| Background:Hepatic ischemia/reperfusion(I/R)injury is a severe phenomenon during the treatment process of liver traumatic,hepatic resections and liver transplantation,which usually caused traumatic hemorrhagic shock,liver injury or graft dysfunction.In addition,during the process of cell hypoxia/reoxygenation,the formation of excessive reactive oxygen species(ROS)and activation of Kupffer cells induced inflammation and further caused cell apoptosis.Recnetly,the martality rate of I/R was increasingly.However,the underlying mechanisms of hepatic I/R injury have not been illuminated clearly.Thus,to search for the effective drugs for I/R is essential in the recent clinical treatment.Propofol is a rapid and short-acting general anesthetic drug and was widely used in clinical anesthesia,up to now,propofol has been found to have a protective effect in I/R injury in liver.However,the potential molecular mechanism has been poorly understood.Mitogen-activated protein kinase(MAPK)belongs to Ser/Thr protein kinase family,the signaling cascade of which was transferred from cellular to intracellular,and then transferred to the downstream based on the change of protein phosphorylation level.MAPKs were activated in response to oxidative stress,and were involved in the regulation mechanism of gene expression and cell physiological process such as proliferation,development,differentialtion and apoptosis.Recently,mounting evidence indicated that MAPK signaling pathways were activated in I/R injury and thus induced the deterioration of tissue structure and function.mitogen-activated protein kinase 6(MAPK6)is an important family member of MAPK,the overexpression of which was verified to promote cell apoptosis.Evidence revealed that MAPK6 was related to the onset and development of liver cancer,while,whether the expression of MAPK6 was related to the mechanism of I/R was still unclear.MicroRNA(miRNA)was a set of small non-coding RNA with the length of less than 22 nt.miRNA was conservation in biological evolution process,and was widely existed in the genome of bacteria,viruses,as well as plant and animal.Studies have reported that miRNA regulated the expression of many genes,and involved in the regulation processes of cell growth,diffferntiation and apoptosis.mounting evidence has revealed that the I/R injury was mediated by miRNAs,and miR-133a regulated the injury level of liver I/R.MIR-133a acted as an protective moleculars in myocardium I/R injury by inhibiting the expression of death associated protein kinase 2(DAPK2)and thus inhibiting myocardial cell apoptosis.Furthermore,miR-133a-5p promoted cell apoptosis of liver cancer that induced by targeting FSCN1.While,whether miR-133a-5p mediated the mechanism of liver I/R was still unknown.In this study,the online database predicted that miR-13 3a-5p can bind the MAPK6 3’UTR,which indicated that MAPK6 might be the downstream molecular of miR-133a-5p.In this study,we firstly establimed the mice model of liver I/R and pre-treated with propofol,Automatic biochemical analyzer method was used to detecte the level of liver injury;real-time PCR and western blot were used to detect the expression level of miR-133a-5p and MAPK6.Secondly,the hypoxia/reoxygenation model of liver cell was constructed to explore the protective mechanism of propofol on liver I/R.Finally,in vivo experiment was performed to further verify the protective mechanism of propofol.The aim of this study was to clairfy the protective effectiveness of propofol on liver I/R injury,the study provided experimental basis and theoretical guidance for the clinical application of propofol.Part I Protective effects of propofol on hepatic ischemia reperfusion injury in rats and its effect on miR-133a-5p/MAPK6 expressionObjectives:To investigate the protective effect of propofol on hepatic ischemia reperfusion(I/R)injury in rats and its regulation of miR-133a-5p/MAPK6 expression.Methods:1.A rat model of hepatic I/R injury was established and pre-treated with propofol.Serum of hepatic I/R group,propofol pre-treatment group and sham operation group were collected the levels of AST and ALT in the serum were measured by Automatic biochemical analyzer.2.All experimental rats were sacrificed and liver tissue was taken.The expression levels of miR-133a-5p and MAPK6 mRNA in liver tissue were detected by qRT-PCR,while the expression of MAPK6 protein was detected by western blot.3.Human normal liver cells QSG-7701 were cultured in RPMI 1640 with 10%fetal bovine serum(FBS)at 37℃ with 5%CO2.H/R group was treated with