Dong-ng Li

Differentiating characterization of human umbilical cord blood‐derived mesenchymal stem cells in vitro

It has been demonstrated that the number and differentiating potential of bone marrow mesenchymal stem cells (MSCs) decrease with age. Therefore, the search for alternative sources of MSCs is of significant value. In the present study, MSCs were isolated from umbilical cord blood (UCB) by combining gradient density centrifugation with plastic adherence. Cultured cells were treated with ascorbate acid‐2‐phosphate, dexamethasone, β‐glycerophosphate dexamethasone, insulin, 1‐methyl‐3‐isobutylxamthine, indomethacin, β‐mercaptoethanol, butylated hydroxyanisole, FGF‐4 and HGF. Differentiating characterization of UCB‐derived MSCs were detected by cytochemistry, immunocytochemistry, radioimmunoassay, RT‐PCR and urea assay. The results showed UCB‐derived MSCs could differentiate into osteoblasts, adipocytes and neuron‐like cells. When MSCs were cultured with FGF‐4 and HGF, approximately 63.6% of cells became small, round and epithelioid on day 28 by morphology. Compared with the control, levels of AFP in the supernatant liquid increased significantly from day 12 and were higher on day 28 (P < 0.01). Albumin increased significantly from day 16 (P < 0.01). Urea was first detected on day 20 (P < 0.01), and continued to increase on day 28 (P < 0.01). Cells first expressed CK‐18 on day 16 through immunocytochemistry analysis. RT‐PCR analysis showed that differentiated cells could express a number of hepatocyte‐specific genes in a time‐dependent manner. Glycogen storage was first seen on day 24. Our results suggest that UCB‐derived MSCs can differentiate not only into osteoblasts, adipocytes and neuron‐like cells, but also into hepatocytes. Human UCB‐derived MSCs are a new source of cell types for cell transplantation and therapy.

Acetylcholine inhibits hypoxia‐induced tumor necrosis factor‐α production via regulation of MAPKs phosphorylation in cardiomyocytes

Recent findings have reported that up‐regulation of tumor necrosis factor‐alpha (TNF‐α) induced by myocardial hypoxia aggravates cardiomyocyte injury. Acetylcholine (ACh), the principle vagal neurotransmitter, protects cardiomyocytes against hypoxia by inhibiting apoptosis. However, it is still unclear whether ACh regulates TNF‐α production in cardiomyocytes after hypoxia. The concentration of extracellular TNF‐α was increased in a time‐dependent manner during hypoxia. Furthermore, ACh treatment also inhibited hypoxia‐induced TNF‐α mRNA and protein expression, caspase‐3 activation, cell death and the production of reactive oxygen species (ROS) in cardiomyocytes. ACh treatment prevented the hypoxia‐induced increase in p38 mitogen‐activated protein kinase (MAPK) and c‐Jun N‐terminal kinase (JNK) phosphorylation, and increased extracellular signal‐regulated kinase (ERK) phosphorylation. Co‐treatment with atropine, a non‐selective muscarinic acetylcholine receptor antagonist, or methoctramine, a selective type‐2 muscarinic acetylcholine (M2) receptor antagonist, abrogated the effects of ACh treatment in hypoxic cardiomyocytes. Co‐treatment with hexamethonium, a non‐selective nicotinic receptor antagonist, and methyllycaconitine, a selective alpha7‐nicotinic acetylcholine receptor antagonist, had no effect on ACh‐treated hypoxic cardiomyocytes. In conclusion, these results demonstrate that ACh activates the M2 receptor, leading to regulation of MAPKs phosphorylation and, subsequently, down‐regulation of TNF‐α production. We have identified a novel pathway by which ACh mediates cardioprotection against hypoxic injury in cardiomyocytes. J. Cell. Physiol. 226: 1052–1059, 2011.

Investigation of a comprehensive identification method used in acoustic detection system for GIS

Nowadays, the acoustic detection is widely used for defect diagnosis of gas insulated substations (GIS) in normal operation and factory tests. In this paper in order to develop a data analyzer for acoustic detection system to make an assistant diagnosis, the characteristic of acoustic signals generated by different artificial defects such as protrusions, floating shield, void in spacer and bouncing particles are investigated. Some meaningful parameters behind the detected acoustic signals are extracted and discussed, which are used to distinguish background noise, partial discharge (PD) phenomena or bouncing particles. Based on those works, a comprehensive identification method realized by processing the acoustic pulse sequences qi, ( Δ ti, qi) and (ti, qi) is introduced, which gives a recognition result with noise, PD type or bouncing particles. For the sequence (ti, qi), the backpropagation artificial neural network optimized by genetic algorithm (GA-BPANN) is used as a classifier based on the fingerprint consisting of 24 operators, which are derivate from typical 2D histograms of phase-resolved partial discharge (PRPD). And with considering the trigger source may having a phase difference from the working voltage, identification with phase compensation (IPC) is used as a try to deal with the challenge. Experimental results show that the comprehensive identification method is practical and effective.

