury induced by D-gal. The mechanism may be associated with reducing oxidative damage.

Effects of interleukin-6 in promoting corneal epithelial stem/progenitor cell regeneration and accelerating corneal epithelial wound healing in diabetic mouse

Background Interleukin-6 (IL-6) is a pleiotropic cytokine involving in inflammation and wound healing.Previous report found that IL-6 increases phosphorylated STAT3 (p-STAT3) level and promotes corneal epithelial wound healing by stimulating migration.However, the essential role of IL-6 in corneal epithelial wound healing and the expression changes in diabetic mellitus remains unknown. Objective This study was to explore the roles of IL-6 in corneal epithelial proliferation and wound healing in both normal and diabetic mice. Methods Fifty-two normal C57BL/6 mice were randomized into normal control group (32 mice) and diabetic group (20 mice). Type 1 diabetic mellitus was induced by intraperitoneal injections of 50 mg/kg streptozotocin once per day for consecutive 5 days in the mice of the diabetic group.Whole corneal epithelium was scraped in all mice, and the corneal epithelial defect area was examined by fluorescein staining in 24, 48 and 72 hours after corneal epithelium removal.Recombinant mouse IL-6 or anti-IL-6 blocking antibody of 5 μl were subconjunctivally injected according to the grouping and contrasted with PBS injection group or isotype control antibody group, respectively.TKE2 cells, a mouse corneal epithelial stem/progenitor cell line, were trypsinized and incubated in the KSFM with different concentrations of IL-6 or without IL-6, and colony formation efficency (CFE) was examined by crystal violet staining.The expressions of ΔNP63 and Ki67, specific makers of stem cells, were detected by immunofluorescine technology.The expressions of ΔNP63, Ki67 and p-STAT3 proteins were assayed in the cells by Western blot, respectively.The expression of IL-6 mRNA and protein in the regenerated corneal epithelium was detected by real time quantitative PCR and ELISA.The use and care of the mice complied with the Statement of Association for Research in Vision and Ophthalmology. Results The percentage of residual corneal epithelium defect area with initial detect area was gradually shrinked over time after PBS and IL-6 injection in both normal control mice and diabetic mice, and the percentage of residual corneal epithelium defect area was significantly reduced in the IL-6 injected group compared with the PBS injected group (normal control group: Fgroup=19.982, P<0.01; Ftime =589.350, P<0.01; Diabetic group: Fgroup =25.411, P<0.01; Ftime =334.807, P<0.01). The CFE was (13.23±1.12)%, (15.87±1.30)%, (21.69±1.62)%, (25.33±1.28)% and (18.67±1.54)% in the blank control group and 10, 20, 50, 100 ng/ml IL-6-treated groups, respectively, showing a gradual increase of CFE dependent upon IL-6 concetrations (F=35.547, P<0.01). The expressions of ΔNP63, Ki67, p-STAT3 proteins in the cells were gradually increased over time after 50 ng/ml IL-6 treated for 5, 10, 15, 30 and 60 minutes, and the relative expression level of the cytokines was significnatly higher in the IL-6 cultured groups than that without IL-6 culture group (all at P<0.05). The relative expression of IL-6 mRNA in the regenerated corneal epithelilum was 0.45±0.21 and 1.00±0.16 in the diabetic group and normal control group, respectively, and compared with the normal control group, the expression of IL-6 mRNA reduced by 56% (t=3.42, P=0.03). The content of IL-6 protein in regenerated corneal epithelium of the diabetic group was (257±12) ng/μl, which was significantly lower than (323±17) ng/μl of the normal control group (t=5.60, P<0.01). Conclusions IL-6 promotes the proliferation and regeneration of corneal limbal stem cells to repair defected corneal epithelium by activating STAT3 signaling pathway in both normal and diabetic mice, while the blocking of endogenous IL-6 impairs the corneal epithelial cell activation and wound healing. Key words: Interleukin-6; Corneal injuries; Cell proliferation/drug effects; STAT3 transcription factor/metabolism; Wound healing/drug effects; Diabetes mellitus; Corneal epithelial stem/progenitor cells; Mice, inbred C57BL

