Neurodegenerative diseases have no treatment currently available, and the principal goals for neurodegeneration care are early diagnosis, physical health, optimizing, identifying and treating abnormal behavioral and psychological symptoms. Glutamate is a neurotransmitter that plays critical roles in various physiological as well as pathological brain functions [1
]. Under normal conditions, glutamate is responsible for cell survival, migration, and differentiation during brain development, while an excessive amount of glutamate leads to neuronal cell death through oxidative stress or excitotoxicity [1
]. Reactive oxygen species (ROS) is one major cause of neuronal cell death in chronic neurodegenerative diseases [3
]. Earlier studies showed that an excessive release of glutamate in the extracellular region provoked the accumulation of intracellular ROS by depletion of intracellular glutathione levels through blocking cysteine uptake [5
]. The presence of an antioxidant, such as flavonoids or N
-acetylcysteine, strongly prevented ROS-induced neuronal cell death [6
The use of natural antioxidants for scavenging free radicals and maintaining homeostasis contributes to alleviating neuronal dysfunction [8
]. Typically, natural compounds are multiple-target molecules found mainly in microorganisms and plants and exhibit strong antioxidant activity [8
]. Phenolic compounds from natural sources also exhibit various beneficial effects in cancer, inflammation, and neurodegenerative disorders [9
]. This broad spectrum of biological or pharmacological activities has made phytochemicals suitable candidates for treating multifactorial diseases, such as neurodegenerative diseases [10
]. However, there are also several concerns regarding their poor bioavailability, stability and safety, which must be resolved before effective therapeutics can be developed.
Mitogen-activated protein kinases (MAPKs), known as a family of serine/threonine protein kinases, are well-identified biological molecules involved in glutamate-induced oxidative neuronal cell death. MAPKs are related to multiple cellular functions including differentiation, proliferation, cell survival, inflammation, and cell death [12
]. The presence of excessive glutamate triggers ROS, an increase in intracellular calcium ion concentration, and the activation of MAPKs, including phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38, which are responsible for neuronal cell death in both primary neuronal cultures and neuronal cell lines [13
]. Recently, we found that phytochemicals, such as chebulinic acid and casuarinin, markedly prevented ROS, elevation of intracellular calcium level, and phosphorylation of MAPKs by excessive glutamate-induced HT22 cell death [14
]. Therefore, the inhibition of MAPK phosphorylation is a key mechanism of neuroprotective compounds among the biological molecules involved in glutamate-induced oxidative stress.
Tetrahydrocurcumin (THC) is a major secondary metabolite of curcumin known as a major bioactive compound of Curcuma longa
]. Curcumin is typically metabolized in the intestine to THC which has strong antioxidant activity [17
]. THC is stable at a wide range of pH, and can be easily absorbed through the gastrointestinal tract. Furthermore, THC also plays a critical role in biological effects of curcumin [17
]. It has been reported that THC shows anti-inflammatory, anticarcinogenic activities, and neuroprotective effects [18
]. However, the effect of THC needs to be clarified on glutamate-related neuronal cell death. Therefore, the present study was carried out to demonstrate the possible effect and the protective mechanism of THC on glutamate-mediated neuronal cell death.
2. Results and Discussion
It is well known that natural products including plant materials contain several antioxidative compounds. Curcumin, a major bioactive compound of Curcuma longa
(turmeric), is typically metabolized in the intestine to THC as a major secondary metabolite and has strong antioxidant activity (Figure 1
A). We also confirmed the antioxidant activity of THC through an in vitro 1,1-diphenyl-picryl hydrazyl (DPPH) radical scavenging assay, which is used to evaluate the antioxidant effects. Consistently, our results showed that THC had strong DPPH scavenging activity (Figure 1
Several studies have demonstrated that curcumin and THC have a strong antioxidant effect and prevent neuronal cell death in traumatic brain injuries [20
]. Thus, THC may attenuate neuronal cell death induced by glutamate in HT22 cells. To assess the neuroprotective effects of THC on glutamate-induced oxidative stress, we incubated HT22 cells with 5 mM glutamate in the absence or presence of THC for 24 h. We found that glutamate decreased cell viability, while THC increased cell viability significantly at concentrations of 10 and 20 µM compared to that in glutamate-treated cells (Figure 2
A). Morphologically, THC almost completely inhibited HT22 cell death induced by glutamate (Figure 2
B). Our data suggest that THC is a potent neuroprotectant against glutamate-induced HT22 cell death in neurodegenerative diseases.
