Amnion mesenchymal stem cell metabolites reduce inflammation in diabetic salivary gland defect rat models

Abstract

Objectives

Hyposalivation or xerostomia are well-established intraoral complications of diabetes mellitus (DM). Amniotic Mesenchymal Stem Cell Metabolite Products (AMSC-MPs) are being widely studied for their immunomodulatory action. However, their potential in a salivary gland defect model has yet to be explored. This study aims to determine the potential of AMSC-MPs in modulating the inflammatory response in the salivary glands of rats with persistent hyperglycemia, mimicking the pathogenesis of salivary gland disorders in DM.

Methods

Forty-eight male pre-conditioned diabetic rats were divided into treatment and control groups. The treatment group received AMSC-MPs intraglandular injections, while the control group received phosphate-buffered saline intraglandular injections. Both groups were injected for 3, 5, 7, and 10 consecutive days. Subsequently, the submandibular salivary glands were biopsied and processed for formalin-fixed paraffin-embedded tissue for immunohistochemical assessment. The inflammatory response was assessed by quantifying the expression of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin 10 (IL-10), and transforming growth factor β (TGF-β).

Results

This study found significant downregulation of inflammatory cytokines, TNF-α and IL-6, in the AMSC-MPs intraglandular injections compared to the control (p < 0.05). Moreover, IL-10 and TGF-β expression, which may act as anti-inflammatory cytokines in this pathology, was significantly upregulated compared to the control (p < 0.05).

Conclusion

Our study demonstrated the anti-inflammatory potential of AMSC-MPs intraglandular injections in a diabetes-induced salivary gland defect rat model.

1. Introduction

In 2019, approximately 463 million people worldwide, primarily aged 20–79, were affected by diabetes mellitus (DM), representing a prevalence rate of 9.3 % within this age group. Xerostomia and hyposalivation are conditions that commonly occur in DM patients.¹ Hyposalivation may induce various pathophysiological changes within the oral cavity, such as elevated concentrations of mucin and glucose, reduced synthesis or activity of antimicrobial agents, depletion of the metalloprotein gustin, dysgeusia, oral candidiasis, increased epithelial exfoliation due to impaired lubrication, heightened proliferation of pathogenic microorganisms, coated tongue, and halitosis. Chronic hyperglycemia associated with diabetes mellitus (DM) further exacerbates these conditions, collectively contributing to burning mouth syndrome. Furthermore, DM adversely impacts wound healing in both soft and hard tissues of the oral mucosa.²

Hyposalivation in diabetes mellitus is primarily triggered by chronic hyperglycemia, which induces oxidative stress, accumulation of advanced glycation end-products (AGEs), and downregulation of aquaporins such as AQP5 in salivary acinar cells, thereby impairing water transport and secretory function.³ In addition, osmotic diuresis caused by persistent hyperglycemia contributes to systemic dehydration and further reduces salivary flow. Beyond hyperglycemia, diabetic complications such as microangiopathy, autonomic neuropathy, and chronic inflammation within salivary gland tissue also play significant roles.⁴ Vascular basement membrane thickening limits perfusion, neuropathy diminishes neural stimulation of secretion, and activation of NF-κB/TGF-β signaling promotes inflammatory infiltration and fibrotic remodeling.⁵ Thus, while hyperglycemia represents the central initiating insult, diabetic hyposalivation results from a combination of metabolic, vascular, neural, and inflammatory mechanisms.

Current therapeutic options for salivary gland hypofunction remain largely palliative, relatively ineffective, costly for patients, and unsuitable as long-term solutions. Effective intervention strategies for preventing salivary gland dysfunction should ideally target the inflammatory phase of tissue injury. DM conditions disrupt the balance between proliferation and apoptosis due to the increase in various inflammatory markers. Research has shown that DM, especially when caused by obesity, creates a highly inflammatory microenvironment, which may impede the regeneration processes.⁶

Mesenchymal stem cell (MSCs) is multipotent and isolated from bone marrow, umbilical cord, placenta, or adipose tissue. Among several types of tissue, the amniotic membrane is a suitable material for the development of stem cells because this tissue contains a large number of cells. The amniotic membrane contains stem cells that have differentiation capabilities and low immunogenicity effects, making this material a promising candidate for use for regenerative medicine.^7,8 When Amnion Mesenchymal Stem Cells (AMSCs) are cultured, several bioactive metabolite products, such as various cytokines and growth factors, are secreted into the culture medium and are referred as AMSC Metabolite Products (AMSC-MPs). Previous study showed AMSC-MPs were found to have anti-inflammatory and immunomodulatory functions by inducing suppression of TNF-α and IL-6 levels.⁹ Continuing studies are widely conducted to obtain novel therapeutics approaches in treating salivary gland dysfunction. These efforts include several modes of therapy with various growth factors, gene transfer, artificial saliva, and stem cell transplantation.¹⁰ Our study will assess the capacity of AMSC-MPs injection on intraglandular tissue inflammatory response of diabetes-induced salivary gland defects rat model.

