Betaine alleviates neuronal impairment in glutamate-injured SH-SY5Y neuroblastoma cells via Nrf2 signaling pathway related ferroptosis

doi:10.21203/rs.3.rs-4834086/v1

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Betaine alleviates neuronal impairment in glutamate-injured SH-SY5Y neuroblastoma cells via Nrf2 signaling pathway related ferroptosis

https://doi.org/10.21203/rs.3.rs-4834086/v1

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Alzheimer's disease (AD) is a devastating neurodegenerative disorder that currently lacks efficacious therapeutic interventions. It’s well documented that ferroptosis is extensively involved in the progression and pathogenesis of AD. Betaine, a critical nutrient for mammal health, is reported to possess neuroprotective actions. The objective of the current research was to discuss whether betaine could mitigate neuronal impairments by suppressing ferroptosis in SH-SY5Y neuroblastoma cells injured by glutamate. The results indicate that betaine improved the survival rate and morphology change of glutamate-damaged SH-SY5Y cells. Additionally, betaine reduced the intracellular accumulation of Fe²⁺, MDA, lipid ROS, and LDH release induced by glutamate. And reversal of the decreased GSH content and downregulation of ferroptosis inhibitors GPX4 expression were observed upon betaine administration. Additionally, betaine facilitated the translocation of Nrf2 from the cytoplasm to the nucleus in SH-SY5Y cells induced by glutamate. Molecular docking validated high-affinity binding between betaine and Nrf2. Collectively, betaine could exert neuroprotective effects by alleviating ferroptosis via activation of Nrf2 pathway, thereby positioning it as a potential candidate for AD therapy.

Betaine

Alzheimer’s disease

ferroptosis

Nrf2

·Betaine has ameliorated the AD model of glutamate-induced SH-SY5Y cells.

·Betaine protection against the ferroptosis produced by SH-SY5Y cells induced by glutamate.

·This study investigated the protective effect and mechanism of betaine against ferroptosis in an AD cell model.

Alzheimer’s disease (AD), a gradually progressive neurodegenerative disorder clinically characterized by cognitive decline and memory lapse threatening our aged population globally, imposes a heavy burden on both families and society (Nasb et al., 2024). It’s pathologically characterized by extracellular amyloid-β (Aβ) plaques accumulation, intracellular neurofibrillary tangles (NFTs) resulted by tau protein hyperphosphorylation and selective neuronal loss caused by hippocampal cell death (Morsy & Trippier., 2019). Currently, the treatment approach primarily focuses on symptomatic treatment.

Ferroptosis, characterized by intracellular lipid peroxidation and iron overload, is an iron-dependent non-apoptotic regulated cell death, which was initially described by Dixon in 2012 (Dixon et al., 2012). During the previous decade, plenty evidence has disclosed that ferroptosis promotes the progression of AD, and restraining of ferroptosis ameliorates the neuronal death and cognitive impairments in both in vivo (Bao et al., 2021) and in vitro (Yong et al., 2024) AD models. Hence, targeting to ferroptosis may be a prospective treatment strategy for AD (Ma et al., 2022). Consequently, targeting ferroptosis may represent a promising therapeutic strategy for AD.

Betaine, also known as trimethylglycine (TMG), is a naturally occurring catabolite of choline in mammals (Fig. 1. A&B). It’s a natural compound which is abundant in beet, fish, whole grains, marine products, and vegetables (Zhao et al., 2081). It is nontoxic and has been reported to exhibit beneficial effects on several human diseases, including liver diseases (Wang et al., 2021), heart diseases (Meyer et al.,2017), and cancer (Lee et al., 2010). Moreover, the clinical administration of betaine has demonstrated successful outcomes in the treatment of fatty liver, coronary atherosclerosis, and hyperlipidemia (Kharbanda et al., 2007; Arumugam et al., 2021; Abdelmalek et al., 2001). Recently, studies have shown that betaine exerts neuroprotective effects against AD both in vivo (Nie et al., 2016) and in vitro (Alipourfard et al., 2023) through antioxidant (Ibi et al., 2021), anti-inflammatory (Zhang & Jia, 2023), and regulating gene expression. Previous clinical studies also suggested that betaine could restore hyperhomocysteine expression to normal level in AD patients, which might ameliorate memory deficits (Sun et al., 2017). However, whether betaine can effectively alleviate AD by regulating ferroptosis and the exact molecular mechanism remains to be confirmed.

