Protective Role And Mechanisms Of STVNa On Mitochondria In Ischemia-reperfusion H9c2 Cardiac Myocytes

Mitochondria are important regulators of cell growth,and are highly dynamic organelles involved in fusion and fission in response to ischemia or other types of oxidative stress.Therefore,it is important to explore a mitochondria targeted drug for treating cardiovascular disease.Isosteviol is the acid hydrolysate product of steviol glycoside,and its sodium salt form Isoteviol sodium(STVNa)is more hydrophilic,thus improving the bioavailablity.In recent years,a number of studies have investigated the anti-hyperglycemic,anti-hypertensive,antiinflammatory and anti-tumor effects of isosteviol.The cardioprotective effect of isosteviol in ischemia-reperfusion injury has also been widely researched.But the potential cardioprotective mechanism of isosteviol is not yet clear.In the present study,we aim to research the protective role and potential mechanism of STVNa on H9c2 cardiomyocytes against IR injury through in vitro model.The main contents are as follows:1.H9c2 cells were subjected to 90 min ischemia followed by 90 min reperfusion.STVNa was treated at the onset of reperfusion.Results showed that compared to control group(0.982 ± 0.007),the cell viability of IR group(0.567 ± 0.007)was decreased by 50%,10 ?M STVNa treatment group(0.627 ± 0.013)significantly improved cell viability(P < 0.05).Further studies have found that 1 ?M?10 ?M?100 ?M STVNa can also recover the mitochondrial membrane potential,which from 34.7% ± 0.6% in IR group increased to 51.6% ± 4.3%?62.9% ± 2.8%?80.4% ± 5.9% respectively(relative value,P < 0.05);reduce reperfusion induced ROS overproduction,which from 130.9% ± 2.4% decreased to 110.9% ± 2.0%?103.5% ± 3.1%?101.6% ± 0.8% respectively(relative value,P < 0.05);and eventually inhibit cell apoptosis,the cell apoptosis rate was decreased from 45.7% ± 3.6% in IR group to 34.0% ± 3.4%?25.8% ± 1.4%?21.3% ± 0.9% respectively(relative value,P < 0.05).2.We also researched the regulation role of STVNa on mitochondrial fission and fusion.Results showed that STVNa treatment changed the morphology of mitochondria from fragmented,discontinuous forms to normal elongated,tubular ones.Cyto-immunofluorescence and western blot analysis revealed that STVNa inhibited mitochondrial fission proteins dynamic related protein 1(Drp1),and mitochondrial fission 1(Fis1).Compared to control group(1.00 ± 0.10?1.00 ± 0.07),the expression of Drp1 and Fis1 protein was up-regulated to 1.74 ± 0.06,1.76 ± 0.06 respectively.10 ?M,100 ?M STVNa treatment group decreased Drp1 and Fis1 protein level to 1.21 ± 0.06,1.15 ± 0.08 and 1.47 ± 0.05,1.25 ± 0.04 respectively(P < 0.05),therefore protecting myocardial ischemia reperfusion induced mitochondrial injury.Conclusion: STVNa plays a protective role on myocardial ischemia reperfusion induced mitochondrial dysfunction.The underlying mechanism may be related to its inhibition on mitochondrial scission,scavenge intracellular excessive ROS and decrease cell apoptosis.