The Function Of Cholinergic Anti-inflammatory Pathway In Dexmedetomidine Against Myocardial Ischemia-reperfusion Injury

Background:Myocardial ischemia-reperfusion injury(myocardium ischemic reperfusion injury, MIRI) refers to myocardial ischemia result in tissue injury in a short period of time, when blood flow is restored, the phenomenon of myocardial ischemic injury is more serious, Commonly occurred in cardiac surgery, coronary artery bypass graft, viscera recanalization after blood supply infarction, etc., which have become the largest obstacles of get the best curative effect from the ischemic myocardial reperfusion therapy[1]. Myocardial ischemia-reperfusion injury often accompanies a body inflammatory reaction and tissue injury, its mechanisms include the generation of oxygen free radicals, calcium overload, myocardial energy metabolic disorders, apoptosis, neutrophil activation and inflammatory factor release, etc., and the process of MIRI is also a process of rising and deteriorating inflammation reaction [2-5].Studies have found that the cholinergic anti-inflammatory pathway(CAP) is probably one of the protective mechanism of MIRI [6].CAP is a nerve- immune regulating pathway, it plays roles through vagus nerve and the neurotransmitter acetylcholine interact with the immune system, participate in anti-inflammatory. Compared with the traditional humoral inflammatory mechanisms, CAP play roles by endogenous neural feedback regulation mechanism to achieve regulate inflammation reaction, alleviate inflammatory damage effect, Owing to its characteristics of faster and easier to control, CAP have more advantages in the inflammatory response control [7-9].Dexmedetomidine is a new type of high selective alpha 2- adrenergic agonist, widely applied in the intensive care unit(ICU) and in clinical anesthesia [10]. Study found that dexmedetomidine has a protective effect on lung, kidney and other organs injury, which can reduce apoptosis, reduce inflammatory reaction [11-13]. Recent studies have shown that dexmedetomidine has heart protection. Its heart protection may involve against ischemia-reperfusion injury, anti-arrhythmic and reduce postoperative cardiac complications[14-16] etc,. Dexmedetomidine has the characteristics of exciting vagus nerve, slowing heart rate and a strong anti-inflammatory effects. All of these pharmacological properties, are similar with those of CAP on acting on central nervous system, exciting vagus nerve and inhibiting the inflammatory response effectively. In addition, as a central sedative analgesics, the target of dexmedetomidine is central nervous system. Therefore, based on myocardial ischemia reperfusion in rats model, combined with research and analysis of clinical samples, the purpose of this topic is to discuss the CAP mechanism of dexmedetomidine anti-inflammatory and against myocardial ischemia-reperfusion injury. Objective:By establishing rat myocardial ischemia reperfusion model and analysis of clinical samples, explore the CAP mechanism of dexmedetomidine anti-inflammatory and against myocardial ischemia-reperfusion injury. It can provide theoretical support for its clinical application if the mechanism of dexmedetomidine against myocardial ischemia-reperfusion injury can be clarified. Methods:Part 1. Dexmedetomidine protection research of myocardial ischemia reperfusion1. The rat myocardial ischemia reperfusion model: 60 healthy rats were randomly divided into four groups, 15 in each group. Group one is pseudo surgery group; Group two is myocardial ischemia reperfusion model(control group), saline preconditioning 10 min before ischemic; A third group is myocardial ischemia reperfusion model(Dex), intravenous dexmedetomidine pretreatment for 10 min before ischemia. The fourth group is myocardial ischemia reperfusion model(experimental group), intravenous dexmedetomidine pretreatment for 10 min before ischemic, and cut off the vagus nerve. After anesthesia, animal is intubed by left femoral artery for arterial blood pressure, inserted by needle electrode into the limbs subcutaneous for standard Ⅱ lead electrocardiogram. By