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Effects of Fluorine on Neutrophil Extracellular Trap Formation through Regulating AMPK/p38 Signaling Pathway.Abstract
Fluorine is an important trace element that is widely dispersed, and studies showed that fluorine could cause severe toxicity to fish. The aim of this study was to investigate the effects of fluorine on neutrophil extracellular trap (NET) formation in common carp and clarify the possible mechanism. The neutrophils were isolated and exposed to 0.25, 0.5, or 1?mM sodium fluoride (NaF). The results showed that NaF could induce the formation of NETs which exhibited a DNA-based network structure modified with histones and myeloperoxidase (MPO). Furthermore, NaF led to the production of reactive oxygen species (ROS) in neutrophils. Western blot results showed that NaF significantly increased the phosphorylation of AMPK and p38. In addition, our results showed that NaF-induced NET formation could be inhibited by an AMPK or p38 inhibitor. In conclusion, our results showed that NaF induced NET formation in neutrophils through regulation of the AMPK/p38 signaling pathway.
Excerpt:
1. Introduction
Fluoride is one of the essential microelements in the body. Long-term excessive intake of fluoride will cause systemic physiological and pathological changes [1]. Fluoride can not only damage bone organs but also accumulate in brain tissue through the blood-brain barrier, affecting the shape and function of brain nerve cells [2, 3]. In recent years, fluoride pollution is serious due to the development of industry. Meanwhile, increased fluorine in water will cause a certain toxic effect on aquatic animals [4]. Fish can absorb fluorine directly in water, which is an aquatic organism vulnerable to fluorine pollution in the water [5]. A previous study showed that sodium fluorine could affect the injury of brain tissues and behaviour of zebrafish in vivo [6]. Also, fluorine could cause thyroid endocrine disruption in male zebrafish [7]. Furthermore, fluorine could induce the injury of the gills of Cyprinus carpio [8]. In addition, fluorine has been reported to induce head kidney macrophage cell apoptosis in vitro [9].
Neutrophils are the most abundant white blood cells in the peripheral blood. It plays an important role in the innate immune system [10]. It is the first line in defending against the invasion of pathogens. In recent years, neutrophils have been found to have a new mechanism in resisting microbial invasion: the formation of neutrophil extracellular traps (NETs) [11]. NETs play an important role in pathogen infection, and its huge network structure can adhere, restrict, and kill pathogenic microorganisms [12]. However, the release of NETs is a double-edged sword, which not only destroys pathogenic microorganisms but also damages the organism [13]. Studies showed that NETs were involved in the pathological process of many diseases, such as lung injury, acute pancreatitis, inflammatory bowel disease, and arthritis [14–17]. Meanwhile, recent studies demonstrated that sodium arsenic, cadmium chloride, or nanosilver could induce the formation of NETs [18–20]. However, whether fluorine could induce the formation of NETs has not been reported. In the present study, the effects of fluorine on NET formation and its possible mechanism were investigated.
4. Discussion
NETs are a double-edged sword. Excessive production of NETs could lead to tissue injury. Experimental evidence suggests that NETs participate in the pathogenesis of autoimmune and inflammatory disorders. Recently, NETs have been reported to be involved in the pathological process of toxicant-induced injury. The production of NETs could aggravate poison-induced damage. Fluorine, an important environmental toxicant, is widely dispersed in the aquatic environment. However, whether fluorine could induce NET formation has not been reported. In the present study, we investigated the new effects of NaF on the NET formation of common carp neutrophils. Our results suggested that NaF-induced NET formation was through regulating the AMPK/p38 signaling pathway. These results can enrich the toxicological effect of fluoride.
In recent years, more and more reports showed that NETs could not only eliminate pathogenic microorganisms but also cause tissue damage and participate in the development of many diseases [13]. Furthermore, recent studies demonstrated that NETs were involved in the pathological process of toxicant-induced injury [20]. A previous study showed that di(2-ethylhexyl)phthalate (DEHP) could induce the formation of NETs in vitro [23]. Also, it has been reported that cadmium chloride could induce the formation of NETs, and inhibition NET formation had protective effects against cadmium chloride-induced lung injury in mice [19]. However, the effects of NaF on NET formation of common carp neutrophils have not been reported. In this study, our results showed that NaF could induce the formation of NETs in a concentration-dependent manner.
It has been reported that ROS and PAD4 are involved in the formation of NETs [24]. The formation of NETs is closely related to ROS, which is the product of NADPH oxidase activation [25]. Previous studies have shown that pretreatment with DPI, a NADPH oxidase inhibitor, significantly inhibited the formation of NETs [26]. ROS can activate the mitogen-activated protein kinase (MAPK) signal pathway and its downstream p38, thus promoting the production of NETs [27]. And a previous study showed that inhibition of p38 phosphorylation could prevent NET formation [22]. In this study, we found that NaF exposure significantly increased the production of ROS. Meanwhile, NaF could activate AMPK and p38 signaling pathways. To further clarify the formation mechanism of NETs, AMPK and p38 inhibitors were added to the cells. The results showed that NaF-induced NET formation was inhibited by these inhibitors, suggesting that NaF-induced NET formation was dependent on AMPK and p38 signaling pathways.
Taken together, the results of this study demonstrated that NaF significantly increased the release of NETs of neutrophils. The mechanism was through regulating the AMPK/p38 signaling pathway. The results of the present study could enrich the toxicity effects of fluorine on the immune system of the common carp.
*The full-text study is online at https://www.hindawi.com/journals/omcl/2021/6693921/