Protective Effects of Fangchinoline and Tetrandrine on Hydrogen Peroxide-Induced Oxidative Neuronal Cell Damage in Cultured Rat Cerebellar Granule Cells

Abstract

The present study was performed to examine the neuroprotective effects of fangchinoline (FAN) and tetrandrine (TET), bis-benzylisoquinoline alkaloids, which exhibit the characteristics of Ca²⁺ channel blockers, on H₂O₂-induced neurotoxicity using cultured rat cerebellar granule neurons. H₂O₂ produced a concentration-dependent reduction of cell viability, which was blocked by (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), an N-methyl-D-aspartate (NMDA) receptor antagonist, verapamil, an L-type Ca²⁺ channel blocker, and N ^G-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor. Pretreatment with FAN and TET over a concentration range of 0.1 to 10 μM significantly decreased the H₂O₂-induced neuronal cell death as assessed by a trypan blue exclusion test, a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and the number of apoptotic nuclei. In addition, FAN and TET inhibited the H₂O₂-induced elevation of glutamate release into the medium, elevation of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_c), and generation of reactive oxygen species (ROS). These results suggest that FAN and TET may mitigate the harmful effects of H₂O₂-induced neuronal cell death by interfering with the increase of [Ca²⁺]_c, and then by inhibiting glutamate release and generation of ROS.

Abbreviations

AP5:D(-)-2-amino-5-phosphonopentanoic acid

DMSO:dimethyl sulfoxide

FAN:fangchinoline

H₂DCF-DA:2′,7′-dichlorodihydrofluorescin diacetate

MK-801:(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,20-imine

MTT:3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide

L-NAME:N ^G-Nitro-L-arginine methyl ester

NMDA:N-methyl-D-aspartate

TET:tetrandrine

Key words

Fangchinoline - tetrandrine - H₂O₂ - neurotoxicity - cytosolic free Ca²⁺ concentration - cerebellar granule cells

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