Protective Effects of 6,7,4′-Trihydroxyflavanone on Hypoxia-Induced Neurotoxicity by Enhancement of HO-1 through Nrf2 Signaling Pathway

Since hypoxia-induced neurotoxicity is one of the major causes of neurodegenerative disorders, including the Alzheimer’s disease, continuous efforts to find a novel antioxidant from natural products are required for public health. 6,7,4′-trihydroxyflavanone (THF), isolated from Dalbergia odorifera, has been shown to inhibit osteoclast formation and have an antibacterial activity. However, no evidence has reported whether THF has a protective role against hypoxia-induced neurotoxicity. In this study, we found that THF is not cytotoxic, but pre-treatment with THF has a cytoprotective effect on CoCl2-induced hypoxia by restoring the expression of anti-apoptotic proteins in SH-SY5y cells. In addition, pre-treatment with THF suppressed CoCl2-induced hypoxia-related genes including HIF1α, p53, VEGF, and GLUT1 at the mRNA and protein levels. Pre-treatment with THF also attenuated the oxidative stress occurred by CoCl2-induced hypoxia by preserving antioxidant proteins, including SOD and CAT. We revealed that treatment with THF promotes HO-1 expression through Nrf2 nuclear translocation. An inhibitor assay using tin protoporphyrin IX (SnPP) confirmed that the enhancement of HO-1 by pre-treatment with THF protects SH-SY5y cells from CoCl2-induced neurotoxicity under hypoxic conditions. Our results demonstrate the advantageous effects of THF against hypoxia-induced neurotoxicity through the HO-1/Nrf2 signaling pathway and provide a therapeutic insight for neurodegenerative disorders.


Introduction
Since continuous provision of enormous amounts of oxygen to the brain is required for its proper function, the brain is easily affected by the limited oxygen condition called hypoxia. Neuronal cytotoxicity is generally induced under hypoxic condition because insufficient supply of oxygen to the brain enhances the mortality and disability of neurons [1,2]. It has been reported that hypoxia-induced neurotoxicity causes brain damage and leads to neurodegenerative diseases, including Alzheimer's disease, vascular dementia, and Parkinson's disease [3,4].
Hypoxia inducible factor 1α (HIF1α) has been elucidated as the representative transcription factor of hypoxic condition that can be accumulated in cerebral cortex and hippocampus [5,6]. Several studies have reported that HIF1α induces the expression of various genes associated with cell survival, angiogenesis, or glucose uptake, including p53, vascular endothelial growth factor (VEGF), and GLUT1 to overcome hypoxic challenges [7][8][9]. These altered microenvironments promote oxidative stress by generating reactive oxygen species (ROS) in neuronal cells, leading to neuronal toxicity. Therefore, the importance is gradually elevated to develop a modulator in the neurotoxic condition induced by hypoxia.
Most cells have developed an endogenous self-defense strategy against ROS-induced damage. One of the most widely studied mechanisms is the promotion of heme oxygenase-1 (HO-1) expression via the nuclear transcription factor erythroid 2-like factor 2 (Nrf2) pathway [10,11]. The cytoprotective role of Nrf2 pathway has been reported to significantly reduce the cytotoxicity under hypoxic conditions [12]. Nonetheless, the protective effect of HO-1 via the Nrf2 pathway is important, and little evidence has been reported on whether bioactive molecules isolated from natural products induce the HO-1/Nrf2 pathway to reduce neurotoxicity induced by hypoxia. 6,7,4 -trihydroxyflavanone (THF), isolated from Dalbergia odorifera, is a flavonoid categorized in the flavanone family. D. odorifera has been studied to mainly live in Southern China, including Hainan, Fujian, Guangdong, and Zhejiang [13]. In East Asia, including Korea and China, D. odorifera extract has long been used as a therapeutic agent for rheumatic and epigastric pain, blood stagnation syndrome, ischemia, and swelling [14]. Accumulating evidences demonstrate that THF exhibits effective regulation of osteoclastogenesis by controlling bone resorption and protective role against methamphetamine-induced cytotoxicity on T cells [15,16]. In particular, it has been reported that THF effectively suppresses the NF-κB pathway in two studies to possess anti-osteoclastogenesis and cytoprotective effect against methamphetamine exposure. However, naringenin, one of the flavanone that has a similar structure with THF, has been elucidated to have a radical scavenging activity and protect liver tissue by acting as antioxidant [17,18], no evidence has been reported if THF possesses an antioxidant effect in CoCl 2 -induced hypoxia condition. In this study, we investigated whether treatment with THF has a cytoprotective role by inducing HO-1 expression through the Nrf2 pathway. In addition, we also explored whether induction of HO-1 by pre-treatment with THF attenuates hypoxia and oxidative stress to suppress neurotoxicity under hypoxic conditions.

