| Objective: To investigate the effects of BanXiaXieXin Decoction on KM transplanted tumor cell S180 in mice in vivo and its mechanisms. To study the effects of inhibiting proliferation of medicament blood serum of BanXiaXieXin Decoction on human gastric cancer line BGC-823 and its mechanisms,so as to find a feasible method for the cure of gastric cancer.Methods: 1 Animal experiment: fifty healthy male KM mice were choosed and the S180 cells were calculated for 1×107 cells per milliliter, the S180 cells which were grown in the abdominal cavity of mice. Then each of mice was injected of S180 tumor cells (0.2ml per mice) by right armpit. After that, all the tumor-bearing mice were randomly divided into five groups at tomorrow, ten mice of each group. The control group, the low-dose BX group(1g/ml), the middle-dose BX group(2g/ml), the high-dose BX group(3g/ml), and the cyclophamide group. The mice in five groups were treated by Sodium Chloride (0.15ml/10g.d), low-dose BX(0.15ml/10g.d),middle-dose BX(0.15ml/10g.d),high-dose BX(0.15ml/10g.d) once per day respectively ,and the cyclophamide(0.3mg/10g.d) once two days. Fourteen days later, all mice were weighted before killed. After that all tumors tissues and all spleen were taken and weighted, the inhibition ratio of tumor growth and the spleen index were calculated. At the same time , the expression of VEGF,Bcl-2 protein was studied by immmunocytochemistry staining and was semi-quantitately examined by flow cytometry. Apoptosis and distribution of cell cycle were also examined by flow cytometry. 2 In vitro: Selecting human gastric cancer BGC-823 cell line , using MTT assay to detect the growth rate among medicament blood serum of BanXiaXieXin Decoction, the low-dose BX group(0.77g/ml), the middle-dose BX group (1.54g/ml), the high-dose BX group(3.08g/ml), and treateded for different time (24h,48h,72h), in order to choose a proper BX Decoction concentration action time. According to the results of MTT, the expression of VEGF,Bcl-2 protein was studied by immmunocytochemistry staining and was semi-quantitately examined by flow cytometry in BGC-823 cell line treated with different doses of medicament blood serum of BX Decoction for 48 hours. Apoptosis and distribution of cell cycle were also examined by flow cytometry in BGC-823 cell line treated with different doses of medicament blood serum of BX Decoction for 48 hours. .Results: 1 Animal experiment (1) Fourteen days later, all the mice were killed,weighted the tufmor and the spleen .Then the inhibition ratio of tumor growth and the spleen index of every group were calculated. The inhibition ratio of tumor of the low-dose BX group,the middle-dose BX group,the high-dose BX group and the cyclophamide group were 22.2%,45.9%,57.8% and 73.0% respectively . Furthemore, the inhibition ratio was stepping up as the BX Decoction concentration increasing. The spleen index of control group,the low-dose BX group,the middle-dose BX group,the high-dose BX group and the cyclophamide group were 8.183,8.380,8.986,9.798 and 8.380 respectively . There was statistically significant difference between control group and the high-dose BX group in spleen index (P < 0.05). (2) Immunocytochemistry staining results: the staining of VEGF,Bcl-2 protein was positive in transplanted S180 tumor cell line in control group and cyclophamide group , and the buffy stain can be seen in endochylema or cell membrance. While, each of the BX group weakened the stain degree of VEGF and Bcl-2 protein. (3) FCM assay results: compared with control group, the number of the transplanted S180 tumor cells in the three BX groups in G0/G1 phase increased gradually, while the number of the transplanted S180 tumor cells in S phase and G2/M phase decreased gradually. The result suggested that BX Decoction can induce an arrest of cell cycle in G0/G1 phase by a dose-dependent manner. In addition, the apoptotic percentage of control group,the low-dose BX group,the middle-dose BX group,the high-dose BX group and the cyclophamide group were 2.52%,15.92%,23.04%,28.54%,31.34% sperately. The typical apoptotic peak which enhanced gradually with the increasing concentration of BX Decoction and the cyclophamide were also observed and analysis on the expression of VEGF and Bcl-2 protein by FCM showed that : the FI values of VEGF of control group,the low-dose BX group,the middle-dose BX group,the high-dose BX group and the cyclophamide group were 2.783,2.219,2.025,1.368 and 2.449 sperately. The FI values of Bcl-2 of control group,the low-dose BX group,the middle-dose BX group,the high-dose BX group and the cyclophamide group were 2.166,1.822,1.639,1.232 and 1.984 sperately. The FI values of VEGF and Bcl-2 in each group were decreased after treated with the different doses of BX Decoction. To the FI values of VEGF and Bcl-2 protein ,there were statistically significant difference between control group and each of treatment group (P<0.05). 