The Neuroprotective Effect of Sophocarpine against Glutamate-Induced HT22 Cell Cytotoxicity.

Abstract

Neuronal cell death and dysfunction of the central nervous system can be caused by oxidative stress, which is associated with the development of neurodegenerative diseases. Sophocarpine, an alkaloid compound derived from Sophora moorcroftiana (Benth.) Baker seeds, has a wide range of medicinal value. This study sought to determine how sophocarpine exerts neuroprotective effects by inhibited oxidative stress and apoptosis in mouse hippocampus neuronal (HT22) cells. 20mM glutamate-induced HT22 cells were used to develop an in vitro model of oxidative stress damage. The Cell Counting Kit-8 (CCK-8) assay was used to assess cell viability. According to the instructions on the kits to detect reactive oxygen species (ROS) levels and oxidative stress indicators. HT22 cells were examined using immunofluorescence and Western Blotting to detect Nuclear Factor Erythroid 2-related Factor 2 (Nrf2) expression. The expression of proteins and messenger RNA (mRNA) for heme oxygenase-1 (HO-1) was examined by Western Blotting and Quantitative real time polymerase chain reaction (qRT-PCR). Mitochondrial membrane potential (MMP) and Cell apoptosis were used by 5, 5', 6, 6'-Tetrachloro-1, 1', 3, 3'-tetraethyl-imidacarbocyanine iodide (JC- 1) kit and Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay kit, respectively. Finally, the expression of pro-apoptotic proteins was detected by Western Blotting. The result demonstrated that sophocarpine (1.25 μM-10 μM) can significantly inhibit glutamate-induced cytotoxicity and ROS generation, improve the activity of antioxidant enzymes. Sophocarpine increased the expression of HO-1 protein and mRNA and the nuclear translocation of Nrf2 to play a cytoprotective role; however, cells were transfected with small interfering RNA targeting HO-1 (si-HO-1) reversed the above effects of sophocarpine. In addition, sophocarpine significantly inhibited glutamate induced mitochondrial depolarization and further inhibited cell apoptosis by reducing the expression level of caspase-related proteins.