The mechanism of submandibular gland dysfunction after menopause may be associated with the ferroptosis

Serum sex-hormone concentration and expression of sex-hormone receptors

The level of serum E2 concentration significantly decreased in the OVX4 group (p<0.01) and the OVX12 group (p<0.001) compared to the CON group (Figure 1A). Expression of estrogen receptors were not significantly different in the OVX group compared to the CON group. To evaluate the effect of sex hormone on sex hormone receptor, we performed real time PCR for the expression of estrogen receptors (ERs) such as ERα, ERβI, and ERβII in submandibular gland. ERα did not exist on submandibular gland. Although the ERβI and ERβII were expressed in submandibular gland, the expression of these receptors showed no significant differences in the OVX groups compared with the CON groups (Figure 1B). Compared with CON group, food intake was increased in OVX group and body weight was increased as a result (Figure 1C, 1D).

Figure 1. Sex hormone and body weight change after ovariectomy. (A) The level of serum estradiol concentration in the CON and OVX groups. (B) The expression of estrogen receptors in the CON12 and OVX12 group. (C), (D) Food intake and body weight in the CON and OVX group. Two-way ANOVA test. *p<0.05, **p<0.01 and ***p<0.001. ER = estrogen receptor, CON = control, OVX = ovariectomy.

Lipid deposition in submandibular gland

To measure the deposition of lipid in submandibular gland, we examined with H&E stain (40X). In morphometric analysis, the number of lipid vacuoles of submandibular gland in the OVX4 group (80.16±9.56) and the OVX12 group (122.65±17.64) was significantly higher compared with the CON4 group (24.32±6.88) and the CON12 group (40.44±11.36) (p<0.05 and p<0.01) (Figure 2A, 2B). This result means lipid deposition in the submandibular gland tissue occurred by menopause.

Figure 2. Lipid deposition in submandibular gland and lipogenesis analysis. (A) Lipid vacuoles (yellow circle) of submandibular gland detected by H & E staining. (B) Morphometric analysis of lipid vacuoles in the CON and OVX groups. (C), (D) Real time PCR and western blots of lipogenesis –related gene; ACC alpha and transcription factors; SREBP-1C and ChREBP expression in the CON and OVX groups. Two-way ANOVA test. *p<0.05, **p<0.01 and ***p<0.001. CON = control, OVX = ovariectomy, ACC = Acetyl-CoA carboxylase, SREBP = sterol regulatory element-binding protein, ChREBP = carbohydrate response element-binding protein.

Lipid metabolism

We investigated the expression of Acetyl-CoA carboxylase (ACC), β-actin, carbohydrate response element-binding protein (ChREBP), and sterol regulatory element-binding protein (SREBP) for lipogenesis by rear time PCR and western blot. ACC was significantly increased in the OVX4 group (p<0.05) and the OVX 12 group (p<0.01) compared to the CON group. In western blot, p-ACCα expression was increased in the OVX group compared to the CON group. However, β-actin expression was not significantly different between two groups (Figure 2C). ChREBP was significantly increased in the OVX4 group (p<0.01) and the OVX12 group (p<0.01) compared to the CON group (Figure 2D). SREBP-1 was significantly increased in the OVX12 group (p<0.05) compared to the CON group. In western blot, SREBP-1C and ChREBP were increased in the OVX group compared to the CON group (Figure 2D).

Electron microscopy

Electron microscopy shows shrunken mitochondria, decreased cristae and ruptured outer membrane. Unlike CON4 and CON12 group, OVX4 and OVX12 groups show swollen mitochondria and a progressive loss of cristae. In the OVX groups, a disintegrated dead cell is presented (Figure 3A).

Figure 3. Mitochondria dysfunction. (A) Electron microscopy of submandibular gland in the CON and OVX groups. (B) Western blotting of antioxidant enzyme; SOD and catalase and control VDAC in the CON and OVX groups. (C) ROS generation in the CON and OVX groups. CON = control, OVX = ovariectomy, SOD = superoxide dismutase, CAT = catalase, VDAC = voltage-dependent anion channel.

Generation of ROS

In western blot, superoxide dismutase (Mn-SOD) and catalase, which can detoxify excess ROS, were decreased in the OVX group compared to the CON group (Figure 3B). As a result, it was confirmed that ROS generation was significantly increased in the OVX4 group (p<0.05) and the OVX12 group (p<0.01) compared to the CON group (Figure 3C).