Liraglutide reduces body weight by upregulation of adenylate cyclase 3

Objective:According to recent studies, adenylate cyclase 3 (AC3) is associated with obesity. Liraglutide reduces blood glucose levels and body weight (BW). We performed a 2 × 2 factorial experiment to study the relationships among AC3, liraglutide and obesity and to obtain a more comprehensive understanding of the mechanisms underlying the physiological effects of liraglutide on obesity.Methods:A high-fat diet was used to induce obesity in C57BL/6J mice. Both the normal and obese mice were treated with liraglutide (1 mg kg−1) or saline twice daily for 8 weeks. The hepatic levels of the AC3 and glucagon-like peptide receptor (GLP-1R) mRNAs and proteins were measured by quantitative real-time PCR and western blotting, respectively. The serum AC3 levels were detected using a rat/mouse AC3 enzyme-linked immunosorbent assay kit.Results:The administration of liraglutide significantly decreased the BW in obese mice and normal control mice. The BW of obese mice exhibited a more obvious decrease. Hepatic AC3 mRNA and protein levels and serum AC3 levels were significantly reduced in obese mice compared with those in normal control mice. The administration of liraglutide significantly increased the hepatic expression of the AC3 and GLP-1R mRNAs and proteins and serum AC3 levels. The hepatic expression of the AC3 mRNA and protein and serum AC3 levels were negatively correlated with BW loss in the liraglutide-treated group. Pearson’s correlation coefficients for these comparisons are r=−0.448, P=0.048; r=−0.478, P=0.046; and r=−0.909, P=0.000, respectively.Conclusions:Based on our research, liraglutide reduces BW, possibly by increasing the expression of AC3.

Anti-inflammatory effect of cinnamaldehyde and linalool from the leaf essential oil of Cinnamomum osmophloeum Kanehira in endotoxin-induced mice

Cinnamomum osmophloeum Kanehira is a Taiwan native plant that belongs to genus Cinnamomum and is also known as pseudocinnamomum or indigenous cinnamon. Its leaf is traditionally used by local people in cooking and as folk therapy. We previously demonstrated the chemical composition and anti-inflammatory effect of leaf essential oil of Cinnamomum osmophloeum Kanehira of linalool chemotype in streptozotocin-induced diabetic rats and on endotoxin-injected mice. The aim of the present study is to evaluate whether cinnamaldehyde and linalool the active anti-inflammatory compounds in leaf essential oil of Cinnamomum osmophloeum Kanehira. Before the injection of endotoxin, C57BL/6 mice of the experimental groups were administered cinnamaldehyde (0.45 or 0.9 mg/kg body weight) or linalool (2.6 or 5.2 mg/kg body weight), mice of the positive control group were administered the leaf essential oil (13 mg/kg body weight), and mice of the negative group were administered vehicle (corn oil, 4 mL/kg body weight) by gavage every other day for two weeks. All mice received endotoxin (i.p. 10 mg/mL/kg body weight) the next day after the final administration and were killed 12 h after the injection. Normal control mice were pretreated with vehicle followed by the injection with saline. None of the treatment found to affect body weight or food or water intake of mice before the injection of endotoxin. Cinnamaldehyde and linalool were found significantly reversed endotoxin-induced body weight loss and lymphoid organ enlargement compared with vehicle (P < 0.05). Both compounds also significantly lowered endotoxin-induced levels of peripheral nitrate/nitrite, interleukin (IL)-1β, IL-18, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and High-mobility group box 1 protein (HMGB-1), and levels of nitrate/nitrite, IL-1β, TNF-α, and IFN-γ in spleen and mesenteric lymph nodes (MLNs) (P < 0.05). Endotoxin-induced expression of toll-like receptor 4 (TLR4), Myeloid differentiation primary response gene 88 (MyD88), myeloid differentiation protein 2 (MD2), Nod-like receptor family, pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), and caspase-1 in spleen and mesenteric lymph nodes (MLNs) were inhibited by all tested doses of cinnamaldehyde and linalool (P < 0.05). Subsequently, the activation of nuclear factor (NF)-κB and the activity of caspase-1 in spleen and MLNs were also suppressed by these two compounds (P < 0.05). In addition, cinnamaldehyde and linalool at the dose equivalent to their corresponding content in the tested dose of the leaf essential oil, which was 0.9 mg/kg and 5.2 mg/kg, respectively, showed similar or slightly less inhibitory activity for most of these inflammatory parameters compared with that of the leaf essential oil. Our data confirmed the potential use of leaf essential oil of Cinnamomum osmophloeum Kanehira as an anti-inflammatory natural product and provide evidence for cinnamaldehyde and linalool as two potent agents for prophylactic use in health problems associated with inflammations that being attributed to over-activated TLR4 and/or NLRP3 signaling pathways.

