Effect And Mechanism Of Propofol Preconditioning On Hepatic Ischemia-reperfusion Injury

[Objective]To explore the feasibility and mechanism of propofol in the prevention of hepatic ischemia-reperfusion(I/R)injury,and explore a new type of protective drug for liver ischemia reperfusion injury with small side effects.[Background]Liver surgery often requires temporary blocking of the liver blood supply.When the blood supply is restored,a large number of free radicals and lipid peroxidation may result in the I/R damage of the liver,which is a severe problem in the liver surgery and liver transplantation.Hepatic I/R injury is one of the main causes of liver transplantation failure and multiple organ failure,which is caused by the releasing of large number of toxic metabolites and inflammatory mediators after transient ischemia of the liver followed by the recovery of blood supply,and can lead to serious metabolic disorders,aggravating the liver injury and dysfunction.Hepatic I/R injury is related to multiple mechanisms,such as ROS production,inflammation and apoptosis.Therefore,rational selection of drugs which can interfere with free radical production,inhibit inflammatory reaction and apoptosis may reduce liver I/R damage.Propofol is a common clinical intravenous anesthesia drug,and because its chemical structure is similar to vitamin E,it has been reported to have extensive antioxidant,anti-inflammatory and anti-apoptotic effects.A large number of literatures have reported that propofol can effectively alleviate multiple organ damage induced by I/R through multiple molecular mechanisms.However,despite a few reports,the role of propofol in the liver I/R damage species and its exact mechanism are still unclear,and the protective effect of propofol is still lacking in clinical trials.On the basis of this,this study systematically studies the protective effect of I/R damage in the liver of propofol in vitro,animal and clinical trials in vitro,and discusses its mechanism.[Methods]1.Effect and mechanism of propofol preconditioning on hypoxia/reperfusion hepatocytes.Human in vitro cultured hepatocytes HL-7702 were divided into control group,I/R group(recovery of oxygen supply culture to establish I/R injury model after 8h hypoxia),propofol group(addition of different concentrations of propofol 5、10、20、40μmol/L,30 minutes before cell hypoxia operation).The cells were observed after 24 hours of preconditioning with different concentrations of propofol after the cells were restored to oxygen,the cell proliferation activity was detected by MTT method.Annexin-V/PI staining was used to determine the cell apoptosis by the flow cytometry.Western Blot was performed to detect the expression of apoptosis related protein Caspase-3 and Bcl-2.JC-1 fluorescence probe and cell ATP content were used to evaluate the damage of structure and function of mitochondria.The function of hepatocyte was evaluated by aminotransferase from cell lysate by reagent kit and biochemical analyzer,and the levels of SOD,MDA and CAT were measured to evaluate the level of cell oxidation.2.Protective effect and mechanism of propofol on hepatic ischemia/reperfusion injury in rats.Wistar rats were divided into control group(n=10),sham operation(n=10,laparotomy),I/R group(n=10,abdominal surgery,blood supply was restored after brief clamping of the left and middle lobes of the liver while retaining the blood vessels in the right and caudate lobe of the liver,and liver I/R injury model was established.),propofol group(n=10,50 mg/kg propofol intraperitoneal injection 30 minutes before ischemia).The serum levels of AST,ALT,ALB and TBIL were measured for the evaluating of liver function at 24 h.The liver tissue sections were stained with H&E or observed by electron microscopy to measure the degree of liver injury.The concentrations of MDA,SOD and CAT were measured for investigation of antioxidant levels of the rat liver tissue homogenat.The levels of ATP in the liver tissue were detected to study the energy metabolism level of the rats.Furthermore,inflammatory factors TNF-a,IL-6,IL-10and TGF-β in liver tissue homogenate were detected for the research of inflammatory reaction.TUNEL staining and Western Blot used to detect the expression of apoptosis related proteins in liver tissues.3.Effect of propofol on I/R injury in patients undergoing liver transplantation.A total of 42 patients with liver transplantation were selected from the Department of hepatobiliary surgery,Shandong Provincia hospital,Shandong University in May 2018 May 2017,of which 18 patients(PPC)were pretreated with propofol intraoperative before liver ischemia,and 24 patients(NPC)without propofol treatment.The transplant was collect at 3 h after reperfusion,and the liver pathology was observed by H&E staining.The serum aminotransferase,total prothrombin time(PT)and bilirubin(TBIL)levels were measured before and after the operation.The liver function was evaluated,the level of serum MDA and H2O2 was detected,and the level of oxidative stress in the patients was evaluated.