| With the aging of the population,the incidence of cerebrovascular diseases in China is getting higher and higher.Brain tissue is highly sensitive to ischemia.Once ischemia occurs,pathological changes will occur rapidly,and it will soon enter I/R reversible damage state.After blood flow recovery,it will easily trigger a series of complex cascades and neuronal apoptosis,necrosis and delayed neurological dysfunction,ie ischemia-reperfusion injury(I/RI).Large release of oxygen free radicals,calcium overload,no excessive release,excitatory amino acids,and release of inflammatory factors may be involved in cerebral ischemia-reperfusion injury.After ischemia-reperfusion injury,it is easy to cause long-term nervous system dysfunction,and learning,memory and cognition will be affected.Clinically,transient cerebral ischemia-reperfusion injury usually occurs after first-aid measures such as cardiopulmonary resuscitation,cardiac arrest,and rescue of shock patients,perioperative neurosurgery,cardiopulmonary bypass is also prone to occur.Cholinergic anti-inflammatory pathway(CAP)is a neuro-immune regulatory pathway,whose activation can effectively reduce the release of pro-inflammatory factors and has an obvious inflammatory inhibitory effect.It is an endogenous neurofeedback regulatory mechanism:Immune stimulation information received central nervous,the project signal to the vagus nerve nuclei,activate the vagus nerve,make the peripheral nerve endings release Ach,acetylcholine as one of the main cell signaling molecules,after the release and epithelial cells,endothelial cells,skin cells,immune cells and secretory cells or other expression of acetylcholine receptors,especially with alpha 7 subunits of specific binding of n-type acetylcholine receptor(nicotinic acetylcholine receptors alpha 7,alpha 7 nachR),by intracellular signaling pathways,Participate in many interrelated biological processes,such as proliferation,differentiation,adhesion,migration,and apoptosis.Apoptosis is a kind of programmed cell death,which mainly includes three stages:signal transduction,gene regulation and apoptosis.The activation of signal transduction is the initiator of apoptosis.p38MAPK/NF-κB is an important signal transduction pathway in the cell and plays an important role in cell proliferation and apoptosis.Mitogen-activated protein kinases(MAPKs)are serine-threonine protein kinases involved in many physiological processes such as cell growth,proliferation,differentiation and apoptosis.p38MAPK is an important member of the MAPKs family.It mainly mediates signaling such as inflammation,stress,and injury.It plays a role in regulating inflammation,neurodegenerative changes,and neuronal apoptosis.Abnormal overactivation of the p38 pathway cause cells growth stagnation and apoptosis.Dexmedetomidine(Dex)is a sedative drug commonly used in clinical practice.It also has analgesic and anxiolytic effects,mainly acting on the a-2 receptor.Recent studies have confl/Rmed that dexmedetomidine can activate mitogen-activated protein kinase(MAPK)in rats to protect against brain injury induced by subarachnoid hemorrhage,and also inhibit the release of pro-inflammatory factors and reduce calcium overload.Dexmedetomidine also prevents myocardial,liver,intestinal,lung,kidney and brain damage caused by ischemia-reperfusion injury.At the same time,it can also inhibit mitochondrial apoptosis,inhibit oxygen sugar deprivation,and inhibit calcium overload to maintain the function of mitochondria.The above studies showed that dexmedetomidine had anti-apoptotic,anti-inflammatory,and brain protective effects,but the doses of dexmedetomidine were very different and were not compared.Based on this,we hypothesized that dexmedetomidine has a protective effect on cerebral ischemia-reperfusion injury,and the protective effect is dose-related.Dexmedetomidine can inhibit the sympathetic nerve,indirectly stimulate the parasympathetic nerve,and slow down the heart rate.Similar to the vagus nerve,this paper intends to explore the brain protective effect of dexmedetomidine from the perspective of cholinergic anti-inflammatory pathway.This study includes the following two aspects:1.Whether the dose-related effects of dexmedetomidine in different dosages on the reduction of brain injury and inhibition of apoptosis of nerve cells.2.To investigate whether the cholinergic anti-inflammatory pathway is involved in the neuroprotective effect of dexmedetomidine.Part One:The effect of different doses of dexmedetomidine on hippocampal nerve in rats with ischemia-reperfusion injuryObjective:To investigate the