A novel assay to determine acetylcholinesterase activity: The application potential for screening of drugs against Alzheimer's disease.

Acetycholinesterase (AChE) that regulates hydrolysis of acetylcholine (ACh) in the brain, is an important target for treatment of Alzheimer's disease (AD), a feature of which is ACh deficiency. However, the methods to precisely determine AChE activity are still under development. We developed a new method to exploit acetylcholine-d4 as a surrogate substrate of ACh and measure product choline-d4 via liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay detected activity of AChE present in the normal mouse brain, which is consistent with the standard Ellman assay that determines products spectrophotometrically. In AD mouse models, the result of LC-MS/MS assay showed significant higher AChE activity than that seen in control normal mice, while treatment of AD mice with an AChE inhibitor, huperzine A, led to partial decreases in AChE activity. Our results suggest that this surrogate-based LC-MS/MS method is a new, sensitive and convenient assay for the determination of AChE activity, providing a useful means for screening active compounds that target AChE.

Annals of Neurology: Volume 81, Number 4, April 2017

ON THE COVER: A photomicrograph of neuromuscular junctions in a normal control mouse triangular sterni muscle. Acetylcholine receptors on the postsynaptic membrane are stained red with alphabungarotoxin, and the motor axons are stained green with neurofilament NF150 and synaptotagmin‐2 antibodies. In mice with mutations of the Vamp1 (vesicle associated membrane protein 1), which causes a congenital myasthenic syndrome similar to humans, the neuromuscular junctions are smaller. See Salpietro et al, pp. 597‐603, this issue. Ann Neurol 2017;81:1–1

Reduced Klotho expression contributes to poor survival rates in human patients with ovarian cancer, and overexpression of Klotho inhibits the progression of ovarian cancer partly via the inhibition of systemic inflammation in nude mice.

Klotho is a recently discovered anti‑aging gene, which has been reported as a tumor suppressor in numerous human malignancies; however, the role of Klotho in human ovarian cancer remains to be elucidated. The aim of the present study was to detect the expression of Klotho and evaluate its association with the progression of human ovarian cancer. A clinical follow‑up study of 120patients with ovarian cancer and 78normal controls was conducted. The expression levels of Klotho were determined by western blotting and immunohistochemistry. The results demonstrated that high Klotho expression levels were detected in all normal controls, whereas the positive rate of Klotho was 61.6% in the ovarian cancer group, which was significantly decreased compared with in the control group (P<0.01). Furthermore, reduced Klotho expression was significantly correlated with decreased survival rates in patients with ovarian cancer (P=0.025). Subsequently, Klotho levels were detected in seven human ovarian cancer cell lines by western blotting. The results demonstrated that the highest levels of Klotho were detected in CaOV3 cells, medium levels of Klotho were detected in CaOV4 and SKOV‑3 cells, and almost no Klotho was detected in the other four cell lines: OVCA432, OVCAR‑5, OVCAR‑8 and A2780 cells. The association between Klotho levels and cell proliferation was determined by MTT assay, and the results indicated that higher levels of Klotho inhibited the proliferation of A2780 and OVCAR‑5 cells, whereas reduced Klotho expression promoted cell growth of CaOV3 and SKOV‑3 cells. In addition, the plasma levels of inflammatory cytokines in tumor‑bearing mice and normal control mice were detected by enzyme‑linked immunosorbent assay, and plasma interleukin (IL)‑6 and IL‑1β levels were elevated in all tumor‑bearing mice. Notably, the mRNA expression levels of IL‑6 were significantly higher in the liver, ovaries and kidneys of Klotho‑/‑ mice compared with in wild type mice (P<0.01), thus indicating that aberrant Klotho expression may contribute to systemic inflammation in Klotho‑/‑ mice. Finally, the invivo antitumor role of aberrant Klotho expression was determined in a nude mice model. A2780 cells were transfected with pCMV6‑Klotho and the stably transfected cells were screened; the mice were injected with the stably transfected cells. The results indicated that tumor volume and tumor weight were significantly decreased in the pCMV6‑Klotho group compared with in the pCMV6 vector group (P<0.01). These findings suggest that overexpression of Klotho may suppress tumor growth in animal models. In conclusion, Klotho was demonstrated to act as a potent tumor suppressor in human ovarian cancer cells. Reduced Klotho expression was detected in the specimens of patients with ovarian cancer, and overexpression of Klotho significantly inhibited cell proliferation of human ovarian cancer cells. Therefore, Klotho may be considered a useful key target for the molecular therapy of human ovarian cancer.

