摘要
目的探讨艾司氯胺酮预处理对脑缺血再灌注损伤(IRI)大鼠的神经保护作用及与Akt/mTOR信号通路的关系。方法成年雄性SD大鼠60只随机分为假手术组、模型组、艾司氯胺酮组和雷帕霉素组各15只。模型组、艾司氯胺酮组、雷帕霉素组采用改良线栓法阻塞大脑中动脉制备脑IRI大鼠模型,假手术组仅分离右侧颈总动脉、颈外动脉和颈内动脉,不插入线栓。雷帕霉素组造模前腹腔注射雷帕霉素1 mg/kg,连续3 d。缺血90 min时,艾司氯胺酮组和雷帕霉素组经尾静脉注射艾司氯胺酮1.6 mg/kg,模型组和假手术组注射等体积生理盐水。再灌注24 h,4组大鼠采用Longa评分和贴纸实验评估神经行为学改变;之后4组各随机取6只大鼠,腹腔注射戊巴比妥钠150 mg/kg麻醉后取缺血区脑组织,采用实时荧光定量PCR法检测诱导型一氧化氮合酶(iNOS)、白细胞介素(IL)-1β、精氨酸酶-1(Arg-1)、IL-10 mRNA相对表达量,采用Western blot法检测p-Akt、Akt和Beclin-1蛋白相对表达量。再灌注72 h,4组各随机取6只大鼠处死,采用TTC染色法测定脑梗死体积;处死各组剩余的3只大鼠,采用TUNEL染色定量分析凋亡细胞百分比。结果(1)假手术组、模型组、艾司氯胺酮组和雷帕霉素组Longa评分[0、(2.73±0.46)、(1.40±0.51)、(1.27±0.46)分]、碰触贴纸时间[(11.53±1.06)、(58.73±4.78)、(36.13±3.64)、(18.60±2.35)s]和移除贴纸时间[(26.73±1.53)、(96.33±9.71)、(58.73±4.74)、(39.40±4.84)s]比较差异均有统计学意义(F=110.784、619.710、388.023,P均<0.001)。模型组、艾司氯胺酮组、雷帕霉素组Longa评分均高于假手术组(P<0.05),碰触贴纸时间和移除贴纸时间均长于假手术组(P<0.05);模型组Longa评分高于艾司氯胺酮组和雷帕霉素组(P<0.05),碰触贴纸时间和移除贴纸时间均长于艾司氯胺酮组和雷帕霉素组(P<0.05);艾司氯胺酮组碰触贴纸时间、移除贴纸时间均长于雷帕霉素组(P<0.05),Longa评分与雷帕霉素组比较差异无统计学意义(P>0.05)。(2)假手术组、模型组、艾司氯胺酮组和雷帕霉素组缺血脑组织iNOS mRNA(0.96±0.10、2.83±0.18、1.83±0.06、1.24±0.10)、IL-1βmRNA(1.00±0.02、2.99±0.14、1.90±0.13、1.37±0.04)、Arg-1 mRNA(1.00±0.08、0.48±0.04、0.74±0.06、0.95±0.08)、IL-10 mRNA(1.00±0.15、0.45±0.05、0.69±0.06、0.85±0.08)相对表达量比较差异均有统计学意义(F=291.509、488.656、78.288、37.457,P均<0.001)。模型组、艾司氯胺酮组和雷帕霉素组缺血脑组织iNOS、IL-1βmRNA相对表达量均高于假手术组(P<0.05),模型组均高于艾司氯胺酮组和雷帕霉素组(P<0.05),艾司氯胺酮组均高于雷帕霉素组(P<0.05);模型组、艾司氯胺酮组Arg-1、IL-10 mRNA相对表达量均低于假手术组(P<0.05),模型组均低于艾司氯胺酮组和雷帕霉素组(P<0.05),艾司氯胺酮组均低于雷帕霉素组(P<0.05)。(3)假手术组、模型组、艾司氯胺酮组和雷帕霉素组缺血脑组织p-Akt/Akt(1.02±0.07、3.11±0.15、2.03±0.17、1.39±0.09)、Beclin-1蛋白相对表达量(1.00±0.09、0.45±0.02、0.76±0.05、0.88±0.05)比较差异均有统计学意义(F=306.260、106.976,P均<0.001)。模型组、艾司氯胺酮组和雷帕霉素组p-Akt/Akt均高于假手术组(P<0.05),Beclin-1蛋白相对表达量均低于假手术组(P<0.05);模型组p-Akt/Akt高于艾司氯胺酮组和雷帕霉素组(P<0.05),Beclin-1蛋白相对表达量低于艾司氯胺酮组和雷帕霉素组(P<0.05);艾司氯胺酮组p-Akt/Akt高于雷帕霉素组(P<0.05),Beclin-1蛋白相对表达量低于雷帕霉素组(P<0.05)。(4)假手术组、模型组、艾司氯胺酮组和雷帕霉素组脑梗死体积百分比[0、(29.78±3.15)%、(10.10±0.68)%、(6.40±0.96)%]、凋亡细胞百分比[(0.27±0.02)%、(48.81±4.31)%、(13.29±2.81)%、(6.29±0.65)%]比较差异均有统计学意义(F=349.771、211.405,P均<0.001)。模型组脑梗死体积百分比、凋亡细胞百分比均高于假手术组、艾司氯胺酮组和雷帕霉素组(P<0.05),艾司氯胺酮组均高于假手术组和雷帕霉素组(P<0.05)。结论艾司氯胺酮预处理可通过抑制Akt/mTOR通路增强自噬,促进小胶质细胞向M2极化,减轻神经炎症,对脑IRI发挥保护作用,且艾司氯胺酮与雷帕霉素联合应用的神经保护效果更显著。
Objective To investigate the role of esketamine perconditioning in alleviating cerebral ischemia-reperfusion injury(IRI)in rats and its relationship with Akt/mTOR signaling pathway.Methods Sixty adult male SD rats were randomly allocated into sham,model,esketamine and rapamycin groups,with 15 rats in each group.The model,esketamine and rapamycin groups were performed middle cerebral artery occlusion(MCAO)using the modified intraluminal filament method to establish cerebral IRI models.The sham group only received surgical separation of the right common carotid artery,external