Sodium fluoride promotes myopia progression

To investigate the effect of sodium fluoride on myopia, we divided 4-week-old mice equally into three groups, namely the control group, saline-treated group, and sodium fluoride-treated group. Before initiating the experiment, we measured the axial length and refraction of the mice and excluded those with significant variations. Subsequently, we administered sodium fluoride solution at a concentration of 80 mg/ml onto the ocular surface of the mice, 10 μl each time, once every 8 h for 4 weeks, with the same procedure applied to the saline-treated group. The experimental protocol is detailed in Fig. 1A. After 4 weeks, the refraction of mice in the sodium fluoride-treated group shifted in the direction of myopia, and the axial length increased compared to the control group, indicating that sodium fluoride promotes myopia progression (Figs. 1B and C).

Fig. 1

Sodium fluoride promotes myopia progression. A The schematic diagram of the model of sodium fluoride-induced myopia; B Detection of refraction in mice after treatment with saline or sodium fluoride; C Examination of axial length in mice after treatment with saline or sodium fluoride. Datas were presented as the mean ± SD of ten replicates in (B and C). P values were determined using one-way ANOVA with Bonferroni post hoc testing. (*P < 0.05, **P < 0.01, ***P < 0.001)

Sodium fluoride activates the ferroptosis pathway

To further investigate the molecular mechanism of sodium fluoride in the promotion of myopia, we collected retinal tissues from the saline-treated group and sodium fluoride-treated group for proteomic analysis. As illustrated in Fig. 2A, differentially expressed proteins were identified using stringent screening criteria (adjusted p-value < 0.05 and |log2(fold change)|> 2), revealing 181 significantly up-regulated and 496 down-regulated candidates. These proteins were subsequently subjected to functional annotation through Gene Ontology (GO) and pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Notably, we found that a large number of differential proteins were enriched in the ferroptosis pathway, suggesting that sodium fluoride may modulate the ferroptosis pathway (Figs. 2B-C). To elucidate the potential involvement of ferroptosis in sodium fluoride-mediated myopia pathogenesis, we conducted a series of mechanistic investigations to assess pathway activation and its functional consequences. We utilized a ferroptosis assay kit to detect biochemical indicators in mouse tissues and Western blotting to assess ferroptosis-related proteins. Glutathione (GSH) is the most abundant intracellular antioxidant, and its levels are negatively correlated with ferroptosis. Malondialdehyde (MDA), a well-established biomarker of lipid peroxidation, exhibited a significant positive correlation with ferroptotic activity, further supporting the involvement of oxidative lipid damage in this form of regulated cell death. Numerous studies have shown that GPX4 expression is down-regulated and COX2 expression is up-regulated in cells or tissues where ferroptosis occurs. As shown in Figs. 2D-F and S1B-C the sodium fluoride-treated group, compared to the saline-treated group, showed a decrease in GSH content, an increase in MDA content, a reduction in GPX4 expression, and an increase in COX2 expression, indicating that the ferroptosis pathway was activated. Concurrently, we conducted comprehensive assessments of cellular activity and ROS levels in 661W cells following exposure to varying concentrations of sodium fluoride, as presented in Figs. S4A-C. Quantitative analysis demonstrated a concentration-dependent relationship, wherein sodium fluoride significantly enhanced intracellular ROS accumulation while concurrently suppressing cellular viability. In summary, sodium fluoride activates the ferroptosis pathway in the process of promoting myopia.

Fig. 2

Sodium fluoride activates the ferroptosis pathway. A The volcanic map of differential proteins; B The GO analysis of differential proteins; C The KEGG analysis of differential proteins; D Detection of the GSH levels in the retina of mice after treatment with saline or sodium fluoride; E Detection of the MDA levels in the retina of mice after treatment with saline or sodium fluoride; F Western blotting analysis of COX2 and GPX4 in the retina of mice after treatment with saline or sodium fluoride. Datas were presented as the mean ± SD of three replicates in (D-F). P values were determined using one-way ANOVA with Bonferroni post hoc testing. (*P < 0.05, **P < 0.01, ***P < 0.001)