MTT Assay
Seeded SH-SY5y cells (1 × 10 4 /well, 96-well plate) were treated with the indicated concentration of THF (0-40 µM) for 24 h. The supernatants were discarded and 500 µg/mL of MTT was incubated with the cells for 1 h. Supernatants were removed and generated formazan crystals were dissolved in 170 µL of DMSO. Plate was read to gain the absorbance at 540 nm and cell viability was determined by comparing with absorbance of control cells (% of control).

Determination of Dead Cell Population by AnnexinV Staining
Seeded SH-SY5y cells (1 × 10 4 /well, 96-well plate) were stained with 1× AnnexinV staining reagent for IncuCyte for 30 min. Then cells were treated with the indicated concentration of THF (0-40 µM) for 24 h or pre-treated with the indicated concentration of THF (0-40 µM) for 6 h and incubated with 0.5 mM CoCl 2 for 24 h. After incubation, the intensity of AnnexinV was assessed by IncuCyte imaging system and DIC images were obtained with AnnexnV fluorescence (green). Integrated intensity of AnnexinV was determined by comparing with control cells (% of control).

Western Blot Analysis
SH-SY5y cells cultured in the indicated conditions were harvested for lysis in RIPA buffer with 1× phosphatase inhibitor at 4 • C for 20 min. Lysates were centrifuged at 13,500 rpm at 4 • C for 20 min and 30 to 50 µg of the lysate was separated on 8-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. Proteins were transferred onto PVDF membranes, which were then blocked with 5% skimmed milk for 1 h. After blocking, membranes were incubated with the respective primary antibodies in 3% skim milk overnight (1:1000 ratio). Excess primary antibodies were removed by washing the membrane four times with TBS-T and incubated with 0.1 µg/mL peroxidaselabeled secondary antibodies (against rabbit or mouse) for 1 h. After four washes with TBS-T, bands were detected with ECL Western blot detection reagents with an ImageQuant LAS 4000 (GE Healthcare, Chicago, IL, USA).

Apoptosis Assay
Apoptotic neurotoxicity of SH-SY5y cells was assessed by a double staining experiment using AnnexinV and PI. After incubation of SH-SY5y neuroblastoma cells treated with the indicated conditions, cells were suspended in 1× trypsin-ethylenediaminetetraacetic acid (EDTA) buffer. After washing with cold PBS, cells were resuspended in 100 µL of 1× binding buffer containing AnnexinV (20 µg/mL) and PI (1 µg/mL) for 15 min at RT. Stained cells were acquired on a BD FACSVerse (BD Biosciences, San Diego, CA, USA), and the population of AnnexinV + cells or AnnexinV + /PI + cells was presented in the bar graph with plots.

