| Objective:Alcoholic liver disease(alcoholic liver disease,ALD)is a toxic liver disease caused by excessive consumption of alcohol.Long-term heavy drinking may induce hepatic steatosis,steatohepatitis,alcoholic liver fibrosis(alcoholic liver fibrosis,ALF),cirrhosis and even tumour.As is known that the cause of ALF is clear,however,the mechanism driving the disease progression is incompletely understood.In the present study,the activation and proliferation of hepatic stellate cells(hepatic stellate cells HSCs)represent a critical event in fibrosis.Recent studies show that hypoxia inducible factor-1(hypoxia inducible factor-1,HIF-1)acts as housekeeping gene of controlling the responding to hypoxic.It is a heterodimer consisting of HIF-1? and an HIF-?.HIF-1? is highly regulated by micro-environmental oxygenation,whereas HIF-? is constitutively expressed.HIF-1? plays an important role in the transcription activity of HIF-1.HIF-1? is regulated by oxygen dependent and independent signals.HIF-1? may be involved in activation and proliferation of HSCs via target genes.Some researchers discovered that the level of HIF-1?,?-SMA and Collagen ? were increased in human hepatic stellate cell line LX-2 treated with LPS compared with control group.Meanwhile,?-SMA,Collagen ? which were markers of activation and proliferation in HSCs were suppressed in HIF-1?siRNA-transfected cells upon LPS stimulation.In the rat model of ALD our group discovered that the expressions of HIF-1? increased gradually with the progress of ALD.HIF-1? may play an important role in the etiopathogenesis of ALD.This study chooses HSC-T6 cells of rat stimulated by acetaldehyde;HIF-1? is knocked down to observe the proliferation of HSC-T6 cells and the expression of ?-SMA,Collagen ?.The study aimed to illuminate HIF-1? influence on the expression of ?-SMA,Collagen ? at the cellular level and to discuss the function of HIF-1? in the pathogenesis of ALF.Methods: The new resuscitated HSC-T6 cells were plated at density of 2×105 cells per well in 6-well plates with HD-DMEM medium supplemented with 10% fetal bovine serum(FBS)and 1% penicillin and 1% sodium pyruvate and cultured at 5% CO2,37?.When the logarithmic phase cells fused completely to 60%-70% confluence,they were divided to normal control group,the group stimulated by acetaldehyde(final concentration of 200?mol/L),HIF-1?siRNA group(siRNA100nm/L+acetaldehyde with final concentration of 200?mol/L),HIF-1?siRNA NC group(NC siRNA+ acetaldehyde with final concentration of 200?mol/L).This study chooses HSC-T6 cells of rat stimulated with acetaldehyde;HIF-1? is knocked down by ribonucleic acid interference;HIF-1?siRNA and NC siRNA were respectively transfected to HSC-T6 cells using LipofectamineTM 2000 following manual instruction.The cells were collected for detecting the proliferation of hepatic stellate cell by CCK-8 and measuring the expression of HIF-1?,?-SMA,collagen ? mRNA by RT-qPCR and protein by Western blot.Meanwhile,the cells were fixed by 4% neutral formaldehyde stained by immunofluorescence staining to observe the expression of ?-SMA.Results:1 Knock down HIF-1? gene to detect the proliferation of rat hepatic stellate Cells.The rat hepatic stellate cells were transfected with HIF-1? si RNA using LipofectamineTM 2000.48 hours later,the cell proliferation was inhibited.The result of CCK-8 bioassay showed that as the group stimulated by acetaldehyde compared with normal control group,cell activity appeared higher.The difference was statistically significant(P<0.01).In HIF-1?siRNA group the cell proliferation was inhibited compared with the group stimulated by acetaldehyde.The difference was statistically significant(all P<0.01).As HIF-1?si RNA NC group compared with the group stimulated by acetaldehyde,there is no statistical difference(all P>0.05)2 Changes of mRNA expression of HIF-1?,?-SMA and Collagen ? in HSC-T6 cells by RT-qPCR.The expression of HIF-1?,?-SMA and Collagen ? was lesser in normal control group.As the group stimulated by acetaldehyde compared with normal control group the expression of HIF-1?,?-SMA,collagen ? were up-regulated(all P<0.01).Knocking down HIF-1? gene,as HIF-1?si RNA group compared with the group stimulated by acetaldehyde,the expression of HIF-1? decreased and the expression of ?-SMA and Collagen ? decreased as well(all P<0.01);As HIF-1?siRNA NC group compared with the group stimulated by acetaldehyde,there is no statistical difference in the expression of ?-SMA and Collagen ?(all P>0.05).3 Changes of protein expression of HIF-1?,?-SMA and Collagen ? in HSC-T6 cells by Western blot.The expression of HIF-1?,?-SMA and Collagen ? was lesser.As the group stimulated by acetaldehyde compared with normal control group the expression of HIF-1?,?-SMA,collagen ? were up-regulated(all P<0.01).Knocking down HIF-1? gene,as HIF-1?siRNA group compared with the group stimulated by acetaldehyde,the expression of HIF-1? decreased and the expression of ?-SMA and Collagen ? decreased as well(all P<0.01);As HIF-1?siRNA NC group compared with the group stimulated by acetaldehyde,there is no statistical difference in the expression of ?-SMA and Collagen ?(all P>0.05).4 Immunofluorescence staining to observe expression of ?-SMA in HSC-T6 cells: The expression of ?-SMA in the control group was lesser.Compared with normal control group,the expression of ?-SMA were up-regulated in the group stimulated by acetaldehyde;Knocking down HIF-1? gene,the expression of ?-SMA protein decreased compared with the group stimulated by acetaldehyde.Conclusions:Acetaldehyde can promote proliferation and activation of hepatic stellate cells.Knocking down HIF-1? can inhibit the proliferation and activation of hepatic stellate cells.HIF-1? may be the potential therapeutic target for alcohol liver fibrosis. |
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