Hence, this study investigates the protective effects of betaine on restoring ferroptosis induced by glutamate in SH-SY5Y neuroblastoma cells, along with the underlying mechanisms. The results may provide valuable insights into the application of betaine in the adjuvant therapy of AD.

Cell Culture

SH-SY5Y neuroblastoma cells, which were as a gift from Prof. Lili Cui, Guangdong Medical University, were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, USA) supplemented with1% penicillin-streptomycin (Solarbio, Beijing, China) and 10% fetal bovine serum (Zhejiang Tianhang Biotechnology Co. LTD, Zhejiang, China). The cells were cultured in 5% CO₂, 37°C and passaged as 70–80% confluency. Refresh the culture medium every other day.

Determination of Cell Viability

Seed SH-SY5Y cells at a density of 5×10³ cells/well/100 µL in 96-well plates and incubated overnight. Following administrated with 25 mM glutamate for 24 h, the cells were treated with betaine for 12, 24, and 36 h. Cell Counting Kit-8 (CCK-8) was applied to check cell viability (Dojindo, Shanghai, China) accordingly.

Measurement of Malonaldehyde (MDA) Content

MDA content, which serves as a marker for lipid peroxidation, was measured by utilizing a commercially available assay kit from Beyotime Institute of Biotechnology (Shanghai, China) accordingly.

Detection of Reactive Oxygen Species (ROS) Formation

ROS-sensitive dye 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was applied to measure the intracellular ROS formation in SH-SY5Y cells.

Determination of Antioxidant Enzyme Activities

After the treatment, the levels of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in SH-SY5Y cells were quantified by specific detection kits (Beyotime Institute of Biotechnology, Shanghai, China).

Measurement of Intracellular Iron Level

The quantification of intracellular iron levels in cell lysates was performed using an iron assay kit from Applygen Technologies in Beijing, China, following the instructions provided by the manufacturer.

Mitochondrial Membrane Potential (MMP)

MMP was assessed by JC-1 staining following the guidelines provided by Beyotime Institute of Biotechnology in Shanghai, China. Images were observed using fluorescence microscopy.

Western Blot

Equal quantities of proteins were separated using SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes, which were then blocked with 5% BSA. Subsequently, the membranes were exposed to primary antibodies targeting GPX4, Nrf2, GAPDH, and Tubulin at 4°C overnight. Following washing, they were exposed to secondary antibodies for 40 min at room temperature. Visualization of the blots was achieved using enhanced chemiluminescence (ECL, Biotool) and then quantified using Image J software (Rawak Software, Inc. Germany).

Molecular Docking Analysis

The molecular structure of catalpol was retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), while the crystal structures of Nrf2 and GPX4 were obtained from the PDB database (http://www.rcsb.org/). Docking studies involving primary targets and potential compounds were conducted using AutoDock Tools (ADT, version 4.2) and AutoDock Vina (version 1.1.2) to determine binding energies. Catalpol and protein molecules underwent various operations such as hydrogenation, charge calculations, and were saved as PBDQT files. PyMoL software (version 2.2.0) was used by visualize the docking results.

Statistical Analysis

GraphPad Prism 8.0.2 (GraphPad Software, USA) was employed for statistical analysis and data representation. Results are presented as mean ± standard deviation (SD). One-way ANOVA tests or student’s t-test were conducted to access differences between groups, with a significance level set at p < 0.05.