RT-PCR and Realtime Quantitative RT-PCR
Total RNA was isolated from cells treated with the indicated conditions using TRIZOL reagent and reverse transcription of the RNA to cDNA was performed. Primers used for each gene were as follows (forward and reverse primers, respectively). human For quantitative PCR analysis, amplification was performed in a DNA Engine Opticon 1 continuous fluorescence detection system (MJ Research, Waltham, MA, USA) using SYBR Premix Ex Taq. The total reaction volume was 10 µL containing 0.1 µg of cDNA and each PCR reaction was performed using the following conditions: 95 • C for 30 s, 60 • C for 30 s, and plate read for 40 cycles followed by 7 min of extension at 72 • C. Melting curve analysis was performed to characterize the dsDNA product by slowly raising the temperature (0.15 • C/s) from 60 • C to 95 • C with fluorescence data collected at 0.15 • C intervals. mRNA levels of genes were normalized with the mRNA levels of GAPDH and were presented as "% of maximum". The "% of maximum" was calculated using the following equation: % of maximum = 2 −∆∆CT × 100, where ∆∆CT = (CTtarget−CTgapdh) at maximum−(CTtarget−CTgapdh).

Reactive Oxygen Species (ROS) Measurement
SH-SY5y cells incubated with the indicated conditions were stained with 2 µM DCF-DA for 20 min in the dark. Generated fluorescence was assessed using the IncuCyte imaging system. The intensity of 2',7'-dichlorofluorescin Diacetate (DCF-DA) was obtained from IncuCyte software and the % of maximum was calculated and presented in bar graph.

Detection of Nrf2 Nuclear Translocation
To detect Nrf2 nuclear translocation in SH-SY5y cells after treatment with THF, cells were incubated with the indicated concentration of THF for 1 h and collected. Nuclear extracts and cytosolic extracts were separated from the whole lysate using the NE-PER Kit. For SDS-PAGE, 20 µg of nuclear extract and 50 µg of cytosolic extract was loaded in 8% SDS gel. Nuclear extracts and cytosolic extracts were normalized with the level of LaminB and β-actin, respectively.

Statistics
Mean values ± SEM were evaluated from the data obtained from three independent experiments performed on separate days and presented as bar graphs. One-way ANOVA was used to determine significance (p value). * indicates differences from the mock-treated group or between two indicated groups considered significant at p < 0.05.

THF Does Not Induce Cell Death and Apoptosis in SH-SY5y Cells
Since it has been previously reported that THF is not cytotoxic to RAW 264.7 cells [15], we first confirmed whether THF shows cytotoxicity in SH-SY5y neuronal cells. Figure 2A shows that treatment with up to 40 µM of THF does not induce cell death. The measurement of intensity of AnnexinV also exhibited that THF does not affect cell death in SH-SY5y cells ( Figure 2B). The changes of cell number was not shown in cells incubated with THF up to 40 µM ( Figure 2C). To investigate whether THF is associated with apoptosis in SH-SY5y cells, the changes in expression of apoptosis-related proteins after THF treatment were determined. As shown in Figure 2D, the expression of Bcl2 and caspase family, which are highly involved in apoptosis, were not altered by THF treatment. The results from the apoptosis assay also demonstrated that SH-SY5y cells treated with up to 40 µM THF did not undergo apoptotic pathway ( Figure 2E). These data suggest that THF does not induce cell death and apoptosis in SH-SY5y cells.

THF Protects SH-SY5y Cells from CoCl 2 -Induced Cytotoxicity in Hypoxic Condition
It has been widely established that treatment with cobalt chloride (CoCl 2 ) induces hypoxia in neuronal cells [20]. To understand whether CoCl 2 leads to neurotoxicity in SH-SY5y cells, MTT assay was performed. Figure 3A showed that cellular viability was reduced in dose-dependent manner. To evaluate whether pre-treatment with THF has a protective effect on the neuronal cytotoxicity induced by treatment with CoCl 2 , cell viability was assessed by MTT assay. Figure 3B shows that the viability of SH-SY5y cells pre-treated with THF was significantly restored in a dose-dependent manner compared to cells pre-treated with mock. The intensity of AnnexinV was also partially inhibited by pretreatment with THF in CoCl 2 -induced hypoxia ( Figure 3C). Cell number was also confirmed that pre-treatment with THF preserves cell viability in CoCl 2 -induced hypoxia condition ( Figure 3D). To confirm if THF pre-treatment protects SH-SY5y cells from CoCl 2 -induced apoptosis, the population of AnnexinV-and PI-positive cells was determined by flow cytometry. As shown in Figure 3E, exposure to CoCl 2 led to the apoptotic pathway in SH-SY5y cells, but THF pre-treatment partially restored the CoCl 2 -induced apoptosis in a dosedependent manner. These data demonstrate that CoCl 2 treatment induces neurotoxicity in SH-SY5y cells, but THF pre-treatment effectively preserves cellular death and apoptosis in a dose-dependent manner.

