Introduction
The increasing societal burden of neurodegenerative diseases has led to growing interest in the development of strategies to reduce the risk of neurodegeneration in the elderly population. The progressive loss of a relatively vulnerable population of neurons is the primary clinical feature of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease [1]. Although neuronal cell degeneration is regarded as a major causative factor, the molecular mechanisms underlying these neurological disorders are not completely understood. Oxidative stress and excitotoxicity are considered major causes of neuronal dysfunction and damage [2].
Glutamate plays an important role as an excitatory neurotransmitter in the cerebral cortex and is involved in the learning and memory processes [3,4]. In the case of overactivation, an excess amount of extracellular glutamate can cause uncontrolled continuous depolarization of neurons, which is called excitotoxicity, resulting in neuronal cell death [5]. In addition to excitotoxicity, high intercellular glutamate causes oxidative stress in neurons due to the dysregulation of cysteine glutamate antiporters, resulting in a reduction in cellular glutathione concentration [6]. Insufficient cellular glutathione leads to the accumulation of cellular reactive oxygen species (ROS), which can trigger Bid truncation and apoptosis inducing factor (AIF)- dependent apoptosis [7-9]. In high cellular oxidative stress conditions, Ca2+ ion influx is dramatically increased [10]. High concentrations of Ca2+ ions and oxidative stress activate cellular signaling pathways, including mitogen-activated protein kinases (MAPKs) [8,11,12].
According to recent literature, natural products can act as major neuroprotective factors and aid in the recovery of cognitive function in in vivo and in vitro models of neurodegenerative disorders. These natural products significantly alleviate the deterioration of neuronal cells caused by oxidative stressmediated neurotoxicity [13]. Therefore, further discovery of natural products with neuroprotective effects is beneficial for the control of neurological disorders. Corydaline, which is purified from the tubers of the Chinese medicinal plant Corydalis yanhusuo, is an isoquinoline alkaloid. It is used in traditional medicine for the treatment of allergies, nociception, gastrointestinal problems, and certain parasitic infections [14,15]. It shows potential for the treatment of indigestion dyspepsia and is currently under a clinical trial [16]. In a previous report, alkaloids from Corydalis tubers showed moderate inhibitory effects on acetylcholine esterase (AChE) [17]. The pharmacological inhibition of AChE has shown neuroprotective effects in an Alzheimer’s disease model [18-20]. However, corydaline is considered to have a weak inhibitory effect on AChE, with an IC50 of 226 μM [21]. To date, the neuroprotective effects of corydaline on oxidative stress-induced neurons have not been studied. Therefore, in the current study, we examined the molecular mechanisms underlying the neuroprotective effects of corydaline in HT22 cells treated with glutamate.
Materials and Methods
1. Cell culture
HT22 hippocampal neuronal cells (obtained from the Korean Cell Line Bank) were grown in Dulbecco’s modified Eagle’s medium high-glucose media (Hyclone; SH30243.01) with 10% fetal bovine serum (Gibco-Thermo Fisher Scientific) and 1% penicillin/streptomycin (Gibco-Thermo Fisher Scientific) and incubated at 37℃ in a 5% CO2 incubator. The seeding density was 5 × 105 cells in a 10 cm dish. The cells were grown for 48 hours to reach confluence.
2. Cell viability assay
HT22 cells (3,000 cells/well) were seeded in 96-well plates and incubated for 24 hours. The cells were then treated with corydaline for 2 hours, followed by treatment with 5 mmol/L glutamate. After 8 hours, the viability of the cells was measured by a live cell assay using the fluorescent dye calcein AM. Briefly, 1 μM calcein AM was added to stain live cells. Live cells were counted using the Operetta high content imaging system and counted with Harmony 3.5 software (PerkinElmer).
3. Measurement of ROS
HT22 cells (5,000 cells/well) were seeded in 96-well black wall clear-bottom plates and grown for 24 hours. After treatment with corydaline and 5 mmol/L glutamate for 8 hours, the medium was removed, and the plate was washed with Dulbecco’s phosphate buffered saline (DPBS). Then, 10 μM 2’ 7’-dichloro-dihydrofluorescein diacetate prepared in serumfree medium was added and incubated for 30 minutes. The solution was removed and the cells were washed with DPBS. Thereafter, fluorescence was measured and the images were analyzed using the Operetta high-content imaging system.
4. Measurement of cellular Ca2+ concentration
HT22 cells were seeded (5,000 cells/well) in a 96-well plate and incubated for 24 hours. Following treatment with the indicated concentrations of corydaline and 5 mmol/L glutamate for 8 hours, the medium was removed and treated with 2 μM of Fluo-3 AM prepared in Hanks’ Balanced Salt solution buffer for 15 minutes. Thereafter, the cells were washed with DPBS and analyzed using the Operetta imaging system.
