| Objective: Coronary artery bypass grafting(CABG)is widely used in cardiovascular surgery.It is mainly used in surgical treatment of coronary heart disease,which has been at a high incidence rate.Myocardial ischemia-reperfusion injury(MIRI)is difficult to avoid during CABG surgery.The occurrence of MIRI can be accompanied by the activation of a series of inflammatory factors,which can reach distant target organs through the circulation of the body and play a harmful role,especially in the central nervous system.Nowadays,more and more studies have elucidated the relationship and the occurrence mechanism between myocardial ischemia-reperfusion and brain injury.It has also been documented that myocardial ischemia-reperfusion injury is able to cause postoperative cognitive dysfunction(POCD).In the daily clinical surgical treatment,we also found that with the rapid and continuous improvement of modern surgical technology and surgical instruments,the survival rate of CABG has been greatly improved,and the postoperative complications have been effectively controlled,but the occurrence of postoperative neurological damage and POCD is increasingly apparent.Kallikrein-kinin system(KKS)is a well-established inflammatory regulatory system,in which Bradykinin B1 receptor(B1R)and Bradykinin B2 receptor(B2R)play a pivotal role as upstream key targets of angiogenic and inflammatory signaling pathways.In the normal physiological state of the body,the expression level of B1 R is very low;but when the tissue function is damaged,the expression level of B1 R rises rapidly,and further plays its biological role,so B1 R is called the injury-induced receptor.Dexmedetomidine(Dex),a potent alpha 2 adrenergic receptor agonist,is a commonly used anesthetic adjunct in many clinical departments.Various studies have recently shown that it has sedative,analgesic,anxiolytic effects and is able to improve postoperative patients' cognitive function,but the exact mechanism still needs further study.Mitogen activated protein kinase(MAPK)is a family of protein kinases containing serine/threonine residues.The p38 MAPK pathway is one of the members of the MAPK signaling pathway,it has a serious effect when the inflammation occurs to the body,and it is also an acceleration part in promoting apoptosis.Recently,it has been speculated that B1 receptor is mainly related to the activation of p38 MAPK pathway.Therefore,we speculated that changes in the level of inflammatory factors during the process of MIRI may activate KKS and trigger a series of pathophysiological changes.This topic through the establishment of rat MIRI model to study far distant organ functional fault caused by myocardial ischemia reperfusion injury and explore dynamic changes in the organization of B1 R in myocardial ischemia-reperfusion injury,and by using dexmedetomidine to inhibit inflammatory reaction and apoptosis in order to reach the role of protective effect in central nervous system,and further study the possible mechanism for the clinical work after cardiac surgery in the treatment of patients with cognitive dysfunction.Methods: 1.Seventy-two Sprague-Dawley(SD)SPF rats weighing 200±20g were selected and randomly divided into 2 groups: 36 rats per group.To the rats in the MIRI group,they were performed thoracotomy and to expose the heart,then we ligature the left anterior descending coronary artery.While in the other group,only thoracotomy was performed to expose the heart.The rats were subjected to Morris water maze behavior experiment at the 7th day of reperfusion.At the time node of 2h,6h,24 h,72h,7d,we randomly choose six rats from the two groups,and then they were anesthetized.After they were anesthetized,blood was collected via abdominal aortic puncture and used to detect IL-1 ?,IL-6,and TNF-? levels in serum by ELISA.After blood collection,whole brain tissue was collected,the whole brain was aliquoted along the longitudinal fissure of the brain.We choose the left brain,the expression of B1 R,p38MAPK in brain tissue was detect by immunofluorescence method.The right half brain tissue was isolated from the frontal and temporal cortices,frozen in liquid nitrogen.After that we transport the brain tissue into a-80°C freezer,they were stored there for later experiment.ELISA is used to detect inflammatory factors content,respectively Western blot is used to detect the expression levels of B1 R,p38 MAPK and p-p38 MAPK.2.Select thirty-six Sprague-Dawley(SD)SPF rats weighing 200±20g and randomly divide them into three groups as follows: sham,MIRI+ normal saline(NS)and MIRI +dexmedetomidine(DEX)groups.The MIRI rat model was replicated as before.Then anesthetize rats in each group with 4% sodium pentobarbital by intraperitoneal injection.After anesthetized,rats in the MIRI+NS group were given a bolus of normal saline 1ml via the tail vein before surgery and then continuously pumped into NS at a rate of 1ml/h using a micro-injector pump until