Fluoride-induced male reproductive toxicity: Dual-tissue pyroptosis in the gut-testis axis mediated by inflammatory cytokines.

Fluorine predominantly occurs as compounds in nature, and adequate intake of fluoride is indispensable for sustaining human health. However, long-term excessive fluoride intake can result in damage to multiple tissues and organs [1], [2]. Numerous studies have demonstrated that fluoride exerts non-negligible reproductive toxicity, especially on the male reproductive system [3], [4], [5], [6]. It can penetrate the blood-testis barrier (BTB), impairing the histological structure and male reproductive function [7]. Although extensive researches have been conducted on the male reproductive toxicity of fluoride [8], [9], [10], its underlying molecular mechanisms remain incompletely elucidated.

Existing research has investigated oxidative stress, endoplasmic reticulum stress, inflammatory pathways, and epigenetic modifications as primary mechanisms mediating fluoride-induced reproductive impairment. Fluoride exposure increases reactive oxygen species (ROS) production, decreases the activities of antioxidant enzymes in testicular tissue, and elevates malondialdehyde (MDA) levels, leading to sperm membrane lipid peroxidation and spermatogenic dysfunction [11]. At the cellular level, fluoride induces apoptosis in testicular germ cells through the mitochondrial pathway (cytochrome c release, caspase-3 activation) and the death receptor pathway (Fas/FasL, caspase-8) [12]. These studies mainly focused on the overall direct impact of fluoride on the reproductive system, while the indirect reproductive damage caused by systemic biological effects after fluoride exposure has not received adequate attention. It is widely recognized that ingestion via the digestive tract is the exposure route for drinking water-derived fluorosis. As an extrinsic substance, fluoride can trigger intestinal microbiota dysbiosis and intestinal wall disruption [13], [14]. Subsequently, the function of distal tissues might be affected by inflammation, immunity, and metabolite-mediated pathways. The regulatory network through which gut microbiota influences male reproductive health via metabolites, immunity, and inflammation is termed the gut-testis axis. Nevertheless, the potential role of the gut-testis axis in fluoride-induced male reproductive impairment has not been elucidated.

Inflammatory factors secreted by the intestine may be important mediators of reproductive damage caused by fluoride through the gut-testis axis. A study [15] has shown that excessive fluoride intake led to significant alterations of gut microbiota and aggravated colitis in rats. Similarly, another animal experimental evidence has shown that fluoride induces the destruction of intestinal structure and increased permeability in mice. These alterations subsequently trigger the release of pro-inflammatory cytokines and initiate intestinal pyroptosis [16]. Furthermore, fluoride has been shown to induce pyroptosis in testicular tissue through upregulation of IL-17A expression and activation of the caspase-1/11-dependent pathway, ultimately impairing spermatogenesis [17]. Additionally, upregulated nuclear factor-_kB (NF-_kB) expression promotes testicular inflammation and triggers testicular ferroptosis [18]. Collectively, these observations highlight that inflammation plays a pivotal role in intestinal and testicular tissue damage, and may serve as the key mediator linking fluoride exposure to male reproductive impairment via the gut-testis axis.

Based on the above research background and hypothesis, this study established in vivo models involving sodium fluoride (NaF)-treated and fecal microbiota transplanted (FMT) rats, combined with an indirect co-culture of human colorectal adenocarcinoma cells (Caco-2) and immortalized human Sertoli cells (IHSCs) in vitro. By integrating in vivo and in vitro experimental approaches, this study aimed to systematically explore the regulatory role of the gut-testis axis in fluoride-induced male reproductive impairment, with a specific focus on identifying and validating the core underlying mechanism. Ultimately, these findings may provide a scientific reference for the prevention and control of fluoride-induced male reproductive damage.