Acetylcholine Inhibits Tumor Necrosis Factor α Activated Endoplasmic Reticulum Apoptotic Pathway via EGFR‐PI3K Signaling in Cardiomyocytes

Previous findings have shown that acetylcholine (ACh) decreased hypoxia‐induced tumor necrosis factor alpha (TNF α) production, thus protected against cardiomyocyte injury. However, whether and how ACh affects TNF α‐induced endoplasmic reticulum (ER) stress and cell apoptosis remain poorly defined. This study was aimed at determining the effect of ACh in H9c2 cells after TNF α stimulation. Presence of ER stress was verified using the ER stress protein markers glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP). Cell apoptosis was shown by caspase‐3 activation and terminal deoxynucleotidyl transferase mediated dUTP‐biotin nick end labeling. Exogenously administered ACh significantly decreased these TNF α‐induced changes. Moreover, when the cells were exposed to nonspecific muscarinic receptor (M AChR) inhibitor atropine, methoctramine (M2 AChR inhibitor) or the epidermal growth factor receptor (EGFR) inhibitor AG1478, the cardioprotection elicited by ACh was diminished. Furthermore, the above effects were also blocked by M2 AChR or EGFR siRNA, indicating that EGFR transactivation by M2 AChR may be the major pathway responsible for the benefits of ACh. In addition, LY294002, a phosphatidylinositol‐3‐kinase (PI3K) inhibitor, displayed the similar trends as AG1478, suggesting that PI3K/Akt signaling may be the downstream of EGFR in ACh‐elicited anti‐apoptotic property. Together, these data indicate that EGFR‐PI3K/Akt signaling is involved in M2 AChR‐mediated ER apoptotic pathway suppression and the subsequent survival of H9c2 cardiomyocytes. We have identified a novel pathway underlying the cardioprotection afforded by ACh. J. Cell. Physiol. 230: 767–774, 2015.

Amlodipine ameliorates endothelial dysfunction in mesenteric arteries from spontaneously hypertensive rats.

1. Endothelial dysfunction plays a critical role in the development and progression or pathogenesis of hypertension. Amlodipine, a calcium channel blocker, is an effective antihypertensive agent. We investigated the effects of amlodipine on endothelial dysfunction in mesenteric arteries from spontaneously hypertensive rats (SHR).

Vagal nerve stimulation improves mitochondrial dynamics via an M3 receptor/CaMKKβ/AMPK pathway in isoproterenol-induced myocardial ischaemia.

Mitochondrial dynamics—fission and fusion—are associated with ischaemic heart disease (IHD). This study explored the protective effect of vagal nerve stimulation (VNS) against isoproterenol (ISO)‐induced myocardial ischaemia in a rat model and tested whether VNS plays a role in preventing disorders of mitochondrial dynamics and function. Isoproterenol not only caused cardiac injury but also increased the expression of mitochondrial fission proteins [dynamin‐related peptide1 (Drp1) and mitochondrial fission protein1 (Fis‐1)) and decreased the expression of fusion proteins (optic atrophy‐1 (OPA1) and mitofusins1/2 (Mfn1/2)], thereby disrupting mitochondrial dynamics and leading to increase in mitochondrial fragments. Interestingly, VNS restored mitochondrial dynamics through regulation of Drp1, Fis‐1, OPA1 and Mfn1/2; enhanced ATP content and mitochondrial membrane potential; reduced mitochondrial permeability transition pore (MPTP) opening; and improved mitochondrial ultrastructure and size. Furthermore, VNS reduced the size of the myocardial infarction and ameliorated cardiomyocyte apoptosis and cardiac dysfunction induced by ISO. Moreover, VNS activated AMP‐activated protein kinase (AMPK), which was accompanied by phosphorylation of Ca2+/calmodulin‐dependent protein kinase kinase β (CaMKKβ) during myocardial ischaemia. Treatment with subtype‐3 of muscarinic acetylcholine receptor (M3R) antagonist 4‐diphenylacetoxy‐N‐methylpiperidine methiodide or AMPK inhibitor Compound C abolished the protective effects of VNS on mitochondrial dynamics and function, suggesting that M3R/CaMKKβ/AMPK signalling are involved in mediating beneficial effects of VNS. This study demonstrates that VNS modulates mitochondrial dynamics and improves mitochondrial function, possibly through the M3R/CaMKKβ/AMPK pathway, to attenuate ISO‐induced cardiac damage in rats. Targeting mitochondrial dynamics may provide a novel therapeutic strategy in IHD.

Alterations of muscarinic acetylcholine receptors-2, 4 and α7-nicotinic acetylcholine receptor expression after ischaemia / reperfusion in the rat isolated heart

1. Cardiac acetylcholine receptors are involved in the negative inotropic effect of the vagus and the protection of the stimulated vagal nerve against myocardial ischaemic injury. Acetylcholine receptors consist of five types of muscarinic acetylcholine receptors (M AChR) and several nicotinic acetylcholine receptors (nAChR). Notably, ischaemic heart disease is accompanied by substantial withdrawal of vagal activity. However, it is not entirely clear what the changes of M2,4 AChR and α7‐nAChR expression are after cardiac ischaemia/reperfusion (I/R) injury.

