| Objective:Ischemia-reperfusion injury is a common clinical surgery pathophysiologic process,such as the body trauma and uncontrolled hemorrhagic shock,heart coronary ischemia and extracorporeal circulation,as well as the liver and kidney resection and transplantation surgery,which can seriously affect the process of surgery and the prognosis of the disease.Currently,we are lack of deep understanding about the pathophysiological process of ischemia-reperfusion injury.And its mechanism is very complicated,which mainly related to the large generation of reactive oxygen species,inflammatory damage and microcirculation disturbance.And the key which starts the links is the disorder of the cell energy metabolism.Mitochondrion,which can produce energy through oxidative phosphorylation,is an important energy-producing organelle.The regulatory mechanism of mitochondrial function closely associates with the ischemia-reperfusion injury.Prostaglandin E2(PGE2)is an important endogenous organism regulating factor.When liver occur ischemia-reperfusion,PGE2 plays an important role in regulating the inflammatory reaction and microcirculation perfusion.Cyclooxygenase-2(COX-2)is an important rate-limiting enzyme to regulate the production of PGE2,and it has been proved that COX-2 plays an important role in liver I/R injury.Inhibiting COX-2 signal can reduce the I/R injury,which through a cellular protective mechanism,namely inhibiting the mitochondrial membrane permeability transition pore(MPTP).But the specific molecular mechanism is unclear.PGE2 has four kinds of G protein coupled receptors,namely the prostaglandin E2 receptor 1(EP1),prostaglandin E2 receptor 2(EP2),prostaglandin E2 receptor 3(EP3)and prostaglandin E2 receptor 4(EP4).EP4 is the important G protein coupled receptor in the COX-2 downstream signaling.Recent studies also confirmed that EP4 may directly mediate the liver protective effect of PGE2 during the I/R injury.However,the relationship between the EP4 signal and the mitochondrial protection is unclear.In this experiment,we used specific EP4 agonist pretreatment rats,and studied the effect and mechanism of EP4 signal in hepatic I/R injury.The liver blood vessel blocking method,which is classic and reversible,is used to establish the hot liver ischemia-reperfusion model.And then used serum enzymology tests,histopathological observation,the determination of mitochondrial calcium capacity and used western blot to detect mitochondrial signaling pathways to study EP4 receptor mediated hepatic ischemia-reperfusion mitochondrial protective effect and its mechanism.Methods:Male Sprague-Dawley(SD)rats(220g-230g)were randomly divided into 6 groups1.sham operation group(sham group): only opening the rat abdominal cavity,separating the blood vessels,and don’t clip them;2.ischemia-reperfusion group(I/R group): 70% liver warm ischemia 60 min,and then reperfusion;3.ischemic preconditioning and ischemia-reperfusion group(IPC group): giving a 10 min ischemia and reperfusion 10 min,and then 70% liver warm ischemia 60 min,and then reperfusion;4.COX-2 inhibitors NS-398 pretreatment and ischemia-reperfusion group(NS-398group): giving COX-2 inhibitor NS-398(30mg/kg,intraperitoneal injection)10 min before ischemia,and then 70% liver warm ischemia 60 min,and then reperfusion;5.ischemia-reperfusion and EP4 agonist CAY10598 treatment group(CAY group):70% liver warm ischemia 60 min and then reperfusion,and in 1.5 h,30 min of ischemia and reperfusion instantly intraperitoneal injecting EP4 agonist CAY10598(the total dose of 1 mg/kg);6.ischemia-reperfusion and EP4 Agonist and Carboxy Atr group(CATR group): 70%liver warm ischemia 60 min and then reperfusion,and in 1.5 h,30 min of ischemia and reperfusion instantly intraperitoneal injecting EP4 agonist CAY10598(the total dose of 1mg/kg),and intraperitoneal injecting the mitochondrial membrane permeability transition pore opening agent Carboxy Atr(5 mg/kg)before 30 min