[Results]1.Propofol preconditioning with different concentrations(5,10,20,40 μmol/L)improved the decrease of the proliferation activity of HL-7702 cells induced by hypoxia/reperfusion(about 10%-17%,p<0.05)significantly.By inhibiting the expression of apoptotic protein Caspase-3 and promoting the expression of anti apoptotic protein Bcl-2,the apoptosis induced by hypoxia/reperfusion was reduced to a certain extent in the cells pretreated with propofol(the total apoptosis rate was reduced about3%,p>0.05).Propofol preconditioning could significantly reverse the decrease of mitochondrial membrane potential(p<0.01)induced by hypoxia/reperfusion and promote the formation of ATP(2-3.5 flods than that in I/R group,p<0.05),which showing a significant dose dependence.8h hypoxia/reperfusion could significantly increase the ALT(1.8 times)and AST levels(1.47 times,p<0.01)in HL-7702 cells,and the concentration of AST and ALT in the hypoxic/reperfusion cell lysate could be effectively reduced by preconditioning with different concentrations of propofol,with a significant dose dependence(p<0.05).Hypoxia/reperfusion could aggravate the degree of lipid peroxidation of liver cells(the level of MDA increased by about 2.7 times,p<0.001),and decrease the level of SOD(2.1 times)and CAT(2.3 times),while the preconditioning with different concentrations of propofol could effectively reverse the decrease of the level of antioxidant level of cells induced by hypoxia/reperfusion(p<0.05).2.Liver I/R treatment can significantly induce the increase of serum ALT and AST levels in rats(5.1 times and 2.6 times,respectively,p<0.001).Intraperitoneal injection of propofol before operation could significantly reduce the level of serum ALT and AST induced by I/R(1.6 times and 1.7 times compared with I/R group,p<0.01),and reduce the level of serum Alb in rats(about 30%,p<0.01)and increase the level of serum TBIL in rats(about 1.67 times higher,p<0.01).Propofol can effectively reverse the liver dysfunction induced by I/R(p<0.05).H&E staining and electron microscopy showed that propofol preconditioning could reduce the degree of liver damage caused by I/R.,I/R treatment aggravated the degree of lipid peroxidation within the liver tissue(MDA increased 1.87 times,p<0.01),and inhibited the level of SOD and CAT(about 1.67 and 2.9 times,respectively,p<0.01).Propofol could effectively reverse the decrease of antioxidant levels in rats induced by liver I/R(compared with I/R group,the level of MDA decreased by 28%,SOD and CAT increased respectively.1.52 and 1.95 times,p<0.01).The content of TNF-alpha and IL-6 in the liver tissue of I/R rats was significantly higher than that in the control group(about 3.38 and 3.77 times,respectively,p<0.01),while the concentration of IL-10 and TGF-beta was significantly lower than that of the control group(about 3.14 and 2.43 times,respectively,p<0.01),and propofol preconditioning could effectively reverse the secretion of the above inflammatory factors(p<0.01).Propofol treatment can alleviate the I/R induced apoptosis in rat liver tissue by promoting the expression of Caspase-3 and inhibiting the expression of Bcl-2 protein.3.Propofol preconditioning could significantly reduce the I/R induced liver damage in patients with liver transplantation(compared with group NPC,the liver pathological score of PPC group was reduced by 19.2%,p<0.01).The levels of ALT and AST in the patients at 6 h,24 h,and 2 D PPC after operation were significantly lower than those in NPC group(p<0.05),and PT significantly lower than those in the NPC group at 24 h after operation.After perfusion of 3h and 1 day after operation,the serum MDA level of patients in PPC group was significantly lower than that in group NPC(p<0.01),and the serum H202 level at 3 h in PPC group was also significantly lower than that of the NPC group(p<0.01)[Conclusions]1.Propofol preconditioning can attenuate the mitochondrial damage,promote the recovery of energy metabolism,increase the level of cell antioxidant levels,reduce the lipid peroxidation,regulate the expression of apoptosis related proteins,and reduce the inflammatory reaction in the liver induced by liver I/R,thus promote the proliferation of liver cells,reduce the apoptosis and eventually reduce the apoptosis,leading to the promotion of liver injury and liver function recovery.2.Propofol preconditioning can effectively reduce the damage of I/R-induced cell and rat liver structure and function by reducing liver transaminase,regulating serum ALB and TBIL levels.It also down-regulated the levels of pro-inflammatory factors TNF-a and IL-6 and up-regulated the levels of anti-inflammatory factors IL-10 and TGF-β,thereby alleviating the damage of I/R-induced inflammatory response to rat liver.3.Propofol preconditioning can increase the antioxidant levels in liver transplantation patients,thereby effectively alleviate liver injury and promote liver function recovery in patients with I/R induced liver transplantation.