| Stilbene glycosideis one of the medicinal components of Polygonum multiflorum Thunb.It has been used in many Chinese herbal medicines.Studies have confirmed that stilbene glycoside can treat cardiovascular diseases,anti-tumor,anti-inflammatory and neuroprotective effects.It has also been proved to be one of the effective components in the treatment of myocardial ischemia-reperfusion.However,how stilbene glycoside protects cardiomyocytes and what signaling pathways it mediates are seldom reported.In this study,we established an ischemia-reperfusion model in vitro of stilbene glycoside extracted and purified from Polygonum multiflorum Thunb.We tried to explore the possible signaling pathway of stilbene glycoside in myocardial cell protection..The specific research contents and results are as follows:(1)In this paper,the structure of the compound extracted from Polygonum multiflorum Thunb.was analyzed by LC/MS and NMR,and was determined to be stilbene glycoside.(2)The stability of stilbene glycoside in cardiomyocyte culture medium and whether its activity is affected by its interaction with the components of culture medium should be investigated before cell level study.A method for determination of stilbene glycoside based on high performance liquid chromatography was established.It was found that stilbene glycoside was stable within 7 hours.The degradation rates of DMEM F12 and DMEM 1x(low sugar)medium were 2.5% and 6.7%.(3)The proliferation and toxicity of stilbene glycoside were measured by WST-1 assay.It was proved that stilbene glycoside could protect myocardial cells in the range of 1?M/L to 100?M/L,and that might be toxic and side effectsover 100?M/L.(4)Mitochondria were stained with mito-tracker-Green,nucleus were stained with Hoechst33342,and apoptosis-like symptoms were found in the nucleus of the ischemia reperfusion model by laser confocal microscopy,further analysis was performed by flow cytometry and the apoptotic rates of the normal group,model group,1,20 and 100?M /L stilbene glycoside group were 2.77%±0.09,8.14%±0.15,5.44%±0.01,3.73%±0.33,4.12%±0.18.Significant difference was seen between the normal group and model group(P < 0.05),suggesting that the model was successfully constructed.Significant difference were seen among model group and 1,20,100 mu M/L group(P < 0.05),suggesting that stilbene glycoside has anti-apoptotic effect.(5)DCFH-DA probe assay showed that stilbene glucoside could significantly reduce ROS in ischemia-reperfusion model groupin a concentration-dependent manner,and there was a concentration difference,and stilbene glucoside could also reduce the inhibition rate of SOD enzyme.(6)Western bolting results showed that the total AKT remained unchanged,the expression of AKT phosphorylated protein p-AKT increased and the expression of GSK-3beta(downstream of p-AKT),suggesting that stilbene glycoside may play a role through PI3K/AKT signaling pathway,which was further verified by LY294002(PI3K/AKT inhibitor).After stilbene glycoside activated PI3K/AKT pathway,the activity of apoptotic protein Caspase 8 decreased..In this study,we demonstrated that stilbene glycoside can inhibit myocardial apoptosis induced by ischemia-reperfusion in vitro.The mechanism may be related to its activation of PI3K/AKT signal,which provides a theoretical basis for the development of stilbene glycoside-related drugs. |
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