| Objective:To investigate signaling pathway underlying mechanism of autophagy of Hep G2 cells induce by Apelin-13.Methods:1.Cultivate Hep G2 cells with Apelin-13 by concentrations as follow 0μmol/L(control),0.0001 μmol/L,0.001 μmol/L,0.01 μmol/L, 0.1 μmol/L for 24 h. And SP600125 group are treated as 10%FBS(control),Apelin-13(0.1μmol/L),Apelin-13(0.1μmol/L)+ SP600125( 20 μ mol/L),SP600125( 20 μ mol/L),DMSO(solvent).2. JNK,p JNK,Bcl-2,p Bcl-2 and Beclin1 protein expression were detected by Western blot. 3. Expression of Beclin1 m RNA were detected by q RT-PCR. 4. Observing the autophagosome in cytoplasm by using fluorescent microscope after monodansylcadaverine(MDC) staining.Results: 1.Western blot:The relative expression levels of p JNK in cells were 0.333±0.007,0.425±0.015,0.529±0.019,0.883±0.036,1.554± 0.021,respectively,which treated with 10% FBS and with 0.0001 μmol/l, 0.001 μmol/l, 0.01 μmol/l and 0.1 μmol/l Apelin-13.Statistics data were significant compare to each group(P<0.01),the relative expression levels of p Bcl-2 were 0.241±0.038, 0.475±0.027, 1.224±0.032, 1.753±0.052,3.072± 0.016,respectively,which were significant compare to each group(P<0.01).The relative expression levels of Beclin1 protein were 0.281±0.007,0.485±0.015,1.540±0.015,2.061±0.010,2.931±0.012, respectively,Compared with negative control group,the levels of Beclin1 expression of which cells treated with Apelin-13 had significant higher(P<0.05). After treated Hep G2 cells with JNK inhibitor SP600125, expression of p JNK were 0.446±0.020, 1.018±0.012, 0.132±0.006, 0.138±0.079,0.462±0.018;and the expression level of p Bcl-2 were 0.029±0.002、1.878±0.036、0.217±0.010、0.196±0.015、0.031±0.024.Both expression of p JNK and p Bcl-2 in group of the Apelin-13(0.1μmol/L)+ SP600125(20μmol/L)and SP600125(20μmol/L)were significantly lower than Control group and Apelin-13(0.1μmol/L) group.Respectively the relative expression levels of Beclin1 protein were 0.145±0.012,1.387±0.017,0.449±0.038,0.119±0.018,0.158±0.007;compar ed with others treated, After treated with SP600125, Beclin1 expression of cells were in significantly lower levels(P<0.05). 2. q RT-PCR:The relative expression level of Beclin1 m RNA were calculated as 1.011 ±0.010,1.735±0.115,2.269±0.007,3.137±0.113, 4.157±0.235 in cells as treated with Control,0.0001 μmol/l, 0.001 μmol/l, 0.01 μmol/l and 0.1 μmol/l Apelin-13.Statistics data were significant compare to each group(P< 0.05). In the SP600125 group the relative expression levels of Beclin1 m RNA were 1.054 ±0.074,7.865 ±0.219,1.563 ±0.102,0.813±0.012,1.012 ±0.093, compared with others treated, cells treated with Apelin-13 and SP600125 had significantly higher levels of Beclin1expression(P<0.05).3. MDC:Fluorescence of Hep G2 cells stained with MDC. It is observed that under inverted fluorescence microscopy that the number of blue and green luminescence autophagosomes were accrue with the increasing of Apelin-13 concentration.After treated by inhibitor SP600125,the autophagosomes in Hep G2 decreased significantly than Apelin-13-treated cells.Conclusion:Aeplin-13 induce the autophagy of Hep G2 cell by enhance the phosphorylation level of Bcl-2 via JNK signaling pathwaydependent on-regulation. |
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