Submandibular Gland Cells Research Articles

The impact of alternate-day fasting on the salivary gland stem cell compartments in non-obese diabetic mice with newly established Sjögren's syndrome.

Intermittent fasting exerts a profound beneficial influence on a spectrum of diseases through various mechanisms including regulation of immune responses, elimination of senescent- and pathogenic cells and improvement of stem cell-based tissue regeneration in a disease- and tissue-dependent manner. Our previous study demonstrated that alternate-day fasting (ADF) led to alleviation of xerostomia and sialadenitis in non-obese diabetic (NOD) mice, a well-defined model of Sjögren's syndrome (SS). This present study delved into the previously unexplored impacts of ADF in this disease setting and revealed that ADF increases the proportion of salivary gland stem cells (SGSCs), defined as the EpCAMhi cell population among the lineage marker negative submandibular gland (SMG) cells. Furthermore, ADF downregulated the expression of p16INK4a, a cellular senescence marker, which was concomitant with increased apoptosis and decreased expression and activity of NLRP3 inflammasomes in the SMGs, particularly in the SGSC-residing ductal compartments. RNA-sequencing analysis of purified SGSCs from NOD mice revealed that the significantly downregulated genes by ADF were mainly associated with sugar metabolism, amino acid biosynthetic process and MAPK signaling pathway, whereas the significantly upregulated genes related to fatty acid metabolic processes, among others. Collectively, these findings indicate that ADF increases the SGSC proportion, accompanied by a modulation of the SGSC property and a switch from sugar- to fatty acid-based metabolism. These findings lay the foundation for further investigation into the functionality of SGSCs influenced by ADF and shed light on the cellular and molecular mechanisms by which ADF exerts beneficial actions on salivary gland restoration in SS.

ORAI2 is Important for the Development of Early-Stage Postirradiation Fibrosis in Salivary Glands

PurposeAlthough post-irradiation hyposalivation significantly impairs patient quality of life, the underlying mechanisms driving radiation-induced salivary gland fibrosis and hyposalivation remain poorly understood. This study aims to explore the role of calcium-mediated signaling pathways in radiation-induced salivary gland fibrosis. Materials and MethodsPrimary human submandibular gland (SG) cells and C57BL/6J female mouse SGs were exposed to irradiation to model fibrosis development. Following 15 Gy irradiation exposure, RNA sequencing and bioinformatic analysis were conducted on mouse SGs. The effects of Store-Operated Calcium Entry (SOCE) inhibition using SKF96365 and YM58483 on fibrosis markers were assessed in vitro and in vivo. Additionally, the involvement of ORAI2 protein and the newly identified JNK/NFAT1/TGF-β1 signaling axis in SG fibrosis was explored. ResultsWe identified that the calcium release-activated calcium modulator ORAI2 was important in promoting early-stage post-irradiation fibrosis in SGs. Calcium channel signaling was activated in both human patients and irradiated C57BL/6J female mice SGs. Inhibition of SOCE signaling effectively blocked fibrosis in an ORAI2-dependent manner 30 days after irradiation. Our mechanistic studies revealed a novel ORAI2/JNK/NFAT1 axis within the SOCE pathway critical in driving TGF-β1-mediated fibrogenesis. Encouragingly, pharmacological inhibition of NFAT1 significantly mitigated radiation-induced SG fibrosis and restored saliva flow to 84.61% of normal levels in treated mice 30 days after irradiation, without detectable side effects. ConclusionsOur findings highlight the significance of the ORAI2-mediated calcium signaling pathway, specifically via the ORAI2/JNK/NFAT1 axis, in promoting TGF-β1 expression and contributing to the development of early-stage salivary gland fibrosis following irradiation exposure. Targeting the ORAI2/JNK/NFAT1 axis emerges as a promising therapeutic strategy to alleviate radiation-induced hyposalivation and fibrosis, potentially improving the quality of life for patients undergoing radiotherapy.

Tert-expressing cells contribute to salivary gland homeostasis and tissue regeneration after radiation therapy.

