| Background:Hepatic ischemia-reperfusion often occurs in the process of liver surgery such as liver transplantation,which can lead to liver injury and distal organ injury,especially the heart injury.However,at present,there is no effective treatment for hepatic ischemia-reperfusion injury and its induced distal myocardial injury.Autonomic nervous system plays an important regulatory role in both liver and cardiovascular system.Previous studies have pointed out that when ischemia-reperfusion injury occurs in various organs,the sympathetic nervous system is overactivated,resulting in the imbalance of autonomic nervous system and aggravating tissue injury.Vagus nerve stimulation is an important measure of neuroregulation,which is often used in the clinical treatment of epilepsy and depression,and its efficacy and safety are considerably clear.Many studies have shown that vagus nerve stimulation can correct the imbalance of autonomic nervous system and play a significant protective role in heart,brain,kidney and skeletal muscle ischemia-reperfusion injury.However,the role of vagus nerve stimulation in hepatic ischemia-reperfusion injury and distal myocardial injury has not been elucidated.Objectives:1.To explore the effect of vagus nerve stimulation on acute hepatic ischemia-reperfusion injury.2.To explore the mechanisms of vagus nerve stimulation in the treatment of acute hepatic ischemia-reperfusion injury.3.To explore the effect of vagus nerve stimulation on myocardial injury induced by acute hepatic ischemia-reperfusion.4.To explore the mechanisms of vagus nerve stimulation in the treatment of myocardial injury induced by acute hepatic ischemia-reperfusion.Methods:The animals used in this study were SPF grade adult male Sprague-Dawley(SD)rats,weighing 220-260 g.All rats were fed in SPF barrier environment with an alternating 12-hour light/dark cycle.Before the experiment,the rats were under no restriction on food and water for 7 days to adapt to the environment.All experimental animals were fasted for 8 hours before operation and were anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 40 mg/kg body weight.During the whole operation,the body temperature of rats was maintained normal with heating pad.The operation of the experiment strictly abided by the guidelines for the Care and use of Experimental Animals.1.SD rats were randomly divided into three groups:(1)sham operation group(Sham group): n=6;the left cervical vagus nerve trunk was separated and connected with the vagus nerve stimulator,but the stimulator switch was not turned on;the hepatic pedicle innervating the left lobe and middle lobe of the liver was separated but not clamped;(2)hepatic ischemia-reperfusion injury group(IR group): n=6;the left cervical vagus nerve trunk was separated and connected with the vagus nerve stimulator,but the stimulator switch was not turned on;the hepatic pedicle innervating the left lobe and middle lobe of the liver was separated and clamped;(3)vagus nerve stimulation group(VNS group): n=6;the left cervical vagus nerve trunk was separated and connected with the vagus nerve stimulator,and the hepatic pedicle innervating the left lobe and middle lobe of the liver was separated and clamped.The vagus nerve stimulator was switched on when ischemia began,and the vagus nerve was stimulated continuously throughout the ischemia-reperfusion process,and the stimulation intensity was adjusted to a level that reduced the heart rate to about 90%of baseline.In IR group and VNS group,the vascular clamp was loosened to restore blood perfusion after 1 hour of ischemia,and the reperfusion time was 6 hours.At the end of the ischemia-reperfusion process,venous blood was taken from the portal vein to determine the levels of aspartate transaminase,alanine transaminase and lactate dehydrogenase in serum.The left lobe of the liver was taken.The pathological changes and hepatocyte apoptosis were observed by hematoxylin-eosin staining and terminal deoxynucleotidyl transferase d UTP nick end labeling.2.The procedure of grouping and experimental operation of SD rats was the same as that of part 1.At the end of the experiment,the left lobe of the liver was taken and the contents of malondialdehyde(MDA),glutathione(GSH)and the activities of superoxide dismutase(SOD)and catalase(CAT)in liver homogenate were detected by kits,and the m RNA expression levels of interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in liver tissue were measured by real-time fluorescence quantitative polymerase chain reaction.Western blotting was used to detect the relative protein levels of B-cell lymphoma-2(Bcl-2),Bcl2-associated X(Bax),nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB)p65,NF-κB phospho-p65(p-p65)and heme oxygenase 1(HO-1)in liver lysates and nuclear factor erythroid-2-related factor 2(Nrf2)in nuclear lysates.The