of ischemia.We take the liver tissues of the Sham group,I/R group,IPC group and the NS-398 group,using Polymerase Chain Reaction(PCR)and western blot techniques to detect the expression of EP4 receptors at different time points.And Taking the liver tissues and the serum of the Sham group,I/R group,CAY group and CATR group,and then used serum enzymology tests,histopathological observation,the determination of mitochondrial calcium capacity to verify the MPTP regulation and the liver injury protection of EP4 signal.And using western blot technique to detect the expression of GSK-3β,p-GSK-3β,ERK1/2,p-ERK1/2,JAK2,p-JAK2,STAT3 and p-STAT3,to study the molecular mechanism of the regulation of mitochondrial function and the liver injury protection of EP4 signal.Results:1.PCR technology found that EP1,EP2,EP3 and EP4 genes have different degree of expression in normal liver tissues.Compared with the sham group,EP1,EP2 and EP4 receptor m RNA expression were significantly increased in 2 hours after reperfusion,and IPC can significantly reduce EP2 and EP4 receptor gene expression,and NS-398 pretreatment down-regulates gene expression of EP4 receptor.Compared with the sham group,I/R can obviously promote the expression EP4(P < 0.05),and compared with I/R group,NS-398 treatment can obviously decrease the expression of EP4 protein(P < 0.05).2.The levels of AST and ALT after 2 h and 6 h of reperfusion display that compared with I/R group,the levels of AST and ALT on the time of 2 h and 6 h reperfusion of CAY group significantly decreased(P < 0.05).Liver tissue pathology finds that compared with I/R group,the liver cell necrosis of CAY group was significantly reduced,and apoptotic cells decreased significantly,and few hepatocyte nuclear condensation and mitochondrial morphology improved significantly.Using differential centrifugation to prepare mitochondria,and the results of CRC shown that compared with I/R group,the CRC on the time of 2 h and 6 h reperfusion of CAY group significantly increased.3.The levels of c AMP and ROS on the time of 2 h and 6 h reperfusion display that compared with sham group,the levels of c AMP and ROS of I/R group significantly increased(P < 0.01).CAY10598 can further improve the concentration of c AMP.But compared with I/R group,CAY10598 can significantly decrease the levels of ROS(P <0.05).Western blot displays that after the reperfusion of 2 hours and 6 hours,compared with the Sham group,I/R and CAY10598 can significantly increase p-ERK1/2 expression(p < 0.05).After the reperfusion of 2 hours,compared with the Sham group,I/R and CAY10598 can significantly increase p-GSK-3β expression,but after the reperfusion of 6hours,compared with the I/R group,CAY10598 can significantly increase p-GSK-3βexpression(P < 0.05).After the reperfusion of 2h and 6h,I/R,CAY10598 and CATR group can significantly increase the expression of p-JAK2 in different extent.And it increased most significantly in 2 h;compared with I/R group,CAY10598 can inhibit the expression of p-JAK2(P < 0.05).After the reperfusion of 2h and 6h,I/R,CAY10598 and CATR group can significantly increase the expression of p-STAT3;compared with sham and I/R groups,CAY10598 can significantly decrease the expression of p-STAT3 in the reperfusion of 2h,and it increase gradually in the reperfusion of 6h(P < 0.05);this reminder that CAY10598 can delay the increase of p-STAT3.Conclusion:1.As an important regulatory site of the COX-2 signal downstream site,EP4 is directly involved in the I/R injury process of occurrence and development.2.Using EP4 agonist CAY10598 to activate EP4 signal can inhibit MPTP opening,reduce mitochondrial damage,and reduce liver cell necrosis and apoptosis.3.EP4 signal can regulate c AMP-ERK1/2 signals to affect GSK-3β and JAK2-STAT3 signal,and then regulates the permeability of MPTP,effecting the generation of ROS,and to regulate the liver I/R injury. |
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