Methyl lucidone exhibits neuroprotective effects on glutamate-induced oxidative stress in HT-22 cells via Nrf-2/HO-1 signaling
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Oxidative stress causes neuronal cell death in various neurodegenerative diseases, such as Alzheimer’s disease, ischemia, and Parkinson’s disease. Therefore, reducing intracellular reactive oxygen species (ROS) has been evaluated as an effective treatment strategy for neurodegenerative disorders. Methyl lucidone (MLC) extracted from Lindera erythrocarpa Makino (Lauraceae) has been previously reported to exhibit microglial-mediated neuroprotective effects via inhibiting neuroinflammation. However, the antioxidant effects of MLC are still unclear. The aim of this study was to determine the neuroprotective mechanism of MLC in HT-22 neurons against oxidative stress induced by glutamate. In results, the pretreatment of MLC significantly enhanced the viability of HT-22 cells under glutamate-induced oxidative conditions, suggesting that MLC has a neuronal mechanism to protect neurons without microglial regulation. Also, the glutamate effect to increase ROS production was effectively blocked by MLC without any free radical scavenging activity. To induce this antioxidant effect, MLC upregulated the expression of heme oxygenase 1 (HO-1) and nuclear translocation of nuclear factor-E2-related factor 2 (Nrf-2), known as an intracellular antioxidant enzyme, and its transcription factor. Additionally, Akt phosphorylation regulating Nrf-2 was confirmed to be involved in the neuroprotective signaling activated by MLC. These results indicate that MLC may play a role as an antioxidant agent to inhibit neurodegenerative processes via activating antioxidant signaling pathways that include Nrf-2 and phosphatidylinositol 3-kinase (PI3K).
KeywordsMethyl lucidone HO-1 Nrf-2 ROS Neuroprotection PI3K Antioxidant
Neurotoxicity in glutamatergic neurons is the major cause to induce cell death in neurodegenerative diseases, such as Alzheimer’s disease, ischemia, and Parkinson’s disease [1, 2, 3]. Excessive extracellular glutamate in the central nervous system (CNS) lowers glutathione levels, inhibiting cystine/glutamate transporters, which cause the accumulation of reactive oxygen species (ROS) and neuronal cell death. Thus, it is called oxidative glutamate toxicity .
To moderate oxidative damage and maintain cellular redox homeostasis, many types of mammalian cells possess a variety of antioxidant systems . One of the important intracellular enzymes for antioxidant signaling is heme oxygenase 1 (HO-1). Nuclear factor-E2-related factor-2 (Nrf-2) regulates the expression of genes encoding phase ΙΙ detoxification enzymes and antioxidants, including HO-1 . Because Nrf-2 is negatively regulated by Kelch-like ECH-associated protein1 (Keap1) and Nrf-2 phosphorylation is required for its dissociation from Keap1, various kinases, such as phosphatidylinositol 3-kinase (PI3K), also participate in the HO-1-mediated antioxidant signaling pathway [7, 8].
Lindera erythrocarpa Makino (Lauraceae), a deciduous shrub, is widely distributed in Korea, Taiwan, Japan, and China. The fruit of L. erythrocarpa is used in traditional medicine as an analgesic, antibacterial agent, antidote, digestive, and diuretic agent . The extract of the fruit includes four cyclopentenediones, linderone, lucidone, methyl linderone and methyl lucidone (MLC). Of them, lucidone has been reported to inhibit human farnesyl protein transferase (FPTase) activity and lucidone and MLC effectively suppressed NO production . We also reported that MLC had a neuroprotective effect via inhibiting microglia-mediated neurotoxicity . However, it is still unclear if MLC contributes to the activation of antioxidant signaling in neurons without microglial involvement. To investigate this, we tested the effect of MLC on the HO-1-mediated antioxidant signaling pathway in HT-22 cells.
The results showed that MLC preserved the survival of HT-22 cells under glutamate-induced oxidative conditions via activating the HO-1 antioxidant enzyme and that the activation of PI3K/Akt signaling was required for the antioxidant effect. Our findings in this study suggest that MLC may be a potent antioxidant agent to protect neurons and inhibit the progression of neurodegenerative diseases.
Materials and methods
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gibco BRL (Grand Island, NY, USA). The antibody against phospho-Akt was purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Akt and Nrf-2 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The antibodies against heme oxygenase-1 (HO-1) and TATA binding protein (TBP) were purchased from Millipore (Temecula, CA, USA) and Abcam (Cambridge, UK), respectively. The antibody against β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) from Amresco (Solon, OH, USA). 2′,7′-Dichlorofluorescin diacetate (DCF-DA) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). LY294002 and Tin protoporphyrin IX dichloride (SnPP) were purchased from Tocris Bioscience (Bristol, UK). MLC was given by Dr. BM Kwon (Korea Research Institute of Bioscience and Biotechnology, South Korea).
