目的:PRAS40是mTORC1复合物的一个重要抑制亚基,调节自噬。提示二仙汤可通过自噬途径治疗脊髓损伤。然而,EXD是否通过PRAS40发挥作用的机制尚不清楚.方法:借助固定化PRAS40、等温滴定量热法(ITC)和分子对接,对EXD中的生物活性代谢物进行了筛选。建立体外SCI模型,将PC12细胞暴露于过氧化氢(H2O2),然后用鉴定的EXD物质处理。此外,进行Western印迹测定以鉴定涉及的潜在分子机制。为了评估体内代谢物的作用,SCI模型大鼠首先用或不用代谢物预处理,然后进行免疫组织化学(IHC)染色,巴索,Beattie&Bresnahan(BBB)运动量表,和H&E染色。结果:固定化PRAS40分离出吲哚,4-硝基苯酚,对苯二甲酸,巴马汀,芥子醛,和3-氯苯胺作为与PRAS40结合的潜在配体。此外,通过ITC阐明了巴马汀和吲哚的缔合常数为2.84×106M-1和3.82×105M-1。分子对接结果也进一步证明了巴马汀与PRAS40结合的机制。PC12细胞的Western印迹分析表明,巴马汀通过与PRAS40结合来抑制p-mTOR的表达,通过显着增加LC3来激活自噬通量。注射巴马汀(10μM和20μM)表明SCI大鼠模型中BBB评分显著增加。此外,通过IHC染色观察到LC3的剂量依赖性增加。结论:本研究证明EXD包含PRAS40拮抗剂,和确定的代谢物,巴马汀,可能通过激活自噬通量治疗SCI。
Objective: The
PRAS40 is an essential inhibitory subunit of the mTORC1 complex, which regulates autophagy. It has been suggested that Erxian Decoction (EXD) could treat spinal cord injury (SCI) via the autophagy pathway. However, the mechanism of whether EXD acts through
PRAS40 remains unclear. Methods: With the help of immobilized
PRAS40, isothermal titration calorimetry (ITC) and molecular docking, the bioactive metabolites in the EXD were screened. To establish in vitro SCI models, PC12 cells were exposed to hydrogen peroxide (H2O2) and then treated with the identified EXD substances. Furthermore, Western blot assay was carried out to identify potential molecular mechanisms involved. For assessing the effect of metabolites in vivo, the SCI model rats were first pretreated with or without the metabolite and then subjected to the immunohistochemistry (IHC) staining, Basso, Beattie & Bresnahan (BBB) locomotor rating scale, and H&E staining. Results: The immobilized PRAS40 isolated indole, 4-nitrophenol, terephthalic acid, palmatine, sinapinaldehyde, and 3-chloroaniline as the potential ligands binding to
PRAS40. Furthermore, the association constants of palmatine and indole as 2.84 × 106 M-1 and 3.82 × 105 M-1 were elucidated via ITC due to the drug-like properties of these two metabolites. Molecular docking results also further demonstrated the mechanism of palmatine binding to
PRAS40. Western blot analysis of PC12 cells demonstrated that palmatine inhibited the expression of p-mTOR by binding to PRAS40, activating the autophagic flux by markedly increasing LC3. The injection of palmatine (10μM and 20 μM) indicated notably increased BBB scores in the SCI rat model. Additionally, a dose-dependent increase in LC3 was observed by IHC staining. Conclusion: This research proved that EXD comprises PRAS40 antagonists, and the identified metabolite, palmatine, could potentially treat SCI by activating the autophagic flux.