Studies On The Myocardial Protective Effect Of δ Opioid Receptor Agonists Postconditioning In Rats With Acute Hemorrhagic Shock

Opioid receptor antagonists have been used for the treatment of shock for many years since the level of serumalβ-endorphin was found markedly elevated during shock. However, the effect was not always satisfied to the clinical application especially for hemorrhagic shock because therapeutic effect was varied with dose and animal species when naloxone was used. So, opioid receptor antagonists were not used extensively for the treatment of hemorrhagic shock. Recently, studies showed thatδopioid receptor agonist could recovery blood pressure, improve cardiac function, attenuate myocardial injury and prolong survive time. There are two subtypes ofδopioid receptor in brain and peripheral circulatory system. Which subtype activated could elicit effects of myocardial protection during hemorrhagic shock?Mitochondrial permeability transition pore (mPTP), as a nonspecific channel, remains closed during the ischemic period and only opens in the first few minutes of reperfusion. The open of mPTP can lead to mitochondrial swelling and efflux of cytochrome C, which induces apoptosis in the setting of ischemia/refusion (I/R) injury. Postconditioning (IPost) is taking reperfusion as point of cut-in to against I/R injury and relationship between IPost and opioid receptor has not been reported previously.In this study, acute hemorrhagic shock animal model was established according to a modification of Wiggers'method. The blood pressure, myocardial systolic function, myocardial morphological structure, mitochondrial damage score, and A540 change of myocardial mitochondria were observed to explore protective effects ofδ opioid receptor agonists (TAN-67 and Deltorpinâ…¡) on myocardial injury induced by acute hemorrhagic shock, and investigated the role of myocardial mPTP in the protection viaδ opioid receptor activation, and provided experimental basis for myocardial protection in acute hemorrhagic shock.Part 1 Myocardial protective effect ofδ₁ opioid receptor agonist in rats with acute hemotthagic shock Methods1. Twenty-four adult male rats weighting 250-300g were randomized into four groups (n=6): group NS, group TAN-67 (3mg/kg i.v.), group BNTX (10mg/kg i.v.), group BNTX+TAN-67. On the day of the experiment, rats were anesthetized with intraperitoneal injection of 30mg/kg sodium pentobarbital and then fixed on an operation table. After tracheotomy and placement of an endotracheal tube, spontaneous breathing was preserved. The left femoral artery was catheterized for bleeding and infusion shed blood. The right femoral artery was catheterized for measurement arterial blood pressure. The right carotid was isolated, and a catheter was inserted into left ventricle for hemodynamic measurement. The left jugular vein was catheterized for injection drugs and fluid. Catheters to artery and vein were all using PE-10 tubing. After arteries and vein were cannulated and electrocardiograms (ECG) were monitored, then each rat was injected with 3mg/kg heparin and stabilized for 15min. Animal model of hemorrhagic shock was established by a modification of Wiggers'method. Rats were bled from left femoral arterial catheter in 10 minute until MAP of～40mmHg was obtained. This level of blood pressure was maintained for 60min by withdrawal further blood or infusion shed blood. Then the total amount of blood withdrawn was recorded and shed blood were infused. HR, MAP, LVP, +dp/dtmax and–dp/dtmax were monitored continuously by using a transducer connected to a multichannel physiological monitor RM-6200.2. At the end of the experiment, a segment of myocardial tissue (1mm×1mm×1mm) was taken for observing ultrastructure of myocardial tissues by JEM-2000EX transmission electron microscope.3. FlaMeng'semi-quantitative analysis of mitochondria was used to analyze electron microscope specimens. Higher scores indicated more serious damage.4. At the end of experiment, myocardium was homogenated and gradient centrifugated at low temperature (4℃), myocardial mitochondria were isolated and absorbance at 540nm was measured by spectrometer. Decrease in light scattering and percentage of mitochondrial population undergoing permeability transition were closely parallel to each other.Results1. Hemodynamics of animals treated with group TAN-67 increased more than group NS. BNTX could eliminate the protective effect. 2. Myocardial ultrastructure in all electron microscopy specimens showed hypoxic injury to a greater or lesser degree. Myofilaments and mitochondria were roughly normal in group TAN-67. Myofilaments and mitochondria were serious injury in group BNTX.3. Score using FlaMeng'semi-quantitative method in group TAN-67 was less than group NS (P<0.05). BNTX could aggravate mitochondria damage.4. Changes in absorbance at A540 in group TAN-67 decreased significantly less compared with group NS (P<0.05). BNTX could eliminate the protective effect and changes in absorbance at A540 in group BNTX increased significantly compared with group NS (P<0.05).Part 2 Myocardial protective effect ofδ₂ opioid receptor agonist in rats with acute hemotthagic shockMethods1. Twenty-four rats weighting 250-300g were randomly divided into 4 groups: group DMSO, group Deltorpinâ…¡(1mg/kg i.v.), group NTB (2mg/kg i.v.) and group NTB+Deltorpinâ…¡.2. Animal model of hemorrhagic shock was established by a modification of Wiggers'method. HR, MAP, LVP, +dp/dtmax and–dp/dt_max were monitored continuously by using a transducer connected to a multichannel physiological monitor RM-6200. Ultrastructure of myocardial tissues was observed by JEM-2000EX transmission electron microscope. FlaMeng'semi-quantitative analysis of mitochondria was used to analyze electron microscope specimens. Myocardial mitochondria were isolated and absorbance at 540nm was measured by spectrometer. Results1. Hemodynamics of animals treated with Deltorpinâ…¡increased more than those of group DMSO. NTB could eliminate the protective effect.2. Myocardial ultrastructure protective effect of Deltorpinâ…¡was better than DMSO. Myocardial damage was more serious using NTB.3. Score in group Deltorpinâ…¡was less than group DMSO (P<0.05). Score in group NTB was higher than group DMSO (P<0.05).4. Changes in absorbance at A540 in group Deltorpinâ…¡decreased significantly compared with group DMSO (P<0.05). Deltorpinâ…¡could inhibit opening of the mPTP, and NTB could eliminate the effect.Conclusions1.δ₁ andδ₂ opioid receptor agonist could improve hemodynamics and attenuate myocardial injury and maintain normal structure of myocardium in acute hemorrhagic shock.δ opioid receptor agonist was an effective method to relieve myocardial damage in acute hemorrhagic shock based on fluid resuscitation.2.δ₁ andδ₂ opioid receptor agonist produced myocardial protection against ischemia/reperfusion (I/R) and improved function of mitochondrial in rats with hemorrhagic shock.3. The improvement of mitochondrial function was probably mediated by inhibiting opening of mPTP.