Dataset Information

Feasibility Analysis of Oxygen-Glucose Deprivation-Nutrition Resumption on H9c2 Cells In vitro Models of Myocardial Ischemia-Reperfusion Injury.

ABSTRACT:

Background

Oxygen-glucose deprivation-nutrition resumption (OGD-NR) models on H9c2 cells are commonly used in vitro models of simulated myocardial ischemia-reperfusion injury (MIRI), but no study has assessed whether these methods for establishing in vitro models can effectively imitate the characteristics of MIRI in vivo. This experiment was designed to analyze the feasibility of six OGD-NR models of MIRI.

Methods

By searching the PubMed database using the keywords "myocardial reperfusion injury H9c2 cells," we obtained six commonly used OGD-NR in vitro models of MIRI performed on H9c2 cells from more than 400 published papers before January 30, 2017. For each model, control (C), simulated ischemia (SI), and simulated ischemia-reperfusion (SIR) groups were assigned, and cell morphology, lactate dehydrogenase (LDH) release, adenosine triphosphate (ATP) levels, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and inflammatory cytokines were examined to evaluate the characteristics of cell injury. Subsequently, a coculture system of cardiomyocyte-endothelial-macrophage was constructed. The coculture system was dealt with SI and SIR treatments to test the effect on cardiomyocytes survival.

Results

For models 1, 2, 3, 4, 5, and 6, SI treatment caused morphological damage to cells, and subsequent SIR treatment did not cause further morphological damage. In the models 1, 2, 3, 4, 5 and 6, LDH release was significantly higher in the SI groups than that in the C group (P < 0.05), and was significantly lower in the SIR groups than that in the SI groups (P < 0.05), except for no significant differences in the LDH release between C, SI and SIR groups in model 6 receiving a 3-h SI treatment. In models 1, 2, 3, 4, 5, and 6, compared with the C group, ATP levels of the SI groups significantly decreased (P < 0.05), ROS levels increased (P < 0.05), and MMP levels decreased (P < 0.05). Compared with the SI group, ATP level of the SIR groups was significantly increased (P < 0.05), and there was no significant ROS production, MMP collapse, and over inflammatory response in the SIR groups. In a coculture system of H9c2 cells-endothelial cells-macrophages, the proportion of viable H9c2 cells in the SIR groups was not reduced compared with the SI groups.

Conclusion

All the six OGD-NR models on H9c2 cells in this experiment can not imitate the characteristics of MIRI in vivo and are not suitable for MIRI-related study.

SUBMITTER: Yang GZ

PROVIDER: S-EPMC6166467 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Feasibility Analysis of Oxygen-Glucose Deprivation-Nutrition Resumption on H9c2 Cells <i>In vitro</i> Models of Myocardial Ischemia-Reperfusion Injury.

Yang Gui-Zhen GZ Xue Fu-Shan FS Liu Ya-Yang YY Li Hui-Xian HX Liu Qing Q Liao Xu X

Chinese medical journal 20181001 19

<h4>Background</h4>Oxygen-glucose deprivation-nutrition resumption (OGD-NR) models on H9c2 cells are commonly used in vitro models of simulated myocardial ischemia-reperfusion injury (MIRI), but no study has assessed whether these methods for establishing in vitro models can effectively imitate the characteristics of MIRI in vivo. This experiment was designed to analyze the feasibility of six OGD-NR models of MIRI.<h4>Methods</h4>By searching the PubMed database using the keywords "myocardial re ...[more]

