Research On The Apoptosic Action In Human Hepatocellular Carcinoma Cell Of Ginsenoside Rg3Alone And When Combined With Oxaliplatin And Sorafenib

Objective:To observe the effects of the apoptosic action in human hepatoma Cell lines SMMC-7721by experiment in vitro using Ginsenoside Rg3alone and when combined with Oxaliplatin and Sorafenib, and to investigate the related mechanism,so as to supply theoretical and experimental bases for the combination of traditional Chinese medicine、chemotherapy and molecular targeting drugs.Methods:Select the appropriate single agent concentration of Ginsenoside Rg3、 Oxaliplatin and Sorafenib, and use the above concentration of single agent alone and combined with each other and observe the changes in hepatoma cells through a variety of experimental methods.(1) Determing the inhibition of proliferation by MTT assay;(2) Detecting the early apoptosis of human hepatoma Cell lines SMMC-7721by flow cytometry;(3)Detecting the middle and late apoptosis of human hepatoma Cell lines SMMC-7721through TUNEL assay;(4) Measuring the gene/protein expression of Bax、 Bcl-2by RT-qPCR and Western blotting;(5)Measuring the protein expression of Bax、Bcl-2、Caspase-3by immunofluorescence cytochemistry technology.Results:(1) Both the single agent and the combinations of Ginsenoside Rg3、 Oxaliplatin and Sorafenib could inhibit the proliferation of SMMC-7721lines,and the triplet combination was superior to doublet combination which was superior to single agent(P<0.01);there was additive effect for these combinations;(2)The results detected by flow cytometry showed that there was apoptosis occured in each experimental groups,singnificant apoptosis showed in triplet combination compared with doublet combination and in doublet combination compared with single agent(P<0.01);(3)Results of TUNEL assay showed that apoptotic cells were hardly found in negative control group with the nucleus stained into lavender;while nucleus of positive control group were stained nearly completely by deeply brown;the nucleus of the experimental groups turned out to be pyknosis and steined into dark brown;both the single agent and the combinations showed significant increase in the apoptosis index (AI)(P<0.01);(4)The results detected by RT-qPCR showed that there was great increase of Bax mRNA in experimental groups, significant increase ourred in the triplet combination compared with doublet combination and in doublet combination compared with single agent (P<0.01或P<0.05),besiding no significant difference of Bax mRNA showed between the Ginsenoside Rg3group and Ginsenoside Rg3plus Sorafenib group;the expression of Bcl-2mRNA showed a decreasing trend among the single agent and the combinations; there was a great increase in Bax/Bcl-2ratio in experimental groups,significant increase ourred in the triplet combination compared with doublet combination and in doublet combination compared with single agent (P<0.01);(5) The results of immunofluorescence cytochemistry technology shows that the protein expression of Bax、Caspase-3and the Bax/Bcl-2ratio increased in experimental groups, significant cincrease ourred when using the combinations(P<0.01or P<0.05); The protein expressions of Bcl-2was decreased,and there was a significant decrease in the triplet combination compared with doublet combination and in doublet combination compared with single agent(P<0.01or P<0.05);(6) The results of Western blotting showed that:there was a increasing trend in the protein expression of Bax and the Bax/Bcl-2ratio,while with no significant trend of Bcl-2protein expression.Conclusion:Both the single agent and the combinations of Ginsenoside Rg3^Oxaliplatin and Sorafenib could inhibit the proliferation and induce apoptosis of human hepatoma Cell lines SMMC-7721,the mechanism of inducing apoptosis might be achieved by up-regulating the expressions of Bax and Caspase-3and Bax/Bcl-2ratio and down-regulating the expressions of Bcl-2. The triplet combination was superior to doublet combination which was superior to single agent,and additive effects showed in the combinations.The results of experiment is of great value in guiding rational combinations of clinical drugs、suppling experimental bases for draftting an optimized combined treatment guiding the clinical therapy and improving the clinical effect of Hepatocellular Carcinoma patients.