the end of reperfusion.Rats in the MIRI+ DEX group received a bolus injection of dexmedetomidine 5?g/kg(5?g/ml)via the tail vein preoperatively and then continuously pumped at 5?g/kg/h using a micro-syringe pump until the end of reperfusion.After 24 h of reperfusion,the samples were fixed as described above.Western blot analysis is used to test the protein expression levels of p38 MAPK,p-p38 MAPK,ERK1/2,p-ERK1/2,NF-?B,p-NF-?B and B1 R.The levels of those inflammatory factors as above were detected by ELISA,B1 R expression were detected by immunofluorescence,and the levels of apoptosis in brain tissues were determined by TUNEL assay.After 7 days of reperfusion,Morris water maze behavioral experiments were performed.3.Thirty-six Sprague-Dawley(SD)SPF rats weighing 200±20g were selected to replicate the same previous MIRI rat model,then the MIRI model rats were randomly divided into six groups: MIRI + NS group,MIRI+DEX group,MIRI+SB203580 group,MIRI+DEX+SB203580 group,MIRI+des-arg9-BK group,and MIRI+DEX+des-arg9-BK group.Rats in the MIRI+NS group and the MIRI+DEX group were administered the same way as before,supplemented by the fact that rats in the MIRI +NS group were reinjected with NS 1ml via tail vein at 12 h of reperfusion after surgery.The rats in the MIRI+DEX group were reinjected with dexmedetomidine at 5?g/kg(5?g/ml)via tail vein at 12 h of postoperative reperfusion,the rats in the MIRI+SB203580 group were reinjected with SB203580(5mg/kg)intravenously before reperfusion,and the rats in the MIRI+DEX +SB203580 group were reinjected with SB203580(5mg/kg)via tail vein at 12 h of postoperative reperfusion.The rats in the MIRI+DEX+SB203580 group received dexmedetomidine(5mg/kg)intravenously before reperfusion,SB203580(5mg/kg)was reinjected via the tail vein 12 h after reperfusion,followed by dexmedetomidine 5?g/kg(5?g/ml)was reinjected via the tail vein 20 min apart.In the MIRI+des-arg9-BK group,des-arg9-BK(100nmol/kg)was intravenously administered before reperfusion,and des-arg9-BK(100nmol/kg)was reinjected via the tail vein 12 h after reperfusion.IN dex+des-arg9-BK group:intraoperative administration of dexmedetomidine as above;before reperfusion,des-arg9-BK(100nmol/kg)was administered intravenously and after 12 h of reperfusion,des-arg9-BK(100nmol/kg)was administered via the tail vein followed by a second injection of dexmedetomidine 5?g/kg(5?g/ml)via the tail vein at 20 min intervals.The rats from each group were anesthetized at 24 h of reperfusion as described above.Inflammatory factor content was assessed by ELISA,and protein expression was assessed by immunofluorescence and Western blot as described above.Results: 1.The water maze test showed that the rats in the MIRI,MIRI+NS,MIRI+DEX groups had increased escape latency,decreased number of crossing the platform,caused learning and memory dysfunction compared with the sham group,while the learning and memory abilities were improved after DEX intervention.2.The protein expression levels of B1 R,p38,and p-p38 increased in the MIRI group at each time point,reaching the highest level at 24 h.3.The levels of IL-1 ?,IL-6 and TNF-? in brain tissue of MIRI rats at each time point were significantly increased,as well as the level in serum.The time of peak is 24 hours after reperfusion;the levels in the groups at 72 h and 7d after reperfusion significantly decreased than those in the group of peak time group.4.The levels of inflammatory factors in the MIRI+NS group and MIRI+DEX group were significantly increased compared with those in the group who were not ligatured,and the expression levels of p38 MAPK,p-p38 MAPK,ERK1/2,p-ERK1/2,NF-?B,p-NF-?B and B1 R in brain tissue of rats in the MIRI+DEX group were significantly decreased than those in the MIRI+NS group.5.Levels of inflammatory factors in the MIRI+DEX,MIRI+SB203580,and MIRI+SB203580+DEX groups had significantly reduced,as well as p38 MAPK,p-p38 MAPK,ERK1/2,p-ERK1/2,NF-?B and p-NF-?B in brain tissue.Moreover,the protein expression levels of p-NF-?B and B1 R were significantly decreased,but increased more in the MIRI+des-arg9-BK group than the MIRI+DEX group and the MIRI+SB203580group.The MIRI+des-arg9-BK group compared with the MIRI+DEX group and the MIRI+des-arg9-BK group compared with the MIRI+SB203580+DEX group,significantly higher levels of the above-mentioned inflammatory factors were detected in the brain and serum.There are the same rusults in the des-arg9-BK+DEX group compared with the MIRI+NS group.Conclusion: Myocardial ischemia-reperfusion is able to cause brain tissue damage,and at 24 h after reperfusion,the inflammatory response and B1 R receptor expression levels in the brain peak,and the p38 MAPK signaling pathway is activated.Dexmedetomidine may have anti-inflammatory effect on MIRI rats by down-regulating the expression of B1 R,and then inhibiting the over-activation of p38 MAPK signaling pathway. |
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