Salivary gland homeostasis and regeneration after radiotherapy depend significantly on progenitor cells. However, the lineage of submandibular gland (SMG) progenitor cells remains less defined compared with other normal organs. Here, using a mouse strain expressing regulated CreERT2 recombinase from the endogenous Tert locus, we identify a distinct telomerase-expressing (TertHigh) cell population located in the ductal region of the adult SMG. These TertHigh cells contribute to ductal cell generation during SMG homeostasis and to both ductal and acinar cell renewal 1 year after radiotherapy. TertHigh cells maintain self-renewal capacity during in vitro culture, exhibit resistance to radiation damage, and demonstrate enhanced proliferative activity after radiation exposure. Similarly, primary human SMG cells with high Tert expression display enhanced cell survival after radiotherapy, and CRISPR-activated Tert in human SMG spheres increases proliferation after radiation. RNA sequencing reveals upregulation of "cell cycling" and "oxidative stress response" pathways in TertHigh cells following radiation. Mechanistically, Tert appears to modulate cell survival through ROS levels in SMG spheres following radiation damage. Our findings highlight the significance of TertHigh cells in salivary gland biology, providing insights into their response to radiotherapy and into their use as a potential target for enhancing salivary gland regeneration after radiotherapy.

Intracellular signaling pathways involved in the regulation of gene expression by pilocarpine

ObjectivesPilocarpine is commonly used clinically to treat dry mouth. The long-term administration of pilocarpine reportedly improves salivary secretion more effectively than short-term administration. Therefore, we hypothesized that pilocarpine alters gene expression in salivary glands via muscarinic receptor stimulation. This study aimed to investigate the effects of pilocarpine use on gene expression mediated by mitogen-activated protein kinase (MAPK) activity. MethodsThe effects of pilocarpine on gene expression were investigated in rats and human salivary gland (HSY) cells using several inhibitors of intracellular signaling pathways. Gene expression in the rat submandibular gland and HSY cells was determined using reverse transcription–quantitative polymerase chain reaction analysis of total RNA. ResultsIn animal experiments, at 7 days after pilocarpine stimulation, Ctgf and Sgk1 expressions were increased in the submandibular gland. In cell culture experiments, pilocarpine increased Ctgf expression in HSY cells. The mitogen-activated protein kinase kinase inhibitor trametinib, the Src inhibitor PP2, and the muscarinic acetylcholine receptor antagonist atropine suppressed the effect of pilocarpine on gene expression. ConclusionsPilocarpine enhances Ctgf and Sgk1 expressions by activating Src-mediated MAPK activity. Although further studies are required to fully understand the roles of Ctgf and Sgk1, changes in gene expression may play an important role in improving salivary secretions.

Submandibular Gland Pathogenesis Following SARS-CoV-2 Infection and Implications for Xerostomia.

Although SARS-CoV-2 induces mucin hypersecretion in the respiratory tract, hyposalivation/xerostomia has been reported by COVID-19 patients. We evaluate the submandibular gland (SMGs) pathogenesis in SARS-CoV-2-infected K18-hACE2 mice, focusing on the impact of infection on the mucin production and structural integrity of acini, ductal system, myoepithelial cells (MECs) and telocytes. The spike protein, the nucleocapsid protein, hACE2, actin, EGF, TNF-α and IL-1β were detected by immunofluorescence, and the Egfr and Muc5b expression was evaluated. In the infected animals, significant acinar hypertrophy was observed in contrast to ductal atrophy. Nucleocapsid proteins and/or viral particles were detected in the SMG cells, mainly in the nuclear membrane-derived vesicles, confirming the nuclear role in the viral formation. The acinar cells showed intense TNF-α and IL-1β immunoexpression, and the EGF-EGFR signaling increased, together with Muc5b upregulation. This finding explains mucin hypersecretion and acinar hypertrophy, which compress the ducts. Dying MECs and actin reduction were also observed, indicating failure of contraction and acinar support, favoring acinar hypertrophy. Viral assembly was found in the dying telocytes, pointing to these intercommunicating cells as viral transmitters in SMGs. Therefore, EGF-EGFR-induced mucin hypersecretion was triggered by SARS-CoV-2 in acinar cells, likely mediated by cytokines. The damage to telocytes and MECs may have favored the acinar hypertrophy, leading to ductal obstruction, explaining xerostomia in COVID-19 patients. Thus, acinar cells, telocytes and MECs may be viral targets, which favor replication and cell-to-cell viral transmission in the SMG, corroborating the high viral load in saliva of infected individuals.