Redox imbalance and lipid peroxidation products

GPX4 is an antioxidant enzyme that protects cells against membrane lipid peroxidation. GSH is a cofactor of GPX4 to catalyze the reduction of lipid peroxides. GPX4 activity and GSH level was significantly decreased in the OVX4 group (p<0.05) and the OVX12 group (p<0.05) compared to the CON group (Figure 4A, 4B). GSSH level was significantly increased in the OVX4 group (p<0.01) and the OVX12 group (p<0.001) compared to the CON group (Figure 4C). To confirm the increase of lipid peroxidation, the concentrations of MDA and 4-HNE, the lipid peroxidation products, were measured. MDA concentration in the serum was significantly increased in the OVX4 group (p<0.05) and the OVX12 group (p<0.05) compared to the CON group (Figure 4D). MDA and 4-HAE concentration in the salivary gland tissue was significantly increased in the OVX12 group (p<0.001) compared to the CON12 group (Figure 4E, 4F).

Figure 4. Lipid peroxidation. (A–C) GPX activity (A), GSH level (B) and GSSH level (C) in the CON and OVX groups. (D) MDA concentration in the serum in the CON and OVX groups. (E) MDA concentration in the submandibular gland tissue in the CON and OVX groups. (F) MDA and HAE concentration in the submandibular gland tissue in the CON and OVX groups. Two-way ANOVA test. *p<0.05, **p<0.01 and ***p<0.001. CON = control, OVX = ovariectomy, GPX = Glutathione peroxidase, GSH = glutathione, GSSG = oxidized GSH, MDA = malondialdehyde, HAE = hydroxyalkenals

Iron overload in submandibular gland

To explore whether ovariectomy is associated with iron overload, we measured stained iron in tissue and cytosolic iron concentrations on submandibular gland. The number of stained iron of submandibular gland tissue in the OVX4 group (2.00±0.82) and the OVX12 group (4.00±0.82) was significantly higher compared with the CON4 group (0.75±0.96) and the CON12 group (2.50±1.29) (p<0.05) (Figure 5A, 5B). The cytosolic iron levels of the OVX groups were upregulated compared to the CON groups (p<0.05) (Figure 5C). These results indicate that the submandibular gland is overloaded with iron after ovariectomy.

Figure 5. Iron deposition in submandibular gland. (A) The number of stained iron (black circle) detected by Prussian Blue iron staining. (B) Morphometric analysis of stained iron in the CON and OVX groups. (C) Cytosolic iron content in the CON and OVX groups. Two-way ANOVA test. *p<0.05, **p<0.01 and ***p<0.001. CON = control, OVX = ovariectomy.

Inflammation cytokines secretion and fibrosis

Excess ROS causes oxidative stress which is associated with inflammation. The secretion of tumor necrosis factor alpha (TNF-α) was significantly increased in the OVX4 group (p<0.05) and the OVX12 group (p<0.01) compared to the CON group (Figure 6A). The secretion of interleukin 1 beta (IL-1β) was significantly increased in the OVX4 group (p<0.05) compared to the CON4 group (Figure 6A).

Figure 6. Increased salivary gland fibrosis and decreased salivary gland function. (A) Real time PCR of TNFα and IL-1β mRNA expression in the CON and OVX groups. (B) Fibrosis of submandibular gland detected by Masson and trichrome's staining. (C) Morphometric analysis of fibrosis in submandibular gland tissue in the CON and OVX groups. (D) Collagen type I expression of submandibular gland detected by immunohistochemistry. (E) Morphometric analysis of expression of collagen type I in the CON and OVX groups. (F) Saliva secretion in the CON and OVX groups. Two-way ANOVA test. *p<0.05, **p<0.01 and ***p<0.001. CON = control, OVX = ovariectomy, TNF = tumor necrosis factor, IL = interleukin.

Morphometric analysis was performed to confirm fibrosis of submandibular gland tissue. In MT stain, fibrosis in submandibular gland tissue was significantly increased in the OVX4 group (p<0.05) and the OVX12 group (p<0.001) compared to the CON group (Figure 6B, 6C). In IHC, the expression of collagen type I was significantly increased in the OVX4 group (p<0.05) and the OVX12 group (p<0.01) compared to the CON group (Figure 6D, 6E).

Saliva secretion

Saliva secretion was decreased in the OVX4 group and OVX12 group compared to the CON group. However, saliva secretion was significantly decreased only in the OVX12 group (p<0.05) (Figure 6F).

Microscopic findings of human submandibular gland tissue

In morphometric analysis, the number of lipid vacuoles of submandibular gland in the postmenopause group (62year; 44.16±6.65, 69year; 63.36±7.02 and 80year; 48.15±3.85) was significantly higher compared to the premenopause group (26year; 22.15±7.12, 32year; 18.72±4.65 and 34year; 11.84±9.33) (Figure 7A, 7B). The number of stained iron of submandibular gland in the postmenopause group (62year; 13.25±2.00, 69year; 16.25±5.00 and 80year; 11.25±2.00) was significantly higher compared to the premenopause group (26year; 1.15±0.50, 32year; 1.55±0.25 and 34year; 1.50±0.02) (Figure 7C, 7D).