Amnion-derived mesenchymal stem cells (AMSCs) provide unique advantages compared to other MSC sources such as bone marrow or adipose tissue. Unlike bone marrow or adipose MSCs, which require invasive harvesting procedures, AMSCs can be isolated from discarded amniotic membranes in a non-invasive and ethically acceptable manner.¹¹ They exhibit lower immunogenicity due to reduced expression of MHC-II and costimulatory molecules, along with higher proliferative capacity compared to bone marrow MSCs.^11,12 Importantly, AMSCs secrete a rich metabolite profile and secretome containing angiogenic and anti-inflammatory factors such as vascular endothelial growth factor (VEGF), insulin growth factor 1 (IGF-1), epithelial growth factor (EGF), transforming growth factor β (TGF-β), and interleukin 8 (IL-8), which play critical roles in promoting angiogenesis, reducing fibrosis, and modulating inflammation.^12,13

Previous studies have demonstrated that bone marrow MSCs or their exosomes can ameliorate salivary gland dysfunction in diabetic rats by modulating the TGFβ/Smad3 pathway,⁵ and that AMSC-derived exosomes promote repair in radiation-damaged salivary glands by enhancing acinar regeneration and reducing fibrosis.¹⁴ However, the role of Amniotic Mesenchymal Stem Cell Metabolite Products (AMSC-MPs) in diabetic salivary gland regeneration has not yet been investigated. In this study, we provide the first evidence that AMSC metabolites, when locally injected into diabetic salivary glands, reduce inflammation and improve salivary secretion, suggesting a novel therapeutic strategy for diabetes-related glandular dysfunction.

2. Material and methods

2.1. DM model in rats

Forty-eight male Wistar rats, each weighing between 250 g and 350 g and aged 3 months, were used in this study. The animals were housed in individual cages under a 12-h light-dark cycle at a constant temperature of 23 °C. They were fed a standard pellet diet and given tap water ad libitum at a temperature of 22 °C ± 2 °C. The standard pellet diet (Oriental Yeast Co., Tokyo, Japan) contained 22 % protein, 5 % fat, 5 % fiber, and 53 % carbohydrates, primarily in the form of complex polysaccharides.

The rats were fasted for approximately 12 h before induction to empty their stomachs and facilitate the onset of diabetes mellitus (DM). Fasting blood glucose levels were first measured using a glucometer to establish baseline values, and the data were recorded. DM was then induced using alloxan monohydrate at a dose of 75 mg/kg body weight, diluted in distilled water to a concentration of 20 mg/mL, and injected intraperitoneally. To prevent sudden hypoglycemia, the rats were provided with a 10 % dextrose solution during the first night post-induction. Hyperglycemia was confirmed by re-measuring fasting blood glucose levels, with values ≥ 100 mg/dL considered diabetic.

2.2. AMSC-MPs intraglandular injection

AMSC-MPs were prepared following the protocol by Wicaksono et al., 2023.¹⁵ AMSC-MPs were injected into the left submandibular salivary glands of the rats in a volume of 100 μL once a day for 3, 5, 7, and 10 consecutive days. The control group received phosphate-buffered saline intraglandular injections. After the designated time, each rat was euthanized using a combination of ketamine (60 mg/kg body weight) and xylazine (30 mg/kg body weight). The left submandibular gland was collected and fixed in 10 % neutral buffered formalin, embedded in paraffin, and processed into histological sections with a thickness of 4 μm.

2.3. Cytokine expression analysis

Indirect immunohistochemistry was conducted using the standard streptavidin-biotin-peroxidase complex method to bind primary antibodies, employing the LSAB System Universal Kit. Diaminobenzidine tetrahydrochloride served as the chromogen, and Mayer's hematoxylin was applied for counterstaining. The primary monoclonal antibodies used were TNF-α (1:500 dilution; TNF-α mouse monoclonal, Santa Cruz Biotechnology), IL-6 (1:500 dilution, IL-6 mouse monoclonal, Santa Cruz Biotechnology), TGF-β (1:1000 dilution, TGF-β mouse monoclonal, Santa Cruz Biotechnology), and IL-10 (1:1000 dilution, IL-10 mouse monoclonal, Santa Cruz Biotechnology). Two observers manually assessed cytokine expression in five separate fields of view using a Nikon H600L light microscope at magnifications of 40× and 100X.