The Role of Muscarinic Receptors in the Beneficial Effects of Adenosine against Myocardial Reperfusion Injury in Rats

Adenosine, a catabolite of ATP, displays a wide variety of effects in the heart including regulation of cardiac response to myocardial ischemia and reperfusion injury. Nonetheless, the precise mechanism of adenosine-induced cardioprotection is still elusive. Isolated Sprague-Dawley rat hearts underwent 30 min global ischemia and 120 min reperfusion using a Langendorff apparatus. Both adenosine and acetylcholine treatment recovered the post-reperfusion cardiac function associated with adenosine and muscarinic receptors activation. Simultaneous administration of adenosine and acetylcholine failed to exert any additive protective effect, suggesting a shared mechanism between the two. Our data further revealed a cross-talk between the adenosine and acetylcholine receptor signaling in reperfused rat hearts. Interestingly, the selective M2 muscarinic acetylcholine receptor antagonist methoctramine significantly attenuated the cardioprotective effect of adenosine. In addition, treatment with adenosine upregulated the expression and the maximal binding capacity of muscarinic acetylcholine receptor, which were inhibited by the selective A1 adenosine receptor antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) and the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME). These data suggested a possible functional coupling between the adenosine and muscarinic receptors behind the observed cardioprotection. Furthermore, nitric oxide was found involved in triggering the response to each of the two receptor agonist. In summary, there may be a cross-talk between the adenosine and muscarinic receptors in ischemic/reperfused myocardium with nitric oxide synthase might serve as the distal converging point. In addition, adenosine contributes to the invigorating effect of adenosine on muscarinic receptor thereby prompting to regulation of cardiac function. These findings argue for a potentially novel mechanism behind the adenosine-mediated cardioprotection.

Low-dose celecoxib improves coronary function after acute myocardial ischaemia in rabbits

The role of celecoxib in cardiovascular events remains contentious. The aim of the present study was to investigate the effects of celecoxib in acute myocardial ischaemia (AMI) in rabbits in comparison with those of another non‐steroidal anti‐inflammatory drug, namely aspirin. Male New Zealand white rabbits were divided into four groups: (i) a sham‐operated group; (ii) an AMI group, in which the left anterior descending coronary arteries were occluded for 60 min; (iii) the celecoxib + AMI group, pretreated with 3 mg/kg celecoxib, twice a day, for 3 days before AMI induction; and (iv) the aspirin + AMI group, pretreated with 12.5 mg/kg aspirin, twice a day, for 3 days before AMI induction. Haemodynamic parameters were monitored using a multichannel physiological recorder. Serum levels of creatine kinase (CK), malondialdehyde (MDA), cyclo‐oxygenase‐2 (COX‐2), tumour necrosis factor (TNF)‐α, total nitrate/nitrite (NOx), nitric oxide synthase (NOS) and myocardial infarct size were determined. Changes in isometric tension of isolated coronary rings were recorded by a myograph system. Compared with the sham group, the AMI group had lower blood pressure, higher left ventricular (LV) end‐diastolic pressure, depressed maximum dP/dt of LV pressure, a larger infarct size and higher CK and MDA levels. Celecoxib, but not aspirin, pretreatment significantly ameliorated these effects of AMI. Celecoxib reversed AMI‐induced increases in COX‐2 levels to a similar extent as aspirin. Pretreatment with celecoxib resulted in a significant reduction in TNF‐α levels and an increase in NOx and NOS levels compared with the AMI group. The dysfunctional vasoconstriction and vasodilation of coronary arteries were ameliorated by celecoxib administration. 4. In conclusion, the experimental evidence suggests that celecoxib exerts its protective effects in a COX‐independent manner.

Adenine Sulfate Improves Cardiac Function and the Cardiac Cholinergic System After Myocardial Infarction in Rats

Recent studies have shown that vagal activation may have an important therapeutic implication for myocardial infarction (MI), but effective strategies remain unexplored. Here, we investigate whether adenine sulfate can preserve cardiac function and the cholinergic system against MI. Rats were treated with adenine sulfate for three weeks after coronary ligation. Cardiac function was assessed by hemodynamics. The muscarinic M2 receptor and cholinesterase-positive nerves were semi-quantified by immunochemical and histochemical staining. The maximal binding capacity (Bmax) of muscarinic receptors, determined by radioligand binding assay, showed that cardiac function was impaired in MI rats. Adenine sulfate reversed MI-induced reduction of mean artery pressure and left ventricular systolic pressure and elevation of left ventricular end-diastolic pressure. Moreover, adenine sulfate also increased nitric oxide (NO) and nitric oxide synthase (NOS) activity. The amelioration was accompanied by a reversal of the infarction-induced reduction of cholinesterase-positive nerves and M2-receptor expression and Bmax in the adenine sulfate high dose group. Meanwhile, adenine sulfate treatment corrected the disorder of cardiac redox state by reduction in maleic dialdehyde and increase in superoxide dismutase. In conclusion, adenine sulfate exerts cardioprotection against MI and ameliorates NO production. Changes in cardiac vagal distribution density and M2-receptor expression raise the possibility that improvement of the cardiac cholinergic system is involved in adenine sulfate-induced cardioprotective effects.