THF Blocks the Cleavage of Caspase Family in CoCl 2 -Induced Hypoxia Condition
It has been evaluated that the fate of cells undergoing apoptotic pathway is tightly controlled by the expression of apoptosis-related proteins [21]. To elucidate the changes in the expression of apoptosis-related proteins after CoCl 2 treatment of SH-SY5y cells, the expression of Bcl2 and caspase family was determined by Western blot analysis. Figure 4A shows that CoCl 2 treatment downregulates the expression of Bcl2 and cleavage of caspase3 and 7 in SH-SY5y cells in a dose-dependent manner. To validate whether THF pre-treatment blocks reduction of the apoptosis-related proteins in CoCl 2 -induced hypoxic condition, Western blot analysis was performed. As shown in Figure 4B, THF pre-treatment partially restored the suppressed expression of Bcl2 and led to cleavage of caspase3 and caspase7 by CoCl 2 treatment. These data suggest that THF pre-treatment preserves the expression of anti-apoptotic proteins but suppresses the active caspase family in CoCl 2 -induced hypoxic condition.

THF Inhibits CoCl 2 -Induced Hypoxia-Related Genes in SH-SY5y Cells
To investigate the underlying mechanism of how THF pre-treatment protects the cells from neurotoxicity induced by CoCl 2 treatment in SH-SY5y cells, we first tested whether THF pre-treatment blocks hypoxia induced by CoCl 2 treatment. As shown in Figure 5A, CoCl 2 exposure induced the mRNA level of HIF1a, a marker of hypoxia, in a dose-dependent manner. Under hypoxic condition, we found that THF pre-treatment significantly inhibited the induction of HIF1a by treatment with 0.5 mM CoCl 2 ( Figure 5B).
We also checked if THF pre-treatment reduced the mRNA levels of hypoxia-related genes, including p53, VEGF, and GLUT1. Quantitative RT-PCR results showed that THF pretreatment significantly suppressed the induction of p53, VEGF, and GLUT1 expression ( Figure 5B). The regulatory effects of THF pre-treatment on the induction of hypoxiarelated genes were also confirmed by Western blot at the protein level ( Figure 5C). These results clearly demonstrate that THF pre-treatment attenuates the CoCl 2 -induced hypoxic condition in SH-SY5y cells.

THF Attenuates the CoCl 2 -Induced Oxidative Stress in SH-SY5y Cells
One of the well-defined cytotoxic mechanisms of hypoxia is the induction of oxidative stress [22]. To explore whether THF pre-treatment is associated with the inhibition of CoCl 2 -induced oxidative stress, we assessed the ROS generation in THF pre-treated and CoCl 2 -exposed SH-SY5y cells. Figure 6A revealed that enhanced ROS generation by CoCl 2 exposure was significantly suppressed by THF pre-treatment in a dose-dependent manner. Since oxidative stress, including ROS generation by CoCl 2 , is highly involved in the reduced expression of SOD and CAT, which are antioxidant proteins, we also determined if THF pre-treatment preserves them in CoCl 2 -induced hypoxia. Quantitative RT-PCR analysis showed that the mRNA levels of SOD and CAT in CoCl 2 -exposed SH-SY5y cells were downregulated, which was restored by THF pre-treatment ( Figure 6B). It was also confirmed on the protein level by Western blot analysis that the expressions of SOD and CAT are preserved by THF pre-treatment ( Figure 6C). These results suggest that THF pre-treatment mitigates the CoCl 2 -induced oxidative stress by restoring the expression of antioxidant proteins.

