Effect Of Black Cohosh Extract On Proliferation And Apoptosis Of Human Hepatocellular Carcinoma Cell HepG2

Objective:Hepatocellular carcinoma(HCC)has the sixth highest incidence of cancer in the world and the fourth highest number of cancer deaths.Currently,surgical resection is the first choice of treatment for early HCC,but most patients reach the late stage of HCC at the initial diagnosis,with low surgical resection rate,high postoperative recurrence rate and poor prognosis.Other palliative treatments,such as hepatic arterial chemotherapy-infusion therapy,were not effective.Therefore,it is urgent to develop drugs for HCC that can effectively improve survival,and it is emphasized that the drug-related pathways and target proteins can provide new solutions for the prevention and treatment of liver cancer.The purpose of this study was to preliminarily explore the effects of black cohosh extract on the proliferation and apoptosis of HepG2 cell line and human normal liver cell line HL7702 and the related mechanisms,and to provide new targeted drugs for regulating the apoptosis gene expression of liver cancer cells.Methods:1.Cytotoxicity detection:cck-8 assay was used to observe the inhibitory effect of different concentrations of black cohosh extracts(0.110,20,30,40,50 μg/ml)on HepG2 cell proliferation,and IC50 was determined.2.DAPI staining was used to observe the morphological changes of BepG2 and HL7702 cells in the control group(black cohosh drug concentration 0 μg/ml)and the intervention group(10,20.32μg/ml)with different concentrations of black cohosh extract after treatment for 48h.3.Annexin v-fitc/PI double staining treatment and flow cytometry analysis were performed to detect the apoptotic cell ratios of HepG2 and H17702 cell lines in the control group and the black cohosh extract treatment group.4.Western blot analysis of the expressions of apoptosis-related proteins caspase-3,caspase-8 and caspase-9 in each group.Results:The cell proliferation inhibition rates of 0,10,20,30,40,50 μg/ml groups were 0%,(12.51±9.31)%,(27.85 ±1.51)%,(53.20±5.55)%,(67.42±3.54)%,(81.43±0.73)%,respectively.The difference between the groups was statistically significant(P<0.05),and the inhibition rate of cell proliferation was gradually increased with the increase of drug concentration in the 10～50μg/ml groups(P<0.05).The IC50 of black cohosh extract in HepG2 cell lines was 32.010 μg/ml.In the DAPI experiment,the morphological changes of HL7702 and HepG2 cells in the groups treated with 10,20,32 μg/ml black cosh were observed intuitively:The early apoptotic cells showed nuclear concentration and staining deepening,or the chromatin was concentrated in the side of nuclear membrane in a crescent shape.The late apoptotic cells showed nuclear fragmentation into round bodies of different sizes,which were surrounded by cell membrane and formed apoptotic bodies.Flow cytometry indicated that in human normal hepatocyte HL7702,the apoptotic cell ratios in groups of 0,10,20 and 32μg/ml were:(6.62±1.27)%,(12.35±0.59)%,(11.59±0.88)%,(10.34±0.12)%,respectively.The percentage of apoptosis in 10,20,32μg/ml black cohosh extract group was up-regulated compared with that in 0 μg/ml group(P<0.05).In human hepatocellular carcinoma cell line HepG2,the ratios of apoptotic cells in the extracts of 0,10,20,32 μg/ml black cohosh were:(5.17±0.22)%,(20.74±0.38)%,(27.44±1.23)%,(43.83±2.53)%,respectively.The extract of black cohosh could effectively induce apoptosis,and the percentage of apoptotic cells increased with the increase of the dosage concentration in the concentration of 10～32 g/ml(P<0.05).In addition,the expression of Cleaved caspase-3 protein in HepG2 cells was up-regulated by 20μg/ml and 32 μg/ml concentrations of black cohosh extract compared with the group of 0μg/ml.The expression of caspase-8 protein in HepG2 cells was increased in 32μg/ml group compared with 0 μg/ml group(P<0.05).Conclisions:1.Extracts of black cohosh at different concentrations can inhibit the proliferation of HepG2 in liver cancer cells;2.Extract of black cohosh can induce apoptosis in HepG2 cell line,but normal hepatocyte HL7702 cell line can be induced into apoptosis at the same concentration.3.The effect of black cohosh extract on HepG2 apoptosis in hepatocellular carcinoma cells is at least partially dependent on the exogenous death receptor pathway.