Experimental Study Of Extract Of Hedyotis Diffusa Willd To Enhance The Radiosensitivity Of Liver Cancer Cells

Objective:The proliferation and apoptosis of human liver cancer cell lines were observed by culturing human liver cancer cell lines Huh-7 and SMMC7721 in vitro,and the effect of Extract of Hedyotis Diffusa Willd(EHDW)on the proliferation and apoptosis of human liver cancer cell lines,as well as the effect of EHDW at different concentration gradients on the radiosensitivity of human liver cancer cell lines.Methods:Human liver cancer Huh-7 and SMMC-7721 cells in logarithmic growth phase were selected as the subjects of this experiment,and the cells were treated with various concentration gradients(0,20,40,60 and 80 μg/ml)of EHDW individually for 24,48 and 72 hr,and the cell survival and survival curves were measured.Cell viability was observed,and survival curves were depicted after 24,48,and 72hr of individual treatment of cells with six dose gradients of 0,2,4,6,8,and 10Gy of X-rays.The optimal concentration gradient of EHDW and the optimal radiation dose were determined,respectively.In this experimental study,we proposed to randomly divide two kinds of human liver cancer cell lines,Huh-7 and SMMC-7721,into blank control group,radiation alone group(IR group),EHDW alone group(EHDW group),and radiation+EHDW group(IR+EHDW group),and observe the effect of EHDW on h human liver cancer cells Huh-7 and SMMC-7721 by in vitro culture experiment using cell survival fraction(SF)as the index.SMMC7721 by in vitro culture experiments to observe the effects of EHDW on the proliferation and radiosensitization effects of human liver cancer cells Huh-7 and SMMC-7721;GraphPad Prism V9.0 software was used to fit the survival curves of human liver cancer cells using one-click multi-target model and L-Q model,respectively,and Sigma Plot V14.5 software was used to calculate the radiobiological parameters of each group of cells,including extrapolation number(N),mean lethal dose(D0),quasi-threshold dose(Dq),cell survival fraction(SF2)at 2 Gy dose irradiation and radiotherapy sensitization ratio(SER).The effects of EHDW on apoptosis rate and cell cycle of human liver cancer Huh-7 and SMMC-7721 cells were analyzed by flow cytometry.Results:1.The linear regression analysis of the number of human liver cancer cells and the mean OD value was performed,and the linear equations were obtained as Y=0.56-0.1X and Y=0.58-0.1X,respectively.The coefficients of determination were 0.964(Huh-7)and 0.983(SMMC-7721),respectively,and the goodness of fit was considered significant for the linear relationship between the mean OD value and the number of human liver cancer cells.2.In the concentration range of 0-80 μg/ml,the growth inhibition of human liver cancer Huh-7 and SMMC-7721 cells by EHDW was more and more obvious with increasing concentrations and showed a significant linear relationship(dose-dependent relationship)with a statistically significant growth inhibition rate(F=40.427,p=0.000004<0.01;F=38.920,p=0.000005<0.01).When EHDW acts on human liver cancer Huh-7 cells and SMMC-7721 cells after 48hr to reach the half lethality,its IC50 is about 40μg/ml,which is the optimal tumor suppressive concentration.EHDW can block the cell cycle of human liver cancer Huh-7 and SMMC-7721 cells,with an increase in G1 phase cells.3.In the radiation dose range of 0-10Gy,human survival of human liver cancer Huh7 and SMMC-7721 cells decreased significantly with increasing irradiation dose intensity and showed a significant linear relationship(time-and dosedependent)with statistically significant inhibition rate(p<0.01).Since there was no statistically significant difference between the cell activity after 10Gy and 8Gy radiation(p>0.05),no further higher dose radiation experiments were performed,so 8Gy was selected as the optimal radiation dose.The activities of human liver cancer Huh-7 and SMMC-7721 were 21.10%and 45.58%,respectively,after 5 min of radiation by 8 Gy radiation dose.4.The results of flow cytometry showed that the apoptosis rates of Huh-7 cells in the control,IR,EHDW and IR+EHDW groups were 20.75%±1.48%,54.27%±1.58%,and the apoptosis rates of SMMC-7721 cells in the control,IR,EHDW and IR+EHDW groups were 24.69%± 2.18%,35.95%± 1.19%,52.66%± 1.04%and 77.77%± 0.89%,respectively,with p-values<0.05,all p-values were<0.05 when compared between groups.5.After treatment with EHDW groups at IC50 concentration for 12hr,2-8Gy radiation was performed,respectively,and the cell clone formation rates were calculated afterwards for Huh-7 cells:66.13%,35.73%,21.63%,10.09%and SMMC-7721 cells:63.68%,38.92%,26.16%,and 14.82%.The SER values of human liver cancer Huh-7 and SMMC-7721 cells in IR+EHDW group were 1.324 and 1.281,respectively,under the condition of 8Gy radiation dose.It is evident that EHDW can increase the radiosensitivity of human liver cancer Huh-7 and SMMC-7721 cells.Conclusions:1.In vitro experiments,EHDW extract and radiation at a certain concentration range could inhibit the growth of human liver cancer Huh-7 and SMMC-7721 cells;2.The best tumor-inhibiting concentration IC50 of EHDW was 40 μg/ml and the best radiation dose was 8 Gy;3.The inhibitory effects of EHDW and radiation on human liver cancer cells were mainly through the induction of apoptosis pathway;4.EHDW had a blocking effect on the cell cycle of human liver cancer Huh-7 and SMMC-7721 cells,with an increase in G1 phase cells predominantly;5.EHDW improves the radio sensitivity of human liver cancer cells.