Hua Shiyao, Wang Bing, Chen Rong, Zhang Yuanbin, Zhang Yiwei, Li Tingting, Dong Lin, Fu Xueyan
School of Pharmacy, Ningxia Medical University, Yinchuan, China.
School of Pharmacy, Nanjing University of Chinese Medical, Nanjing, China.
J Stroke Cerebrovasc Dis. 2019 Mar;28(3):751-760. doi: 10.1016/j.jstrokecerebrovasdis.2018.11.018. Epub 2018 Dec 8.
Piper nigrum L. and Piper longum L. consist a classic formula in traditional Chinese Hui medicine and are widely used in treatment of stroke. To examine the therapeutic effect of neuron injury after apoplexy, we used a permanent middle cerebral artery occlusion model in rats to investigate the effects of dichloromethane fraction (DF) of Piper nigrum L. and Piper longum L.
After subjecting the rats to permanent middle cerebral artery occlusion, DF (100 and 200 mg/kg) were administered for 14 days. Neurological deficits and the degree of cerebral tissue injury was detected by 2,3,5-Triphenyltetrazolium Chloride Staining Hematoxylin and eosin staining and Nissl staining. Postsynaptic density protein 95 (PSD-95), synapsin-I (syn-I), and α-synuclein (α-syn) were stained by immunohistochemistry. PSD-95, Ca/calmodulin (CaM)-dependent protein kinase II (CaMK II), phosphorylated CaMK II (p-CaMK II), CaM, N-methyl D-aspartate receptor subtype 2B (NR2B) expression were detected by Western blot. Meanwhile, phytochemical profile of DF was determined through ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS).
DF alleviated neurological deficits and markedly prevented ischemia-induced cellular damage. Immunohistochemical micrographs revealed that PSD-95 and syn-I proteins increased, and α-syn presented reduced expression in brain samples from the sham group. Western blot analyses revealed that the model group exhibited a noticeable reduction in PSD-95, p-CaMK II, CaM, and NR2B. The DF-treated model group exhibited increased PSD-95, p-CaMK II, CaM, and NR2B. UPLC-Q-TOF/MS analysis revealed eight main components of DF, of which piperine accounted for the largest proportion.
胡椒和荜茇是中国传统回医药中的经典配方，广泛用于中风治疗。为研究中风后神经元损伤的治疗效果，我们采用大鼠永久性大脑中动脉闭塞模型，研究胡椒和荜茇二氯甲烷部位（DF）的作用。
大鼠永久性大脑中动脉闭塞后，给予DF（100和200mg/kg），持续14天。通过2,3,5-氯化三苯基四氮唑染色、苏木精-伊红染色和尼氏染色检测神经功能缺损和脑组织损伤程度。采用免疫组织化学法检测突触后致密蛋白95（PSD-95）、突触素-I（syn-I）和α-突触核蛋白（α-syn）。通过蛋白质免疫印迹法检测PSD-95、钙/钙调蛋白（CaM）依赖性蛋白激酶II（CaMK II）、磷酸化CaMK II（p-CaMK II）、CaM、N-甲基-D-天冬氨酸受体2B亚型（NR2B）的表达。同时，通过超高效液相色谱-四极杆飞行时间质谱（UPLC-Q-TOF/MS）测定DF的植物化学图谱。
DF减轻了神经功能缺损，显著预防了缺血诱导的细胞损伤。免疫组织化学显微照片显示，假手术组脑样本中PSD-95和syn-I蛋白增加，α-syn表达降低。蛋白质免疫印迹分析显示，模型组PSD-95、p-CaMK II、CaM和NR2B明显降低。DF治疗的模型组PSD-95、p-CaMK II、CaM和NR2B增加。UPLC-Q-TOF/MS分析显示DF有8种主要成分，其中胡椒碱占比最大。
