Heme Oxygenase 1 Attenuates Hypoxia-Reoxygenation Injury In Mice Liver Sinusoidal Endothelial Cells

Background:Ischemia reperfusion injury(IRI)is a common pathophysiological process that occurs during liver transplantation.Lysis and apoptosis of cells are caused by ischemia and hypoxia,which seriously affects the survival rate of liver transplantation after recovery of the blood flow.The mechanisms of early reperfusion injury include activation of Kupffer cells,hepatocyte swelling,and microcirculatory dysfunction of the liver.Among them,liver sinusoidal endothelial cells(LSECs)play a major key in the regulation of hepatic microcirculation.Many studies have shown that LSEC is particularly prone to lose normal function in the liver IRI,and is the initiation site of hepatic injury.The damage of LSEC structure and function in early stages is the root for the development of liver IRI pathology.Therefore,effective protection of LSEC activity and function is the key to attenuate IRI in the process of hepatic ischemia-reperfusion injury during liver transplantation.In recent years,heme oxygenase-1(HO-1)with cytoprotection has become a hot research.HO-1,heat shock protein 32,is the most sensitive indicator of cellular oxidative stress response.It is up-regulated during inflammation,hypoxia,and radiation,and can be involved in the resolution of inflammation by modulating cytokine expression and apoptotic cell death.Previous studies have shown that the induction of high expression of HO-1 can significantly reduce hepatic ischemia-reperfusion injury,and its mechanism may be to protect the liver by inhibiting the secretion of inflammatory mediators and anti-apoptosis in Kupffer cells.However,the expression of HO-1 in hepatic sinusoidal endothelial cells in hepatic ischemia reperfusion injury is not clear at this stage,especially the study on the protective effect of HO-1 on hepatic sinusoidal endothelial cells.Objective:In this study,we transfected with adenovirus vectors encoding mice HO-1 gene(Ad-HO-1)in vitro according to their characteristics including anti-inflammation,anti-oxidant,and anti-apoptosis.A model of LSEC ischemia-reperfusion injury in vitro was established to investigate the protective effect of HO-1 on hepatic sinusoidal endothelial cells after hypoxia-reoxygenation injury.Method:1.Study groups:The LSECs were randomly divided into the following 3 groups for the in vitro study:the enhanced green fluorescent protein(EGFP)group;the HO-1 group,in which cells were transfected with adenoviral vector encoding the mice HO-1 gene;the Control group,which were not infected with any viral vectors during the incubation period.2.Transfection efficiency detection:the LSECs reached 80%to 90%confluence,the transfection efficiency was examined by the analysis of LSECs transfected with adenoviral vector encoding EGFP and HO-1 using an inverted fluorescence microscope after 48 hours of transfection.And the mRNA and protein expression level of HO-1 after transfection was detected by RT-qPCR and Western blot.3.Experimental model establishment:when the cell growth confluence is greater than 80%,the low serum(<5%)cell culture medium was replaced into each well,hypoxia-reoxygenation(H-R)injury of the cultured cells was conducted in a closed chamber at 37?,the LSECs were subjected to hypoxic conditions in an N2/CO2(95:5)gaseous mixture for 6 hours and reoxygenated with an air/CO2(95:5)gaseous mixture for 24 hours.The control cells were exposed to normoxic conditions(air/C02,95:5).4.Measurement of cytokine release by ELISA:the production of the inflammatory mediators IL-6 and TNF-a levels in the supernatant were determined with mouse-specific enzyme linked immunosorbent assays kit in accordance with the manufacturer's instructions.5.Flow cytometric detection of apoptosis of LSECs:the supernatant was discarded after experimental model,to each sample,Annexin V/Alexa Fluor 647 were added.The solutions were transferred into flow cytometry tubes for analysis.6.Determination of cell viability by CCK-8:the LSECs viability was determined by using the Cell Counting Kit 8 test.The CCK-8 method for monitoring in vitro cell viability was applied in accordance with the manufacturer's instructions.7.Real-Time Quantitative Polymerase Chain Reaction(RT-qPCR):the mRNA expression level of HO-1 was detected by RT-qPCR in mice LSECs.8.Western blot:the protein expression level of HO-1 was detected by Western blot in mice LSECs.9.Statistical Analysis:All data were expressed as the mean ± SEM.The statistical analysis was performed by using SPSS Version 13.0 for Windows.The differences between the experimental groups were analyzed by using two-way analysis of variance or Student's t test.For all comparisons,a value of P less than 0.05 was considered statistically significant.Results:1.Transfection efficiency:the LSECs infected at an MOI of 80 at 48 hours with a transfection efficiency greater than 80%,and the cell morphology remained stable.The level of HO-1 mRNA expression in LSEC cultures was greater than that in the control group and the EGFP group;a stronger positive band was detected in HO-1 group than in the EGFP and control groups.The difference was statistically significant(P<0.05).2.ELISA results:the H-R groups(control,EGFP,and HO-1 groups)showed increased levels of the cytokines IL-6 and TNF-a as compared with the normoxic groups(control,EGFP,and HO-1groups)(n=6 per group,*P<0.05,**P<0.01).The HO-1 groups had significantly lower cytokine levels in comparison with the EGFP groups(###P<0.001)?3.Flow cytometric detection:apoptotic cell numbers were reduced after H-R injury in the HO-1 group versus the EGFP group(n = 3 per group,**P<0.01).4.CCK-8 detection:the cell viability of EGFP and HO-1 groups were lower than that of the control groups(n = 5 per group,**P<0.01,****<0.001)after transfection and H-R injury.Significantly increased cell viability was observed in the HO-1 group compared to the EGFP group after H-R injury(**P<0.01).5.RT-qPCR results:the level of HO-1 mRNA expression in LSEC cultures was greater than that in the control group and the EGFP group,and expression levels of HO-1 were found to be significantly downregulated in the H-R groups(including control,EGFP,and HO-1 groups)(n = 6 per group,***P<0.001).6.Western blot results:a stronger positive band was detected in HO-1 group than in the EGFP and control groups,and a clear downregulation of HO-1 was observed after H-R injury in comparison with the normoxic groups(control,EGFP,and HO-1 groups)(n = 3 per group,*P<0.05,**P<0.01,***P<0.001).Conclusion:1.The method of in vitro adenoviral vectors established the transfection of the HO-1 gene into LSECs is feasible and stable;low serum,hypoxic-reoxygenation culture method of mice LSEC in vitro ischemia-reperfusion injury model is consistent with the pathophysiological process of clinical liver ischemia-reperfusion injury,it is an experimental model with strong stability and operability.2.The cellular stress response induced by hypoxia-reoxygenation injury model can downregulate the expression of HO-1 in LSECs.The mechanism of HO-1 downregulation in mice LSECs after early reperfusion remains unknown.One possible explanation is related to the hepatic microcirculatory failure partially caused by the disruption of the LSEC microstructure.3.Upregulation of HO-1 can attenuate H-R injury in mice LSECs by inhibiting proinflammatory cytokine release and diminishing apoptotic cell death.