HT-22 neurons, an immortalized hippocampal neuronal cell line , were a generous gift from Dr. B. H. Lee (Gachon University of Medicine and Science, South Korea). The cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, and incubated at 37 °C under 5% CO2.
Measurement of cell viability
Neuronal viability of HT-22 cells was determined by hiring MTT assay. HT-22 cells were first seeded at a density of 5 × 104 cells/well in a 24-well plate. After 12 h, cells were treated with MLC at various concentrations for 1 h and then washed with DMEM. Next, the cells were again treated with 5 mM glutamate for 12 h. For MTT measurement, 100 µl of MTT solution (2 mg/ml) was added to each well and cells were incubated again at 37 °C for 2 h. After the incubation, the culture supernatant was removed and the MTT formazan crystals were dissolved with 300 µl of DMSO. The absorbance was measured at 550 nm wave length using a microplate reader (Model 550, Bio-Rad, USA).
Measurement of intracellular ROS level
The intracellular ROS level was measured by using DCF-DA. HT-22 cells were seeded at a density of 5 × 104 cells/well in a 24-well plate and incubated for 12 h. The cells were treated with MLC at several non-toxic concentrations for 1 h and then washed with DMEM. Next the cells were again treated with 5 mM glutamate for 12 h. For ROS measurement, the cells were loaded with 50 µM DCF-DA for 15 min. The fluorescence intensity was detected by using a fluorescence reader (Spectra Fluor, Tecan, Austria) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
Measurement of free radical scavenging effect
DPPH assay was performed to measure a free radical scavenging activity of MLC. MLC and N-acetyl cysteine (NAC) (10 µl) were added to 190 µl DPPH (0.15 mM) in each well (96-well plate) and mixed vigorously. The mixture was incubated at room temperature for 1 h in the dark by covering with aluminum foil. The absorbance was detected at 517 nm wavelength using a microplate reader (VersaMax, Molecular devices, USA).
Preparation of cytoplasmic and nuclear protein
The preparation of cytoplasmic and nuclear proteins was performed by using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Invitrogen, USA) according to manufacturer’s protocols. HT-22 cells were seeded at a density of 1 × 106 cells/dish in 100 mm dish. The cells were treated with MLC during the time indicated in results. Then, they were washed twice and collected with cold phosphate-buffered saline (PBS; pH 7.4). After centrifuging, cell pellets were resuspended in a Cytoplasmic extraction reagent. After centrifuging them again, the supernatant cytoplasmic extract was transferred to a new tube. And then, the nuclear pellets were resuspended in a nuclear extraction reagent, and centrifuged. The supernatant nuclear protein extract was transferred to a new tube and stored at − 80 °C until using them.
Western blot analysis
Cell extracts were separated with 12% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, CA, USA). After blocking with 5% skim milk in TBS (25 mM Tris, pH 7.4, 150 mM NaCl), membranes were probed with anti-Nrf-2 (1:200), anti-HO-1 (1:1000), anti-phospho-Akt (1:1000), anti-Akt (1:200), anti-β-actin (1:5000) or anti-TBP (1:1000). Blots were washed three times with TBS including 1% tween-20 and incubated with HRP-conjugated anti-mouse or anti-rabbit antibody. Then the blots were detected using an enhanced chemiluminescence reagent according to the manufacturer’s protocol. Optical densities of the band were quantified by using an Image J program.
The data were presented as the mean value ± SEM through at least 3 independent experiments. Statistical analysis was performed using Student t test and one-way ANOVA. The differences between groups were considered to be statistically significant when p < 0.05 or p < 0.01.
MLC increases neuronal viability under glutamate-induced oxidative condition
MLC inhibits glutamate-induced ROS production in HT-22 neurons
MLC activates Nrf-2/HO-1 signaling transduction
The PI3K/Akt pathway is involved in the neuroprotective effects of MLC
The major finding of the present study was that MLC, a cyclopentenedione isolated from Lindera erythrocarpa Makino, had neuroprotective effects on glutamate-induced oxidative stress in HT-22 cells without microglial regulation. This was confirmed by observing the enhanced neuronal viability, reduced ROS production, increased HO-1 expression and enhanced Nrf-2 transcription in MLC-pretreated cells under glutamate-induced oxidative conditions. The antioxidant effect of MLC required the activation of PI3K/Akt signaling, as a PI3K inhibitor completely blocked the ability of MLC to increase HO-1 expression and cell survival rates. Together with our previous report , these results provide evidence that MLC may enhance neuronal viability under oxidative conditions associated with neurodegenerative pathogenesis by activating neuronal antioxidant signaling, as well as microglial neuroinflammatory processing.
