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Objective To investigate the effect of intrathecal morphine preconditioning (ITMP) on the excitability of substantia gelatinosa (SG) neurons in the dorsal horn of the spinal cord in a rat model of myocardial ischemia-reperfusion (I/R).Methods Thirty-six adult male Sprague-Dawley rats,weighing 200-300 g,in which intrathecal catheters were successfully placed without complications,were randomly divided into 3 groups (n =12 each) using a random number table:sham operation group (group S),group I/R,and group ITMP.Myocardial I/R injury was produced by occlusion of the left anterior descending branch of the coronary artery for 30 min followed by 120 min reperfusion.In group ITMP,the rats received intrathecal morphine 3 μg/kg (10 μl) by three cycles of 5 min infusions interspersed with 5 min infusion-free periods starting from 30 min before ischemia,and the equal volume of normal saline was given instead of morphine in group I/R.At 10 min of reperfusion,6 rats randomly selected in each group were sacrificed,and the T2-6 segments of the spinal cords were acutely isolated to prepare spinal cord slices.The resting potential,threshold of action potential (APT),peak of action potential (APP) and action potential duration in SG neurons in the dorsal horn of spinal cord slices were determined using the whole-cell patch-clamp technique,and the number of action potentials evoked by currents of 40,60,80 and 100 pA was recorded.At 120 min of reperfusion,6 rats randomly selected in each group were sacrificed,and myocardial specimens were obtained for determination of myocardial infarct size (IS) and area at risk (AAR),and IS/AAR ratio was calculated.The expression of c-fos in the T2-5 dorsal horns of the spinal cords was detected by Western blot.Results Compared with group S,the IS/AAR ratio was significantly increased,the expression of c-fos was up-regulated,the number of action potentials in SG neurons in dorsal horns of spinal cord was increased,APT was decreased,and APP was increased in group I/R (P＜0.05).Compared with group I/R,the IS/AAR ratio was significantly decreased,the expression of c-fos was down-regulated,the number of action potentials in SG neurons in dorsal horns of spinal cord was decreased,APT was increased,and APP was decreased in group ITMP (P＜0.05).Conclusion The mechanism by which ITMP attenuates myocardial I/R injury is related to decrease in the excitability of SG neurons in the dorsal horn of the spinal cord and reduction of responses to nociceptive stimuli in rats.
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Objective To investigate the role of p38 mitogen-activated protein kinase (p38MAPK) signaling pathway in reduction of myocardial ischemia-reperfusion (I/R) injury by morphine preconditioning in the rats with chronic heart failure in vitro.Methods Adult male Sprague-Dawley rats,weighing 200-230 g,aged 6-7 weeks,in which doxorubicin 2 mg/kg was injected via the tail vein once a week for 6 consecutive weeks to induce chronic heart failure,were studied.At the end of 8th week,30 rats with chronic heart failure were randomly divided into 5 groups (n =6 each) using a random number table:sham operation group (group Sham),I/R group,morphine preconditioning group (group MPC),SB203580 (p38MAPK inhibitor) + morphine preconditioning group (group SBM),and SB203580 group (group SB).The hearts were quickly excised and passively perfused in a Langendorff apparatus and subjected to 30 min of occlusion of the anterior descending branch of the left coronary artery followed by 120 min of reperfusion to establish the model of myocardial I/R injury.After equilibration,the hearts were subjected to 3 cycles of 5 min perfusion with K-H solution containing morphine 1 μmol/L at 5-min intervals before ischemia in group MPC.In group SBM,the hearts were perfused with K-H solution containing SB203580 (5 μmol/L) for 45 min starting from l0 min before morphine preconditioning until 5 min of ischemia.In group SB,morphine preconditioning was not performed,and the hearts were only perfused with K-H solution containing SB203580 (5 μmol/L) starting from 40 min before ischemia until 5 min of ischemia.At 15 min of equilibration (baseline),5 and 10 min of reperfusion,the coronary effluent was collected to detect the activity of lactate dehydrogenase (LDH) using the chemical colorimetry.At 10 min of reperfusion,the expression of phosphor-p38MAPK (p-p38MAPK) in the myocardium was determined by Western blot in Sham,I/R and MPC groups.At 120 min of reperfusion,the area at risk (AAR),total areas of right and left ventricles (LV+RV),and infarct size (IS) were measured,and the IS/AAR ratio was calculated.Results Compared with group Sham,the LDH activity in coronary effluent during reperfusion and IS/AAR ratio were significantly increased in the other groups,and the expression of p-p38MAPK was significantly up-regulated in I/R and MPC groups (P＜0.05).Compared with group I/R,the LDH activity in coronary effluent during reperfusion was significantly decreased,the expression of p-p38MAPK was significantly up-regulated,and the IS and IS/AAR ratio were significantly decreased in group MPC (P＜0.05),and no significant change was found in the LDH activity in coronary effluent,IS and IS/AAR ratio in SBM and SB groups (P＞0.05).Compared with group MPC,the LDH activity in coronary effluent during reperfusion was significantly increased,and the IS and IS/AAR ratio were significantly increased in group SBM (P＜0.05).Conclusion The mechanism by which morphine preconditioning reduces myocardial I/R injury is related to activation of p38MAPK signaling pathway in the rats with chronic heart failure in vitro.
