N-acetylcysteine blocked hypoxia-reoxygenation induced apoptosis through ROS-p38 MAPK signaling pathway in neonatal rat cardiomyocytes

Objective Previous investigations have shown that N-acetylcysteine （NAC） could regulate diverse cell type＇s apoptosis. The purpose of this study was to evaluate the mechanism of NAC reversed apoptosis of cardiomyocytes induced by hypoxia-reoxygenation （H/R）. Methods Cardiomyocytes were treated with hypoxia 6 h and reoxygenation 72 h in the absence and presence of NAC （100/2mol/ L）. The ROS was assayed by using Image-iTTM LIVE green reactive oxygen species detection kit. The viability of cell was assayed with trypan blue. Early stages ofapoptosis were assessed by flow cytometry using Annexin V, and late stages of apoptosis were assessed using TUNEL system. Bcl2 and bax mRNA levels were determined by real-time quantitative PCR. Bcl2, bax, p38 and pp38 protein levels were determined by western blot. Results We found that H/R could markedly increase ROS generation and induce the apoptosis of cardiomyocytes （P〈0.01）. NAC （10012 mol/L） significantly reduced the generation of ROS and apoptosis （P all 〈0.01）. NAC also significantly reduced the protein ratio of pp38 and p38 and increased the RNA and protein ratio of bcl2 and bax （P all 〈0.01）. Conclusion The results showed that NAC significantly reduced apoptosis through inhibiting the phosphorylation of p38 signal pathway, which has potential value for clinical cardiac diseases （J Geriatr Cardio12009; 6：168-172）.

Effects and mechanism of glucagon-like peptide-1 on injury of rats cardiomyocytes induced by hypoxia-reoxygenation

Background Although the insulinotropic role of glucagon-like peptide-1 （GLP-1） in type 2 diabetes mellitus has been substantiated, its role in cardioprotection remains largely unknown. This study aimed to determine the effects of GLP-1 on injury of rats cardiac myocytes induced by hypoxia-reoxygenation （H/R） and the possible mechanisms. Methods The cultured neonatal rats cardiac myocytes were randomly divided into seven groups： the normal control group, the H/R group, the GLP-1＋H/R group, the GLP-1＋H/R＋UO126 （the p42/44 mitogen-activated protein kinase （MAPK） inhibitor） group, the GLP-1＋H/R＋LY294002 （phosphatidylinositol 3-kinase （PI3K） inhibitor） group, the H/R＋UO126 group, and the H/R＋LY294002 group. The lactate dehydrogenase （LDH） activity, apoptosis rate of cardiac myocytes, and caspase-3 activity were detected after the injury of H/R. Results Compared with the normal control group, the activity of LDH, cardiac myocyte apoptosis rate, and caspase-3 activity all increased significantly in the H/R group （P 〈0.01）. Compared with the H/R group, these three indices all decreased in the H/R＋GLP-1 group （P 〈0.01）. However, the changes of LDH activity, apoptosis rate, and caspase-3 activity were inhibited by LY294002 and UO126 respectively. Conclusions GLP-1 can directly act on cardiac myocytes and protect them from H/R injury mainly by inhibiting their apoptosis. Its mechanism may be through the PI3K-Akt pathway and the MAPK signaling pathway.

Shexiang Tongxin Dropping Pill（麝香通心滴丸）Protects against Na2S2O4-Induced Hypoxia-Reoxygenation Injury in H9c2 Cells

Objectives: To investigate the protective effects of Shexiang Tongxin Dropping Pill(麝香通心滴丸,STP) on Na2S2O4-induced hypoxia-reoxygenation injury in cardiomyoblast H9c2 cells. Methods: The cell viability and levels of mRNA and protein expression in H9c2 cells were determined following Na2S2O4-induced hypoxia using Hoechst staining, annexin V/propidium iodide(PI) flow cytometry, real-time polymerase chain reaction and Western blot analysis. Results: STP pretreatment signi?cantly increased the viability and inhibited aberrant morphological changes in H9c2 cardiomyoblast cells induced by Na2S2O4 treatment(P<0.05). In addition, STP pretreatment attenuated Na2S2O4-induced hypoxic damage, down-regulated the expression of pro-apoptotic Bax,and up-regulated the expression of anti-apoptotic Bcl-2 in H9c2 cells(P<0.05). Conclusions: STP was strongly cardioprotective in hypoxia-reoxygenation injury by preventing hypoxic damage and inhibiting cellular apoptosis.These results further support the use of STP as an effective drug for the treatment of ischemic heart disease.