In mammalian nervous systems, the excessive production of intracellular ROS causes a variety of diseases by damaging cellular components, such as DNA, lipids, and proteins. In particular, ROS-induced neuronal cell death is irreversible, so finding new antioxidant agents has been the focus of studies to research neurodegenerative diseases mediated with oxidative signaling. Glutamate-mediated toxicity is well known as a major cause to induce dysfunctions in many types of neurons via activating oxidative signaling [4, 19, 20, 21, 22]. Also, HT-22 neurons are commonly used to study oxidative glutamate toxicity because they are highly responsive to extracellular glutamate, which suppresses cystine uptake via inhibiting glutamate/cystine antiporters, resulting in oxidation-mediated neuronal cell death [4, 20, 23]. In this study, 5 mM glutamate treatment was used to increase intracellular ROS production and cause cell death in HT-22 cells (Figs. 1, 2). Under this oxidative condition, the cells would have activated several antioxidant systems, including endogenous antioxidants (e.g., glutathione and bilirubin) and antioxidant enzymes (e.g., catalase, superoxide dismutase, glutathione reductase, and HO-1) to maintain ROS homeostasis . Therefore, the MLC effects observed here may be specific for the antioxidant signaling pathways in neurons that express many types of glutamate receptors.
We previously reported that the microglia-mediated neuroprotective effects of MLC were due to the inhibition of neuronal inflammation . However, it was not clear if MLC might directly protect neurons against oxidative stress without microglia regulation. In the present study, MLC certainly suppressed glutamate-induced neuronal death, even in the absence of microglia, strongly suggesting that neuronal signaling may be sufficient for MLC to protect neuronal cells from oxidative stress (Fig. 1). Even though it is not an endogenous antioxidant factor, the neuroprotective effect of MLC seems to be due to the suppression of glutamate-increased ROS production (Fig. 2). These results are also consistent with previous studies reporting that phytochemicals exerted protective effects by activating cytoprotective proteins, including HO-1 [25, 26, 27]. Furthermore, the antioxidant effects of MLC to directly regulate HO-1 expression, as well as Nrf-2 activation, were experimentally confirmed in this study (Fig. 3). The transcription factor Nrf-2, which is normally distributed in the cytoplasm and activated by being translocated to the nucleus, actively regulates HO-1 expression by binding to ARE . Taken together, our results provide evidence that the enhanced HO-1 expression by MLC may be a key factor to inhibit glutamate-induced oxidative stress.
How does MLC regulate HO-1 expression and Nrf-2 activation? In the previous study, it has been reported that the activation of Nrf-2 was controlled by its upstream regulators, such as PI3K . The PI3K/Akt pathway is a well-known signaling cascade that regulates cellular growth and inhibits apoptotic processing in response to extracellular signals . In this pathway, PI3K has a role to phosphorylate phosphatidylinositol-(4,5) bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and activate Akt . Then, PI3K-activated Akt sequentially phosphorylates Nrf-2 to promote its translocation from the cytoplasm to the nucleus . Those reports support our results showing that MLC treatment significantly increased the expression of phosphorylated Akt, as well as Nrf-2 accumulation in the nucleus (Fig. 5). The requirement of Akt phosphorylation for the MLC effects was additionally confirmed by showing that a PI3K inhibitor (LY294002) blocked the enhancement of both HO-1 and Nrf-2 expression and reduced the neuronal viability in cells pretreated with MLC (Fig. 6). These results strongly suggest that the activation of the PI3K/Akt pathway should be preceded to activate Nrf-2 signaling which is absolutely required for antioxidant effects of MLC, consistent with previous reports [18, 28]. However, LY294002 did not completely block the MLC effects, as shown in Fig. 6, indicating that additional functions of MLC to affect another antioxidant signaling pathway may exist in neurons. Mitogen-activated protein kinases (MAPKs) and ubiquitin–proteasomes are also known to be involved in a major pathway activating Nrf2/HO-1 signaling, but their involvement was not investigated in this study [29, 30].
JYP and KA who were equally contributed to this study, mainly performed experiments related with cell culture, protein detection and ROS signalings. YJC, JHL and JW performed and supported the additional experiment for protein detection. YSY supported data analysis and statistical evaluation. SYE supported the experimental design and supervised the current study. SCJ mainly supervised the current study as a corresponding author. All authors read and approved the final manuscript.
The work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea government (MEST) (No. NRF-2016R1D1A1B0101086)
The authors declare that they have no competing interests.
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