Novel mechanism of fluoride induced cardiovascular system injury by regulating p53/miR200c-3p during endothelial dysfunction.

Fluorosis results from excessive fluoride exposure in specific geographical areas, primarily through chronic intake via water, food or air, which could adversely affect multiple bodily systems, including the skeletal, liver, nervous system and cardiovascular systems [1], [2], [3], [4]. Drinking water is the most prevalent source of fluoride, with over 180 million people worldwide consuming water containing elevated fluoride levels. This issue is particularly widespread in regions such as western North America, eastern Brazil, and various parts of Africa and Asia. China is notably one of the countries most severely impacted by endemic fluorosis related to drinking water, making it a significant public health concern ^[5]. Epidemiological studies have identified fluoride as a critical risk factor for cardiovascular diseases (CVD), whose underlying mechanisms remain poorly understood, and effective therapeutic options are lacking ^[6]. Consequently, this study aims to investigate the potential mechanisms linking fluoride exposure to CVD, and explore intervention targets. Based on this study, we hope to provide a scientific foundation for the prevention and treatment of endemic fluorosis.

Fluoride is recognized as an independent risk factor for CVD, including conditions such as hypertension and atherosclerosis [7], [8]. Epidemiological studies have shown that consuming water with high fluoride levels significantly increases the incidence of primary hypertension, displaying a positive correlation with elevated blood pressure. Yousefi et al. reported a higher prevalence of hypertension among residents who drank fluoride-rich water ^[9]. In addition, the risk of primary hypertension increasing in endemic fluorosis areas of China as water fluoride concentrations rose ^[10]. In addition, populations exposed to industrial fluoride exhibited a markedly higher proportion of atherosclerosis compared to control groups ^[11]. In adults residing in endemic fluorosis regions, the prevalence of carotid atherosclerosis was positively associated with the intake of high-fluoride water ^[12]. Endothelial cells (ECs), which form the vascular endothelium, are essential in maintaining the functional integrity of the cardiovascular system ^[13]. Recent studies have demonstrated that fluoride exposure can lead to ECs injury. NaF has been shown to inhibit its target gene, Fap-1, through the upregulation of miR-200c-3p, resulting in structural damage to ECs ^[14]. Additionally, fluoride has been found to disrupt endothelial amino acid metabolism, indicated that it exerts direct endothelial toxicity ^[15]. Despite these findings, much of the research to date has primarily focused on the structural and metabolic damage caused by fluoride to ECs. However, little attention has been given to its effects on endothelial cell function, which plays a crucial role in the early diagnosis of CVD. Vascular endothelial cells (VECs) secrete various bioactive substances involved in functions such as vasodilation, adhesion, and anticoagulation. Nitric oxide (NO), a vasodilator produced by endothelial nitric oxide synthase (eNOS), is crucial for regulating vascular dilation, while Angiotensin Converting Enzyme (ACE) and Endothelin-1 (ET-1) help maintain vascular tone. Furthermore, Inter Cellular Adhesion Molecule-1 (ICAM-1), Vascular Cell Adhesion Molecule-1 (VCAM-1), and von Willebrand Factor (vWF) are indicators of endothelial antiadhesion and anticoagulation activity ^[16]. Evaluating the impact of fluoride on the levels of these endothelial-derived bioactive substances can provide valuable insights into the broader cardiovascular effects of fluoride exposure.

Among the proposed mechanisms for systemic injuries caused by fluorosis, oxidative stress stands out as the most comprehensive and reasonable explanation for the multisystem injury linked to fluoride exposure. Fluoride, being chemically reactive, enhances the production of Reactive Oxygen Species (ROS) within the body, triggering oxidative stress ^[17]. At present, the occurrence of oxidative stress is evaluated by detecting the changes of Malondialdehyde (MDA), Super Oxide Dismutase (SOD), Total Antioxidant Capacity (T-AOC) and Glutathione Peroxidase 4 (GPx4). Research has demonstrated that excessive ROS production is a key factor in ECs death, contributing to the disruption of the vascular barrier, vasoconstriction, and endothelial dysfunction—all of which are closely associated with the progression of CVD ^[18]. The tumor suppressor p53 is a redox-sensitive transcription factor that mediates the effects of oxidative stress on the expression of downstream target genes. Under oxidative stress conditions, p53 expression is upregulated, largely through the accelerated translation of p53 mRNA ^[19]. Notably, in animal models of fluorosis, p53 expression in liver and kidney cells was significantly higher than that in control groups [3], [20]. However, the role of p53 in fluoride-induced endothelial injury has yet to be fully investigated. p53 could bind to promoter regions of various genes, including miR-200c-3p, thereby influencing their transcriptional activity ^[21]. It was found that ROS can increase the expression of miR-200, and miR-200c-3p belongs to the miR-200 family ^[22]. At present, most of the studies on miR-200 focus on the field of cancer, and there are few studies on its role in CVD. As CVD is a disease closely related to age, the study found that aging can lead to increased expression of miR-200c in the femoral artery of mice [23], [24]. It has also been found that miR-200 can promote arterial inflammation and inhibit angiogenesis, which may be related to vascular complications of diabetes [25], [26], [27]. At the same time, miR-200c can directly target the 3 ‘UTR region that binds eNOS, reduce NO synthesis, and induce apoptosis of endothelial cells [24], [25]. Our previous study found that the expression of miR-200c-3p in the serum of patients with skeletal fluoride was significantly increased ^[28]. Therefore, combined with the above research evidence, we speculated that miR-200c-3p may play an important role in fluoride-induced CVD. Furthermore, in a model of fluoride-induced ECs injury, miR-200c-3p was markedly overexpressed and played a role in inducing endothelial apoptosis ^[14]. Nevertheless, whether fluoride contributes to endothelial dysfunction through the p53/miR-200c-3p axis remains a subject for further investigation.

Based on the above facts, this study first conducted a comprehensively analyzed the basic situations, blood samples, blood pressure and other relevant data of residents in the fluoride-exposed areas, and further pointed out that fluoride-exposed was an independent risk factor for hypertension, and fluoride could cause oxidative stress in the body. Subsequently, in vivo experiments were conducted by grouping fluoride-infected wistar rats and in vitro ECs experiments to further confirm whether fluoride caused endothelial dysfunction and oxidative stress. Next, in vitro cell experiments were conducted to investigate whether p53/miR-200c-3p affected the damage effect of fluoride on ECs function. We investigated for the first time that fluoride could generate oxidative stress and activation of p53/miR-200c-3p lead to ECs dysfunction. These findings will provide theoretical research for the formulation of endemic fluorosis control strategies.