Effect of deferoxamine and ferrostatin-1 on salivary gland dysfunction in ovariectomized rats

Abstract

The mechanism underlying xerostomia after menopause has not yet been fully elucidated. This study aimed to investigate the mechanism of xerostomia and the effect of the ferroptosis inhibitors deferoxamine (DFO) and ferrostatin-1 (FER) on salivary gland dysfunction in a postmenopausal animal model. Twenty-four female Sprague-Dawley rats were randomly divided into four groups: a SHAM group (n = 6, sham-operated rats), an OVX group (n = 6, ovariectomized rats), an FER group (n = 6, ovariectomized rats injected intraperitoneally with FER), and a DFO group (n = 6, ovariectomized rats injected intraperitoneally with DFO). GPX4 activity, iron accumulation, lipid peroxidation, inflammation, fibrosis, and salivary gland function were analyzed. Recovery of GPX4 activity and a decrease in iron accumulation and cytosolic MDA + HAE were observed in the DFO group. In addition, collagen I, collagen III, TGF-β, IL-6, TNF-α, and TGF-β levels were decreased in the DFO group compared to the OVX group. Recovery of GPX4 activity and the morphology of mitochondria, and reduction of cytosolic MDA + HAE were also observed in the FER group. In addition, decreased expression of inflammatory cytokines and fibrosis markers and increased expression of AQP5 were observed in both the DFO and FER groups. Postmenopausal salivary gland dysfunction is associated with ferroptosis, and DFO and FER may reverse the postmenopausal salivary gland dysfunction after menopause. DFO and FER are hence considered promising treatments for postmenopausal xerostomia.

Keywords: deferoxamine; ferroptosis; ferrostatin-1; menopause; xerostomia.

Figure 1

Food intake, body weight, and sex hormone change. Food intake (A) and body weight (B) in each of the four groups. Food intake and body weight increased in the OVX group compared with the SHAM group, but there was no difference in the DFO and FER groups compared with the OVX group. (C) The level of serum estradiol concentration in each of the four groups. The serum estradiol levels were lower in the OVX group than in the SHAM group. Neither DFO nor FER treatment affected the serum estradiol level. (D) Quantitative real-time PCR analysis of ErβI and ErβII gene expression in the submandibular gland. The expression of ErβI and ErβII did not differ between the groups. Two-way ANOVA was performed. ^***p < 0.001 vs. SHAM group. Abbreviations: NS: not significant; SHAM: sham surgery group; OVX: ovariectomized group; DFO: deferoxamine injection group; FER: ferrostatin-1 injection group; ER: estrogen receptor.

Figure 2

Lipid deposition in the submandibular gland. (A) Lipid deposition in the submandibular gland (H&E staining, 40×). Yellow circles indicate lipid vacuoles. The lipid distribution increased in the OVX group and decreased in the FER group, but there was no significant difference in the DFO group. (B) Morphometric analysis of lipid vacuoles in the SHAM, OVX, DFO, and FER groups. Lipid vacuoles were significantly increased in the OVX group than in the SHAM group. The FER group exhibited decreased lipid vacuoles compared with the OVX group. Two-way ANOVA was performed. ^***p < 0.001 vs. SHAM group, ^#p < 0.05, vs. OVX group. Abbreviations: NS: not significant; SHAM: sham surgery group; OVX: ovariectomized group; DFO: deferoxamine injection group; FER: ferrostatin-1 injection group.

Figure 3

Inhibitory effect on ferroptosis. Cytosolic MDA (A) and cytosolic MDA + HNE (B) concentrations in the submandibular gland tissue. Compared to the SHAM group, the cytosolic MDA and MDA + HAE concentrations in the submandibular gland increased in the OVX group (p < 0.001) and decreased in the DFO group (MDA + HAE; p < 0.01 and MDA; p < 0.05) and FER group (MDA + HAE; p < 0.001 and MDA; p < 0.01) (Figure 3A and 3B). (C) GPX4 activity. GPX4 activity was reduced in the OVX group (p < 0.001), while the DFO (p < 0.05) and FER (P < 0.01) groups exhibited increased GPX4 activity. (D) Cytosolic iron content. Compared to the SHAM group, the cytosolic iron level increased in the OVX group and significantly decreased in the DFO group. (E) Electron microscopy images of mitochondria in the submandibular gland. The yellow arrow for indicating mitochondrial swelling, and blue arrow for indicating mitochondrial degeneration In the SHAM group, there was clear condensation of mitochondria and the shape was normal, but in the OVX group, the shape was irregular and the integrity was decreased. In the DFO and FER groups, mitochondrial recovery could be confirmed morphologically. Two-way ANOVA was performed. ^**p < 0.01, ^***p < 0.001 vs. SHAM group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. OVX group. Abbreviations: NS: not significant; SHAM: sham surgery group; OVX: ovariectomized group; DFO: deferoxamine injection group; FER: ferrostatin-1 injection group; MDA: malondialdehyde; HAE: 4-hydroxynonenal; GPX4: glutathione peroxidase 4.

