The Study Of Verbascoside On Invasion And Migration Of Hepatocarcinoma Cells Through ERK5 Signaling Pathway

Objective: To explore the effect of Verbascoside on invasion and migration of hepatoma cells and its molecular mechanism.Method: Different concentrations(0μM,40μM,60μM,80μM,100μM,120μM)of Verbascoside were added to HepG2 hepatoma cells for 24 hours,48 hours and 72 hours respectively.The morphological effects of Verbascoside on HepG2 hepatoma cells were observed under inverted microscope.The effects of Verbascoside on hepatoma cell proliferation were detected by CCK8 method,and the next drug experimental concentration was determined.Cell scratch test was used to detect the effect of Verbascoside on the motility of HepG2 hepatocarcinoma cells,Transwell chamber test was used to detect the effect of Verbascoside on the invasion and migration of HepG2 hepatocarcinoma cells,Western blot was used to detect the effect of Verbascoside on the expression of ERK5,p-erk5,matrix metalloproteinases MMP-9 and MMP-2.Result:1.The Liver cancer cell morphological changes: With the increase of the concentration of Verbascoside,the number of HepG2 cells decreased significantly,the morphology became round gradually,and the adhesiveness became poor.More floating necrotic cells could be seen,the connections between cells were sparse,the boundary was also unclear,and some cells with incomplete morphology were also seen.2.CCK8: With the increase of Verbascoside concentration and time,the proliferation was inhibited in varying degrees,and showed a certain concentration and time-dependent tolerance.The IC50 was about 98μM,so the drug concentrations were60μM and 100μM in the follow-up experiments.3.The results of wound healing:Verbascoside has a visibly restrain effect on the motility of Human liver cancer HepG2 cells,which is positively correlated with the increase of concentration.3.Cell scratch test: Compared with the blank control group,the experimentalgroup(60 μ m,100 μ m)could inhibit the motility of HepG2 cells(P < 0.01).Compared with the positive control group,60 μ M group had statistical difference(P< 0.05),100 μ M group had no significant difference(P > 0.05).4.Transwell chamber experiments:a.Impact on migration:The number of cell migration was 435.80 ± 32.13,241.07 ± 23.64,150.50 ± 6.04 and 99.82 ± 9.01 in blank control group,experimental group(60 μ m,100 μ m)and positive control group,respectively.Compared with the blank control group,the number of liver cancer cells passing through the small chamber in the experimental group was significantly reduced(P < 0.01).Compared with the positive control group,there was a difference(P < 0.05)in the 60 μ M group and a significant change(P > 0.05)in the 100 μ M group;B.the impact on invasion: the number of invasion cells in the blank control group,the experimental group(60 μ m,100 μ m)and the positive control group were419.80 ± 19.1,211.07 ± 15.64,respectively 103.2 ± 16.04 and 88.50 ± 11.5.Among them,compared with the blank control group,the ability of the experimental group to pass through the ependyma was significantly weakened(P < 0.01),compared with the positive control group,the 60 μ M group had a difference(P < 0.05),and the 100 μ M group had no significant change(P > 0.05).5.Western Blot: Compared with the blank control group,the expression of p-erk5,MMP-9(P < 0.01)and MMP-2(P < 0.05)in the experimental group decreased significantly with the increase of drug concentration.Compared with the positive control group,the expression of 60 μ M group was statistically significant(P< 0.05),while that of 100 μ M group was not statistically significant(P > 0.05).The expression of ERK5 protein had no significant difference between groups(P > 0.05).Conclusion:1.Verbascoside can inhibit the proliferation of HepG2 cells in a concentration and time-dependent manner.2.Verbascoside inhibits the invasion and migration of HepG2 cells through ERK5 signaling pathway.