Iguratimod Alleviates Experimental Sjögren's Syndrome by Inhibiting NLRP3 Inflammasome Activation.

Iguratimod (T-614) is a compound widely used as anti-rheumatic drug. This study investigated the effect and underlying mechanism of T-614 on experimental Sjögren's syndrome (ESS). ESS mice model was established by injection of submandibular gland protein. Mice were randomly divided into control, experimental Sjögren's syndrome (ESS), ESS + T-614 (10 mg/kg), ESS + T-614 (20 mg/kg), and ESS + T-614 (30 mg/kg) groups. Human submandibular gland (HSG) were cultured with 0, 0.5, 5, or 50 μg/ml T-614 in the absence or presence of interferon-α (IFN-α). Haematoxylin and eosin (H&E) and cytokine levels were used to detect immune cells activation in submandibular glands. Apoptosis in submandibular glands tissues and cells was determined by TUNEL and flow cytometry. Apoptosis and NLRP3 inflammasome-related proteins were detected by western blotting. T-614 treatment attenuated submandibular gland damage in ESS mice. T-614 administration inhibited submandibular gland cell apoptosis in ESS mice. Furthermore, T-614 blocked inflammatory factor levels and NLRP3 inflammasome activation in the submandibular glands. In vitro, results corroborated that T-614 could protect HSG cells from IFN-α-induced cell apoptosis and inflammation by inhibiting NLRP3 inflammasome activation. Our results expounded that T-614 alleviated ESS by inhibiting NLRP3 inflammasome activation.

Balancing Risks versus Benefits: Vitamin C Therapy versus Copper Oxide Nanoparticles Toxicity in Albino Rats' Submandibular Salivary Gland.

This study aimed to examine the suppressive effect of the natural antioxidant vitamin C (VC) against submandibular gland toxicity induced by copper oxide nanoparticles (CuO-NPs). Three groups of 30 mature male albino rats (4 weeks old) weighing between 150 and 200 g were selected. The rats were randomly assigned for 6 weeks to receive: intraperitoneal injection (IP) of vehicle (control group); IP of 2.5 mg/kg body weight (bw) of CuO-NPs (CuO-NPs group); and IP of 2.5 mg/kg bw of CuO-NPs, combined with a daily oral dose of 100 mg/kg bw of VC in drinking water via gavage (CuO-NPs/VC group). The rats were euthanized, and their submandibular glands were dissected for histological evaluation, including hematoxylin and eosin staining and immunohistochemistry for Ki-67 and caspase-3. The area expression for Ki-67 and caspase-3 was statistically analyzed using GraphPad Prism. Following analysis of variance analysis, Tukey's post hoc was used for multiple comparisons. The significance level was set at p < 0.05. CuO-NPs caused significant cytotoxic effects on submandibular salivary gland cells in albino rats. This led to an increase in Ki-67 and caspase-3 levels compared with the control group. VC administration improved tissue histology and reduced Ki-67 and caspase-3 levels in the VC/CuO-NPs group compared with rats treated with CuO-NPs alone. The study revealed significant cytotoxic effects of CuO-NPs on the submandibular salivary gland of albino rats. VC effectively mitigated these toxic effects, suggesting its potential as a readily available antioxidant.

F-actin/DRP1 axis-mediated mitochondrial fission promotes mitophagy in diabetic submandibular glands.

