ABSTRACT
OBJECTIVE：To study the improvement effects and mechanism of Polygonum orientale flower extract on hypoxia- reoxygenation injury of H 9c2 cardiomyocytes. METHODS ：H9c2 cardiomyocytes were divided into normal control group ，model group and low- ，medium- and high-concentrations groups of P. orientale flower extract （20，40，80 μg/mL）. Except for normal control group ，other groups were given 800 μmol/L CoCl2 to induce hypoxia-reoxygenation injury model. Cell apoptosis was observed. The levels of Ca 2+（in cytoplasm ），mitochondrial membrane potential （MMP），ATP enzyme （Na+-K+-ATP enzyme ，Ca2+-Mg2+-ATP enzyme） activities， the ratio of cytochrome c （Cyto c ）， protein in cytosol to mitochondria ，phosphorylation levels of reperfusion injury salvage kinase （RISK） signaling pathwayrelated protein [protein kinase B （Akt）and extracellular signal regulated kinase 1/2（ERK1/2）] as well as protein expression of HIF- 1 α were detected respectively. In addition，the cells were divided into normal control group ，model group and P. orientale flower extract group （80 μ g/mL），PI3K inhibitor LY294002+CoCl2 group（15 μmol/L LY294002+80 μmol/L ，LY294002+P. orientale flower extract group （15 μmol/L LY294002+80 μg/mL P. orientale flower extract ），MEK inhibitor PD98059+CoCl2 group（25 μmol/L PD98059+800 μmol/L CoCl2），PD98059+P. orientale flower extract group （25 μmol/L PD98059+80 μg/mL P. orientale flower extract ）. After cultured by the same method ，the phosphorylation levels of Akt protein and ERK1/2 protein in the cells were measured to verify the activation of P. orientale flower extract to RISK signaling pathway. RESULTS：Compared with model group ，nuclear pyknosis and the number of apoptotic bodies were reduced in different concentrations groups of P. orientale flower extract. ROS level ，Ca2+ level（except for low-concentration group ），MMP，ratio of Cyto c in cytoplasm to Cyto c in mitochondria ，protein expression of HIF- 1α were decreased significantly（P＜0.05 or P＜0.01）； the activity of ATP enzyme （except for the low-concentration group ），Akt protein and ERK 1/2 protein phosphorylation level were significantly increased （P＜0.01）. After treated with PI 3K inhibitor LY 294002 and MEK inhibitor PD 98059，Akt protein and ERK 1/2 protein phosphorylation level in cadiomyocyte were decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：P. orientale flower extract can improve hypoxia-reoxygenation injury of H 9c2 cardiomyocytes，the mechanism of which may be associated with inhibiting cardiomyocyte apoptosis ，improving ATPase activity ，protecting mitochondria ，regulating RISK signaling pathway related proteins and HIF- 1α protein expression.
ABSTRACT
To investigate the protective effects and mechanism of Polygonum orientale flower extract on H₂O₂-induced oxidative damage of human umbilical vein endothelial cells (HUVEC), H₂O₂ was used to induce the oxidativestress damage on HUVEC cells and efforts were made to screen the low, medium and high drug concentrations of P.orientale flower extract. Cell viability was detected by the MTS assay. The content of lactate dehydrogenase (LDH) and malondialdehyde (MDA), and the activities of superoxidedimutase (SOD) and catalase (CAT) were detected by biochemical kits. The mRNA and protein levels of Bax, Bcl-2 were detected respectively by quantitative real time polymerase chain reaction (qRT-PCR) and Western blot. The protein level of cleaved caspase-3 was detected by Western blot. According to the results, the viability of HUVEC cells was reduced to around 55% after being treated with 120 μmol·L⁻¹ H₂O₂ for 0.5 h. Treatment of H₂O₂ also could increase LDH leakage rate and MDA content and attenuate the activities of SOD and CAT, up-regulate the expression level of Bax and cleaved caspase-3, and down-regulate the expression level of Bcl-2. As compared with H₂O₂ model group, P.orientale flower extract of 50-200 mg·L⁻¹ could increase the viability of HUVEC cells, reduce LDH release and MDA content, enhance the activities of SOD and CAT, down-regulate pro-apoptotic protein cleaved caspase-3 and Bax, and up-regulate apoptosis inhibitory protein Bcl-2. In summary, P.orientale flower extract showed a protective effect on H₂O₂-induced HUVEC cells injury, which may result from enhancing the cell capability of clearing the oxygen free radial, decreasing the production of lipid peroxidation and inhibiting apoptosis.