摘要
目的:探讨臭牡丹(Clerodendrum bungei Steud.,CBS)的镇痛作用是否与N-甲基-D-天冬氨酸(N-methyl-Daspartate,NMDA)受体相关。方法:雄性成年SD大鼠24只,随机分为假手术组(Sham组)、坐骨神经分支选择性损伤(Spared nerve injury,SNI)模型组(SNI组)和30 g·kg^(-1)CBS治疗组(SNI+CBS组)。SNI+CBS组于SNI术后第7 d灌胃给予CBS,持续21 d,Sham组和SNI组给予同等体积的生理盐水灌胃。于术后第26~28 d检测大鼠步态;第28 d取脊髓,qPCR方法检测NMDA受体亚基GluNR1、GluN2A、GluN2B和GluN3A mRNA表达,Western blot和免疫荧光方法检测GluN2A和GluN2B蛋白水平,分子对接方法模拟CBS中主要成分与GluN2B的亲和力。结果:与Sham组相比,SNI模型组大鼠患肢的站立时间、最大接触时的最大强度、足印长度、足印宽度和爪印面积降低,最大接触时间百分比升高,脊髓GluN2A、GluN2B和GluN3A mRNA表达降低,GluN2A和GluN2B蛋白水平升高。与SNI组相比,SNI+CBS组大鼠患肢的站立时间、最大接触时的最大强度、足印长度和足印宽度升高,最大接触时间百分比降低,脊髓GluN2A、GluN2B和GluN3A mRNA表达升高,GluN2A和GluN2B蛋白水平降低。各组GluNR1 mRNA表达差异无统计学意义(P>0.05)。分子对接实验结果显示,原始配体Ro25-6981与GluN2B对接的结合自由能(△G)为−11.72 KJ·mol^(-1),估计抑制常数(Ki)为2.58 nmol;CBS主要成分α-香树脂醇、赪桐酮和木栓酮与GluN2B对接的△G及Ki与Ro25-6981接近,甚至优于Ro25-6981;从对接构象上看,Ro25-6981与木栓酮占据了GluN1B结合位点,α-香树脂醇和赪桐酮则占据了GluN2B结合位点,呈现出较强的NMDA受体抑制作用。结论:CBS对SNI模型所致神经病理性疼痛具有缓解作用,其机制可能是通过抑制NMDA受体来实现。
Objective:To investigate whether the analgesic effect Clerodendrum bungei Steud.(CBS)was related to N-methyl-D-aspartate(NMDA)receptors.Methods:A total of 30 adult male SD rats were randomly divided into Sham operation group,SNI model group and 30 g·kg^(-1)CBS treatment group.Rats of treatment group were dealt with CBS by intragastric administration once a day from 7 th to 28 th day after SNI,and the Sham and SNI groups were treated with same volume of normal saline.The CatWalk gait of rats was detected at 26 th-28 th day after operation,and the extraction of spinal core was done on the 28 th day.The mRNA expressions of NMDA receptor subunits,including GluNR1,GluN2A,GluN2B,and GluN3A were detected by qPCR,and the protein levels and localization of GluN2A and GluN2B were detected by Western blot and immunofluorescence.The affinity between the main components of CBS and GluN2B was simulated by molecular docking method.Results:Compared with sham group,SNI model group showed decreases in the standing time,maximum strength at maximum contact,footprint length,footprint width and paw print area of the affected limb,an increase in the percentage of maximum contact time,decreases in the mRNA expressions of GluN2A,GluN2B and GluN3A,and increases in the protein levels of GluN2A and GluN2B.Compared with SNI group,SNI+CBS group showed better CatWalk gait parameters including standing time of the affected limb,the maximum intensity at the maximum contact time,the ratio of the maximum contact time to the duration,and the length and width of the footprints,but had no significant change on the paw print area.CBS treatment also increased GluN2A,GluN2B and GluN3A mRNA in spinal cord,and decreased GluN2A and GluN2B protein levels.There was no statistical difference in GluNR1 mRNA expression in all groups(P>0.05).The results of molecular docking experiments showed that the binding free energy(△G)of the original ligand Ro25-6981 was^(-1)1.72 KJ·mol^(-1)and the estimated inhibition constant(Ki)was 2.58 nmol.The main components of CBS,alpha-balsaminol,clerodone and suberone,were close to and even better than Ro25-6981 in△G and Ki of GluN2B.By contrast,Ro25-6981 and suberone occupied the GluN1B binding site,and alpha-balsaminol and clerodone occupied the GluN2B binding site,showing strong NMDARs inhibition.Conclusion:CBS treatment can improve the CatWalk gait of rats with neuropathic pain induced by SNI,which may be achieved by inhibitingNMDA receptors.
作者
温婷
虞子宁
鞠艺楠
黄成炬
王安琪
黄诚
胡志苹
WEN Ting;YU Zi-ning;JU Yi-nan;HUANG Cheng-ju;WANG An-qi;HUANG Cheng;HU Zhi-ping(The First Clinical Medical School of Gannan Medical University;The Neuroinflammation Lab of Ganzhou,Gannan Medical University;School of Basic Medicine,Gannan Medical University,Ganzhou,Jiangxi 341000)
出处
《赣南医学院学报》
2023年第9期877-884,共8页
JOURNAL OF GANNAN MEDICAL UNIVERSITY
基金
国家自然科学基金资助项目(30060213)
江西省教育厅科学技术研究项目(GJJ170863)
赣南医学院重点科研项目(ZD201704)
赣南医学院创新团队项目(TD201705,TD2021JC06)。
