微透析—高效液相色谱—化学发光法动态监测6—羟基多巴帕金森病模型大鼠纹状体内多巴胺水平  

Microdialysis sampling-high performance liquid chromatography- chemiluminescence method for dynamic monitoring of dopamine levels in the striatum of 6-hydroxydopamine-induced Parkinson’s disease model rats

作　　者：吴丹 孙志军 张俊婷 郑琴 程文明[1] 谢晋[1] 张群林[1] WU Dan;SUN Zhi-jun;ZHANG Jun-ting;ZHENG Qin;CHENG Wen-ming;XIE Jin;ZHANG Qun-lin(Anhui Medical University,School of Pharmacy,Anhui Laboratory of Inflammatory and Immune Diseases,Hefei 230032,China;Key Laboratory of Modern Preparation of Traditional Chinese Medicine,Ministry of Education,Jiangxi University of Traditional Chinese Medicine,Nanchang 330004,China)

机构地区：[1]安徽医科大学药学院,炎症免疫性疾病安徽省实验室,合肥230032 [2]江西中医药大学现代中药制剂教育部重点实验室,南昌330004

出　　处：《药物分析杂志》2023年第1期133-140,共8页Chinese Journal of Pharmaceutical Analysis

基　　金：江西中医药大学现代中药制剂教育部重点实验室开放基金资助项目(201802);安徽省学术和技术带头人及后备人选学术科研活动资助经费资助项目(2018H203)。

摘　　要：目的:建立动态监测自由清醒大鼠纹状体内多巴胺水平的微透析-高效液相色谱-化学发光分析法,研究天然冰片联合左旋多巴对6-羟基多巴诱导的帕金森病(PD)模型大鼠纹状体内多巴胺水平的影响。方法:雄性SD大鼠随机分为正常组、6-羟基多巴PD模型组、左旋多巴组和天然冰片联合左旋多巴组。模型组大鼠麻醉后于右侧前脑内侧束缓慢注入4μg·μL^(-1)6-羟基多巴,术后4周腹腔注射0.5 mg·kg^(-1)阿扑吗啡,筛选旋转圈数≥7 r·min^(-1)(或每30 min 210 r)的大鼠作为6-羟基多巴诱导的PD模型动物。采用脑微透析活体采样联用高效液相色谱-化学发光法(HPLC-CL)监测自由清醒大鼠纹状体内多巴胺水平的动态变化。HPLC分离系统包括Shim pack ODS色谱柱(250 mm×4.6 mm, 5μm),三水合乙酸钠缓冲液-乙腈(80∶20)作为流动相,流速为1.0 mL·min^(-1),柱温为37℃。CL检测系统包括3μmol·L^(-1)鲁米诺溶液、稀释1 000倍的纳米金溶液和50μmol·L^(-1)Ag(Ⅲ)溶液。结果:建立的HPLC-CL法分析多巴胺在0.05~50 ng·mL^(-1)范围内线性关系良好,检测限为0.014 ng·mL^(-1)。与左旋多巴组相比,天然冰片联合左旋多巴组大鼠脑微透析液中游离多巴胺药时曲线下面积(AUC0-t)从3.042 h·ng·mL^(-1)增加到4.395 h·ng·mL^(-1),达峰时间(Tmax)从1.516 h缩短为1.028 h;峰浓度(Cmax)从0.435 ng·mL^(-1)升高到0.792 ng·mL^(-1),更接近于正常组大鼠脑透析液中游离多巴胺水平(0.971 ng·mL^(-1))。结论:天然冰片可促进左旋多巴快速入脑并转化为多巴胺,这可能与天然冰片提高左旋多巴的血脑屏障通透性有关。本研究结果可为天然冰片联合左旋多巴用药临床治疗PD提供科学依据。Objective: To establish a microdialysis sampling-high performance liquid chromatography-chemiluminescence method for dynamic monitoring of dopamine levels in the striatum of free awake rats and study the effect of natural borneol combined with levodopa on the dopamine levels in the striatum of 6-hydroxydopamine-induced Parkinson’s disease(PD) model rats. Methods: Male SD rats were randomly divided into control group, 6-hydroxydopamine-induced PD model group, levodopa group and natural borneol combined with levodopa group. PD model rats were slowly injected with 4 μg·μL^(-1)6-hydroxydopamine in right medial forebrain bundle(MFB) after anesthesia 0.5 mg·kg^(-1)apomorphine was then injected intraperitoneally 4 weeks after operation, and rats with the number of rotations ≥7 r·min^(-1)(or 210 r 30 min) were selected as 6-hydroxydopa-induced PD model rats. High performance liquid chromatography-chemiluminescence(HPLC-CL) method combined with in vivo brain microdialysis sampling was developed for the detection of free dopamine in the striatum of free awake rats. The HPLC separation system included Shim pack ODS column(250 mm×4.6 mm, 5 μm), sodium acetate trihydrate buffer-acetonitrile(80∶20) as mobile phase, flow rate of 1.0 mL·min^(-1), column temperature of 37 ℃. CL detection system included 3 μmol·L^(-1)luminol solution, 1 000 times diluted gold nanoparticle solution and 50 μmol·L^(-1)Ag(Ⅲ) solution. Results: The linear range of dopamine of HPLC-CL method was 0.05-50 ng·mL^(-1), and the detection limit was 0.014 ng·mL^(-1). The pharmacokinetic results showed that compared with levodopa group, the AUC0-tof dopamine in natural borneol combined with levodopa group increased from 3.042 h·ng·mL^(-1)to 4.395 h·ng·mL^(-1), Tmaxdecreased from 1.516 h to 1.028 h, and the Cmaxincreased from 0.435 ng·mL^(-1)to 0.792 ng·mL^(-1), which was close to that of control group(0.971 ng·mL^(-1)). Conclusion: Natural borneol promotes the rapid transformation of levodopa into the brain and conversion to dopam

关 键 词：帕金森病 天然冰片 左旋多巴 多巴胺 微透析 高效液相色谱 化学发光 

分 类 号：R917[医药卫生—药物分析学]