carotid artery,and internal carotid artery,with no filament insertion.The rapamycin group received intraperitoneal rapamycin injection(1 mg/kg)for 3 consecutive days prior to modeling.At 90 min post-ischemia,the esketamine and rapamycin groups received esketamine injection(1.6 mg/kg)via tail vein,while model and sham groups received injection of equivalent volume of saline.At 24 h post-reperfusion,neurological deficits were assessed in all groups using Longa scoring and double-sided sticker experiment.Subsequently,6 randomly selected rats per group were anesthetized with intraperitoneal injection of pentobarbital sodium 150 mg/kg,followed by collection of ischemic brain tissue.Real-time quantitative PCR was used to measure the relative expressions of inducible nitric oxide synthase(iNOS),interleukin(IL)-1β,arginase-1(Arg-1)and IL-10 mRNAs,and Western blot was used to quantify the relative expressions of p-Akt,Akt,and Beclin-1 proteins.At 72 h post-reperfusion,another 6 randomly selected rats per group were sacrificed for cerebral infarction volume measurement by TTC staining,and the remaining 3 rats per group were sacrificed for quantitative analysis of apoptotic cell percentages via TUNEL staining.Results(1)There were statistically significant differences in the Longa scores(0,2.73±0.46,1.40±0.51,1.27±0.46),sticker contact time[(11.53±1.06),(58.73±4.78),(36.13±3.64),(18.60±2.35)s],and sticker removal time[(26.73±1.53),(96.33±9.71),(58.73±4.74),(39.40±4.84)s]among the sham,model,esketamine and rapamycin groups(F=110.784,619.710,388.023;all P values<0.001).The Longa score was higher in the model,esketamine and rapamycin groups than that in the sham group(P<0.05),was higher in the model group than that in the esketamine and rapamycin groups(P<0.05),and showed no statistically significant difference between the esketamine and rapamycin groups(P>0.05).The sticker contact time and sticker remoual time were longer in the model,esketamine and rapamycin groups than those in the sham group(P<0.05),were longer in the model group than those in the esketamine and rapamycin groups(P<0.05),and were longer in the esketamine than those in the rapamycin group(P>0.05).(2)There were statistically significant differences in the relative expressions of iNOS mRNA(0.96±0.10,2.83±0.18,1.83±0.06,1.24±0.10),IL-1βmRNA(1.00±0.02,2.99±0.14,1.90±0.13,1.37±0.04),Arg-1 mRNA(1.00±0.08,0.48±0.04,0.74±0.06,0.95±0.08),and IL-10 mRNA(1.00±0.15,0.45±0.05,0.69±0.06,0.85±0.08)in the sham,model,esketamine and rapamycin groups(F=291.509,488.656,78.288,37.457;all P values<0.001).The relative expressions of iNOS and IL-1βmRNAs were statistically higher in the model,esketamine and rapamycin groups than those in the sham group(P<0.05),higher in the model group than