coronary ligation method in rats in vivo, give dispose to heart by 30 min ischemia and 180 min reperfusion, blood pressure drop in 20 mm Hg and electrocardiogram(ECG) S-T segment elevation for successful myocardial ischemia; Blood pressure and electrocardiogram(ECG) gradually recover means successful reperfusion. Serum and left ventricular myocardial tissue are rapidly taken after reperfusion.2. Myocardial tissue was processed by formaldehyde fixation, paraffin embedding and slice. After the eosin-wood grain(HE staining), myocardial morphology and inflammatory cell infiltration etc, were observed and myocardial infarction areas were calculated.3. Creatine kinase(CK), lactate dehydrogenase(LDH) levels in myocardial tissue and serum were detected by colorimetry.4. The serum TNF-α、IL-1β、IL-6 and HMGB1 levels are determined by using ELISA method.5. Grind myocardial tissue, then extract protein. Detect apoptosis factors in organization bcl-2、bax、caspase-3 and signaling molecules α7 n ACh R, p65, IkB-a expression by Western Blot.Part 2. The role of the cholinergic anti-inflammatory pathway in dexmedetomidine against myocardial ischemia reperfusion1. Build T calcium channel high expression and low expression in slow virus vector, respectively by intrathecal injection for transfection. T calcium channel high expression and low expression in rats respectively infusion dexmedetomidine(Dex group) and normal saline(control group), use the brain slice technology to detect changes in intracellular calcium ion concentration and activity of acetylcholine after injection drug.2. Take 20 rats with T calcium channel high expression and 20 rats with T calcium channel low expression, build myocardial ischemia reperfusion model respectively. Dexmedetomidine(Dex group) and normal saline(control group) was injected in the ischemia-reperfusion, 10 in each group. Test myocardial injury index each group: myocardial infarction area; lactate dehydrogenase(LDH), creatine kinase(CK) levels in plasma; TNF-α, IL-1β, IL-6 and HMGB1 levels in serum.3. 30 rats were divided into three groups. No processing was done in 10 rats in control group. a7 n Ach receptor antagonist α-BGT was given in 10 rats in low a7 n Ach group. a7 n Ach receptor agonists GTS-21 was given in 10 rats in high a7 n Ach group. Myocardial ischemia reperfusion were build in three groups rats, and dexmedetomidine pretreatment for 10 min in each group. Test myocardial injury index: myocardial infarction area; lactate dehydrogenase(LDH), creatine kinase(CK) levels in plasma; TNF-α, IL-1β, IL-6 and HMGB1 levels in serum.4. LPS induced inflammation of the vascular endothelial cells, si- RNA silenced α7 n ACh R. The detection of inflammatory factors TNF-α, IL-1β, IL-6 and HMGB1 RNA expression; detect apoptosis by flow cytometry; and detect p65, bcl- 2, bax expression.Part 3. Dexmedetomidine protection to myocardial ischemia-reperfusion injury of clinical patients64 patients who underwent elective CABG surgery with CPB from March 2013 to August 2014 were randomly divided into two groups. The patients received either Dex or normal saline. Dex pump infusion was started at a loading dose of 0.5μg kg–1 for 10 mins, followed by a continuous pump infusion of 0.5 μg kg–1 h–1 until the completion of surgery. The control group received the same infusion of normal saline. The mean arterial pressure(MAP) and HR were recorded at the following time points: baseline(before administration of Dex or normal saline)(T1), 15 mins after declamping the aorta(T2), 2 h after declamping the aorta(T3), and 24 h after declamping the aorta(T4).Central venous blood samples were drawn at the same time points. Results:1、Dexmedetomidine protection on myocardial ischemia reperfusion injury in rats(1) Tissue morphology results show: tissue injury caused by myocardial ischemia reperfusion significantly reduce, organizational form relatively intact, tissue apoptosis necrosis and inflammatory cells infiltration significantly reduce, myocardial infarction area significantly reduce after dexmedetomidine treatment.