Dong-Jie Li

Metformin promotes irisin release from murine skeletal muscle independently of AMP‐activated protein kinase activation

Irisin, a novel myocyte‐secreted hormone mediating beneficial effects of exercise on metabolism, is supposed to be an ideal therapeutic target for metabolic disorders such as obesity and diabetes. Here, we investigated the potential effects of metformin and glibenclamide, two antidiabetic medicines, on irisin release in mouse.

The novel exercise-induced hormone irisin protects against neuronal injury via activation of the Akt and ERK1/2 signaling pathways and contributes to the neuroprotection of physical exercise in cerebral ischemia

BACKGROUND Irisin is a novel exercise-induced myokine involved in the regulation of adipose browning and thermogenesis. In this study, we investigated the potential role of irisin in cerebral ischemia and determined whether irisin is involved in the neuroprotective effect of physical exercise in mice. MATERIALS AND METHODS The middle cerebral artery occlusion (MCAO) model was used to produce cerebral ischemia in mice. First, the plasma irisin levels and changes in expression of the irisin precursor protein FNDC5 in skeletal muscle were determined post ischemic stroke. Second, the association between plasma irisin levels and the neurological deficit score, brain infarct volume, or plasma concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β in mice with MCAO were evaluated. Third, the therapeutic effect of irisin on ischemic brain injury was evaluated in vivo and in vitro. Recombinant irisin was injected directly into the tail vein 30min after the MCAO operation, and then the effects of irisin treatment on brain infarct volume, neurological deficit, neuroinflammation, microglia activation, monocyte infiltration, oxidative stress and intracellular signaling pathway activation (Akt and ERK1/2) were measured. Irisin was also administered in cultured PC12 neuronal cells with oxygen and glucose deprivation (OGD). Finally, to assess the potential involvement of irisin in the neuroprotection of physical exercise, mice were exercised for 2weeks and an irisin neutralizing antibody was injected into these mice to block irisin 1h before the MCAO operation. RESULTS The plasma irisin concentration and intramuscular FNDC5 protein expression decreased after ischemic stroke. Plasma irisin levels were negatively associated with brain infarct volume, the neurological deficit score, plasma TNF-α and plasma IL-6 concentrations. In OGD neuronal cells, irisin protected against cell injury. In mice with MCAO, irisin treatment reduced the brain infarct volume, neurological deficits, brain edema and the decline in body weight. Irisin treatment inhibited activation of Iba-1+ microglia, infiltration of MPO-1+ monocytes and expression of both TNF-α and IL-6 mRNA. Irisin significantly suppressed the levels of nitrotyrosine, superoxide anion and 4-hydroxynonenal (4-HNE) in peri-infarct brain tissues. Irisin treatment increased Akt and ERK1/2 phosphorylation, while blockade of Akt and ERK1/2 by specific inhibitors reduced the neuroprotective effects of irisin. Finally, the exercised mice injected with irisin neutralizing antibody displayed more severe neuronal injury than the exercised mice injected with control IgG. CONCLUSION Irisin reduces ischemia-induced neuronal injury via activation of the Akt and ERK1/2 signaling pathways and contributes to the neuroprotective effect of physical exercise against cerebral ischemia, suggesting that irisin may be a factor linking metabolism and cardio-cerebrovascular diseases.

Exercise-stimulated FGF23 promotes exercise performance via controlling the excess reactive oxygen species production and enhancing mitochondrial function in skeletal muscle

