Intervention Mechanism Of Modified YangHe Decoction Drug-containing Serum On Breast Caancer MCF-7 Cell Via PI3K/Akt Signal Pathway

Objective:To observe the influence of proliferation,cell cycle,apoptosis and PI3K/Akt signaling pathway related gene and protein expression by Modified YangHe Decoction serum acting on the breast cancer MCF-7 cell,Preliminary discussion on its intervention mechanism of PI3K/Akt signal pathway,lay a foundation of research for the future to explored yang-warming and reduce phlegm method specific mechanism from the angle of cellular level,broaden its application space.Methods:Conventional cell culture and batches of Breast cancer MCF-7 cells,using different concentrations of Modified YangHe Decoction serum to intervene.○1Observed under inverted microscope each drug-containing serum on in vitro cultured breast cancer MCF-7 cell growth condition and form;○2 MTT method to detect proliferation of Modified YangHe Decoction drug-containing serum on MCF-7 cells;○3 FCM detect Modified YangHe Decoction drug-containing serum of MCF-7 cell cycle and apoptosis rate of breast cancer;○4 Scratches and Transwell cell attacks were used to detect the effects of cell invasion metastases;○5 RT-PCR to detect Modified YangHe Decoction drug-containing serum on breast cancer MCF-7 cell PI3K,Akt,caspase-3,CyclinD₁,Bcl-2 and BaxmRNA expression;○6 Western Blot to detect the experession of PI3K,Akt,p-AKT,MMP-2 and TIMP-2 protein.Results:① Inverted microscope visible groups of cells are different degree of morphological change;Modified YangHe Decoction drug-containing serum with high dose group was obviously inhibit the growth of breast cancer MCF-7 cell;② The MTT method shows that,Cell inhibition increased in all groups;③ According to the results of FCM,Modified YangHe Decoction drug-containing serum on breast cancer MCF-7 cell after 48 h,middle and high dose group of G₀/G₁stage of cell DNA content decreased significantly（P<0.05）,S phase cell DNA content were significantly increased（P<0.05）;At the same time,early apoptosis rate and late apoptosis rate were higher than the negative control group（P<0.05）;④ The results of the attack showed that the breast cancer MCF-7 cells had more membranes and were more aggressive in vitro.After the intervention of the serum containing the serum,the effect of the effect on extracellular invasion was significant.⑤ The results of PCR shows that compared with negative control group,each dose group of cells PI3K,Akt caspase-3,Bax expression values were significantly increased（P<0.05）.On the contrary,the Bcl-2,Cyclin D₁mRNA expression decreased,significant difference between each dose groups（P<0.05）;⑥ According to the results of Western blot,different concentrations of Modified YangHe Decoction drug-containing serum on MCF-7 in breast cancer cells after 24 h,the expression of Akt have no significant change.With the increase of drug concentration,the expression of P13K,P-Akt are decreased,the statistical analysis was carried out on the gray value in the band,compared with the blank group were significantly different（P<0.05）.The high dose group of PI3K,P-Akt content compared with dose group were significantly lower（P<0.05）;At the same time,each dose group of Chinese medicine compound can obviously lower the expression of MMP-2protein（P<0.05）,raising TIMP-2 protein expression（P<0.05）.Conclusion:① Modified YangHe Decoction drug-containing serum can inhibit the growth of breast cancer MCF-7 cell activity,make its different levels of morphological change;② Modified YangHe Decoction drug-containing serum can induce the breast cancer MCF-7 cell had different degrees of apoptosis;③ Modified YangHe Decoction drug-containing serum can affect the cell cycle,by blocking cyclinD1 into the nucleus,hence block cell cycle arrest in S phase;④ Modified YangHe Decoction drug-containing serum can inhibition of cell invasion and metastasis,be through the regulation of MMP-2/TIMP-2 balance is resisting the invasion and metastasis of breast cancer cells;⑤ The treatment breast cancer resistance mechanism of Modified YangHe Decoction drug-containing serum may be through adjusting the PI3K/Akt signaling pathway and downstream related factors of expression.In order to prevent the proliferation,influence the cell cycle and promote apoptotic and anti-invasion transfer ability.