| Objective:1. Through the detection of apoptosis and cell cycle changes, learn the effect of Aitongxiao Prescription and its split minor prescriptions on primary liver cancer (PLC.), and to explicit the status of the three class of drugs in Aitongxiao Prescription;2. To study the mechanisms of three class of drugs in Aitongxiao Prescription on primary liver cancer3. To study the mechanisms of Aitongxiao Prescription on primary liver cancer。Methods:10male Wistar rats from67s, randomly selected for the normal diet group, fed with normal diet, the liver tissue as control group for the end of the experiment; The remaining57rats were transplanted hepatocellular carcinoma model,eight days after the modeling and randomly selected seven (2normal rats,5models) to verify the success of modeling. Then the50model rats were randomly divided into four groups, namely, the ATXP group, the Blood Circulation (split Prescription I) group, the Detoxification (split Prescription II) group, the Pei-Yuan (split PrescriptionⅢ) group, which all were given the corresponding concentration of the liquid (both human equivalent dose by10ml of·kg·d-1orally, the Model group and Normal Control group was given the same volume of distilled water, twice daily, continuous administration of14d.24hours withdrawal at the end of the experiment, cardiac blood sampling to test Hemorheology; and rats were killed, the line flow cytometry Antlexin-v/PI double staining method were used to detect circulating hepatocellular apoptosis rate and cell cycle; by RT-PCR, immunohistochemistrychemical assay HCC tissue/normal liver of Bcl-2, survivin, Fas, FasL and Caspase-3mRNA and protein expression detected by immunohistochemical staining of VEGF in HCC tissue protein.Results:1. The apoptosis rates of The ATXP group and Detoxification group was close to the normal group, the difference between the two groups were not statistically significant (P>0.05); but, The apoptosis rates of the Blood Circulation group and Pei-Yuan group were significantly lower than the Normal Control group, the difference between the groups were significant (P<0.05). In the impact of the cell cycle, the majority of liver cancer cells of The ATXP group and Detoxification group remain in the stationary phase (GO phase) or G1phase, just a very small number of liver cancer cells into S phase. While a large portion of liver cancer cells in the Blood Circulation group and Pei-Yuan group into S phase and G2phase.2. The differences of test results of the whole blood viscosity, plasma viscosity, hematocrit, erythrocyte sedimentation equation K value of hemorheology in all groups by analysis of variance, were statistically significant (P<0.01or P<0.05). The ATXP group was lower than the model group in all these indicators, the differences between two groups were statistically significant (P<0.01or P<0.05); In the split minor prescriptions, the Blood Circulation group approach to The ATXP group, no significant difference between the groups (P>0.05). While the Detoxification group and Pei-Yuan group in the index are similar to the model group, no significant differences among three groups (P>0.05). The MVD value of the ATXP group and Blood Circulation group which hadn’t no significant difference between them (P>0.05) were significantly lower than the model group, the differences of pairwise comparisons were highly significant (P<0.01). In addition, the Detoxification group, Pei-Yuan group and model group pairwise comparison, the differences of MVD vale were no differencestatistically significant (P>0.05).3. The ATXP group can significantly inhibit the expression of Bcl-2, Survivin mRNA and protein in primary liver cancer cells. In the split minor prescriptions, the efficacy of the Detoxification group is most significantly, in which the effect on the expression of Bcl-2, Survivin mRNA and protein is close to the ATXP group, the difference between the groups was not statistically significant (P>0.05); but, the Pei-Yuan group had no significant effect on it (P>0.05); It is worth noting that the Bood Circulation group can significantly Upregulate the expression of Bcl-2, Survivin mRNA and protein (P<0.01or P<0.05).Fas and FasL mRNA and protein highestly expressed in the ATXP group, the differences of all the groups were extremely significantly (P<0.01); these mRNA and protein also highly expression in the Detoxification group (P<0.05); but no significant effect on the expression in the Blood Circulation group (P>0.05).It should be noted that in the Pei-Yuan group, the expression of Fas and FasL protein and FasL mRNA were no significant changes (P>0.05), but Fas mRNA was high expression (P <0.05). In all groups, caspase-3mRNA and protein highestly expressed in the ATXP group, as same, it also highly expressed in the Detoxification group, compared with the two goups, the difference was not significant (P>0.05). In addition, Caspase-3mRNA are highly expressed in the Blood Circulation group and Pei-Yuan group (P<0.05), but Caspase-3protein in the difference between the expression of the two groups with the model group group no significant (P>0.05).Conclusions:1. TCM Herb for clearing heat and detoxicating of ATXP can obviously promote apoptosis and inhibit proliferation of hepatoma cells, compared with the other groups, the efficacy of it is the most significant. From short-term efficacy to evaluate the function of TCM Herb for clearing heat and detoxicating is almost equivalent to the whole ATXP, so it plays a major therapeutic role in the treatment of Primary Liver Cancer2. The blood circulation herb of ATXP is the olny one which can affect blood rheology of tumor-bearing body, improving the Microcirculation to reduce the formation of tumor thrombus, to improve the shear force, therefore, it can inhibit the growth and metastasis of PLC. It is fully affirmed that the blood circulation herb is necessity in the treatment of PLC. Meanwhile, the Blood Circulation drugs can also significantly reduce the MVD value to effectively inhibit tumor angiogenesis. Therefore, we suggest that the blood circulation herb of ATXP fight against Yin pathogen (plegm and blood stasis), to inhibit tumor growth and metastasis by changing tumor survival environment.3. The ATXP can significantly affect the apoptotic signal transduction regulatory factors in PLC, the performance is down-regulating the apoptosis suppressor genes Bcl-2and survivin expression; at the same time, it can up-regulated the expression of apoptosis promoting factor, such as Fas, FasL and caspase-3, in which the effect of TCM Herb for clearing heat and detoxicating is most significant, followed by the tonic Herb; but the blood circulation drugs of ATXP had no significant effect on the expression of the apoptosis signal transduction regulatory factors. Therefore, we suggest that TCM Herb for clearing heat and detoxicating of ATXP fight against Yang pathogen (Un-Vital Qi) to adjust the transmission of the apoptotic signal, thereby promoting apoptosis and inhibiting proliferation of hepatoma cells. the blood circulation herb of ATXP fight against Yin pathogen, to inhibit tumor growth and metastasis by changing tumor survival environment.4. The ATXP can fight against PLC from two aspects of Yin and Yang, which can improve blood rheology, and inhibit tumor vascular proliferation; the same time, can also affect the apoptotic signal transduction regulatory factors, the performance is down-regulating the apoptosis suppressor genes Bcl-2and survivin expression; in additon, it can up-regulated the expression of apoptosis promoting factor, such as Fas, FasL and caspase-3, thus play the role of anti-tumor. |
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