The Effect Of Rho-kinase On PI3-kinase/Akt In Myocardial Ischemia Reperfusion Injury And The Intervention Effect Of Pterostilbene

Part Ⅰ The effect of Rho-kinase on PI3-kinase/Akt in myocardial ischemia reperfusion injury and ischemic preconditioningBackgroundCardiovascular disease has been keeping a high incidence, of which coronary heart disease and acute myocardial infarction are with the highest morbidity and mortality. Myocardial ischemia reperfusion therapy is a clinical strategy to alleviate the ischemic symptoms and reduce the myocardial infarction area. But ischemia reperfusion can often worsen myocardial injury, i.e. ischemia and reperfusion injury(I/R). I/R injury can cause apoptosis and necrosis of the myocardial cells, decrease of cardiac systolic function, arrhythmia and other cardiac dysfunction, coronary flow decline. Rho-kinase, a serine/threonine kinase, has been identified as one of the effectors of the small GTP-binding protein Rho. Rho-kinase takes part in various cellular functions, and plays an important role in cardiovascular diseases such as hypertension, heart failure, myocardial infarction and atherosclerosis. A large number of studies have confirmed that Rho-kinase is closely related to the occurrence and development of myocardial ischemia and reperfusion injury.Myocardial ischemic preconditioning (IPC) is one of the protective mechanisms of myocardial ischemia reperfusion injury, which can obviously protect the subsequent long-term ischemia after repeated transient myocardial ischemia. IPC can decrease the intracellular calcium overload during I/R, and reduce myocardial infarction area and cardiomyocyte apoptosis, and improve myocardial contraction. Previous researches have found that overexpression of Rho-kinase in rat heart I/R can aggravate myocardial injury and increase apoptosis; on the contrary, IPC can reduce the apoptosis by inhibiting the activity of Rho-kinase.Therefore, this study intends to explore the changes of Rho-kinase in I/R and IPC, and explain its possible role in signaling pathway, in order to demonstrate the role and mechanism of Rho-kinase in IPC.ObjectiveTo investigate the effect of Rho-kinase on PI3-kinase/Akt in I/R and IPC.Methods1 Healthy female Wistar rats were randomly divided into four groups:(1) control group (sham group)(n=12);(2) I/R group (n=12):the group of model of myocardial ischemia reperfusion; (3) I/R+fasudil group (n=12):the group was given fasudil(Rho-kinase inhibitor)before I/R modeling; (4) IPC group (n=12):the group of myocardial ischemic preconditioning.2 Rat heart ischemia and reperfusion model was prepared and established. The I/R rats were created by ligating the left anterior descending branch (LAD) for 30 min and releasing the ligature for 180 min.3 Rat myocardial ischemic preconditioning model was prepared and established: two cycles of 5 min ligation of the LAD and 5 min reflow before I/R.4 Determination of myocardial infarct size:Evans blue and Nitro blue tetrazolium was used to evaluate the infarcted and noninfarcted areas. Infarct size was expressed as the percentage of the area at risk(AAR).5 TUNEL technique was used to evaluate myocardial apoptotic activity.6 Immunohistochemical staining was used to evaluate p-Akt in the myocardium of different groups of rats.7 Western blot analysis was performed to evaluate p-Akt and Rho-kinase activity. Rho-kinase activity was assessed by examining phosphorylation of MYPT-1, a well established Rho-kinase specific substrate.Results1 P-MYPT-1 (myosin phosphatase targeting subunit) increased in myocardial I/R group. This increase in MYPT-1 phosphorylation was reversed in IPC and I/R+fasudil groups, demonstrating that Rho-kinase activity decreased in IPC.2 Inhibition of Rho-kinase during I/R decreased cardiomyocyte apoptosis. The number of TUNEL positive cells was significantly increased in I/R group (32.78 ± 5.1%). The TUNEL positive cells were significantly reduced to (18.5 ± 4.2%)in I/R+ fasudil group and (17.29 ± 0.84%) in IPC group (P<0.05 vs I/R group). These data suggest that inhibition of Rho-kinase activity in I/R reduces cardiomyocyte apoptosis and cell apoptosis was also attenuated in IPC.3 IPC and Rho-kinase inhibition decreased myocardial infarct size. The AAR and infarct size of the heart were (52.62 ± 2.73%) and (59.89 ± 3.83%), respectively in I/R group. Administration of fasudil,the AAR and infarct size were(32.07 ± 3.0%) and (38.62 ± 2.66%), suggesting that inhibition of Rho-kinase activity reduces myocardial infarct size in I/R injury. In IPC, the AAR and infarct size were(27.92 ± 1.54%) and(29.16 ± 1.08%) (P<0.05 vs I/R group). These data suggest that AAR and infarct size were attenuated in IPC.4 IPC and Rho-kinase inhibition during I/R increased the activation of Akt. The expression of p-Akt decreased in I/R group (P<0.05 vs sham group).But p-Akt was clearly increased in the heart in IPC group and I/R+fasudil group (P<0.05 vs I/R group). These data suggest that Akt was strongly activated in IPC.Conclusion1 Rho-kinase activity increased significantly during I/R, which can aggravate cardiomyocyte apoptosis and increase myocardial infarction area.2 Inhibition of Rho-kinase activity has protection on myocardium by decreasing the cardiomyocyte apoptosis and infarct size in I/R.3 Rho-kinase can affect cardiomyocyte apoptosis by regulating p-Akt.4 IPC can reduce myocardial apoptosis and infarct size in I/R.5 Rho-kinase inhibition by IPC is a major mechanism in reduced cardiomyocyte apoptosis. The inhibition of Rho-kinase leads to cardiovascular protection might be mediated by the activation of PI3-kinase/Akt during IPC. This may provide new treatment possibilities for patients suffering coronary heart diseases in the future.Part II The effect of pterostilbene pretreatment on rat myocardial ischemia reperfusion injuryBackgroundMyocardial ischemia reperfusion injury can cause severe heart damage, structural and functional abnormalities, resulting in heart and other organ failure. I/R injury is often the key node of a series of pathological chain reactions. Myocardial I/R injury often leads to reperfusion arrhythmias (RA), especially ventricular arrhythmias (VA), including ventricular premature beat (VPB) and ventricular tachycardia (VT). RA is one of the most common types of arrhythmia, sometimes leading to sudden death of patients suffering coronary heart diseases. Pterostilbene has the effect of anti- fungal, anti cell proliferation, preventing oxidative stress, anti-inflammation, reducing blood lipid and so on. Pterostilbene can improve ischemia reperfusion injury.ObjectiveTo investigate the effect of pterostilbene on rat myocardial ischemia reperfusion injuryMethods1 Healthy female Wistar rats were randomly divided into four groups:(1) control group (sham group) (n=12);(2) I/R group(n=12):the group of model of myocardial ischemia reperfusion;(3)and(4) I/R+pterostilbene groups (respectively n=12): before the rat model of I/R, rats were given 20mg/kg or 40mg/kg pterostilbene respectively, once a day, totally 2 weeks.2 Rat myocardial ischemia reperfusion model were prepared. The I/R rats were created by ligating the left anterior descending branch (LAD) for 30 min and releasing the ligature for 180 min.3 Incidence of arrhythmia, ventricular premature beat (VPB) and ventricular tachycardia (VT) were recorded by ECG. VA score was recorded.4 TNF-α, IL-1β and SP in serum was detected by ELISA.5 Content of ROS was detected.6 Activity of Glutathione(GSH) in myocardial tissue was analyzed.7 SOD in myocardial tissue was analyzed.8 Western blot analysis was performed to evaluate Caspase-3 activity.Results1 The incidence of arrhythmia, VPB and VT in I/R group was significantly increased (P< 0.05 vs sham group); but pterostilbene pretreatment can reduce the incidence of arrhythmia, VPB and VT (P< 0.05 vs I/R group). And compared with the low dose group, changes in high dose group were more significantly (P< 0.05).2 VA score increased significantly in I/R group (P< 0.05 vs sham group); but pterostilbene pretreatment can significantly reduce the VA score (P< 0.05 vs I/R group).These data suggest that pterostilbene can reduce the incidence of reperfusion arrhythmias.3 ROS production increased significantly and the content of SOD decreased in I/R group (P< 0.05 vs sham group). Pterostilbene pretreatment can inhibit ROS production and increase SOD content (P< 0.05 vs I/R group), and the change was more obvious with higher dose.4 GSH expression in myocardial tissue was high in sham group, and it was significantly lower in I/R rats (P< 