effects of different doses of dexmedetomidine on neuronal cells in ischemic reperfusion rats by establishing(a rat model of middle cerebral artery occlusion)(MCAO)with different doses of dexmedetomidine.Methods:Forty rats were divided into 5 groups,8 in each group:sham group(S),ischemia-reperfusion group(I/R),Dex high-dose group(DH),and Dex medium-dose group(DM),Dex low dose group(DL).In group S,only vascular separation was performed in rats,and no tying plug was performed.In the I/R group and DEX group,the cerebral ischemia-reperfusion animal model was established after occlusion of the right middle cerebral artery for 2 hours and reperfusion for 24 hours.The IR group did not undergo drug intervention.Dex high dose group(DH,60 μg/kg),Dex medium dose group(DM,6 μg/kg),Dex low dose group(DL,0.6 μg/kg)were given different doses of Dex after ischemia.Nerve damage was detected by staining with 2,5,3,5-triphenyltetrachlorotetrachloride(TTC).The contents of water,and y-aminobutyric acid were measured by wet-dry weighting and HPLC-mass spectrometry,respectively.In addition,TUNEL staining and flow cytometry were used to detect hippocampal neuronal apoptosis.In addition,mRNA and protein expression of caspase-3,Bcl-2 and Bax were detected by real-time quantitative PCR and Western blot.Results:1.Effects of different doses of dexmedetomidine on neurological function scores in I/R ratsCompared with the sham operation group,the neurological function scores of the I/R group were significantly lower.Compared with the I/R group,the neurological scores of the low,middle and high dex rats were significantly higher than those of the DL group.The neurological scores of the rats in the group were significantly increased.Compared with the DM group,the neurological scores of the DH group were significantly higher(p<0.05).2.Effects of different doses of dexmedetomidine on cerebral infarction area and water content in I/R ratsCompared with the sham operation group,the cerebral infarct size and water content of the I/R group were significantly increased.Compared with the I/R group,the cerebral infarct size and water content of the low,middle and high dex rats were significantly reduced,compared with the DL group.Compared with the DM group,the infarct size and water content of the rats in the DM group were significantly decreased(p<0.05).Compared with the DM group,the infarct size and water content of the DH group were significantly decreased,and the difference was statistically significant.(p<0.05).3.Effects of different doses of dexmedetomidine on the content of gaba in brain tissues of I/R ratsCompared with the sham operation group,the content of γ-aminobutyric acid in the hippocampus of the I/R group was significantly decreased.Compared with the I/R group,the content of γ-aminobutyric acid in the hippocampus of the low,medium and high DEX groups was significantly increased.Compared with the DM group,the content of gamma-aminobutyric acid in the hippocampus of rats in the DL group was significantly decreased,while the content of gamma-aminobutyric acid in the hippocampus of rats in the DH group was significantly increased(P<0.05).4.Effects of different doses of dexmedetomidine on apoptosis in I/R ratsCompared with the sham operation group,apoptosis of hippocampal neurons in the I/R group was significantly increased.Compared with the I/R group,hippocampal neuron apoptosis was significantly reduced in the middle and high DEX groups.Compared with the DM group,hippocampal neuron apoptosis was significantly increased in the DL group and decreased in the DH group(P<0.05).5.Effects of different doses of dexmedetomidine on caspase-3 protein in hippocampal tissues of I/R ratsCompared with the sham operation group,the caspase-3 protein level in the hippocampus of the I/R group were significantly increased.Compared with the I/R group,the levels of caspase-3 protein in the hippocampus of the low,medium and high DEX groups were.Compared with the DM group,the level of caspase-3 protein in the hippocampus of rats in the DL group was significantly increased,and the level of caspase-3 protein in the hippocampus of rats in the DH group was significantly decreased(P<0.05).6.Effects of different doses of dexmedetomidine on the mRNA content and protein expression level of bcl-2/Bax in the hippocampus of I/R ratsCompared with the sham operation group,the mRNA and protein levels of bcl-2/Bax in the hippocampal tissues of the I/R group were significantly reduced.Compared with the I/R group,the mRNA and protein levels of bcl-2/Bax in the hippocampal tissues of rats in the