Diabetes is accompanied by a high prevalence of hyposalivation, causing severe damage to oral and systemic health. Mitochondrial dynamics play important roles in the pathogenesis of various diabetic complications; however, little is known about their roles in diabetic hyposalivation. A diabetic mouse model and a high glucose (HG)-induced diabetic submandibular gland (SMG) cell model were employed. More mitochondria surrounded by autophagosomes and higher expression of mitophagy-related proteins were detected in the SMGs of diabetic mice and HG-treated SMG cells. In diabetic SMGs, dynamin-related protein 1 (DRP1) was upregulated, whereas mitofusin-2 was downregulated both invivo and invitro. Shortened mitochondria and impaired mitochondrial functions were observed in the HG group. A DRP1-specific inhibitor, mdivi-1, suppressed mitochondrial fission and mitophagy, as well as restored mitochondrial functions in the HG condition. Moreover, the interaction of F-actin and DRP1 was enhanced in the diabetic group. Inhibiting F-actin with cytochalasin D repaired the injured effects of HG on mitochondrial dynamics and functions. Conversely, the F-actin-polymerization-inducer jasplakinolide aggravated mitochondrial fission and dysfunction. F-actin contributes to HG-evoked mitochondrial fission by interacting with DRP1, which induces mitophagy and impairs mitochondrial function in SMG cells, ultimately damaging the SMG.

Dicliptera chinensis-derived polysaccharide enhanced the growth activity of submandibular gland cells in vitro after radiotherapy

ObjectiveRadiotherapy for head and neck can damage the salivary gland cells, which can easily result in xerostomia. No effective treatment for radiation-induced salivary gland dysfunction currently exists. Thus, we aimed to study the protective effect of Dicliptera chinensis polysaccharides (DCP) on the prevention of submandibular gland (SMG) cell damage caused by radiotherapy in Sprague-Dawley rats. DesignMechanical enzyme digestion was used to extract primary rat SMG cells. A radiation injury model was established by treating these cells with a dose of 8 Gy, followed by intervention using different DCP concentrations. The cell counting kit 8 assay was used to determine the inhibition rate of SMG cells in each group. The rates of apoptosis and cell cycle progression were detected using flow cytometry. Expression of the Mre11/Rad50/Nbs1 complex (MRN) was detected using western blotting. ResultsDCP increased the proliferation of SMG cells after irradiation, and cell growth activity positively correlated with polysaccharide concentration. Flow cytometry analysis of SMG cell apoptosis revealed that DCP markedly reduced the total apoptosis rate after irradiation, especially the early apoptosis rate. Cell cycle results suggested that DCP reduced the number of cells in the S and G2 phases after irradiation and alleviated the S and G2 blocks. Western blot results indicated that the expression of Mre11, Rad50, and Nbs1 decreased in the radiation-injured group, whereas their expression increased after DCP treatment. ConclusionsDCP can protect the rat SMG cells after radiation and be used as a protective agent against salivary gland cell damage caused by radiotherapy.

PANoptosis induced by inflammatory cytokines promotes salivary glands radiation injury.

Salivary glands are frequently damaged in patients undergoing radiotherapy for head and neck cancer. Whether PANoptosis, which is characterized by pyroptosis, apoptosis, and necroptosis, occurs during radiation injury to the salivary glands and its role remain unclear. Radiation-induced injury models of mouse submandibular gland, as well as primary acinar cells and HSG cell lines were established to determine the presence of radiation-induced PANoptosis. Several programmed cell death inhibitors, PFTα, disulfiram, Nec-1 and zVAD, were used to compare the effects of different cell death pathway on radiation injury. The LEGENDplex™ Human Inflammation Panel was used to characterize the inflammatory landscape secreted by salivary gland cells after radiotherapy. Single 15Gy or 8Gy radiotherapy triggered PANoptosis in mouse submandibular gland or salivary gland cells. Compared to the suppression of pyroptosis, apoptosis, or necroptosis alone, the inhibition of PANoptosis is more effective in preventing radiation injury to the salivary glands (p < 0.0001). The levels of multiple inflammatory cytokines were significantly up-regulated in the supernatants of HSG cells within 48 h after IR. Neutralizing inflammatory cytokines are capable of inhibiting salivary glands PANoptosis. Inhibition of PANoptosis induced by inflammatory cytokines can effectively prevent radiation injury of salivary glands.

