Abstract：
BACKGROUND: Anshen Dingzhi prescription (ADP), documented in \"Yi Xue Xin Wu\", is a famous prescription for treating panic-related mental disorders such as post-traumatic stress disorder (PTSD). However, the underlying mechanism remains unclear.
OBJECTIVE: This study aimed to investigate the mechanisms by which ADP intervened in PTSD-like behaviors.
METHODS: A mouse model of single prolonged stress (SPS) was established to evaluate the ameliorative effects and mechanisms of ADP on PTSD. Behavioral tests were used to assess PTSD-like behaviors in mice; transmission electron microscopy was used to observe changes in the ultrastructure of hippocampal synapses, and western blot, immunofluorescence, and ELISA were used to detect the expression of hippocampal deleted in colorectal cancer (DCC) and downstream Ras-related C3 botulinum toxin substrate 1 (Rac1) - P21-activated kinase 1 (PAK1) signal, as well as levels of synaptic proteins and inflammatory factors. Molecular docking technology simulated the binding of potential brain-penetrating components of ADP to DCC.
RESULTS: SPS induced PTSD-like behaviors in mice and increased expression of hippocampal netrin-1 (NT-1) and DCC on the 14th day post-modeling, with concurrent elevation in serum NT-1 levels. Simultaneously, SPS also decreased p-Rac1 level and increased p-PAK1 level, the down-stream molecules of DCC. Lentiviral overexpression of DCC induced or exacerbated PTSD-like behaviors in control and SPS mice, respectively, whereas neutralization antibody against NT-1 reduced DCC activation and ameliorated PTSD-like behaviors in SPS mice. Interestingly, downstream Rac1-PAK1 signal was altered according to DCC expression. Moreover, DCC overexpression down-regulated N-methyl-d-aspartate (NMDA) receptor 2A (GluN2A) and postsynaptic density 95 (PSD95), up-regulated NMDA receptor 2B (GluN2B) and increased neuroinflammatory responses. Administration of ADP (36.8 mg/kg) improved PTSD-like behaviors in the SPS mice, suppressed hippocampal DCC, and downstream Rac1-PAK1 signal, upregulated GluN2A and PSD95, downregulated GluN2B, and reduced levels of inflammatory factors NOD-like receptor protein 3 (NLRP3), nuclear factor kappa-B (NF-κB) and interleukin-6 (IL-6). Importantly, DCC overexpression could also reduce the ameliorative effect of ADP on PTSD. Additionally, DCC demonstrated a favorable molecular docking pattern with the potential brain-penetrating components of ADP, further suggesting DCC as a potential target of ADP.
CONCLUSIONS: Our data indicate that DCC is a key target for the regulation of synaptic function and inflammatory response in the onset of PTSD, and ADP likely reduces DCC to prevent PTSD via modulating downstream Rac1-PAK1 pathway. This study provides a novel mechanism for the onset of PTSD and warrants the clinical application of ADP.
OBJECTIVE: This study aimed to investigate the mechanisms by which ADP intervened in PTSD-like behaviors.
METHODS: A mouse model of single prolonged stress (SPS) was established to evaluate the ameliorative effects and mechanisms of ADP on PTSD. Behavioral tests were used to assess PTSD-like behaviors in mice; transmission electron microscopy was used to observe changes in the ultrastructure of hippocampal synapses, and western blot, immunofluorescence, and ELISA were used to detect the expression of hippocampal deleted in colorectal cancer (DCC) and downstream Ras-related C3 botulinum toxin substrate 1 (Rac1) - P21-activated kinase 1 (PAK1) signal, as well as levels of synaptic proteins and inflammatory factors. Molecular docking technology simulated the binding of potential brain-penetrating components of ADP to DCC.
RESULTS: SPS induced PTSD-like behaviors in mice and increased expression of hippocampal netrin-1 (NT-1) and DCC on the 14th day post-modeling, with concurrent elevation in serum NT-1 levels. Simultaneously, SPS also decreased p-Rac1 level and increased p-PAK1 level, the down-stream molecules of DCC. Lentiviral overexpression of DCC induced or exacerbated PTSD-like behaviors in control and SPS mice, respectively, whereas neutralization antibody against NT-1 reduced DCC activation and ameliorated PTSD-like behaviors in SPS mice. Interestingly, downstream Rac1-PAK1 signal was altered according to DCC expression. Moreover, DCC overexpression down-regulated N-methyl-d-aspartate (NMDA) receptor 2A (GluN2A) and postsynaptic density 95 (PSD95), up-regulated NMDA receptor 2B (GluN2B) and increased neuroinflammatory responses. Administration of ADP (36.8 mg/kg) improved PTSD-like behaviors in the SPS mice, suppressed hippocampal DCC, and downstream Rac1-PAK1 signal, upregulated GluN2A and PSD95, downregulated GluN2B, and reduced levels of inflammatory factors NOD-like receptor protein 3 (NLRP3), nuclear factor kappa-B (NF-κB) and interleukin-6 (IL-6). Importantly, DCC overexpression could also reduce the ameliorative effect of ADP on PTSD. Additionally, DCC demonstrated a favorable molecular docking pattern with the potential brain-penetrating components of ADP, further suggesting DCC as a potential target of ADP.
