ABSTRACT
Farrerol is a flavonoid found in plants with a wide range of pharmacological effects, including protection and enhancement of nerve cell function, as well as antioxidant and antibacterial properties, among others. Neurodegenerative diseases are irreversible neurological disorders resulting from the loss of neuronal cells in the brain and spinal cord. In this experiment, we investigated the neuroprotective and antioxidant effects of farrerol on glutamate-induced HT22 cells. Our results showed that farrerol inhibited reactive oxygen species expression, apoptosis, mitochondrial damage, and the activation of caspases 3 and 9 in HT22 cells induced by glutamate. Additionally, farrerol potentially regulated the Nrf2/heme oxygenase-1 (HO-1) signaling pathway, as it attenuated the nuclear translocation of Nrf2 and promoted the expression of HO-1. These findings suggest that farrerol has potential as a new therapeutic option.
The mechanism of farrerol inhibiting cell apoptosis.
Neurodegenerative diseases are a group of irreversible neurological disorders characterized by neuronal dysfunction resulting from the death of brain and spinal cord neurons (Panchal and Tiwari 2019; Salis et al.2020). Hippocampal neuronal cells are mainly affected by neuronal dysfunction in the central nervous system (Kim et al.2022). When hippocampal neuronal cells are stimulated by glutamate and other excitatory neurotransmitters, an excess of reactive oxygen species (ROS) is produced, leading to oxidative stress (OS) and cell death, which in turn results in neuronal dysfunction and the emergence of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS; Wang et al.2019; Yang et al.2022).
Oxidative stress is a significant contributor to the emergence of neurodegenerative disorders (Park et al.2019; Sabogal-Guáqueta et al.2019). Oxidative stress is a process in which the body's redox becomes unbalanced in response to certain environmental stimuli, producing free radicals that overwhelm the body's antioxidant defenses and leading to the buildup of reactive oxygen and nitrogen species in cells and organs, resulting in oxidative damage (Daenen et al.2019; Ornatowski et al.2020). The antioxidant enzyme heme oxygenase-1 (HO-1) is widely distributed in cerebral tissue and significantly reduces cellular damage (Facchinetti 2020). Drugs that increase HO-1 expression protect cells against the effects of OS and neurodegenerative disorders (Huang et al.2018). The essential cellular transcription factor Nrf2 regulates and promotes the expression of several detoxification and antioxidant genes (Hao et al.2022). The Nrf2 signaling pathway controls OS, protects cells by enhancing the antioxidant activity of HO-1, and reduces the formation of ROS (Bellezza et al.2018).
Oryzanol, mecobalamin, and amantadine are some of the medications used to treat neurodegenerative disorders (Ikeda, Iwasaki and Kaji 2015; Zhang et al.2019; Binvignat and Olloquequi 2020). However, these medications are associated with numerous side effects, such as gastrointestinal problems, depression, anxiety, and others (Ma et al.2022). Consequently, there is growing interest in developing new protective compounds from natural sources to address these concerns. One such compound is farrerol, a flavonoid found in plants (Ma et al.2021). The chemical structural formula is shown in Figure 1. Flavonoids have been shown to possess a range of pharmacological activities, including the protection and improvement of nerve cell function and antioxidant and antibacterial properties, among others. Many studies have shown that farrerol has a strong neuroprotective effect (Lai et al.2016). However, research on the mechanism of the neuroprotective properties of farrerol is very limited. Thus, this study aims to investigate the molecular basis of its neuroprotective effects.
Chemical structure of farrerol.
Materials and methods
Chemicals and reagents
Farrerol (purity >98%) was purchased from Sichuan Weikeqi Biotechnology Co., Ltd, CAS: 24211-30-1. All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). DCFH-DA, mitochondrial membrane potential (JC-1), and apoptosis (TUNEL) measurement kits were used from Beyotime Biotechnology (Shanghai, China). Tissue culture supplies, including Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and other products, were purchased from GIBCO BRL Co. (Grand Island, NY, USA). The primary and secondary antibodies, including mouse/rabbit anti-caspase-3, anti-caspase-9, cleaved caspase-3, cleaved caspase-9, HO-1, and Nrf2, were provided by Cell Signaling Technologies (Beverly, MA, USA).
Cell culture
HT22 cells were obtained from the China Center for Type Culture Collection (Wuhan, China) and maintained in DMEM with 10% FBS at 37 °C, and 80% confluent cells were rendered quiescent by incubation for 24 h before treatment with farrerol or glutamate.