2.4. Statistical analysis

Data analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 20.0. A t-test was conducted to assess significant differences in the expression of TNF-α, IL-6, IL-10, and TGF-β between the groups (p < 0.05).

3. Results

3.1. TNF-α expression

TNF-α, a pro-inflammatory cytokine, the intraglandular injection of AMSC-MPs resulted in a significant reduction in its expression from day 5 to day 10 post-injection (p < 0.05), indicating a delayed but potent anti-inflammatory response. Interestingly, no significant change in TNF-α expression was observed at day 3, suggesting that the anti-inflammatory action requires time to manifest (Fig. 1).

Fig. 1.

(A) TNF-α expression in submandibular salivary gland using indirect immunohistochemistry under 400x and 1000x magnification. (B) TNF-α expression was significantly downregulated after 5, 7 and 10 days after injection compared control groups. Asterix indicated significant differences using Independent t-test with number of each sample was 6. ∗p < 0.05. Scale bar 100 μm. Yellow arrow showed TNF-α expression. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3.2. IL-6 expression

Similarly, IL-6, another key pro-inflammatory cytokine, exhibited a marked decrease in expression from day 5 to day 10 post-injection, mirroring the trend seen with TNF-α (Fig. 2). This indicates that AMSC-MPs consistently suppress inflammatory signaling pathways over time.

Fig. 2.

(A) IL-6 expression in submandibular salivary gland using indirect immunohistochemistry under 400x and 1000x magnification. (B) IL-6 expression was significantly downregulated after 5, 7 and 10 days after injection compared control groups. Asterix indicated significant differences using Independent t-test with number of each sample was 6. ∗p < 0.05. Scale bar 100 μm. Yellow arrow showed IL-6 expression. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3.3. IL-10 expression

In contrast, the expression of the anti-inflammatory cytokine IL-10 showed an early and sustained increase, starting as early as day 3 and continuing through day 10 (p < 0.05). This rapid upregulation of IL-10 suggests that AMSC-MPs actively promote an anti-inflammatory environment soon after administration (Fig. 3).

Fig. 3.

(A) IL-10 expression in submandibular salivary gland using indirect immunohistochemistry under 400x and 1000x magnification. (B) IL-10 expression was significantly downregulated after 3, 5, 7 and 10 days after injection compared control groups. Asterix indicated significant differences using Independent t-test with number of each sample was 6. ∗p < 0.05. Scale bar 100 μm. Yellow arrow showed IL-10 expression. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3.4. TGF-β expression

Furthermore, the regenerative marker TGF-β demonstrated a significant elevation in expression beginning at day 3 and persisting through day 10 (p < 0.05) (Fig. 4). This indicates that AMSC-MPs not only mitigate inflammation but also facilitate tissue repair and regeneration within the diabetic salivary gland model.

Fig. 4.

(A) TGF-β expression in submandibular salivary gland using indirect immunohistochemistry under 400x and 1000x magnification. (B) TGF-β expression was significantly downregulated after 3, 5, 7 and 10 days after injection compared control groups. Asterix indicated significant differences using Independent t-test with number of each sample was 6. ∗p < 0.05. Scale bar 100 μm. Yellow arrow showed TGF-β expression. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

4. Discussion

Diabetes mellitus remains one of the most prevalent global burdens today, particularly in Indonesia.^16,17 The degenerative implications of this chronic disease frequently lead to organ impairment. Diabetes-induced hyposalivation is one of the most undesirable outcomes due to its potential to cause a domino effect of further complications, such as dental decay, periodontal disease, and fungal infection. While the exact mechanisms of this disease are not completely understood, a key factor is the damage to the glandular cells of the salivary glands, resulting in their impaired ability to produce saliva.^18,19 Our study explores the potential of AMSC-MPs intraglandular injection as a regenerative strategy to restore glandular cells of the salivary glands.