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Objective To investigate the effect of intrathecal morphine preconditioning on the expression of nerve growth factor (NGF) in the dorsal root ganglia (DRG) in a rat model of myocardial ischemia-reperfusion (I/R).Methods Thirty healthy adult male Sprague-Dawley rats in which intrathecal catheters were successfully placed without complications,weighing 250-350 g,were randomly divided into 5 groups (n =6 each) using a random number table:sham operation group (S group),I/R group,intrathecal morphine preconditioning group (ITMP group),μ receptor antagonist CTOP + intrathecal morphine preconditioning group (CTOP + ITMP group),and CTOP control group (CTOP group).Myocardial ischemia was induced by 30 min of occlusion of the anterior descending branch of the left coronary artery followed by 120 min of reperfusion in all the groups except S group.Intrathecal morphine preconditioning was produced by 3 cycles of 5 min intrathecal injection of morphine 3 μg/kg (10 μl) at 5 min intervals within 30 min before ischemia in ITMP group.In CTOP+ITMP and CTOP groups,1 μg/μ1 CTOP 10 μl was injected intrathecally at 10 min before morphine preconditioning and 40 min before ischemia,respectively.At 120 min of reperfusion,the rats were sacrificed,and myocardial specimens were obtained for determination of myocardial infarct size,and DRGs were removed for determination of the expression of NGF by using immunohistochemistry and Western blot.Results Compared with S group,the myocardial infarct size was significantly increased,and the expression of NGF in DRGs was significantly up-regulated in I/R group (P＜0.05).Compared with I/R group,the myocardial infarct size was significantly decreased,and the expression of NGF in DRGs was significantly down-regulated in ITMP group (P＜ 0.05),and no significant change was found in the parameters mentioned above in CTOP group (P＞0.05).Compared with ITMP group,the myocardial infarct size was significantly increased,and the expression of NGF in DRGs was significantly up-regulated in CTOP+ITMP and CTOP groups (P＜0.05).Conclusion The mechanism by which intrathecal morphine preconditioning reduces myocardial I/R injury is related to activation of spinal μ receptors,inhibition of NGF expression in DRGs,and reduction of responses to noxious stimulation in the rats.
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Aim To evaluate the effects of morphine preconditioning ( MPC ) on the expression of microR-NAs ( miRNAs ) induced by hypoxia-reoxygenation (H/R) in H9c2 myocardial cells. Methods H9c2 cells were randomly divided into 3 groups ( n=4 each) as follows:control group ( CON) , hypoxia/ reoxygen-ation group ( H/R ) and morphine preconditioning group ( MPC+H/R) . The cells were cultured in nor-mal condition in CON group. The cells were subjected to 5 h hypoxia followed by 1 h reoxygenation in H/R group and MPC+H/R group. Specifically, the cells in MPC+H/R group were preconditioned with morphine with the final concentration of 1 μmol·L-1 for 10 min before H/R. After the treatment, CCK-8 was used to detect cell viability and chemical colorimetry was used to detect lactate dehydrogenase ( LDH ) activity in the culture medium. Cell apoptosis was assessed by An-nexin-V-FITC/PI flow cytometry. Relative expression of Fas protein was detected by Western blot. The ex-pression of miRNA in myocardial cells was analyzed by quantitative reverse transcription polymerase chain re-action ( qRT-PCR ) . Results Compared with CON group, the cell viability was significantly decreased, while the LDH activity, apoptotic rate and Fas protein expression were dramatically increased in group H/R (P<0. 01). However, MPC significantly increased the cell viability, whereas it decreased the LDH activity, apoptotic rate and Fas protein expression induced by H/R injury ( P < 0. 01 ) . The expressions of miR-133a-5p, miR-133b-5p, miR-664-1-5p, miR-6216 and let-7 e-5 p were markedly down-regulated by H/R as compared to CON group ( P <0. 05 ) , while MPC inhibited these miRNAs which were significantly down-regulated by H/R group ( P <0. 01 ) . Conclusion Morphine preconditioning might protect H9 c2 myocar-dial cells against H/R injury by regulating the expres-sion of miRNAs such as miR-133a-5p, miR-133b-5p, miR-664-1-5p, miR-6216 and let-7e-5p.