5. Western blot analysis
HT22 cells (5 × 105 cells) were seeded in 6-well plates and treated with corydaline and 5 mmol/L glutamate. After 8 hours of incubation, the cells were collected and mixed with radioimmunoprecipitation assay cell lysis buffer supplemented with a protease inhibitor cocktail. The isolated protein fraction was quantified using the BCA assay, and an equal amount of protein was resolved in SDS-PAGE gel for 90 minutes at 100 V. The resolved proteins were transferred onto polyvinylidene difluoride membranes for 90 minutes at 100 V. The membrane was incubated with 3% bovine serum albumin blocking buffer for 30 minutes. After washing the membrane with Tris Buffered Saline, with Tween (TBS-T), it was incubated with primary antibodies against ERK, p-ERK, p38, p-p38, JNK, p-JNK, Bcl- 2, Bax, AIF, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) overnight, followed by horseradish peroxidase (HRP)- conjugated secondary antibody treatment for 90 minutes at room temperature. The membranes were then imaged using a gel doc system.
6. Statistical analysis
All experiments in this study were performed at least three times, and graphs are shown as the means ± standard deviations. We performed a t-test using GraphPad PRISM software 9 and a p-value ＜ 0.05 was considered to indicate statistically significant difference.
Results
1. Corydaline ameliorates glutamate-induced neuronal cell death
The structure of corydaline is shown in Fig. 1A. The cytotoxicity of various concentrations of corydaline was also measured. As shown in Fig. 1B, corydaline exerted little cytotoxicity at high concentrations, with a cell viability of 85% at 25 μM and 80% at 50 μM. The effect of corydaline treatment on glutamate-induced neurotoxicity was evaluated in HT22 cells, originated from mouse hippocampus, using an image-based cell viability assay. HT22 cells were treated with the indicated concentrations of corydaline (1.56, 3.12, 6.25, 12.5, 25, and 50 μM) for 2 hours followed by the addition of 5 mmol/L glutamate. Glutamate treatment reduced cell viability to 45%, but corydaline co-treatment with glutamate significantly inhibited cell death and maintained viability to 80% (Fig. 1C). The post treatment of corydaline effects in HT22 cells were evaluated. The neuroprotective effect of corydaline was evaluated in HT22 cells upon 5 mmol/L glutamate treatment followed by the treatment of corydaline. Corydaline was treated at 2 hours interval in the 5 mmol/L glutamate-pretreated HT22 cells. Until 8 hours after treatment, corydaline exerts neuroprotective effect on glutamate-induced HT22 cells (Fig. 1D).
The neuroprotective effect of corydaline was also evaluated in human SH-SY5Y cells treated with high concentration of glutamate (100 mmol/L). 100 mmol/L of glutamate treatment reduced ratio of cell viability up to 68.3% of cell viability. Corydaline co-treatment with glutamate inhibited neuronal cell death and increased ratio of cell viability up to 84.3% in SHSY5Y cells (Supplementary Fig. 1). Therefore, corydaline treatment showed protective effects against oxidative induced cell death in both mouse and human cell line.
Flow cytometry analysis using 7-Aminoactinomycin D (7- AAD) and annexin V showed that glutamate-treated HT22 cells showed a high apoptotic or dead cell (annexin V +/7-AAD – to annexin V +/7-AAD +) ratio of 49.14% (Supplementary Fig. 2B). However, corydaline treatment decreased the ratio of apoptotic or dead cells to 2.65%, which was similar to the ratio observed with the dimethyl sulfoxide control (Supplementary Fig. 2A and 2C–2E). These observations suggest that corydaline protected the cells from glutamate-induced apoptosis.
To date, corydaline has been shown to protect against glutamate- induced cell death from 2 hours of pretreatment with corydaline. Therefore, we also tested its neuroprotective effects in corydaline post-treatment and glutamate-pretreated wells. To study the corydaline rescue time frame, cells were treated with 12.5 μM corydaline after glutamate-induced cell damage at 2-hour time intervals in glutamate-pretreated wells until 24 hours. As a result, corydaline protected against glutamate-induced cell death until 6 hours post treatment; however, after 6 hours, it failed to rescue glutamate-induced cell death. This indicates that corydaline must be administered within 6 hours of glutamate treatment to prevent cell death (Fig. 1D).