Betaine exhibits neuroprotective effects in glutamate-induced SH-SY5Y cells

MTT assay was used to assess the effects of glutamate or betaine on the viability of SH-SY5Y cells. As shown in Fig. 1C, betaine (1-150 µM) did not cause any discernible cytotoxic effects in SH-SY5Y cells (Fig. 1C). Subsequently, SH-SY5Y cells were exposed to a gradient of glutamate concentrations at different temporal intervals. The results demonstrated that the viability of cells decreased significantly in a concentration-dependent manner upon exposure to glutamate (Fig. 1D). The viability of the cells was diminished to 50.59 ± 2.97% upon exposure to 25 mM glutamate for 24 h. Consequently, a 24-hour incubation with 25 mM glutamate was employed in this study to elicit injury in SH-SY5Y cells. To investigate the protective effects of betaine against glutamate-induced neurotoxicity in SH-SY5Y cells, cells incubated with 25 mM glutamate 24 h with or without betaine for 12, 24, and 36 h. Glutamate markedly diminished cell vitality and induced cytotoxicity which was reversed by betaine in SH-SY5Y cells (Fig. 1E). Hence, 50 µM betaine administrated for 12 h was selected in subsequent studies. Additionally, the observation of cellular morphology revealed that glutamate-treated SH-SY5Y cells exhibited a loss of the neurite-like shape and displayed widened intercellular gaps. Conversely, treatment with 25 µM betaine prevented these morphological alterations induced by glutamate. Cells administrated with betaine showed extensive branching and intact intercellular connections (Fig. 1F). These findings suggest that betaine possesses the ability to mitigate the neurotoxic effects caused by glutamate in SH-SY5Y cells.

Betaine ameliorates glutamate-induced ferroptosis in SH-SY5Y cells

Initially, we investigate whether the protective effect of betaine in alleviating the cytotoxicity was associated with ferroptosis. As shown in Fig. 2A, Fer-1, a ferroptosis specific inhibitor, effectively prevented glutamate-induced cytotoxicity in SH-SY5Y cells, suggesting that glutamate caused ferroptosis in SH-SY5Y cells. Moreover, the cytotoxicity induced by erastin, a ferroptosis specific inducer, was eliminated by betaine. Other ferroptosis parameter were also measured. The elevated intracellular iron and MDA contents in SH-SY5Y cells administrated with glutamate or erastin were markedly reduced by betaine (Fig. 2B&C). In addition, betaine administration markedly reversed MMP and GSH content in glutamate or erastin-treated SH-SY5Y cells (Fig. 2D&E). Furthermore, the reduced GPX4 expression caused by glutamate or erastin, a vital enzyme for preventing ferroptosis, was recovered by betaine or Fer-1. The findings presented herein provide compelling evidence supporting the potential of betaine in mitigating glutamate-induced ferroptosis in SH-SY5Y cells.

Betaine attenuates glutamate-induced oxidant stress in SH-SY5Y cells

As demonstrated in Fig. 3A-C, the levels of antioxidative defense system enzymes, including SOD, CAT, and GPx, were evaluated. The activities of SOD, CAT, and GPx were reduced in the glutamate administrated group, which were reversed by betaine and Fer-1. Moreover, the accumulation of ROS induced by glutamate was significantly decreased by treated with betaine in SH-SY5Y cells. These findings demonstrate that betaine effectively mitigates the oxidative stress induced by glutamate in SH-SY5Y cells.

Betaine simulates the translocation of Nrf2 from the cytoplasm into the nucleus in glutamate-induced SH-SY5Y cells

To explore whether betaine can activate Nrf2, the expression of nuclear Nrf2 was detected by western blot. Herein, glutamate showed inhibitory effects on nuclear Nrf2 expression in SH-SY5Y cells, which was upregulated by betaine. In SH-SY5Y cells subjected to betaine, a comparable induction of Nrf2 in nuclear fractions was observed as with Nrf2 inducer cheirolin. ML385, the specific Nrf2 inhibitor, effectively inhibited the nuclear translocation of Nrf2 prompted by both betaine and cheirolin. Furthermore, the translocation of Nrf2 into the nucleus, induced by both betaine and cheirolin, consistently exhibited a positive correlation with an enhancement in cellular viability. The protective impact of betaine against glutamate-induced cytotoxicity was nullified by ML385. These findings suggest that betaine may ameliorate ferroptosis by activating Nrf2 pathway.

Molecular docking

Nrf2 and betaine had a Docking Score of less than − 3.1kcal/mol. Figure 5 illustrated that betaine was bound with Nrf2 by amino acid residues (VAL523, ASP530) through carbon-hydrogen bonds and amino acid residue (GLU4, PHE39, GLU526, ASP530) through hydrophobic interactions. The above results confirmed that betaine could alleviate AD via Nrf2 which suggested that it has the potential to become a lead drug for the treatment of AD.

