远端肢体缺血预处理对氯胺酮致发育期大鼠大脑皮层HMGB1和COX-2表达的影响

Effects of Remote Limb Ischemic Preconditioning on HMGB1 and COX-2 in the Cerebral Cortex of Developmental Rats during Ketamine Anesthesia

目的研究远端肢体缺血预处理(remote limb ischemic preconditioning,RIPC)对氯胺酮致发育期大鼠大脑皮质中神经因子高迁移率族蛋白B1(HMGB1)和环氧合酶-2(COX-2)表达水平的影响,探讨其保护作用。方法将48只5日龄SD大鼠随机分为4组:对照组(C组)7日龄时经腹腔注射生理盐水8 mL·kg^(-1),每2 h注射1次,共6次;RIPC组5日龄时接受右下肢缺血预处理,5 min缺血,5 min再灌注,交替进行4个循环,出生第7天给予生理盐水注射,方法同C组;氯胺酮(KTM)组7日龄时经腹腔注射氯胺酮20 mg·kg^(-1)(用生理盐水将氯胺酮溶液稀释至2.5 mg·mL^(-1),注射容量为8 mL·kg^(-1)),每2 h注射1次,共6次;氯胺酮+RIPC组(K+R组):5日龄时行右下肢RIPC,出生第7天时进行氯胺酮注射,方法同KTM组。末次药物注射完成后12 h经心脏采血,采用ELISA法检测HMGB1和COX-2在大鼠血清中的浓度。采血后处死大鼠,取大脑组织,分别采用免疫组化法、Western blot法、qRT-PCR法测定大鼠大脑皮质中HMGB1和COX-2的表达水平。结果与C组相比,RIPC组大脑皮质中HMGB1、COX-2阳性细胞占比、mRNA、蛋白表达水平和血清中含量差异均无统计学意义(P均>0.05);与C组相比,KTM组和K+R组大脑皮质中HMGB1、COX-2阳性细胞占比、mRNA、蛋白表达水平和血清中含量均升高(P均<0.05);与KTM组相比,K+R组大脑皮质中HMGB1、COX-2阳性细胞占比、mRNA、蛋白表达水平和血清中含量均减少(P均<0.05)。结论反复多次、大剂量氯胺酮暴露可诱导发育期大鼠大脑皮质神经因子HMGB1、COX-2表达增高,与发育期神经损伤有关;远端缺血预处理可下调氯胺酮所致HMGB1、COX-2高表达,是其发挥神经保护作用机制之一。

Objective To study the protect effect of remote limb ischemic preconditioning on high mobility group protein B1(HMGB1)and cyclooxygenase-2(COX-2)in the cerebral cortex of developmental rats during ketamine anesthesia.Methods A total of 485-day-old SD rats weighing 7 to 13 g were randomly divided into 4 groups(n=12),the control group(C group)was injected intraperitoneally with normal saline at a dose of 8 mL·kg^(-1)once every 2 h,a total of 6 times.The distal limb ischemia preconditioning group(RIPC group),the newborn 5-day-old rats received right lower limb root ischemic preconditioning,ischemia for 5 min,and reperfusion for 5 min,and then underwent 4 cycles in turn.The rats were injected with normal saline on the 7th day after birth,using the same method as in group C.The ketamine group(KTM group),the newborn 7-day-old rats were intraperitoneally injected with 20 mg·kg-1 ketamine hydrochloride,once every 2 h,for a total of 6 times.Ketamine+remote limb ischemic preconditioning group(K+R group),Newborn 5-day-old rats underwent distal limb ischemia-preconditioning,and ketamine injection was performed on the 7th day after birth,using the same method as the KTM group.Blood samples were drawn from the heart 12 h after the last dose,and the changes of HMGB1 and COX-2 in the serum of rats were observed by ELISA.After blood collection,rats were sacrificed and brain tissues were taken,and the expression levels of HMGB1 and COX-2 in the cerebral cortex of rats were determined by immunohistochemistry,Western blot and qRT-PCR.Results Compared with C group,there were no statistically significant differences in the proportion of HMGB1 and COX-2 positive cells,protein content,mRNA expression level and serum HMGB1 and COX-2 expression levels in the cerebral cortex of RIPC group(P all>0.05).Compared with the C group,the proportion of HMGB1 and COX-2 positive cells,protein content,mRNA expression level and serum expression levels of HMGB1 and COX-2 in the cerebral cortex of the KTM group and the K+R group were significantly increased(P all<0.05).However,compared with the KTM group,the proportion of HMGB1 and COX-2 positive cells,protein content,mRNA expression level and serum HMGB1 and COX-2 expression levels in the cerebral cortex of the K+R group were significantly reduced(P all<0.05).Conclusion Repeated and high-dose ketamine exposure can increase the expression levels of nerve factors HMGB1 and COX-2 in cerebral cortex and serum of developmental rats,which are related to nerve injury during development.Remote limb ischemic preconditioning can reduce the expression levels of neural factors HMGB1 and COX-2 induced by ketamine,which is one of its neuroprotective mechanisms.