The effects of intermittent hypoxia on hepatic expression of fatty acid translocase CD36 in lean and diet-induced obese mice.

BACKGROUND: Both obstructive sleep apnea (OSA) and non-alcoholic fatty liver disease (NAFLD) are prevalent within obese individuals. We aimed to investigate the effects of intermittent hypoxia (IH), a clinical feature of OSA, on hepatic expression of fatty acid translocase (CD36) in relation to liver injury in lean and diet-induced obese mice. METHODS: Four-week-old male C57BL/6J mice were randomized to standard diet (SD) or high fat (HF) diet groups. At 13-week-old, all mice were exposed to either air or IH (IH30; thirty hypoxic episodes per hour) for four weeks. We assessed liver injury through lipid profile, oxidative and inflammatory stress, histological scoring and hepatic CD36 expression. RESULTS: In lean mice, IH elevated serum and hepatic triglyceride and free fatty acid (FFA) levels, in line with upregulation of hepatic CD36 expression and myeloperoxidase (MPO)-positive cells in support of inflammatory infiltrates along with increase in serum malondialdehyde (MDA), C-X-C motif chemokine ligand 1(CXCL-1) and monocyte chemoattractant protein-1 (MCP-1). In diet-induced obese mice, an increase in hepatic alanine transaminase (ALT) activity, serum and hepatic levels of lipid parameters and inflammatory markers, serum MDA level, hepatic expressions of CD36 and α-smooth muscle actin (α-SMA), and MPO-positive cells was observed. IH potentiated hepatic ALT activity, serum CXCL-1 and hepatic interleukin-6 (IL-6), in line with inflammatory infiltrates, but paradoxically, reduced hepatic FFA level and hepatic CD36 expression, compared to obese mice without IH exposure. However, IH further augmented diet-induced liver steatosis and fibrosis as shown by histological scores. CONCLUSION: This study contributes to support that IH featuring OSA may lead to liver injury via differential regulation of hepatic CD36 expression in lean and diet-induced obese mice.

Differential metabolic and inflammatory responses to intermittent hypoxia in substrains of lean and obese C57BL/6 mice.

AIMS: This study was to investigate the degree of susceptibility to intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), between the two mice inbred lines C57BL/6N (6N) and C57BL/6J (6J). MATERIALS AND METHODS: Four-week old male mice of 6N and 6J substrains (n = 8) were randomized to standard diet (SD) group or high fat (HF) diet group. At the age of 13-week, all two groups of mice were subjected to either air or IH (IH30; thirty hypoxic events per hour) for one week. KEY FINDINGS: All mice fed with HF diet exhibited obesity with more body weight and fat mass (percentage to body weight) gain. IH reduced serum LDL, HDL and total cholesterol levels in lean 6J mice. In obese mice, IH lowered obesity-induced serum total cholesterol level in 6J substrain but raised further in 6N substrain. Furthermore, IH caused elevation of serum FFA and MDA levels, and pro-inflammatory cytokines MCP-1 and IL-6 levels in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) of lean 6J but not lean 6N mice. There was reduced number of adipocytes and elevation of macrophages in SAT and VAT of HF-induced obese mice of both substrains. IH led to increased number of adipocytes and macrophages in SAT of lean 6J mice. SIGNIFICANCE: The genetic difference between 6N and 6J mice may have direct impact on metabolic and inflammatory responses after IH. Therefore, attention must be given for the selection of C57BL mice substrains in the experimental IH-exposed mouse model.

Induced Pluripotent Stem Cells-Derived Mesenchymal Stem Cells Attenuate Cigarette Smoke-Induced Cardiac Remodeling and Dysfunction.

