Neuroprotective Effect of Fructus Ligustri Lucidi in Spinal Cord Neuron Injury during H2O2-Mediated Oxidative Stress

Lee, Junseon; Kim, Hyunseong; Jeon, Wan-Jin; Yeo, Changhwan; Kim, Hyun; Hong, Jin Young; Baek, Seung Ho; Lee, Yoon Jae; Ha, In-Hyuk

doi:https://doi.org/10.1155/2024/6679695

Journal of Food Biochemistry

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Abstract Introduction Materials and Methods Results Discussion Conclusions Data Availability Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2024 | Article ID 6679695 | https://doi.org/10.1155/2024/6679695

Neuroprotective Effect of Fructus Ligustri Lucidi in Spinal Cord Neuron Injury during H₂O₂-Mediated Oxidative Stress

Junseon Lee,¹Hyunseong Kim,¹Wan-Jin Jeon,¹Changhwan Yeo,¹Hyun Kim,¹Jin Young Hong,¹Seung Ho Baek,²Yoon Jae Lee,¹and In-Hyuk Ha¹

Academic Editor: Jae Young Je

Received07 Sept 2023

Revised05 May 2024

Accepted10 May 2024

Published23 May 2024

Abstract

Oxidative stress-induced neuronal damage is a significant factor contributing to spinal cord injury. Although previous research has shown that Fructus Ligustri Lucidi (FLL) has neuroprotective benefits in SH-SY5Y, BV2, and PC12 cells, its impact on primary spinal cord neurons, which more accurately reflect the characteristics of central nervous system neurons, remains unexplored. This research investigated how FLL can protect rat primary spinal cord neurons from injury triggered by hydrogen peroxide (H₂O₂)-mediated oxidative stress. Cell viability, generation of reactive oxygen species (ROS), upregulation of inducible nitric oxide synthase (iNOS), activation of the Nrf2/HO-1 antioxidant pathway, and mitochondrial superoxide were assessed. Rat primary spinal cord neurons were not adversely affected by concentrations of FLL extract up to 100 μg/mL. Furthermore, FLL extract showed a significant protective effect against H₂O₂-induced neuronal toxicity at 10–100 μg/mL. Fluorescence-activated cell sorting analysis revealed that FLL extract inhibited H₂O₂-induced ROS generation in a dose-dependent manner. Immunocytochemistry and gene expression analysis confirmed that FLL extract reduced the overexpression of iNOS induced by H₂O₂ and enhanced the stimulation of the Nrf2/HO-1 pathway, crucial for antioxidant responses. In conclusion, FLL extract demonstrated neuroprotective effects on rat primary spinal cord neurons against the oxidative stress induced by H₂O₂. FLL extract effectively preserved cell viability, reduced ROS generation, suppressed iNOS overexpression, and activated the Nrf2/HO-1 antioxidant pathway. These results highlight the capacity of FLL extract as a neuroprotective agent against oxidative stress-related spinal neuron damage.

1. Introduction

Spinal cord injury (SCI) refers to permanent motor, sensory, and autonomic dysfunction resulting from physical damage to the central nervous system (CNS) [1]. In the physiological processes of SCI, oxidative stress is a critical component of the secondary phase of injury [2]. Oxidative stress is due to a disruption in the equilibrium between the production of reactive oxygen species (ROS) and the body’s ability to counteract them with antioxidant defense mechanisms. Under normal conditions, the body has antioxidant systems that neutralize the excess ROS generated and maintain cellular redox homeostasis; however, during SCI, there is a disruption in the equilibrium between the generated ROS and cellular antioxidants, resulting in an overabundance of ROS [3, 4]. Excessive ROS generation leads to severe mitochondrial problems, negatively affecting the electron transport chain, reducing ATP synthesis, and damaging mitochondrial proteins, lipids, and DNA [5–7]. This cascade of events, triggered by SCI, induces secondary neuroinflammatory responses, neuronal death, and axonal dieback. These issues are particularly pronounced due to the high oxygen demand of nerve cells, their limited antioxidant defenses, and the presence of easily oxidizable fatty acids [8, 9]. Therefore, antioxidants are a crucial component of SCI treatment to manage and protect the nervous system. The use of medicinal herbs and natural bioactive compounds, with their excellent antioxidant properties, is increasingly attractive due to fewer side effects compared with conventional chemical agents [10].