Interleukin-17A exacerbates high-fat diet-induced hepatic steatosis by inhibiting fatty acid β-oxidation

There is a growing body of evidence that the interleukin-17A (IL-17A) signaling pathway contributes to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). However, the mechanism by which IL-17A signaling induces hepatocyte injury is unclear. The aim of the present study was to investigate the significance of the IL-17A axis in NAFLD and to explore the role of IL-17A in high-fat diet (HFD)-induced NAFLD in C57BL/6 mice and oleic acid (OA)-induced lipid accumulation in hepatocytes. Firstly, Consistent upregulation of IL-17A was observed in the HFD-induced steatosis mice but not the normal chow-fed control mice. Administration of IL-17A impaired liver function, aggravated hepatic lipid accumulation by inhibiting fatty acid oxidation in the HFD mice. Conversely, inhibition of IL-17A using an anti-IL-17A monoclonal antibody (mAb) significantly attenuated HFD-induced liver injury. Furthermore, IL-17A accelerated hepatic steatosis through activation of the JNK-PPARα pathway in the HFD mice and OA-preloaded hepatocytes. ConclusionThe present study demonstrated that a high fat diet induces IL-17A expression, which exacerbates the progression of NAFLD by inhibiting fatty acid β-oxidation and promoting the accumulation of triglycerides (TG).

Osteogenic differentiation potential of adipose-derived stem cells from ovariectomized mice.

Osteoporosis (OP) is a systemic disease caused by imbalance between bone resorption and bone formation, commonly resulting from post-menopausal oestrogen deficiency. Although osteogenic differentiation potential of adipose-derived stem cells (ASCs) has been demonstrated, the effect of OP on osteogenic differentiation of ASCs remains unclear. Here, our work has been designed to compare proliferative capacity and osteogenic differentiation ability of ASCs obtained from osteoporotic mice and normal control mice. Twenty 14-week-old female C57BL/6 mice were randomly divided into two groups: one, the ovariectomy (OVX) group (n=10), the other being the sham operated (Sham) group (n=10). ASCs and OP-ASCs were obtained from subcutaneous fat of female inguinal sites. Cells were passaged three times prior to subsequent experimentation. The xCELLigence system was used to monitor cell adhesion and proliferation. Mineralized nodules of differentiated ASCs and OP-ASCs were analysed using Alizarin red staining after osteogenic induction. Expressions of osteogenic-specific genes including osteopontin (Opn) and runt-related transcription factor 2 (Runx2) were assessed by real-time PCR and expression of bone-related proteins was detected by Western blotting. Numbers of cells in all groups increased steadily for 6days; rate of cell proliferation in the Sham group was found to be higher than in the OVX group after 48hours. Mineralized bone nodular structures were significantly more concentrated in the Sham group than in the OVX group by day 21, and mRNA levels of Runx2 in the OVX group were significantly lower than in the Sham group. Transcript levels of genes coding for Opn showed a similar pattern to those of Runx2. Western blot results indicated that protein expression levels of OPN and RUNX2 in the OVX group were lower than those in the Sham group, at each time point. These results indicated that the proliferative capacity and osteogenic potential of ASCs were significantly impaired in osteoporotic mice compared to normal controls. However, use of autologous transplantation of modified OP-ASCs for treatment of OP, or combination of composite scaffolds and modified OP-ASCs for repair of osteoporotic bone defects, can overcome shortcomings of other methods.

