Malk Eun Pak1, Yeo Jin Park2, Hye Jin Yang1, Youn-Hwan Hwang3, Wei Li1, Younghoon Go4. 1. Korean medicine (KM)-Application Center, Korea Institute of Oriental Medicine, Daegu 41062, Republic of Korea. 2. Korean medicine (KM)-Application Center, Korea Institute of Oriental Medicine, Daegu 41062, Republic of Korea; Korean Convergence Medicine, University of Science and Technology, Daejeon 34054, Republic of Korea. 3. Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon 34054, Republic of Korea. 4. Korean medicine (KM)-Application Center, Korea Institute of Oriental Medicine, Daegu 41062, Republic of Korea. Electronic address: gotra827@kiom.re.kr.
Abstract
BACKGROUND: Samhwangsasim-tang (SST) is a traditional medicine used to treat hypertension and arteriosclerosis. Additionally, due to the effects of its constituent herbs, SST is considered effective for memory-related disorders. PURPOSE: We investigated the effects of SST on neuronal survival and memory in glutamate-induced hippocampal cells and in a mouse model of scopolamine-induced memory impairment. METHODS: SST components were identified using 3D-ultra performance liquid chromatography (3D-UPLC). In vitro, we induced glutamate-induced excitotoxicity in HT22 cells after SST pretreatment. We used a cell counting kit-8 and cell cytotoxicity assay, flow cytometry, and western blotting to test the protective effects of SST on neuronal death. In vivo, C57BL/6J mice were administered with 150 and 300 mg/kg SST once daily for 7 days and then intraperitoneally injected with 1 mg/kg scopolamine for 7 days to induce cognitive impairment. We then measured cognitive behavior using a novel object recognition test (NORT) and passive avoidance test (PAT) and analyzed the histological and protein changes. RESULTS: Our results showed that treatment with 50 and 100 μg/ml SST provided significant protection against glutamate-induced cell death. Flow cytometry and western blotting results suggested that 100 μg/ml SST treatment reduced oxidative stress and mitochondrial dysfunction. SST treatment also increased brain-derived neurotrophic factor (BDNF), its receptor, TrkB receptor, and cAMP-response element binding protein (CREB) activation while reducing the P75NTR and JNK signaling activation. Our in vivo results showed that SST administration improved cognitive impairment, similar to donepezil treatment (as a positive control), in NORT and PAT. SST and donepezil decreased neuronal cell death and apoptosis, and acetylcholine levels were increased in the scopolamine-treated hippocampus. Additionally, SST promoted CREB phosphorylation and BDNF maturation while reducing JNK and P75NTR activation; in contrast, donepezil did not alter levels of these proteins in the scopolamine-treated mouse hippocampus. CONCLUSION: Our results suggest that SST has neuroprotective effects to attenuate neuronal cell death and oxidative stress through CREB/JNK signaling via BDNF activation. SST may regulate endogenous survival factors in the hippocampus, which may be a safe and potential clinical treatment for cognitive impairment in AD.
BACKGROUND: Samhwangsasim-tang (SST) is a traditional medicine used to treat hypertension and arteriosclerosis. Additionally, due to the effects of its constituent herbs, SST is considered effective for memory-related disorders. PURPOSE: We investigated the effects of SST on neuronal survival and memory in glutamate-induced hippocampal cells and in a mouse model of scopolamine-induced memory impairment. METHODS: SST components were identified using 3D-ultra performance liquid chromatography (3D-UPLC). In vitro, we induced glutamate-induced excitotoxicity in HT22 cells after SST pretreatment. We used a cell counting kit-8 and cell cytotoxicity assay, flow cytometry, and western blotting to test the protective effects of SST on neuronal death. In vivo, C57BL/6J mice were administered with 150 and 300 mg/kg SST once daily for 7 days and then intraperitoneally injected with 1 mg/kg scopolamine for 7 days to induce cognitive impairment. We then measured cognitive behavior using a novel object recognition test (NORT) and passive avoidance test (PAT) and analyzed the histological and protein changes. RESULTS: Our results showed that treatment with 50 and 100 μg/ml SST provided significant protection against glutamate-induced cell death. Flow cytometry and western blotting results suggested that 100 μg/ml SST treatment reduced oxidative stress and mitochondrial dysfunction. SST treatment also increased brain-derived neurotrophic factor (BDNF), its receptor, TrkB receptor, and cAMP-response element binding protein (CREB) activation while reducing the P75NTR and JNK signaling activation. Our in vivo results showed that SST administration improved cognitive impairment, similar to donepezil treatment (as a positive control), in NORT and PAT. SST and donepezil decreased neuronal cell death and apoptosis, and acetylcholine levels were increased in the scopolamine-treated hippocampus. Additionally, SST promoted CREB phosphorylation and BDNF maturation while reducing JNK and P75NTR activation; in contrast, donepezil did not alter levels of these proteins in the scopolamine-treated mouse hippocampus. CONCLUSION: Our results suggest that SST has neuroprotective effects to attenuate neuronal cell death and oxidative stress through CREB/JNK signaling via BDNF activation. SST may regulate endogenous survival factors in the hippocampus, which may be a safe and potential clinical treatment for cognitive impairment in AD.