OBJECTIVE Physical exercise induces many adaptive changes in skeletal muscle and the whole body and improves metabolic characteristics. Fibroblast growth-factor 23 (FGF23) is a unique member of the FGF family that acts as a hormone regulating phosphate metabolism, calcitriol concentration, and kidney functions. The role of FGF23 in exercise and skeletal muscle is largely unknown yet. MATERIALS AND METHODS C57BL/6J mice were exercised on a motor treadmill. Mice serum FGF23 levels; FGF23 mRNA expression in various organs including the liver, heart, skeletal muscle tissue, and thyroid; and FGF23 receptor Klotho mRNA expression were examined using enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and immunoblotting, respectively, after a single bout of acute exercise (60min), exhaustive exercise, and chronic prolonged exercise (60min every day for one week). C57BL/6J mice were injected with recombinant FGF23 (100mg/kg, twice per day, i.p.) or vehicle control (saline) for 3days, and then the exercise performance, reactive oxygen species (ROS), H2O2 production, and mitochondrial functional biomarkers in muscle (gene expression of sirtuin 1, PPAR-δ, PGC-1α and mitochondrial transcription factor A [TFAM], and citrate synthase activity) were assayed. RESULTS Three forms of exercise, acute exercise, exhaustive exercise, and chronic exercise, increased serum FGF23 levels. However, only chronic exercise upregulated FGF23 mRNA and protein expression in skeletal muscle. FGF23 mRNA expression in the heart, liver, and thyroid was not affected. FGF23 protein was mainly located in the cytoplasm in skeletal muscle tissue and the localization of FGF23 was not altered by exercise. Exogenous FGF23 treatment significantly extended the time to exhaustion and reduced the exercise-induced ROS and H2O2 production. FGF23 treatment increased the mRNA level of PPAR-δ and citrate synthase activity, but did not influence the mRNA expression of sirtuin 1, PGC-1α, and TFAM in skeletal muscle. CONCLUSION These results demonstrate that exercise-stimulated FGF23 promotes exercise performance via controlling the excess ROS production and enhancing mitochondrial function in skeletal muscle, which reveals an entirely novel role of FGF23 in skeletal muscle.

High-salt diet enhances hippocampal oxidative stress and cognitive impairment in mice

Previous evidence suggests that a high-salt (HS) diet may increase oxidative stress and contribute to the development of hypertension that is already present. Oxidative stress is thought to play a critical role in the development of neurodegenerative diseases. Lower dietary sodium intake putatively contributes to a lower rate of cognitive impairment; however, the specific effects of HS diet on cognitive function remain poorly understood. In this work, C57BL/6J mice were administered a normal-salt (NS) diet (0.4% NaCl) or a HS diet (7.0% NaCl) for 12 weeks, and cognitive ability and oxidative stress in the brain were measured. It was found that the HS diet significantly impaired retention of spatial memory. Additionally, superoxide anion production in the hippocampus was significantly increased in the HS diet mice compared with that in the NS mice. Interestingly, the antioxidant defense capacities for HS diet mice were markedly reduced in the hippocampus, but not in the cerebral cortex, compared with the NS mice. Taken together, these data demonstrate that HS diet directly impairs retention of spatial memory, which may be related to the increased oxidative stress observed in the hippocampus.

α7 Nicotinic Acetylcholine Receptor Relieves Angiotensin II–Induced Senescence in Vascular Smooth Muscle Cells by Raising Nicotinamide Adenine Dinucleotide–Dependent SIRT1 Activity

Objective—&agr;7 nicotinic acetylcholine receptor (&agr;7nAChR) is a subtype of nAChR and has been reported to be involved in hypertension end-organ damage. In this study, we tested the role of &agr;7nAChR in angiotensin II (Ang II)–induced senescence of vascular smooth muscle cells (VSMCs). Approach and Results—Expression of &agr;7nAChR was not influenced by Ang II. Ang II induced remarkable senescent phenotypes in rodent and human VSMCs, including increased senescence-associated &bgr;-galactosidase activity, phosphorylation of H2A.XSer139, phosphorylation of Chk1Ser317, reduced replication, and downregulation of proliferating cell nuclear antigen. Activation of &agr;7nAChR with a selective agonist PNU-282987 blocked Ang II–induced senescence in cultured VSMCs. Moreover, PNU-282987 treatment attenuated the Ang II infusion–induced VSMC senescence in wild-type but not in &agr;7nAChR−/− mice. PNU-282987 reduced the Ang II–enhanced reactive oxygen species, lipid peroxidation, and the expression of NADPH oxidase 1, NADPH oxidase 4, and p22phox in cultured VSMCs isolated from wild-type but not in &agr;7nAChR−/− mice. Furthermore, PNU-282987 diminished Ang II–induced prosenescence signaling pathways, including p53, acetyl-p53, p21, and p16INK4a. Finally, although &agr;7nAChR activation by PNU-282987 did not affect the Ang II–induced downregulation of sirtuin 1 (SIRT1), it significantly increased intracellular NAD+ levels, and thereby enhanced SIRT1 activity in an AMP-dependent protein kinase–independent manner. Depletion of SIRT1 by knockdown or SIRT1 inhibitor EX527 abrogated the antisenescence effect of &agr;7nAChR against Ang II. Conclusions—Our results demonstrate that activation of &agr;7nAChR alleviates Ang II–induced VSMC senescence through promoting NAD+–SIRT1 pathway, suggesting that &agr;7nAChR may be a potential therapeutic target for the treatment of Ang II–associated vascular aging disorders.