The strong relationship between cigarette smoking and cardiovascular disease (CVD) has been well-documented, but the mechanisms by which smoking increases CVD risk appear to be multifactorial and incompletely understood. Mesenchymal stem cells (MSCs) are regarded as an important candidate for cell-based therapy in CVD. We hypothesized that MSCs derived from induced pluripotent stem cell (iPSC-MSCs) or bone marrow (BM-MSCs) might alleviate cigarette smoke (CS)-induced cardiac injury. This study aimed to investigate the effects of BM-MSCs or iPSC-MSCs on CS-induced changes in serum and cardiac lipid profiles, oxidative stress and inflammation as well as cardiac function in a rat model of passive smoking. Male Sprague-Dawley rats were randomly selected for exposure to either sham air (SA) as control or 4% CS for 1 h per day for 56 days. On day 29 and 43, human adult BM-MSCs, iPSC-MSCs or PBS were administered intravenously to CS-exposed rats. Results from echocardiography, serum and cardiac lipid profiles, cardiac antioxidant capacity, cardiac pro- and anti-inflammatory cytokines and cardiac morphological changes were evaluated at the end of treatment. iPSC-MSC-treated group showed a greater effect in the improvement of CS-induced cardiac dysfunction over BM-MSCs-treated group as shown by increased percentage left ventricular ejection fraction and percentage fractional shortening, in line with the greater reversal of cardiac lipid abnormality. In addition, iPSC-MSCs administration attenuated CS-induced elevation of cardiac pro-inflammatory cytokines as well as restoration of anti-inflammatory cytokines and anti-oxidative markers, leading to ameliorate cardiac morphological abnormalities. These data suggest that iPSC-MSCs on one hand may restore CS-induced cardiac lipid abnormality and on the other hand may attenuate cardiac oxidative stress and inflammation via inhibition of CS-induced NF-κB activation, leading to improvement of cardiac remodeling and dysfunction. Thus, iPSC-MSCs may be a promising candidate in cell-based therapy to prevent cardiac complications in smokers.

Intermittent hypoxia-induced NF-κB and HO-1 regulation in human endothelial EA.hy926 cells.

Intermittent hypoxia (IH) is a hallmark feature in obstructive sleep apnea (OSA) which is increasingly recognized as an independent risk factor for atherosclerosis. Oxidative stress, inflammation, and cell apoptosis are major pathological events initiating or accelerating atherogenesis. This study addressed whether IH would affect these proatherogenic factors in endothelial cells and the mechanistic pathways involved. EA.hy926 cells were exposed to intermittent normoxia or IH for different numbers of cycles (32, 64, or 96). IH exposure time-dependently raised cellular GSSG/GSH ratio, increased production of IL-6 and IL-8, and accelerated cell apoptosis and death, concurrent with activation of NF-κB and inhibition of Nrf2/HO-1 pathways. At 64 cycles, inhibition of NF-κB attenuated IH-induced cellular oxidative stress and accumulation of inflammatory cytokines in cell culture medium but aggravated IH-induced cell apoptosis, while stimulation of HO-1 suppressed IH-induced cellular oxidative stress and cell apoptosis without affecting accumulation of inflammatory cytokines in cell culture medium. We demonstrated that early stage of exposure to IH-induced oxidative and inflammatory stresses leading to acceleration of cell apoptosis via NF-κB and Nrf2/HO-1 pathways in endothelial cells, suggesting the potential mechanisms for IH-induced vascular pathogenesis, in resemblance to OSA.

Cigarette smoking accelerated brain aging and induced pre-Alzheimer-like neuropathology in rats.

Cigarette smoking has been proposed as a major risk factor for aging-related pathological changes and Alzheimer's disease (AD). To date, little is known for how smoking can predispose our brains to dementia or cognitive impairment. This study aimed to investigate the cigarette smoke-induced pathological changes in brains. Male Sprague-Dawley (SD) rats were exposed to either sham air or 4% cigarette smoke 1 hour per day for 8 weeks in a ventilated smoking chamber to mimic the situation of chronic passive smoking. We found that the levels of oxidative stress were significantly increased in the hippocampus of the smoking group. Smoking also affected the synapse through reducing the expression of pre-synaptic proteins including synaptophysin and synapsin-1, while there were no changes in the expression of postsynaptic protein PSD95. Decreased levels of acetylated-tubulin and increased levels of phosphorylated-tau at 231, 205 and 404 epitopes were also observed in the hippocampus of the smoking rats. These results suggested that axonal transport machinery might be impaired, and the stability of cytoskeleton might be affected by smoking. Moreover, smoking affected amyloid precursor protein (APP) processing by increasing the production of sAPPß and accumulation of ß-amyloid peptide in the CA3 and dentate gyrus region. In summary, our data suggested that chronic cigarette smoking could induce synaptic changes and other neuropathological alterations. These changes might serve as evidence of early phases of neurodegeneration and may explain why smoking can predispose brains to AD and dementia.

Inhibitory effect of Chinese green tea on cigarette smoke-induced up-regulation of airway neutrophil elastase and matrix metalloproteinase-12 via antioxidant activity.

