Research On The Mechanism Of Nrf2-ARE Pathway Activated By Hypoxic Postconditioning And Pharmacological Postconditioning

Objectives: To establish a hypoxia reoxygenation injury model on adult SD rat myocardialcells, and to observe the effect of hypoxic/emulsified isoflurane(EI)/pinacidil(P)postconditioning on rat myocardial cells. Objective One: Targeting the Nrf2, this project isto discuss the NF-E2-related factor2-antioxidant response elements (Nrf2-ARE)myocardial protection mechanism of oxygen/emulsified isoflurane(EI) postconditioning. Itis to compare the groups with and without the active oxygen scavengerN-2-mercaptopropionyl glycine(MPG) and observe the changes of mRNA and amount ofprotein expression in the following products(HO-1、Nqo-1、SOD-1) of Nrf2-ARE, in orderto clarify the effect of reactive oxygen species(ROS) in the mycardial protection with thepostconditioning ways mentioned above, which is the proper mechanism activated byNrf2-ARE; Objective Two: With determining the concentration of ROS, this project is toobserve the effects of the ischemic postconditioning, the pharmacological postconditioningof two kinds of drugs with varied concentration and fat emulsion group on ROS level,which is to demonstrate whether the ROS level induced by the above postconditioningways relates to the effect of mycardial protection.Methods:1. Cardiomyocytes were isolated from the left ventricle of male adult Sprague-Dawleyrat(250-300g) with Langendorff-perfusion and collagenase-II digestion, on which ahypoxia reoxygenation injury model was established. With the objective that this project isto study activation mechanism of Nrf2-ARE, the cardiomyocytes were randomly dividedinto9groups(n=9, each group), i.e. the normal group (group N), the control group (groupH/R), the ischemic postconditioning group (group HPO), isoflurane(1.68mmol/L)postconditioning group(group EI2), fat emulsion postconditioning group(group FAT),Pinaddil (50μmol/L) postconditioning group(group P50), hypoxic postconditioning andMPG postconditioning group (group HPO+MPG), emulsified isoflurane postconditioning and MPG postconditioning group (group EI+MPG) and Pinaddil postconditioning groupand MPG postconditioning group (group P+MPG). The separated myocardial cells werecultured20h. Excepting the continuous training of N group for110min, the other groupswere cultured in hypoxia for45min. and then the N/R group was in reoxygenation for60min; Group HPO, after cultured in hypoxia for45min, repeated the cycles of hypoxia/reoxygenation for three times, with each step lasting for5min. Then, it was continuouslycultured in hypoxia for60min; Group EI2, after cultured in reoxygenation for45min, waspostconditioned with EI(1.68mmol/L) for5min, and then in reoxygenation for60min;Group FAT, after cultured in hypoxia for45min, was postconditioned with fat emulsionfor5min, and then in reoxygenation for60min; Group P50, after cultured in hypoxia for45min, was postconditioned with Pinaddil (50μmol/L) for5min, and then in reoxygenationfor60min; Group HPO+MPG, after in the cycle of hypoxia/reoxygenation, each step for45min, was postconditioned with MPG for10min, and then in reoxygenation for60min;Group P+MPG, after postconditioned with Pinaddil for5min, MPG for10min and was inreoxygenation for60min.2. Confocal Laser Scanning Microscopy(CLSM) was used to detect the level ofintracellular calcium and Nrf2nuclear translocation at the end of reoxygenation.3. Transmission electron microscope was used to observe the ultrastructure ofmyocardial cells after hypoxia/reoxygenation injury and to evaluate the mitochondria.4. Real-Time PCR and western-blot were applied to detect the quantities of mRNAexpressions and protein expressions of Nrf2, HO-1, SOD1andNQO1in cardiomyocytes atthe end of reoxygenation.5. With the second objective that this project is to study the correlation of theconcentration of ROS and the activated degree of pathway by postconditioning.Enzyme-linked immune adsorbent(ELISA) was applied for the determination of activeoxygen content in groups without MPG, groups postconditioned with varied concentrationof EI (0.84、1.68and2.52mmol/L, groupEI1, groupEI2, groupEI3) and in groupspostconditioned with varied concentration of Pinaddil(25、50and100μmol/L，groupP25，groupP50，groupP100) at the point of5min and60min, respectively, during reoxygenation. And the content of ROS at point of60min was compared with the expression quantity ofmRNA and protein in Nrf2, respectively.Results:1. Intracellular calcium：Compared with group N, the intracellular calcium contentin other groups are obviously higher (P <0.05). Compared with group H/R, intracellularcalcium content in other groups are obviously lower (P <0.05), among which theintracellular