We aimed to identify a medicinal herb capable of enhancing antioxidant defenses to produce a neuroprotective effect. This quest is a key aspect of our broader research, which focuses on discovering potential therapeutic agents that could effectively treat SCI.

Fructus Ligustri Lucidi (FLL) refers to the dried, mature fruit of the broad-leaf privet (Ligustrum lucidum Aiton) or wax-leaf privet (Ligustrum japonicus Thunb) used in traditional Korean and Chinese herbal medicine therapy. Its traditional known effects include hepatoprotection, cardiac function enhancement and protection, and pain relief; it also has an anticancer effect [11]. FLL contains compounds with antioxidant activity, such as oleanolic acid, ursolic acid, flavonoids, secoiridoid glucosides, and phenolic compounds, which show potential as ROS scavengers [12, 13]. In particular, previous in vitro research demonstrated that FLL has neuroprotective properties; it increased caspase-3 and antioxidant enzyme levels in SH-SY5Y cells treated with hydrogen peroxide (H₂O₂) [14]. Furthermore, FLL treatment reduced intracellular ROS in PC12 cells exposed to 1-methyl-4-phenylpyridinium (MPP+), an agent known to induce Parkinson’s disease [15]. In a BV2 cell line-based neuroinflammation model, FLL decreased the expression of proinflammatory cytokines and suppressed NF-κB, inducible nitric oxide synthase (iNOS), and heme oxygenase 1 (HO-1), indicating anti-inflammatory and antioxidative properties [16]. In animal models, FLL has also demonstrated potential neuroprotective benefits in conditions such as Parkinson’s disease, Alzheimer’s disease, lumbar disc herniation, and depression. It has been found to boost glutathione peroxidase activity, reduce amyloid β-levels, inhibit γ-secretase activity, and enhance cognitive function [17]. Furthermore, FLL activates a key memory-enhancing factor and reduces inflammation, leading to improvement in depressive symptoms and decreased inflammation in disc herniation [18–20].

Although FLL has demonstrated neuroprotective and antioxidant effects in neuron-like cell lines, such as BV2, PC12, and SH-SY5Y, as well as in various models of neurological diseases, it has not yet been evaluated in SCI models or in cell models mimicking these conditions. The regenerative capacity of the nerve cells that constitute the CNS, including the spinal cord, is limited [21]. Neurons cultured directly from spinal cord tissue mimic the conditions of the actual spinal cord, allowing researchers to systematically explore the complex interactions and pathways related to SCI and directly observe effects on spinal neurons.

Mature spinal cord neurons, cultured for 7 d, were exposed to three concentrations of FLL and H₂O₂ for a 24-hour period. We investigated iNOS expression, nuclear factor E2-related factor 2 (Nrf2)/HO-1 signaling pathway regulation, and cell viability to assess the antioxidant and neuroprotective effects of FLL. Additionally, the study assessed the impact of FLL on the regulation of MitoSOX expression, an indicator of mitochondrial superoxide.

2. Materials and Methods

2.1. Preparation of FLL Extracts

Dried FLL (manufacturing number: CK20-C112-3-345, origin: China) was procured from CK Pharm Co., Ltd. (Seoul, Korea). It was subsequently stored in a desiccator until it was ready for use. FLL was extracted using the hot-water extraction method. FLL (30 g) was added to distilled water (300 ml), and the mixture was warmed to a temperature of 100°C for 3 h. After extraction, the resulting solution was allowed to cool to 25°C, filtered using a filter paper (HA-030; Hyundai Micro, Seoul, Korea), and stored in a deep freezer at −80°C. The extracted solution was lyophilized to obtain a powder (6.397 g) with a freeze dryer (Ilshin BioBase, Gyeonggi-do, Korea). FLL extraction yield was 21.32%. For analysis, a stock solution was prepared by combining the freeze-dried extract with phosphate-buffered saline (PBS) to reach a concentration of 10 mg/mL.