THF Promotes HO-1 Expression by Leading Nrf2 Translocation in SH-SY5y Cells
HO-1 has been widely reported as an important product of the antioxidant signaling pathway [23]. To evaluate whether THF treatment is associated with HO-1 induction, we detected the expression of HO-1 in THF-treated SH-SY5y cells in a dose-dependent manner. Figure 7A clearly demonstrates that the expression of HO-1 is induced by THF treatment. The time-dependent experiment showed that the expression of HO-1 was inducible in cells treated with 40 µM THF for 6 h ( Figure 7B). Since the Nrf2 pathway is known to be a major signaling pathway for the induction of HO-1, we checked whether treatment with THF leads to the nuclear translocation of Nrf2 into the nucleus in SH-SY5y cells. Figure 7C shows that Nrf2 is translocated into the nucleus by a dose-dependent THF treatment in SH-SY5y cells. Besides, we performed the Western blot assay to explore whether both CoCl 2 and THF affect to Nrf2 nuclear translocation and HO-1 expression. As shown in the Figure 7D, HO-1 expression is induced by exposure to CoCl 2 but pre-treatment with THF promotes more HO-1 expression compared to CoCl 2 exposure only. We also found that exposure to CoCl 2 induces the Nrf2 nuclear translocation but pre-treatment with THF boosts it in SH-SY5y cells ( Figure 7E). These data suggest that THF treatment enhances the expression of HO-1 through Nrf2 nuclear translocation and exposure to CoCl 2 stimulates the defense mechanism inside cells but pre-treatment with THF improves defense pathway against toxicity including CoCl 2 in SH-SY5y cells.

Enhancement of HO-1 by THF Pre-treatment Protects SH-SY5y Cells from CoCl 2 -Induced Neurotoxicity in Hypoxic Condition
Since the expression of induced HO-1 by antioxidants has been reported to protect cells against cytotoxic conditions, including hypoxia, we investigated whether HO-1 induction by THF treatment is involved in the protective role of THF under hypoxic conditions. To remove the cytoprotective effect of HO-1 induced by THF pre-treatment, cells were pretreated with 20 µM SnPP to inhibit the activity of HO-1, then the cell viability was assessed. Figure 8A shows that pre-treatment with SnPP significantly mitigates the protective effect of THF in SH-SY5y cells. SH-SY5y cells pre-treated with SnPP also revealed undiminished mRNA levels of hypoxia-related genes, including HIF1a, p53, VEGF, and GLUT1 ( Figure 8B). To confirm whether pre-treatment with SnPP removes the antioxidative effect of THF in CoCl 2 -induced hypoxia, generated ROS were measured in SH-SY5y cells pre-treated with SnPP and THF and exposed to CoCl 2 . Figure 8C shows that the suppressive effect of THF pre-treatment on ROS generation was mitigated in SH-SY5y cells. Interstingly, treatment with 20 µM SnPP does not affect to cell viability, mRNA level of hypoxia-related genes and ROS production. These data demonstrated that induction of HO-1 expression by THF pre-treatment protects SH-SY5y cells from cytotoxicity induced by CoCl 2 treatment. of THF for 6 h. After treatment with 0.5 mM CoCl 2 for 24 h, cells were incubated with 2 µM of DCF-DA for 20 min in dark. Generated ROS were detected by IncuCyte imaging system. The mean value of three experiments ± SEM is presented. * p < 0.05 between two indicated groups.