Cell viability assay
Cell viability was determined using the MTT assay. Briefly, HT22 cells were seeded at a density of 8000 cells per well in a 96-well growth plate. Farrerol of varying concentrations was preincubated for 12 h before treatment. Cells were then treated with glutamic acid (20 m m) and cultivated for 24 h. Next, 50 µL of MTT (2.5 mg/mL) was added, and cells were cocultivated for an additional 4 h at 37 °C. The insoluble purple carbazan crystals formed by living cells were dissolved with dimethyl sulfoxide, and absorbance (OD) was measured at a wavelength of 490 nm. Results were expressed as a percentage of the OD value compared to untreated cells (100%).
Monitoring ROS formation in cells
The fluorescent probe 2,7-dichlorodihydrofluorescein-diacetate (DCFH-DA) was used to detect ROS production in cells according to the manufacturer's instructions (S0033, Beyotime Biotechnology, Shanghai, China). Briefly, cells were exposed to farrerol for 12 h and then incubated with diluted DCFH-DA probes for 20 min, washed 3 times in a serum-free medium, treated with glutamate (20 m m) for 30 min, and washed 3 times in PBS. The observation was carried out using a laser confocal microscope.
Cytosolic and nuclear fraction preparation
The cytoplasmic fraction of HT22 cells was prepared by centrifuging them in Pierce Biotechnology's (Rockford, IL, USA) PER mammalian protein extraction buffer. The cytoplasm and nucleus of HT22 cells were separated using NE-PER nuclear and cytoplasmic extraction reagents following the manufacturer's instructions (Pierce Biotechnology). The protein concentration was determined using the Bradford Protein Determination Kit (Solarbio Science & Technology Co., Ltd, Beijing, China).
Determining mitochondrial membrane potential
Mitochondrial depolarization was assessed using the JC-1 Test Kit (C2006, Beyotime Biotechnology, Shanghai, China). After exposure to glutamic acid and/or farrerol treatments, cells were washed with PBS and stained for 30 min at 37 °C in the dark with diluted JC-1 staining solution. The supernatant was collected and added to the culture medium, and a fluorescent microscope was used to observe it. Red fluorescence indicates healthy mitochondria, while green fluorescence indicates that the cell's mitochondria have become depolarized. ImageJ software was used to analyze the relative ratio of red and green fluorescence to calculate the depolarization ratio of mitochondria.
Apoptosis detection
To identify apoptosis, a TUNEL kit (C1088, Beyotime, Shanghai, China) was employed. After fixing the HT22 cells with 4% paraformaldehyde for 30 min, they were washed with PBS, exposed to 0.3% Triton X-100 at room temperature for 5 min, washed with PBS, and then diluted with the TUNEL detection solution at 37 °C for 60 min. They were washed with PBS, sealed with 4′,6-diamino-2-phenylindole (DAPI)-containing anti-fluorescence quenching sealing solution, and viewed under a fluorescence microscope. TUNEL was used to label apoptotic cells in green, and DAPI was used to designate nuclei in blue, according to the fluorescence signals. The proportion of apoptotic cells was determined by the ratio of TUNEL-positive cells, which were randomly chosen from 10 random fields and magnified at the same magnification.
Western blot analysis
To identify protein expression, cell lysates containing an equal amount of protein were separated using electrophoresis on 10%-15% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membrane was sealed using TBST containing 5% nonfat dry milk for 2 h. Following 3 washes with TBST, the membrane was exposed to a diluted primary antibody (dilution rate: 1:1000) for 1.5 h at room temperature. The membrane was then washed again at room temperature, treated with either goat anti-rabbit antibody or rabbit anti-mouse antibody (dilution rate: 1:5000), conjugated with horseradish peroxidase for 1 h, and finally exposed to ECL Western Blotting Substrate for visualization (Amersham Bioscience, Buckinghamshire, UK). The intensity of the proteins was measured using the ChemiDoc image analyzer (Tanon 4600, Tanon, China).
Immunofluorescence
To determine the location of Nrf2, HT22 cells were cultured on Lab-Tek II chamber slides. After farrerol treatment, the cells were fixed with formalin, and then made permeable with cold acetone. A secondary antibody (Santa Cruz Biotechnology) that recognizes Nrf2 and is fluorescently labeled with fluorescein isothiocyanate (FITC) was used to probe the cells (Alexa Fluor 488; Invitrogen, Carlsbad, CA, USA). Additionally, DAPI at 1 mg/mL in PBS and 50 µL VectaShield were applied to the cells to visualize their nuclei (Vector Laboratories, Burlingame, CA, USA). Stained cells were viewed and captured using a Provis AX70 fluorescent microscope (Olympus Optical, Tokyo, Japan).