2.2. Primary Rat Spinal Cord Neuron Cultures

Primary spinal cord neurons were harvested from embryonic 15 d Sprague-Dawley rats obtained from Dae Han Bio Link (Chungju, Korea). The primary spinal cord neuron culture was conducted with slight modifications [22]. After a prompt cesarean section, the embryos were placed in Petri dishes containing Leibovitz’s L-15 medium (Gibco-BRL, Grand Island, NY), and the spinal cord was carefully isolated by removing the immature spines while positioning the embryonic body with the abdomen facing upwards. The meninges and dorsal root ganglia were carefully excised from the separated spinal cord with fine forceps under magnification. Thereafter, the spinal cords were rinsed once with L-15 medium. For enzymatic digestion, the isolated spinal cords were subjected to the Neural Tissue Dissociation Kit and the gentleMACS™ Dissociator (both Miltenyi Biotec, Bergisch Gladbach, Germany) at 37°C for 20 min. Following dissociation, the supernatants were removed through centrifugation for 3 min at . The resulting cell pellet was suspended in 1 mL of neurobasal medium, supplemented with B27, GlutaMAX, and 1% penicillin/streptomycin (Gibco-BRL), before being cultured on coated plates.

2.3. H₂O₂-Induced Oxidative Injury and FLL Treatment

Following a 24-hour period of acclimatization/stabilization, primary spinal cord neurons were cultured for 7 d to promote neuronal cell maturation. To induce oxidative stress, the existing culture medium was replaced with either a medium containing 500 μM of H₂O₂ or a medium containing 500 μM of H₂O₂ along with various concentrations (25, 50, or 100 μg/mL) of FLL extract and cultured for 24 h. The samples were separated into five groups as follows: the blank group without treatment, the H₂O₂ group with H₂O₂-only treatment, the FLL 25 group with cotreatment of FLL 25 μg/mL + H₂O₂, the FLL 50 group with cotreatment of FLL 50 μg/mL + H₂O₂, and the FLL 100 group with cotreatment of FLL 10 μg/mL + H₂O₂. A comprehensive overview of our experimental procedures is shown in Scheme 1.

2.4. Cell Viability Assay

Using 2-fold increased doses of FLL, with and without H₂O₂ exposure, the viability of the neurons was evaluated with the Cell Counting Kit-8 assay (CCK-8; Dojindo, Kumamoto, Japan). After a 7-day incubation period, each well’s cells were exposed to CCK-8 solution and left to incubate for 4 h. Absorbance at 450 nm was quantified using the Epoch Microplate Reader (BioTek, Winooski, VT). Neuronal survival rates were calculated by comparing the absorbance of treated neurons to that of control neurons, which were considered to have 100% viability.

2.5. Live/Dead Assay

For visual confirmation, live and dead cells were visualized using a live/dead cell imaging kit (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s guidelines. Cells were mixed with fluorescent live/dead assay dye and incubated at 37°C for 15 min. To measure the quantity of living and dead cells accurately, six pictures per test group were randomly selected and captured with a confocal microscope (Eclipse C2 Plus; Nikon, Tokyo, Japan) with a 10x objective.

