School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, 750004, China.
School of Clinical Medicine, Ningxia Medical University, Yinchuan, 750004, China.
J Ethnopharmacol. 2022 Sep 15;295:115309. doi: 10.1016/j.jep.2022.115309. Epub 2022 May 19.
Piperine (PIP), a main active component isolated from Piper nigrum L., exerts neuroprotective effects in a rat model of ischemic stroke (IS). However, studies on the effects of PIP on neuroprotection and autophagy after IS are limited.
This study aimed to prove the protective effects of PIP against brain IS and elucidate its underlying mechanisms.
Specific pathogen-free male Sprague-Dawley rats were selected to establish a permanent middle cerebral artery occlusion model. The experiment was randomly divided into six groups: sham group, model group, PIP intervention group (10, 20, and 30 mg/kg group), and nimodipine group (Nimo group, 12 mg/kg). Neurological function score, postural reflex score, body swing score, balance beam test, and grip strength test were used to detect behavioral changes of rats. The area of cerebral infarction was detected by TTC staining, and the number and morphological changes of neurons were observed by Nissl and HE staining. In addition, the ultrastructure of hippocampal dentate gyrus neurons was observed using a transmission electron microscope. Western blot was used to detect the expression of PI3K/AKT/mTOR signaling pathway proteins and autophagy-related proteins, namely, Beclin1 and LC3, in the hippocampus and cortex. Cell experiments established an in vitro model of oxygen-glucose deprivation (OGD) with the HT22 cell line to verify the mechanism. The experiment was divided into five groups: control group, OGD group, OGD + PIP 20 μg/mL group, OGD + PIP 30 μg/mL group, and OGD + PIP 40 μg/mL group. CCK-8 was used to measure cell activity, and Western blot was used to measure the expression of PI3K/AKT/mTOR signaling pathway proteins and autophagy-related proteins (Beclin1 and LC3).
Compared with the model group, the neurological function scores, body swing scores, and postural reflex scores of rats in the 10, 20, and 30 mg/kg PIP intervention groups and Nimo groups decreased, whereas the balance beam score and grip test scores increased (all p < 0.05). After 10, 20, and 30 mg/kg PIP and Nimo intervention, the cerebral infarction area of pMCAO rats was reduced (p < 0.01), and Nissl and HE staining results showed that the number of neurons survived in the 30 mg/kg PIP and Nimo intervention groups increased. Cell morphology and structure were significantly improved (p < 0.05). Most of the hippocampal dentate gyrus neurons and their organelles gradually returned to normal in the 30 mg/kg PIP and Nimo intervention groups, with less neuronal damage. The expression levels of p-mTOR, p-AKT, and p-PI3K in the hippocampus and cortex of the 30 mg/kg PIP and Nimo intervention groups decreased, whereas the expression level of PI3K increased (all p < 0.05). In addition, the expression level of autophagy-related proteins, namely, Beclin1 and LC3-II, in the 30 mg/kg PIP and Nimo intervention groups decreased (all p < 0.05). Results of CCK-8 showed that after 1 h of OGD, the 30 and 40 μg/mL PIP intervention groups had higher cell viability than the OGD group (p < 0.01). Western blot results showed that compared with the OGD group, the expression level of p-mTOR, p-AKT, and p-PI3K in the 30 and 40 μg/mL PIP intervention groups decreased, and the expression level of PI3K increased (all p < 0.05). Moreover, the expression level of autophagy-related proteins, namely, Beclin1 and LC3-II, in the 30 and 40 μg/mL PIP intervention groups decreased (all p < 0.05).
This study shows that PIP is a potential compound with neuroprotective effects. PIP can inhibit the PI3K/AKT/mTOR pathway and autophagy. Its inhibition of autophagy is possibly related to modulating the PI3K/AKT/mTOR pathway. These findings provide new insights into the use of PIP for the treatment of IS and its underlying mechanism.
胡椒碱（PIP）是从黑胡椒（Piper nigrum L.）中分离出的主要活性成分，在缺血性中风（IS）大鼠模型中具有神经保护作用。然而，关于 PIP 对 IS 后神经保护和自噬的影响的研究有限。
本研究旨在证明 PIP 对脑 IS 的保护作用，并阐明其潜在机制。
选择特定病原体的雄性 Sprague-Dawley 大鼠建立永久性大脑中动脉闭塞模型。实验随机分为六组：假手术组、模型组、PIP 干预组（10、20 和 30mg/kg 组）和尼莫地平组（Nimo 组，12mg/kg）。通过神经功能评分、姿势反射评分、体摆评分、平衡梁试验和握力试验检测大鼠的行为变化。TTC 染色检测脑梗死面积，尼氏和 HE 染色观察神经元数量和形态变化。此外，用透射电子显微镜观察海马齿状回神经元的超微结构。Western blot 检测海马和皮质中 PI3K/AKT/mTOR 信号通路蛋白和自噬相关蛋白（Beclin1 和 LC3）的表达。细胞实验采用 HT22 细胞系建立体外氧葡萄糖剥夺（OGD）模型，验证其机制。实验分为五组：对照组、OGD 组、OGD+PIP 20μg/mL 组、OGD+PIP 30μg/mL 组和 OGD+PIP 40μg/mL 组。CCK-8 法检测细胞活性，Western blot 法检测 PI3K/AKT/mTOR 信号通路蛋白和自噬相关蛋白（Beclin1 和 LC3）的表达。
与模型组相比，10、20 和 30mg/kg PIP 干预组和尼莫地平组大鼠的神经功能评分、体摆评分和姿势反射评分降低，而平衡梁评分和握力测试评分升高（均 p<0.05）。10、20 和 30mg/kg PIP 和尼莫地平干预后，pMCAO 大鼠的脑梗死面积减少（p<0.01），Nissl 和 HE 染色结果显示，30mg/kg PIP 和尼莫地平干预组存活神经元数量增加，细胞形态和结构明显改善（p<0.05）。30mg/kg PIP 和尼莫地平干预组海马齿状回神经元及其细胞器逐渐恢复正常，神经元损伤较少。30mg/kg PIP 和尼莫地平干预组海马和皮质中 p-mTOR、p-AKT 和 p-PI3K 的表达水平降低，而 PI3K 的表达水平升高（均 p<0.05）。此外，30mg/kg PIP 和尼莫地平干预组自噬相关蛋白 Beclin1 和 LC3-II 的表达水平降低（均 p<0.05）。CCK-8 结果显示，OGD1h 后，30 和 40μg/mL PIP 干预组细胞活力高于 OGD 组（p<0.01）。Western blot 结果显示，与 OGD 组相比，30 和 40μg/mL PIP 干预组 p-mTOR、p-AKT 和 p-PI3K 的表达水平降低，PI3K 的表达水平升高（均 p<0.05）。此外，30 和 40μg/mL PIP 干预组自噬相关蛋白 Beclin1 和 LC3-II 的表达水平降低（均 p<0.05）。
本研究表明 PIP 是一种具有神经保护作用的潜在化合物。PIP 可抑制 PI3K/AKT/mTOR 通路和自噬。其自噬抑制可能与调节 PI3K/AKT/mTOR 通路有关。这些发现为 PIP 治疗 IS 及其潜在机制提供了新的见解。
