柴胡皂苷A对脑外伤大鼠海马神经元自噬及AMPK-mTORC1通路的影响

Effects of saikosaponin A on autophagy and AMPK-mTORC1 pathway in hippocampal neurons of rats with traumatic brain injury

目的探讨柴胡皂苷A对脑外伤大鼠腺苷酸活化蛋白激酶(AMPK)–雷帕毒素靶蛋白复合物1(m TORC1)信号通路及海马神经元自噬的影响。方法 60只SD大鼠随机分为假手术组、模型组及柴胡皂苷A低剂量(5 mg/kg)、中剂量(10 mg/kg)、高剂量(20 mg/kg)组,和阳性对照(自噬抑制剂3-甲基腺嘌呤,15 mg/kg)组,每组10只。除假手术组外,其余各组大鼠均采用皮质撞击法构建脑外伤大鼠模型,造模成功后,柴胡皂苷A各剂量组、阳性对照组ip相应药物,假手术组和模型组ip等量生理盐水,持续14 d。采用Morris水迷宫测定大鼠的认知功能,以苏木精-伊红染色检测各组大鼠海马组织病理变化,采用酶联免疫吸附试剂盒检测大鼠海马组织脑源性神经生长因子(BDNF)、白细胞介素-6(IL-6)、肿瘤坏死因子(TNF-α)水平。透射电镜观察海马组织自噬情况,采用蛋白免疫印迹法检测海马组织中微管相关蛋白1轻链3(LC3)、Beclin-1及AMPK-mTORC1相关通路蛋白表达。结果与假手术组相比,模型组大鼠的潜伏期显著延长,穿台次数显著降低,海马组织神经细胞损坏严重,呈现细胞破裂、细胞核固缩等状态,海马组织BDNF含量显著降低,IL-6、TNF-α表达显著升高,海马组织自噬小泡、LC3Ⅱ/LC3Ⅰ、Beclin-1、p-AMPK/AMPK蛋白表达水平显著升高,p-mTORC1/mTORC1表达水平显著下降。与模型组相比,柴胡皂苷A各剂量组的潜伏期呈现逐渐缩短的趋势,穿台次数呈现逐渐升高的趋势,BDNF含量显著升高,IL-6、TNF-α表达显著降低,海马组织自噬小泡、LC3Ⅱ/LC3Ⅰ、Beclin-1、p-AMPK/AMPK蛋白表达水平显著降低,p-mTORC1/mTORC1表达水平显著升高,且均呈现一定的剂量相关性。结论柴胡皂苷A可调节脑外伤大鼠海马神经元自噬情况,可能通过抑制AMPK,促进m TORC1表达实现。

Objective To investigate the effects of saikosaponin A on the signal pathway of AMPK-mTORC1, and autophagy of hippocampal neurons in rats with traumatic brain injury. Methods A total of 60 SD rats were randomly divided into sham operation group, model group, saikosaponin A low-dose group(5 mg/kg), medium-dose group(10 mg/kg), and high-dose group(20 mg/kg),positive control group(autophagy inhibitor 3-methyladenine, 15 mg/kg), with 10 rats in each group. Except for the sham operation group, the rats in other groups were established with cortical impact method, and after successful modeling, saikosaponin A groups and positive control group were intraperitoneally injected with corresponding drugs, while the sham operation group and model group were injected with the same amount of normal saline for 14 d. Morris water maze was used to measure the cognitive function of rats,hematoxylin-eosin staining was used to detect the pathological changes of hippocampus, enzyme-linked immunosorbent assay was used to detect the levels of BDNF, IL-6,and TNF-α. Observation of autophagy of hippocampus by transmission electron microscopy,and LC3, Beclin-1, and AMPK-mTORC1 related pathway proteins in hippocampus were detected by Western blotting. Results Compared with sham operation group, the latency of rats in the model group was significantly extended, the times of crossing the platform was significantly lower, hippocampal neurons were seriously damaged, showing cell rupture and nuclear pyknosis, the content of BDNF in hippocampus was significantly decreased, the expression of IL-6 and TNF-α was significantly increased, autophagic vesicles and the protein expression levels of LC3Ⅱ/LC3Ⅰ, Beclin-1, and p-AMPK/AMPK in hippocampus were significantly increased, but the expression level of p-mTORC1/mTORC1 was significantly decreased. Compared with those in the model group, the latency of rats in saikosaponin A groups decreased gradually, the times of crossing the platform gradually increased, the content of BDNF in hippocampus increased gradually, the expression of IL-6 and TNF-α decreased gradually, autophagic vesicles and the protein expression levels of LC3Ⅱ/LC3Ⅰ, Beclin-1 and p-AMPK/AMPK in hippocampus decreased gradually, the expression level of pm TORC1/mTORC1 increased gradually, and there was a dose-dependent manner. Conclusion Saikosaponin A can regulate the autophagy of hippocampal neurons in rats with traumatic brain injury, which may be achieved by inhibiting AMPK and promoting the expression of mTORC1.