2.6. Fluorescence-Activated Cell Sorting Analysis of ROS Generation

2′,7′-Dichlorodihydrofluorescein diacetate (DCFDA) is widely used to directly evaluate cellular redox states, enabling the assessment of free radical production [23]. The DCFDA/H2DCFDA-Cellular ROS Assay Kit (Abcam, Cambridge, UK) was used for conducting DCFDA assays. Briefly, following the manufacturer’s guidelines, spinal cord neuron cells cultured for 7 d were exposed to a 0.05% trypsin-EDTA solution for 2–3 min at 37°C to obtain a cell suspension. [24]. Then, after washing once with PBS by centrifugation, DCFDA (20 μM with 1X buffer) was stained for 30 min at 37°. The stained cells were carefully separated into single cells using a pipette and were immediately analyzed by fluorescence-activated cell sorting (FACS; Accuri C6 Plus Flow Cytometer; BD Biosciences, Franklin Lakes, CA).

2.7. Immunocytochemistry

Following a 30-minute fixation period using 4% paraformaldehyde (PFA; Biosesang, Seongnam, Korea), the cells underwent three washes with PBS (Gibco-BRL), each lasting 5 min. The cells were treated with a 0.2% Triton X-100 solution and then washed twice with PBS to facilitate cell permeability. The cells were subsequently incubated with 2% normal goat serum (NGS) in PBS for 1 h to prevent nonspecific binding. Primary antibodies, such as mouse anti-iNOS (R&D Systems, Minneapolis, MN, USA), guinea pig anti-MAP2 (Synaptic Systems, Goettingen, Germany), rabbit anti-NRF2 (Abcam), and mouse anti-HO-1 (Enzo Life Sciences, Farmingdale, NY, USA), were diluted in a PBS solution containing 2% NGS and applied at 4°C overnight. The cells were incubated for 2 h with secondary antibodies labeled with FITC or rhodamine (Jackson ImmunoResearch Laboratories, West Grove, PA). Following three 5-minute PBS washes, the cells were mounted using a fluorescence mounting medium (Dako, Santa Clara, CA, USA), and confocal microscopy (Eclipse C2 Plus; Nikon) was used to acquire images.

2.8. Mitochondrial Superoxide Staining

MitoSOX-based assays are commonly employed in live-cell studies to quantify ROS, specifically mitochondrial superoxide [25]. After incubating for 7 d, the cells were exposed to MitoSOX Red, a fluorescent probe used for detecting mitochondrial superoxide levels, for 30 min at a temperature range of 20–25°C. After rinsing with warm PBS, the stained samples were carefully mounted onto glass slides using Dako Mounting Medium (Dako).

2.9. RNA Isolation and RT-qPCR

We evaluated the impact of FLL on antioxidant activity by using RT-qPCR to analyze the expression of antioxidant-related genes in primary spinal cord neurons induced with H₂O₂. Initially, total RNA was extracted with TRIzol reagent (Ambion, Austin, TX, USA), followed by cDNA synthesis using random hexamer primers and AccuPower RT PreMix (Bioneer, Daejeon, Korea). Primer sets for RT-qPCR were designed by using information from the UCSC Genome Bioinformatics and NCBI databases (Primer-Blast, National Institutes of Health); the details are provided in Table 1. RT-qPCR was conducted on a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA) using SYBR Green Supermix (Bio-Rad). Each RT-qPCR analysis was performed in triplicate. The levels of the target genes were adjusted based on GAPDH expression and are shown as fold changes compared with the blank group.

3. Results

3.1. FLL Suppressed Cell Viability Reduction against H₂O₂-Induced Oxidative Stress Injury in Rat Primary Spinal Cord Neurons

