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Sirtuin 3 (SIRT3) mediates cellular resistance toward various forms of stress. SIRT3 expression in the developing brain, especially its localization in various glial cell types, has not been fully explored. This study aimed to determine SIRT3 expression in the brain of neonatal rats subjected to hypoxia. By immunohistochemistry, immunofluorescence and Western blotting, we show here that SIRT3 expression in the periventricular white matter was up-regulated in hypoxia group compared with the control group at the corresponding time points. Intense SIRT3 expression was detected in microglia at early time points after hypoxia whose cell number was increased with reduced ramifications in hypoxia group compared with the control group. Furthermore, SIRT3 immunoreactivity was obviously enhanced at 24 h, 3 and 7d, but was declined at 14d after hypoxia so that SIRT3 expression between the two groups was comparable. SIRT3 immunofluorescence was also localized in astrocytes labeled with GFAP which was augmented at different time points in hypoxia group. GPAP positive astrocytes exhibited long extending processes being most pronounced at 3d. SIRT3 was moderately expressed at 24 h, 3 and 7d, but was markedly increased at 14d after hypoxia. Moderate SIRT3 expression was also localized in oligodendrocytes labeled with CNPase in the control group. The incidence of CNPase positive oligodendrocytes showing colocalization of SIRT3 increased significantly at 24 h, 3 and 7d after hypoxia. In conclusion, SIRT3 expression was differentially up-regulated in all three major glial cell types following hypoxia. It is suggested that increased SIRT3 expression in the respective glial cell types following hypoxia is involved in different signaling pathways that protect against hypoxic stress in the developing brain. [ABSTRACT FROM AUTHOR]

The toxic effects of lead on normal rat kidney epithelial cells (NRK cells) may occur via various pathways. However, the role of intrinsic mitochondrial pathway in Lead-induced apoptosis in NRK cells has not been investigated. The purpose of our study was to investigate cytotoxic responses and cell apoptosis mediated by lead in NRK cells. NRK cells were treated with different concentrations of Lead acetate for 12 h to determine the cytotoxicity of lead. Mitochondrial transmembrane potential was also analyzed using a fluorescence spectrophotometer. Moreover, the activities of caspase-3 and caspase-9 were detected in the presence of lead. Finally, the lead-induced cell apoptosis was evaluated by flow cytometry in the present of caspase inhibitors Z-VAD-FMK and Ac-LEHD-FMK, respectively. The results would contribute to clarify the role of Lead in proliferation and apoptosis of NRK cells, and help to understand the underlying mechanism responsible for lead-induced cell apoptosis. [ABSTRACT FROM AUTHOR]

The seasonality of depressive symptoms is prevalent in children and adolescents. However, the mechanisms that underlie such susceptibility to seasonal influences on mood disorders are unclear. We examined the effects of a short photoperiod condition on the susceptibility to subchronic unpredictable mild stress (SCUS) and rhythmic alterations of plasma corticosterone (CORT), melatonin, and neuropeptide Y (NPY) in adolescent male rats. Compared with the 12 h/12 h light/dark photoperiod control (CON) rats, the 8 h/16 h photoperiod SCUS rats exhibited significant anhedonia, a core symptom of human depression, together with a blunted diurnal rhythm and elevation of 24 h CORT, melatonin, and NPY levels. The 8 h/16 h photoperiod condition also blunted the rhythmicity of CORT, caused a phase inversion of melatonin, and caused a phase delay of NPY compared with 12 h/12 h CON rats. Such abnormalities of plasma CORT, NPY, and melatonin might cause adolescent individuals to present higher stress reactivity and greater vulnerability to stress over their lifetimes. The present study provides evidence of the susceptibility to the seasonality of stress-related disorders in adolescence. [ABSTRACT FROM AUTHOR]

Nowadays, people pay more and more attention to the natural products based on their multiple targets in the antitumor treatment. In our previous research, Rhizoma paridis saponins (RPS) were regarded as potent anticancer agent that elicits programmed cell death and inhibits metastases in murine lung adenocarcinoma in vivo. In the present study, we set up a rat model of diethylnitrosamine (DEN) induced pulmonary adenoma to evaluate the antitumor effects of RPS again. After 20 weeks treatment, rats were sacrificed in order to perform histopathological examinations, blood biochemistry, immunohistochemistry, western blot, PCR and metabonomics. As a result, DEN induced pulmonary adenoma generation in the lungs and damaged hepatocytes and hepatoma formation in the livers. RPS effectively attenuated hepatotoxic and inhibited pulmonary adenoma through down-regulating expression of MMP-9 and up-regulating level of TIMP-2 in DEN-induced rats. Meanwhile, RPS remarkably decreased energy metabolism, and glycine, serine and threonine metabolism to block the tumor growth. In conclusion, RPS would be a potent anticancer agent used in the prospective application. [ABSTRACT FROM AUTHOR]

A highly selective and sensitive method for simultaneous quantitation of osthole, bergapten and isopimpinellin in rat plasma and tissues was developed by liquid chromatography–tandem quadrupole mass spectrometry (LC–MS/MS). After liquid–liquid extraction of samples with methyl tert-butyl ether, the analytes and dextrorphan (internal standard, IS) were separated by a Hypersil GOLD AQ C18 column with gradient elution of acetonitrile and water containing 0.5‰ formic acid. Three determinands were detected using an electrospray ionization (ESI) tandem mass spectrometry in the multiple reaction monitoring (MRM) modes with positive electrospray ionization. Calibration curves were recovered over the concentration ranges of 1–200 ng/ml, 1–500 ng/ml, 0.25–200 ng/ml for osthole, bergapten and isopimpinellin in plasma; 1–100 ng/ml, 1–500 ng/ml, 0.5–100 ng/ml for osthole, bergapten and isopimpinellin in tissues, respectively. The intra-day precision (R.S.D.) was within 13.90% and the intra-day accuracy (R.E.) was within −6.27 to 6.84% in all biological matrixes. The inter-day precision (R.S.D.) was less than 13.66% and the inter-day accuracy (R.E.) was within −10.64 to 13.04%. Then the method was successfully applied to investigate plasma pharmacokinetic study and tissue distribution of osthole, bergapten and isopimpinellin in rats after oral administration of Fructus Cnidii extraction, especially for testis/uterus tissue distribution. The results demonstrated that osthole, bergapten and isopimpinellin were absorbed and eliminated rapidly with wide distributions in rats. Distribution data of these three bioactive components in testis/uterus tissues could offer useful information for the further preclinical and clinical studies of Fructus Cnidii in the treatment of genital system disease. [ABSTRACT FROM AUTHOR]

Abstract: Preclinical studies suggest that propofol may cause damage to immature neurons. However, the effect of maternal propofol exposure on the neuronal development of the offspring is largely unknown. In this study, pregnant rats were assigned to receive continuous infusion of saline (control) or propofol for 1h (1HP) or 2h (2HP) on gestational day 18. An additional group (lipid) was assigned to receive continuous infusion of intralipid fat emulsion (vehicle of propofol) for 2h. Pups were then tested on the appearance and progression of sensory and physical motor abilities between postnatal day 1 (P1) and P28. The brain and body weights of pups from 2HP group on P10 were significantly lower than those from the saline control group, although they were the same in all four groups at birth (P0). Pups from 1HP and 2HP groups, but not lipid group, showed slower maturation of eyes (delayed opening) and several neurological reflexes (hindlimb reflex, righting reflex); they also showed delayed improvement in execution on gait reflex and inclined board tests. The forelimb reflex and negative geotaxis were also delayed in 2HP group. All parameters examined except body weight of 2HP pups recovered to normal levels by P28. We conclude that administration of propofol to pregnant rats leads to retardation in physical and neurological reflex development in their offspring. [Copyright &y& Elsevier]

Background and Aims: With the developing and widely used electromagnetic field (EMF) technology, more and more studies are focusing on the relationship between EMF and Alzheimer’s disease (AD). Electromagnetic pulse (EMP) is one type of widely used EMF. This study aimed to clarify whether EMP exposure could induce cognitive and memory impairment, thus finding a possible relationship between EMP and AD. Methods: Forty healthy male Sprague Dawley rats were randomly divided into four groups. Animals, respectively, received 100, 1000, and 10,000 pulses EMP (field strength 50 kV/m, repetition rate 100 Hz) exposure and sham exposure when 2 months old. Monthly Morris water maze (MWM) was used to test the changes of cognitive and memory ability. Superoxide dismutase (SOD) activity and glutathione (GSH) content were used as oxidative stress indexes. Expressions of some types of Alzheimer’s disease-related proteins were also detected. Results: After exposure, EMP exposure caused clear cognitive and memory impairment compared with sham exposure group (p <0.05). Determination of oxidation indexes showed decreased SOD activity and GSH content in exposure groups compared with sham group. Immunohistochemical (IHC) staining showed increased beta amyloid protein (Aβ) in EMP exposure groups compared with sham group. Western blot experiments showed increased expressions of Aβ oligomer and beta amyloid protein precursor (APP) in EMP exposure groups. Increased expression of microtubule-associated protein 1 light chain 3-II (LC3-II) was also found. Conclusions: The present results showed that EMP exposure can cause long-term impairment in impaired cognition and memory of rats, resulting in AD-like symptoms. This may be induced by enhancing oxidative stress and is related to autophagy dysfunction. [ABSTRACT FROM AUTHOR]

