| Objective: As a common clinic physiopathologic process,hepatic ischemia reperfusion injury(HIRI)is the main cause for primary liver dysfunction and organ failure after liver transplantation,liver resection and hemorrhagic shock.Although the precise sequence of events leading to HIRI has not been completely elucidated,it is believed that several mediators such as reactive oxygen species,proinflammatory cytokines,chemokines,adhesion molecules,and excess NO contribute to this injury.There is,however,substantial evidence that much of hepatic injury and dysfunction following I/R is the result of transcription factor NF-?B signaling pathway activation.In unstimulated cells,NF-?B proteins are sequestered in the cytoplasm as latent complexes by ?B inhibitor proteins(I?B).A wide variety of stimuli activate classical NF-?B signaling,including proinflammatory cytokines,bacterial lipopolysaccharide(LPS)and viral infection,all of which converge at the I?B kinase(IKK)complex.Activated IKK phosphorylates I?B proteins,triggering their ubiquitination and degradation by the proteasome,thus allowing NF-?B to translocate to the nucleus and initiate the transcription of target genes,such as immune related receptor genes,chemokines genes and adhesion molecules genes.As a critical regulator of proinflammatory genes,NF-k B is very important in the physiopathologic process of HIRI.The zinc finger protein A20(also known as TNFAIP3),one of target proteins of NF-k B,is an immediate early response protein that plays a critical role in the negative regulation of NF-?B signaling pathway by inhibiting the activation of IKK.It has been demonstrated,A20 could protect several organs(kidney,liver)from IRI through the inhibitory effect on NF-?B signaling pathway.However,the regulatory mechanisms involved in the NF-?B inhibitory effect of A20 are not clear.Long non-coding RNAs(lnc RNAs)are a group of non-coding RNAs with a length of more than 200 nucleotides,which usually do not encode proteins.However,lnc RNA has biological functions related to apoptosis,inflammation and autophagy.It is reported that lnc RNA HOX antisense RNA(HOTAIR)activates autophagy by increasing the expression of autophagy-related gene 7(ATG7)in hepatocellular carcinoma.However,whether HOTAIR regulates autophagy in liver I/R damage is still unclear.These results strongly suggest that HOTAIR may inhibit inflammation by inhibiting NF-kappa B signaling pathway,and may regulate ischemia-reperfusion injury.Therefore,the role of HOTAIR in hepatic ischemia-reperfusion injury and hypoxia-reoxygenation model in rats was preliminarily observed,and the molecular mechanism of autophagy regulation in this process was discussed.Materials and Methods: 1.The cephalic branch of hepatic vessel supplying the left lateral,left medial lobe was clipped by an atraumatic clip to built hemi-hepatic warm ischemia reperfusion injury model of rat.2.Morphological analysis of livers was used by light microscope and transmission electron microscopy.3.The levels of liver enzymes and proinflammatory cytokines were determined by ELISA method.4.Protein expression was determined by Western blot analysis.5.The expression of protein was detected by Western blot.6.Trypan blue exclusion assay was used to measure cell viability.7.Apoptosis was detected by flow cytometry and PI staining.8.Plasmid transfection technology was used to intervene the gene expression level of mouse liver cells in vitro.9.The molecular mechanism of HOTAIR and mi R-20b-5p binding sites was detected by luciferase.10.Statistical analysis: All values were expressed as means ± SEM.The differences of the results between two groups were evaluated by Student's t-test.P<0.05 was considered to be statistically significant.Results: 1.Experiment 1: After hemi-hepatic ischemia 1 h following reperfusion 1 h,hepatic cell of rat began to swell and apoptosis,and the levels of serum AST and ALT began to rise.In addition,changes in serum TNF-? and IL-6 levels increased significantly,reaching the peak value 6 h after reperfusion,which is responsible for the pathophysiological changes in I/R injury.Expression of p-IKK 1 h after reperfusion was increased significantly then decreased 6 h after reperfusion,nearly returned to the normal level,while IKK level didn't change obviously.The expression of Itch and A20 increased slightly 1 h after reperfusion,which increased solidly with reperfusion period prolonging and reached the peak value 24 h after reperfusion.With the increase of the expression of Itch and A20,the activation of IKK was inhibited obviously.2.Experiment 2: The changes of NF-kappa B signaling pathway in hepatic ischemia-reperfusion injury in rats were further elucidated by the hemi-hepatic ischemia-reperfusion injury model.To elucidate the changes of NF-kappa B signal during ischemia-reperfusion injury and to detect the expression of IKK and p-IKK in rat liver cells at different time points after reperfusion.The results showed that the expression of p-IKK increased significantly after 1 hour of reperfusion,decreased at 6 hours and returned to normal at 24 hours.However,there was no significant change in IKK level before and after ischemia-reperfusion.3.Experiment 3: Studies have shown that the expression of HOTAIR increases in I/R liver and directly regulates mi R-20b-5p.In addition,we combine ATG7 with ATG7 mRNA's 3'UTR to make sure that ATG7 is the target of mi R-20b-5p inhibition.Through remedial experiments,we found that HOTAIR regulates autophagy through the mi R 20b-5p/ATG7 axis.Our research provides new clues for the role of HOTAIR in regulating autophagy in liver I/R damage.Conclusion: 1.The hemi-hepatic ischemia reperfusion injury of rat was feasible.Serum levels of liver enzymes and proinflammatory cytokines were responsible for the pathophysiological changes in I/R injury.2.NF-?B signaling pathway was one of the main mechanisms of HIRI.The expression of p-IKK was elevated after ischemia following reperfusion,which was responsible for the pathophysiological changes and serum levels of liver enzymes and proinflammatory cytokines.3.The expression of lnc RNA-HOTAIR was up-regulated after hepatic ischemiareperfusion in rats.After hypoxia-reoxygenation,the expression of p-IKK in BRL3 A cells overexpressing lnc RNA-HOTAIR was significantly decreased compared with that in blank vector transfection group,indicating that lnc RNA-HOTAIR could inhibit the activation of NF-kappa B signaling pathway in hepatocytes after hypoxia-reoxygenation and protect hepatocytes in rats.Effect.4.After hypoxia-reoxygenation treatment,the death rate and apoptosis rate of BRL3 A cells overexpressing lnc RNA-HOTAIR were significantly lower than those of blank vector transfection group,which indicated that lnc RNA-HOTAIR could inhibit the death and apoptosis of liver cells after hypoxia-reoxygenation,and had protective effect on rat liver cells.5.the expression of HOTAIR was upregulated and the level of autophagy was increased in hepatocyte anoxia model.6.in liver cells,mi R-20b-5p can inhibit ATG7 expression through targeting binding to ATG7 3'UTR.7.HOTAIR mediated I/R injury by targeting inhibition of the up-regulation of ATG7 expression by micro RNAs-20b-5p in hypoxic hepatocytes in vitro. |
PDF Full Text Request