A cytotoxicity evaluation was conducted to assess the potential harm that FLL could cause to primary spinal cord neurons. The primary spinal neurons were initially exposed to different concentrations of FLL for 24 h without H₂O₂ treatment. The results from the CCK-8 assays showed that doses of FLL up to 100 μg/mL did not have any harmful impacts on spinal cord neurons cultured in vitro (Figure 1(a)). Further tests revealed that the tested concentrations of FLL (ranging from 10 to 100 μg/mL) displayed a notable ability to protect against neuronal toxicity induced by H₂O₂. Therefore, we set the optimal concentration of FLL as 25–100 μg/mL, a concentration at which FLL exhibits a neuroprotective effect against H₂O₂ (Figure 1(b)). The live/dead cell assay, conducted under identical culture conditions, exhibited a pattern comparable to that observed in the CCK-8 assay. Interestingly, the group treated with H₂O₂ showed a notably elevated proportion of red-stained dead neurons, in contrast to that of the control group. In contrast, neurons cotreated with H₂O₂ and FLL exhibited a significantly lower proportion of dead cells, compared with the controls, in a dose-dependent manner (Figures 1(c) and 1(d)).

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(b)

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Figure 1

Effect of FLL extract on the viability of spinal cord neurons under oxidative stress induced by H₂O₂. (a) CCK-8 results of spinal cord neurons treated with various concentrations of FLL for 24 h without H₂O₂ exposure and (b) cotreated with FLL and H₂O₂. (c) Dead cells as a percentage of total cells in the live/dead assay. (d) Visualization of live (green) and dead (red) neurons using a live/dead imaging assay kit. Scale bar = 200 μm. Data are expressed as means ± SEM. Significant differences are indicated as ^# and ^#### compared with the blank group. and vs. the H₂O₂ group were analyzed via one-way ANOVA with Tukey’s post hoc test.

These findings indicate that FLL successfully preserves neurons, which survive in simulated ROS-producing in vitro environments. As a result, FLL has the ability to offer neuroprotection from oxidative stress and can be used safely, as concentrations of up to 100 μg/mL did not induce cellular toxicity.

3.2. FLL Reduced H₂O₂-Induced Overexpression of ROS and the iNOS Signaling Pathway

Using FACS analysis, we investigated the potential antioxidant effects of FLL by assessing its ability to inhibit ROS generation induced by H₂O₂. Exposure to H₂O₂ caused a significant increase in ROS production, but cotreatment with FLL resulted in a gradual dose-dependent decrease in ROS generation (Figures 2(a) and 2(c)). Furthermore, we assessed iNOS expression by immunocytochemistry to confirm the antioxidant effect of FLL treatment on H₂O₂-treated spinal cord neurons. When exposed to H₂O₂, the relative fluorescence intensity of iNOS exhibited a notable elevation compared to that of the blank group. When quantified, all the FLL groups demonstrated dose-dependent decreases compared with the H₂O₂ group, but significant decreases were found only in the FLL 50 and 100 μg/mL groups (Figures 2(b) and 2(d)). Additionally, the evaluation of the mRNA levels of iNOS gene expression after FLL treatment under the H₂O₂-insulting condition in spinal cord neurons revealed a significant upregulation in the H₂O₂ group compared to that in the blank group, whereas a concentration-dependent decrease was confirmed after FLL treatment. However, the two other FLL (50 and 100 μg/mL) groups revealed significantly downregulated iNOS mRNA levels compared to the H₂O₂ group (Figure 2(e)).

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(b)

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Figure 2

Effect of FLL extract on ROS accumulation and iNOS expression. (a) Flow cytometric dot plot depicting the assessment of reactive oxygen species (ROS) in spinal cord neurons using DCFDA. (b) MAP2 (red) and iNOS (green) expression immunocytochemistry images with confocal microscopy. White scale bar = 200 μm. Yellow scale bar = 50 μm. (c) Flow cytometric quantification of ROS-positive cells, challenged with 500 μM H₂O₂. (d) Quantitative evaluation of each group’s relative iNOS expression fluorescence intensity. (e) Expression of the iNOS gene was measured in spinal cord neurons after cotreatment with FLL and H₂O₂ for 24 h. Data are expressed as means ± SEM. Significant differences are indicated as ^#### compared with the blank group. , , and vs. the H₂O₂ group were analyzed via one-way ANOVA with Tukey’s post hoc test.