Abstract: Many clinical studies have shown that circadian rhythmic abnormalities are strongly associated with major depression. Meanwhile a large number of researches have indicated that physical exercise could effectively reduce the stress impairment and improve one’s mood, cognition and self-concept. We examined the effects of chronic unpredictable mild stress (CUMS) on rodent behaviors and the circadian rhythm of hormone and peptide levels in the plasma. We collected blood samples at 4-h intervals for 24h to examine circadian rhythmic expression of corticosterone, melatonin and vasoactive intestinal peptide (VIP). We found that CUMS led to depressive-like behaviors and inversed rhythm in corticosterone and blunted circadian rhythms in melatonin and VIP compared to control group. Levels of corticosterone showed no differences from control group. But levels of melatonin were decreased and VIP was increased compared to control group, respectively. Swimming exercise could not alleviate the depressive-like behaviors and the blunted circadian rhythmic expression of blood hormone and peptide, also could not rescue levels of melatonin and VIP. These findings indicate that swimming exercise as a physical exercise, unexpectedly, may not act as a protect factor rather than as an additional stress in the present study. It has no effect on the depressive-like behaviors and alterations of blood hormone and peptide whether in circadian rhythm or in blood levels induced by CUMS. [Copyright &y& Elsevier]

Abstract: Although there is increasing clinical acceptance of acupuncture and electroacupuncture (EA) as a treatment of substance abuse-related disorders, our understanding of this treatment remains incomplete. Previous clinical and pre-clinical studies have shown that acupuncture and EA are effective in reducing ethanol consumption. Recent studies have shown that Sprague-Dawley (SD) rats under an intermittent-access two-bottle choice drinking procedure (IE procedure) voluntarily drank high amounts of ethanol. However, an effect of EA on ethanol consumption of the SD rats under this drinking procedure has not been demonstrated. In the present study, we demonstrated that SD rats escalated their ethanol intake and subsequently developed ethanol dependence under the IE procedure. A single low (2Hz), but not high frequency (100Hz) EA treatment applied at the bilateral acupoint Zusanli (ST36), but not at the tail reduced voluntary intake of, and preference for ethanol, but not sucrose. Furthermore, repeated EA treatments decreased the intake of and preference for ethanol, without resulting in a rebound increase in ethanol intake when the EA treatments were terminated. These observations indicate that EA may be a useful treatment for alcohol abuse. [Copyright &y& Elsevier]

Abstract: Aim: Autopsy evidence suggests that the presence of both Alzheimer''s disease (AD) and cerebral infarction pathology is associated with more severe cognitive impairment than that produced by AD pathology alone. This study aims to investigate the effect of cerebral ischemia on cognitive function in rats with AD constructed by hippocampal injection and to determine its underlying mechanism, which is proposed to be of significance to the treatment of AD. Main methods: AD was modeled by injection of aggregated Aβ(1–40), either alone or followed by hippocampal endothelin-1 injection to mimic cerebral ischemia in hippocampus, into the right dentate gyrus (DG) of rats. The Morris water maze was used to evaluate cognitive function. Aβ deposition, neuronal loss and phosphorylated tau expression in hippocampus were examined by Congo red staining, Nissl''s staining and immunohistochemistry, respectively. Reactive astrocytes, IL-1β and TNF-α expressions were measured by immunohistochemistry, in situ hybridization and reverse transcription–polymerase chain reaction. Key findings: Compared with rats treated with either Aβ or endothelin alone, rats treated with both Aβ and endothelin showed more aggravated cognitive impairment and more Aβ deposits, neuron loss, phosphorylated tau expression, reactive astrocytes, IL-1β and TNF-α expressions in hippocampus. Significance: Hippocampal ischemia aggravates cognitive impairment of AD rats by increasing Aβ deposits, neuron loss and tau phosphorylation in hippocampus. The enhanced inflammatory response may be responsible for cerebral ischemia-induced aggravation of cognitive impairment in AD rats. Based on these findings, prevention and treatment of cerebral ischemia may improve clinical symptoms of AD and suppress the progression of AD. [Copyright &y& Elsevier]

Abstract: Short-term sleep deprivation (SD) has been shown to enhance cortical activity. However, alterations in the cellular excitability of cortical neurons following SD are not yet fully understood. The present study investigated the effects of 4-hour SD on pyramidal neurons in the prefrontal cortex (PFC) of rats using whole-cell patch-clamp recording. SD led to an increase in the initial slope of firing frequency–current curve and a decrease in frequency adaptation, which were reversed by recovery sleep (RS). Correspondingly, the total afterhyperpolarization (AHP) was reduced in the SD group and returned in the RS group. Furthermore, the component of AHP changed after SD seemed to be sensitive to Ca2+. These observations indicate an enhancement in intrinsic excitability due to short-term SD, and suggest a role for Ca2+-dependent AHP in this change. The findings of the present study may provide a possible explanation for the SD-induced increase in cortical activity. [Copyright &y& Elsevier]

Abstract: Immunological changes initiated by major operative injury may result in inflammatory responses in both peripheral and central nervous system, which may lead to organ dysfunction. Recent studies indicate that β-adrenergic receptors (β-ARs) may mediate production of pro-inflammatory cytokines in the brain. In the present study propranolol (β-AR antagonist), but not prazosin (α1-AR antagonist), antagonized surgical trauma induced pro-inflammatory cytokine production in microglia cells isolated from rats. β-AR activation in the absence of pro-inflammatory stimuli increased IL-1β, TNF-α and IL-6 mRNA and protein expressions in the primary microglia cell culture. Isoproterenol (β-AR agonist) treatment induced a time- and concentration-dependent increase of IL-1β in cells. Both ERK1/2 and P38 MAPK inhibitor, but not PKA and JNK1/2 inhibitor abrogated isoproterenol-induced IL-1β and IL-6 production in microglia cells. In conclusion, the results suggest that β-ARs possess pro-inflammatory properties by modulating the functions of microglia cell. [Copyright &y& Elsevier]

Abstract: We compared the expression of chemokine receptor CCR2 protein in the dorsal root ganglia (DRG) injured by the chronic constriction injury (CCI), the spinal nerve ligation (SNL) and a chronic compression of DRG (CCD). Each of these injuries produced the same significant increase in CCR2 protein in the DRG, as assessed by Western blot analyses. Whole-cell patch-clamp recordings revealed that CCL2, a ligand for CCR2 receptor, depolarized nociceptive DRG neurons from rats of the all three models. A greater percentage of these neurons was depolarized by CCL2 after CCD than after either of the other injuries. Furthermore, CCL2 significantly lowered current threshold only in CCD neurons but not in CCI or SNL neurons. CCL2 significantly lowered the net whole-cell potassium currents in neurons after CCD but not after CCI or SNL. Thus, the injury-induced effects of CCL2 in increasing the excitability of the cell bodies of DRG neurons depend on the site of the injury – with greater effects occurring after an injury of the ganglion than after an injury of the spinal or peripheral nerve. [Copyright &y& Elsevier]

Abstract: Berberine can improve insulin resistance, lower blood glucose, and regulate lipid metabolism disorders which cause endothelial dysfunction, leading to vascular complications of type 2 diabetes mellitus. The aim of the present study was to investigate the effects of berberine on endothelial dysfunction of aortas in type 2 diabetes mellitus rats and its mechanism. Wistar rats were randomly divided into four groups: diabetic rats, control rats, diabetic rats treated with berberine (100 mg/kg), and control rats treated with berberine. The serum fasting blood glucose, insulin, total cholesterol, triglyceride and nitric oxide (NO) levels were tested. Acetylcholine-induced endothelium-dependent relaxation and sodium nitroprusside induced endothelium-independent relaxation were measured in aortas for estimating endothelial function. The expression of endothelial nitric oxide synthase (eNOS) mRNA was measured by RT-PCR, and the protein expressions of eNOS and NADPH oxidase (NOX4) were analyzed by western blot. The results showed that berberine significantly decreased fasting blood glucose, and triglyceride levels in diabetic rats. Berberine also improved endothelium-dependent vasorelaxation impaired in aorta. The expressions of eNOS mRNA and protein were significantly increased, while NOX4 protein expression was decreased in aortas from diabetic rats with berberine treatment. Moreover, serum NO levels were elevated after berberine treatment. In conclusion, berberine restores diabetic endothelial dysfunction through enhanced NO bioavailability by up-regulating eNOS expression and down-regulating expression of NADPH oxidase. [Copyright &y& Elsevier]