2 experiment in vitro (1) MTT assay results: within the concentration of medicament blood serum of BX Decoction (the low-dose BX,the middle-dose BX and the high-dose BX),BX Decoction can obviously inhibit proliferation BGC-823 cell in vitro. After treated with medicament blood serum of BX Decoction for 24h,48h,72h,compared with the control group,the OD values of BX Decoction groups decreased , and there were statistically significant difference between control group and each of treatment group (P<0.05). Meanwhile, with the increasing concentration of BX Decoction and prolonging of treatment time, the OD values decreased gradually. The highest inhibition ratio was 52.9% in the high-dose BX group for 72h.The results suggested that: BX Decoction could inhibit the proliferation BGC-823 cells in a dose-dependent and time-dependent manner. (2) Immunocytochemistry staining results: the staining of VEGF,Bcl-2 protein was positive in BGC-823 cells which untreated with BX Decoction, and the buffy stain can be seen in endochylema or cell membrance. While, the stain degree of VEGF and Bcl-2 protein were weakened in BGC-823 cells that treated with medicament blood serum of BX Decoction for 48h. (3) FCM assay results: after treated BGC-823 cell line with medicament blood serum of BX Decoction for 48h, all the BGC-823 cells were collected for the analysis on tistribution of cell cycle and apoptosis by flow cytometry. Comparing with control group, the number of BGC-823 cells in BX group in G0/G1 phase increased gradually, while the number of BGC-823 cells in S phase and G2/M phase decreased gradually. The result showed that BX Decoction can induce an arrest of cell cycle in G0/G1 phase by a dose-dependent manner. Moreover, the typical apoptotic peak which enhanced gradually with the increasing concentration of BX Decoction were observed and the apoptotic percentage were 1.31%,1.66%,7.66%,13.16%,23.24% respectively. Analysis on the expression of VEGF and Bcl-2 protein by FCM showed that : the FI values of VEGF of the normal group,the control group,the low-dose BX group,the middle-dose BX group and the high-dose BX group were 3.231,3.326,2.966,2.798 and 2.631 sperately. The FI values of Bcl-2 of the normal group,the control group,the low-dose BX group,the middle-dose BX group and the high-dose BX group were 3.268,3.471,3.136,3.083 and 2.801 sperately. The FI values of VEGF and Bcl-2 in each treatment group cells treated with medicament blood serum of BX Decoction for 48h were decreased. To the FI values of VEGF and Bcl-2 protein ,there were statistically significant difference between control group and every treatment group (P<0.05). There also was the most obvious reduction in the FI values of VEGF and Bcl-2 protein of high-dose BX group.Conclusions: 1 BX Decoction had the effect of anti-transplanted tumor cell S180 in mice within a certain BX Decoction concentration,and it was in a dose-dependent manner. Furthermore, the high-dose BX group could improve the spleen index of S180 mice. The result suggests that BX Decoction has an improvement of immunity and antitumor ability of S180 mice. 2 BX Decoction could inhibit proliferation of BGC-823cells in vitro by a dose-dependent and time-dependent manner within a certain BX Decoction concentration. 3 BX Decoction could induce an arrest of cell in cycle in G0/G1 phase as well as apoptosis in the transplanted tumor cell S180 in mice and the BGC-823 cells, it also was in a dose-dependent manner. 4 BX Decoction might directly inhibit angiogenesis,repress proliferation,induce apoptisis to bring into play the antitumor effect by down-regulation of the expression of VEGF and Bcl-2. 5 The results provide basic theory to the BX Decoction application in inhibiting the transplanted tumor cell S180 in mice and gastric cancer. 6 The results may provide some theoretic support for the Traditional Chinese medicine's theory of dialectic debate and therapy at molecular level. |
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