[Compound matrine injection reduces morphine tolerance of the mice with lung cancer by inhibiting expression of multidrug resistance gene 1 and P-glycoprotein].

Objective: To investigate the effect of compound matrine injection on morphine tolerance in mice with lung cancer in situ and the expressions of multidrug resistance gene 1 (MDR1) and P-glycoprotein (P-gp). Methods: A mouse model of lung cancer in situ and morphine tolerance mode was established. The mice were injected with gradient concentration of compound matrine. The pain thresholds under different conditions were measured by thermal radiation tail-flick method. The mRNA level of MDR1 was tested by reverse transcription polymerase chain reaction (RT-PCR) and the protein level of P-gp was detected by western blot. The DNA binding activity of cyclophosphoadenosine response element binding protein (CREB) to the promoter of MDR1 gene was detected by electrophoretic mobility shift assay (EMSA). Results: The maximum analgesic percentage (MPE) of the mice in the morphine group was (85.21±6.53)% on the 8th day, and decreased to (38.45±5.52)% and (28.14±4.52)% on the 10th and 12th day, respectively, which indicated the morphine tolerance of mice with lung cancer in situ.The MPE of the mice in the group treated with morphine and compound matrine injection (300 mg/kg) was (79.34±6.50)% on the 8th day, and decreased to (62.16±5.53)% and (40.20±4.50)% on the 10th and 12th day, respectively.The results of RT-PCR assay showed that the relative expression levels of MDR1 mRNA in the brain tissues of mice in the morphine group, saline group, morphine combined with compound matrine injection (300 mg/kg) group and compound matrine injection (200 mg/kg) group were 2.33±0.79, 1.04±0.38, 1.37±0.38, and 1.43±0.53, respectively. There were statistically significant differences between the morphine group and the normal saline group, the morphine group and the morphine combined with compound matrine injection (300 mg/kg) group (P<0.05). There was no significant difference between the normal saline group and the compound matrine injection (200 mg/kg) group (P=0.05). The results of western blot showed that the relative expression levels of P-gp protein in the brain tissue of mice in the morphine group, saline group, and morphine combined with compound matrine injection (300 mg/kg) group were 1.86±0.40, 1.00±0.23, and 1.27±0.27, respectively. The expression of P-gp protein in the morphine group was significantly higher than those of the normal saline group and the morphine combined with compound matrine injection (300 mg/kg) group (P<0.05). The DNA-binding activity of CREB in the saline group was (0.23±0.07) Pu, significantly lower than (0.89±0.23) Pu of morphine combined with naloxone group and (0.80±0.23) Pu of morphine group (P<0.05). While the CREB DNA binding activity of morphine combined with compound matrine injection (300 mg/kg) group was (0.79±0.21) Pu, implicated that compound matrine had marginal effect on the DNA-binding activity of CREB (P>0.05). Conclusion: Compound matrine injection can significantly improve morphine tolerance and drug resistance of lung cancer through inhibiting the upregulations of MDR1 and P-gp induced by morphine.目的： 研究复方苦参注射液对原位肺癌小鼠吗啡耐受的影响，检测其对原位肺癌小鼠模型吗啡激活的多药耐药基因1(MDR1)介导的P糖蛋白(P-gp)过表达的关系。 方法： 建立原位肺癌小鼠吗啡耐受模型，采用梯度浓度的复方苦参注射液处理，热辐射甩尾法测定不同条件下痛阈值；采用实时荧光定量聚合酶链反应(RT-PCR)和Western blot法，检测MDR1 mRNA和P-gp蛋白的表达；采用凝胶迁移技术检测MDR1基因上游启动子环磷腺苷效应元件结合蛋白(CREB)的DNA结合活性的改变。 结果： 吗啡组小鼠在第8天的最大镇痛百分率(MPE)为(85.21±6.53)%，第10天和第12天分别下降至(38.45±5.52)%和(28.14±4.52)%，表明原位肺癌小鼠已经形成吗啡耐受。而吗啡＋300 mg/kg复方苦参注射液组小鼠在第8天的MPE为(79.34±6.50)%，第10天和第12天分别下降至(62.16±5.53)%和(40.20±4.50)%。RT-PCR检测结果显示，吗啡组、生理盐水组、吗啡＋300 mg/kg复方苦参注射液组和200 mg/kg复方苦参注射液组小鼠脑组织中MDR1 mRNA的相对表达量分别为2.33±0.79、1.04±0.38、1.37±0.38和1.43±0.53。吗啡组与生理盐水组比较，差异有统计学意义(P<0.05)；生理盐水组与200 mg/kg复方苦参注射液组比较，差异无统计学意义(P＝0.05)；吗啡组与吗啡＋300 mg/kg复方苦参注射液组比较，差异有统计学意义(P<0.05)。Western blot法检测结果显示，吗啡组、生理盐水组、吗啡＋300 mg/kg复方苦参注射液组小鼠脑组织中P-gp蛋白的相对表达量分别为1.86±0.40、1.00±0.23和1.27±0.27。吗啡组P-gp蛋白的表达量明显高于生理盐水组(P<0.05)；吗啡＋300 mg/kg复方苦参注射液组与吗啡组比较，P-gp蛋白的表达亦明显下降(P<0.05)。吗啡＋纳络酮组CREB的DNA结合活性为(0.89±0.23)Pu；生理盐水组CREB的DNA结合活性为(0.23±0.07)Pu，明显低于吗啡组[(0.81±0.23)Pu，P<0.05]和吗啡＋纳络酮组(P<0.05)；而吗啡＋300 mg/kg复方苦参注射液组CREB的DNA结合活性为(0.79±0.21)Pu，对吗啡依赖所诱导的CREB的DNA结合活性升高未显示调节作用(P>0.05)。 结论： 复方苦参注射液能明显改善吗啡耐受，同时可抑制被吗啡激活的MDR1 mRNA和P-gp蛋白的表达，这与其延缓吗啡耐受的机制有关。.