0.05). Pterostilbene pretreatment can increase GSH significantly (P< 0.05 vs I/R group).5 TNF-α and IL-1β expression significantly increased in I/R group, compared with sham group (P< 0.05). Pretreatment of pterostilbene, TNF-α and IL-1β were reduced (P< 0.05 vs I/R group). And the change was more obvious with a higher dose of pterostilbene.6 Caspase-3 expression increased significantly in I/R group (P< 0.05 vs sham group).Pterostilbene pretreatment can significantly reduce the expression of Caspase-3 (P< 0.05 vs I/R group).7 SP secretion decreased in I/R group (P< 0.05vs sham group). Pterostilbene pretreatment can significantly promote the secretion of SP (P< 0.05 vs I/R group).Conclusions1 Pterostilbene preconditioning can reduce the incidence of ventricular arrhythmias in I/R.2 Pterostilbene plays a role in myocardial protection by inhibiting the expression of inflammatory factors in myocardial ischemia reperfusion injury.3 Pterostilbene preconditioning can improve myocardial ischemia and reperfusion injury by regulation of oxidant/antioxidant balance and increasing the expression of SP.Part Ⅲ The effect of pterostilbene pretreatment on Rho-kinase in myocardial ischemia reperfusion injuryBackgroundRho-kinase takes part in various cellular functions, and plays an important role in myocardial ischemia and reperfusion injury. The overexpression of Rho-kinase can aggravate cardiomyocyte apoptosis and heart damage in I/R. Pterostilbene can improve ischemia reperfusion injury. Whether pterostilbene protect myocardial I/R by affecting Rho-kinase has not been elucidated, so this study intends to investigate the effect of pterostilbene on Rho-kinase in myocardial ischemia reperfusion injury.ObjectiveTo investigate the effect of pterostilbene pretreatment on Rho-kinase in I/RMethods1 Healthy female Wistar rats were randomly divided into 3 groups:(1) control group (sham group) (n=12); (2)I/R group(n=12):the group of model of myocardial ischemia reperfusion;(3) I/R+pterostilbene group(n=12):before the rat model of I/R, rats were given 40mg/kg pterostilbene, once a day, totally 2 weeks.2 Rat myocardial ischemia reperfusion model were prepared. The I/R rats were created by ligating the left anterior descending branch (LAD) for 30 min and releasing the ligature for 180 min.3 Determination of myocardial infarct size:Evans blue and Nitro blue tetrazolium was used to evaluate the infarcted and noninfarcted areas. Infarct size was expressed as the percentage of the area at risk(AAR).4 ROCK1 and ROCK2 expression were detected by Real time PCR.5 Western blot analysis was performed to evaluate p-Akt and Rho-kinase activity. Rho-kinase activity was assessed by examining phosphorylation of MYPT-1, a well established Rho-kinase specific substrate.Results1 In myocardial I/R group, p-MYPT-1 increased significantly. This increase in p-MYPT-1 was reversed in I/R+ pterostilbene group, demonstrating that pterostilbene can reduce Rho-kinase activity.2 The AAR and myocardial infarct size were(54.17±3.73%)and (61.22±4.51%), respectively in I/R group. In I/R+pterostilbene group, the AAR and infarct size were (22.17±3.50%) and (41.11±4.65%) (P< 0.05 vs I/R group),suggesting that pterostilbene can reduce the infarct size in I/R.3 ROCK1 expression increased significantly in I/R group (P< 0.05 vs sham group). Pretreatment of pterostilbene, the ROCK1 expression was decreased (P< 0.05 vs I/R group).4 ROCK2 expression increased in I/R group (P< 0.05 vs sham group). But amounts of ROCK2 were similar between the I/R and I/R+pterostilbene groups and there was no statistically difference. These data suggest that pterostilbene preconditioning didn’t change ROCK2 expression.5 The level of p-Akt was significantly inhibited in I/R group(P< 0.05 vs sham group). Pretreatment of pterostilbene, p-Akt was significantly increased (P< 0.05 vs I/R group). These data indicated that pterostilbene can increase the activity of Akt.Conclusions1 Pterostilbene preconditioning can reduce the apoptosis of cardiomyocytes and infarct size by inhibiting Rho-kinase activity and reducing ROCK1 expression in myocardial ischemia reperfusion injury.2 Pterostilbene can increase the activity of p-Akt, indicating that the procection of pterostilbene in I/R might be mediated by PI3-kinase/Akt signaling pathway.