川芎内酯通过 ULK1/NCOA4 通路抑制浦肯野细胞铁蛋白自噬，从而减轻丙戊酸诱导的自闭症特征。

Ligustilide inhibits Purkinje cell ferritinophagy via the ULK1/NCOA4 pathway to attenuate valproic acid-induced autistic features.

机构信息

Chongqing Institute for Brain and Intelligence, Guangyang Bay Laboratory, Chongqing, 400064, China.

Department of Military Cognitive Psychology, School of Psychology, Army Medical University, Chongqing, 400038, China.

出版信息

Phytomedicine. 2024 Apr;126:155443. doi: 10.1016/j.phymed.2024.155443. Epub 2024 Feb 15.

DOI:10.1016/j.phymed.2024.155443

PMID:38394737

Abstract

BACKGROUND

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder in which social impairment is the core symptom. Presently, there are no definitive medications to cure core symptoms of ASD, and most therapeutic strategies ameliorate ASD symptoms. Treatments with proven efficacy in autism are imminent. Ligustilide (LIG), an herbal monomer extracted from Angelica Sinensis and Chuanxiong, is mainly distributed in the cerebellum and widely used in treating neurological disorders. However, there are no studies on its effect on autistic-like phenotypes and its mechanism of action.

PURPOSE

Investigate the efficacy and mechanism of LIG in treating ASD using two Valproic acid(VPA)-exposed and BTBR T + Itpr3tf/J (BTBR) mouse models of autism.

METHODS

VPA-exposed mice and BTBR mice were given LIG for treatment, and its effect on autistic-like phenotype was detected by behavioral experiments, which included a three-chamber social test. Subsequently, RNA-Sequence(RNA-Seq) of the cerebellum was performed to observe the biological changes to search target pathways. The autophagy and ferroptosis pathways screened were verified by WB(Western Blot) assay, and the cerebellum was stained by immunofluorescence and examined by electron microscopy. To further explore the therapeutic mechanism, ULK1 agonist BL-918 was used to block the therapeutic effect of LIG to verify its target effect.

RESULTS

Our work demonstrates that LIG administration from P12-P14 improved autism-related behaviors and motor dysfunction in VPA-exposed mice. Similarly, BTBR mice showed the same improvement. RNA-Seq data identified ULK1 as the target of LIG in regulating ferritinophagy in the cerebellum of VPA-exposed mice, as evidenced by activated autophagy, increased ferritin degradation, iron overload, and lipid peroxidation. We found that VPA exposure-induced ferritinophagy occurred in the Purkinje cells, with enhanced NCOA4 and Lc3B expressions. Notably, the therapeutic effect of LIG disappeared when ULK1 was activated.

CONCLUSION

LIG treatment inhibits ferritinophagy in Purkinje cells via the ULK1/NCOA4-dependent pathway. Our study reveals for the first time that LIG treatment ameliorates autism symptoms in VPA-exposed mice by reducing aberrant Purkinje ferritinophagy. At the same time, our study complements the pathogenic mechanisms of autism and introduces new possibilities for its therapeutic options.

摘要

背景

自闭症谱系障碍（ASD）是一种神经发育障碍，其核心症状为社交障碍。目前，尚无明确的药物可以治愈 ASD 的核心症状，大多数治疗策略仅能改善 ASD 症状。具有明确疗效的 ASD 治疗方法迫在眉睫。藁本内酯（LIG）是从当归和川芎中提取的一种草药单体，主要分布在小脑，广泛用于治疗神经疾病。然而，目前尚无关于其对自闭症样表型的影响及其作用机制的研究。

目的

使用两种丙戊酸（VPA）暴露和 BTBR T + Itpr3tf/J（BTBR）自闭症小鼠模型，研究 LIG 治疗 ASD 的疗效和机制。

方法

用 LIG 治疗 VPA 暴露和 BTBR 小鼠，通过行为实验检测其对自闭症样表型的影响，包括三箱社交测试。随后，对小脑进行 RNA 测序（RNA-Seq），观察生物变化以搜索目标途径。通过 Western blot（WB）检测筛选出的自噬和铁死亡途径，并通过免疫荧光染色和电子显微镜检查对小脑进行染色。为了进一步探索治疗机制，使用 ULK1 激动剂 BL-918 阻断 LIG 的治疗效果，以验证其靶向作用。

结果

我们的工作表明，从 P12-P14 开始给予 LIG 治疗可改善 VPA 暴露小鼠的自闭症相关行为和运动功能障碍。同样，BTBR 小鼠也表现出相同的改善。RNA-Seq 数据表明，LIG 通过调节 VPA 暴露小鼠小脑中的铁蛋白自噬，将 ULK1 作为其在小脑中的作用靶点，自噬被激活，铁蛋白降解增加，铁超载和脂质过氧化。我们发现，VPA 暴露诱导的铁蛋白自噬发生在浦肯野细胞中，NCOA4 和 Lc3B 的表达增强。值得注意的是，当激活 ULK1 时，LIG 的治疗效果消失。