Abstract: Chronic alcoholics are predisposed to the development of a systemic inflammatory response syndrome, which is usually triggered in the gut. This study aimed to investigate in rats the role of intestinal epithelial inflammatory responsiveness in the susceptibility of alcoholics to excessive inflammation. Thirty Wistar rats were randomly divided into three groups: 10 rats killed immediately after acclimation (baseline control), 10 rats treated with 25% (vol/vol) ethanol for 6 months (ethanol group), and 10 rats given double-distilled water until killed simultaneously with the ethanol group (9-month control). The intestinal microflora, the epithelial histology and ultrastructure, the level of Toll-like receptor 4 (TLR4), TANK-binding kinase-1 (TBK1), and activated nuclear factor-κB (NF-κB) in the intestinal mucosa, and somatostatin (SST) levels in plasma and small intestine were evaluated in each group. Isolated intestinal epithelia from each rat were used to examine lipopolysaccharide (LPS) responsiveness with or without SST pretreatment by quantification of TLR4, TBK1, activated NF-κB, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). Compared with both the control groups, the amount of mucosal Escherichia coli in the ethanol group was not changed, whereas the number of intestinal lactobacilli in the ethanol group was significantly reduced (P <.05). Mild inflammatory injury and upregulation of TLR4 and TBK1 were observed in the intestinal mucosa of the ethanol group. The LPS-enhanced in vitro expression of TLR4, TBK1, and production of IFN-γ and TNF-α in isolated intestinal epithelia of the ethanol group were significantly higher than those in either control group (P <.05) and were dramatically inhibited by SST (P <.05), whereas NF-κB was activated by LPS only in the control groups. The plasma and intestinal levels of SST in the ethanol group were significantly lower than those in either control group (P <.05). These findings suggest that impairment of intestinal SST production by chronic ethanol administration leading to upregulation of the TLR4–TBK1 pathway may be one of the mechanisms underlying the LPS hypersensitivity of intestinal epithelia. [Copyright &y& Elsevier]

Abstract: Ischemia/reperfusion (I/R) injury mainly caused by oxidative stress plays a major role in cardiac damage. The extent of the I/R injury is also an important factor that determines the function of a transplanted heart. This study first examined whether hemoglobin-based oxygen carriers (HBOCs) could protect isolated rat heart from I/R injury and then elucidated the underlying mechanism. Using the Langendorff model, isolated Sprague–Dawley rat hearts were arrested and stored at 4°C for 8 h and then reperfused for 2 h. Compared with St. Thomas'' solution (STS) and rat self blood in STS, polymerized placenta hemoglobin (PolyPHb) in STS greatly improved heart contraction and decreased infarction size. The extent of myocardial apoptosis was also significantly decreased, which was related to reduced iNOS-derived nitric oxide production, increased protein ratio of Bcl-2/Bax, and reduced caspase-3 activity and cleavage level. Furthermore, PolyPHb in STS did not increase malondialdehyde, peroxynitrite, or mitochondrial hydrogen peroxide formation, but greatly elevated superoxide dismutase activity and preserved mitochondrial ATP synthesis, which served to maintain redox homeostasis in I/R heart. In conclusion, our results demonstrate that HBOCs protected isolated heart from I/R injury and this protection was associated with attenuation of NO-mediated myocardial apoptosis and restoration of the nitroso-redox balance. [Copyright &y& Elsevier]

Autophagy has been implicated in the developmental toxicity of multiple organs in offspring caused by adverse environmental conditions during pregnancy. We have previously found that prenatal caffeine exposure (PCE) can cause fetal overexposure to maternal glucocorticoids, leading to chondrodysplasia. However, whether autophagy is involved and what role it plays has not been reported. In this study, a PCE rat model was established by gavage of caffeine (120 mg/kg.d) on gestational day 9–20. The results showed that reduced cartilage matrix synthesis in male fetal rats in the PCE group was accompanied by increased autophagy compared to the control group. Furthermore, the expression of mTOR, miR-421–3p, and glucocorticoid receptor (GR) in male fetal rat cartilage of PCE group was increased. At the cellular level, we confirmed that corticosterone inhibited matrix synthesis in fetal chondrocytes while increasing autophagic flux. However, administration of autophagy enhancer (rapamycin) or inhibitor (bafilomycin A1 or 3-methyladenine) partially increased or further decreased aggrecan expression respectively. At the same time, we found that corticosterone could increase the expression of miR-421–3p through GR and target to inhibit the expression of mTOR, thereby enhancing autophagy. In conclusion, PCE can cause chondrodysplasia and autophagy enhancement in male fetal rats. Intrauterine high corticosterone activates GR/miR-421–3p signaling and down-regulates mTOR signaling in fetal chondrocytes, resulting in enhanced autophagy, which can partially compensate for corticosterone-induced fetal chondrodysplasia. This study confirmed the compensatory protective effect of autophagy on the developmental toxicity of fetal cartilage induced by PCE and its epigenetic mechanism, providing novel insights for exploring the early intervention and therapeutic target of fetal-originated osteoarthritis. • Prenatal caffeine exposure (PCE) can cause fetal chondrocyte dysplasia and enhance chondrocyte autophagy. • Autophagy can partially compensate for PCE-induced chondrodysplasia in male fetal rats. • GR/miR-421–3p/mTOR pathway mediates the enhancement of autophagy in chondrocytes. [ABSTRACT FROM AUTHOR]

Prenatal caffeine exposure (PCE) induces developmental toxicity of multi-organ and susceptibility to multi-disease in offspring. However, the effects of PCE on osteoarthritis susceptibility in adult offspring and its intrauterine programming mechanism remain to be further investigated. Here, we found that PCE induced susceptibility to osteoarthritis in male adult offspring rats, which was related to the inhibited function of cartilage matrix synthesis from fetuses to adults. Meanwhile, PCE consistently downregulated the H3K9ac and expression levels of transforming growth factor β receptor 1 (TGFβR1), and then blocked TGFβ signaling pathway, which contributed to the suppressed cartilage matrix synthesis. Moreover, the high level of corticosterone caused by PCE reduced the H3K9ac level on TGFβR1 promoter region through acting on glucocorticoids receptor (GR) and recruiting histone deacetylase 2 (HDAC2) into the nucleus of fetal chondrocytes. Taken together, PCE induced osteoarthritis susceptibility in male adult offspring rats, which was attributed to the low-functional programming of TGFβR1 induced by corticosterone via GR/HDAC2 signaling. • Prenatal caffeine exposure (PCE) caused osteoarthritis susceptibility in offspring. • The low-expressional TGFβR1 contributed to the inhibited cartilage matrix synthesis. • PCE induced-high corticosterone decreased H3K9ac of TGFβR1 via GR/HDAC2 signaling. [ABSTRACT FROM AUTHOR]

Open vascular reconstructions such as bypass are common treatments for cardiovascular disease. Unfortunately, neointimal hyperplasia (IH) follows, leading to treatment failure for which there is no approved therapy. Here we combined the strengths of tailoring nanoplatforms for open vascular reconstructions and targeting new epigenetic mechanisms. We produced adhesive nanoparticles (ahNP) that could be pen-brushed and immobilized on the adventitia to sustainably release pinometostat, an inhibitor drug selective to the epigenetic writer DOT1L that catalyzes histone-3 lysine-79 dimethylation (H3K79me2). This treatment not only reduced IH by 76.8% in injured arteries mimicking open reconstructions in obese Zucker rats with human-like diseases but also avoided the shortcoming of endothelial impairment in IH management. In mechanistic studies, chromatin immunoprecipitation (ChIP) sequencing revealed co-enrichment of the histone mark H3K27ac(acetyl) and its reader BRD4 at the gene of aurora kinase B (AURKB), where H3K79me2 was also enriched as indicated by ChIP-qPCR. Accordingly, DOT1L co-immunoprecipitated with H3K27ac. Furthermore, the known IH driver BRD4 governed the expression of DOT1L which controlled AURKB's protein level, revealing a BRD4- > DOT1L- > AURKB axis. Consistently, AURKB-selective inhibition reduced IH. Thus, this study presents a prototype nanoformulation suited for open vascular reconstructions, and the new insights into chromatin modulators may aid future translational advances. [Display omitted] • Bioadhesive nanoparticle is pen-brushed on arteries for adventitia-localized delivery. • A DOT1L inhibitor drug (Pino) so delivered abates neointima in obese Zucker rats. • Unlike rapamycin, Pino does not impair the recovery of injured endothelium in vivo. • The epigenetic axis BRD4->DOT1L->AURKB regulates smooth muscle cell phenotype. • AURKB-selective inhibition hampers neointimal hyperplasia in injured rat arteries. [ABSTRACT FROM AUTHOR]

