Compound Xishu Granules Inhibit Proliferation of Hepatocellular Carcinoma Cells by Regulating Ferroptosis
Objective:To study the mechanism of compound Xishu granules(CXG)in inhibiting the proliferation of hepatocellular carcinoma cells by regulating ferroptosis.Methods:The transplanted tumor model of human Huh7 was established with nude mice and the successfully modeled mice were randomized into model,Fufang Banmao(0.21 g·kg-1),low-dose(1.87 g·kg-1)CXG,medium-dose(3.74 g·kg-1)CXG,and high-dose(7.49 g·kg-1)CXG groups.Mice were administrated with drinking water or CXG for 28 days,and the body weight and tumor volume were measured every 4 days.Hematoxylin-eosin staining was employed to observe the histopathological changes of tumors.The cell-counting kit-8(CCK-8)was used to examine the survival rate of Huh7 cells treated with different concentrations(0,31.25,62.5,125,250,500,1 000 mg·L-1)of CXG for 24 h and 48 h.CA-AM,DCFH-DA,and C11-BODIPY581/591 fluorescent probes were used to determine the intracellular levels of ferrous ion(Fe2+),reactive oxygen species(ROS),and lipid peroxide(LPO),respectively.The colorimetric method was employed to measure the levels of glutathione(GSH)and superoxide dismutase(SOD).Western blot was employed to determine the protein levels of glutathione peroxidase 4(GPX4),transferrin receptor 1(TFR1),and ferritin heavy chain 1(FTH1),respectively.Results:In the animal experiment,compared with the model group,the drug treatment groups showed reductions in the tumor volume from day 12(P＜0.01).After treatment,the Fufang Banmao and low-,medium-,and high-dose CXG groups had lower tumor volume,relative tumor volume,and tumor weight than the model group(P＜0.05),with tumor inhibition rates of 48.99％,79.93％,91.38％,and 97.36％,respectively.Moreover,the CXG groups had lower tumor volume and relative tumor volume(P＜0.05 in all the three dose groups)and lower tumor weight(P＜0.05 in medium-dose and high-dose groups)than the Fufang Banmao group.Compared with the model group,the drug treatment groups showed reduced number of tumor cells,necrotic foci with karyopyknosis,nuclear fragmentation,and nucleolysis,and the high-dose CXG group showed an increase in the proportion of interstitial fibroblasts.In the cell experiment,compared with the blank group,CXG reduced the survival rate of Huh7 cells in a dose-dependent manner after incubation for 24 h and 48 h(P＜0.05).Compared with the blank group,the RSL3 group and the low-,medium-,and high-dose CXG groups showed a decrease in the relative fluorescence intensity of CA-AM and increases in the fluorescence intensity of DCFH-DA and fluorescence ratio of C11-BODIPY581/591,which indicated elevations in the levels of Fe2+(P＜0.01),ROS(P＜0.05),and LPO(P＜0.01),respectively.Compared with the blank group,the RSL3 and low-,medium-,and high-dose CXG groups showed lowered levels of GSH and SOD(P＜0.05).In addition,the RSL3 group and the medium-and high-dose CXG groups showed down-regulated expression of GPX4 and FTH1(P＜0.05),and the low-and high-dose CXG groups presented up-regulated expression of TFR1(P＜0.05).Conclusion:CXG suppresses the proliferation of hepatocellular carcinoma cells by inducing ferroptosis via downregulating the GSH-GPX4 signaling axis and increasing intracellular Fe2+and LPO levels.
compound Xishu granuleshepatocellular carcinomaferroptosisproliferationtraditional Chinese medicine
NETL
NSTL
万方数据