Sodium fluoride promotes PIEZO1 expression in vivo and in vitro

Protein enrichment analysis within the ferroptosis pathway revealed that PIEZO1 is significantly implicated in sodium fluoride-induced ferroptosis, suggesting a potential regulatory role in this process (Fig. 3A). This is because PIEZO1 is strongly associated with ferroptosis (Hirata 2023) and has been shown to promote myopia progression in guinea pigs (Zhong et al. 2023). We quantitatively evaluated PIEZO1 expression dynamics post-sodium fluoride exposure, employing a multimodal analytical approach to assess both transcriptional and translational profiles. As depicted in Figs. 3B, D and S1A, quantitative analyses demonstrated significant upregulation of PIEZO1 expression in sodium fluoride-exposed samples, with concomitant elevation in both mRNA and protein levels relative to untreated controls. Meanwhile, we treated 661W cells with varying concentrations of sodium fluoride and collected their RNA and protein to assess PIEZO1 expression. As depicted in Figs. 3C and E, the mRNA and protein expression levels of PIEZO1 increased in a dose-dependent manner. These data suggest that sodium fluoride promotes PIEZO1 expression.

Fig. 3

Sodium fluoride promotes PIEZO1 expression in vivo and in vitro. A The visual presentation of genes enriched into ferroptosis pathway; B The detection of Piezo1 mRNA expression in mice after saline or sodium fluoride treatment; C The detection of Piezo1 mRNA expression in 661W cells after saline or sodium fluoride treatment; D The expression of PIEZO1 in mice after saline or sodium fluoride treatment; E The expression of PIEZO1 in 661W cells after saline or sodium fluoride treatment; Datas were presented as the mean ± SD of three replicates in (B-E). P values were determined using one-way ANOVA with Bonferroni post hoc testing. (*P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001)

Sodium fluoride promotes myopia progression by activating ferroptosis via PIEZO1

To further investigate the function of PIEZO1 in myopia, we injected the PIEZO1 inhibitor (GsMTx4) into the vitreous cavity of mice, employed a morphologic deprivation model of myopia, and subsequently examined the refraction and axial length of the mice. As shown in Fig. S2B, GsMTx4 inhibited PIEZO1 expression compared to the control group. As depicted in Figs. S2C and S2D, compared to the saline group, the refraction of mice in the GsMTx4 group shifted towards orthokeratology, and axial length growth was delayed. Conversely, pharmacological activation of PIEZO1 using Yoda1 significantly exacerbated myopia progression, as evidenced by the experimental data presented in Fig. S3. These collective findings strongly suggest that PIEZO1 plays a promotive role in myopia pathogenesis. Next, we investigated whether sodium fluoride activates the ferroptosis pathway via PIEZO1, thereby promoting myopia progression. We treated mice in the saline and sodium fluoride groups with either saline or the PIEZO1 inhibitor (GsMTx4) and measured ferroptosis markers, including glutathione (GSH) and malondialdehyde (MDA). As shown in Figs. 4A-C, compared to the control, retinal tissues from GsMTx4-treated mice demonstrated decreased expression of PIEZO1 and COX2, increased expression of GPX4, elevated GSH content, and reduced MDA content. These findings suggest that sodium fluoride activates the ferroptosis pathway through PIEZO1. Subsequently, we examined the refraction and axial length of mice in the four groups: Saline/Saline, Sodium fluoride/Saline, Saline/GsMTx4, and Sodium fluoride/GsMTx4. As shown in Figs. 5B and C, the shift in refraction towards orthokeratology and delayed axial length growth in the Sodium fluoride/GsMTx4 group compared to the Sodium fluoride/Saline group demonstrated that sodium fluoride can influence myopia progression via PIEZO1. In conclusion, sodium fluoride promotes myopia progression by activating ferroptosis via PIEZO1.

Fig. 4

Sodium fluoride activates the ferroptosis pathway via PIEZO1. A Western blotting analysis of PIEZO1, COX2 and GPX4 in the retina of mice after different groups of treatments (saline/saline; sodium fluoride/saline; saline/GsMTx4; sodium fluoride/ GsMTx4); B Detection of the GSH levels in the retina of mice after different groups of treatments (saline/saline; sodium fluoride/saline; saline/GsMTx4; sodium fluoride/ GsMTx4); C Detection of the MDA levels in the retina of mice after different groups of treatments (saline/saline; sodium fluoride/saline; saline/GsMTx4; sodium fluoride/ GsMTx4); Datas were presented as the mean ± SD of three replicates in (A-B). P values were determined using one-way ANOVA with Bonferroni post hoc testing. (*P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001)

