Compound anisodine affects the proliferation and calcium overload of hypoxia-induced rat retinal progenitor cells and brain neural stem cells via the p-ERK1/2/HIF-1α/VEGF pathway.

As a Traditional Chinese Medicine, compound anisodine (CA) has previously been shown to regulate the vegetative nervous system, improve microcirculation and scavenge reactive oxygen species, and has been commonly utilized as a neuroprotective agent to treat ischemic optic neuropathy and choroidoretinopathy. The present study aimed to investigate the neuroprotective effects of CA on the proliferation and calcium overload of hypoxia-induced rat retinal progenitor cells (RPCs) and brain neural stem cells (BNSCs) harvested from neonatal Sprague-Dawley rats. Cells were treated with CA at 0.126, 0.252, 0.505 or 1.010 g/l for four hours prior to or after hypoxia (<1% oxygen) for four h, followed by re-oxygenation for four hours; a normal control group and a CA-untreated hypoxia model group were also included. An MTT assay demonstrated that the cell viability was markedly improved following treatment with 0.126-1.010 g/l CA, compared with that in the hypoxia model group (P<0.05). Bromodeoxyuridine (BrdU) immunocytochemical staining and flow cytometry indicated that after culture in hypoxia for 4 h, the number of BrdU RPCs and BNSCs was significant decreased, as well as the cell population in S+G2 phase of the cell cycle, which was significantly attenuated by treatment with 1.010 g/l CA for 4 h prior to hypoxia (P<0.05). Furthermore, laser scanning confocal microscopy showed that the intracellular calcium concentration in hypoxia-cultured RPCs and BNSCs was markedly increased, which was attenuated by 0.126-1.010 g/l CA in a concentration-dependent manner (P<0.05). Furthermore, western blot analysis demonstrated that after hypoxia, the protein levels of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) were upregulated in RPCs and BNSCs, whereas phosphorylated extracellular signal-regulated kinase (phospho-ERK 1/2) and Cyclin D1 were downregulated; of note, treatment with 1.010 g/l CA significantly attenuated these changes (P<0.05). The results of the present study suggested that CA may improve the proliferation and inhibit calcium overload in hypoxia-induced RPCs and BNSCs by altering the protein levels of Cyclin D1 as well as signaling through the p-ERK1/2/HIF-1α/VEGF pathway.