摘要
目的观察电针对阿片类药物耐受大鼠机械刺激缩足反应阈值(Paw withdrawal mechanical thresh⁃old,PWMT))、海马组织G蛋白偶联受体(G Protein-Coupled Receptors,GPCRs)及环磷酸腺苷(cyclic AMP,cAMP)、cAMP反应元件结合蛋白(cyclic-AMP response binding protein,CREB)表达的影响,探索G蛋白-谷氨酸受体-cAMP通路是否为电针治疗阿片类药物耐受的可能机制。方法选取60只SPF级SD大鼠,雌雄各半,按随机数字表法分为生理盐水对照组、药物耐受组、谷氨酸受体抑制剂+药物耐受组、电针+药物耐受组及电针+谷氨酸受体抑制剂+药物耐受组,每组各12只。通过对药物耐受组、谷氨酸受体抑制剂+药物耐受组、电针+药物耐受组及电针+谷氨酸受体抑制剂+药物耐受组正常大鼠连续腹腔注射吗啡9 d,2次/d,制备吗啡耐受(Mor⁃phine tolerance,MT)模型,建模成功后,4组继续给予连续7 d腹腔注射吗啡,谷氨酸受体抑制剂+药物耐受组在予腹腔吗啡注射前0.5 h,予腹腔注射谷氨酸受体抑制剂;电针+药物耐受组在予腹腔吗啡注射后0.5 h,予双侧“合谷”“内关”“三阴交”及“足三里”穴位电针干预,断续波,频率2 Hz,2次/d,每次干预时间为10 min,连续7 d;电针+谷氨酸受体抑制剂+药物耐受组给予谷氨酸受体抑制剂和吗啡后,再进行同电针+药物耐受组相同的电针干预。各组分别于造模前、造模第1天、3天、6天、9天和建模成功后第1天、3天、5天、7天检测左足PWMT;采用蛋白质免疫印迹(Western Blot,WB)法检测大鼠海马组织GPCRss、cAMP、CREB蛋白表达,采用酶联免疫吸附测定法(ELISA)检测血浆β-内啡肽(β-EP)含量。结果与生理盐水对照组比较,药物耐受组、谷氨酸受体抑制剂+药物耐受组、电针+药物耐受组及电针+谷氨酸受体抑制剂+药物耐受组大鼠注射吗啡后第1天、3天、6天PWMT明显升高,差异有统计学意义(P<0.01),但注射吗啡9 d后降低至基础水平,与生理盐水对照组比较,差异无统计学意义(P>0.05),说明MT模型造模成功;与药物耐受组比较,谷氨酸受体抑制剂+药物耐受组、电针+药物耐受组、电针+谷氨酸受体抑制剂+药物耐受组大鼠在第3天、5天、7天的PWMT升高,差异有统计学意义(P<0.01);与电针+谷氨酸受体抑制剂+药物耐受组比较,电针+药物耐受组大鼠在第3天、5天、7天的PWMT明显升高,差异有统计学意义(P<0.01)。与生理盐水对照组比较,药物耐受组、谷氨酸受体抑制剂+药物耐受组、电针+谷氨酸受体抑制剂+药物耐受组大鼠海马组织中CREB、cAMP的相对表达均升高,GPCRss的表达相对下降,差异有统计学意义(P<0.01);与药物耐受组比较,谷氨酸受体抑制剂+药物耐受组、电针+药物耐受组和电针+谷氨酸受体抑制剂+药物耐受组大鼠海马组织中CREB、cAMP的相对表达均有所下降,差异有统计学意义(P<0.01),电针+药物耐受组大鼠海马组织中GPCRss的表达相对升高,差异有统计学意义(P<0.01);与电针+谷氨酸受体抑制剂+药物耐受组比较,电针+药物耐受组大鼠海马组织中CREB、cAMP的相对表达均降低,GPCRs的表达相对升高,差异有统计学意义(P<0.01)。结论电针可明显改善MT模型大鼠阿片类药物耐受效应,其机制可能与下调大鼠海马组织中cAMP、CREB蛋白表达,促进β-EP表达,进而减弱G蛋白-谷氨酸受体-cAMP通路上调有关。
Objective To investigate the effects of electroacupuncture on paw-withdrawal mechanical threshold(PWMT),and the expression of hippocampal G protein-coupled receptors(GPCRs),cyclic adenosine monophosphate(cAMP)and cAMP response element-binding protein(CREB)in opioid-resistant rats..To explore whether G protein-glutamate receptor-cAMP pathway is involved in the mechanism for electroacupuncture in the treatment of opioid toler⁃ance.Methods Sixty SPF-grade SD rats,half male and half female were selected and divided into 5 groups by using random number table,namely,saline control group(group A),drug-tolerant group(group B),glutamate receptor inhibitor+drug-tolerant group(group C),electroacupuncture+drug-tolerant group(group D)and electroacupuncture+glutamate receptor inhibitor+drug-tolerant group(group E),with 12 rats in each group.The rats in groups B,C,D and E were con⁃tinuously intraperitoneally injected with morphine twice a day for 9 days to prepare a morphine tolerance(MT)model.After successful modeling,the rats in four groups were given morphine intraperitoneally for 7 consecutive days,and the rats in glutamate receptor inhibitor+drug-tolerant group were given intraperitoneal injection of glutamate receptor inhibitor 0.5 h before morphine injection.In the drug-tolerant group,0.5 h after intraperitoneal morphine injection,electroacupuncture intervention was applied on bilateral Hegu Neiguan Sanyinjiao and Zusanli acupoints for 10 min twice a day for 7 consecu⁃tive days,with intermittent wave and frequency of 2 Hz.After