Effect Of Diallyl Trisulfide On Bcl-2 Protein Expression Of Hepatic Stellate Cells In Rat

Objective: To investigate the effects of diallyl trisulfide(DATS)on cell proliferation of rat hepatic stellate cells(HSCs)and the expression of Bcl-2 and to preliminarily explore the relevant apoptosis inducing mechanisms of DATS.Methods: Cells were cultured in DMEM containing 10% fetal bovine serum(FBS),1×105IU/L penicillin,and 100 mg/L streptomycin at 37℃ in a humidified 5% CO2 incubator.The HSC-T6 cells were treated with DATS 25,50,100 and 200 μmol/L for 0,6,12,24 and 48 h in vitro,respectively.Changes of morphological and number of cells were determined by inverted phase contrast microscope and transmission electron microscope(TEM).Cell proliferations were determined using MTS assay.The relative amount of Bcl-2 mRNA were detected by realtime quantitative polymerase chain reaction(qRT-PCR).The relative expression levels of apoptosis associated protein Bcl-2,Bax and cleaved caspase-3 were detected by western blotting analysis.All qualitative data were expressed as mean ±standard deviation(SD),and were statistically analyzed using one-way analysis of variance(ANOVA)and least significant difference(LSD)test to determine significant differences between groups,with SPSS17.0 software.The differences were marked as statistical significance if P value was less than 0.05 or 0.01.Results:1.The effect of DATS on HSC-T6 cell morphology.After being incubated with DATS with different concentrations for 48 h,HSCs in blank control group were denser and showed a long fusiform shape while HSCs in DATS group showed a smaller cell density and volume,polygonal pseudopodia were reduced or even disappeared,and HSCs showed a ellipse shape with the increase of drug concentrations.After being incubated with DATS 100 μmol/L for some times,both the number and volume of HSCs were less and polygonal pseudopodia were reduced as the extension of time.2.The inhibiting effects of DATS on cell proliferation.The survival rate of HSCs was restrained in a dose-and time-dependent manner after being treated with different concentrations of DATS for various time intervals.The survival rate of HSCs were significantly reduced when they were incubated with DATS 25 μmol/L for 12、24 and 48 h,respectively(P<0.01).An apparent decline in HSCs survival rate were found when they were treated with DATS 50,100 and 200 μmol/L for 6,12,24 and 48 h,respectively(P<0.05 or P<0.01).3.The ultrastructure changes of HSC-T6.After being treated with DATS 100 μmol/L for 12 h,HSCs in blank control group showed a complete structure with even distribution of nuclear chromatin in nucleus whlie HSCs in DATS groups showed a structure disturbance ofkaryopyknosis,nuclear chromatin pyknosis and nuclei karorrhexis even apoptotic body were found.4.The effects of DATS on Bcl-2 gene expressions of HSC-T6.The relative amounts of Bcl-2gene were down-regulated gradually with the increased of DATS concentrations after incubated with DATS 25,50,100 and 200 μmol/L for 48 h.Relative gene expressions of Bcl-2 were significantly down-regulated in drug groups compared with control group(P<0.01).5.The effects of DATS on expressions of protein Bcl-2,Bax and cleaved caspase-3 in HSC-T6.Western blotting analysis of Bcl-2,Bax and cleaved caspase-3 in HSCs treated with DATS 25,50,100 and 200 μmol/L for 48 h showed that levels of Bax,cleaved caspase-3 and Bax/Bcl-2 ratio were gradually increased while Bcl-2 was gradually decreased with the elevation of drug concentrations.The expression of Bax and cleaved caspase-3 were up-regulated with the increase of DATS concentrations while being treated with DATS 25,50,100 and 200 μmol/L for 48 h(P<0.05 or P<0.01).The expression of Bcl-2 was gradually down-regulated and Bax/Bcl-2 ratio gradually increased with the increase of DATS concentrations while being treated with DATS 50,100 and 200 μmol/L for 48 h(P<0.05 or P<0.01).Conclusion:1.DATS could significantly inhibit HSC-T6 proliferation in a dose-and time-dependent manner.2.DATS could promote the apoptosis of HSC-T6 by down-regulating the protein expressions of Bcl-2 and up-regulating the protein expressions of Bax and cleaved caspase-3,which might be involved in its anti-fibrosis mechanism.