DM can induce salivary gland damage through several well-documented mechanisms. Chronic hyperglycemia, a defining characteristic of DM, leads to oxidative stress and the formation of advanced glycation end products (AGEs), causing both structural and functional damage to glandular cells. Hyperglycemia induces inflammation through the activation of the nuclear factor Kappa-B (NF-κB) pathway, which stimulates the release of various pro-inflammatory cytokines such as TNF-α and IL-6, both systemically and locally within salivary glands. This prolonged local inflammatory reaction results in tissue inflammation, apoptosis, and ultimately, salivary gland defects, significantly reducing saliva production. Additionally, diabetic neuropathy can impair the neural regulation of salivary secretion, further exacerbating glandular dysfunction. Collectively, these factors lead to decreased saliva production, manifesting as hyposalivation and xerostomia.^2,20,21

Monitoring the levels of pro-inflammatory and anti-inflammatory cytokines provides insights into the healing process and effectiveness of treatments.²² Our experimental models demonstrated that intraglandular injection of AMSC-MPs downregulates pro-inflammatory cytokines, TNF-α and IL-6, while upregulating anti-inflammatory cytokines IL-10 and TGF-β within diabetic-induced submandibular salivary gland defects.^23,24 These cytokines are known to act through key signaling pathways, including TGF-β/Smad, NF-κB, and MAPK, which collectively reduce acinar cell apoptosis, limit fibrosis, and promote proliferation of functional acinar and ductal cells. Previous studies have shown that stimulation of TGF-β signaling promotes regeneration of submandibular gland tissue in diabetic rats, while increased IL-10 levels reduce acinar cell vacuolization and support tissue repair.^25,26 The bioactive content of AMSC-MPs, including immunomodulatory molecules such as IL-10, TGF-β, IL-1 receptor antagonist, SDF-1/CXCL12, MCP-1/CCL2, IL-8, and IL-13, acts synergistically to restore a non-inflammatory microenvironment conducive to regeneration.^23,24 Additionally, AMSC-MPs contain pro-regenerative and anti-apoptotic factors such as VEGF, IGF-1, bFGF, NGF, and PGF, which enhance vascularization, acinar regeneration, and secretory function.^27,28 Together, these mechanisms suggest that AMSC-MPs not only modulate the inflammatory microenvironment but also promote physiological and functional recovery of the salivary gland, including restoration of acinar structure, ductal integrity, and saliva secretion. This comprehensive mechanism supports the potential of AMSC-MPs as a therapeutic strategy for diabetes-related salivary gland dysfunction.

This study has several limitations. First, we focused exclusively on the submandibular gland, which is the largest and most accessible salivary gland in rats, and therefore the findings may not fully extrapolate to other salivary glands with distinct structures and functions. Second, the AMSC-MPs were not characterized for total protein content or specific growth factor concentrations, limiting precise dose standardization and mechanistic insight. Third, the study was conducted over a defined period in a rat model of alloxan-induced diabetes, and long-term efficacy, safety, and translatability to humans remain to be determined. Fourth, while PBS-injected diabetic controls allowed us to account for injection effects, untreated diabetic and non-diabetic controls were not included for broader baseline comparisons. Although AMSC-MP injection alleviated inflammatory markers, we did not perform longitudinal functional assessments of salivary secretion, and it remains unclear whether glandular damage is fully reversible or if persistent diabetic conditions could lead to recurrence. Finally, clinical translation will require careful consideration of dosing, formulation, controlled local delivery, and regulatory safety, all of which remain to be investigated. Future studies should address these limitations by including protein quantification, functional measurements before and after therapy, long-term follow-up, evaluation of other salivary glands, and strategies for safe and effective clinical application.

5. Conclusion

Our study demonstrated substantial experimental evidence of the anti-inflammatory effect of the intraglandular injection of AMSC-MPs. However, further study is needed to investigate the rate of glandular cell restoration using morphological markers and other molecular regeneration biomarkers to strengthen the evidence of the regenerative potential of AMSC-MPs in the context of salivary gland disease.

Patient's/Guardian's consent

This study did not involve human participants, and therefore, informed consent were not required.

Ethical approval

All animal-related experimental procedures adhered to the National Health Institute guidelines for laboratory animal care and use. The protocol of the research was registered and proved by the Animal Experiments Board at the Faculty of Dental Medicine, Universitas Airlangga number 048/HRECC.FODM/I/2023.

Declaration of generative AI and AI-assisted technologies in the writing process

In the course of preparing this work, the author utilized ChatGPT for language review. After employing this tool, the author carefully reviewed and edited the content as necessary and assumes full responsibility for the publication's content.

Source of funding

This work was supported by Airlangga Research Fund, Universitas Airlangga in 2024 with number assignment 672/UN3/2024.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Diah Savitri Ernawati reports financial support was provided by Airlangga University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.