glutamate receptor inhibitor and morphine were given in elec⁃troacupuncture+glutamate receptor inhibitor+drug-tolerant group,the same electroacupuncture intervention was per⁃formed on the first day,the 3rd days,the 6th day,and the 9th day before modeling and the first day,the 3 day,the 5th days,and the 7th day after modeling,respectively.Western Blot(WB)was used to detect the GPCRs,cAMP CREB protein expression in the hippocampus of rats,and enzyme-linked immunosorbent assay(ELISA)was used to detect the plasmaβ-endorphin(β-EP)content.Results Compared with the saline control group,the PWMT of rats in the drug-tolerant group,glutamate receptor inhibitor+drug-tolerant group,electroacupuncture+drug-tolerant group and electroacupunc⁃ture+glutamate receptor inhibitor+drug-tolerant group was significantly increased on the 1st,3rd and 6th days after mor⁃phine injection(P<0.01),but it decreased to the basal level after 9 days of injection,and compared with the saline con⁃trol group,the difference was not statistically significant(P>0.05),indicating that the MT model was successfully estab⁃lished.Compared with the drug-tolerant group,the PWMT of rats in the glutamate receptor inhibitor+drug-tolerant group,electroacupuncture+drug-tolerant group,electroacupuncture+glutamate receptor inhibitor+drug-tolerant group was significantly increased on days 3,5 and 7(P<0.01),and compared with the electroacupuncture+glutamate receptor inhibitor+drug-tolerant group,the PWMT of rats in the electroacupuncture+drug tolerance group was significantly in⁃creased on days 3,5 and 7,and the difference was statistically significant(P<0.01).Compared with the saline control group,the relative protein expression of CREB and cAMP in rat hippocampal tissues in the drug-tolerant group,glutamate receptor inhibitor+drug-tolerant group,electroacupuncture+glutamate receptor inhibitor+drug-tolerant group was in⁃creased,and the expression of GPCRs was decreased significantly(P<0.01).The relative expression of cAMP was de⁃creased significantly(P<0.01),and the expression of GPCRs in hippocampal tissues of rats in the electroacupuncture+drug-tolerant group was statistically higher(P<0.01),and compared with the electroacupuncture+glutamate receptor inhibitor+drug-tolerant group,the relative expression of CREB and cAMP in the hippocampal tissues of rats in the elec⁃troacupuncture+drug-tolerant group was reduced,and the expression of GPCRs was significantly elevated(P<0.01).Conclusion Electroacupuncture can significantly improve the opioid tolerance in MT model rats,and its mechanism may be related to the down-regulation of the protein expression of cAMP and CREBs in hippocampal tissue of rats,the promo⁃tion ofβ-EP expression,and the attenuation of the up-regulation of G-protein-glutamate receptor-cAMP pathway.
作者
刘春
于波
王铁东
LIU Chun;YU Bo;WANG Tie-dong(Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,Shenyang Liaoning 110032)
出处
《世界中西医结合杂志》
2023年第10期1982-1988,共7页
World Journal of Integrated Traditional and Western Medicine
基金
辽宁省教育厅科学技术研究项目(L202002)。