(2) Serum lactate dehydrogenase(LDH), creatine kinase(CK) levels decreased obviously in myocardial ischemia-reperfusion rats after dexmedetomidine treatment.(3) Serum TNF-α, IL-1β, IL-6 and HMGB1 levels significantly reduced, statistically significant differences in myocardial ischemia-reperfusion rats after dexmedetomidine treatment.(4) Myocardial tissue apoptosis factors in the expression of bax and apoptosis protein caspase-3 decreases significantly, while the expression of antiapoptotic factor bal-2 significantly increased in myocardial ischemia-reperfusion rats after dexmedetomidine treatment.2. The cholinergic anti-inflammatory pathway mediate dexmedetomidine anti-inflammatory against myocardial ischemia-reperfusion injury.(1) In the group of dexmedetomidine treatment but cut off vagus nerve myocardial ischemia reperfusion model, result shows that the action of dexmedetomidine against myocardial ischemia-reperfusion injury decreased significantly(myocardial infarction area is increased than Dex group, LDH and CK levels rise than Dex group; TNF-α, IL-1β, IL-6 and HMGB1 levels rise than Dex group).(2) In the group of dexmedetomidine treatment but cut off vagus nerve myocardial ischemia reperfusion model, the expression ofα7 n ACh R and Ik B-α clearly down-regulated, the expression of p65 up-regulated in the myocardial tissue of rats.(3) Similiar to dexmedetomidine pre-treatment, T calcium channel high expression also improve intracellular calcium ion concentration and enhance activity of acetylcholine; While in T calcium channel low expression rats, intracellular calcium ion concentration and acetylcholine activity obviously decrease.(4) Similiar to dexmedetomidine pre-treatment, T calcium channel high expression also have the same role against myocardial ischemia-reperfusion injury; While in T calcium channel low expression rats, myocardial ischemia-reperfusion injury obviously increase.(5) When use a7 n Ach receptor antagonist α-BGT processing, the action of dexmedetomidine against myocardial ischemia-reperfusion injury obviously decreased(myocardial infarction area is increased than Dex group, LDH and CK levels rise than Dex group; TNF-α, IL-1β, IL-6 and HMGB1 levels rise than Dex group); Similiar to dexmedetomidine processing, a7 n Ach agonists GTS-21 processing have the same action against myocardial ischemia-reperfusion injury.(6) LPS induced inflammation in vascular endothelial cells, silence after α7 n ACh R, inflammatory factors TNF-α, IL-1β, IL-6 and HMGB1 levels were found obviously increases; Apoptosis percentage increased obviously; Promote the expression of bax apoptosis factors and apoptosis protein caspase-3 decreases significantly, while the expression of antiapoptotic factor bal-2 significantly increased; NF-kappa B signaling pathway molecules P65 expression raised obviously, while the expression of inhibitory molecules Ik B-α clearly down-regulated.3. Dexmedetomidine protection on myocardial ischemia-reperfusion clinical patients(1) Compared with control group, MAP and HR are at relative normal levels in experimental group patients.(2) Compared with control group, c Tn I and CK levels are lower in experimental group.(3) Compared with control group, serum TNF-α, IL-6, IL-8, IL-10 levels are lower in experimental group. Conclusion:In this paper, by using the model of myocardial ischemia-reperfusion injury in rats, and dexmedetomidine pre-treatment, detect and analysis myocardial tissue, inflammatory reaction and apoptosis, choline signal pathway of rats after ischemia reperfusion injury. Draw the following conclusion:(1) Dexmedetomidine can significantly reduce rat myocardial tissue injury induced by myocardial ischemia reperfusion.(2) Dexmedetomidine through the activation of T type calcium channel to improve the intracellular calcium ion concentration and the vagus nerve excitement, activating cholinergic anti-inflammatory pathway, enhance the activity of acetylcholine to alleviate myocardial ischemia-reperfusion injury in rats.(3) Dexmedetomidine may through activating cholinergic anti-inflammatory pathway to alleviate myocardial ischemia-reperfusion injury clinical patients.