Protective effects of anthocyanins on the nervous system injury caused by fluoride-induced endoplasmic reticulum stress in rats.

Abstract

Highlights

Long-term fluoride exposure can produce neurotoxicity. Anthocyanins, as antioxidants, have a certain protective effect in nerve damage. This study aimed to investigate the protective role of anthocyanins in fluoride-induced neurological damage due to endoplasmic reticulum stress (ERS). Using a fluoride-exposed Wistar rat model, we assessed learning memory capacity and pathologic and ultrastructural injury. The level of oxidative stress (OS) in vivo was detected by colorimetric method, the level of ERS was analyzed by immunohistochemistry, and the apoptosis of neuronal cells was observed by TUNEL staining. The results showed that fluoride exposure could decrease the learning and memory ability in rats, and led to histopathological and ultrastructural damage in the hippocampal CA1, CA3 and cortical regions. Fluoride exposure-induced OS in vivo, which further activates ERS, which was manifested by increased levels of ERS-related proteins GRP78, Caspase 12, and Caspase 3 in hippocampal CA1, CA3, and cortical regions, and eventually led to a significant increase in neuronal apoptosis rate. Notably, after anthocyanins treatment, pathological and ultrastructural damage was restored, the level of OS and ERS were significantly restored, and the apoptosis rate of neuronal cells was significantly reduced. In summary, as nutritional interventions, anthocyanins exert a protective role in fluoride-induced neurological injury.

Introduction

Fluorine is closely related to health, but its effect on the human body is bidirectional. When fluoride intake exceeds the body’s safety limit, it will cause damage to bones, teeth, the nervous system, the immune system, the reproductive system, and the cardiovascular system^[1]. Nearly 200 million people are thought to be at risk from excessive levels of fluoride exposure in drinking water in nations including China, India, Sri Lanka, and many more^[2].

Fluoride has been shown to cross the blood-brain barrier and accumulate in neurons, causing neurotoxicity3, 4. Fluoride-induced neurotoxicity is mainly characterized by cognitive dysfunction and a reduced capacity for learning and memory. The prevalence of neurological disorders is strongly correlated with regional levels of fluoride exposure. In an ecological study of children aged 4–17 years in the United States found that the prevalence of childhood attention deficit hyperactivity disorder (ADHD) increased from 7.8% in 2003 to 11.0% in 2011, with a positive correlation between the prevalence of ADHD and the rate of state water fluoridation^[5]. A survey conducted in Alappuzha District, Kerala, India, found that 20% of children with dental fluorosis were rated as intellectually impaired, while no intellectual impaired was found in the control group, suggesting that fluorosis is associated with cognitive impairment in children ^[6]. In a prospective Canadian cohort, children aged 3 to 4 years had poorer IQ scores when exposed to higher levels of fluoride during pregnancy ^[7]. Several studies have found that high levels of fluoride exposure in drinking water are associated with reduced IQ and cognitive levels in children8, 9.Thus the adverse effects of fluoride on the nervous system have attracted a great deal of attention. Fluoride-induced neurotoxicity has far-reaching public health implications, and chronic fluoride exposure negative effects both learning ability and neurodevelopment in children. Previous studies have identified high water fluoride exposure as a risk factor for cognitive dysfunction in older adults, with higher risks observed in areas with highly fluoridated water^[10]. While the mechanisms underlying fluoride-induced neurotoxicity have been extensively documented, effective and universally applicable interventions remain limited. Thus, identifying suitable therapeutic strategies based on a deeper understanding of its mechanistic pathways is crucial for public health.

In vivo experiments revealed that 50 mg/L and 100 mg/L NaF were able to interfere with neurogenesis through Notch1 signaling, resulting in reduced hippocampal neurogenesis, lower dendritic spine density, inhibition of synapse growth, and a significant decrease in spatial learning and memory abilities in the rat^[8]. Chronic fluoride exposure also promotes GPX4 degradation via mtROS chaperone-mediated autophagy, leading to hippocampal neuronal iron death and cognitive deficits^[11]. Fluoride exposure during pregnancy alters the expression of key proteins of the ERK/CREB signaling pathway in the hippocampus and cerebral cortex of rat offspring, affecting spatial learning and memory abilities and leading to neurodevelopmental impairments^[12]. All of these studies have found that fluoride is capable of causing damage to the nervous system. However, the underlying mechanisms have not been clarified.

Research has demonstrated that endoplasmic reticulum stress (ERS) can impact the function of the nervous system and is associated with various central nervous system disorders^[13]. The endoplasmic reticulum is the primary intracellular site responsible for protein synthesis, lipid generation, and regulation of cellular stress responses, playing a crucial role in the folding, processing, and trafficking of diverse proteins. Disruption by certain biochemical and physiological stimuli may lead to the accumulation of unfolded or misfolded proteins within the endoplasmic reticulum, followed by excessive production of reactive oxygen species (ROS), triggering oxidative stress (OS) and ultimately the unfolded protein response (UPR), which results in ERS14, 15, 16. The accumulation of unfolded proteins and ROS in the endoplasmic reticulum lumen can activate downstream UPR signaling pathways, thereby initiating the ERS-mediated apoptotic pathway and ultimately leading to cell death17, 18. Whether fluoride can induce ERS and thereby cause neurotoxicity has not been clearly reported in the literature, and identifying drugs and therapeutic approaches to ameliorate and alleviate fluoride-induced neurotoxicity remains an important challenge.

Research has found that antioxidants possess a protective effect on the nervous system^[19]. Anthocyanidins are a class of natural, water-soluble flavonoid pigments found in plants^[20] and have been demonstrated to possess physiological activities such as free radical scavenging and antioxidant properties^[21]. The neuroprotective effects of anthocyanins are primarily attributed to their potent antioxidant and free radical scavenging properties. Studies show that anthocyanins protect brain tissues from fluoride-induced damage by mitigating oxidative stress, reducing malondialdehyde (MDA) levels, and enhancing superoxide dismutase (SOD) activity. These mechanisms collectively contribute to the attenuation of histopathological alterations and the suppression of neuronal apoptosis^[22]. Furthermore, anthocyanins modulate inflammatory pathways, significantly reducing neuroinflammation in BV-2 microglia by downregulating key mediators like Cyclooxygenase-2, p38-MAPK, and NF-?B^[23]. Anthocyanins also reduce ischemia/reperfusion-induced neuronal apoptosis by modulating Bcl-2 family proteins, reducing cytochrome c and caspase 3, and inhibiting the JNK/p53 pathway, thereby providing neuroprotection^[24]. Furthermore, anthocyanidins have been shown to ameliorate Alzheimer’s disease, aging, amyloid ?, and improve learning and memory functions, thus exhibiting a degree of neuroprotective effects25, 26, 27.

Based on the above research background, we hypothesized that anthocyanins may play a protective role in the neurological injury caused by fluoride-induced ERS. This study will investigate the relationship between ERS and fluoride-induced nervous system injury in an animal model, as well as confirm the neuroprotective effects of anthocyanidins against fluoride-induced nervous system damage. This will provide new evidence for the neurotoxicity of fluoride compounds and offer new approaches for the prevention and treatment of fluorosis.