Our previous in vivo studies showed that prenatal caffeine exposure (PCE) could restrain the development of chondrogenesis, which may delay fetal articular cartilage development and increase susceptibility to osteoarthritis in adults. So, the goal of the current study is to clarify theincreasing susceptibility to adult osteoarthritis in caffeine-exposed female offspring and its’mechanism. Pregnant rats were treated with 120 mg/kg·d caffeine or equal volumes of saline from gestational day (GD) 9 to 20. knee joints were collected from GD20 female fetuses and 18-week old female offspring which was treated with strenuous running for 6 weeks (55 min/day at 20 m/min) load to induce osteoarthritis. Knee joints from GD20 fetuses and adult offspring were collected for histochemistry and immunohistochemistry. Next, chondrocytes were isolated from 1-day-old newborn rats and in vitro studies were conducted where the cells in primary culture were exposed to 1, 10, and 100 μM caffeine and 250, 500, and 1,250 nM corticosterone. Insulin-like growth factor 1 (IGF-1) signal pathway genes’ expression levels in fetal chondrocytes were studied, and IGF-1 histone acetylation was detected in vitro. Immunohistochemical results showed low expression levels of IGF-1 signaling genes (IGF-1, IRS-1, AKT, and COL2A1) both in fetal and adult cartilage with PCE. For adult offspring, histological results and Mankin score revealed increased cartilage destruction and accelerated osteoarthritis progression in PCE group with strenuous running exercise. Analysis in vitro revealed that caffeine and corticosterone impeded the expression of IGF-1 signaling pathway aggrecan and COL2A1 genes, but only corticosterone decreased H3K9 and H3K27 acetylation in the IGF-1 promoter region. In concluson, PCE low functional programmed cartilage IGF-1 by histone acetylation modification via overexposure to corticosterone and delayed articular cartilage development from fetus to adults. Then, the delayed cartilage development increased susceptibility to osteoarthritis in offsprings. [ABSTRACT FROM AUTHOR]

The present study investigated the antidepressant potential of curcumin in olfactory bulbectomy and forced swimming test models of depression in male albino rats under chronic treatment. The experimental animals were divided into four groups, and curcumin was administered for 45 days. Our results showed that the curcumin significantly reduced olfactory bulbectomy-induced behavioral abnormalities including deficits in step-down passive avoidance, increased activity in the open area and immobility time. Chronic administration of curcumin significantly reversed levels of 3, 4-dihydroxyphenylacetic acid, noradrenaline, serotonin and 5-hydroxyindoleacetic acid in the hippocampus region of male albino rats. Also, curcumin normalizes the levels of dopamine, noradrenaline, and 5-hydroxyindoleacetic acid in the frontal cortex of rats. Taking all these results together, it may suggest that curcumin is potent compound acting against the depression in the male albino rats. [ABSTRACT FROM AUTHOR]

A specific high performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS method) was established for determining the concentration of protodioscin (PG) in rat plasma after intragastric administration of its standard form. Ginsenoside Rb 1 was selected as the internal standard (IS). The plasma sample was prepared using one-step deproteinization procedure by adding three parts of acetonitrile to precipitate proteins. The chromatographic separation was accomplished on an Inersil ODS-3 C 18 column (250 × 4.6 mm, 5 μm) with a mobile phase composed with acetonitrile and water containing 0.1% formic acid under a gradient elution mode at a flow rate of 1 mL min −1 . A 3:1 portion of the eluent after a microsplit was detected on a triple quadrupole tandem mass spectrometer coupled with electrospray ionization (ESI) in positive ion and multiple reaction monitoring (MRM) scanning modes. The mass transitions were selected as 888.1 → 1050.2 for PG and 948.2 → 1110.3 for IS, respectively. After careful validation, the plasma samples were always stable under different storage conditions. These analytical results rendered sensitive, selective, and reliable values by this established method which displayed high accuracy, adequate extracted recoveries, and almost negligible matrix effects. This method was applied to the pharmacokinetic studies on PG level in the rat plasma and its pharmacokinetic effect. The results of our studies suggest that the present method may be a useful tool for further clinical study of PG. [ABSTRACT FROM AUTHOR]

Aims Tanshinone IIA (TSA) has been reported to protect neurons and microvascular endothelial cells against ischemic injury. However, the effect of TSA on astrocytes in response to ischemic injury is not clear. The aims of this study were to evaluate the effect of TSA on astrocytes following ischemic insult. Main methods Primary cultured rat astrocytes were treated with oxygen–glucose deprivation (OGD) followed by recovery. Cell death was measured by lactate dehydrogenase assay. Astrocyte proliferation was determined by cell viability assay, cell counting and 5-bromo-2-deoxyuridine incorporation assay. The glial fibrillary acidic protein (GFAP) expression was assessed by immunocytochemistry. Stromal cell-derived factor-1 (SDF-1) secretion from astrocytes was measured by enzyme linked immunosorbent assay. The expressions of hypoxia inducible factor-1α (HIF-1α), SDF-1, GFAP and the phosphorylation of Akt and extracellular regulated protein kinase (Erk) 1/2 were evaluated by immunoblot assay. Quantitative RT-PCR was used to assess the mRNA expression of HIF-1α and SDF-1. Key findings Mild OGD (2 h-OGD) did not induce astrocyte injury but proliferation at 48 and 72 h after recovery. Mild OGD also induced the accumulation of HIF-1α, the subsequent expression and secretion of SDF-1, resulting in the phosphorylation of Erk1/2 and Akt. TSA attenuated astrocyte proliferation and significantly decreased the OGD-induced accumulation of HIF-1α and SDF-1, then blocked the downstream signaling, Erk1/2 and Akt activation. Significance TSA inhibits the astrocyte proliferation induced by sub-lethal ischemic insult via blocking the activation of HIF-1α/SDF-1 pathway. This finding suggests that TSA may be a potential therapeutic option for gliosis after cerebral ischemia. [ABSTRACT FROM AUTHOR]

Abstract: A sensitive, reliable and accurate reversed-phased liquid chromatography with tandem mass spectrometry (LC⬜MS/MS) in negative ion mode was developed and validated for the quantification of tenuifolin in rat plasma and tissue. A single step protein precipitation by methanol was used to prepare plasma and tissue homogenate samples. Tenuifolin and polydatin (internal standard, IS) were separated by HPLC using a C18 column and an isocratic mobile phase consisted of acetonitrile and water containing 0.05% formic acid (42:58, v/v) running at a flow rate of 0.2 ml/min for 6 min. Detection and quantification were performed using a mass spectrometer by the multiple reaction monitoring (MRM) in negative electrospray ionization mode. The transition monitored were m/z [M↙H]↙ 679.4 ⠙ 455.4 for tenuifolin and m/z [M↙H]↙ 389.0 ⠙ 227.2 for IS, respectively. Calibration curves were recovered over a concentration range of 0.5⬜1000 ng/ml for plasma, heart, liver, lung and kidney, 0.5⬜200 ng/ml for spleen, and 0.5⬜50 ng/ml for brain, respectively. The lower limit of quantification was 0.5 ng/ml for plasma and tissue homogenates. The inter-day precision (R.S.D.) was less than 12.9% and intra-day precision R.S.D. was less than 13.4%, while the inter-day accuracy (R.E.) was ranged from ↙7.20 to 6.87% and intra-day accuracy (R.E.) was ranged from ↙6.20 to 8.04% in plasma and tissue homogenates. This method was successfully applied to the pharmacokinetic and tissue distribution study of pure tenuifolin in rat. The pharmacokinetic study indicated that poor absorption into systemic circulation was observed after rat was administered orally tenuifolin, and the absolute bioavailability was low (0.83 ± 0.28%). The results of tissue distribution showed the higher tenuifolin concentrations were found in liver, kidney and heart, and the small amount of drug was distributed quickly into the brain tissue at 5 min after the intravenous injection of tenuifolin. The fact that tenuifolin could cross the blood⬜brain barrier provided the material basis for pharmacological action of the tenuifolin in the treatment of memory loss. [Copyright &y& Elsevier]

