Paracrine Effect And Mechanism Of Hepatocellular Carcinoma Cell On LX-2 Cell Proliferation And Collagen Secretion,in Vitro

Objective: To investigate the paracrine effect and mechanism of hepatocellular carcinoma cell on LX-2 cell proliferation and collagen secretion.Methods: Human hepatocellular carcinoma cell line HepG2 was cultured and the conditioned medium was collected after 3 days.Human activated hepatic stellate cell line LX-2 was exposed to culture medium containing different concentrations of conditioned medium(5%,10%,20%,30% and 40%).The cell proliferation rate was determined by the MTT colorimetric assay.The morphological changes were observed by the inverted microscope.The intracellular collagen in LX-2 cells was confirmed by Sirius red staining and Masson staining.The hydroxyproline content in LX-2 culture medium was quantified.The protein levels were detected by Western blotting.The secretion levels of cytokines including IL-6,TNF-? and TGF-?1 in HepG2 conditioned medium and LX-2 culture medium were determined by enzyme-linked immunosorbent assay.Results:(1)HepG2 conditioned medium promoted the proliferation of LX-2 cells.After 24 h,the proliferation rate of LX-2 cells in 40% conditioned medium group was higher than the other groups statistically(P<0.05).After 48 h,the proliferation rate of LX-2 cells in 20% group and 40% group were higher than the control group,with statistically significant(P<0.05).After 72 h,the proliferation rate of LX-2 cells in 20% and 40% conditioned medium group was higher than the control group,the 5% group and the 10% group,with statistically significant(P<0.05).The morphological changes of LX-2 cells were observed under the inverted microscope after 72 h.LX-2 cells in the control group distributed sparsely,while the 20% and 40% groups showed a more densely distribution and a tendency of multilayer growth.(2)HepG2 conditioned medium promoted the collagen synthesis and secretion of LX-2 cells.The Masson staining and Sirius red staining results showed that the collagen staining in the cytoplasm was increased with the increment of the conditioned medium.The hydroxyproline content in LX-2 cell culture medium was increased in conditioned medium treated group in a time-and concentration-dependent manner(P<0.05).After 24 h,there are statistically significant differences between 40% conditioned medium group and the other groups(P<0.05).After 48 h,the hydroxyproline content in the 20% group and 40% group was different from the other groups with statistically significant(P<0.05).After 72 h,the hydroxyproline content in conditioned medium intervention groups was higher than the control group statistically(P<0.05).The hydroxyproline content of 40% conditioned medium group was statistically higher than the other groups(P<0.05).The hydroxyproline content of 20% conditioned medium group was higher than the control group,the 5% group and the 10% group with statistically significant(P<0.05).(3)The molecular mechanisms of the effects of HepG2 conditioned medium on LX-2 cell proliferation and collagen secretion.The protein levels of Col-1? and ?-SMA,two markers of myofibroblast,were increased after added conditioned medium with statistically significant(P<0.05).The expression level of p-P38,p-AKT and BCL-2 was statistically significant increased compared with the control group(P<0.05).There was an increasing trend of the protein levels of P38?PI3K and AKT after added conditioned medium.Differences were observed in 20% group and 40% group when compared with the control group statistically(P<0.05).The cytokine expression of TNF-? and TGF-?1 in the conditioned medium added group was higher than the control group with statistically significant(P<0.05).The IL-6 expression in 10%,20% and 40% conditioned medium group was higher than the control group statistically(P<0.05).Conclusion:(1)The paracrine effect of human hepatocellular carcinoma cell HepG2 could promote the proliferation and induce excessive collagen secretion of human activated hepatic stellate cell LX-2.(2)Paracrine cytokines of human hepatocellular carcinoma cell HepG2 including TNF-?,TGF-?1 and IL-6 may promote the proliferation and induce excessive collagen secretion of human activated hepatic stellate cell LX-2 through the activation of TGF-?/Smad3 pathway,P38 MAPK pathway and PI3K/AKT pathway.