PMID: 30246713

Similar Datasets

Project description:Shuxuetong injection composed of leech (Hirudo nipponica Whitman) and earthworm (Pheretima aspergillum) has been used for the clinical treatment of acute stroke for many years in China. However, the precise neuroprotective mechanism of Shuxuetong injection remains poorly understood. Here, cerebral microvascular endothelial cells (bEnd.3) were incubated in glucose-free Dulbecco's modified Eagle's medium containing 95% N2/5% CO2 for 6 hours, followed by high-glucose medium containing 95% O2 and 5% CO2 for 18 hours to establish an oxygen-glucose deprivation/reperfusion model. This in vitro cell model was administered Shuxuetong injection at 1/32, 1/64, and 1/128 concentrations (diluted 32-, 64-, and 128-times). Cell Counting Kit-8 assay was used to evaluate cell viability. A fluorescence method was used to measure lactate dehydrogenase, and a fluorescence microplate reader used to detect intracellular reactive oxygen species. A fluorescent probe was also used to measure mitochondrial superoxide production. A cell resistance meter was used to measure transepithelial resistance and examine integrity of monolayer cells. The fluorescein isothiocyanate-dextran test was performed to examine blood-brain barrier permeability. Real-time reverse transcription polymerase chain reaction was performed to analyze mRNA expression levels of tumor necrosis factor alpha, interleukin-1β, interleukin-6, and inducible nitric oxide synthase. Western blot assay was performed to analyze expression of caspase-3, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, occludin, vascular endothelial growth factor, cleaved caspase-3, B-cell lymphoma 2, phosphorylated extracellular signal-regulated protein kinase, extracellular signal-regulated protein kinase, nuclear factor-κB p65, I kappa B alpha, phosphorylated I kappa B alpha, I kappa B kinase, phosphorylated I kappa B kinase, claudin-5, and zonula occludens-1. Our results show that Shuxuetong injection increases bEnd.3 cell viability and B-cell lymphoma 2 expression, reduces cleaved caspase-3 expression, inhibits production of reactive oxygen species and mitochondrial superoxide, suppresses expression of tumor necrosis factor alpha, interleukin-1β, interleukin-6, inducible nitric oxide synthase mRNA, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, markedly increases transepithelial resistance, decreases blood-brain barrier permeability, upregulates claudin-5, occludin, and zonula occludens-1 expression, reduces nuclear factor-κB p65 and vascular endothelial growth factor expression, and reduces I kappa B alpha, extracellular signal-regulated protein kinase 1/2, and I kappa B kinase phosphorylation levels. Overall, these findings suggest that Shuxuetong injection has protective effects on brain microvascular endothelial cells after oxygen-glucose deprivation/reperfusion. Moreover, its protective effect is associated with reduction of mitochondrial superoxide production, inhibition of the inflammatory response, and inhibition of vascular endothelial growth factor, extracellular signal-regulated protein kinase 1/2, and the nuclear factor-κB p65 signaling pathway.

Project description:HECT, UBA and WWE domain-containing 1 (Huwe1), an E3 ubiquitin ligase involved in the ubiquitin-proteasome system, is widely expressed in brain tissue. Huwe1 is involved in the turnover of numerous substrates, including p53, Mcl-1, Cdc6 and N-myc, thereby playing a critical role in apoptosis and neurogenesis. However, the role of Huwe1 in brain ischemia and reperfusion injury remains unclear. Therefore, in this study, we investigated the role of Huwe1 in an in vitro model of ischemia and reperfusion injury. At 3 days in vitro, primary cortical neurons were transduced with a control or shRNA-Huwe1 lentiviral vector to silence expression of Huwe1. At 7 days in vitro, the cells were exposed to oxygen-glucose deprivation for 3 hours and reperfusion for 24 hours. To examine the role of the c-Jun N-terminal kinase (JNK)/p38 pathway, cortical neurons were pretreated with a JNK inhibitor (SP600125) or a p38MAPK inhibitor (SB203508) for 30 minutes at 7 days in vitro, followed by ischemia and reperfusion. Neuronal apoptosis was assessed by TUNEL assay. Protein expression levels of JNK and p38MAPK and of apoptosis-related proteins (p53, Gadd45a, cleaved caspase-3, Bax and Bcl-2) were measured by western blot assay. Immunofluorescence labeling for cleaved caspase-3 was performed. We observed a significant increase in neuronal apoptosis and Huwe1 expression after ischemia and reperfusion. Treatment with the shRNA-Huwe1 lentiviral vector markedly decreased Huwe1 levels, and significantly decreased the number of TUNEL-positive cells after ischemia and reperfusion. The silencing vector also downregulated the pro-apoptotic proteins Bax and cleaved caspase-3, and upregulated the anti-apoptotic proteins Gadd45a and Bcl-2. Silencing Huwe1 also significantly reduced p-JNK levels and increased p-p38 levels. Our findings show that downregulating Huwe1 affects the JNK and p38MAPK signaling pathways as well as the expression of apoptosis-related genes to provide neuroprotection during ischemia and reperfusion. All animal experiments and procedures were approved by the Animal Ethics Committee of Sichuan University, China in January 2018 (approval No. 2018013).