Abstract: Baicalin, a major constituent of flavonoids isolated from Scutellariae Radix, has been previously confirmed to decrease the duration of immobility in mice exposed to the forced swimming test (FST) and tail suspension test (TST). However, its antidepressant effects and mechanisms are still seldom studied in chronic mild stress (CMS) model of depression. In the present study, we attempted to investigate the effects of baicalin on the depressive-like behavior, the mRNA expression and activity of cyclooxygenase-2 (COX-2), as well as prostaglandin E2 (PGE2) levels in the frontal cortex and hippocampus. Moreover, the serum corticosterone levels were also examined. We found that CMS procedure not only decreased the sucrose preference and increased serum corticosterone levels, but also elevated the activity and mRNA expression of COX-2, and increased PGE2 levels in rat brain regions. Treatment with baicalin (10, 20, 40mg/kg) prevented these abnormalities induced by CMS. These results confirmed that baicalin exerted antidepressant-like effects, and suggested its mechanisms at least partially related to decease COX-2 activity and expression, subsequently resulted in reduction of PGE2 levels in brain. Our findings may provide a new aspect to understand the antidepressant action of baicalin, which is targeted on the COX-2 system in brain. [Copyright &y& Elsevier]

Abstract: Since the introduction of the thrifty phenotype hypothesis, the potential traits of thrift have been described in increasingly broad terms but biochemical and behavioral evidence of thrift has not been well demonstrated. The objective of our studies was to use a rodent model to identify features of thrift programmed by early life protein restriction. Robust programming of thrifty features requires a thrifty nutritional environment during the entire window of developmental plasticity. Therefore, pregnant rats were exposed to a low protein diet throughout the window of developmental plasticity spanning the period of gestation and lactation and its effects on energy acquisition, storage and expenditure in the adult offspring were examined. Maternal protein restriction reduced birth weight and produced long term reductions in body and organ weights in the offspring. Low protein offspring demonstrated an increased drive to seek food as evidenced by hyperphagia that was mediated by changes in plasma leptin and ghrelin levels. Hyperphagia was accompanied by increased efficiency in converting caloric intake into body mass. The higher feed efficiency was mediated by greater insulin sensitivity. Energy expenditure of low protein offspring in locomotion was not affected either in the light or dark phase. However, low protein offspring exhibited higher resting and basal metabolic rates as evidenced by higher core body temperature in the fed and fasted states. The increased thermogenesis was not mediated by thyroid hormones but by an increased sympathetic nervous system drive as reflected by a lower areal bone mineral density and bone mineral content and lower plasma adiponectin and triglyceride levels. Elevated thermogenesis in the low protein offspring possibly offsets the effects of hyperphagia, minimizes their chances of weight gain, and improves survivability. This constellation of metabolic features in the low protein offspring will maximize survival potential in a post natal environment of nutritional scarcity and constitute a thrifty phenotype. [Copyright &y& Elsevier]

Abstract: Aripiprazole is an atypical antipsychotic drug primarily characterized by partial agonist activity at dopamine (DA) D2 receptors and serotonin-1A (5-hydroxytryptamine, 5-HT1A) receptors and minimal side effects. Based on its pharmacological profile, including stabilization of mesocorticolimbic DA activity (a pathway implicated in drug addiction), we investigated the effects of aripiprazole on relapse to morphine seeking in rats. In experiment 1, rats underwent morphine-induced conditioned place preference (CPP) training with alternate injections of morphine (5 mg/kg, s.c.) and saline (1 ml/kg, s.c.) for 8 consecutive days. To examine the effect of aripiprazole on the expression of morphine-induced CPP, rats received aripiprazole (0, 0.03, 0.1, and 0.3 mg/kg, i.p.) 30 min before testing for the expression of CPP. In experiment 2, rats underwent the same CPP training as in experiment 1 and subsequent extinction training. To examine the effect of aripiprazole on reinstatement of morphine-induced CPP, rats received aripiprazole 30 min before testing for reinstatement of CPP. In experiment 3, to assess the effects of aripiprazole on locomotor activity, aripiprazole was administered 30 min before testing for locomotor activity. Aripiprazole significantly decreased the reinstatement of CPP induced by a priming injection of morphine but had no effect on the expression of morphine-induced CPP or locomotor activity. The D2 and 5-HT1A partial agonist and 5-HT2A antagonist properties of aripiprazole likely account for the blockade of relapse to drug seeking. These findings suggest that aripiprazole may have therapeutic value for reducing craving and preventing relapse to drug seeking. [Copyright &y& Elsevier]

Background: Depression is a common mental disorder with unknown mechanism. Emerging evidence shows that miRNAs play a critical role in the process of depression. Here we reported the cerebrospinal fluid (CSF) miR-16 expression and its association with miR-16 and serotonin transporter (SERT) in the raphe of a rat model of depression.Methods: 20 rats were randomized to the control or CUMS (chronic unpredictable mild stress) group. The rats in the CUMS group underwent CUMS for 21 days, while those in the control group received no treatment. After anesthetization, CSF was collected for the measurement of miR-16. Then raphes from all rats were separated for determination of miR-16 and SERT protein.Results: The expression levels of miR-16 in CSF and raphe of the CUMS group were significantly lower than those of the control group (P = 0.007 and 0.031). However, SERT protein in raphe of the CUMS group was obviously increased as compared that of the control group (P = 0.005). There was a positive correlation between CSF miR-16 and raphe miR-16 (r = 0.95, P = 0.000). Meanwhile, negative correlations between miR-16 and SERT protein in raphe (r = -0.70 P = 0.02), between CSF miR-16 and raphe SERT protein (r = -0.86, P = 0.002) were observed in the CUMS group.Limitations: We have not explored the reason why CSF miR-16 was decreased in the rat model of depression and only tested the association of miR-16 between CSF and raphe.Conclusions: CSF miR-16 was involved in the pathogenesis of depression via reflecting raphe miR-16 level, and thus affecting raphe SERT expression. [ABSTRACT FROM AUTHOR]

Objectives The aim of this study was to use DKI to detect the microstructural change of the discs in matured normal rats. Methods Total 24 normal SD rats (12 males/12 females) underwent DWI/DKI and T2 sequences with a 3T MRI scanner to get the values of ADC, FA, MD, Da, Dr, MK, Ka and Kr. The discs were categorized using a five-grade degeneration grading system in the T2-images. The height of the discs and the parameters in DWI/DKI were measured to compare between the different grades and sexes. The histological images and the images of fiber tracking were also done in the discs. Results There were 30 Grade 1 and 18 Grade 2 in the discs. Compared with Grade 1, decreased ADC, increased FA and MK values were observed in Grade 2 (P < 0.05). By the ROC analysis of grades of the discs, there was low diagnostic accuracy in ADC value, while FA and MK showed higher accuracy. In Grade 1, there were lower ADC value, lower Dr, higher MK, Ka and Kr in male's group than them in female's group. There were no differences in the parameters except the ADC value in the two sexes in Grade 2. The different microstructure of the normal discs in the male and female rats had been proved by the histological images and the images of fiber tracking. Conclusion DKI is a noninvasive and sustainable means to test the changes of intervertebral discs. The discs in Grade 2 were also found in the normal matured SD rat tails. The assessment of the grade of the discs in T2-images should be done before the experimental management. There was microstructural difference in the nucleus pulposus in the discs in Grade 1 and 2. FA and MK showed higher diagnostic accuracy. The laboratory rats should be the same sex because the microstructure of the normal discs weren't the same. [ABSTRACT FROM AUTHOR]

Data from clinical investigations and laboratory fundings have provided preliminary evidence for the effectiveness and safety of acupuncture therapy in depression. However, the mechanisms underlying the antidepressant response of acupuncture are not fully elucidated. To elucidate the potential effects of acupuncture for depression on the hippocampal genome-wide transcriptome at the molecular level, we evaluated the transcriptomic profile of depression rats under treatment of acupuncture, and fluoxetine. We identified a very significant effect of acupucture intervention, with 107 genes differentially expressed in acupuncture vs. model group; while 41 genes between fluoxetine vs. model group. Notably, the 54 differentially expressed genes between acupuncture and fluoxetine showed the significantly different effect between acupuncture and fluoxetine. Through GO (gene ontology) functional term and KEGG (kyoto encyclopedia of genes and genomes) pathway analysis, we identified that the upregulation of gene sets were related to inflammatory response, innate immunity and immune response. We found that toll-like receptor signalling pathway and NOD like receptor signalling pathway were associated with the function of inflammatory response, innate immunity and immune response. Importantly, acupuncture reversed the upregulation of gene sets that were related to inflammatory response, innate immunity and immune response (including toll-like receptor signalling pathway and NOD like receptor signalling pathway), which might be critical for the pathogenesis of depression and provide evidence for the antidepressive effects of acupuncture by regulating inflammatory response, innate immunity and immune response via toll-like receptor signalling pathway and NOD like receptor signalling pathway. [ABSTRACT FROM AUTHOR]

