ABSTRACT
OBJECTIVE： To study the protective effects of Pericarpium Trichosanthis extract（TPE） on H9c2 myocardial cells injured by hypoxia/reoxygenation（H/R）. METHODS： H/R injury model of H9c2 myocardial cells was established by using sodium dithionite （Na2S2O4） as chemical hypoxia agent. The modeling conditions of different concentrations of Na2S2O4 （0.625-10 mmol/L） and different time of hypoxia （10-60 min） and reoxygenation （2-8 h）， as well as the concentration of TPE （12.5-400 μg/mL） were screened. The cultured H9c2 myocardial cells were randomly divided into normal group， model group， TPE different dose groups （TPE low-dose， medium-dose and high-dose groups， 25， 50， 100 μg/mL） and positive control group （quercetin， 25 μmol/L）. They were pre-treated with relevant medicine for 24 h， and then H/R injury model was established. Cell viability was measured by MTT assay. The levels of LDH， CK-MB， SOD and MDA in cells were tested by ELISA. Apoptosis of H9c2 myocardial cells were observed by flow cytometry. Western blotting was used to detect the protein expression of Bax and Bcl-2 in cells. RESULTS： The condition of H/R injury modeling included modeling concentration of Na2S2O4 2.5 mmol/L， hypoxia time 30 min， reoxygenation time 4 h. 12.5-400 μg/mL TPE showed no toxicity to H9c2 myocardial cells. After treatment， compared with blank group， survival rate and apoptotic rate of H9c2 myocardial cells in model group were increased significantly； the levels of CK-MB， LDH and MDA were increased significantly， while SOD level was decreased significantly； protein expression of Bax and Bax/Bcl-2 ratio were increased significantly， while that of Bcl-2 was decreased significantly （P＜0.05 or P＜0.01）. Compared with model group， above changes of H9c2 myocardial cells were reversed in all dose groups of TPE （P＜0.05 or P＜0.01）. CONCLUSIONS： TPE can protect H9c2 myocardial cells against H/R injury. Its mechanism may be associated with inhibiting the increase of lipid peroxide， improving the ability of scavenging reactive oxygen free radicals， up-regulating the protein expression of Bcl-2 or down-regulating protein expression of Bax， so as to inhibit the cell apoptosis.
ABSTRACT
Objective To explore the protective effect and molecular mechanism of dl-3-n-butylphthalide (NBP) against oxygen-glucose deprivation induced by sodium hydrosulfite in PC12 cells at proteomic level.Methods PC12 cells impaired by serum-free culture media with sodium hydrosulfite were used as the cell models ofhypoxia,and the protective effects of NBP on hypoxic cells were observed.Methyl thiazolyl tetrazolium (MTT) assay was used to measure the viabilities of PC12 cells.Proteomic technique was employed to identify the differential expression proteins.Results Following the increment of NBP concentrations,the cell absorbance (A) value increased gradually (the PC12 cell apoptosis reduced gradually); when the NBP concentration reached 5 mol/L,their cell A value was significantly different as compared with that of cells without adding NBP (P＜0.05).By using proteomics methods,17 differentially-expressed protein spots in the cells without adding NBP and cells with NBP were identified; most of proteins were cytoskeleton proteins,apoptosis related proteins and oxidative stress related proteins.Conclusion NBP can reduce neuronal apoptosis induced by hypoxia,and cytoskeletal proteins,apoptosis-related proteins and oxidative stress related proteins may role in protection of NBP on ischemic neurons.