Project description:Autophagy and the ubiquitin proteasome system (UPS) are two major protein degradation pathways involved in brain ischemia. Autophagy can compensate for UPS impairment‑induced cellular dysfunction. HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1 (Huwe1), an E3 ubiquitin ligase, serves critical roles in nervous system plasticity, regeneration and disease. However, the role of Huwe1 in autophagy in brain ischemia/reperfusion (I/R) injury remains unknown. The aim of the present study was to investigate the crosstalk between autophagy and the UPS in brain ischemia. The present study established an oxygen‑glucose deprivation and reperfusion (OGD/R) model in rat primary cortex neurons in vitro. Lentiviral interference was used to silence the expression of Huwe1. An autophagy promoter (rapamycin), an autophagy inhibitor (wortmannin) and a JNK pathway inhibitor (SP600125) were also used in the current study. Cellular autophagy‑related proteins, including Beclin‑1, autophagy related (ATG) 7, ATG5, ATG3 and microtubule associated protein 1 light chain 3 α, and apoptosis‑related proteins, such as P53, cleaved caspase 3, Bax and Bcl2, were detected via western blotting and immunocytochemistry. Neuronal apoptosis was evaluated using a TUNEL assay. The results demonstrated that silencing Huwe1 increased the expression levels of autophagy‑related proteins at 24 h after OGD/R. Treatment with a JNK inhibitor or cotreatment with Huwe1 shRNA significantly increased autophagy. Rapamycin increased apoptosis under OGD/R conditions. However, treatment with Huwe1 shRNA decreased the number of TUNEL‑positive cells at 24 h after OGD/R. Cotreatment with Huwe1 shRNA and wortmannin alleviated neuronal apoptosis under OGD/R conditions compared with cotreatment with DMSO. Collectively, the present results suggested that silencing Huwe1 was accompanied by a compensatory induction of autophagy under OGD/R conditions. Furthermore, the JNK pathway may be a key mediator of the interaction between Huwe1 and autophagy in response to UPS impairment.

Project description:Ischemia-reperfusion (I/R) could cause heart irreversible damage, which is tightly combined with glucose metabolism disorder. It is demonstrated that GLUT4 (glucose transporter 4) translocation is critical for glucose metabolism in the cardiomyocytes under I/R injury. Moreover, DRD4 (dopamine receptor D4) modulate glucose metabolism, and protect neurocytes from anoxia/reoxygenation (A/R) injury. Thus, DRD4 might regulate myocardial I/R injury in association with GLUT4-mediated glucose metabolism. However, the effects and mechanisms are largely unknown. In the present study, the effect of DRD4 in heart I/R injury were studied ex vivo and in vitro. For I/R injury ex vivo, DRD4 agonist (PD168077) was perfused by Langendorff system in the isolated rat heart. DRD4 activated by PD168077 improved cardiac function in the I/R-injured heart as determined by the left ventricular developed pressure (LVDP), +dp/dt, and left ventricular end diastolic pressure (LVEDP), and reduced heart damage evidenced by infarct size, the release of troponin T (TNT) and lactate dehydrogenase (LDH). DRD4 activation diminished I/R injury induced apoptosis and enhanced cell viability impaired by I/R injury in cardiomyocyte, showed by TUNEL staining, flow cytometer and CCK8 assay. Furthermore, DRD4 activation did not change total GULT4 protein expression level but increased the membrane GULT4 localization determined by western blot. In terms of mechanism, DRD4 activation increased pPI3K/p-AKT but not the total PI3K/AKT during anoxia/reoxygenation (A/R) injury in vitro. Interestingly, PI3K inhibitor, Wortmannin, blocked PI3K/AKT pathway and depleted the membrane GULT4, and further promoted apoptosis showed by TUNEL staining, flow cytometer, western blot of cleaved caspase 3, BAX and BCL2 expression. Thus, DRD4 activation exerted a protective effect against I/R injury by promoting GLUT4 translocation depended on PI3K/AKT pathway, which enhanced the ability of glucose uptake, and ultimately reduced the apoptosis in cardiomyocytes.