Effect Of Yangyin Huayu Jiedu Decoction On Proliferation And Apoptosis Of Colon Cancer Cell Under Normoxic And Hypoxic Conditions

ObjectiveProliferation and escape from cell death are signs of tumor formation.This topic is based on the etiology and pathogenesis of heat,deficiency,and stasis of colon cancer in Chinese medicine.To investigate the mechanism of Yangyin Huayu Jiedu Decoction（YYHYJDD）drug-containing serum on colon cancer cell proliferation and apoptosis under normal hypoxia environment,and provide theoretical and practical support for clinical treatment of colon cancer.Methods1.Test for the proliferation and apoptosis of colon cancer cells with serum containing Yangyin Huayu Jiedu Decoction under normoxic conditions:（1）The effect of YYHYJDD drug-containing serum on the proliferation of colon cancer HCT116 cells under normoxic conditions was measured by MTS colorimetry;（2）AOEB staining was used to observe the effect of drug-containing serum on morphological changes of HCT116 cells;（3）Annexin V-FITC/PI double staining and flow cytometry to detect the effects of drug-containing serum on the apoptosis of colon cancer HCT116 cells;（4）Western Blot to detect the expression of B-lymphoma-2（Bcl-2）,B-lymphoma-2-related X protein（Bax）,and pro-proteinase-3（pro-Caspase-3）apoptosis-related proteins in colon cancer cells.2.Detection of colon cancer HCT1116 cell proliferation with different oxygen concentrations:（1）Establish（1% O₂,5% O₂,20% O₂）oxygen concentration models,and use MTS to detect the effect of different oxygen concentrations on the proliferation of colon cancer HCT116 cells.Screen out the strongest and slowest oxygen concentration of HCT116 cell proliferation activity;（2）Detect Proliferation cycle,the apoptosis of HCT116 cells by Annexin V-FITC/PI double staining with the selected oxygen concentration;（3）Detection of HIF-1αm RNA gene expression in colon cancer HCT116 cells under different oxygen concentrations by real-time quantitative PCR.（4）Protein detection The expression of HIF-1αprotein in colon cancer HCT116 cells under two oxygen concentrations.3.Mechanism of YYHYJDD drug-containing serum on colon cancer HCT116 cell proliferation and apoptosis under hypoxic conditions:（1）The effect of YYHYJDD drug-containing serum on the proliferation and apoptosis of colon cancer cells under hypoxic conditions was measured using the MTS colorimetric method;（2）AOEB staining to observe the morphological changes of apoptosis of colon cancer HCT116 cells with drug-containing serum;（3）Annexin V-FITC/PI double staining was used to detect the apoptosis of HCT116 cells by drug-containing serum after flow cytometry;（4）Western Blot was used to detect the expression of hypoxia-inducible factor 1α（HIF-1α）and downstream Bcl-2,Bax,and Pro-Caspase-3 related target proteins.Results1.The test results of Yangyin Huayu Jiedu Decoction on the proliferation and apoptosis of colon cancer cells under normoxic conditions:（1）Compared with the blank serum control group,the activity inhibition of the low-medium high-dose group containing Yangyin Huayu Jiedu Decoction showed a significant trend from 48 h,and the inhibition rate was the highest at 72 h（P<0.05,P<0.01）;（2）Intervening 48 h AOEB stained cells showed apoptotic morphology at different stages;（3）Flow cytometry showed that the low-medium-high-dose medicated serum group interfered with HCT116 cells late apoptosis and the total apoptosis rate was statistically significant compared with the blank control group（P<0.01）;（4）Western Blot showed that the drug-containing serum down-regulated the protein expression of Bcl-2 and pro-Caspase-3 in the high-dose group,the medium-high dose group up-regulated the expression of Bax,and the Bcl-2/Bax ratio down-regulated（P<0.05,P<0.01）.2.Test results of three different oxygen conditions of 1% O₂,5% O₂,and 20% O₂:（1）HCT116 cells colon cancer cells increased from 24 h to 96 h.Compared with the conditions of 1% O₂ and 20% O₂,Colon cancer cells proliferated fastest under 5% O₂（P<0.05,P<0.01）,Compared to 1% O₂ and 5% O₂,Colon cancer proliferation was relatively slow at 20% O₂（P<0.05,P<0.01）;（2）Flow cycle and apoptosis detection:Colon cancer cells with 5% O₂ colon cancer cells in the S phase and G2/M phase ratio are greater than 20% O₂ colon cancer cells（P<0.05,P<0.01）,there is no statistical difference in the apoptosis rate between the two（P<0.05）;（3）Western Blot results,compared with 20% O₂,HIF-1αprotein levels were up-regulated under 5% O₂conditions（P<0.01）.3.Hypoxic test results of Yangyin Huayu Jiedu Decoction’s serum on colon cancer cell proliferation and apoptosis:（1）Compared with the hypoxic blank control group,the inhibition rate of YYHYJDD drug-containing serum on HCT116 cells was the most significant at 48 h and 72 h（P<0.05,P<0.01）;（2）The intervention of AOEB staining drug-containing serum in colon cancer cell apoptosis increased with the concentration of drug-containing serum（P<0.05,P<0.01）;（3）Flow cytometry results:YYHYJDD drug-containing serum can promote the apoptosis of HCT116 cells（P<0.05,P<0.01）;（4）Western Blot results:The medicated serum of the prescription can increase the expression of Bax in the low,medium and high dose groups（P<0.01）,and can decrease the expression of Bcl-2 in the medium dose group（P<0.05）.the high-dose concentration group has a tendency to down-regulate pro-Caspase-3,HIF-1αexpression could be down-regulated in the high-dose concentration group（P<0.05）.Conclusions1.The serum containing Yangyin Huayu Jiedu Decoction-containing serum can inhibit the proliferation of colon cancer HCT116 cells under normoxic conditions,It is possible to play a pro-apoptotic role by down-regulating the Bcl-2/Bax ratio and the expression of the pro-Caspase-3 protein.2.5% O₂ oxygen concentration has the fastest proliferation rate of colon cancer cells.The possible mechanism is to keep colon cancer cell proliferation in S,G2/M phases,and up-regulate the expression of HIF-1αprotein at the molecular level.3.Yangyin Huayu Jiedu Decoction-containing serum can also have a certain time-effectiveness under hypoxic conditions,inhibiting the proliferation of colon cancer HCT116 cells,and change the apoptotic morphology and apoptosis rate,and play a role in promoting apoptosis.It is related to down-regulating the expression of HIF-1a and its downstream related signal molecule Bcl-2/Bax ratio,and the expression of pro-Caspase-3 related protein.