Statistical analysis
Data are presented as the mean ± SD. The statistical analysis was conducted using GraphPad Prism version 6.0 (GraphPad Prism Software Inc., San Diego, CA, USA). One-way ANOVA with post hoc Tukey's test was used to analyze the experimental data. P values < .05 were considered statistically significant. All experiments were repeated at least 3 times.
Results
Farrerol effects on glutamate-induced cytotoxicity and production of ROS in HT22 cells.
The cytoprotective effects and ROS inhibitory activities of farrerol were investigated in glutamate-induced HT22 cells to assess its neuroprotective properties. Compared to untreated cells, the viability of HT22 cells after glutamate treatment decreased dramatically (Figure 2). Farrerol pretreatment improved HT22 cell viability in a concentration-dependent manner (Figure 2a). Glutamate significantly increased intracellular ROS production in HT22 cells. However, the production of ROS induced by glutamate was dramatically reduced following pretreatment with farrerol (Figure 2b). As a positive control for its antioxidant activities, trolox (50 µm) demonstrated considerably enhanced cytoprotective impact and ROS scavenging activity. The results showed that farrerol reduced the cytotoxicity and ROS production induced by glutamate in HT22 cells.
Effects of farrerol on glutamate-induced oxidative neurotoxicity and reactive oxygen species production in HT22 cells. HT22 cells were pretreated with various concentrations (1.25, 2.5, 5, and 10 µm) of farrerol (a, b) and then incubated for 24 h with glutamate (20 m m). Trolox (50 µm) was used as a positive control. Cell viability (a) and ROS (b) production were measured by MTT assay and DCF fluorescence measurement, respectively. Quantitative analysis of ROS (c) using ImageJ software. Values are calculated as percentages of untreated cells. Each bar represents the mean ± SD (n = 3). ##P < .01 compared to the control group. *P < .05 or **P < .01 compared to the group treated with 20 m m glutamate. The “+” and “−” signs indicate the presence or absence.
Farrerol effects on glutamate-induced apoptosis
To determine whether farrerol's ability to suppress apoptosis is responsible for its protective properties, we used a TUNEL study. TUNEL can detect apoptotic cells emitting green fluorescence due to DNA strand breakage. As demonstrated, farrerol (1.25-10 µm) significantly decreased glutamate (20 m m)-induced apoptosis in a dose-dependent manner (Figure 3).
Effect of farrerol on glutamate-induced apoptosis in HT22 cells. The apoptosis rate of HT22 cells induced by glutamate (20 m m) for 24 h with pretreatment of farrerol (1.25, 2.5, 5, and 10 µm) was detected by TUNEL staining in each group, and the apoptotic cells showed green fluorescence (200×). Values are calculated as percentages of untreated cells. Each bar represents the mean ± SD (n = 3). ##P < .01 compared to the control group. *P < .05 or ** P < .01 compared to the group treated with 20 m m glutamate. The “+” and “−” signs indicate the presence or absence.
Farrerol effects on glutamate-induced mitochondrial membrane potential and apoptosis in HT22 cells.
The mitochondrial membrane potential was assessed using the JC-1 staining approach, where green fluorescence indicated depolarized mitochondria, while red fluorescence indicated cells with normal mitochondria. The red-to-green fluorescence ratio can indicate the degree of mitochondrial depolarization. As seen in Figure 4a, glutamate administration increased the amount of depolarized mitochondria, but pretreatment of cells with farrerol (2.5-10 µm) greatly reduced this increase. Farrerol was found to reduce glutamate-induced HT22 cell mitochondrial damage. Cleaved caspase-3 and cleaved caspase-9 are essential components of the apoptotic machinery that can activate a variety of cellular target proteins to cause cell death. Therefore, we investigated how farrerol affected the cleavage of caspases in HT22 cells stimulated with glutamate. As shown in Figure 4b, pretreatment with farrerol (5-10 µm) for 24 h substantially and dose-dependently decreased the cleavage of caspases 3 and 9 induced by glutamate.