Chloride intracellular channel 1 regulates colon cancer cell migration and invasion through ROS/ERK pathway

AIM: To investigate the mechanisms of chloride intracellular channel 1 (CLIC1) in the metastasis of colon cancer under hypoxia-reoxygenation (H-R) conditions.

Effects of L-THP on Ca^(2+) Overload of Cultured Rat Cardiomyocytes during Hypoxia and Reoxygenation

The effects of L-tetrahydropalmatine (L-THP) on the cultured rat cardiomyocytes during hypoxia and reoxygenation and the mechanism of L-THP treating reperfusion-arrythmias were stud- ied. The concentration of intracellular free calcium ([Ca2+]i) of single cultured ventricular myocyte was determined by using EPC-9 light-electricity measurement system. It was found that L-THP (100μmol/L) could reduce the [Ca2+]i augmentation in single cultured ventricular myocyte during hypoxia and reoxygenation. Verapamil (10 μmol/L ) had the similar effect. It was concluded that L- THP could inhibit the Ca2+ overload of cultured rat cardiomyocytes during hypoxia and reoxygena- tion.

Nitric oxide and oxygen radicals induced apoptosis via bcl-2 and p53 pathway in hypoxia-reoxygenated cardiomyocytes

Neonatal rat cardiomyocytes were subjected to 24 h of hypoxia 95%N2/5%CO2 and 24 h of hypoxia plus 4 h of reoxygenation 95%O2/5%CO2. 24 h of hypoxia increased the levels of NO, --23NO/NO, TBARS and LDH. 24 h of hypoxia plus 4 h of reoxygenation decreased the levels of NO, --23NO/NO, but further increased TBARS and LDH. The hypoxia up-regulated the expression of bcl-2, p53 and p21/waf1/cip1 but the reoxygenation down-regulated the expression of bcl-2, and further up-regulated p53 and p21/waf1/cip1. The hypoxia increased cell apoptosis and reoxygena-tion further increased both apoptotic and necrotic cell death. NO, -23NO/NO, TBARS, DNA frag-mentation and cell apoptosis were enhanced by SNP and inhibited by L-NAME respectively. In addition, SOD/catalase down-regulated the expression of p53, p21/wafl/cipl and TBARS but up-regulated bcl-2 and increased indirectly the level of NO, --23NO/NO, and inhibited DNA frag-mentation. The results suggest that hypoxia-induced cell death is associated with the activation of NO, bcl-2 and p53 pathway, while hypoxia-reoxygenation induced cell death via the generation of reactive oxygen species and activation of p53 pathway. The present study clarified that NO may be an initiative signal to apoptotic cell death and the activation of bcl-2, p53 and p21/waf1/cip1 path-way in hypoxic and hypoxia-reoxygenated cardiomyocytes.

Platelet-mediated adhesion facilitates leukocyte sequestration in hypoxia-reoxygenated microvessels

Leukocyte transendothelial migration and sequestration are two distinct outcomes following leukocyte adhesion to endotheli- um during ischemia-reperfusion injury, in which platelets may play a pivotal role. In the present study, we established an in vitro hypoxia-reoxygenation model to mimic ischemia-reperfusion injury and found platelet pre-incubation significantly in- creased leukocyte adhesion to endothelial cells after hyoxia-reoxygenation （over 67%）. Blockade of endothelial-cell-expressed adhesion molecules inhibited leukocyte direct adhesion to endothelial cells, while platelet-mediated leukocyte adhesion was suppressed by blockade of platelet-expressed adhesion molecules. Further experiments revealed platelets acted as a bridge to mediate leukocyte adhesion, and platelet-mediated adhesion was the predominant pattern in the presence of platelets. However, platelet pre-incubation significantly suppressed leukocyte transendothelial migration after hypoxia-reoxygenation （over 31%）, which could be aggravated by blockade of endothelial-cell-expressed adhesion molecules, but alleviated by blockade of plate- let-expressed adhesion molecules. This would indicate that platelet-mediated adhesion disrupted leukocyte transendothelial migration. An in vivo meseuteric ischemia-reperfusion model demonstrated leukocyte transfusion alone caused mild leukocyte adhesion to reperfused vessels and subsequent leukocyte infiltration, while simultaneous leukocyte and platelet transfusion led to massive leukocyte adhesion and sequestration within reperfused microvessels. Our studies revealed platelets enhanced leu- kocyte adhesion to endothelial cells, but suppressed leukocyte transendothelial migration. Overall, this leads to leukocyte se- questration in hypoxia-reoxygenated microvessels.