Study Of Delayed Preconditioning On Rat Myocardium Induced By Fentanyl Or Lipopolysaccharide

IntroductionIt has long believed that any kind of ischemia will do great harm on heart muscle,no matter how short the ischemia will last. But Murry etc. found that many times ofintermittent ischemia would not add together to injury heart cells. Murry presented theconcept "ischemic preconditioning" which means that transit innoxious ischemicpreconditioning will get heart cell the tolarence following long term ischemia.Perioperative Myocardia ischemia is a mostly seen pathophysiology. Especiallyin cardiac surgery or in non-cardiac surgical patient with coronary heart disease.Cardiac protection is very much importment in anesthesia performance andperioperative patient safety. Researchers were very much interested in ischemicpreconditioning from the very beginning and did a lot of experiments. It has beenproven that ischemic preconditioning has a lot of biological effects:①extenuatenecrosis following ischemic reperfusion.②Reduce fetal arrhythmia after ischemicreperfusion.③Is good for cardiac recovery from ischemicpreconditioning/reperfusion.④Promote myocardial energy metabolism. So far notonly IPC protection been observed both in animal and human subjects, but also it hasbeen regarded as the most effective protection for heart cells.Apart from ischemia, hot stress, rapid ventricular pacing, physical exercise,many medicine like endotoxin, IL-1, TNF-a, adenosine agonist, opioid agonist, etc.all can have protection effect like IPC.IPC has two phases. The first phase happens in 1-2 hours after preconditioningwhich is early preconditioning. The second phase happens in the 24-72 hours afterpreconditioning which is later or delayed preconditioning, or the second window of preconditioning. It is well accepted now that IPC triggers a diphase protection. Theearly phase is strong but short. The later phase is weak but long. The later phase,although weak, but can last as long as 24-72 hour, It has much more clinicalsignificance.Later preconditioning is the result of a successive events of heart muscle afterstress, there are two kinds of factors: trigger appears in the early phase which act asan initiator. Mediator appears at 24-72 hours after stress which act as the effector ofthe delayed protection. Signal cascade is started by trigger and result in the expressionof the mediator.A lot of triggers can start intracellular signal cascade which will finally lead tothe expression of mycardia protective genes. Accordingly the protein synthesisincreases. For example, PKC, protein tyrosine kinase(PTK), mitomycin activatedprotein kinase(MAPK), all can be the important pathway in the late preconditioningcascade.MAPK a kind of serine/threonine which lies ubiquitously in eukaryotic cell. Itmainly has three families: p44/p42 MAPK(ERK), p38 MAPK and p46/p54MAPK(JNK). It has been reported that LPC may trigger above mentioned threesubfamilies. Among these three MAPK may play a role in the signal delivery of LPC.But the true mechanism is still unknown.It has been studied that apioid like morphrine and fentanyl may have amyocardial protection like LPC. Low dose lipopolysaccharide(LPS) may also haveischemia-reperfusion protection.In my study we established a heart muscle ischemia-reperfusion model in rat.Compare fentany and LPS as trigger and mediator. Compare the hemodynamic, thearea of myocardial infarction, heart cell apoptosis, expression of p38 MAPK, inorder to study the mechanism of IPC and the role of MAPK.Materials1. experimental animals: healthy male SD rat, provided by china medical university laboratory animal center, weigh 250-350g.2. chemicals and reagents: 3% pentobarbital sodium(shanghai) Evans blue(shanghai) Lipopolysaccharide(escheruchia coli 055:B5, sigrna) Nalotone(Renfu pharmaceutical company, Yichang, Hubei) fentanyl(Renfu pharmaceutical company, Yichang, Hubei) TUNEL kit(Wuhan boster company) Caspase-3 kit:Beyotime Institute of Biotechnology Cytochrom C Antibody: New MarkerMonoclonal antibody of mouse Anti-rat p38 MAPK antigen and monoclonalAntibody of mouse Anti-rat phosphor p38 MAPK antigen: Beyotime Institute ofBiotechnologyWestern blotting 2nd Antibody:Santa Cruz3. Experimental instruments animal ventilator (HX-300, Chengdu Taimeng Scientific tech.) Datex anesthetic gas monitor (Finland) Hewlett-packard