middle and high DEX groups were significantly increased,and the difference was statistically significant.Compared with the DM group,the content and protein level of bcl-2/Bax mRNA in the hippocampus of rats in the DL group were significantly decreased,while the content and protein level of bcl-2/Bax mRNA in the hippocampus of rats in the DH group were significantly increased(P<0.05).Conclusion:1.Dexmedetomidine can reduce nerve function damage and cerebral infarction area in I/R rats.2.Dexmedetomidine can up-regulate the expression of bcl-2/Bax protein and has a significant protective effect on the brain cells of I/R rats.3.The brain protective effect of dexmedetomidine was dose-dependent,and the protective effect increased with the increase of dose.Part Two:The cholinergic anti-inflammatory pathway-mediated dexmedeto--midine on the neuroprotective effects of hippocampus in rats with ischemia-reperfusionObjective:1.To investigate whether the protective effect of dexmedetomidine on hippocampal nerve cells in I/R rats is related to the vagus nerve.2.To clarify whether the cholinergic anti-inflammatory pathway mediates the protective effect of dexmedetomidine on hippocampal nerve cells in I/R rats.Experiment Ⅰ:Methods:40 rats were randomly divided into 5 groups(8 in each group):sham operation group(S group),ischemia reperfusion group(I/R group),dexmedetomidine group(Dex group),Dex+vagotomy group(D+V group)and vagotomy group(V group).All rats were subjected to model building intervention.Rats in group S were not inserted into thread plug and were sewn normally.Wire plug was removed after 2 hours of ischemia in I/R group.The first intravenous infusion dose of the Dex group before MCAO preparation was the Dex(6ug/kg),the vagus nerve was cut off before the D+V group was given Dex(6ug/kg),and the vagus nerve was cut off before the model preparation in the vagus nerve cutting group.All rats were sacrificed after perfusion for 24h,and the neurological function score,brain water content and infarct area of each group were determined.The levels of inflammatory factors TNF-α,IL-6 and IL-1β were detected.The apoptosis rate of brain cells and the protein expression of NF-Bp65,Bcl-2,Bax,caspase-3 were determined.Results:1.Effects of vagus nerve amputation on water content,cerebral infarction area and neurological function score in I/R ratsCompared with the sham operation group,the water content,cerebral infarction area and neurological function scores of the I/R group were significantly increased.Compared with I/R group,the brain water content,cerebral infarction area and neurological function scores of dexmedetomidine group were significantly reduced.Compared with dexmedetomidine group,the brain water content,cerebral infarction area and neurological function scores of D+V group were significantly improved,with statistically significant differences(P<0.05).2.Effects of vagus nerve transection on TNF-α,IL-6 and IL-1β levels in brain tissues of I/R ratsCompared with the sham group,TNF-α,IL-6 and IL-1β levels in rat brain homogenate were significantly increased in the I/R group.Compared with I/R group,TNF-α,IL-6 and IL-1β levels in brain homogenate of dexmedetomidine group were significantly reduced.Compared with dexmedetomidine group,TNF-α,IL-6 and IL-1β levels in homogenate of rat brain tissues in D+V group were significantly increased,and the difference was statistically significant(P<0.05).3.Effect of vagus nerve amputation on apoptosis rate of hippocampal cells in I/R ratsCompared with the sham group,the apoptosis rate of I/R group was significantly increased.Compared with I/R group,the apoptosis of dexmedetomidine group was significantly reduced.Compared with dexmedetomidine group,the apoptosis rate of D+V group was significantly increased,and the difference was statistically significant(P<0.05).4.Effect of vagus nerve amputation on expression level of apoptotic protein in hippocampal cells of I/R ratsCompared with the sham group,the expression of Bax and caspase-3 in the I/R group was significantly increased,while the expression of bcl-2 was decreased.Compared with the I/R group,the expression of Bax and caspase-3 in hippocampal tissues of dexmedetomidine group was significantly decreased,and the expression of bcl-2 was increased.Compared with dexmedetomidine group,the expressions of Bax,caspase-3 and bcl-2 in the hippocampal tissues of D+V group were decreased,with statistically significant differences(P<0.05).5.Effects of vagus nerve amputation on the expression levels of p-p38,CytC and p65 proteins in I/R ratsCompared with the sham group,the