Effect And Significance Of Lipopolysaccharide On IL-6/STAT3 Signal Pathway In Human Intrahepatic Biliary Epithelial Cells

Objective To investigate whether lipopolysaccharide(LPS)stimulation can activate interleukin-6(IL-6)/ signal transduction and the activation of transcription activator 3(STAT3)signal pathway and its effect on the expression of downstream factor c-Myc and myeloid leukemia-1(Mcl-1)of this signal pathway in human intrahepatic bile duct epithelial cells(HIBECs).To investigate the effect of LPS on the proliferation of HIBEC and possible mechanisms.Methods(1)The Hi BECs were cultured in vitro.The cells were stimulated by LPS at different concentrations(0,0.1,1,2,4 and 8?g/ml)and at different time points(24h,48 h and 72h).The expression level of IL-6 at each concentration and each time point was measured by enzyme linked immunosorbent assay(ELISA)to determine the optimal concentration and checkpoint of LPS treated.Then we chose the optimal concentration and time point of LPS treated to intervene the cells in further experiments.(2)The HIBECs were stimulated with the measured LPS,and a control group with non-treated cells was set up.The expression of phosphorylated signal transducer and activator of transcription 3(p-STAT-3)and STAT3 protein was detected by Western blot to determine the proportion of p-STAT3/STAT3 protein,and to investigate whether IL-6/STAT3 signal pathway could be activated by LPS.(3)The HIBECs were treated withthe measured LPS,and a control group with non-treated cells was set up in the meanwhile.The expression levels of c-Myc,Mcl-1 m RNA and protein were measured by real-time quantitative polymerase chain reaction(RT-q PCR)and Western blot respectively to investigate the effect of LPS stimulation on the expression of downstream factor of IL-6/STAT3 signal pathway.(4)The HIBECs were treated with the measured LPS,and meanwhile a control group with non-treated cells and a blank control group were set up.The proliferation of HIBECs was identified by MTT assay to investigate the effect of LPS stimulation on the proliferation of HIBECs.(5)The abovementioned results were analyzed by SPSS16.0 statistical software.Results(1)ELISA results showed that LPS could stimulate HIBECs to secret IL-6,which is related to time-effect and dosage-effect to a certain extent.It was observed that the concentration of IL-6 gradually got higher with the increase of the concentration of LPS in a certain range,and the concentration of IL-6 reached its peak when the concentration of LPS was 4?g/ml(F=16.492,P<0.001)and the treatment lasted for 24h(F=17.763,P<0.01).Then the concentration of IL-6 gradually decreased with the increase of the concentration of LPS and the treatment time.Therefore,the following experiments were performed using LPS 4?g/ml and treatment time for 24 h.(2)Western blotting results showed that compared to the control group with non-treated cells,the ratio of pSTAT3/STAT3 protein increased(t=6.022,P<0.05)after the HIBECs were stimulated by LPS.(3)RT-q PCR results showed that the m RNA expression levels of c-Myc and Mcl-1 were significantly higher after the treatment of LPS than that of the control group.The expression of c-Myc m RNA increased by about 36 times(t=14.59,P<0.01)and the expression of Mcl-1 m RNA increased by about 2.4 times(t=19.10,P<0.01).Western blotting results showed that compared to the control group with non-treatedcells,the expression of c-Myc(t=46.45,P<0.001)and Mcl-1(t=5.383,P<0.05)protein increased after the cells were stimulated by LPS.(4)MTT results showed that LPS could promote the proliferation of Hi BECs in vitro(t=10.66,P<0.01).Conclusion LPS stimulation could activate the IL-6 / STAT3 signal pathway in HIBECs,increase the expression of downstream factors c-Myc and Mcl-1of this signal pathway at m RNA and protein levels and could promote the proliferation of HIBECs through IL6/STAT3 signal pathway.