those in the esketamine and rapamycin groups(P<0.05),and higher in the esketamine group than those in the rapamycin group(P<0.05).The relative expressions of Arg-1 and IL-10 mRNAs were significantly lower in the model and esketamine groups than those in the sham group(P<0.05),lower in the model group than those in the esketamine and rapamycin groups(P<0.05),and lower in the esketamine group than those in the rapamycin group(P<0.05).(3)There were statistically significant differences in the relative expressions of p-Akt/Akt(1.02±0.07,3.11±0.15,2.03±0.17,1.39±0.09)and Beclin-1 protein(1.00±0.09,0.45±0.02,0.76±0.05,0.88±0.05)in the sham,model,esketamine and rapamycin groups(F=306.260,106.976;all P values<0.001).The relative expression of p-Akt/Akt was statistically higher in the model,esketamine and rapamycin groups than that in the sham group(P<0.05),higher in the model group than that in the esketamine and rapamycin groups(P<0.05),and higher in the esketamine group than that in the rapamycin group(P<0.05).The relative expression of Beclin-1 protein was statistically lower in the model,esketamine and rapamycin groups than that in the sham group(P<0.05),lower in the model group than that in the esketamine and rapamycin groups(P<0.05),and lower in the esketamine group than that in the rapamycin group(P<0.05).(4)There were statistically significant differences in the percentages of cerebral infarction volume[0,(29.78±3.15)%,(10.10±0.68)%,(6.40±0.96)%]and apoptotic cells[(0.27±0.02)%,(48.81±4.31)%,(13.29±2.81)%,(6.29±0.65)%]in the sham,model,esketamine and rapamycin groups(F=349.771,211.405;all P values<0.001).The percentages of cerebral infarction volume and apoptotic cells were higher in the model group than those in the sham,esketamine and rapamycin groups(P<0.05),and higher in the esketamine group than those in the sham and rapamycin groups(P<0.05).Conclusions Esketamine perconditioning exerts a neuroprotective effect against cerebral IRI by enhancing autophagy through suppression of Akt/mTOR pathway,promoting microglial polarization toward the M2 phenotype,and alleviating neuroinflammation.Moreover,the combined application of esketamine and rapamycin demonstrates more pronounced neuroprotective effects.
作者
高颖
李璐
程怡
金沐
薛富善
GAO Ying;LI Lu;CHENG Yi;JIN Mu;XUE Fushan(Department of Anesthesiology,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China;Department of Anesthesiology,Beijing Anzhen Hospital,Capital Medical University,Beijing 100029,China;Departmentof Anesthesiology,Fuzhou University Affiliated Provincial Hospital,Fuzhou,Fujian35000l,China)
出处
《中华实用诊断与治疗杂志》
2025年第7期589-596,共8页
Journal of Chinese Practical Diagnosis and Therapy
基金
北京市自然科学基金(719204)
北京市属医院科研培育项目(PX024006)。
作者简介
通信作者:薛富善,E-mail:xuefushan@aliyun.com。
