SILAC-based Proteomic Analysis And Mitochondria Mechanism Researchs On The Different Expression Of Proteins Which On The Hypoxic And Salidroside Intervened Conditions

ABSTRACTObjective: The hypoxic status alters the energy metabolism of cardiomyocytestoinduce cell injury, and thentriggers the occurrence and development of cardiovascular diseases. The previous studies have showed that Salidroside(SAL),a phenylpropanoid glycoside isolated from Rhodiolarosea L, exhibits anti-hypoxic activity. Here we choose H9c2 embryonic rat cardiac cells as a research model to study the protective effects of SAL on Co Cl2-induced hypoxic injuries,and explored the targets and potential molecular mechanisms of SAL actions on H9c2 cells.Methods: H9c2 cells were incubated with H-DMEM medium containing the different dose of Co Cl2 for 24 h as the hypoxic group, or cells were treated with different dose of SAL alone for 12, 24 and 36 h as the positive control group, or pretreated with different dose of SAL for 24 h and followed by treatment with 500 μmol/L Co Cl2 for 24 h as anti-hypoxic group, respectively. The cell viability was measured by MTT assay. We utilized stable isotope labeling with amino acids in cell culture(SILAC) coupled to mass spectrometry to screen the different expression proteins in different groups. Raw data were analyzed with DAVID online software. Apoptosis of H9c2 cells in different conditions were detected by Hoechst33342 staining and FITC-annexin V/PI binding assay.The intercellular Ca2+([Ca2+]i) concentrations were investigated by confocal microscopy. The ATP contents of H9c2 cells were examined by chemiluminiscence assay.The protein expression levels of SUCLG1,SUCLG2,MDH2 and other apoptosis related proteins were investigated by Western Blot.Results:1 After treating with different concentrations of Co Cl2(300, 400, 500,600 and 700μmol/L) for 24 h, the results of MTT showed that the cell viability obviously decreased compared with that of control group, in dose-dependent manner(P<0.05). The IC50 value for Co Cl2 was 500μmol/L in H9c2 cells.2 We also analyzed the effects of different concentrations of SAL(1, 10, 100, and 1000 nmol/L) on the H9c2 cells growth, the results showed that 1000 nmol/L SAL inhibited the cells growth(P<0.05), and other lower concentration SAL had not obvious effects on cells growth(P>0.05).3 The H9c2 cells were pretreated with different concentrations of SAL for 24 h followed by treating with 500 μmol/L Co Cl2. The results of MTT suggested that 10 and 100 nmol/L SAL reduced the 500 μmol/L Co Cl2-induced the hypoxic effects in H9c2 cells.4 A total of 5088 proteins were found(protein level FDR<1%) in the two “forward and reverse” SILAC experiments. Among them, 838 proteins were identified across all these groups. By setting 2-fold change as the threshold for significant up- or down-regulation of proteins in this study, we identified 30 up-regulated and 62 down-regulated proteins in Co Cl2 treatment groups compared with control group, 70 up-regulated and 36 down-regulated proteins in SAL-mediated anti-hypoxic group compared with Co Cl2-induced hypoxic group, respectively.5 Gene ontology(GO) classification indicates that Co Cl2-induced hypoxia is involved in a broad range of cellular processes. The biological process analysis showed that the carbohydrates metabolic, including acetyl-Co A metabolic, TCA cycle and cellular respiration, were heavily affected in Co Cl2-induced hypoxic or SAL-mediated anti-hypoxic group. Among them, the SUCLG1, SUCLG2, MDH1 and MDH2 were heavily involved in these biological processes.6 SAL reduced hypoxia-induced apoptosis of H9c2 cells,and 500 nmol/L of SAL had the significant effects.7 SAL increased the ΔΨm.8 SAL antagonized the increasing [Ca2+]i concentration in H9c2 cells treated with 500 μmol/L Co Cl2.(P<0.05).9 SAL decreased the ROS production.10 SAL increased the ATP level under the Co Cl2 treatment significantly(P<0.05).11 SAL up-regulated the expression of SUCLG1 and SUCLG2, and down-regulated the expression of MDH2 significantly compared with those of Co Cl2-induced hypoxic group(P<0.05).12 SAL down-regulated the levels of caspase-9 and cleaved-caspase-3(P<0.05).13 SAL regulated the ratio of Bcl-2/Bax to protect the H9c2 cells from hypoxic injury(P<0.05).Conclusion: SAL played a critical role in protecting cardiomyocyte against oxidative injury by restoring the TCA cycle, increasing the ΔΨm, inhibiting the excessive consumption of ATP, reducing the mitochondrial Ca2+ overload and ROS production, and attenuating the activation members of the caspase family.