ABSTRACT
Objective To establish the Nifedipine-induced liver cell damage model,and to investigate the cellular or molecular mechanism for children's liver cell damage.Methods The HepG2 cells were utilized to establish liver cell damage models.The optimal concentration and the optimal pretreatment time of Nifedipine-induced liver cell injury were confirmed.Western blot and real-time PCR(RT-PCR)were used to check the alteration in proteins and mRNAs level of the liver function-associated classic markers,which contained alkaline phosphatase(ALP),aspartate amino transferase(AST),glutamyl transpeptidase(γ-GT)and alanine aminotransferase(ALT).Additionally,flow cytometry(FCM),colony formation assay(CFA)and cell wound healing assay(CWHA)were utilized to check the effect of Nifedipine on the cell cycle progression and proliferation of HepG2.Results (1)The optimal concentration of Nifedipine was 20 mg/L and the optimal treatment period was 21 days for liver damage.(2)Western blot:intracellular ALT protein content after Nifedipine group was less than that of control group,while the protein levels of AST,γ-GT and ALP in the culture medium after Nifedipine addition(3.55 ± 0.05,4.91 ± 0.055,3.51 ± 0.05,3.08 ± 0.08) were higher than those of control group(0.96 ± 0.02,1.03 ± 0.02,1.00 ± 0.05,0.90 ± 0.13),and the differences were all significant(t = -85.695,-117.582,-47.371,-33.260,all P<0.05).(3)The findings of RT-PCR showed that the mRNA levels of intracellular ALT,γ-GT and ALP in Nifedipine group(0.26 ± 0.02,0.05 ± 0.04, 0.05 ± 0.02)were lower than those of control group(1.13 ± 0.21,0.94 ± 0.10,1.03 ± 0.06),and the differences were all significant(t=7.233,127.436,25.687,all P<0.05).However,the mRNAs levels in purified culture me-dium in Nifedipine group(5.95 ± 0.05,3.13 ± 0.10,3.32 ± 0.08)were higher than those in control group(1.01 ± 0.08,1.00 ± 0.05,1.00 ± 0.05),and the differences were all significant(t= -92.339,-31.250,-43.007,all P<0.05).(4)The portion of G0/G1 phase in Nifedipine group[(84.09 ± 0.43)%]was more than that of control group[(30.93 ± 0.32)%],which had statistical significance(t=173.084,P=0.000).(5)In contrast with control group,the colony formation of cells in Nifedipine group declined from(97.10 ± 1.17)% to(38.56 ± 1.51)%(t=92.088,P=0.000)and the migration rate of cells wound healing was(56.37 ± 2.06)%,(25.00 ± 1.71)% sepa-rately(t=20.285,P=0.000).Conclusion Nifedipine may promote children's liver injury through regulating cell cycle related proteins.