Experimental Studies On Confidence Test Of YangHe Decoction Treat Breast Cancer

Objective:Respecting the different standpoint of great Qing Dynasty physician WangHongxu and MaPeizhi about whether YangHe(YH) Decoction could cure breast cancer,we carried out the experiment. To probe the effect of female sex hormonelike of YH Decoction on weaned KM mice in uterus experiment. To study the effects of inhibiting proliferation of rat's medicament blood serum of YH Decoction on human breast cancer cell MCF-7 ER(+) and MDA-MB-231 ER(-).Apoptosis and distribution of cell cycle were examined by flow cytometry in MCF-7 cell line and MDA-MB-231 cell line treated with different doses of medicament blood serum of YH Decoction for 48 hours. So as to investigate the feasibility of YH Decoction treat Breast cancer.Methods:1 Experiment in vivo:One hundred healthy female weaned KM mice were chosen (10-12g) which divided into ten groups at random and then be weighed and recorded,ten mice each group. The mice of seven traditional Chinese medicine groups were treated by YH Decoction at seven density:0.5g/kg,1g/kg,2g/kg,4g/kg,7.5g/kg,15g/kg,30g/kg,Qd. The mice of positive control groups were given Progynova (E2)dissolved in PSS,and be divided into high-dose group(0.56mg/kg) and low-dose group (0.28mg/kg),Qd. Negative control group only fed with PSS part. aeq,Qd. Ten days later,all mice were weighted uterus and ovaries and recorded after they had been killed.2 Experiment in vitro:According to the method from documents,selecting human breast cancer MCF-7 cell line and MDA-MB-231 cell line,using MTT assay to detect the growth rate among medicament blood serum of YH Decoction,the low-dose YH group (4.5g/kg);the middle-dose YH group (9g/kg);the high-dose YH group (18g/kg);CTX group(70mg/kg·d); E2 group(0.17mg/kg);PSS group,and treated for different time (24h-120h) , in order to choose a proper YH Decoction concentration action time. According to the results of MTT,apoptosis and distribution of cell cycle were examined by flow cytometry in MCF-7 cell line and MDA-MB-231 cell line treated with different doses of medicament blood serum of YH Decoction for 48 hours.Results:1 Experiment in vivo:Every uterus and ovaries'wet weight and it's organ coefficient in YH groups was less than PSS group's,as well had extremely significant difference (P<0.01).As the dose of YH from low to high ,every uterus and ovaries'wet weight and it's organ coefficient took on a tendency of decrease progressively. It had dose-effect relation with the dose of YH. (Uterus r= - 0.8198 negative correlation P=0.0240<0.05 significant difference , r= - 0.8979 negative correlation P=0.0060<0.01 significant difference). Illustrated that YH had no female sex hormonelike effect,on the contrary,it presented a repressed tendency in the growth and development of uterus and ovaries,and had dose-effect relation obviously (negative correlation).2 Experiment in vitro:2.1 The observation of cell morphology After MCF-7 cell line treated with YH in different doses for 48h,apoptosis cells can be seen,the volume of the cells were diminished,karyopycnosis,became round,chromatin became en plaque,agglutinated,broken to pieces,cells changed obviously after 96h later. Apoptosis cells were increased,desquamated from the bottle. As the density from low to high,the cells amount tapered off contrast with the control group.After MDA-MB-231 cell line treated with YH in different doses for 24h,the change not clear. Later the cells shrank as cambiform gradually or like round .The changes got obvious after 72h. The amount of adherent cells were reduced,cells cast off increased,cell's volume was diminution,density increased,cells pyknosis,karyopycnosis,nuclear chromotin pyknosis,cells multiplication suffered restincreased external rotation along with the YH density increase,cell population taper off. Above mentioned cell morphology alter have the same tendency with the MTT result.2.2 The influence of MCF-7 cell line and MDA-MB-231 cell line treated by medicament blood serum on their growth curve.Cell's growth curve was like an"s"shape for each group. Contrast with PSS group,the turns for the influence from low to high was the low-dose YH,the middle-dose YH,the high-dose YH and the CTX group. E2 group could make the MCF-7 cell's growth curve rose obviously. The extent of high-dose YH descended the curve exceeded the CTX group before 72h,after that,the case is on the contrary. 2.3 The lethal effect for medicament blood serum treat MCF-7 cell line and MDA-MB-231 cell line by MTT.MTT assay results : Within the concentration of medicament blood serum of YH Decoction (the low-dose YH,the middle-dose YH and the high-dose YH),YH Decoction could inhibited MCF-7 cell's proliferation to some extent,could obvious inhibited MDA-MB-231 cell's proliferation in vitro,and both had evident dose-time-dependent relation. From 24h to 120h,the YH dose from low to high,the MCF-7 cell inhibition ratio was from 1.47%-11.06% to 6.29%-20.01%,the MDA-MB-231 cell inhibition ratio was from 15.45-20.19% to 21.24%-45.79%.2.4 The result of apoptosis and distribution of cell cycle were examined by flow cytometry in MCF-7 cell line and MDA-MB-231 cell line treated with different doses of medicament blood serum of YH Decoction for 48h. Comparing with control group , the number of MCF-7 cell and MDA-MB-231 cell in YH group in G0/G1 Phase increased gradually , while the number of MCF-7 cells and MDA-MB-231 cells in s phase and G2/M Phase decreased gradually,proliferation index tapered too. But the figures of MCF-7 cells had no statistical significance contrast with the control group (P>0.05). The figures of G0/G1 Phase and PI of middle-dose YH and high-dose YH were had statistical significance obviously compared with control group (P<0.01).The result showed that YH Decoction can induce an arrest of cell cycle in G0/G1 Phase by a dose-dependent manner,but the effect on MDA-MB-231 cell was stronger than it treated with MCF-7 cell.Apoptosis result showed that:The typical hapodiploid apoptotic peak which enhanced gradually with the increasing concentration of YH Decoction. The apoptosis ratio of control group,the low-dose YH group,the middle-dose YH group,the high-dose YH group and the CTX group of MCF-7 cell were 0.68%,2.36%,3.61%,10.75%,the apoptosis ratio of MDA-MB-231 cell were:3.93%,6.86%,16.89%,39.54%. There were statistically significant difference between control group and middle,high-dose group (P<0.01),the inhibition ratio of MDA-MB-231 cell was higher than that of MCF-7 cell. Conclusion:1 YH Decoction had no female sex hormonelike effect,on the contrary it presented a repressed tendency in the growth and development of uterus and ovaries,and had dose-effect relation obviously (negative correlation).2 YH Decoction could inhibit proliferation of MCF-7 cell and MDA-MB-231 cell in vitro by a dose–dependent and time-dependent manner within a certain concentration. It also could induce an arrest of cell in cycle in G0/G1 Phase as well as apoptosis in the MCF-7 cell and the MDA-MB-231 cell. But the effect on MDA-MB-231 cell was stronger than that on MCF-7 cell.3 YH Decoction was really suitable for the breast cancer which type was ER(-).