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Objective To evaluate the role of microRNA-133b-Sp (miR-133b-Sp) in apoptosis hypoxia/reoxygenation (H/R) induced by in rat cardiomyocytes.Methods Rat myocardial cell line H9c2 was cultured in DMEM/F12 culture medium supplemented with 10％ fetal bovine serum.The cells were seeded in 96-well or 6-well plates and randomly divided into 4 groups (n=64 wells each):control group (group C);group H/R;miR-133b-5p mimic + H/R group (group M+H/R);miR-133b-Sp negative control + H/R group (group NC+H/R).The cells were exposed to 95％ N2-5％ CO2for 5 h at 37 ℃ followed by 1 h reoxygenation in DMEM/F12 culture medium supplemented with 10％ fetal bovine serum in all the groups except group C.The cells were cultured in normal culture atmosphere in group C.In M+H/R and NC+H/R groups,the cells were transfected with miR-133b-5p mimic (final concentration 30 nmol/L) and miR-133b-5p negative control (final concentration 30 nmol/L),respectively,for 24 h before H/R.Total RNA was extracted from cells to detect the expression of miR-133b-5p using quantitative real-time PCR.The cell viability (by CCK-8) and lactic dehydrogenase (LDH) activity in the culture medium were detected.Cell apoptosis was assessed by Annexin V/PI flow cytometry.Apoptotic rate was calculated.Result Compared with group C,the cell viability was significantly decreased,and the LDH activity and apoptotic rate were increased in H/R,M+H/R and NC+H/R groups,the expression of miR-133b-5p was down-regulated in H/R and NC+H/R groups,and the expression of miR-133b-Sp was up-regulated in group M+H/R.Compared with group H/R,the cell viability was significanttly increased,the LDH activity and apoptotic rate were decreased,and the expression of miR-133b-5p was up-regulated in group M+H/R,and no significant change was found in the parameters mentioned above in group NC+H/R.Conclusion H/R in rat cardiomyocytes can induce cell apoptosis possibility through down-regulating the expression of miR-133b-5p
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Objective To evaluate the roles of 1-phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) signaling pathways in reduction of ischemia-reperfusion (I/R) injury to the isolated hearts by morphine preconditioning in the rats with chronic heart failure.Methods Adult male Sprague-Dawley rats,weighing 200-230 g,in which doxorubicin 2.0 mg/kg was injected via the tail vein once a week for 6 weeks to induce chronic heart failure,were studied.At the end of 8th week,42 rats with chronic heart failure were randomly divided into 7 groups (n =6 each) using a random number table:sham operation group (group S),I/R group,morphine preconditioning group (group MP),PD98059 (ERK inhibitor) + morphine preconditioning group (group PD + MP),wortmannin (PI3K inhibitor) + morphine preconditioning group (group WT + MP),PD98059 group (group PD) and wortmannin group (group WT).The hearts were quickly excised and passively perfused in a Langendorff apparatus and subjected to 30 min of occlusion of the left coronary artery followed by 2 h of reperfusion to establish the model of I/R injury.In group S,the hearts were only sutured,but not ligated and were continuously perfused with K-H solution for 195 min.In group I/R,the hearts were perfused with K-H solution for 45 min before ischemia.In group MP,the hearts were perfused with K-H solution for 15 min,with K-H solution containing morphine 1 μmol/L for 5 min and then with K-H solution for 5 min (3 cycles in total) before ischemia.In PD + MP and WT + MP groups,the hearts were perfused with K-H solution containing PD98059 (10 μmol/L) and wortmannin (100 nmol/L),respectively,starting from 10 min before morphine preconditioning until 5 min of ischemia.In PD and WT groups,the hearts were perfused with K-H solution containing PD98059 (10 μmol/L) and wortmannin (100 nmol/L),respectively,starting from 40 min before ischemia until 5 rin of ischemia.At 15 min of equilibration (baseline) and 5 and 10 min of reperfusion,the coronary flow was collected to detect the activity of lactate dehydrogenase (LDH).Infarct size (IS) and area at risk (AAR) were measured at the end of reperfusion and IS/AAR ratio was calculated.Results Compared with group S,LDH activity was significantly increased at 5 and 10 min of reperfusion,IS and IS/AAR ratio were also increased (P ＜ 0.05),and no significant change was found in AAR in group I/R (P ＞ 0.05).Compared with group I/R,LDH activity was significantly decreased at 5 min of reperfusion,IS and IS/AAR ratio were also decreased (P ＜ 0.05),and no significant change was found in AAR in group MP,and no significant change was found in LDH activity,IS,AAR and IS/AAR ratio in WT and PD groups (P ＞ 0.05).Compared with group MP,LDH activity was significantly increased at 5 and 10 min of reperfusion (P ＜ 0.05),and IS and IS/AAR ratio were decreased in group PD + MP,and no significant change was found in LDH activity,IS,AAR and IS/AAR ratio in group WT + MP (P ＞ 0.05).Conclusion Activation of ERK signaling pathway is involved in reduction of I/R injury to isolated hearts by morphine preconditioning in rats with chronic heart failure,however,PI3K signaling pathway has no such effect.