multiple physiology monitor(USA) 2020D gel imaging analytical system(united-biotechnology USA)Methods1. GroupingGrouping in experiment one1. Fen Fen+24hr+I/R2.LPS LPS+24hr+/fR3.NS NS+24hr+I/R4.Nal+Fen Nal+10min+Fen+24hr+I/R5.Nal+LPS Nal+10min+LPS+24hr+I/R6.Nal+NS Nal+10min+NS+24hr+I/R 7.Fen +Nal Fen+24hr+10min+Nal+I/R8.LPS +Nal LPS+24hr+10min+Nal+I/R9.NS +Nal NS+24hr+10min+Nal~I/R10.Con 24hr+I/RGrouping in experiment two1.Fen Fen2.LPS LPS3.NS NS4.Nal +Fen Nal+10min+Fen5.Nal+LPS Nal+10min+LPS6.Nal +NS Nal+10min+NS7. Con no medicationSampling and 30min, 2hr, 24hr after medication and measure the expression ofp38 MAPK, phosphor P38 MAPKDosing:Nalotone: 1mg/kgLPS: 1mg/kgFentanyl: 0.1mg/kg2 Establish of animal modelRats were anesthetized by pentobarbital 60mg/kg. when rats don't react to painstimulation, weigh the rats and fasten rat on the table. Tracheostomy was done andanimals were connected to animal ventilator. Venous and arterial catheterization weredone for infusion and sampling. Needle-like electrodes were inserted subcutaneouslyof extremities for electrocardial monitoring.Open chest via the left margin of the second and third rib. Cut the cardial sac andexpose the heart and great vessels. Chose right coronary vein near the intersection ofpulmonary artery cone and left heart aur as the landmark, put a thread under theramus descendens anterior arteriae coronariae sinistrae. Put a sponge between vessel and thread in case vessel torn while fastening and loosing for ischemia andreperfusion. Fasten the thread for 30min for ischemia, then loose it for reperfusion.The sign of successful ligation is the cyanosis of front heart wall and the ECG changeby ST segment, elevation.Monitoring the hemodynamic through the experiment. Gathering 2ml blood aftersacrifice animal for CK and LDH testing.3 Parameters and measurementsGrouping in experiment one were subjected to these test:1.hemodynamic data: record ECG, MAP, HR at 20min stable, 30min, 2hr and24hr after ligation.2.lab testing: measure the change of CK and LDH3.SOD and MDA measurement of heart tissue by spectrofluorimeter.4.infarction size: evans blue and TTC treat the tissue, dry and weigh the leftventricle, the area at risk and the infarction size. The ratio of infarction size andischemic area is the infarction scope.5.TUNEL method of measurement of heart cell apoptosis.6.Actavity of caspase-37.Cytochrome C, p38 MAPK and phospho P38 MAPK expression were testedby western blot after sacrifice the animal.Grouping in experiment two: test the p38 MAPK and phosphor P38 MAPKexpression by western blot at 30min, 2hr and 24hr after the last medication.4 Statistic analysisData were presented as mean SE and subjected to one-way analysis fo varianceusing SPSS 13.0 checked by students-Newman-Keuls test. Test within group ischecked by student t test. P＜0.05 is regarded as significant difference.Rrsults1. Hemodynamic MAP and HR don't have significant difference between groupsc. At 120min afterreperfusion in LPS group, 30min, 60min, 120min after reperfusion in LPS +Nal group,60min, 120min after reperfusion in Nal +LPS group, MAP has significant differencecompared with control level(P＜0.05). At 120min after reperfusion in LPS group,60min after reperfusion in Nal +LPS group, MAP was significantly going downcompared with Fen group. 120min after reperfusion in Fen +Nal group, 120min afterreperfusion in LPS+Nal group, HR is significantly going down compared with controllevel. MAP and HR were going down with time during experiment, especially ingroups with LPS(P＜0.05).2. CK and LDHCK and LDH are much lower in Fen and LPS group compared with Fen group.CK is lower in Nal +LPS group than in NS group(P＜0.05). CK and LDH is lower inNal +Fen group and in Fen +Nal group than in Fen group(P＜0.05). CK and LDH ishigher in LPS +Nal group than in LPS group(P＜0.05).3. SOD and MDA in heart tissue.SOD is lower in Fen, LPS and Nal +LPS group than in NS group(P＜0.05). SODis higher in Nal +Fen and Fen +Nal group than in Fen group(P＜0.05). SOD is higherin LPS +Nal group than in LPS group(P＜0.05). MDA change is the opposite to SOD.4.Infarction sizeinfarction size in Fen, LPS and Nal +LPS group is smaller than that in NSgroup(P＜0.05). Infarction size in Nal +Fen and Fen +Nal group is larger than that inFen group(P＜0.05). Infarction size in LPS +Nal group is larger than that in LPSgroup(P＜0.05).5. Cell apoptosisApoptosis in Fen, LPS, Nal +LPS group is fewer thanthat in NS group(P＜0.05).Apoptosis in Nal +Fen and Fen +Nal group is greater than that in Fen group(P＜0.05).apoptosis in LPS +Nal group is greater than that in LPS group(P＜0.05). 