甲氨蝶呤对大鼠急性脊髓损伤后的神经保护作用及对PTEN/AKT/mTOR信号通路的影响

Neuroprotective effect of methotrexate on acute spinal cord injury in rats and its effect on PTEN/AKT/mTOR signaling pathway

目的探讨甲氨蝶呤通过调控PTEN/AKT/mTOR信号通路对大鼠急性脊髓损伤(ASCI)后的神经保护作用。方法将30只大鼠随机分为假手术组、模型组和甲氨蝶呤组,制备大鼠脊髓损伤模型,假手术组只移除椎板,甲氨蝶呤组在ASCI后立即腹腔注射0.5 mg/kg甲氨蝶呤,假手术组和模型组注射等量的生理盐水,持续治疗2周,采用脊髓运动功能(BBB)评分评定大鼠脊髓损伤情况,测定脊髓中白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)和丙二醛(MDA)水平,检测脊髓中张力蛋白同源物(PETN)、丝氨酸-苏氨酸蛋白激酶(AKT)、磷酸化丝氨酸-苏氨酸蛋白激酶(p-AKT)、雷帕霉素靶蛋白(mTOR)、雷帕霉素靶蛋白(p-mTOR)、BCL2关联X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)和半胱氨酸蛋白酶-3(Caspase-3)水平的变化及脊髓细胞凋亡情况。结果与假手术组相比,模型组和甲氨蝶呤组BBB评分降低,细胞凋亡率增加(P<0.05);与模型组相比,甲氨蝶呤组BBB评分增加,细胞凋亡率降低(P<0.05);与假手术组相比,模型组和甲氨蝶呤组脊髓组织中IL-6、TNF-α和MDA水平增加,PETN、Bax和Caspase-3水平增加,Bcl-2、p-AKT和p-mTOR水平降低(P<0.05);与模型组相比,甲氨蝶呤组脊髓组织中IL-6、TNF-α和MDA水平降低,PETN、Bax和Caspase-3水平降低,Bcl-2、p-AKT和p-mTOR水平增加(P<0.05)。各组大鼠脊髓组织中AKT和mTOR水平变化不显著(P>0.05)。结论甲氨蝶呤通过调控PTEN/AKT/mTOR信号通路对ASCI大鼠神经发挥保护作用。

Objective To investigate the methotrexate to play the neuroprotective effect on acute spinal cord injury in rats by regulating PTEN/AKT/mTOR signaling pathway.Methods Thirty rats were randomly divided into the sham operation group,model group and methotrexate group.The rat spinal cord injury model was prepared.The vertebral plate was removed only in the sham operation group.The methotrexate group was injected intraperitoneally with 0.5 mg/kg methotrexate immediately after acute spinal cord injury(ASCI).The sham operation group and model group were injected with the same amount of normal saline for 2-week persistent treatment.The spinal cord injury situation was assessed by adopting the BBB score.The levels of IL-6,TNF-αand MDA in spinal cord were measured.The changes of PETN,AKT,p-AKT,mTOR,p-mTOR,Bax,Bcl-2 and Caspase-3 protein levels and apoptosis of spinal cord cells were detected.Results Compared with the sham operation group,the BBB score in the model group and methotrexate group was decreased and cellular apoptotic rate was increased(P<0.05).Compared with the model group,the BBB score in the methotrexate group was increased and the cellular apoptotic rate was decreased(P<0.05).Compared with the sham operation group,the levels of IL-6,TNF-αand MDA in spinal cord tissue of the model group and methotrexate group were significantly increased,the levels of PETN,Bax and Caspase-3 were increased,while the levels of Bcl-2,p-AKT and p-mTOR were decreased(P<0.05).Compared with the model group,the levels of IL-6,TNF-αand MDA in spinal cord tissue of the methotrexate group were decreased,the levels of PETN,Bax and Caspase-3 were decreased,while the levels of Bcl-2,p-AKT and p-mTOR were increased(P<0.05).The levels of AKT and mTOR in rat spinal cord tissue of each group had no significant change(P>0.05).Conclusion Methotrexate plays the neuroprotective effect to ASCI rats by regulating PTEN/AKT/mTOR signaling pathway.