Neuroprotective Effect of Dichloromethane Extraction From Piper nigrum L. and Piper longum L. on Permanent Focal Cerebral Ischemia Injury in Rats

Abstract

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, Ca2+/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.