Fig. 5

Sodium fluoride promotes myopia progression by activating ferroptosis. A The schematic diagram of the model of sodium fluoride-induced myopia; B Detection of refraction in mice after treatment with sodium fluoride or GsMTx4; C Examination of axial length in mice after treatment with sodium fluoride or GsMTx4. Datas were presented as the mean ± SD of ten replicates in (B and C). P values were determined using one-way ANOVA with Bonferroni post hoc testing. (PIEZO1 inhibitor: GsMTx4, *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001)

PIEZO1 is a drug target of baicalin

To pharmacologically investigate whether sodium fluoride affects the development of myopia through PIEZO1, we reviewed extensive literature and found that baicalin can affect the ferroptosis pathway (Wen et al. 2023). However, the relationship between baicalin and PIEZO1, as well as its role in myopia, remains unknown. Therefore, we hypothesized that PIEZO1 is the drug target of baicalin. To test this hypothesis, we conducted molecular docking modeling and cellular experiments to explore whether PIEZO1 is the target of baicalin. As shown in Fig. 6A, the molecular docking model indicated that PIEZO1 can bind to baicalin. Then, we found that the degradation rate of PIEZO1 protein was reduced in the baicalin group compared to the saline group, as determined by CETSA assay (Fig. 6B), indicating that baicalin targets PIEZO1. Finally, we treated mice and 661W cells with baicalin and, compared to the control group, observed reduced mRNA and protein expression levels of PIEZO1 both in vivo and in vitro (Figs. 6C-F). These data suggest that PIEZO1 is a potential target of baicalin and that baicalin inhibits PIEZO1 expression both in vivo and in vitro.

Fig. 6

PIEZO1 is a drug target of Baicalin. A The molecular docking simulation of Baicalin binding to PIEZO1; B The detection of PIEZO1 expression in 661W cells after treatment with Saline or Baicalin at different temperature; C The detection of PIEZO1 expression in 661W cells after Saline or Baicalin treatment; D The detection of PIEZO1 expression in mice after Saline or Baicalin treatment; E The detection of Piezo1 mRNA expression in 661W cells after Saline or Baicalin treatment; F The detection of Piezo1 mRNA expression in mice after Saline or Baicalin treatment. Datas were presented as the mean ± SD of three replicates in (B-F). P values were determined using one-way ANOVA with Bonferroni post hoc testing. (*P < 0.05, **P < 0.01, ***P < 0.001)

Baicalin inhibits myopia progression through PIEZO1

Finally, we administered saline and baicalin treatments to the mouse morphologic deprivation myopia model, respectively, and examined the refraction and axial length of the mice to investigate whether baicalin could delay myopia progression. As shown in Figs. S5B and S5C, the refraction shifted towards orthokeratology and the ocular axial length decreased in the baicalin group compared to the control group, suggesting that baicalin could delay myopia progression. We conducted a comparative analysis of myopia-related parameters between the GsMTx4-treated and Baicalin-treated groups, as illustrated in Fig. S6. Quantitative assessment revealed that the Baicalin treatment group demonstrated superior efficacy in myopia suppression compared to the GsMTx4 treatment group. Meanwhile, we administered sodium fluoride and saline drops to the ocular surface of the mice, followed by gavage administration of baicalin and saline, respectively. As shown in Fig. 7B, baicalin attenuated the sodium fluoride-induced upregulation of PIEZO1 compared to the control group. Finally, we examined the myopic parameters of mice in the different treatment groups described above. As shown in Fig. 7C and D, baicalin attenuated sodium fluoride-induced myopia progression compared to the control group. In summary, baicalin inhibited sodium fluoride-induced myopia via PIEZO1.

Fig. 7

Baicalin inhibits myopia progression through PIEZO1. A The schematic diagram of sodium fluoride-induced myopia model; B Detection of PIEZO1 expression after different groups of treatments (saline/saline; sodium fluoride/saline; saline/baicalin; sodium fluoride/ baicalin); C Detection of refraction in mice after different groups of treatments (saline/saline; sodium fluoride/saline; saline/baicalin; sodium fluoride/ baicalin) D Examination of axial length in mice after different groups of treatments (saline/saline; sodium fluoride/saline; saline/baicalin; sodium fluoride/ baicalin). Datas were presented as the mean ± SD of three replicates in (B) and ten replicates in (C and D). P values were determined using one-way ANOVA with Bonferroni post hoc testing. (*P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001)