In this study, we aimed to develop a safe and stable form of human growth hormone (hGH) and to refine PEGylation methods for therapeutic proteins via genetic code expansion. Through this precise approach, a series of polyethylene glycol (PEG) moieties and sites were combined in various ways. Additionally, the effects of combinatorial PEGylation on the biological, pharmacological, and immunogenic properties of hGH in vitro and vivo were analyzed. Our results showed that combinatorial PEGylation at Y35, G131, and K145 significantly reduced immunogenicity and improved pharmacokinetic (PK) profiles compared with mono-PEGylation, while retaining biological activity. Upon re-examination of the pharmacodynamics in hypophysectomized rats, multi-PEGylated hGH was found to be much more stable than mono-PEGylated hGH. Thus, this method for combinatorial, precise PEGylation may facilitate the development of next-generation, long-acting hGH with low immunogenicity. [ABSTRACT FROM AUTHOR]

Type 2 diabetes mellitus(T2DM) is a risk factor of Alzheimer’s disease (AD), which is most likely linked to impairments of insulin signaling in the brain. Hence, drugs enhancing insulin signaling may have therapeutic potential for AD. Lixisenatide, a novel long-lasting glucagon-like peptide 1 (GLP-1) analogue, facilitates insulin signaling and has neuroprotective properties. We previously reported the protective effects of lixisenatide on memory formation and synaptic plasticity. Here, we describe additional key neuroprotective properties of lixisenatide and its possible molecular and cellular mechanisms against AD-related impairments in rats. The results show that lixisenatide effectively alleviated amyloid β protein (Aβ) 25-35-induced working memory impairment, reversed Aβ25-35-triggered cytotoxicity on hippocampal cell cultures, and prevented against Aβ25-35-induced suppression of the Akt-MEK1/2 signaling pathway. Lixisenatide also reduced the Aβ25-35 acute application induced intracellular calcium overload, which was abolished by U0126, a specific MEK1/2 inhibitor. These results further confirmed the neuroprotective and cytoprotective action of lixisenatide against Aβ-induced impairments, suggesting that the protective effects of lixisenatide may involve the activation of the Akt-MEK1/2 signaling pathway and the regulation of intracellular calcium homeostasis. [ABSTRACT FROM AUTHOR]

Objective The study aims to find regulatory microRNA(s) responsible for down-regulated insulin receptor (InsR) in the liver of HFD-MetS E3 rats with insulin resistance. Methods: Firstly, hepatic insulin resistance in HFD-MetS E3 rats was evaluated by RT-qPCR, western blotting, immunohistochemistry and PAS staining. Secondly, the candidate miRNAs targeting rat InsR were predicted through online softwares and detected in the liver of HFD-MetS E3 rats with insulin resistance. Then, the expression of InsR, phosphorylated IRS-1 (pIRS-1) at Tyr632, phosphorylated AKTs (pAKTs) at Ser473 and Thr308, phosphorylated GSK-3β (p GSK-3β) at Ser9, phosphorylated GS (pGS) at Ser641 and the glycogen content were detected in CBRH-7919 cells treated with 100 nM insulin for different time periods by western blotting or PAS staining respectively, after transient transfection with miR-497 mimics or inhibitors for 24 h. Lastly, the relation between miR-497 and InsR was further determined using dual luciferase reporter assay. Results: Elevated miR-497 was negatively related with down-regulated InsR in the liver of HFD-MetS E3 rats with insulin resistance. Comparing with the mNC group, glycogen content and the expression of InsR, pIRS-1 (Tyr632), pAKTs (Ser473 and Thr308) and pGSK-3β (Ser9) decreased significantly in CBRH-7919 cells, while pGS (Ser641) increased significantly, after transient transfection with miR-497 mimics for 24 h and treatment with 100 nM insulin for corresponding time periods, counter to those results in CBRH-7919 cells after similar procedures with miR-497 inhibitors and insulin. In addition, dual luciferase reporter assay further confirmed that miR-497 can bind to the 3′UTR of rat InsR. Conclusion: Insulin receptor is the target gene of miR-497, and elevated miR-497 might induce hepatic insulin resistance in HFD-MetS E3 Rats through inhibiting the expression of insulin receptor and confining the activation of IRS-1/PI3K/Akt/GSK-3β/GS pathway to insulin. [ABSTRACT FROM AUTHOR]

In this study, we aimed to assess the neuroprotective effect of sevoflurane preconditioning in a cerebral focal ischemia–reperfusion rat model. Sixty Sprague Dawley rats were divided into six groups: sham operated group, cerebral focal ischemia–reperfusion (CIR) group, CIR + sevoflurane preconditioning (SP) (2%) group, CIR + sevoflurane preconditioning (2.5%) group, CIR + sevoflurane preconditioning (3%) group, and CIR + sevoflurane preconditioning (3.5%) group. All subjects were euthanized 2 days post-surgery and their hippocampus tissues were removed. Tissue malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GSH-Px) levels were measured and hippocampus tissue samples were examined histopathologically. Results showed that significant difference in antioxidant, immunity indexes, and apoptosis-related protein expression was detected in hippocampus tissue between sham-operated control and CIR groups. Sevoflurane preconditioning significantly dose-dependently reduced MDA, IL-1β, IL-6, IL-10 and TNF-α levels and enhanced antioxidant enzyme activities in hippocampus tissue of CIR + SP groups compared to CIR group. In addition, sevoflurane preconditioning significantly dose-dependently upregulated PI3K, p-Akt and Bcl-2 levels and downregulated caspase-3 and Bax levels in hippocampus tissue of CIR + SP groups compared to CIR group. It can be concluded that sevoflurane preconditioning demonstrates a strong and ameliorative effect on cerebral I/R damage in rats. The neuroprotective mechanisms of sevoflurane preconditioning are associated with its properties of anti-apoptosis and anti-oxidation as well as regulation of PI3K and p-Akt signal activation. [ABSTRACT FROM AUTHOR]

Multiple mitochondrial dysfunction (MMD) can lead to complex damage of mitochondrial structure and function, which then lead to the serious damage of various metabolic pathways including cerebral abnormalities. However, the effects of MMD on heart, a highly mitochondria-dependent tissue, are still unclear. In this study, we use iron-sulfur cluster assembly 1 (Isca1), which has been shown to cause MMD syndromes type 5 (MMDS5), to verify the above scientific question. We generated myocardium-specific Isca1 knockout rat (Isca1 flox/flox / α-MHC-Cre) using CRISPR-Cas9 technology. Echocardiography, magnetic resonance imaging (MRI), histopathological examinations and molecular markers detection demonstrated phenotypic characteristics of our model. Immunoprecipitation, immunofluorescence co-location, mitochondrial activity, ATP generation and iron ions detection were used to verify the molecular mechanism. This study was the first to verify the effects of Isca1 deficiency on cardiac development in vivo , that is cardiomyocytes suffer from mitochondria damage and iron metabolism disorder, which leads to myocardial oncosis and eventually heart failure and body death in rat. Furthermore, forward and reverse validation experiments demonstrated that six-transmembrane epithelial antigen of prostate 3 (STEAP3), a new interacting molecule for ISCA1, plays an important role in iron metabolism and energy generation impairment induced by ISCA1 deficiency. This result provides theoretical basis for understanding of MMDS pathogenesis, especially on heart development and the pathological process of heart diseases, and finally provides new clues for searching clinical therapeutic targets of MMDS. • First study to verify the effects of Isca1 deficiency on cardiac development in vivo. • Isca1 deficiency in myocardium causes mitochondria and iron metabolism damage. • Isca1 deficiency leads to myocardial oncosis and eventually HF and body death in rat. • STEAP3 is involved in iron metabolism disorder induced by Isca1 deficiency. • Provides new clues for searching clinical therapeutic targets of MMDS. [ABSTRACT FROM AUTHOR]

Oxidative stress plays an important role in the neuronal damage induced by epilepsy. The present study assessed the possible neuroprotective effects of astaxanthin (ATX) on neuronal damage, in hippocampal CA3 neurons following amygdala kindling. Male Sprague–Dawley rats were chronically kindled in the amygdala and ATX or equal volume of vehicle was given by intraperitoneally. Twenty-four hours after the last stimulation, the rats were sacrificed by decapitation. Histopathological changes and the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and reduced glutathione (GSH) were measured, cytosolic cytochrome c (CytC) and caspase-3 activities in the hippocampus were also recorded. We found extensive neuronal damage in the CA3 region in the kindling group, which was preceded by increases of ROS level and MDA concentration and was followed by caspase-3 activation and an increase in cytosolic CytC. Treatment with ATX markedly attenuated the neuronal damage. In addition, ATX significantly decreased ROS and MDA concentrations and increased GSH levels. Moreover, ATX suppressed the translation of CytC release and caspase-3 activation in hippocampus. Together, these results suggest that ATX protects against neuronal loss due to epilepsy in the rat hippocampus by attenuating oxidative damage, lipid peroxidation and inhibiting the mitochondrion-related apoptotic pathway. [ABSTRACT FROM AUTHOR]