AD is a chronic neurodegenerative disorder that primarily affects the elderly, characterized by progressive cognitive and memory decline. It is the most common cause of dementia worldwide and has emerged as a significant global health concern (Nasb et al., 2024). The currently available clinically interventions for AD exhibit suboptimal. Therefore, it’s necessary to further investigate the specific pathogenesis of AD or to identify potential therapeutic targets for AD (Ahmad et al., 2024). The critical nutrient betaine, which plays a vital role in mammal health, has been the subject of numerous studies that provide compelling evidence of its significant health benefits (Wang et al., 2021). The glutamate-induced cell injury model of AD has been extensively employed for investigating the pathogenesis of AD. In this study, we have elucidated for the first time that betaine significantly mitigates neurotoxicity in glutamate-induced SH-SY5Y cells. SH-SY5Y cells stimulated by glutamate alone revealed significantly decreased cell survival rates and cellular morphology changes, which were efficiently reversed by betaine pretreatment (Fig. 1D&E). Therefore, the essential role of betaine in the beneficial effects observed in AD suggests its potential as a promising clinical therapeutic agent.

Ferroptosis is a distinctive form of regulated cell death, which is iron-dependent and mediated by lipid peroxidation (Dixon et al., 2012). Emerging research evidence strongly manifests that ferroptosis plays a pivotal role in the pathogenesis of neurodegenerative disorders, including AD. In fact, the biological characteristics observed in the brains of AD are similar to those associated with ferroptosis, such as the accumulation of lipid peroxide, depletion of GSH and GPX4, and dysregulated iron metabolism (Lewerenz et al., 2018; Lane et al., 2021; Masaldan et al., 2019). Clinical trial evidence supports the rationale for targeting ferroptosis in AD. A Phase II clinical trial conducted over a span of two years demonstrated that the iron chelator desferrioxamine effectively mitigated cognitive deficits in individuals with AD (Crapper McLachlan et al., 1991). Currently, the iron chelator deferiprone, which is brain-penetrant and orally active and is approved to treat β-thalassemia, is going through a Phase II clinical trial to evaluate its potential benefits in AD (Nikseresht et al., 2019). α-Lipoic acid, a naturally occurring enzyme cofactor, has been shown to improve cognitive function in patients with AD, potentially through its ability to suppress ferroptosis (Liu et al., 2021). Therefore, targeting ferroptosis represents an effective approach to mitigate the multifaceted aspects of AD. In our current study, SH-SY5Y cells exhibited characteristic ferroptosis changes following glutamate stimulation, manifested as elevated formation of MDA, accumulation of Fe²⁺, and release of LDH. Additionally, there was a decrease in MMP, reduced levels of GSH content, and downregulation of GPX4 expression. The observed alterations closely resemble those induced by the specific ferroptosis inducer erastin. Moreover, administration of betaine and the ferroptosis inhibitor Fer-1 effectively reversed all these changes, thereby confirming that betaine exerts a neuroprotective effect through inhibition of ferroptosis in AD.

The brains of patients with AD exhibit an abundance of polyunsaturated fatty acids, which significantly amplifies ROS signaling and leads to the production of the toxic derivative MDA, rendering them particularly susceptible to oxidative damage (Dei et al., 2002). Inhibition of oxidative stress has been recognized as a proactive approach for alleviating AD pathology (Anekonda & Reddy., 2005). The first-line defense mechanism is the most efficient and involves antioxidant enzymes such as SOD, CAT, and GPx, which play an indispensable role in dismutation superoxide radicals (O2•-) and hydrogen peroxide (H₂O₂) (Dammak et al., 2023). In this study, we verified that glutamate incubation was observed to induced the formation of ROS and decrease the enzyme activities of SOD, CAT and GPx, which were improved by betaine or Fer-1 treatment. The oxidative damage resulting from glutamate administration was abolished by betaine or Fer-1 treatment. The aforementioned observation highlights the pivotal role of betaine in protection against oxidative stress, owing to its capacity to induce anti-oxidative enzyme activity.