Our recent study has indicated that Chinese green tea (Lung Chen), in which epigallocatechin-3-gallate (EGCG) accounts for 60% of catechins, protected cigarette smoke-induced lung injury. We now hypothesized that Lung Chen tea may also have potential effect on lung oxidative stress and proteases/anti-proteases in a smoking rat model. Sprague-Dawley rats were exposed to either sham air (SA) or 4% cigarette smoke (CS) plus 2% Lung Chen tea or water by oral gavage. Serine proteases, matrix metalloproteinases (MMPs) and their respective endogenous inhibitors were determined in bronchoalveolar lavage (BAL) and lung tissues by gelatin/casein zymography and biochemical assays. Green tea consumption significantly decreased CS-induced elevation of lung lipid peroxidation marker, malondialdehyde (MDA), and CS-induced up-regulation of neutrophil elastase (NE) concentration and activity along with that of α(1)-antitrypsin (α(1)-AT) and secretory leukoproteinase inhibitor (SLPI) in BAL and lung. In parallel, significant elevation of MMP-12 activity was found in BAL and lung of the CS-exposed group, which returned to the levels of SA-exposed group after green tea consumption but not CS-induced reduction of tissue inhibitor of metalloproteinase (TIMP)-1 activity, which was not reversed by green tea consumption. Taken together, our data supported the presence of local oxidative stress and protease/anti-protease imbalance in the airways after CS exposure, which might be alleviated by green tea consumption through its biological antioxidant activity.

Up-regulation of crystallins is involved in the neuroprotective effect of wolfberry on survival of retinal ganglion cells in rat ocular hypertension model.

Wolfberry (fruit of Lycium barbarum Linn) has been known for balancing 'Yin' and 'Yang' in the body, nourishing the liver and kidney, improving visual acuity for more than 2,500 years in oriental countries. The active components in wolfberry include L. barbarum polysaccharide (LBP), zeaxanthine, betaine, cerebroside and trace amounts of zinc, iron, and copper. Each of them confers distinct beneficial effects and together they help to explain widespread use of wolfberry in the eastern world. Earlier study reported the neuroprotective effects of LBP on retinal ganglion cell (RGC) in an experimental model of glaucoma and the underlying in vivo cellular mechanisms of LBP neuroprotection deserve further exploration. In this study, we adopted proteomics, functional genomics, to evaluate pharmacological effects of LBP on the neuronal survival pathways. Among the significantly changed proteins induced by LBP feeding on ocular hypertension (OH) retinas, only proteins in crystallin family were focused in this study. The proteomic results were further confirmed using the Western blotting of the retinas and immunohistochemical staining of the retinal sections. We demonstrated that neuroprotective effect of-wolfberry extract-LBP on the survival of RGCs may be mediated via direct up-regulation of neuronal survival signal betaB2-crystallin.

Modulation of morphological changes of microglia and neuroprotection by monocyte chemoattractant protein-1 in experimental glaucoma.

Monocyte chemoattractant protein-1 (MCP-1)/CCL2 is a C-C chemokine involved in the activation and recruitment of monocytic cells to injury sites. MCP-1/CCL2 can induce either neuroprotection or neurodestruction in vitro, depending on the experimental model. We aim to use MCP-1/CCL2 as an experimental tool to investigate the morphological changes of microglia when loss of healthy retinal ganglion cells (RGCs) is exacerbated or attenuated in an experimental glaucoma model. While a high concentration (1000 ng) of MCP-1/CCL2 and lipopolysaccharide (LPS)-exacerbated RGC loss, 100 ng MCP-1/CCL2 provided neuroprotection towards RGC. Neuroprotective MCP-1/CCL2 (100 ng) also upregulated insulin-like growth factor-1 (IGF-1) immunoreactivity in the RGCs. The neuroprotective effect of MCP-1/CCL2 was not due to the massive infiltration of microglia/macrophages. Taken together, this is the first report showing that an appropriate amount of MCP-1/CCL2 can protect RGCs in experimental glaucoma.

Modulation of microglia by Wolfberry on the survival of retinal ganglion cells in a rat ocular hypertension model.

