王康恒, 王毅. 基于mTOR/ULK1/ATG13通路探讨绞股蓝总苷对抑郁症大鼠海马神经元过度自噬的保护作用[J]. 蚌埠医学院学报, 2023, 48(10): 1333-1338. DOI: 10.13898/j.cnki.issn.1000-2200.2023.10.002
    引用本文: 王康恒, 王毅. 基于mTOR/ULK1/ATG13通路探讨绞股蓝总苷对抑郁症大鼠海马神经元过度自噬的保护作用[J]. 蚌埠医学院学报, 2023, 48(10): 1333-1338. DOI: 10.13898/j.cnki.issn.1000-2200.2023.10.002
    WANG Kang-heng, WANG Yi. Protective effect of gypenosides on excessive autophagy of hippocampal neurons in depression rats based on mTOR/ULK1/ATG13 pathway[J]. Journal of Bengbu Medical College, 2023, 48(10): 1333-1338. DOI: 10.13898/j.cnki.issn.1000-2200.2023.10.002
    Citation: WANG Kang-heng, WANG Yi. Protective effect of gypenosides on excessive autophagy of hippocampal neurons in depression rats based on mTOR/ULK1/ATG13 pathway[J]. Journal of Bengbu Medical College, 2023, 48(10): 1333-1338. DOI: 10.13898/j.cnki.issn.1000-2200.2023.10.002

    基于mTOR/ULK1/ATG13通路探讨绞股蓝总苷对抑郁症大鼠海马神经元过度自噬的保护作用

    Protective effect of gypenosides on excessive autophagy of hippocampal neurons in depression rats based on mTOR/ULK1/ATG13 pathway

    • 摘要:
      目的探讨绞股蓝总苷(Gyp)对抑郁症大鼠海马神经元自噬及雷帕霉素靶蛋白(mTOR)/自噬激活激酶1(ULK1)/自噬相关蛋白13(ATG13)通路的影响。
      方法取SD大鼠,随机分为正常对照组、模型组、Gyp低(25 mg/kg)剂量组、高(100 mg/kg)剂量组、自噬激活剂-雷帕霉素(Rap)组(1 mg/kg)、Gyp+Rap组(100 mg/kg+1 mg/kg),每组10只。用慢性轻度不可预见性应激法制备大鼠抑郁模型,各组连续给药4周,1次/天。用旷场、糖水偏好及强迫游泳试验评估大鼠抑郁样行为,取海马组织,透射电镜观察海马神经元结构变化及自噬小体、自噬溶酶体形成情况;TUNEL法检测神经元凋亡状况;免疫组织化学染色法检测ULK1磷酸化水平(p-ULK1)及p-ATG13在海马组织中阳性表达水平;Western blotting检测总mTOR、p-mTOR、核糖体S6蛋白(S6)及p-S6、ULK1特定位点Ser757蛋白(ULK1 Ser757)及p-ULKl Ser757、自噬相关蛋白Beclin1、微管相关蛋白轻链3(LC3)蛋白、凋亡相关蛋白-半胱天冬酶-3(Caspase-3)及多聚腺苷酸二磷酸核糖聚合酶(PARP)蛋白表达水平。
      结果与正常对照组相比,模型组大鼠旷场实验中央探索时间、糖水摄取百分比均降低(P < 0.01),强迫游泳不动时间延长(P < 0.01),海马神经元核膜破坏严重、自噬小体形成较多,凋亡率、凋亡相关蛋白及Beclin1、LC3蛋白表达均升高(P < 0.01),mTOR/ULK1/ATG13通路蛋白磷酸化水平均降低(P < 0.01)。与模型组相比,Gyp剂量越高,大鼠抑郁样行为缓解(P < 0.01)、海马神经元凋亡及自噬水平减弱,mTOR/ULK1/ATG13通路蛋白磷酸化水平升高(P < 0.01);Rap组大鼠海马组织mTOR/ULK1/ATG13通路蛋白磷酸化水平进一步降低(P < 0.01),海马神经元凋亡及自噬水平进一步升高(P < 0.01),大鼠抑郁样行为进一步加重(P < 0.01)。Gyp+Rap组大鼠上述指标变化与Gyp组相反(P < 0.01)。
      结论Gyp可能通过激活mTOR磷酸化活性水平,促进ULK1及ATG13磷酸化来抑制自噬,防止神经元自噬性死亡,发挥抗抑郁作用。

       

      Abstract:
      ObjectiveTo investigate the effects of gypenosides (Gyp) on hippocampal neuron autophagy and pathway of mammalian target of rapamycin (mTOR)/autophagy-activated kinase 1 (ULK1)/autophagy-related protein 13 (ATG13) in depression rats.
      MethodsSD rats were randomly divided into normal control group, model group, Gyp low (25 mg/kg), high (100 mg/kg) dose group, autophagy activator-rapamycin (Rap) group (1 mg/kg), and Gyp+Rap group (100 mg/kg+1 mg/kg), with 10 rats in each group.Chronic mild unpredictable stress was used to prepare rat depression models, and each group was administered for 4 weeks, once daily.Open field, sugar water preference and forced swimming tests were used to assess depression-like behavior in rats, transmission electron microscope was used to observe the changes in hippocampal neuron structure and the formation of autophagosomes and autophagolysosomes.TUNEL method was used to detect neuronal apoptosis.Immunohistochemical staining was used to detect the phosphorylation level of ULK1 (p-ULK1) and the positive expression level of p-ATG13 in the hippocampus.Western blotting was used to detect the total mTOR, p-mTOR, ribosomal S6 protein (S6), p-S6, ULK1 specific site Ser757 protein (ULK1 Ser757) and p-ULKl Ser757, autophagy-related protein Beclin1, microtubule-related protein Light chain 3 (LC3) protein, apoptosis-related protein-Caspase-3 (Caspase-3) and polyadenylic acid diphosphate ribose polymerase (PARP) protein expression levels.
      ResultsCompared with the normal control group, the central exploration time in the open field experiment and the percentage of sugar water intake in the model group were reduced, the time of forced swimming immobility was prolonged, the depressive behavior was severe, the nuclear membrane of hippocampal neurons was severely damaged, and autophagosomes were formed more, the apoptosis rate, apoptosis-related protein, Beclin1 and LC3 protein expression were increased, the phosphorylation level of mTOR/ULK1/ATG13 pathway protein was decreased, and the changes of each index were significantly different (P < 0.01).Compared with the model group, with the Gyp dose increased, the depressive behavior of rats was relieved, and the hippocampal neuronal apoptosis and level of autophagy were decreased, the phosphorylation level of mTOR/ULK1/ATG13 pathway protein was increased (P < 0.01);the phosphorylation level of mTOR/ULK1/ATG13 pathway protein in the hippocampus tissue of the Rap group was further reduced, the levels of hippocampal neuron apoptosis and autophagy were further increased, and the depression-like behavior in rats was further aggravated (P < 0.01).The changes of the above indicators in the Gyp+Rap group were opposite to those in the Gyp group (P < 0.01).
      ConclusionsGyp may inhibit autophagy by activating the activity level of mTOR phosphorylation and promoting the phosphorylation of ULK1 and ATG13, so as to prevent autophagic death of neurons, and exert antidepressant effect.

       

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