Potential application of neogalactosylalbumin in positron emission tomography evaluation of liver function.

AIMTo investigate the evaluation of neogalactosylalbumin (NGA) for liver function assessment based on positron emission tomography technology.METHODSFemale Kunming mice were assigned randomly to two groups: fibrosis group and normal control group. A murine hepatic fibrosis model was generated by intraperitoneal injection of 10% carbon tetrachloride (CCl4) at 0.4 mL every 48 h for 42 d. 18F-labeled NGA ([18F]FNGA) was synthesized and administered at a dosage of 3.7 MBq/mouse to both fibrosis mice and normal control mice. Distribution of [18F]FNGA amongst organs was examined, and dynamic scanning was performed. Parameters were set up to compare the uptake of tracers by fibrotic liver and healthy liver. Serologic tests for liver function were also performed.RESULTSThe liver function of the fibrosis model mice was significantly impaired by the use of CCl4. In the fibrosis model mice, hepatic fibrosis was verified by naked eye assessment and pathological analysis. [18F]FNGA was found to predominantly accumulate in liver and kidneys in both control group (n = 21) and fibrosis group (n = 23). The liver uptake ability (LUA), peak time (Tp), and uptake rate (LUR) of [18F]FNGA between healthy liver (n = 8) and fibrosis liver (n = 10) were significantly different (P < 0.05, < 0.01, and < 0.05, respectively). LUA was significantly correlated with total serum protein level (TP) (P < 0.05). Tp was significantly correlated with both TP and glucose (Glu) concentration (P < 0.05 both), and LUR was significantly correlated with both total bile acid and Glu concentration (P < 0.01 and < 0.05, respectively).CONCLUSION[18F]FNGA mainly accumulated in liver and remained for sufficient time. Functionally-impaired liver showed a significant different uptake pattern of [18F]FNGA compared to the controls.

Effect of histone deacytylase inhibitors on allergic airway inflammation in mouse model anesthetized with ketamine

Background Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. The chronic inflammation is associated with airway hyper-responsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning. These episodes are usually associated with variable airflow obstruction within the lung that is often reversible either spontaneously or with treatment. Objective The present study was designed to examine the modulatory effects of the histone deacytelase inhibitor [valproic acid (VPA)] on ovalbumin (OVA)-induced airway inflammation in mice. Methods Airway inflammation was induced by means of intraperitoneal injection of 83.33 μg of OVA in 2.92 mg of alum on days 1, 11, and 14 and then challenged with 1% w/v OVA aerosol in PBS on days 15–18. However, normal control mice received PBS instead. The animals were then divided into three groups: the normal group, the untreated asthmatic group, and the VPA-treated group, which received 300 mg/kg of VPA on days 15–18 1 h before each nebulization. At the end of the experimental period (day 19), each animal was anesthetized with 80 mg/kg of ketamine intraperitoneally and then bronchoalveolar lavage fluid was collected and centrifuged. The collected supernatants of bronchoalveolar lavage were used to assess interleukin-4, whereas the cell pellets were collected and resuspended for cytological examination. Results Treatment with VPA resulted in a significant reduction in interleukin-4 level, total cell count, and neutrophil percentage in bronchoalveolar lavage fluid (P Conclusion The treatment with VPA reduces inflammatory cellular infiltration, mainly neutrophils, in a mouse model of asthma.