The Mechanisms Of Mitochondria On 6’-Hydroxy Justicidin A Inducing HepG2 Cell Apoptosis

Objective:To investigate the effects of 6’-hydroxy justicidin A inducing human hepatocellular carcinoma HepG2 cell apoptosis and the potential mechanisms involving mitochondria of 6’-hydroxy justicidin A-induceded HepG2 cell apoptosis. To provide scientific experimental evidence and theoretical basis for the clinical application of JR6 inducing tumor cell apoptosis.Methods: ①The proliferation inhibition of HepG2 cells exposed to JR6 0～196μmol·L-1 and for 48 h were measured by MTT assay. ② The morphological changes of apoptosis was obseverd;nucleus morphological changes stained by Hoechst 33258 were observed under fluorescent inverted microscope;The rate of cell apoptosis was evaluated by Annexin V-FITC/PI double staining method. ③The changes of mitochondrial membrane potential (△ψm) were measured by JC-1 staining throughhigh content imaging systemand flow cytometry;The changes of intracellular Ca2+ concentration were detected by Fluo-3 AM through the high content imaging system. ④The expressions of the apoptosis related protein were analyzed by Western blot.Results:①MTT results showed that compared with control group, JR6 and 5-FU groups could significantly inhibit the proliferation of HepG2 cells(P< 0.05), the IC50 value of JR6 and 5-FU were 74.9 μmol·L-1 and 49.75μmol·L-1. ②The morphology of Cells connect disappear and count decreases when observed after treated with JR6 and 5-FU for 48 h;Hoechst 33258 staining indicated cell shrinkage, nuclear condensation in HepG2 cells treated with JR6 and 5-FU; Flow cytometry analysis indicated that the ratio of cell apoptosis was significantly higher than control group (P<0.05, P<0.01), JR6 induced apoptosis in a concentration-dependent manner. ③ After being treated with different concentration of JR6 and 5-FU for 48 h, the analysis of red/greenfluorescence ntensity suggested that the mitochondrial transmembrane potential in HepG2 cells decreased significantly,comparing with control group (P<0.01). At the same time, the results of flow cytometry compared with control group showed that the ratio of mitochondrial membrane potential decreased after treated with 49 and 196μmol·L-1 JR6 (P<0.05, P<0.01); Simultaneously, Ca2+ fluorescence intensity in JR6 treatment groups was more higher than those in the control group (P<0.01) ￤estern blot results showed that after exposure with JR6 for 48 h. the expression of Procaspase-3,9 was decreased, while Cleavedcaspase-3,9 was increased. Meanwhile, the protein expression of Bax increased whereas Bcl-2 decreased, leading to an increase in the Bax/Bcl-2 ratio. In mitochondria, the expression of mitochondrial cytc, caspase-independent pathway AIF and EndoG decreased, while those of increased in the cytoplasm.Conclusion:JR6 significantly inhibited the proliferation of human hepatocellular carcinoma HepG2 cells and obviously induced apoptosis by affecting mitochondrial membrane potential, intracellular Ca2+ concentration and the expression of apoptosis related protein. Those suggested that mitochondrial pathway was involved in the apoptosis of HepG2 cells.