Rutin alleviates Sjogren's syndrome via CaR/NLRP3/NF-κB signal pathway.

Sjogren's syndrome (SS) is an autoimmune disease. Its mechanism and treatment methods are unclear. The purpose of this study was to investigate the effects of rutin (Ru) on SS. Proteomics was used to detect differential proteins in the submandibular glands of normal mice and SS mice. Salivary secretion (SAS) and salivary gland index (SGI) were detected. Oxidative stress and inflammatory cytokine in submandibular glands were detected. The levels of NLRP3, ASC, Caspase-1, IL-1β, and p-NF-κBp65 in submandibular gland tissues and submandibular gland cells of overexpressedcalcium-sensing receptor (over-CaR) mice and overexpressed CaR primary submandibular gland cells (over-CaR-PSGs) were detected. In total, 327 differential proteins were identified in the submandibular gland tissues of SS mice compared to control mice. CaR was one of the most differential proteins and significantly increased compared to control mice. Ru could significantly increase SGI and SGI, and inhibit oxidative stress and inflammatory cytokine in submandibular glands. In addition, Ru was shown to further improve SS via regulation of the CaR/NOD-like receptor thermal protein domain associated protein 3 (NLRP3)/nuclear factor kappa-B (NF-κB) signal pathway. Overexpression of CaR counteracted partial activity of Ru. CaR may be an important target for the treatment of SS. In addition, Ru improved the SS via the CaR/NLRP3/NF-κB signal pathway. This study provides a basis for the treatments for SS.

Abstract 5442: Terthighcells: key players in salivary gland homeostasis and regeneration after radiation therapy in adult mice

Abstract Salivary gland homeostasis and regeneration post-radiotherapy depend significantly on stem/progenitor cells. However, the lineage of SMG progenitor cells is not as well defined as in other normal organs, and better understanding of this population would be important for their future clinical application for tissue regeneration. Using a mouse strain expressing regulated CreERT2 recombinase from the endogenous Tert locus, we identify a distinct Terthigh cell population that are located in the ductal region throughout the submandibular gland (SMG), express ASCL3 (another progenitor cell marker) and can give rise to ductal cells during homeostasis. In contrast, Terthigh cells are found to repopulate both ductal and acinar cells at one year of tracing after radiotherapy. Terthigh cells from adult SMG retain self-renewal capacity when subjected in vitro culture, are resistant to radiation cell kill, and display enhanced proliferative activity post-radiation. Similarly, primary human SMG cells with high Tert expression display enhanced cell survival after radiotherapy. RNA sequencing reveals upregulation of the cell cycling and oxidative stress response pathways these cells after radiation. Mechanistically, Tert appears to modulate cell survival and ROS level in SMG spheres following radiation damage. Collectively, these data suggest that Terthigh cells constitute a novel subset of SMG progenitor cells located throughout the ductal region of the gland that can help to repopulate both ductal and acinar cells after RT damage. Citation Format: Li Guan, Vignesh Viswanathan, Sivakamasundari V, Hongbin Cao, Yuyan Jiang, Junfei Zhao, Deana R. Colburg, Patrick T. Neuhoefer, Yu Xu, Eyiwunmi E. Laseinde, Steven Artandi, Quynh-Thu Le. Terthighcells: key players in salivary gland homeostasis and regeneration after radiation therapy in adult mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5442.

Effect of silencing SSB1 gene on the expression of NBS1 in irradiated rat submandibular gland cells.