Vagus Nerve Attenuates Hepatocyte Apoptosis upon Ischemia–reperfusion via α7 Nicotinic Acetylcholine Receptor on Kupffer Cells in Mice

Background:Hepatic ischemia–reperfusion (HIR) injury is a complication of liver surgery. As much as 50% of hepatocytes undergo apoptosis within the first 24 h of reperfusion. The neurotransmitters of the vagus nerve can activate &agr;7 nicotinic acetylcholine receptor (&agr;7nAChR) on macrophages. The function of Kupffer cells (KCs) determines HIR injury. We hypothesize that the vagus nerve could attenuate HIR-induced hepatocyte apoptosis by activating &agr;7nAChR on KCs. Methods:Hepatic vagotomized C57BL/6J mice, KC-eliminated C57BL/6J mice, and &agr;7nAChR−/− mice were used for HIR. Primary KCs and hepatocytes were subjected to hypoxia/reoxygenation (HR). Liver injury, hepatocyte apoptosis, reactive oxygen species (ROS) production, and soluble CD163 were measured. Results:Hepatic vagotomy and &agr;7nAChR−/− caused higher levels of alanine transaminase and liver caspase-3 and -8 activity by HIR. Activating &agr;7nAChR attenuated these changes in wild-type but not in the &agr;7nAChR−/− mice. Furthermore, activating &agr;7nAChR diminished hepatic injury and reduced liver apoptosis by HIR in vagotomized mice. In vitro, activating &agr;7nAChR reduced apoptosis of hepatocytes cocultured with KCs that suffered HR. Similar to the effects by catalase, activating &agr;7nAChR on KCs reduced ROS and H2O2 by HR. The supernatant from KCs, with &agr;7nAChR activated or catalase treated, prevented hepatocyte apoptosis by HR. Finally, KC elimination reduced HIR-induced H2O2 production in mice. Activating &agr;7nAChR significantly attenuated soluble CD163 both in mice by HIR (serum: 240 ± 34 vs. 446 ± 72; mean ± SD; n = 8; P < 0.01) and in KCs by HR (supernatant: 4.23 ± 0.06 vs. 5.60 ± 0.18; n = 3; P < 0.01). Conclusions:The vagus nerve could minimize HIR-induced liver apoptosis through activating &agr;7nAChR on KCs possibly by preventing their excessive ROS production.

Retraction. Fenofibrate improves the impaired endothelial progenitor cell function through inhibiting eNOS uncoupling in diabetic mice.

The above article, published online on 24th December 2014 as an Accepted Article in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1111/bph.13054/abstract), has been retracted by agreement between the authors, the journal Editor in Chief, Professor J. McGrath and John Wiley & Sons Limited. The retraction has been agreed owing to duplication of some images. In this paper, there is a figure duplication in Figure 8A (‘Control’ is from an identical tissue slice as ‘STZ+FF’). There are also partially overlapping figures in supplemental figures S3 and S5.

Cholinergic anti-inflammatory pathway inhibits neointimal hyperplasia by suppressing inflammation and oxidative stress