Discussion
It has been widely studied that the expression of VEGF and GLUT1 are regulated by HIF1α under hypoxic conditions. The biological function of VEGF has been reported to promote angiogenesis and increase the permeability of blood vessels under hypoxic conditions [24,25]. GLUT1 is a part of glucose transporter family that is located on the cell membrane and induces glucose transport to the cells. Several previous studies have demonstrated that cells induce the expression of VEGF and GLUT1 to absorb more oxygen in situations where oxygen is limited [7][8][9]. In addition, it has been shown that HIF1α binds to a specific sequence in target genes of hypoxia-responsive promoters, including p53, VEGF, and GLUT1, depending on the concentration of oxygen [26]. In this study, we investigated whether CoCl 2 -induced hypoxia induces the expression of HIF1α and THF pre-treatment suppresses this increment in SH-SY5y cells ( Figure 5). We also showed that THF pre-treatment reduced the expression of VEGF and GLUT1 at the mRNA and protein levels. Results from the inhibitor assay confirmed that induction of HO-1 by THF pre-treatment plays a critical role in cytoprotection under hypoxic conditions (Figure 8). These results suggest that pre-treatment with THF indirectly regulates the expression of HIF1α via promotion of the HO-1/Nrf2 pathway rather than direct regulation in vitro.
Further studies should include whether THF is directly involved in the transcription of HIF1a, p53, VEGF, and GLUT1 genes by performing EMSA assay.
To maintain normal cellular signaling response, ROS generation is tightly regulated in brain tissue through the expression of antioxidant enzymes, including SOD and CAT. In particular, strategies to reduce oxidative stress in brain tissue have been considered promising for the development of therapeutics for neurodegenerative diseases. One of the main factors inducing oxidative stress in the brain is the limited concentration of oxygen that causes a hypoxic environment. Excessive ROS generated in hypoxic conditions leads to apoptosis, DNA damages, and cell death. We confirmed whether exposure to CoCl 2 augments ROS generation in SH-SY5y cells, but THF pre-treatment effectively suppresses generated ROS ( Figure 6A). In addition, the expression of SOD and CAT was significantly upregulated in a THF dose-dependent manner ( Figure 6B,C). Figure 8C confirmed that HO-1 induced by THF pre-treatment is highly involved in the regulatory role of THF in the generation of ROS under hypoxic conditions. These data suggest that promotion of HO-1 expression by THF pre-treatment effectively protects neuronal cells from neurotoxicity induced by hypoxic condition.
Anti-apoptotic or cytoprotective effects of flavonoids have been widely elucidated. Quercetin, the most studied flavonoid, has been investigated that it partially blocks H 2 O 2induced apoptosis through the regulation of activator protein 1 (AP-1) transcription factor [27]. In particular, flavanone compounds have been studied as anti-apoptotic activity. Treatment with hesperetin and its metabolites, 5-nitro-hesperetin has shown a protective effect on neuronal cell death by modulation of ERK/PI3K pathway and naringenin possesses an anti-apoptotic activity in ischaemic stroke damage via Nrf2/HO-1 signaling pathway [28,29]. In the present study, we explored the anti-apoptotic effect of THF, one of flavanone compounds, in CoCl 2 -induced hypoxia condition through induction of Nrf2/HO-1 cytoprotective pathway. Our study suggests that a flavanone family including naringenin, hesperetin and THF has a potential to be developed as a source of therapeutic for neurodegenerative diseases.

Conclusions
In this study, we evaluated the cytoprotective effect of THF on CoCl 2 -induced neurotoxicity by promoting the HO-1/Nrf2 pathway. We showed that THF pre-treatment effectively enhanced the expression of HO-1 through the Nrf2 pathway in SH-SY5y cells and induced HO-1 suppresses the expression of hypoxia-related genes induced by CoCl 2 treatment. This reduced hypoxic condition by THF pre-treatment mitigates oxidative stress and leads to protection of SH-SY5y cells from neurotoxicity by CoCl 2 treatment.