The active component of Wolfberry (Lycium barbarum), lycium barbarum polysaccharides (LBP), has been shown to be neuroprotective to retinal ganglion cells (RGCs) against ocular hypertension (OH). Aiming to study whether this neuroprotection is mediated via modulating immune cells in the retina, we used multiphoton confocal microscopy to investigate morphological changes of microglia in whole-mounted retinas. Retinas under OH displayed slightly activated microglia. One to 100 mg/kg LBP exerted the best neuroprotection and elicited moderately activated microglia in the inner retina with ramified appearance but thicker and focally enlarged processes. Intravitreous injection of lipopolysaccharide decreased the survival of RGCs at 4 weeks, and the activated microglia exhibited amoeboid appearance as fully activated phenotype. When activation of microglia was attenuated by intravitreous injection of macrophage/microglia inhibitory factor, protective effect of 10 mg/kg LBP was attenuated. The results implicated that neuroprotective effects of LBP were partly due to modulating the activation of microglia.

Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research.

Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line which has been used as an in vitro model for neurotoxicity experiments. Although the neuroblastoma is usually differentiated by all-trans-retinoic acid (RA), both RA-differentiated and undifferentiated SH-SY5Y cells have been used in neuroscience research. However, the changes in neuronal properties triggered by RA as well as the subsequent responsiveness to neurotoxins have not been comprehensively studied. Therefore, we aim to re-evaluate the differentiation property of RA on this cell line. We hypothesize that modulation of signaling pathways and neuronal properties during RA-mediated differentiation in SH-SY5Y cells can affect their susceptibility to neurotoxins. The differentiation property of RA was confirmed by showing an extensive outgrowth of neurites, increased expressions of neuronal nuclei, neuron specific enolase, synaptophysin and synaptic associated protein-97, and decreased expression of inhibitor of differentiation-1. While undifferentiated SH-SY5Y cells were susceptible to 6-OHDA and MPP+, RA-differentiation conferred SH-SY5Y cells higher tolerance, potentially by up-regulating survival signaling, including Akt pathway as inhibition of Akt removed RA-induced neuroprotection against 6-OHDA. As a result, the real toxicity cannot be revealed in RA-differentiated cells. Therefore, undifferentiated SH-SY5Y is more appropriate for studying neurotoxicity or neuroprotection in experimental Parkinson's disease research.

Erratum: Modulation of microglia by Wolfberry on the survival of retinal ganglion cells in a rat ocular hypertension model.

The active component of Wolfberry (Lycium barbarum), lycium barbarum polysaccharides (LBP), has been shown to be neuroprotective to retinal ganglion cells (RGCs) against ocular hypertension (OH). Aiming to study whether this neuroprotection is mediated via modulating immune cells in the retina, we used multiphoton confocal microscopy to investigate morphological changes of microglia in whole-mounted retinas. Retinas under OH displayed slightly activated microglia. One to 100 mg/kg LBP exerted the best neuroprotection and elicited moderately activated microglia in the inner retina with ramified appearance but thicker and focally enlarged processes. Intravitreous injection of bacterial endotoxin lipopolysaccharide (LPS) decreased the survival of RGCs at 4 weeks, and the activated microglia exhibited amoeboid appearance as fully activated phenotype. When activation of microglia was attenuated by intravitreous injection of macrophage/microglia inhibitory factor, protective effect of 10 mg/kg LBP was attenuated. The results implicated that neuroprotective effects of LBP were partly due to modulating the activation of microglia.[This corrects the article on p. in vol. .].

Retrograde labeling of retinal ganglion cells by application of fluoro-gold on the surface of superior colliculus.

Retinal ganglion cell (RGC) counting is essential to evaluate retinal degeneration especially in glaucoma. Reliable RGC labeling is fundamental for evaluating the effects of any treatment. In rat, about 98% of RGCs is known to project to the contralateral superior colliculus (SC) (Forrester and Peters, 1967). Applying fluoro-gold (FG) on the surface of SC can label almost all the RGCs, so that we can focus on this most vulnerable retinal neuron in glaucoma. FG is taken up by the axon terminals of retinal ganglion cells and bilaterally transported retrogradely to its somas in the retina. Compare with retrograde labeling of RGC by putting FG at stump of transected optic nerve for 2 days, the interference of RGC survival is minimized. Compare with cresyl violet staining that stains RGCs, amacrine cells and endothelium of the blood vessel in the retinal ganglion cell layer, this labeling method is more specific to the RGC. This video describes the method of retrograde labeling of RGC by applying FG on the surface of SC. The surgical procedures include drilling the skull; aspirating the cortex to expose the SC and applying gelatin sponge over entire dorsal surface of SC are shown. Useful tips for avoiding massive intracranial bleeding and aspiration of the SC have been given.