Expertmental Study On The Effect Of Ruanganyin On Human Hepatoma Cells HepG2 And Hu-7

ObjectiveRuanganyin was applied to human hepatoma HepG2 and hu-7 cells to observe the inhibition rate of cell proliferation,the apoptotic effect of the cells and its effect on the migration of hepatoma cells,and to observe the expression of apoptosis related proteins,as well as p-c-raf,p-c-mek,p-c-myc,p-c-fos hepatoma gene proteins regulated by the classical MAPK pathway.To provide a theoretical basis for the treatment of liver cancer(HCC).MethodRuanganyin were probed using CCK-8 assay with drug containing serum at different concentrations(50%,40%,30%,20%,10%,5%,2.5%)for various times(24 h,48 h and 72 h)to determine the proliferation inhibition rate of the compounds in human HCC cells(HepG2,hu-7)and to find out the optimal inhibition rate group by contrast;Setting groupings: liver cancer HepG2,hu-7 cell group,normal serum control group,drug serum group with optimal inhibition rate(50%)of Ruanganyin,and positive inhibitor erlotinib group;Flow cytometry was performed to evaluate the effect of the optimal inhibitory concentration(50%)on the cell cycle of two different cell lines(HepG2,hu-7)treated for 48 h with hepatoma cells as previously described;Transwell migration assay was performed to detect the migration ability of human HCC cells(HepG2,hu-7)treated with the optimal concentration of a TCM compound Ruanganyin WB was performed to evaluate the expression results of Caspase-3,PARP apoptotic proteins in the cells of each group and to detect the expression of p-c-raf,p-c-mek,p-c-myc,p-c-fos,genes regulated by MAPK pathway in HCC cells.ResultThe results of CCK-8 assay suggested that the compound Ruanganyin inhibited the proliferation of human HCC cells(HepG2,hu-7)in a concentration-and time-dependent manner;The results of flow cycle experiments showed that Ruanganyin effectively interfered with DNA replication in the S-phase in hepatoma HepG2,hu-7cell lines,while G2 / M phase appeared retarded and blocked division and replication in hepatoma cells;Flow cytometric apoptosis assays suggested that Ruanganyin could promote the hepatoma HepG2 and hu-7 cell lines to undergo necroptosis,which initiated the early apoptotic program and markedly accelerated the late apoptotic program in hepatoma HepG2 and hu-7 cells;The results of Transwell migration assay showed that the migration ability of the cells was significantly inhibited by the compound Ruanganyin drug serum group compared with the liver cancer HepG2 and hu-7 cell groups(P < 0.05);The results of WB experiment showed that the optical density values of proteins related to apoptosis of Caspase-3 and PARP were continuously enhanced in the liver cancer cell group,normal serum control group,Ruanganyin containing serum group,positive inhibitor group in turn(P < 0.05);The expressions of proto oncogene proteins p-c-raf,p-c-mek,p-c-myc,p-c-fos mediated by MAPK pathway in WB results were constantly decreased in liver cancer cells group,normal serum control group,soft drinking(rge)medicated serum group,positive inhibitor group in turn(P < 0.05).Conclusion1.The traditional Chinese medicine Ruangan Yin(RGY)has a certain degree of inhibitory effect on the proliferation and replication of HepG2 and Hu-7 cell lines in liver cancer;And it may promote the initiation of the apoptosis program of liver cancer cells,leading to cell apoptosis;Cell experiments suggest that Ruangan Yin can block the migration and metastasis of HepG2 and Hu-7 cells in liver cancer,and may play a role in inhibiting tumor cell metastasis and regeneration.2.The traditional Chinese medicine compound Ruanganyin may activate MAPK signal channel by regulating the expression of related oncogenes p-c-raf,p-c-mek,p-c-myc and p-c-fos,so as to inhibit tumor proliferation,replication and metastasis.