Glutamate induces oxidative stress-mediated neuronal cell death in both acute brain injuries as well as neurodegenerative disease [5
]. Because oxidative stress is a major event during neuronal cell death, preventing ROS is a possible strategy for attenuating neuronal cell death. Thus, we investigated whether THC could reduce glutamate-induced accumulation of intracellular ROS in HT22 cells. The cells were exposed to 5 mM glutamate with 10 or 20 µM THC for 8 h and then stained with H2DCF-DA to evaluate intracellular ROS levels. Our results showed that THC markedly prevented the accumulation of intracellular ROS increased by glutamate treatment, and quantitative analysis showed that treatment with glutamate in HT22 cells increased the ROS production measured by fluorescent intensity of DCF to 2.54-fold, but the fluorescent intensity significantly reduced by THC (Figure 3
A,B). Previously, it has been suggested that intracellular Ca2+
) is a characteristic of neuronal cell death by glutamate-induced oxidative stress [22
]. Therefore, we also assessed the levels of [Ca2+
using Fluo-4 AM, a membrane-permeable fluorescent indicator for Ca2+
. Our results showed that THC also prevented the glutamate-triggered elevation of [Ca2+
from 3.22-fold increases in glutamate-treated cells to 2.0- or 1.33-fold in 10 or 20 μM THC-treated cells, respectively (Figure 3
C,D). Taken together, these results suggest that THC can protect HT22 cell from glutamate toxicity through the inhibition of oxidative stress and the increase in [Ca2+
Glutamate is known to induce both necrotic and apoptotic cell death. Previously, glutamate was shown to induce apoptotic cell death during early stages and necrotic cell death at a later time [24
]. Therefore, we mainly focused on early apoptotic cell death induced by glutamate. To investigate the effect of THC on glutamate-induced apoptosis in HT22 cells, cells were treated with 5 mM glutamate in the presence and absence of 10 or 20 μM THC for 12 h. We first stained HT22 cells with Hoechst 33342 to see a chromatin condensation, which is a morphological feature of apoptotic cell death [26
]. The result showed that the chromatin condensation was observed in glutamate-treated HT22 cells, while THC completely prevented those effects of glutamate (Figure 4
A). In addition, we quantitatively analyzed the proportion of apoptotic cell population. HT22 cells were stained with propidium iodide (PI) and annexin V and quantitatively analyzed using image-based cytometric analysis. Our results showed that glutamate significantly increased the percentage of annexin V-positive cells, indicating apoptotic cells, whereas PI-positive cells were not detected (Figure 3
A). This suggested that most HT22 cell death caused by glutamate occurred through the apoptotic pathway. However, THC markedly reduced the percentage of annexin V-stained cells (Figure 4
B). Quantitative analysis results showed that 54.12% of glutamate-treated cells were annexin V-positive, while only 21.22% and 12.35% annexin V-positive cells were observed among glutamate-treated cells treated with 10 and 20 μM THC, respectively (Figure 3
B). This shows that THC significantly reduced the number of apoptotic cell deaths after treatment with glutamate in HT22 cells.
Inhibition of MAPK phosphorylation is a protective mechanism against glutamate-induced neuronal cell death [27
]. MAPK activation is responsible for cell survival as well as death. It has been also reported that preventing the intracellular ROS blocked the phosphorylation of MAPKs and cell death induced by hydrogen peroxide activation, indicating the involvement of ROS-mediated phosphorylation of MAPKs in neuronal cell death [12
]. In addition, treatment with a specific inhibitor of ERK, U0126, prevented glutamate-induced phosphorylation of ERK in primary cortical neurons and HT22 cells [12
]. Therefore, we examined whether THC blocked MAPK activation. We treated HT22 cells with 5 mM glutamate and 10 or 20 μM THC for 8 h and performed Western blot analysis to detect MAPKs phosphorylation. The result showed that glutamate increased the activation of ERK, JNK, and p-38 by 2.52-, 3.57-, and 3.04-fold, respectively. In contrast, THC significantly diminished the phosphorylation of MAPKs induced by glutamate to control levels (Figure 5
A,B). These results suggest that the inhibition of phosphorylation of MAPKs is a molecular mechanism of the THC-mediated neuroprotective effect against HT22 cell death induced by glutamate.
The present study demonstrated that THC strongly prevented HT22 cell death induced by glutamate. Its protective mechanisms occur through inhibition of accumulation of intracellular ROS as an antioxidant property, blockage of MAPK activation, and prevention of apoptotic cell death of HT22 cells by glutamate. Therefore, THC may be useful as a potent neuroprotective metabolite of curcumin against glutamate-mediated neuronal death. However, as is the case with most natural products, poor oral bioavailability limits the therapeutic effects of phytochemicals in vivo. In addition to the problems regarding bioavailability, the results of the cell experiment may not be reflected at the organism level. An important direction for future investigation is the further optimization of the bioavailability of natural products in vivo. However, natural products are relatively safe and exhibit various beneficial effects. Therefore, combinatorial strategies which target multiple mechanisms, such as reducing oxidative stress and increasing anti-inflammation effects, may offer a better chance for clinically meaningful prevention. In addition, extensive efforts should be simultaneously made to solve challenges such as bioavailability, optimization of the lead material, and securing efficacy in clinical trials.