Effect of farrerol on mitochondrial membrane potential in glutamate-induced HT22 cells. (a) HT22 cells were exposed to glutamate (20 m m) for 24 h with pretreatment of farrerol (1.25, 2.5, 5, and 10 µm). Mitochondrial membrane potential was detected by JC-1 staining (200×). The green fluorescence indicates that the mitochondria of the cell were depolarized, and the red fluorescence implies that the mitochondria were normal. (b) Quantitative analysis of mitochondrial membrane potential. (c) The expressions of cleaved caspase-9, caspase-9, cleaved caspase-3, and caspase-3 were detected by western blotting. (d) Quantitative analysis of cleaved caspase-9, caspase-9, cleaved caspase-3, and caspase-3. Values are the mean ± SD of 3 independent experiments with triplicate samples. #P < .05 or ##P < .01 compared to the control group; *P < .05 or **P < .01 compared to the group treated with 20 m m glutamate.
Farrerol effect on HO-1 expression in HT22 cells
To ascertain the influence of farrerol on HO-1, we examined HO-1 expression at the protein level following farrerol treatment. The HO-1 inducer Cobalt protoporphyrin (CoPP) was used as a positive control and significantly increased HO-1 expression at the protein level (Figure 5a). Farrerol increased the expression of HO-1 at the protein level in a concentration-dependent manner (Figure 5a and b). Farrerol (10 µm) treatment also elevated HO-1 protein levels in a time-dependent manner (Figure 5b).
Effects of farrerol on HO-1 protein expression in HT22 cells. HT22 cells were incubated for 12 h with various concentrations (1.25, 2.5, 5, and 10 µm) of farrerol (a). HT22 cells were incubated with 10 µm of farrerol for various periods (b). CoPP (20 µm) was used as a positive control. The expression of HO-1 protein (a) was assessed by Western blot. Protein expression results for HO-1 were normalized to actin. Each bar represents the mean ± SD (n = 3). ##P < .01 compared to the control group. *P < .05 or **P < .01 compared to the group treated with CoPP. The “+” and “−” signs indicate the presence or absence.
Farrerol effects on Nrf2 translocation in HT22 cells
Nrf2 plays a crucial role in inducing HO-1 expression, and thus, we investigated the effect of farrerol on Nrf2 translocation in HT22 cells. As shown in Figure 6b, farrerol (10 µm) incubation resulted in a time-dependent increase in nuclear Nrf2 expression. Conversely, cytosolic Nrf2 expression decreased correspondingly (Figure 6a). Immunofluorescence microscopy revealed similar results (Figure 6c).
Effects of farrerol on Nrf2 translocation in HT22 cells. HT22 cells were incubated with 10 µm of farrerol for 0.5, 1, 1.5, or 2 h. The cytosolic and nuclear extracts were fractionated using PER-Mammalian Protein Extraction buffer. Nrf2 protein expression was detected by Western blot in cytosolic and nuclear extracts (a, b). Nrf2 translocation was detected by immunofluorescence (c). The levels of cytosolic and nuclear Nrf2 were normalized to β-actin and lamin B, respectively. *P < 0.05 compared to the control group. Values are the mean ± SD of 3 independent experiments with triplicate samples. *P < .01 compared to the control group.
Farrerol effects on HO-1 expression induced by glutamate in HT22 cells
We examined whether the neuroprotective effects of farrerol are mediated through HO-1 induction by blocking the HO-1 pathway with tin protoporphyrin (SnPP). Farrerol significantly reduced glutamate-induced cytotoxicity (Figure 7a) and ROS production (Figure 7b). SnPP treatment substantially reversed farrerol's inhibitory effect on cytotoxicity and ROS production induced by glutamate (Figure 7a and b).
Effects of farrerol-induced HO-1 on glutamate-induced cytotoxicity and reactive oxygen species generation in HT22 cells. HT22 cells were pretreated with 10 µm farrerol in the presence or absence of 50 µm SnPP and then exposed to glutamate (20 m m) for 24 h. Cell viability (a) and ROS (b) production were measured by the MTT assay and DCF fluorescence measurement, respectively. Each bar represents the mean ± SD (n = 3). #P < 0.05 or ##P < .01 compared to the control group. *P < 0.05 compared to the group treated with 20 m m glutamate. The “+” and “−” signs indicate the presence or absence.
