| ObjectiveThis trial aims to study the therapeutic mechanism of Sen Tao Ruan Gan Capsules (STRGC) for hepatoma from three perspectives, including in vivo, in vitro and Immunohistochemistry.Methods1. In vitro studyWe prepared low, medium and high dosage of drug serums and tumor cells in increased logarithmic phase. Six groups were established in the trial, including normal control group, normal serum group, three drug serum groups (low, medium and high dosages) and positive control group. With MTT T assay, inhibition ratios of drug serums was determined in the cells of hepatoma HepG2, respectively;with Flow cytometry. effect of drug serum on early apoptosis in human hepatoma HepG2cells; with RT-PCR, effect of drug serum on levels of bax and bcl-2mRNA in human hepatoma HepG2cells; with wb, effect of drug serum on levels of bax, bcl-2and activated caspase-3proteins in cells.2. In vivo studyThe tumor-bearing mouse models were randomized into five groups,8in each, with the aim to explore anti-hepatoma mechanism of STRGC. The negative control group was assigned to saline; low, medium and high dosage groups to STRGC Suspension0.35g/kg,0.7g/kg and1.4g/kg, respectively; positive control group to Cisplatin Injection2.Omg/kg; twice daily, three weeks in total. The samples were harvested24hours after the withdrawal. We observed the drug effect on tumor inhibition and apoptosis in tumor cells in mice and determine the contents of TNF-a, IL-1β and IL-6in serums and tumor tissues of mice as well as the killing quality of splenic lymphocytes on H22cells, the proportion of CD4+and CD8+in peripheral blood and splenic lymphocytes and drug effect on mice’s immunity. In tumor tissues, with immunohistochemistry, the levels of CD34and VEGF and activated caspase-3were determined; with RT-PCR, the levels of bcl-2and bax-RNA; wi th western blot, the levels of bcl-2, activated caspase-3proteins.Results1. In vitro study1.1After the administration of STRGC-containing serum, morphology change was noted in the human hepatoma HepG2cells as well as a decline in cell number.1.2The role of drug serum in hepatoma cell proliferation:the normal mouse serum plays’no role in the hepatoma cell proliferation (P>0.05). Low, medium and high dosage of drug serum and Cisplatin showed significant inhibitory action (P<0.05). The inhibitory action grew with the increased concentration of STRGC in serum in three drug serum groups (low, medium and high dosages) and had dose-dependent quality.1.3Flow cytometry revealed that:normally cultured human hepatcma hepG2cells have5%-10%natural apoptosis; normal mouse serum has no remarkable effect on apoptosis of hepG2cells; low, medium and high dosage of drug serum and Cisplatin had significant role in inducing apoptosis of hepG2cells.1.4RT-PCR showed that:normal mouse serum had no significant effect on the levels of Bax and Bcl-2mRNA in human hepatoma HepG2cells (P>0.05). Low, medium and high dosage drug serums and positive chemo drug played a significant role in inducing the transcription of Bax gene (P<0.01), inhibiting the transcription of Bcl-2gene (P<0.01) and elevating the value of Bax/Bcl-2. The increase in the Bax mRNA level and decrease in Bcl-2mRNA level in low, medium and high dosage drug serum groups were to some extent dependent upon the STRGC concentration.1.5western blot suggested that:normal mouse serum had no significant effect on the levels of Bax, Bcl-2and caspase-3proteins in human hepatoma HepG2cells (P>0.05). Low, medium and high dosage drug serums and positive chemo drug played a significant role in inducing the expression of Bax protein, inhibiting the expression of Bcl-2protein, elevating the ratio of Bax/Bcl-2and activating caspase-3. This action grew with the increased concentration of STRGC in low, medium and high dosage groups.2. In vivo study2.1The maximum tumor size and weight were noted in model group while the minimum in positive control group with the low, medium and high dosage group in between. The anti-tumor effect grew with the increased concentration of drug and compared to the model group, a significant reduction was noted in tumor weight in all other groups, which is significantly different (P<0.01).2.2The mouse tumor tissue in model group has2%-5%natural apoptosis. The low, medium and high dosage of STRGC and peritoneal injection of positive chemo drug played a significant role in inducing the apoptosis of tumor cells in vivo. The apoptosis ratio was significantly elevated and the highest of which was noted in positive control group (50%-70%), followed successively by high dosage group (40%-60%), medium dosage group (30%-50%), and the low dosage group (20%-30%).2.3The content of cytokine in serum of tumor-bearing mouse, which was obviously lower than that in the normal control group (P<0.01), was significantly elevated with the increased concentration of STRGC, which is significantly different (P<0.01), compared to the model group. No improvement was made in the cytokine content in serum of mouse managed with positive chemo drug (P>0.05). The cytokine content in the tumor tissue of mouse held the trend that was similar to blood serum. The STRGC was able to significantly elevate the content of cytokine in local tumor tissue, thus improving the immunity, but not the positive chemo drug.2.4Compared with the normal mouse, a reduction was noted in CD4+cells and an increase in CD8+cells in the peripheral blood and splenic T lymphocytes, resulting in a reduction in the value of CD4+/CD8+.The low, medium and high dosage of STRGC was capable of significantly improving the cellular immunity in mice (P<0.01), but not the positive chemo drug (P>0.05).2.5Compared with the normal mouse, the ability of splenic lymphocytes to kill the H22cells in tumor-bearing mouse is significantly weakened. The low, medium and high dosage of STRGC can significantly enhance the killing ability of splenic lymphocytes (P<0.01) which is stronger than that in normal mouse, but not the positive chemo drug (P>0.05).2.6Compared with the model group, a significant reduction in the MVD counts was noted in the positive control group, low, medium and high dosage group, which is statistically significant (P<0.01). There’s no definite difference in VEGF expression level between groups.2.7STRGC in low, medium and high dosage groups and positive chemo drug played a significant role in inducing the transcription of Bax gene (P<0.01), inhibiting the transcription of Bcl-2gene (P<0.01) and elevating the value of Bax/Bcl-2. The increase in the Bax mRNA level and decrease in Bcl-2mRNA level in low, medium and high dosage drug serum groups were to some extent dependent upon the STRGC concentration.2.8The minimum proportion of activated caspase-3cell was noted in model group while the maximum in positive group, with low, medium and high dosage group in between, which was to some extent dependent upon the dosage.2.9Low, medium and high dosage of STRGC and positive chemo drug played a role in inducing the expression of Bax protein, inhibiting the expression of Bcl-2protein and elevating the value of Bax/Bcl-2. In low, medium and high dosage groups, these effects grew with the increased concentration of STRGC. The tread of expression of activated caspase-3protein in tumor tissues of mice was similar to that in the immunohistochemistry study and compared with the model group, a significant increase in the level of activated caspase-3protein was noted in the tumor tissues of mice in the low, medium and high dosage groups (depending on the dosage) and positive group.Conclusions1. STRGC-containing serum could play a role in inhibition and apoptosis of the human hepatoma HepG2cells.2. STRGC could play a role in tumor growth inhibition and apoptosis of tumor cells in H22hepatoma-bearing mouse.3. STRGC could contribute to improving the immunity.4. STRGC could play a role in inhibition of tumor angiogenesis.5. STRGC could play a role in activating caspase-3protein for suppress tumor by modulating the ratio of Bax/Bcl-2. |
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