3.3. FLL Exerts Nrf2/HO-1-Related Neuroprotective and Antioxidant Effects

We further examined the alterations in the levels of Nrf2 and HO-1 expression, which play a role in antioxidant activity, using immunocytochemical assays. Initially, the blank group showed robust Nrf2 expression, while the H₂O₂ group exhibited significantly suppressed Nrf2 expression in response to H₂O₂ treatment. In contrast, FLL 50 and 100 μg/mL treatments resulted in a significant increase in Nrf2 immunoreactivity (Figures 3(a) and 3(c)). Nrf2 gene expression was significantly increased only at an FLL concentration of 100 μg/mL (Figure 3(d)). HO-1 immunoreactivity was found to be the lowest in the blank group, with no significant variance compared with the H₂O₂ group. However, a significant increase in HO-1 immunoreactivity was observed after FLL 50 or 100 μg/mL treatment (Figures 3(b) and 3(e)). Similarly, HO-1 gene expression matched the pattern observed by immunocytochemistry, the exception being the H₂O₂ group, which showed a significant increase compared with the blank group (Figure 3(f)).

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Figure 3

Antioxidant effect of FLL extract by upregulation of the Nrf2/HO-1 pathway. (a and b) Nrf2 and HO-1 immunocytochemistry images with confocal microscopy. White scale bar = 200 μm. Yellow scale bar = 50 μm. (c) Quantitative evaluation of each group’s relative Nrf2 expression fluorescence intensity. (d) Expression of the Nrf2 gene was measured in spinal cord neurons after cotreatment with FLL and H₂O₂ for 24 h. (e) Quantitative evaluation of each group’s relative HO-1 expression fluorescence intensity. (f) Expression of the HO-1 gene was measured in spinal cord neurons after cotreatment with FLL and H₂O₂ for 24 h. Data are expressed as means ± SEM. Significant differences are indicated as ^# and ^#### compared with the blank group. , and vs. the H₂O₂ group were analyzed via one-way ANOVA with Tukey’s post hoc test.

3.4. FLL Attenuates Mitochondrial ROS Production

We evaluated the expression levels of MitoSOX in spinal cord neurons exposed to oxidative stress induced by H₂O₂ (Figure 4(a)). We demonstrated a strong and notable elevation in cytoplasmic MitoSOX expression levels in spinal cord neurons exposed to H₂O_2, compared to those of the control group, suggesting a significant increase in mitochondrial ROS generation. However, when neurons exposed to H₂O₂ were treated with FLL, the proportion of MitoSOX-positive neurons decreased significantly in response to all FLL concentrations (Figure 4(b)). These results validate that FLL has a defensive impact against oxidative stress caused by H₂O₂ in spinal cord neurons; this was accomplished by suppressing mitochondrial ROS generation in a dose-dependent manner.

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Figure 4

Effect of FLL extract on reducing mitochondrial reactive oxygen species production. (a) Representative pictures of spinal cord neurons stained with MitoSOX after treatment with FLL in spinal cord neurons induced by H₂O₂. White scale bar = 200 μm. Yellow scale bar = 50 μm. (b) Quantification of the percentage of MitoSOX-labeled cells/total cells in each group. Data are expressed as means ± SEM. Significant differences are indicated as ^#### compared with the blank group. and vs. the H₂O₂ group were analyzed via one-way ANOVA with Tukey’s post hoc test.

4. Discussion

In the study of SCI, the critical role of oxidative stress during the secondary phase of injury is well-documented. Research into traditional herbal medicines, known for their antioxidant properties, often reveals benefits in treating SCI [26]. Notably, an olive leaf extract from Olea europaea of the Oleaceae family, similar to FLL, shows promising results in reducing inflammatory markers such as IL-6 and TNF-α and boosting neuroprotective BDNF in a chronic SCI model in rabbits [27]. Our study builds on this foundation by hypothesizing that FLL could similarly act as an antioxidant and neuroprotector. Uniquely, our research uses primary spinal cord neurons isolated directly from spinal cord tissue, offering a more accurate representation of tissue morphology and function than previous studies, thereby providing reliable insights into neurophysiological and functional characteristics.