calcium content in group HPO is apparently lower than group H/R and groupFAT. The intracellular calcium content in group HPO, EI2, P50has no significant disparity(P>0.05). However, the intracellular calcium content group HPO, EI2, P50is lower thanthat in group HPO+MPG, EI2+MPG, P50+MPG (P <0.05).2. Subcellular distribution of Nrf2: The Nrf2fluorescence intensity in the nuclei ofgroup N was low, compared with which, that of H/R is higher (P <0.05). Compared withH/R group, the Nrf2fluorescence intensity in other groups are significant higher (P <0.05),among which the Nrf2fluorescence intensity in group HPO, EI2, P50has no significantdisparity (P>0.05), but all are higher than FAT (P <0.05). The Nrf2fluorescence intensityin group HPO, EI2, P50is higher than that in group HPO+MPG, EI2+MPG, P50+MPG (P<0.05).3. Mitochondria construction of myocardial cells: The electron microscopy showsthat the mitochondria construction of myocardial cells in N group is normal. Themitochondria construction in H/R group is with the worst damage. The mitochondriaconstruction in group HPO, EI2, P50is with little damage, apparently less than that ofgroup FAT (P<0.05). And the mitochondria construction in FAT is less damaged than thatof H/R (P<0.05). Besides, the mitochondria construction in group HPO, EI2, P50is lessdamaged than that in group HPO+MPG, EI2+MPG, P50+MPG (P <0.05).4. The quantity of mRNA expressions of Nrf2, HO-1, SOD1and NQO1：Comparedwith group N, the quantities of mRNA expressions of Nrf2, HO-1, SOD1and NQO1inother groups are obviously lower (P<0.05). Compared with group H/R, the quantities ofmRNA expressions in other groups are apparently higher (P<0.05). The quantities ofmRNA expressions in group HPO, EI2, P50have no significant disparity(P>0.05), which, however, are all higher than that of group FAT(P<0.05). The quantities of mRNAexpressions in group HPO, EI2, P50are higher than that in group HPO+MPG, EI2+MPG,P50+MPG (P<0.05).5. The quantity of protein expressions of Nrf2, HO-1, SOD1and NQO1:Compared with group N, the quantities of protein expressions of Nrf2, HO-1, SOD1andNQO1in other groups are apparently lower(P<0.05). Compared with group H/R, thequantities of protein expressions are higher(P>0.05). The quantities of protein expressionsin HPO, EI2, P5O have no significant disparity(P>0.05), which, however, are all higherthan that of group FAT(P<0.05). The quantities of protein expressions in group HPO, EI2,P50are higher than that in group HPO+MPG, EI2+MPG, P50+MPG (P<0.05).6. The reactive oxygen species(ROS) content: During the procedure ofreoxygenation, at5min and60min, the ROS content in each group is higher than that ingroup N. Compared with group H/R, the ROS content in other groups is lower(P<0.05).The ROS content in group HPO,EI2, P50has no significant disparity(P>0.05), which,however, is lower than that in FAT(P<0.05). The ROS content in group FAT is slightlower than that in H/R, but without apparent disparity (P>0.05). The ROS content in groupEI1, EI2, EI3is increasing with the rise of their concentration at5min duringreoxygenation (P<0.05). The ROS content in group EI1, EI3has no apparent disparity at60min during reoxygenation (P>0.05), but, which is significantly higher than that in EI2(P<0.05). Within group EI1, EI2, EI3, the ROS content is lower at5min than that at60minduring reoxygenation(P<0.05). The ROS content in group P25, P50, P100is increasingwith the rise of their concentration at5min during reoxygenation (P<0.05). And the ROScontent in group P25, P100has no obvious disparity at60min (P>0.05), which, however, isapparently higher than that in group P50(P<0.05). Within the group P25, P50, P100, theROS content is higher at5min than that at60min during reoxygenation (P<0.05). The ROScontent, in the H/R cardiomyocytes being postconditioned by emulsifiedisoflurane/pinacidil, presents negative linear correlation with the quantity of mRNAexpressions and protein expressions(P<0.01).Conclusions: 1. The ROS produced during reperfusion in hypoxic postconditioning/EIpostconditioning/P postconditioning activates the pathway of Nrf2-ARE, the expressionsof whose following peroxiredoxins and phaseⅡdetoxifying enzymes are up-regulated,which realizes the effect of anti-H/R injury in myocardial cells.2. The ROS with different level, produced during the early stage of reperfusion, inpostcoditioned by Pinacidil and Emulsified Isoflurane with varied concentration, activatesthe pathway of Nrf2-ARE, which realizes the effect of protection on myocardium tovarious extents. The ROS content, produced during the early stage of reperfusion, inpostcoditioned by Pinacidil(50μM) and Emulsified Isoflurane(1.68mM) is optimum torealize the best effect of protection on myocardium.