鞣花酸对吗啡镇痛耐受的影响及机制  

Effect of ellagic acid on morphine analgesic tolerance and its mechanism

作　　者：肖勇 陈渔 陆巍 XIAO Yong;CHEN Yu;LU Wei(Department of Pain,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China;School of Anesthesiology,Guizhou Medical University,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学附属医院疼痛科,贵州贵阳550004 [2]贵州医科大学麻醉学院,贵州贵阳550004

出　　处：《贵州医科大学学报》2024年第6期826-835,共10页Journal of Guizhou Medical University

基　　金：贵州省普通高等学校青年科技人才成长项目(黔教合KY[2022]239);贵州省研究生科研基金立项课题(黔教合YJSKYJJ[2021]148);贵州省卫生健康委科学技术基金(gzwkj2023-395)。

摘　　要：目的探讨鞣花酸(EA)对大鼠吗啡镇痛耐受的影响和作用机制。方法动物实验,将40只健康雄性SD大鼠随机分为生理盐水(NS)组、吗啡(MT)组、鞣花酸(EA)组、鞣花酸+吗啡(EA+MT)组,后3组吗啡鞘内注射给药建立大鼠吗啡镇痛耐受模型、4组大鼠灌胃生理盐水或相应药物,采用温水甩尾法和Von Frey法测定各组大鼠给药前和给药第1~7天的甩尾潜伏期(TFL)和机械缩足反射阈值(MWT);大鼠造模结束后麻醉处死并取中脑导水管周围灰质(PAG)组织,Western blot检测PINK1、LC3、P62蛋白的表达,硫代巴比妥酸显色法测定丙二醛(MDA)含量,透射电镜观察线粒体完整性;细胞实验,通过鞣花酸和吗啡处理人神经母细胞瘤SH-SY5Y细胞,设置对照(SHAM)组、吗啡(Mo)组、鞣花酸(Ea)组、鞣花酸+吗啡(Ea+Mo)组,采用MTT法测定细胞活力、ELISA法测定cAMP的含量、活性氧荧光探针检测ROS水平,Western blot测定PINK1、LC3、P62蛋白的表达。结果动物实验结果显示,与MT组比较,EA+MT组大鼠TFL和MWT下降趋势较缓慢,差异具有统计学意义(P<0.05);与NS组比较,MT组PINK1蛋白、LC3Ⅱ/LC3Ⅰ比值、P62蛋白和MDA水平升高,差异具有统计学意义(P<0.05),且线粒体肿胀明显可见较多自噬体;与MT组比较,EA+MT组PINK1蛋白、LC3Ⅱ/LC3Ⅰ比值升高而P62蛋白、MDA水平下降,差异具有统计学意义(P<0.05),线粒体轻度肿胀可见少量自噬体;细胞实验结果显示,与SHAM组比较,Mo组cAMP、ROS、LC3Ⅱ/LC3Ⅰ比值及P62蛋白表达水平均升高、细胞活力和PINK1蛋白表达水平下降,差异具有统计学意义(P<0.05);与Mo组比较,Ea+Mo组细胞活力、PINK1蛋白和LC3Ⅱ/LC3Ⅰ比值升高而cAMP、ROS和P62蛋白表达水平下降,差异具有统计学意义(P<0.05)。结论EA具有一定镇痛作用,可缓解大鼠吗啡镇痛耐受的形成,其作用机制可能与线粒体自噬有关。Objective To investigate the effect and mechanism of ellagic acid(EA)on morphine analgesic tolerance in rats.Methods In vivo experiment,40 healthy male SD rats were randomly divided into the normal saline(NS)group,morphine(MT)group,ellagic acid(EA)group,and ellagic acid+morphine(EA+MT)group.The rat model of morphine analgesic tolerance was established by intrathecal injection of morphine.The latter three groups were administered with morphine,while all the 4 groups were infused with saline or the corresponding drug.Tail flick latency(TFL)was measured by warm water bath method and mechanical withdrawal threshold(MWT)was measured by von Frey method to observe the change trend of analgesic threshold before and after administration on the 1 st-7 th days.After modeling,the subject rats were anesthetized and killed,and the periaqueductal gray(PAG)tissue samples were taken.The expression of PINK1,LC3 and P62 proteins was detected by Western blot.Malondialdehyde(MDA)was determined by thiobarbituric acid method,and mitochondrial integrity was observed by transmission electron microscopy(TEM).In vitro experiments,human neuroblastoma cells(SH-SY5Y cells)were treated with EA and morphine,and divided into the control(SHAM)group,morphine(Mo)group,EA group,and EA+morphine(Ea+Mo)group.The cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.The content of cAMP was determined by enzyme-linked immunosorbent assay(ELISA),the level of ROS was detected by ROS fluorescent probe(DCFH-DA)and the expressions of PINK1,LC3 and P62 protein were determined by Western blot.Results Compared with MT group,TFL and MWT decreased more slowly in EA+MT group,and the difference was statistically significant(P<0.05).Compared with NS group,the levels of PINK1 protein,the ratio of LC3Ⅱ/LC3Ⅰ,P62 protein and MDA in MT group increased,and the difference was statistically significant(P<0.05),and mitochondrial swelling was obviously with more autophagosomes.Compared with MT group,PINK1 protein and the ra

关 键 词：鞣花酸 吗啡 镇痛耐受 线粒体自噬 中脑导水管周围灰质 

分 类 号：R614.2[医药卫生—麻醉学]