2. Corydaline inhibits ROS and cellular Ca2+ accumulation in HT22 cells
Glutamate induces ROS and Ca2+ accumulation, eventually inducing cell death. The glutamate treatment increased intracellular ROS levels after 8 hours. However, corydaline co-treatment at 6.25 μM and 12.5 μM effectively prevented the increased ROS production (Fig. 2). This suggests that the protective activity of corydaline is associated with the regulation of ROS generation. Next, we investigated whether corydaline inhibits the accumulation of cellular Ca2+ ions. The cell-permeable calcium ion fluorescence indicator 4-(6-Acetoxymethoxy- 2,7-dichloro-3-oxo-9-xanthenyl)-4′-methyl- 2,2′(ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester (Fluo 3-AM) was used to measure Ca2+ levels. Glutamate treatment significantly increased the calcium levels, which are associated with oxidative stress. The calcium ion influx by glutamate treatment was increased by 3.5 fold compared with that in the control. However, corydaline co-treatment decreased Ca2+ levels to a level similar to that in the control (Fig. 3). Thus, the neuroprotective effects of corydaline appear to be associated with the regulation of oxidative stress pathways.
3. Corydaline regulates the Bcl-2 and Bax protein levels in HT22 cells
Glutamate treatment activates apoptosis by altering the levels of pro- and anti-apoptotic proteins [22]. Treatment with 5 mmol/L glutamate increased the expression of BCL2 associated X (Bax), which is a pro-apoptotic protein whose activation leads to apoptotic cell death. Furthermore, glutamate reduced the levels of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) (Fig. 4A). In contrast, co-treatment with corydaline and glutamate increased Bcl-2 and reduced Bax expression (Fig. 4A). Together, these findings suggest that corydaline inhibits glutamate-induced oxidative stress-mediated apoptosis.
4. Corydaline suppresses glutamate-induced MAPK activation
Glutamate-induced oxidative stress mediates cell death via activation of the MAPK pathway. MAPK activation is highly associated with the oxidative stress generated inside the cell, and is connected to various cell death pathways. To clarify the mechanism underlying corydaline protection, immunoblotting was performed to study the effect of corydaline on the MAPK pathway. Compared to the control group, glutamate treatment enhanced the phosphorylation of extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNK1/2), and p38. However, co-treatment with corydaline decreased the expression of p-JNK, p-ERK, and p-p38 (Fig. 4B). These results indicate that glutamate-induced phosphorylation of p38, ERK, and JNK was effectively repressed by treatment with corydaline in a dose-dependent manner (Fig. 4B). Thus, corydaline prevents the activation of the MAPK pathway.
5. Corydaline suppresses AIF nuclear translocation
Cell death induced by glutamate is characterized by both apoptosis and necrosis in HT22 cells. Previous studies have shown that glutamate-induced cell death follows a caspaseindependent AIF apoptosis pathway. Therefore, we evaluated whether corydaline prevented AIF-dependent apoptosis mediated by AIF translocation. Immunoblotting was performed on the cytoplasmic and nuclear fractions, and the results showed that glutamate mediated AIF translocation to the nucleus, whereas corydaline co-treatment inhibited the AIF translocation (Fig. 5A). Furthermore, immunostaining data showed that corydaline inhibited AIF translocation in HT22 cells (Fig. 5B). Thus, glutamate-induced death of HT22 cells involves AIFdependent apoptosis, which is abrogated by corydaline.
Discussion
The current study shows that corydaline protects HT22 cells from glutamate toxicity and maintains their viability. In this study, corydaline treatment significantly attenuated the glutamate- induced cellular Ca2+ influx and ROS generation. Glutamate- mediated oxidative stress elevated proapoptotic Bax levels and decreased antiapoptotic Bcl-2 protein levels, which led to apoptosis in HT22 cells, while corydaline co-treatment reversed the Bax and Bcl-2 expression levels. In addition, corydaline treatment decreased MAPK activation induced by glutamate and prevented AIF-dependent apoptosis in HT22 cells.
However, corydaline showed weak neuroprotection against tunicamycin (2.5 ng/mL), which induces endoplasmic reticulum stress (Supplementary Fig. 3A). Furthermore, corydaline showed a 2-fold increase in the NF-E2-related factor 2 antioxidant responsive element (NRF2-ARE) luciferase reporter assay (Supplementary Fig. 3B), which indicated the activation of the NRF2 anti-oxidant pathway. Corydaline is known to inhibit AChE with an IC50 of 226 μM, which could also play a role in the neuroprotective potential of corydaline. To evaluate the association of AChE inhibition with the neuroprotection in our model, we chose a known AChE inhibitor, donepezil, and compared its neuroprotective effect against glutamate. In our study, donepezil showed no neuroprotective effect against glutamate (Supplementary Fig. 4). Therefore, the neuroprotective effects of corydaline appear to be mainly related to the inhibition of cellular Ca2+, ROS, MAPK activation, and AIF translocation.
In conclusion, corydaline showed neuroprotective effects against oxidative stress-mediated cell death in an HT22 neuronal model. Therefore, it can be used as a lead compound for the development of potential neuroprotective agents for the treatment of neurodegenerative diseases.