Our previous investigations have revealed that cerebellar cholinergic innervation is involved in cardiovascular regulation. This study was performed to examine the effects of the muscarinic cholinergic receptor (mAChR) in the cerebellar cortex on blood pressure (BP) modulation in rats. Acetylcholine (ACh, 100 mM), nonselective mAChR agonist (oxotremorine M; Oxo-M, 10, 30 and 100 mM) and 100 mM ACh mixed with nonselective mAChR antagonist atropine (1, 3 and 10 mM) were microinjected into the cerebellar cortex of anesthetized rats. Mean arterial pressure (MAP), maximal decreased MAP (MDMAP), and reaction time (duration required for BP to return to basal values) were measured and analyzed. The results showed that Oxo-M dose-dependently decreased MAP, increased MDMAP, and prolonged reaction time, which displayed a homodromous effect of ACh-mediated blood depressor response; meanwhile, atropine concentration-dependently blocked the effect of ACh on the BP regulation. In conclusion, the present study showed for the first time that mAChRs in cerebellar cortex could modulate somatic BP by participation in ACh-mediated depressor response. [ABSTRACT FROM AUTHOR]

Highlights: [•] Highlight of evaluation of TM208 toxic effects by metabonomics. [•] The biomarkers were identified with metabonomics analytical software and online database. [•] Mechanism of TM208 toxicity was related to tricarboxylic acid (TCA) cycle and lysine metabolism. [ABSTRACT FROM AUTHOR]

Abstract: A novel strontium salt compound strontium fructose 1, 6-diphosphate (FDP-Sr) has been proved to have highly effective for bone loss via dual effects of stimulating bone formation and suppressing bone absorption. In the present study, metabolomic approach was used to identify and study potential biomarkers associated with the effect and safety of FDP-Sr. The metabolomic profiles of bone loss induced by estrogen deficiency in a rat model was described to attain a system-level map of the shift on the metabolic response in plasma using GC/TOF-MS, after FDP-Sr was orally administered at the dose of 110mg/kg/day for the prevention and 220mg/kg/day for the treatment. Meanwhile, bone turnover biomarkers and bone mineral density were investigated to identify the specific changes of potential anti-osteoporosis effects of FDP-Sr. The differences in metabolic profiles between osteoporosis rats and FDP-Sr treated rats were well observed by the partial least squares-discriminant analysis (PLS-DA) to the MS spectra. Some metabolites including homocysteine, arachidonic acid, alanine, and hydroxyproline, which significantly changed during osteoporosis progression could be effectively reversed after FDP-Sr therapy. Of course some metabolites such as uric acid, glyceric acid, octadecadienoic acid, docosahexaenoic acid, oleic acid, and hexadecanoic acid were not found to reverse significantly after FDP-Sr administration. These results delineated the FDP-Sr effects-related metabolic alterations in the bone loss rats, suggesting that metabonomic analysis could provide helpful information on the new potential biomarkers relating to the mechanism of anti-osteoporosis action and side effects of FDP-Sr against estrogen deficiency induced bone loss. [Copyright &y& Elsevier]

Abstract: Eleutheroside B and Eleutheroside E, two kinds of the major bioactive saponins of Eleutherococcus senticosus, play a pivotal role in biologic activity. In this study, a specific and sensitive high performance liquid chromatography–electrospray ionization-tandem mass spectrometry method (HPLC–MS/MS) was developed and validated for simultaneous determination of Eleutheroside B and Eleutheroside E in rat plasma. The analytes were extracted from rat plasma via a simple protein precipitation procedure with methanol and polygonin was used as internal standard. Chromatographic separation was achieved on a C18 column using a gradient elution program with acetonitrile and water containing 0.1% ammonium hydroxide solution as the mobile phase, with a flow rate of 0.2mL/min. The detection was performed on a triple–quadrupole tandem mass spectrometer by multiple reactions monitoring (MRM) mode in a negative ion mode via electrospray ionization (ESI). The transition monitored were m/z 371 [M−H]− →209 for Eleutheroside B, m/z 741[M−H]− →579 for Eleutheroside E and m/z 389[M−H]− →277 for internal standard. Linear calibration curves were obtained in the concentration range of 1–2000ng/mL for both (Eleutheroside B and Eleutheroside E), with a lower limit of quantification of 1ng/mL. Extraction recovery was over 80% in plasma. The intra- and inter-day precision (RSD) values were below 12% and accuracy (RE) was −2.80 to 5.70% at three QC levels for both. The assay was successfully applied to study pharmacokinetics behavior in rats after oral and intravenous administration of the single substances (Eleutheroside B and Eleutheroside E). And further research was performed by comparing the difference in pharmacokinetic behavior between the single substances and an aqueous extract of E. senticosus after oral administration. Significant difference in pharmacokinetic characteristics between the single substances and an aqueous extract was found in rat, which would be beneficial for the pre-clinical research and clinical use of E. senticosus. [Copyright &y& Elsevier]

Abstract: Aluminum (Al) is a low toxicological metal and can accumulate in the liver. The hepatic microsomal cytochrome P450 enzyme system (CYPS) plays important role in the transformation of the toxic materials. It is not clear if the CYPS is affected by Al exposure. Thus, the aim of this study is to investigate the effects of aluminum trichloride (AlCl3) on CYPS in rats. Forty male Wistar rats (5weeks old) weighing 110–120g were randomly allocated and orally exposed to 0, 64.18, 128.36 and 256.72mg/kg body weight (BW) AlCl3 in drinking water for 120days. The body weight (BW) of rats, hepatosomatic index (HSI), hepatic Al content, the concentrations of cytochrome P450 (CYP450), cytochrome B5 (B5), microsomal protein and the activities of NADPH-cytochrome c reductase (CR), aminopyrin N-demethylase (AND), erythromycin N-demethylase (ERND) and aniline-4-hydeoxylase (AH) were assessed at the end of the experiment. The results showed that the increase in Al concentration decreased BW, HIS, concentrations of CYP450, B5, microsomal protein and the activity of CR, AND, ERND and AH in hepatic microsomes. The results revealed that exposure to AlCl3 inhibited the microsomal CYP450 dependent enzyme system of liver. Our findings suggest that long term daily exposure of AlCl3 exerts the suppressive effects and thus may cause dysfunction of hepatic CYP450 dependent enzyme system of rat. [Copyright &y& Elsevier]

Abstract: A simple and sensitive high-performance liquid chromatography–electro-spray ionization tandem mass spectrometry (LC–ESI-MS/MS) method was developed and validated to determine raddeanin A in rat plasma. After precipitation of rat plasma samples with methanol, chromatographic separation was achieved on a BDS Hypersil C18 column (100×2.1mm, 2.4μm) using the mobile phase consisted of acetonitrile and 2mM ammonium acetate with 0.05% formic acid (60:40, v/v). The detection was performed in a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode using negative ionization. The transition monitored were m/z 895.6→455.0 for raddeanin A and m/z 359.3→329.0 for IS, respectively. The method was linear over the concentration range of 2–1000ng/mL for raddeanin A. The intra-day and inter-day assay variations were <9.46%, and the accuracy values were between −2.04% and −6.52% relative error. The extraction recovery of raddeanin A was more than 70%, and the relative matrix effect ranges from 108.52% to 112.36%. The validated method has been successfully applied to determine the pharmacokinetic profile of raddeanin A in rat plasma following oral and intravenous administration. [Copyright &y& Elsevier]

Abstract: The bioaccumulation and immunotoxicity of aluminum (Al) have been previously documented. Al accumulates in the organs of the organism, including spleen. Spleen is a peripheral organ of the immune system. The accumulated Al may alter the immune function. Here, we investigated the bioaccumulation of Al in spleen and its alterations in the immune system. Forty male Wistar rats (5weeks old) weighed 110–120g were orally exposed to aluminum trichloride (AlCl3) (0, 64.18, 128.36 and 256.72mg/kg body weight) in drinking water for 120days. The concentrations of spleen’s Al, iron (Fe), copper (Cu), zinc (Zn), interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) and growth index were examined at the end of the experiment. The results showed that the concentrations of Al and Cu in the spleen were increased in an AlCl3-dose dependent manner, and the concentrations of spleen’s growth index, Fe, Zn, IL-2 and TNF-α were reduced in AlCl3-treated rats. The results suggest that AlCl3 can suppress the growth of spleen, disorder the balance of trace elements and inhibit the immune regulation of cytokines in the spleen. It indicates that AlCl3 suppresses the immune function of spleen. [Copyright &y& Elsevier]

