Abstract
Amarogentin (AMA) is a secoiridoid glycoside isolated from the traditional Chinese medicine, Gentiana rigescens Franch. AMA exhibits nerve growth factor (NGF)-mimicking and NGF-enhancing activities in PC12 cells and in primary cortical neuron cells. In this study, a possible mechanism was found showing the remarkable induction of phosphorylation of the insulin receptor (INSR) and protein kinase B (AKT). The potential target of AMA was predicted by using a small-interfering RNA (siRNA) and the cellular thermal shift assay (CETSA). The AMA-induced neurite outgrowth was reduced by the siRNA against the INSR and the results of the CETSA suggested that the INSR showed a significant thermal stability-shifted effect upon AMA treatment. Other neurotrophic signaling pathways in PC12 cells were investigated using specific inhibitors, Western blotting and PC12(rasN17) and PC12(mtGAP) mutants. The inhibitors of the glucocorticoid receptor (GR), phospholipase C (PLC) and protein kinase C (PKC), Ras, Raf and mitogen-activated protein kinase (MEK) significantly reduced the neurite outgrowth induced by AMA in PC12 cells. Furthermore, the phosphorylation reactions of GR, PLC, PKC and an extracellular signal-regulated kinase (ERK) were significantly increased after inducing AMA and markedly decreased after treatment with the corresponding inhibitors. Collectively, these results suggested that AMA-induced neuritogenic activity in PC12 cells potentially depended on targeting the INSR and activating the downstream Ras/Raf/ERK and PI3K/AKT signaling pathways. In addition, the GR/PLC/PKC signaling pathway was found to be involved in the neurogenesis effect of AMA.
Highlights
Alzheimer’s disease (AD) is a type of progressive neurodegenerative disease that accounts for 60–70% of dementia cases and its symptoms include an initial memory loss, later visual, language and cognitive disorders and a decline in the executive capacity in daily life [1]
The results showed that AMA induced neurite outgrowth in PC12 cells in a dose-dependent manner
These results indicated that AMA exhibited nerve growth factor (NGF)-mimicking and NGF-enhancing activities in PC12 cells
Summary
Alzheimer’s disease (AD) is a type of progressive neurodegenerative disease that accounts for 60–70% of dementia cases and its symptoms include an initial memory loss, later visual, language and cognitive disorders and a decline in the executive capacity in daily life [1]. With its high polarity and large molecule weight, the NGF cannot pass through the blood-brain barrier (BBB) and is difficult to apply as a drug [5] This finding indicates that discovering a small molecule with an NGF-mimicking activity may be a potential alternative for AD treatment. G. rigescens Franch (Jian Long Dan in Chinese), a well-known TCM that is widely distributed in the Yunnan Province, southwest China, is generally utilized for hepatitis, rheumatism, cholecystitis and inflammation treatment [14] This TCM is praised with its anti-aging activity and cognition-improving effect in ‘Sheng Nong’s Herbal Classic’, a classic book on TCM material medica. The GR/PLC/PKC was involved in the neuritogenic activity of AMA in PC12 cells
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