Discussion
The incidence of neurodegenerative diseases increases with age, causing irreversible pathological changes that can be fatal in severe cases (Hou et al. 2019). Thus, appropriate treatment interventions are necessary to prevent the progression of the disease. Natural compounds such as quercetin, isoquercitrin, and hesperetin have demonstrated neuroprotective benefits and offer tremendous potential for treating neurodegenerative illnesses (Shen et al. 2018; Ikram et al. 2019; Park et al. 2021). In this study, we investigated how farrerol protects glutamate-stimulated HT22 cells against neurodegeneration.
Glutamate is a key neurotransmitter in the central nervous system of mammals, regulating the release of neurotransmitters and participating in normal physiological functions (Fernstrom 2018). However, high concentrations of glutamate can cause OS in neurons, leading to neuronal dysfunction (Sutcliffe et al. 2017). Our results showed that farrerol could attenuate glutamate-induced cytotoxicity and ROS production in HT22 cells stimulated by glutamate, suggesting that farrerol has neuroprotective properties.
Apoptosis plays a significant role in the emergence of neurodegenerative disorders (Erekat 2022). High levels of intracellular glutamate cause excess ROS production, which depolarizes mitochondrial membrane potential, alters mitochondrial permeability, and releases cytochrome C, AIF, and endo G from the mitochondria into the cytoplasm, leading to caspase activation (Sabogal-Guáqueta et al. 2019). Cytochrome C in the cytoplasm binds to Apaf-1 and procaspase-9 to form apoptotic bodies, which activate caspase-9, thereby activating caspase-3 and leading to apoptosis (Verma, Lizama and Chu 2022). Our results demonstrate that farrerol not only improves the depolarization of mitochondrial membrane potential but also reduces the activation of caspase-3 and improves apoptosis in glutamate-induced neuronal cells.
HO-1 is a ubiquitous antioxidant defense enzyme that plays a crucial role in combating OS (Zhang et al. 2021). Research has shown that HO-1 has significant neuroprotective effects both in vitro and in vivo (Hu et al. 2022). HO-1 is regulated by Nrf2. In the physiological state, when Nrf2 and Kelch-like ECH-associated protein 1 (Keap1) interact normally, Keap1 negatively regulates Nrf2 and facilitates the proteasome's ability to degrade Nrf2 ubiquitination, keeping Nrf2 levels low (Bellezza et al. 2018). However, when OS or other chemical stimuli occur, Nrf2 and Keap1 rapidly dissociate into the nucleus, combine with related gene promoter sequences, cause downstream gene HO-1 to express, and play an anti-OS role. Nrf2 is a basic-domain Leucine zipper transcription factor that maintains intracellular redox homeostasis by regulating the expression of various antioxidant proteins. Under steady-state conditions, Nrf2 is isolated by the E3 ligase junction Kelch-like ECH-related protein 1 (Keap1) in the cytoplasm, resulting in degradation through the ubiquitin proteasome system. When exposed to OS, Nrf2 escapes Keap1-mediated degradation by dissociating from the Nrf2-Keap1 heterodimer, and then translocates into the nucleus to identify the enhancer sequence, called antioxidant response element (ARE), which encodes the network metabolism of synergistic enzymes involved in antioxidants, including HO-1, GPx, and quinone oxidoreductase-1 (NQO-1). In addition to the Nrf2/HO-1 pathway, other pathways such as NF-KB and MAPK also need to be considered in HT22 cells (Ulasov et al. 2022). Our results suggest that farrerol can promote the nuclear entry of Nrf2, which in turn upregulates the expression of the antioxidant gene HO-1. Furthermore, SnPP experiments showed that farrerol-induced HO-1 expression was synergistic with the neuroprotective effect of HT22 cells. These findings indicate that farrerol may exert antioxidant effects by activating the Nrf2/HO-1 pathway.
In conclusion, our study demonstrates that farrerol can reduce HT22 cell apoptosis and OS caused by glutamate by activating the Nrf2/HO-1 pathway and participating in its protective mechanism. Farrerol shows potential as a neuroprotective agent for preventing and treating oxidative nerve damage and degenerative illnesses.
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Author contribution
L.G., B.L., and S.H. were responsible for the writing of the study; T.W. and L.G. contributed to the in vitro experiments. D.Z. and R.Y. were responsible for the data statistics and analysis.
Funding
This work was supported by National Natural Science Foundation of China (Grant numbers 81960781 and 82074578) and Shandong Provincial Natural Science Foundation (Grant number ZR2021LZY032).
Disclosure statement
No potential conflict of interest was reported by the authors.
