The Anti-hepatoma Effect Of Hydroxysafflor Yellow A And Its Mechanism

Objective:To investigate the effects of hydroxysafflor yellow A and PI3K/Akt inhibitor LY294002 on the proliferation,migration and apoptosis of hepatoma cells,and to determine the possible role and mechanism through the PI3K/Akt signaling pathway.Methods:1.The effects of HSYA and PI3K inhibitor LY294002 on the proliferation of human hepatoma cell HepG2,Hep3B and SMMC7721 were measured by CCK-8 assay.In order to study the role of HSYA in carcinoma cells the proliferative capacity and migration ability of tumor cells,the colony formation assay and the scratch assay test were studied.Flow cytometry was used to detect the effect of HSYA on the apoptosis of HepG2 in human hepatoma cells.Western blot was used to detect the expression of PI3K/Akt signaling pathway related proteins.2.The model of subcutaneous transplantation of nude mice was established by using HepG2 cells.Nude mice were randomly divided into 4 groups:control group,HSYA group(intraperitoneal injection 2 times,0.35 mg/kg),group LY294002(3 times a week,intraperitoneal injection of 25 mg/kg)and group HSYA+LY294002(HSYA daily intraperitoneal injection for 2 times,0.35 mg/kg;3,LY294002 weekly intraperitoneal injection of 25 mg/kg).The state of the nude mice was observed daily,and the body weight of nude mice was measured every 3 days and the volume curve of the tumor was recorded.After 3 weeks,the nude mice were killed and the tumors were weighed.Morphological observation of tumor tissues were performed by HE staining.The expression of VEGF,Ki67,p-Akt and Cleaved Caspase-3 proteins in tumor tissues were detected by immunohistochemistry method.Results:1.HSYA,with a concentration of 100 mol/L,could inhibit the proliferation of HepG2,Hep3B and SMMC7721 in human liver cancer(P<0.05).LY294002 could inhibit the proliferation of human hepatoma cells(P<0.01),and had a dose and time dependence.HSYA can reduce the number of hepatoma cells forming clones(P<0.05)and reduce the migration ability of tumor cells(P<0.05),and HSYA has more obvious effect on HepG2 cells.The results of flow cytometry showed that the apoptotic rates in control group,HSYA group,LY294002 group and combination group were(7.6 ±1.6)%?(9±3.0)%?(17.3±3.8)%?(31.2±3.9)%,respectively,and the difference was statistically significant(P<0.01).HSYA single drug can reduce the level of phosphorylation of Akt and inhibit the expression of MMP-2 and Caspase-3 protein,and the effect of combined LY294002 is more obvious(P<0.01).2.The transplanted tumor model of human hepatocellular carcinoma cell HepG2 was established.Compared with the control group,the tumor volume and weight of the nude mice in the experimental group were significantly less than those in the control group(P<0.05).The tumor suppressor rate of group HSYA,PI3K inhibitor group and combined application group were(33.94:± 2.03)%,(45.62±7.34)%and(58.31±12.15)%respectively.After HE staining,compared with the control group,under the light microscope,cell atypia in group HSYA was reduced,and the number of tumor cells in combination group was significantly reduced,and some regular round cells were observed.Immunohistochemical results showed that compared with the control group,the expression level of Ki67,VEGF and p-Akt protein in group HSYA decreased,and the expression of Cleaved Caspase-3 increased(P<0.05).The difference in combination group was more significant(P<0.01),the difference was statistically significant.Conclusion:1.Hydroxysafflor yellow A can inhibit cell proliferation,migration,apoptosis through the PI3K/Akt pathway.2.Human hepatocellular carcinoma HepG2 cells can be established by subcutaneous injection,HSYA can inhibit the proliferation,angiogenesis and apoptosis of the transplanted tumor cells.