Neointimal hyperplasia as a consequence of vascular injury is aggravated by inflammatory reaction and oxidative stress. The α7 nicotinic acetylcholine receptor (α7nAChR) is a orchestrator of cholinergic anti-inflammatory pathway (CAP), which refers to a physiological neuro-immune mechanism that restricts inflammation. Here, we investigated the potential role of CAP in neointimal hyperplasia using α7nAChR knockout (KO) mice. Male α7nAChR-KO mice and their wild-type control mice (WT) were subjected to wire injury in left common carotid artery. At 4 weeks post injury, the injured aortae were isolated for examination. The neointimal hyperplasia after wire injury was significantly aggravated in α7nAChR-KO mice compared with WT mice. The α7nAChR-KO mice had increased collagen contents and vascular smooth muscle cells (VSMCs) amount. Moreover, the inflammation was significantly enhanced in the neointima of α7nAChR-KO mice relative to WT mice, evidenced by the increased expression of tumor necrosis factor-α/interleukin-1β, and macrophage infiltration. Meanwhile, the chemokines chemokine (C-C motif) ligand 2 and chemokine (CXC motif) ligand 2 expression was also augmented in the neointima of α7nAChR-KO mice compared with WT mice. Additionally, the depletion of superoxide dismutase (SOD) and reduced glutathione (GSH), and the upregulation of 3-nitrotyrosine, malondialdehyde and myeloperoxidase were more pronounced in neointima of α7nAChR-KO mice compared with WT mice. Accordingly, the protein expression of NADPH oxidase 1 (Nox1), Nox2 and Nox4, was also higher in neointima of α7nAChR-KO mice compared with WT mice. Finally, pharmacologically activation of CAP with a selective α7nAChR agonist PNU-282987, significantly reduced neointima formation, arterial inflammation and oxidative stress after vascular injury in C57BL/6 mice. In conclusion, our results demonstrate that α7nAChR-mediated CAP is a neuro-physiological mechanism that inhibits neointima formation after vascular injury via suppressing arterial inflammation and oxidative stress. Further, these results imply that targeting α7nAChR may be a promising interventional strategy for in-stent stenosis.

MiR-135a Protects Vascular Endothelial Cells Against Ventilator-Induced Lung Injury by Inhibiting PHLPP2 to Activate PI3K/Akt Pathway

Background/Aims: Loss of endothelial barrier function plays an important role in the development of ventilator-induced lung injury (VILI). This study aimed to investigate the effects of miR135a on VILI in a model of mechanical stretch (MS)-induced human umbilical vein endothelial cell (HUVEC) injury. Methods: HUVECs were randomly assigned to 7 groups: blank, negative control (NC), NC+MS, miR135a over-expression (mi-miR135a), mi-miR135a + MS, miR135a silencing (si-miR135a) and si-miR135a + MS groups. MS was induced by subjecting cells to cyclic stretch at 20% stretch for 4 h. After 24 h, levels of reactive oxygen species (ROS) were measured by DCFH-DA fluorescence intensity. Apoptosis was measured using annexin V-FITC/propidium iodide assay with flow cytometry. Inflammatory cytokine levels were determined by ELISA. Barrier integrity was determined using FITC-conjugated dextran assay. Expression levels of PI3K, p-PI3K, Akt, p-Akt, Bcl-2 and Bax were examined using western blotting. The interaction between miR135a and PHLPP2 was evaluated by dual-luciferase reporter assay. Results: Our results showed that MS reduced cell numbers, increased the number of apoptotic cells, increased ROS, barrier dysfunction and inflammatory cytokines in HUVECs, and reduced p-PI3K and p-Akt expression; silencing of miR135a worsened MS-induced HUVEC injury. However, miR135a over-expression protected HUVECs against MS-induced increases in apoptotic cells, ROS, barrier dysfunction and inflammatory cytokines, which were accompanied by activation of the PI3K/Akt signaling pathway. Simultaneous silencing of miR135a and PHLPP2 partially salvaged the effects of miR135a silencing, and miR135a was found to interact with PHLPP2. Conclusion: miR135a may protect HUVECs from MS-induced injury by inhibiting PHLPP2 to activate PI3k/Akt signaling pathway.

Nicotinic acetylcholine receptor α7 inhibits platelet-derived growth factor-induced migration of vascular smooth muscle cells by activating mitochondrial deacetylase SIRT3: α7nAChR inhibits PDGF-BB-induced VSMCS migration

PDGF‐BB is an angiogenic factor involved in cardiovascular diseases. Here, we investigated the possible effects of activation of the nicotinic ACh receptor α7 subtype (α7nAChR) on PDGF‐BB‐induced proliferation and migration in vascular smooth muscle cells (VSMCs).