expression of HO-1 protein in liver tissue was detected by immunohistochemical staining.3.SD rats were randomly divided into three groups: sham operation group(Sham group),myocardial injury group(MI group)and vagus nerve stimulation group(VNS group).The acute hepatic ischemia-reperfusion model was established in MI group and VNS group,and the whole vagus nerve stimulation was given in VNS group.At the end of the experiment,venous blood was taken from the inferior vena cava to determine the levels of serum cardiac troponin-I,creatine kinase-MB and lactate dehydrogenase-1.The left ventricular tissue was taken.The pathological changes and cardiomyocyte apoptosis were observed by hematoxylin-eosin staining and terminal deoxynucleotidyl transferase d UTP nick end labeling.4.The procedure of grouping and experimental operation of SD rats was the same as that of part 3.At the end of the experiment,venous blood was taken from the inferior vena cava and myocardial tissue was taken from the left ventricle.The contents of MDA,GSH and the activities of SOD and CAT in both serum and myocardial tissue were measured by kits.The m RNA expressions of IL-1β,IL-6 and TNF-α in myocardium was measured by real-time fluorescence quantitative polymerase chain reaction.The relative protein levels of Bax,Bcl-2,NF-κB p65 and NF-κB p-p65 in myocardium tissue were measured by Western blotting.Results:1.Vagus nerve stimulation significantly improved the liver histological injury induced by acute ischemia-reperfusion and decreased the Suzuki pathological scores of ischemic liver lobes.After acute hepatic ischemia-reperfusion,the serum levels of alanine transaminase,aspartate transaminase and lactate dehydrogenase increased significantly,while vagus nerve stimulation significantly decreased the levels of these serum enzymes.Ischemia-reperfusion injury significantly increased the number of apoptotic hepatocytes in liver tissue,while vagal nerve stimulation significantly decreased the number of apoptotic hepatocytes.All p<0.05.2.Vagus nerve stimulation significantly inhibited the process of apoptosis in ischemic hepatic lobes,increased the expressions of anti-apoptotic protein Bcl-2 and decreased the expressions of pro-apoptotic protein Bax.Vagus nerve stimulation significantly reversed the activation of inflammatory response in ischemic hepatic lobes,inhibited the expressions of pro-inflammatory factors IL-1β,IL-6,TNF-α and NF-κB p-p65 in liver tissue.Ischemia-reperfusion increased the level of oxidative metabolite MDA and decreased the levels of antioxidants GSH,SOD and CAT in liver tissue,while vagus nerve stimulation significantly reversed these changes and inhibited the levels of oxidative stress in ischemic liver lobes.After acute ischemia-reperfusion,the contents of Nrf2/HO-1 protein in liver tissue increased,and vagus nerve stimulation further increased the protein contents of Nrf2/HO-1.All p<0.05.3.Vagus nerve stimulation significantly improved myocardial histological injury caused by acute hepatic ischemia-reperfusion and decreased the scores of myocardial pathological injury.Hepatic ischemia-reperfusion increased the levels of serum cardiac troponin-I,creatine kinase-MB and lactate dehydrogenase-1,while vagus nerve stimulation significantly reduced the levels of myocardial injury markers.Vagus nerve stimulation significantly reversed the increase of apoptotic cardiomyocytes induced by hepatic ischemia-reperfusion.All p<0.05.4.Hepatic ischemia-reperfusion led to the increase of oxidative metabolite MDA and the decrease of antioxidants GSH,SOD and CAT in serum and myocardial tissue,and vagus nerve stimulation significantly reversed these changes.Vagus nerve stimulation significantly reversed the increase of Bax and the decrease of Bcl-2 in myocardial tissue after hepatic ischemia-reperfusion.Vagus nerve stimulation inhibited the expressions of pro-inflammatory cytokines IL-1β,IL-6 and TNF-α and phosphorylation of NF-κB p65 in myocardium after hepatic ischemia-reperfusion,thus alleviating the inflammatory response in myocardium.All p<0.05.Conclusions:1.Vagus nerve stimulation significantly reduced acute hepatic ischemia-reperfusion injury.2.Vagus nerve stimulation markedly inhibited oxidative stress,inflammation and apoptosis in liver tissue after hepatic ischemia-reperfusion,and enhanced Nrf2/HO-1 signal pathway,thus played a protective role in liver tissue.3.Vagus nerve stimulation significantly reduced the remote myocardial injury induced by acute hepatic ischemia-reperfusion.4.Vagus nerve stimulation markedly inhibited the systemic oxidative stress after hepatic ischemia-reperfusion and the process of oxidative stress,inflammation and apoptosis in myocardial tissue induced by hepatic ischemia-reperfusion,thus played a protective role in myocardial tissue. |
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