Figure 7. Microscopic finding of human submandibular gland tissue. (A) Lipid vacuoles of submandibular gland detected by H & E staining in the postmenopause group than premenopause group. (B) The number of lipid vacuoles of submandibular gland in the postmenopause group than premenopause group. (C) Iron accumulation of submandibular gland detected by Prussian Blue iron staining in the postmenopause group than premenopause group. (D) The number of stained iron of submandibular gland in the postmenopause group than premenopause group.

Animals

Forty-eight female Sprague-Dawley rats (Samtako, Osan, Korea) aged nine weeks were used in this study. Animal care and research protocols were based on the principles and guidelines adopted by Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication).

Experimental design

After a week acclimatization period, rats were randomly divided into four groups (12 rats in each group): Group I (4 weeks after sham operation rats as CON4), group II (4 weeks after ovariectomy rats as OVX4), group III (12 weeks after sham operation rats as CON12) and group IV (12 weeks after ovariectomy rats as OVX12). Each animal group was weight matched at the beginning of the study. Rats were sacrificed four weeks (CON4 and OVX4) and twelve weeks (CON12 and OVX12) after surgery.

Establishment of the ovariectomized rat model

The rats were anesthetized using isoflurane inhalation (3% dissolved in oxygen). Sham operation and ovariectomy (OVX) was performed as follows: rats were anesthetized, abdominal incision was made at the midline of the abdomen and bilateral ovaries being revealed. In the OVX groups, the ovaries were ligated and cut off bilaterally followed by closure of the abdominal cavity. In the sham operations groups, the ovaries were exposed and their anatomical position was examined, then the abdomen was closed without excision of ovaries.

Plasma sex hormones analysis

Concentration of estradiol (E2) in serum was measured by rat-specific estradiol Enzyme-linked immunosorbent assay plates coated with biotin-conjugated binding protein kit purchased from Calbiotech Incorporation (Spring Valley, CA, USA).

Histology and morphometric analysis

The submandibular glands were isolated from each rat and prepared for fixation overnight in 4% formalin. We used automatic tissue processor for paraffin embedding (Leica, TP1020, semi-enclosed benchtop tissue processor) and dispensing (Leica EG1150H, Heated paraffin embedding module). Cross-sections (8μm thick) were placed on slide glasses, and sections were prepared for Hematoxylin and Eosin (H&E), Masson and trichrome’s (MT) and Iron staining. Morphometric determination was undertaken at 40X pictures from whole part of submandibular gland tissues by menu of the morphometric method using image analysis system program composed of a light microscope (Leica Basic LAS V3.8 software).

Prussian blue iron staining

The method based on the Prussian Blue stain reaction in which ionic iron reacts with acid ferrocyanide producing a blue color. Prussian blue reaction involves the treatment of sections with acid solutions of ferrocyanides. Any ferric ion present in the tissue combines with the ferrocyanide and results in the formation of a bright blue pigment called Prussian blue, or ferric ferrocyanide. Deparaffinize and rehydrate sections incubate for 3 min in iron stain (mix of potassium ferrocyanide and hydrochloric acid) and rinse in water. Counterstain in nuclear fast red and dehydrate in alcohol and mount.

Tissue preparations

Frozen submandibular gland tissue were homogenized in hypotonic lysis buffer using a tissue homogenizer for 20 sec. Homogenates were kept on ice for 15 min, 125 μl of 10% Nonidet P-40 (NP-40) solution was added and mixed for 15 second, and the mixture was centrifuged at 14,000 g for 2 min. The supernatants were used as the cytosol fraction. The pelleted nuclei were washed once with 400 μl of buffer A plus 25 μl of 10% NP-40, centrifuged, suspended in 200 μl of nuclear buffer, kept on ice for 30 min, and centrifuged at 14,000 g for 10 min. The supernatant (nuclear protein) was harvested and then stored at -80°C (Kim et al. 2010). Protein concentration was measured by the bicinchonic Acid (BCA) assay.

Real time-qPCR

To confirm mRNA expression, we isolated whole submandibular gland tissue and used real time PCR method. Tissue RNA was extracted using the TRIzol system (Life Technologies, Rockville, MD). Reverse Transcription Kit (Applied Biosystems, Foster City. California) was used to perform reverse transcription according to the manufacturer's protocol. Real-time PCR was performed according to the SYBR Green PCR protocol (Applied Biosystems Foster City CA). Gene-specific PCR products were continuously measured by an ABI PRISM 7900 HT Sequence Detection System (PE Applied Biosystem Norwalk, CT). All the primers used for Real Time PCR analysis have been designated using Primer Express software 1.5 (Applied Biosystems, Foster City, CA), and synthesized by Invitrogen Life Technologies (Carlsbad, CA). Primer sequences can be found in Table 1.