Figure 4

Inflammatory cytokine secretion in the submandibular gland. Quantitative real-time PCR analysis of Il-6 (A) and Tnf-α (B) gene expression in the submandibular gland. The expression levels of Il-6 and Tnf-α mRNA were increased in the OVX group and decreased in the DFO and FER groups. Two-way ANOVA was performed. ^**p < 0.01, ^***p < 0.001 vs. SHAM group, ^#p < 0.05, ^##p < 0.01 vs. OVX group. Abbreviations: NS: not significant; SHAM: sham surgery group; OVX: ovariectomized group; DFO: deferoxamine injection group; FER: ferrostatin-1 injection group; Il-6: interleukin 6; Tnf-α: tumor necrosis factor-alpha.

Figure 5

Fibrosis in the submandibular gland. (A) Collagen distribution in the salivary gland tissue. The fibrotic area was significantly larger in the OVX group than in the SHAM group, while in both the DFO and the FER group it was smaller than in the OVX group. (B) Expression of Col1a1 and Col3a mRNAs. The expression of collagen type I alpha 1 chain (Col1a1) and collagen type III alpha chain (Col3a) mRNAs also increased in the OVX group (p < 0.001) and decreased in the DFO (Col1a1; p < 0.001 and Col3a; p < 0.05) and FER (Col1a1; p < 0.001 and Col3a; p < 0.01) groups. (C) Immunohistochemical assessment of TGF-βI levels. The brown staining reflects TGF-βI expression. The TGF-βI staining was higher in the OVX group than in the SHAM group and decreased in both the DFO and the FER group compared with that in the OVX group. (D) Expression of Tgf-βI and Tgf-βII mRNAs. The expression of Tgf-βI and Tgf-βII mRNAs increased in the OVX group (Tgf-βI; p < 0.001 and Tgf-βII; p < 0.01) and decreased in the DFO (Tgf-βI; p < 0.001 and Tgf-βII; p < 0.05) and FER (Tgf-βI; p < 0.001 and Tgf-βII; p < 0.01) groups. Two-way ANOVA was performed. ^**p < 0.01, ^***p < 0.001 vs. SHAM group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. OVX group. Abbreviations: SHAM: sham surgery group; OVX: ovariectomized group; DFO: deferoxamine injection group; FER: ferrostatin-1 injection group; Col1a1: Collagen type I alpha 1 chain; Col3a: Collagen type III alpha chain; TGF: Tumor growth factor.

Figure 6

Aquaporin and amylase in the submandibular gland. (A) Immunohistochemical assessment of AQP5 expression. The brown staining reflects AQP5 expression. The expression AQP5 was lower in the OVX group than in the SHAM group and increased in both the DFO and FER groups compared with that in the OVX group. (B) Expression of Aqp3 and Aqp5 mRNAs. The expression of Aqp3 mRNA was increased in the FER group compared to the OVX group (p < 0.01), but there was no difference in the DFO group, and the expression of Aqp5 mRNA was increased in both the DFO and FER groups compared to the OVX group (p < 0.01). (C) Salivary α-amylase activity and (D) expression of Amy1 mRNA. Salivary α-amylase activity and Amy1 mRNA expression were also significantly increased in the DFO group compared to the OVX group, but there was no difference in the FER group. Two-way ANOVA was performed. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. SHAM group, ^##p < 0.01 vs. OVX group. Abbreviations: NS: not significant; SHAM: sham surgery group; OVX: ovariectomized group; DFO: deferoxamine injection group; FER: ferrostatin-1 injection group.

Figure 7

Schematic diagram of postmenopausal salivary gland dysfunction in ovariectomized rats. After menopause, iron and lipids accumulate in the salivary glands and GPX4 activity decreases. This promotes lipid peroxidation and induces ferroptosis. DFO and FER inhibit the ferroptosis pathway, thus reducing salivary gland dysfunction.