hypoxia and reoxygenation.4.After cell treatment is completed,the expression levels of miR-133a-5p and MAPK6 mRNA in liver cell were detected by qRT-PCR,and the expression of MAPK6 protein was detected by western blot.Results:1.Compared with the sham operation group,the levels of AST and ALT in the serum of the hepatic I/R group were significantly increased.2.In the hepatic I/R group,the expression of miR-133a-5p in hepatocytes was significantly decreased,while expression levels of MAPK6 mRNA and protein were significantly increased.Compared with hepatic I/R group,propofol pre-treatment group showed the opposite trend.3.H/R treatment resulted a significant decrease in the expression of miR-133a-5p in hepatocytes,but a significant increase in MAPK6 mRNA and protein expression levels.Compared with H/R group,the treatment of H/R with propofol could promote miR-133a-5p expression and inhibit MAPK6 mRNA and protein expression.Conclusions:1.Propofol could significantly reduce the serum levels of AST and ALT in hepatic I/R rats,then protect hepatic I/R injury.2.In liver tissue of hepatic I/R rats,the expression of miR-133a-5p was decreased and the expression of MAPK6 was significantly increased.Propofol could reverse this phenomenon.3.In the normal hepatocyte QSG-7701 pathological model,H/R treatment decreased miR-133a-5p expression and increased MAPK6 expression,while he effect of H/R on hepatocyte was reversed by propofol.Part Ⅱ Effect of propofol on H/R-induced hepatocellular apoptosis and its mechanism of regulation of miR-133a-5p/MAPK6 expressionObjectives:To explore the molecular mechanism of propofol protecting hepatic I/R injury in vitro.Methods:1.The sequence of miR-133a-5p and MAPK6 was analyzed by bioinformatics software.The regulatory relationship between miR-133a-5p and MAPK6 was identified by dual-luciferase reporter gene system.2.The hepatocytes were transfected by miR-133a-5p mimic or inhibitor.The expression of MAPK6 in hepatocytes was detected by qRT-PCR and western blot.3.The hepatocyte H/R model was established and transfected with miR-133a-5p mimic.The expression of MAPK6 was detected by qRT-PCR and western blot.4.The hepatocyte H/R model was treated with propofol before H/R treatment and transfected with miR-133a-5p inhibitor.The expression of MAPK6 was detected by qRT-PCR and western blot.5.The hepatocyte H/R model was treated with propofol before H/R treatment and transfected with pcDNA-MAPK6.The level of apoptosis was detected by TUNEL assays.Results:1.MiR-133a-5p negatively regulates MAPK6 expression,i.e.miR-133a-5p overexpression significantly down-regulated the expression level of MAPK6 mRNA and protein,miR-133a-5p knockdown significantly promoted the expression level of MAPK6 mRNA and protein.2.Hepatocyte H/R significantly promoted the expression of MAPK6,whereas miR-133a-5p overexpression significantly reversed the results.3.Propofol significantly inhibited MAPK6 expression and miR-133a-5p knockdown eliminated the effect.4.H/R significantly promoted the apoptosis of hepatocytes.Propofol inhibited I/R-induced hepatocellular apoptosis,whereas MAPK6 overexpression eliminated the protective effect of propofol on hepatocytes.Conclusions:Propofol inhibited H/R-induced hepatocyte apoptosis by up-regulation of miR-133a-5p to inhibit MAPK6 expression.Part III:Effect of miR-133a-5p inhibition on hepatic I/R injury remitted by propofolObjectives:To verify the mechanism of action of propofol on remitting hepatic I/R injury by in vivo.Methods:1.The experiment was grouped into HI/R group,HI/R+propofol group,HI/R+propofol+NC group,HI/R+propofol+miR-133a-5p inhibitor group.The levels of AST and ALT in the serum of different treatment groups were detected by Automatic biochemical analyzer.2.Taken the liver tissue of all rats in the above experiment groups and detected the expression of MAPK6 mRNA and protein in liver tissue by qRT-PCR and western blot.Results:1.The levels of ALT and AST in serum of hepatic I/R rats were significantly up-regulated,and propofol could decrease the levels,while miR-133a-5p inhibitor eliminated the protective effect of propofol on hepatic injury.2.The expression of MAPK6 in hepatic I/R group was significantly increased,and propofol decreased the expression level,whereas miR-133a-5p inhibitor abolished the effect of propofol on MAPK6 expression in injured liver tissue.Conclusion:In vivo experiments confirmed that propofol regulated MAPK6 through miR-133a-5p and then protected hepatic I/R injury in rats. |
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