Western blot

Western blotting was carried out as described previously. The membrane was incubated with specific primary antibody at 25°C for 3 hr, followed by a horseradish peroxidase-conjugated anti-mouse antibody (Santa Cruz, 1:10,000), an anti-rabbit antibody (Santa Cruz, 1:10,000), or an anti-goat antibody (Santa Cruz, 1:10,000) at 25°C for 1 hr. Antibody labeling was detected using West-zol Plus and chemiluminescence Fluorchem^TMSP (Alpha Innotech Corporation, San Leandro, CA, USA).

Quantitation of redox status and lipid peroxidation

ROS generation was measured with specific fluorescence probe. Briefly, 25 μM of 2′, 7′-DCF-DA was added to homogenates to a 250 μl final volume. Changes in fluorescence intensity were measured every 5 min for 30 min on a fluorescence plate reader, GENios (Tecan Instruments, Salzburg, Austria) with excitation and emission wavelengths set at 485 and 530 nm, respectively. For the assay to measure glutathione (GSH) levels, 1 mM EDTA-50 mM phosphate buffer was added to the supernatant of trichloric acid (TCA)-treated homogenates, followed by o-phthaldehyde, and the mixture was incubated for 25 min at room temperature. To assay oxidized GSH (GSSG) level, N-ethylmaleimide was added to the supernatant of TCA-treated homogenates, then incubated for 25 min. Both GSH and GSSG levels were measured at excitation and emission wavelengths set at 360 and 460 nm, respectively. Glutathione peroxidase 4 (GPX4) activity was investigated with a Glutathione Peroxidase Assay Kit (Abcam, USA). Malondialdehyde (MDA)/4-hydroxyalkenals (HAE) concentrations were determined through use of a Bioxytech LPO-586 Assay Kit (OXIS Health Products, Foster, CA, USA). The kit uses a chromatogenic reagent that reacts with the lipid peroxidation products, MDA and 4-HAE, yielding a stable chromophore with maximum absorbance at 586 nm.

Cytosolic iron assay

Concentration of iron in serum was measured by rat-specific colorimetric iron assay kit from Biovision Incorporation (Spring Valley, CA, USA).

Immunohistochemistry (IHC)

De-paraffinized sections were washed with PBS and blocked for 1 hour at room temperature with 2% BSA containing 0.3% Triton X-100 in PBS. They were then incubated for 24 hours at 4°C with the following primary anti-collagen I (Abcam, Cambridge, United Kingdom). After the primary antibody was removed by rinsing, sections were incubated with secondary antibodies for 1 hour at room temperature. The following goat-anti rabbit secondary antibodies were used for double-staining with DAB staining. Incubation with phosphate-buffered saline supplemented with 1% bovine serum albumin instead of the primary antibody served as a negative control. For immunohistochemical analysis of submandibular gland, three nonoverlapping areas were analyzed at 400X, and nine areas were analyzed in each section. Results were expressed as stained area per total area in micrometers squared. Data were expressed as medians and ranges.

Electron microscopy

The material was pre-fixed with 2.5% glutaraldehyde (4°C, phosphate buffer, pH 7.4) and was post-fixed with 1% osmuim tetroxide in the same buffer. The material dehydrated with a series of the graded ethyl alcohol, and embedded in epoxy resin (Epon 812 mixture). Thick sections (1 μm) were stained with 1% toluidine blue for light microscope. Thin sections (50~60 nm) were prepared by using an ultramicrotome (EM UC7, Leica) and were double stained with uranyl acetate and lead citrate. Thin sections were examined with a transmission electron microscope (JEM-1200EXII, JEOL). The morphological feature of mitochondria was observed using electron microscopy.

Saliva secretion

Saliva secretion was induced by subcutaneous injection of pilocarpine (2 mg/kg body weight). The saliva was collected using cotton balls between 5 to 30 min after pilocarpine injection under deep anesthesia with isoflurane inhalation (3% dissolved in oxygen). The total weight of the secreted saliva (difference in the weight of the cotton balls before and after collection) was measured.

Human tissue analysis

In the submandibular gland of the patient who underwent submandibular resection due to submandibular gland disease, a tissue slide of the normal part was analyzed. We observed lipid vacuole and iron deposition in the submandibular tissues of 3 premenopausal women (26, 32 and 34 years old) and 3 postmenopausal women (62, 69 and 80 years old). This study was approved by the Institutional Review Board of Pusan National University Hospital (IRB No. H-1910-031-084) and informed consent was waived.

Statistical analysis

Unless otherwise noted, all quantitative data were reported as the mean standard error of the mean from at least three parallel repeats. Two-way analysis of variance (ANOVA) was used to determine significant differences between groups in which P < 0.05 was considered statistically significant. For Western blotting, one representative blot is shown from independent experiments done in triplicate.