protein expressions of p-p38,CytC and p65 were significantly increased in the I/R group.Compared with group I/R,the expression of p-p38,CytC and p65 in dexmedetomidine group was significantly decreased.Compared with dexmedetomidine group,the protein expressions of p-p38,CytC and p65 in D+V group were significantly increased,and the difference was statistically significant(p<0.05)Experiment Ⅱ:Methods:The 40 rats were randomly divided into 5 groups(8 for each group),namely,sham operation group(S group),ischemia reperfusion group(I/R group),dexmedetomidine group(Dex group),Dex+inhibitor group(D+G group)and inhibitor group(G group).All rats were subjected to model building intervention.Rats in group S were not inserted into thread plug and were sewn normally.The thread plug was removed after 2 hours of ischemia in group I/R.The first intravenous infusion dose of the Dex group before the preparation of MCAO was Dex(6ug/kg),the D+G group was given α-GBT(lug/kg)before the Dex(6ug/kg),and the G group was given a-GBT(lug/kg)before the preparation of the model.All rats were sacrificed after perfusion for 24h,and the neurological function score,brain tissue water content and cerebral infarction area of each group were determined.The levels of inflammatory factors TNF-α and IL-6 and IL-1 were detected.The apoptosis rate of brain cells and the protein expression of NF-Bp65,bcl-2,Bax and caspase-3 and p-p38 were determined.Results:1.Effects of 7nAChR inhibitor on water content,cerebral infarction area and neurological function score in I/R ratsCompared with the sham operation group,the water content,cerebral infarction area and neurological function scores of the I/R group were significantly increased.Compared with I/R group,the brain water content,cerebral infarction area and neurological function scores of dexmedetomidine group were significantly reduced.Compared with dexmedetomidine group,the brain water content,cerebral infarction area and neurological function scores of D+G group were significantly improved,with statistically significant differences(P<0.05).2.Effects of 7nAChR inhibitors on TNF-α,IL-6 and IL-1β levels in brain tissues of I/R ratsCompared with the sham group,TNF-α,IL-6 and IL-1β levels in rat brain homogenate were significantly increased in the I/R group.Compared with I/R group,TNF-α,IL-6 and IL-1β levels in brain homogenate of dexmedetomidine group were significantly reduced.Compared with dexmedetomidine group,TNF-β,IL-6 and IL-1β levels in homogenate of rat brain tissues in D+G group were significantly increased,and the difference was statistically significant(P<0.05).3.Effects of 7nAChR inhibitor on the apoptosis rate of hippocampal cells in I/R ratsCompared with the sham group,the apoptosis rate of I/R group was significantly increased.Compared with I/R group,the apoptosis of dexmedetomidine group was significantly reduced.Compared with dexmedetomidine group,the apoptosis rate of D+G group was significantly increased,and the difference was statistically significant(P<0.05).4.Effects of beta 7nAChR inhibitor on the expression level of apoptotic proteins in hippocampal cells of I/R ratsCompared with the sham group,the expression of Bax and caspase-3 in the I/R group was significantly increased,while the expression of bcl-2 was decreased.Compared with the I/R group,the expression of Bax and caspase-3 in hippocampal tissues of dexmedetomidine group was significantly decreased,and the expression of bcl-2 was increased.Compared with dexmedetomidine group,the expressions of Bax,caspase-3 and bcl-2 in the hippocampal tissues of D+G group were decreased,with statistically significant differences(P<0.05).5.Effects of 7nAChR inhibitors on the expression levels of p-p38,CytC and p65 proteins in I/R ratsCompared with the sham group,the protein expressions of p-p38,CytC and p65 were significantly increased in the I/R group.Compared with group I/R,the expression of p-p38,CytC and p65 in dexmedetomidine group was significantly decreased.Compared with dexmedetomidine group,the protein expressions of p-p38,CytC and p65 in D+G group were significantly increased,and the difference was statistically significant(p<0.05).Conclusion:1.Dexmedetomidine plays a protective role in the brain depending on the integrity of the vagus nerve.2.The cholinergic anti-inflammatory pathway mediates the protective effect of dexmedetomidine on cerebral ischemia-reperfusion injury.3.The realization of brain protective effect of dexmedetomidine may be related to p38MAPK receptor-mediated apoptosis... |
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