We have determined that concentrations of 25, 50, and 100 μg/mL of FLL effectively protect against H₂O₂-induced oxidative stress in these neurons, thereby establishing precise standards for subsequent preclinical investigations. Conversely, prior studies using SH-SY5Y neuron-like cells elucidated a concentration-dependent protective response of FLL against H₂O₂, reaching its peak at 300 μg/mL [14]. While our findings demonstrate efficacy up to 100 μg/mL, a notable decline in cell viability is evident beyond this concentration threshold. This variance in outcomes can be attributed to the distinct cellular substrates used in our experimentation. Unlike primary spinal cord neurons, SH-SY5Y cells represent a neuronal model derived from stem cells, retaining certain stem cell attributes that facilitate proliferation and differentiation. Conversely, primary spinal cord neurons are vulnerable to apoptosis under various environmental, experimental, and technical conditions [28].

To comprehend the antioxidant mechanism underlying FLL’s protection against oxidative stress, we examined the production of ROS and the expression of iNOS, a key enzyme involved in NO production. Oxidative stress induces cellular damage through the elevation of reactive molecules such as free radicals. FLL mitigates this damage by neutralizing the ROS generated by hydrogen peroxide treatment, thereby preventing or minimizing oxidative damage. iNOS contributes to oxidative stress by producing NO, and its activation due to oxidative stress can lead to cellular damage. FLL effectively suppressed the expression of iNOS at both the protein and mRNA levels, indicating its potential to mitigate oxidative stress-induced cellular damage [29, 30]. We analyzed the Nrf2/HO-1 signaling pathway, a key mechanism that promotes antioxidant response and cellular protection in response to oxidative stress. In SCI therapy research, this pathway is considered a potential target for reducing oxidative stress and promoting antioxidant reactions [31]. Additionally, the vitamin gene network, which includes various antioxidant and cellular defense mechanisms, including the Nrf2/HO-1 signaling pathway, has important implications for biological adaptation and response to oxidative stress. Nrf2 triggers the initiation of antioxidant defense mechanisms by upregulating vitagene family members such as HO-1 and Hsp72 to mitigate oxidative stress. [32]. Stimulation of Nrf2-induced HO-1 production increases bilirubin production with antioxidant and neurotrophic-like properties, mitigating cellular oxidative damage and promoting cell survival. Simultaneously, the inhibition of iNOS, which generates high levels of nitric oxide under pathological conditions, reduces oxidative stress responses and neuroinflammation [33]. Research has shown that the generation of vitagenes has neuroprotective effects in conditions characterized by neurodegeneration like Alzheimer’s disease [34]. Thus, by modulating the iNOS and Nrf2/HO-1 signaling pathways, FLL enhances the inherent antioxidant capability of spinal cord neurons, providing neuroprotection against oxidative damage induced by H₂O₂.

In our final investigation, we examined FLL’s ability to inhibit oxidative damage in mitochondria, which are known to play a crucial role in protecting cells from oxidative stress. Mitochondria play a critical role in cellular energy production, underscoring their significance in safeguarding cells against oxidative stress. Nevertheless, under conditions of oxidative stress within cells, mitochondria can overproduce ROS, exacerbating the progression of SCI. Neurons typically possess more mitochondria than other cells in the body because of their higher energy demands, rendering them more susceptible to oxidative damage [35]. Oxidative stress induces mitochondrial ROS production, disrupting mitochondrial energy synthesis and triggering cell death and apoptosis. Elevated calcium influx due to oxidative stress impairs mitochondrial function, opening the nonselective permeability transition pore (mPTP) within the mitochondria [36]. This sequence of events results in mitochondrial swelling, membrane depolarization, and the liberation of apoptotic factors, ultimately culminating in cell demise. Through MitoSOX staining, we validated that FLL effectively suppresses mitochondrial ROS generation in an oxidative stress milieu [37, 38]. Consequently, FLL mitigates mitochondrial dysfunction provoked by oxidative stress, preserving cellular homeostasis and preventing neuronal death in the spinal cord.

