Cleavage of cyclic AMP-responsive element-binding protein H aggravates myocardial hypoxia reperfusion injury in a hepatocyte-myocardial cell co-culture system.
This study aims to determine whether the proinflammatory cytokines have effects on myocardial cells (MC) and hepatocytes during myocardial ischemia to induce cyclic element-binding protein H (CREBH) cleavage AMP-responsive, enabling the acute phase response in the liver, and cause injury MCs.In superimposed on this research, transwell co-culture system hepatocyte-MC is used to investigate the relationship between the injury of hypoxia / reperfusion myocardial and CREBH cleavage.
MC and neonatal rat hepatocytes derived from the ventricles and heart Sprague Dawley rats each. MC inoculated into the lower chamber of the transwell chamber for 12 hours under hypoxia. The level of endoplasmic reticulum stress protein glucose-regulated protein 78 in MC, CREBH in hepatocytes, inflammatory factors (tumor necrosis factor-α and interleukin-6) levels, and cell viability is evaluated.
CREBH influence knockdown was also studied using short hairpin RNA specifically CREBH (Ad-CREBHi) .We found that proinflammatory cytokines affect MC and hepatocytes during myocardial ischemia to induce cleavage CREBH, activate the acute phase response in the liver, and cause injury superimposed on MCs. Expression of CREBH exacerbate the myocardial injury during myocardial ischemia.
Development co-culture model of mouse primary hepatocytes and splenocytes to evaluate the genotoxicity of xenobiotics using medium-throughput Comet assay.
To date, only a limited number of toxicology studies have focused on the establishment and validation of in vitro genotoxicity screening system using primary hepatocytes, and the results of these studies have been inconsistent. Therefore, the aim of this study was to develop a model of effective co-culture of rat primary hepatocytes and splenocytes derived for screening chemicals for medium-throughput genotoxicity using the Comet assay.
Cocultured model is built and verified using known genotoxic and non-genotoxic compounds as positive and negative controls, respectively. Cytotoxicity was measured using Cell Counting Kit-8 and lactate dehydrogenase method. DNA damage was detected using both alkaline and formamidopyrimidine glikosilase DNA test (FPG) Comet. Compared with controls, DNA strand breaks and FPG-sensitive sites showed a significant concentration-dependent increase in group-treated genotoxic agents.
In contrast, DNA damage remains unchanged in groups of non-genotoxic-agent-treated. In addition, various types of DNA lesions lead to genotoxic agents depending on different times.Connection error.
Our results show that the tail DNA% showed both strands of DNA break and FPG-sensitive sites may be an effective marker for predicting chemical-induced DNA damage and oxidative DNA damage using a model cocultured hepatocytes and splenocytes. Collectively, these findings provide reliable experimental data for the establishment of in vitro genotoxicity screening methods.