Nrf2, a redox-sensitive master regulatory transcription factor, plays a pivotal role in mediating the expression of endogenous antioxidant. Recent reports have highlighted that crucial and emerging involvement of Nrf2 in ferroptosis (Dodson et al., 2019). Under normal physiological conditions, Nrf2 is sequestered in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1) to maintain inactive, which facilitates its degradation. The Keap1-sensitive cysteine residues were targeted and attacked under stimulation, leading to the disruption of the Keap1-CUL3 ubiquitination system. As a result, Nrf2 translocates into the nucleus and binds with the ARE domain, initiating the transcription including the antioxidant genes SOD, CAT, and GPx, as well as ferroptosis-associated genes such as the ferroptosis inhibitor GPX4 (Nishizawa et al., 2022). Forsythoside A alleviates neurotoxicity in erastin-stimulated HT22 cells by inhibiting ferroptosis through activation of the Nrf2/GPX4 axis (Wang et al., 2022). Salidroside mitigates neuronal ferroptosis by activating the Nrf2/HO-1 signaling pathway in Aβ₁₋₄₂-administrated AD mice and glutamate-induced HT22 cells (Yang et al., 2022). Therefore, modulation of the Nrf2 pathway is a viable strategy to treat ferroptosis-driven neurodegeneration including AD. Moreover, Nrf2 activation is a potentially effective treatment strategy for AD. Indeed, the expression and translocation of nucleus Nrf2 were significantly reduced in the brains of AD patients compared to healthy elderly people (Ramsey et al., 2007). Previous studies have reported that Nrf2 activation can mitigate Aβ-induced neurological injury in nerve cells and ameliorate cognitive decline as well as AD-like pathological characteristics in AD transgenic animals (Yu et al., 2020; Jiang et al, 2023). Our study demonstrated that betaine activates the Nrf2 pathway in glutamate-induced SH-SY5Y cells, leading to its accumulation and translocation to the nucleus where it activates downstream signaling cascades involving SOD, CAT, GPx and GPX4. However, the protective effect of betaine on the expressions of these proteins was abolished by the Nrf2 inhibitor ML385, thereby indicating that Nrf2 is indeed plays a crucial role in contributing to the antioxidant and anti-ferroptosis effects of betaine. Moreover, molecular docking was employed to validate high-affinity binding between betaine and Nrf2.

The present study provides the first evidence demonstrating the neuroprotective effects of betaine in glutamate-induced SH-SY5Y cells. Mechanistically, betaine exerts its protective effects by promoting Nrf2 nuclear translocation to attenuate oxidative stress and inhibit ferroptosis. These findings highlight the potential of betaine as a promising therapeutic candidate for reducing neuronal ferroptosis in AD. Furthermore, future animal studies are warranted to fully elucidate the protective mechanisms and therapeutic potential of betaine in AD.

Ethical Statement

This work does not contain any studies with human participants or animals performed by any of the authors.

Conflict of Interests

The authors declare no conflict of interests.

Data Availability Declaration

Data not available due to restrictions.

Funding

This work was supported by National Natural Science Foundation of Guangdong Province (2021B1515140011 & 2022A1515010638 & 2022A1515140067), Project of Administration of Traditional Chinese Medicine of Guangdong Province (20231232).

Acknowledgements

This work was supported by National Natural Science Foundation of Guangdong Province, Project of Administration of Traditional Chinese Medicine of Guangdong Province.

Author Contribution

Guihua Wu and Fuwei Wang wrote the main manuscript text and prepared figures 1-6. All authors reviewed the manuscript.

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You are reading this latest preprint version

Betaine alleviates neuronal impairment in glutamate-injured SH-SY5Y neuroblastoma cells via Nrf2 signaling pathway related ferroptosis

Status:

Version 1

Abstract

Figures

Highlights

Introduction

Materials and Methods

Cell Culture

Determination of Cell Viability

Measurement of Malonaldehyde (MDA) Content

Detection of Reactive Oxygen Species (ROS) Formation

Determination of Antioxidant Enzyme Activities

Measurement of Intracellular Iron Level

Mitochondrial Membrane Potential (MMP)

Western Blot

Molecular Docking Analysis

Statistical Analysis

Results

Betaine exhibits neuroprotective effects in glutamate-induced SH-SY5Y cells

Betaine ameliorates glutamate-induced ferroptosis in SH-SY5Y cells

Betaine attenuates glutamate-induced oxidant stress in SH-SY5Y cells

Molecular docking

Discussion

Conclusion

Declarations

Author Contribution

References

Additional Declarations

Status:

Version 1