To the best of our knowledge, this study is the first to discover the molecular mechanisms through which FLL delivers neuroprotective and antioxidant effects in spinal neurons possessing CNS tissue-specific properties. Hence, evaluating the neuroprotective and antioxidant effects of FLL in H₂O₂-induced primary cultured spinal neurons could validate its potential in preventing neuronal damage from oxidative stress. Nonetheless, the current study is limited in its capacity to comprehensively analyze its findings. Firstly, while primary spinal neurons serve as an in vitro model for spinal cord injury, they do not fully reflect the complexity of spinal cord tissue in vivo. Additionally, this study primarily focuses on assessing the acute effects of FLL. Therefore, further studies are necessary to investigate the long-term efficacy and safety profile of FLL in animal models of SCI.

5. Conclusions

This study investigated the neuroprotective effects of FLL on rat primary spinal cord neurons exposed to oxidative stress induced by H₂O₂. The research demonstrated that FLL effectively preserves neuronal viability and reduces oxidative damage by inhibiting mitochondrial ROS production and suppressing iNOS expression. Additionally, FLL enhanced antioxidant defense mechanisms by upregulating Nrf2 and HO-1 expression. The findings suggest that an optimal concentration range of 25–100 μg/mL for FLL offers significant neuroprotection without causing cytotoxic effects. These results support the potential therapeutic use of FLL in treating conditions associated with oxidative stress, such as SCI, highlighting its ability to protect neurons and underscoring the need for further clinical investigation.

Data Availability

The data presented in this study are available upon request from the corresponding author.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This study was supported by the Jaseng Medical Foundation, Korea. This work was also supported by a grant from the Traditional Korean Medicine Research and Development Program of the Korean Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (grant number: HF21C0100).

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Copyright © 2024 Junseon Lee et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Journal of Food Biochemistry

Neuroprotective Effect of Fructus Ligustri Lucidi in Spinal Cord Neuron Injury during H2O2-Mediated Oxidative Stress

Abstract

1. Introduction

2. Materials and Methods

2.1. Preparation of FLL Extracts

2.2. Primary Rat Spinal Cord Neuron Cultures

2.3. H2O2-Induced Oxidative Injury and FLL Treatment

2.4. Cell Viability Assay

2.5. Live/Dead Assay

2.6. Fluorescence-Activated Cell Sorting Analysis of ROS Generation

2.7. Immunocytochemistry

2.8. Mitochondrial Superoxide Staining

2.9. RNA Isolation and RT-qPCR

3. Results

3.1. FLL Suppressed Cell Viability Reduction against H2O2-Induced Oxidative Stress Injury in Rat Primary Spinal Cord Neurons

3.2. FLL Reduced H2O2-Induced Overexpression of ROS and the iNOS Signaling Pathway

3.3. FLL Exerts Nrf2/HO-1-Related Neuroprotective and Antioxidant Effects

3.4. FLL Attenuates Mitochondrial ROS Production

4. Discussion

5. Conclusions

Data Availability

Conflicts of Interest

Acknowledgments

References

Copyright

Neuroprotective Effect of Fructus Ligustri Lucidi in Spinal Cord Neuron Injury during H₂O₂-Mediated Oxidative Stress

2.3. H₂O₂-Induced Oxidative Injury and FLL Treatment

3.1. FLL Suppressed Cell Viability Reduction against H₂O₂-Induced Oxidative Stress Injury in Rat Primary Spinal Cord Neurons

3.2. FLL Reduced H₂O₂-Induced Overexpression of ROS and the iNOS Signaling Pathway