CONCLUSIONS: Our data indicate that DCC is a key target for the regulation of synaptic function and inflammatory response in the onset of PTSD, and ADP likely reduces DCC to prevent PTSD via modulating downstream Rac1-PAK1 pathway. This study provides a novel mechanism for the onset of PTSD and warrants the clinical application of ADP.
摘要：
背景：安神定智方（ADP），记录在“易学新吴”中，是治疗与恐慌有关的精神障碍如创伤后应激障碍（PTSD）的著名处方。然而,潜在机制尚不清楚.
目的：本研究旨在探讨ADP干预PTSD样行为的机制。
方法：建立单一延长应激（SPS）小鼠模型，以评估ADP对PTSD的改善作用和机制。行为测试用于评估小鼠PTSD样行为；透射电子显微镜用于观察海马突触超微结构的变化，和westernblot,免疫荧光，ELISA法检测结直肠癌海马缺失(DCC)及下游Ras相关C3肉毒毒素底物1(Rac1)-P21活化激酶1(PAK1)信号的表达,以及突触蛋白和炎症因子的水平。分子对接技术模拟了ADP潜在的脑穿透性成分与DCC的结合。
结果：SPS在模型后第14天诱导小鼠PTSD样行为，并增加海马netrin-1（NT-1）和DCC的表达，同时血清NT-1水平升高。同时,SPS还降低了p-Rac1水平，增加了p-PAK1水平，DCC的下游分子。慢病毒过表达DCC诱导或加剧对照和SPS小鼠的PTSD样行为,分别,而针对NT-1的中和抗体降低了SPS小鼠的DCC激活并改善了PTSD样行为。有趣的是,下游Rac1-PAK1信号根据DCC表达而改变。此外，DCC过表达下调N-甲基-d-天冬氨酸（NMDA）受体2A（GluN2A）和突触后密度95（PSD95），上调NMDA受体2B（GluN2B）和增加的神经炎症反应。给予ADP（36.8mg/kg）改善了SPS小鼠的PTSD样行为，抑制海马DCC，和下游Rac1-PAK1信号，上调GluN2A和PSD95，下调GluN2B，炎症因子NOD样受体蛋白3（NLRP3）水平降低，核因子κB（NF-κB）和白细胞介素6（IL-6）。重要的是,DCC过表达还可以降低ADP对PTSD的改善作用。此外,DCC与ADP的潜在脑穿透性成分表现出良好的分子对接模式，进一步表明DCC是ADP的潜在靶标。
结论：我们的数据表明，DCC是PTSD发作中调节突触功能和炎症反应的关键靶标，ADP可能通过调节下游Rac1-PAK1途径降低DCC以预防PTSD。这项研究为PTSD的发作提供了新的机制，并保证了ADP的临床应用。
目的：本研究旨在探讨ADP干预PTSD样行为的机制。
方法：建立单一延长应激（SPS）小鼠模型，以评估ADP对PTSD的改善作用和机制。行为测试用于评估小鼠PTSD样行为；透射电子显微镜用于观察海马突触超微结构的变化，和westernblot,免疫荧光，ELISA法检测结直肠癌海马缺失(DCC)及下游Ras相关C3肉毒毒素底物1(Rac1)-P21活化激酶1(PAK1)信号的表达,以及突触蛋白和炎症因子的水平。分子对接技术模拟了ADP潜在的脑穿透性成分与DCC的结合。
结果：SPS在模型后第14天诱导小鼠PTSD样行为，并增加海马netrin-1（NT-1）和DCC的表达，同时血清NT-1水平升高。同时,SPS还降低了p-Rac1水平，增加了p-PAK1水平，DCC的下游分子。慢病毒过表达DCC诱导或加剧对照和SPS小鼠的PTSD样行为,分别,而针对NT-1的中和抗体降低了SPS小鼠的DCC激活并改善了PTSD样行为。有趣的是,下游Rac1-PAK1信号根据DCC表达而改变。此外，DCC过表达下调N-甲基-d-天冬氨酸（NMDA）受体2A（GluN2A）和突触后密度95（PSD95），上调NMDA受体2B（GluN2B）和增加的神经炎症反应。给予ADP（36.8mg/kg）改善了SPS小鼠的PTSD样行为，抑制海马DCC，和下游Rac1-PAK1信号，上调GluN2A和PSD95，下调GluN2B，炎症因子NOD样受体蛋白3（NLRP3）水平降低，核因子κB（NF-κB）和白细胞介素6（IL-6）。重要的是,DCC过表达还可以降低ADP对PTSD的改善作用。此外,DCC与ADP的潜在脑穿透性成分表现出良好的分子对接模式，进一步表明DCC是ADP的潜在靶标。
结论：我们的数据表明，DCC是PTSD发作中调节突触功能和炎症反应的关键靶标，ADP可能通过调节下游Rac1-PAK1途径降低DCC以预防PTSD。这项研究为PTSD的发作提供了新的机制，并保证了ADP的临床应用。