6. Actavity of caspase-3Caspase-3 in Fen, LPS, Nal +LPS group is fewer than that in NS group(P＜0.05).Caspase-3 in Nal +Fen and Fen +Nal group is greater than that in Fen group(P＜0.05).Caspase-3 in LPS +Nal group is greater than that in LPS group(P＜0.05).7. Cytochrome C, p38 MAPK and phosphor P38 MAPKexpressionCytochrome C in Fen, LPS, Nal +LPS group is fewer than that in NSgroup(P＜0.05). Cytochrome C in Nal +Fen and Fen +Nal group is greater than that inFen group(P＜0.05).Cytochrome C in LPS +Nal group is greater than that in LPSgroup(P＜0.05).p38 MAPK expression has no significant difference between groups, phosphorP38 MAPK expression in Fen, LPS, Nal +LPS is more than control level. Expressionin Fen, LPS, Nal +LPS is more than that in NS group(P＜0.05). Expression in Nal+Fen and Fen +Nal is less than that in Fen group(P＜0.05). Expression in LPS +Nalgroup is less than that in LPS group(P＜0.05).8. phosphor P38 MAPK expression at different time point ofGrouping in experiment two.30min after medication, phosphor P38 MAPK expression in Fen, LPS and Nal+LPS group is more than that in NS group(P＜0.05). 2hr and 24hr after medicationphosphor P38 MAPK expression was going down compared with that in NS group,but of no statistic significance(P＞0.05).DiscussionIt has long been an important study area that ischemia or medicinepreconditioning trigger intrinsic protective mechanism to provent injury. So far invivo and in vitro study has concluded that both ischemia and medicinepreconditioning can produce later myocardial protection. It was Schultz who first found that morphrine injection can mimic I/R to reduceinfarction area. So can intrathecal morphrine injection. Morphrine also have laterpreconditioning. Reduction of infarction area by morphrine has been tested in vivoand in vitro in different animal species. There is no doubt that morphrine has I/Rprotection on heart muscle.Fentanyl has wider clinical application that morphrine. But little has beenreported on preconditionging of fentanyl. It might because there are only d andκbutnoμopioid receptors in adult rats. Fentanyl might act on peripheral or central opioidreceptors because heart muscle does not haveμopioid receptors. LPS is the mainendotoxin of G- bacteria, which bind to mononuclear macrophage for biological effect.But sublethal dose of LPS has protective effect similar to I/R preconditioning.In my study, in group fentanyl and LPS, the infarction area is apparently reduced.The reduction has statistical significance which means fentanyl and LPS has laterpreconditioning. It is the same result as literature. We chose phosphocreatinekinase(CK), LDH, heart SOD and MDA to reflect the degree of injury. Results showit correlates to myocardial infarction area.Nalotone is d,κ,μopioid antagonist. In my study, among the four groups,fentanyl/LPS +nalotone and nalotone is applied in the trigger phase, nalotone didn'tstop the protection of LPS that the infarction area is decreasing. But in the other threegroups, the infarction area didn't change. The change of CK is correlated with theinfarction area. The late preconditioning induced by fentanyl can be stopped bynalotone either in trigger or in mediator phase. Nalotone, if applied in the mediatorphase, can also stop the preconditioning. But applied in the trigger phase, nalotonecannot stop the LPC induced by LPS, which means later preconditioning induced byLPS act on opioid receptors in mediator phase but not in trigger phase. We get aconclusion that the later preconditioning induced by different medicine may gothrough different pathway. The complicated mechanism worth further investigation.It has been well tested that cells will necrosis or apoptosis characterized biologically and morphologically after I/R. Experiments in vivo and in vitro also showthat early phase of IPC can decrease cell apoptosis. By far there is little report aboutapoptosis in delayed preconditioning. Baghelai etc. found that heart cell apoptosisdecreased when induce delayed preconditioning in isolated rat heart. In my study,compared with normal saline group, groups of fentanyl, LPS, Nal+LPS have