Abstract: Cognitive dysfunction is commonly observed in epileptic patients. It has been shown that not only epilepsy but also antiepileptic drugs could induce cognitive impairment. Thus, there is an urgent need for drugs that can suppress seizures without causing cognitive deficit. Recent studies have shown that oxidative stress is involved in the pathophysiology of epilepsy, and many antioxidants have an antiepileptic property. Epigallocatechin-3-gallate (EGCG), a catechin polyphenols component, is found to be an effective antioxidant. The purpose of this study was to assess the effect of EGCG against seizures, seizure-induced oxidative stress and cognitive impairment in pentylenetetrazole-induced kindling. Male Sprague-Dawley rats were injected intraperitoneally with a dose of 35mg/kg of pentylenetetrazole (PTZ) once every alternate day for 13 injections. EGCG was administered daily in two doses (25mg/kg and 50mg/kg) intraperitoneally along with alternate-day PTZ. Morris water maze test was carried out 24h after the last injection of PTZ, and the oxidative stress parameters (malondialdehyde and glutathione) were assessed after the completion of the behavioral test. The results showed that EGCG dose-dependently suppressed the progression of kindling. EGCG also ameliorated the cognitive impairment and oxidative stress induced by PTZ kindling. These observations suggest that EGCG may be a potential agent for the treatment of epilepsy as well as a preventive agent against cognitive impairment induced by seizure. [Copyright &y& Elsevier]

Abstract: Background: Previous studies suggested that platelet-activating factor (PAF) plays an important role in ischemic diseases. Apoptosis has been implicated in myocardial infarction-related cell death. The present study was designed to determine whether PAF could induce apoptosis in cardiac myocytes and the underlying mechanisms by which PAF causes apoptosis. Methods: H9c2 cardiac myocytes were used to investigate the effect of PAF on intracellular calcium concentration, cell viability and cell apoptosis. Signaling pathway of caspase-3, cytochrome c and MAPK (ERK, JNK, p38) was determined during the PAF induced apoptosis. Results: First, our results showed that treatment of H9c2 cardiomyocytes with PAF (0.2 to 20 µM) caused apoptosis in these cells and the apoptotic process was suppressed by either BN52021 (an antagonist of PAF receptor) or BAPTA/AM (an intracellular Ca2+ chelator), suggesting an involvement of PAF and its receptor mediated calcium-dependent signaling. Second, we found that activity of p38-MAPK (mitogen-activated protein kinase) and caspase-3 was elevated in the cells treated with PAF, without altering activity of ERK and JNK, and that PAF-induced enhancement of caspase-3 activity was attenuated by application of either BAPTA/AM or SB203580 (p38 inhibitor). Furthermore, PAF-induced apoptosis and release of cytochrome c from mitochondria was blunted by SB203580, and PAF-induced enhancement of p38 activity was also attenuated by BAPTA/AM. Conclusion: Our data implicate that a PAF and its receptor in triggering apoptosis occurs in cultured H9c2 cardiac myocytes via a calcium-dependent p38 MAPK activated cytochrome c/caspase-3 apoptosis signaling pathway. [Copyright &y& Elsevier]

Abstract: Nicotine was reported to reduce the plaque burden and could be used as a possible anti-Alzheimer''s disease agent. However, the effect of nicotine on memory and tau pathology in Alzheimer''s disease has been less studied. The present study investigated the effect of nicotine on tau phosphorylation and cognitive impairment induced by hippocampus injections of amyloid-beta (Aβ) 25–35. Rats were treated with nicotine hydrogen tartrate salt dissolved in normal saline by subcutaneous injection twice per day for 14days. The age and gender matched rats treated with same amount of normal saline were used as the control. Morris water maze was used to detect the cognitive impairment induced by Aβ25–35. Compared to the sham-operated rats, Aβ25–35 injection significantly prolonged the mean escape latency in vehicle-treated rats in the Morris water maze test and increased the number of tau(pS202) and tau(pT231) immunoreactive cells. The data show that nicotine (1mg/kg in base weight) treatment significantly exacerbates cognitive impairment and tau phosphorylation at Ser-202 and Thr-231 in the hippocampus compared with Aβ25–35 injection groups in the Aβ rat model of Alzheimer''s disease. The use of nicotine for treatment of Alzheimer''s disease should be reassessed. [Copyright &y& Elsevier]

Abstract: cAMP is a key modulator for glucose-dependent insulin secretion (GDIS). Members of the phosphodiesterase (PDEs) gene family regulate intracellular levels of cAMP by hydrolyzing cAMP to the corresponding inactive 5′AMP derivative. These studies examined the expression and function of all 18 cAMP-specific PDEs in the rat insulinoma derived INS-1 (832/13) cell and isolated rat islets using quantitative PCR and siRNA-mediated gene-specific knockdown. PDE1C, PDE3B, PDE4C, PDE8B, PDE10A, and PDE11A were significantly expressed in rat islets and INS-1 (832/13) cells at the mRNA level. PDE1C, PDE10A and PDE11A were also expressed in brain, along with PDE3B, PDE4C and PDE8B which were also highly expressed in liver, and PDE3B was present in adipose tissue and PDE4C in skeletal muscle. siRNA mediated knockdown of PDE1C, PDE3B, PDE8B and PDE4C, but not PDE10A and PDE11A, significantly enhanced GDIS in rat INS-1 (832/13) cells. Also, selective inhibitors of PDE3 (trequinsin) and PDE4 (roflumilast and L-826,141) significantly augmented GDIS in both INS-1 (832/13) cells and rat islets. The combination of PDE3 and PDE4 selective inhibitors demonstrate that these enzymes comprise a significant proportion of the cAMP metabolizing activity in INS-1 cells and rat islets. [Copyright &y& Elsevier]

Abstract: A rapid and sensitive method for the simultaneous determination of cryptotanshinone and its active metabolite tanshinone IIA in rat plasma was developed and well validated, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. This method entailed a single step of liquid–liquid extraction with ethyl acetate from a small volume of plasmas. The analytes and internal standard diazepam were baseline separated on a Shim-pack VP-ODS analytical column. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization source operated under selected reaction monitoring (SRM) mode. The method was linear in the concentration range of 1–100ng/ml for both tanshinone IIA and cryptotanshinone. The intra- and inter-day precisions (R.S.D.%) were within 10.2% for both analytes. Deviation of the assay accuracies was within ±12.0% for both analytes. Both analytes were proved to be stable during all sample storing, preparation and analytic procedures. The method was successfully applied to a pharmacokinetic study after an oral administration of cryptotanshinone to rats with a dose of 20mg/kg. With the lower limits of quantification at 1.0ng/ml for tanshinone IIA and 0.2ng/ml for cryptotanshinone, this method was proved to be sensitive enough and reproducible for the pharmacokinetics study of both tanshinones. [Copyright &y& Elsevier]

Chronic manganese (Mn) exposure is related to elevated risks of neurodegenerative diseases, and mitochondrial dysfunction is considered a critical pathophysiological feature of Mn neurotoxicity. Although previous research has demonstrated Mn-induced alpha-synuclein (α-Syn) overexpression, the role of α-Syn in mitochondrial dysfunction remains unclear. Here, we used Wistar rats and human neuroblastoma cells (SH-SY5Y cells) to elucidate the molecular mechanisms underlying how α-Syn overexpression induced by different doses of Mn (15, 30, and 60 mg/kg) results in mitochondrial dysfunction. We found that Mn-induced neural cell injury was associated with mitochondrial damage. Furthermore, Mn upregulated α-Syn protein levels and increased the interaction between α-Syn and mitochondria. We then used a lentivirus vector containing α-Syn shRNA to examine the effect of Mn-induced α-Syn protein on PINK1/Parkin-mediated mitophagy in SH-SY5Y cells. Our data demonstrated that the knockdown of α-Syn decreased the interaction between α-Syn and PINK1. The enhanced level of phosphorylated Parkin (p-Parkin) was due to the decrease of the interaction between α-Syn and PINK1. Moreover, the knockdown of α-Syn increased recruitment of p-Parkin to mitochondria. Collectively, these observations revealed that Mn-induced α-Syn overexpression repressed PINK1/Parkin-mediated mitophagy and exacerbated mitochondrial damage. [Display omitted] • Mn could induce mitochondria-mediated nerve cell injury. • Mn-induced over-expressed α-Syn protein was localized to mitochondria. • Over-expressed α-Syn interfered with the phosphorylation of Parkin by PINK1. • Overexpression of α-Syn disturbed PINK1/Parkin-mediated mitophagy. • Mn-induced over-expressed α-Syn protein aggravated mitochondrial damage. [ABSTRACT FROM AUTHOR]