Protective Effect And Its Mechanisms Of Sufentanil Pretreatment On Hypoxia/Reoxgenation-induced Injury And Apoptosis Of H9c2 Rats Myoblasts Cultured In High-glucose Culture Medium

BackgroudThe ischemia and reperfusion injury is the common cause of CRE in noncardiac surgery perioperative period. Ischemia-reperfusion (IR) injury is a complex and extensive pathophysiology. The IR injury occured during myocardiac recovery increase the myocardiac injury and the chance of arrhythmia, and severe impact of cardiac function recovery after ischemia, it can threat on perioperative quality of life of patients and even life. How to prevent and reduce myocardial ischemia-reperfusion injury is still the focus of medical studies.Diabetes serious threate on the healthy of people, it directly or indirectly lead to the increase of CRE in patients with diabetes. However, the effect of diabetic cardiomyopathy on the IR injury have two viewpoint. Many study reported that high glucose can induce apoptosis of myocardial cell. But another studies show that high glucose play a protective role. Therefore, it may have different effect in different circumstances. The exact study need to be done.Sufentanil, a nonselective opioid receptor agonist, have the strongest analgesic effect and is widely used in the anesthesia and ICU for analgesia. In recent years, mang clinical and animal experiments have been certificated that sufentanil have good protection on myocardial ischemia-reperfusion injury. These studies focus on the changes of creatine kinase(CK), cTnI and lactate dehydrogenase(LDH)content in serum and myocardial infarct size, but the exact protective mechanism is not clear. Its effect on apoptosis in vitro heart and cell line have not been reported. Our study prepared to investigate protective effect and its mechanisms of sufentanil pretreatment on hypoxia/reoxgenation-induced injury and apoptosis of H9c2 rats myoblasts cultured in high-glucose culture mediumObjectiveâ‘ To evaluate the injury of H9c2 cardiomyocytes after hypoxia/reoxygenation.â‘¡To investigate the Protective effect and its mechanisms of sufentanil pretreatment on hypoxia/reoxgenation-induced injury and apoptosis of H9c2 rats myoblasts.â‘¢To investigate the protective effect of sufentanil pretreatment on hypoxia/reoxgenation-induced injury and apoptosis of H9c2 rats myoblasts cultured in high-glucose culture medium.â‘£To investigate the effects of high- glucose culture medium on hypoxia/reoxygenation-induced injury and apoptosis of H9c2 cardiomyocytes.MethodsIn the study, for establishing injury model, rat cardiac myoblasts (H9c2 cells) were exposed in 3 hours of hypoxia followed by 3 hours of reoxygenation (hypoxia-reoxygenation HR). And then the cell injury induced by HR were determineded by cell activity, lactate dehydrogenase release(LDH), superoxide dismutase(SOD)and flow cytometry. The expression of porteins include p-p38, p27, cleaved caspase-3 and p-Akt were determineded by western blot. Cells cultured in plates were individed into several groups according to different objective.Chapter one:Cells cultured in plates were individed into several groups including normal control group(Group NC), H/R group(Group HR) and sufentanil pretreatment group(Group SF). H/R injury was induced in cultured H9C2 myocardial cells by three hours hypoxia followed by three hours of reoxygenation. According to the concentration of sufentanil ranging from 10-llmol/L to 10-6mol/L, Group SF was also individed into six subgroups, including SF11, SF10., SF9, SF8, SF7 and SF6 group(n=6/group). In Group SF, sufentanil was add to cell culture medium 15 minutes before hypoxia.. After 3 hours of hypoxia and 3 hours of reoxygenation, cell activity(detected by MTT) and concentration of LDH in cell culture medium was determined.Chapter Two:Cultured H9c2 cardiomyocytes were divided into four groups: (1) NC group; (2) H/R group:(3) SF group:cells were pretreated with sufentanil(10-9M) 15 minutes before hypoxia. (4) NA group:cells were treated with naloxone(10-6M) 10 minutes before sufentanil preatreatment. Colorimetric assay was used to detect cell viability, superoxide dismutase(SOD) and lactate dehydrogenase (LDH) release to evaluate cell injury. Apoptotic rate of H9c2 cardiomyocytes were determined by flow cytometer. The expression of porteins include p-p38, p27, cleaved caspase-3 and p-Akt were determineded by western blot.Chapter Three:Cultured H9c2 cardiomyocytes were divided randomly into eight groups:normal glucose-cultured control group(Group NC), normal glucose-cultured HR group(Group HR), normal glucose-cultured sufentanil pretreatment group(Group SF), normal glucose-cultured naloxone and sufentanil pretreatment group(Group NA) and high glucose-cultured control group(Group HC), high glucose-cultured HR group(Group HHR),high glucose-cultured sufentanil pretreatment group(Group HSF), high glucose-cultured naloxone and sufentanil pretreatment group(Group HNA). Colorimetric assay was used to detect cell viability and lactate dehydrogenase (LDH) release to evaluate cell injury. Superoxide dismutase (SOD) activity was measured by colorimetric assay. Cleaved caspase-3 protein were determineded by western blot.ResultsIn the study we have firstly established that exposure of rat cardiac myoblasts (H9c2 cells) to 3 hours of hypoxia followed by 3 hours of reoxygenation(hypoxia-reoxygenation HR) resulted in significant cell injury determineded by cell activity, lactate dehydrogenase release(LDH), superoxide dismutase(SOD)and flow cytometry. In the followed study, we found that pretreatting H9c2 cells with sufentanil significantly protects myoblasts against subsequent HR injury exposure in a concentration-dependent manner with maximum protection obtained at and more than 10-9M of sufentanil. With Western blotting, it was observed that sufentanil -protected cells had higher levels of the phosphoserine-473 Akt (activated Akt) compared with HR injured cells. In contrast, HR injured cells had significantly higher levels of the cleaved caspase-3, P27 and p38 compared with sufentanil-protected cells.We also found that opioid antagonist naloxone can abolish sufentanil-induced cytoprotective effect.ConclusionIn all, we concluded that:(1) Hypoxia reoxygenation lead to significant cell injury, the fuction of cell was deteriorationa and apoptosis was increased.(2) Sufentanil pretreament protect cardiomyocytes from H/R-induced injury under high glucose and common glucose culture medium.(3) Sufentanil pretreament protect cardiomyocytes from H/R-induced injury in a concerntration-dependent manner (When the concentration exeed 10-9 mol/L, the protection exeed the highest level).(4) High glucose may result in stress injury in the normal condition, but markedly strengthen the anti-H/R ability of H9c2 cells and also have some cardioprective effects in the hypoxic condition.(5) Sufentanil pretreament can elevate cell activity, suppress oxidative stress and apoptosis and relief cell cycle arrest in HR-induced H9c2 cells, And the protection is mediated, at least in part, via activating the PI3K/Akt pathway, supressing p38MAPK pathway the expression of the cyclin-dependent kinase inhibitor p27 protein. which is a negative regulator of the cell cycle.