muchless cell apoptosis, which is correlated with infarction area. We get a clue thatapoptosis lies in the mechanism of later preconditioning, but people not yet havefurther insight.In the internal pathway of apotosis, cytochrome-C come into cytoplasm throughPTP on mitochondria then activate Caspase. cytochrome-C and Caspase-3 play animportant role in apoptosis. IPC can decrease cardiomyocyte apoptosis rate throughinhibiting permeability of mitochondria, which decrease cytochrome-C leak. IPC alsodecrease apoptosis through inhibiting caspase activity.In this study, Caspase-3 and cytochrome-C in Fen, LPS, Nal+LPS groups is fewerthan that in NS group, which at equal pace with apoptosis rate. This indicate DPCdecrease apoptosis through down-regulation of Caspase-3 and cytochrome-C.Many in vivo or in vitro animal experiments show that ischemia and medicinepreconditioning will induce DPC. Although DPC is as common as EPC. But the realmechanism is still unknown. Current results show it may have something to do withheat shock protein, SOD, aldose reductase, NOase, COX-2 etc. the activation andtransportation of PKC is the key process in delayed preconditioning. Protein tyrosinekinase(PTK) may be the other protein kinase in IPC signal delivery. Activated PKCor PTK will get biological effect by phosphorylate downstream substrate.mitomycin activated protein kinase(MAPK) is a kind of serine/threonineprotein kinase whine lies ubiquitously in eukaryocyte. MAPK is the common pathwayfrom extracellular signal to nuclear effect which includes JNK, p38 MAPK and ERKs.Activated MAPK phosphorylate many substrate like transcription factors, cellskeleton protein and other enzyme to modulate cell physiology. Under pathophysiology MAPK is important nuclear pathway component in gene expressionand nuclear proliferation by multiple extracellular stimuli.Zhao etc. found that p38 participate in the delayed protection induced bymedicine preconditioning activated A1 receptor. He believes that p38 may be theterminal acceptor in delayed protection. Ting etc. found in their experiment inducedby foenicli that p38 is the trigger in delayed protection. Dana etc. found thatmyocardial infarction area was greatly decreasing if preconditioned by adenosineA1 agonist CCPA in rabbit ischemia-reperfusion model, meanwhile p38 activity is 7fold. All those would disappear if PKC or TK antagonist was applied in advance.When studying on mouse heart it is found that antagonist of p38MAPK can erasethe later protection. So we can draw a conclude p38 MAPK participate in the delayedprotection.In my study we can find that in group fentanyl, LPS and Nal+LPS, thephosphorylation of p38MAPK is greater compare with group normal saline. Thechange is correlated with the change of infarction area which imply that p38 MAPKparticipate in the delayed protection. It is the same as literature.In delayed protection little has been studied about the change with time. In mystudy we chose 30min, 2hr, 24hr after medication as time point to measure thephosphorylation of p38 MAPK without ischemia/reperfusion. Results show thephosphorylation of p38 MAPK is greatly going up to climax 30min after medicationin groups fentanyl, LPS and Nal+LPS. The phosphorylation goes down 2hr aftermedication and reach the original level at 24hr. in the three groups with nalotone thephosphorylation doesn't have significant difference compare with group normal saline.We can draw a conclusion that we can test the delayed protection 24hr aftermedication while the phosphorylation has already back to normal level, which meansthat p38 MAPK is not the final effector but rather it play its role through othersubstance.Further research is to investigate which subtype of p38 plays an important role in DPC, and the mutual impact of p38 and ERK or JNK, and the relationship amongsignaling molecule like PKC, PTK, NF-KB.Conclusions1. Both fentanyl and LPS can induce DPC on rat cardiomyocytes.2. As the trigger of DPC, the protection of LPS on heart muscle is independentof opioid receptors.3. Apoptosis cells decrease in DPC induced by fentanyl or LPS.4. There are decrease of Cytochrome C, caspase-3 and increase of phospho p38MAPK in DPC induced by fentanyl or LPS.5. P38 MAPK participate in DPC induced by fentanyl or LPS, thephosphorylation of p38 MAPK is increase at 2 hours and return to baseline at24hours.