川芎嗪对神经病理性疼痛模型大鼠血、脑药代动力学及其镇痛活性的影响

Effects of tetramethylpyrazine on pharmacokinetics in plasma and brain dialysate and neuropathic pain in rat model of spared sciatic nerve injury

研究川芎嗪(tetramethylpyrazine,TMP)对坐骨神经部分损伤(spared sciatic nerve injury,SNI)诱导的神经病理性疼痛模型大鼠血浆和脑透析液中的药代动力学和镇痛活性的影响,考察TMP的镇痛效应与其脑内的血药浓度的相关性。雄性SD大鼠,随机分为假手术组(Sham组)、SNI组和SNI+TMP组,采用von fray纤维丝机械刺激法和冷喷法评价大鼠机械痛敏和冷痛敏行为。另取Sham+TMP组和SNI+TMP组,分别采用大鼠颈总静脉插管和前扣带回脑区(ACC)植入微透析探针,按照80 mg·kg^(-1)单次腹腔注射TMP,应用自动采血和脑内微透析(灌流速度1.0μL·min^(-1))采样系统,分别收集血液和脑细胞外液,连续收集24 h。液相色谱分离采用HSS T3 C18反相色谱柱(2.1 mm×50 mm,2.5μm),流动相为甲醇⁃水(含0.005%甲酸),流速0.25 mL·min^(-1),梯度洗脱;质谱检测采用电喷雾离子源,扫描方式为多反应正离子监测模式。定量分析离子对分别为TMP m/z 137/122,阿司匹林m/z 179/137。利用DAS 2.11软件计算药代动力学参数。TMP镇痛效应和冷痛敏抑制最佳时间均为药后60 min。TMP在SNI大鼠血浆和脑细胞外液中T_(max)为15、30 min,C_(max)为(2866.43±135.39)、(1462.14±197.38)μg·L^(-1),AUC_(0⁃t)为(241463.30±28070.31)、(213115.62±32570.07)μg·min·L^(-1),MRT_(0⁃t)为(353.13±47.73)、(172.16±12.72)min,CLz为0.73、0.36 L·min·kg^(-1)。TMP的镇痛效应与其疼痛敏感区ACC的血药浓度具有明显的相关性,说明该方法适用于大鼠血浆和脑内透析液中TMP的检测,准确可靠、灵敏度高,能够实现“自动采血⁃微透析/PK⁃PD”关联分析理论应用在神经病理性疼痛研究中。

This study aims to explore the effects of tetramethylpyrazine(TMP)on pharmacokinetics in plasma and brain dialysate and neuropathic pain in the rat model of partial sciatic nerve injury(SNI),and to investigate the correlation between the analgesic effect of TMP and its concentrations in the plasma and brain dialysate.Male SD rats were randomized into Sham,SNI,and SNI+TMP groups.Mechanical stimulation with von frey filaments and cold spray method were employed to evaluate the mechanical sensitivity and cold sensitivity of rats.Another two groups,Sham+TMP and SNI+TMP,were used to intubate the common jugular vein and implant microdialysis probes into the anterior cingulate gyrus(ACC),respectively.After intraperitoneal injection of TMP at a dose of 80 mg·kg^(-1),automatic blood collection and intracerebral microdialysis(perfusion rate of 1μL·min^(-1))systems were used to collect the blood and brain dialysate for 24 h.HSS T3 C18 reversed⁃phase chromatographic column(2.1 mm×50 mm,2.5μm)was used for liquid chromatographic separation.Gradient elution was carried out with the mobile phase of methanol⁃water(containing 0.005%formic acid)at a flow rate of 0.25 mL·min^(-1).Electrospray ion source was used for mass spectrometry,and the scanning mode was multi⁃reaction monitoring under the positive ion mode.The ion pairs for quantitative analysis were TMP m/z 137/122 and aspirin m/z 179/137,respectively.DAS 2.11 was used to calculate the pharmacokinetic parameters.The optimal time of TMP to exert the analgesia effect and inhibit cold pain sensitivity was 60 min after treatment.The TMP in the plasma and brain dialysate of SNI rats showed the Tmax of 15 min and 30 min,the Cmax of(2866.43±135.39)and(1462.14±197.38)μg·L^(-1),the AUC_(0⁃t)of(241463.30±28070.31)and(213115.62±32570.07)μg·min·L^(-1),the MRT_(0⁃t)of(353.13±47.73)and(172.16±12.72)min,and the CLZ of 0.73 and 0.36 L·min·kg^(-1),respectively.The analgesic effect of TMP had a significant correlation with the blood drug concentration in the ACC,which indicated that this method was suitable for the detection of TMP in rat plasma and brain dialysate.The method is accurate,reliable,and sensitive and can realize the important value of the application of correlation analysis theory of"automatic blood collection⁃microdialysis/PK⁃PD"in the research on neuropathic pain.