四氢胡椒碱，一种对缺血性中风具有神经保护作用的天然生物碱。

Tetrahydropiperine, a natural alkaloid with neuroprotective effects in ischemic stroke.

作者信息

Ren Hongyan, Yuan Qianqian, Lu Jiayuan, Xi Siyu, Liu Yanbo, Yang Guangyu, Xie Zhixi, Wang Bo, Ma Li, Fu Xueyan, Liu Juan, Zhang Yiwei

机构信息

School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004, China; Key Laboratory of Ningxia Ethnomedicine Modernization, Minority of Education, Ningxia Medical University, Yinchuan 750004, China.

School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004, China.

出版信息

J Chem Neuroanat. 2024 Mar;136:102397. doi: 10.1016/j.jchemneu.2024.102397. Epub 2024 Feb 7.

DOI:10.1016/j.jchemneu.2024.102397

PMID:38331229

Abstract

BACKGROUND

Ischemic stroke (IS) is a life-threatening neurological disease with various pathological mechanisms. Tetrahydropiperine (THP) is a natural alkaloid with protective effects against multiple diseases, such as seizure, and pain. This study was to examine the impact of THP on IS and investigate its potential mechanism.

MATERIAL AND METHODS

We employed network pharmacology and molecular docking techniques to identify the target proteins of THP for intervention in IS. Adult male Sprague-Dawley rats were used to create a permanent middle cerebral artery occlusion model. PC-12 cells were chosen to establish an oxygen-glucose deprivation (OGD) cell model. Disease modeling followed by nimodipine (NIMO); 3-methyladenine (3-MA) and rapamycin (RAP) interventions. Open field test, Longa score, balance beam test, and forelimb grip test were used to measure motor and neurological functions. The degree of neurological damage recovery was assessed through behavioral analysis, and cerebral infarction volume was determined using TTC staining. Morphological changes were examined through HE and Nissl staining, and ultrastructural changes in neurons were observed using transmission electron microscopy. The protein expression of autophagy and related pathways was analyzed through Western blot (WB). The appropriate hypoxia time and drug concentration were determined using CCK-8 assay, which also measured cell survival rate.

RESULTS

The network pharmacology findings indicated that the impact of THP on IS was enhanced in the PI3K/Akt signaling pathway. THP demonstrated robust docking capability with proteins associated with the autophagy and PI3K/Akt/mTOR, as indicated by the molecular docking outcomes. THP significantly improved behavioral damage, reduced the area of cerebral infarction, ameliorated histopathological damage from ischemia, increase neuronal survival, and alleviated ultrastructural damage in neurons (P < 0.05). THP enhanced the survival of PC-12 cells induced by OGD and ameliorated the morphological harm to the cells (P < 0.05). THP was found to elevate the quantities of P62, LC3-Ⅰ, PI3K, P-AKt/Akt, and P-mTOR/mTOR proteins while reducing the levels of Atg7 and Beclin1 proteins. The results of transmission electron microscopy showed no autophagosomes in the THP, 3-MA, and 3-MA + THP groups.

CONCLUSION

The activation of the PI3K/Akt/mTOR signaling pathway by THP inhibits autophagy and provides relief from neurological damage in IS.

摘要

背景

缺血性中风（IS）是一种具有多种病理机制的危及生命的神经疾病。四氢胡椒碱（THP）是一种天然生物碱，对多种疾病如癫痫和疼痛具有保护作用。本研究旨在探讨THP对IS的影响并研究其潜在机制。

材料与方法

我们采用网络药理学和分子对接技术来确定THP干预IS的靶蛋白。成年雄性Sprague-Dawley大鼠用于建立永久性大脑中动脉闭塞模型。选用PC-12细胞建立氧糖剥夺（OGD）细胞模型。疾病建模后进行尼莫地平（NIMO）、3-甲基腺嘌呤（3-MA）和雷帕霉素（RAP）干预。采用旷场试验、Longa评分、平衡木试验和前肢抓握试验来测量运动和神经功能。通过行为分析评估神经损伤恢复程度，使用TTC染色测定脑梗死体积。通过HE和Nissl染色检查形态学变化，使用透射电子显微镜观察神经元的超微结构变化。通过蛋白质印迹法（WB）分析自噬及相关通路的蛋白质表达。使用CCK-8法测定合适的缺氧时间和药物浓度，该方法还可测量细胞存活率。

结果

网络药理学研究结果表明，THP对IS的影响在PI3K/Akt信号通路中增强。分子对接结果表明，THP与自噬及PI3K/Akt/mTOR相关蛋白具有强大的对接能力。THP显著改善行为损伤，减小脑梗死面积，减轻缺血引起的组织病理学损伤，增加神经元存活，并减轻神经元的超微结构损伤（P < 0.05）。THP提高了OGD诱导的PC-12细胞的存活率，并改善了对细胞的形态损伤（P < 0.05）。发现THP可提高P62、LC3-Ⅰ、PI3K、P-AKt/Akt和P-mTOR/mTOR蛋白的含量，同时降低Atg7和Beclin1蛋白的水平。透射电子显微镜结果显示，THP组、3-MA组和3-MA + THP组均未发现自噬体。