The salivary gland (SGS) is a kind of organ vulnerable to ionizing radiation. Radiotherapy is an important treatment for head and neck tumors, but in the process of radiotherapy, tumor cells will be injured by radiation to a certain extent. Infrared-induced DNA double-strand break (IR-DSBs) is one of the most serious DNA damage. DNA repair proteins such as Nymegan rupture syndrome protein 1 (NBS1) play a key role in the identification and repair of DNA damage. but the interaction between SSB1 and NBS1 has not been elucidated. In this study, we irradiated rat submandibular gland (SMG) cells, which were either infected with a rAdE5-SSB1-1p2-shRNA recombinant adenovirus to silence SSB or a control virus, to explore the effect of IR on the expression NBS1 in the absence of SSB. Our results showed that the SSB1 mRNA transcripts and protein expression of SSB1 and NBS1 initially increased and decreased later with increased doses. The relative expression reached the highest levels when the SMG cells were irradiated with 2Gy of IR. Silencing the SSB1 gene suppressed the expression of both SSB1 and NBS1 regardless of irradiation. The expression of NBS1 decreased when the SSB1 gene was silenced. We concluded that IR affected the expression of both SSB1 and NBS1 and there is a synergistic effect on IR-induced NBS1 suppression and DSBs repair in SMG cells. These observations shed light on further investigation and elucidation of IR-caused DNA repair mechanisms.

Total glucosides of paeony alleviates experimental Sjögren's syndrome through inhibiting NLRP3 inflammasome activation of submandibular gland cells.

The mechanisms by which total glucosides of paeony (TGP) mitigates Sjögren's syndrome (SS) remains elusive. In the present study, we aim to explore the relationship between the therapeutic effects of TGP in the treatment of SS and NLRP3 inflammasome activation in submandibular gland (SG) cells. Female non-obese diabetic (NOD) mice were selected as the model of SS. The mice were divided into PBS and TGP treatment group. For treatment, TGP (400mg·kg-1) was administered intragastrically every day for 4 weeks. The SS-like symptoms and pathological changes of the SG of mice were compared between the PBS and TGP group. The activation of NLRP3 inflammasome in SG was detected by RT-qPCR, immunohistochemistry and western blot. The SG cells stimulated by lipopolysaccharide (LPS) and adenosine triphosphate (ATP) for activation of NLRP3 inflammasome were treated with or without TGP. Then, NLRP3 inflammasome activation was assessed. The IL-1β and IL-18 in homogenate of SG, serum and supernatant were detected by ELISA. Compared with balb/c mice, NOD mice showed SS-like symptoms and lymphocyte infiltration in SG, and the expression of NLRP3 inflammasome in SG was significantly increased. The SS-like symptoms were alleviated, and lymphocyte infiltration in SG was reduced, and the level of NLRP3 inflammasome in SG mice was decreased after TGP treatment. TGP also significantly inhibit the activation of NLRP3 inflammasome of SG cells in vitro. Collectively, our results indicated that TGP alleviates SS through inhibition of the activation of NLRP3 inflammasome of SG. These findings clarified the mechanism underlying the therapeutic effects of TGP on SS, and provided new evidence for the further application of TGP in the treatment of SS.

Single-cell analysis reveals the transcriptional alterations in the submandibular glands of aged mice

ObjectivesAging-related salivary gland changes, such as lymphocyte infiltration and acinar cell loss decrease saliva secretion, thereby affecting quality of life. The precise molecular mechanisms underlying these changes remain unclear. MethodsWe here performed single-cell RNA sequencing to clarify gene expression changes in each cell type comprising the submandibular glands (SMGs) of adult and aged mice. ResultsThe proportion of acinar cells decreased in various epithelial clusters annotated with cell type-specific marker genes. Expression levels of the cellular senescence markers, Cdkn2a/p16 and Cdkn1a/p21, were increased in the basal and striated ducts of aged SMGs relative to their levels in those of adult SMGs. In contrast, senescence-associated secretory phenotype-related genes, except transforming growth factor-β, exhibited little change in expression in aged SMGs relative to adult SMGs. Conclusions: Gene Ontology analysis revealed increased expression levels of genes encoding major histocompatibility complex (MHC) class I components in the ductal component cells of aged SMGs. MHC class I expression may thus be associated with salivary gland aging.

Tlx1 regulates acinar and duct development in mouse salivary glands.

Tlx1 encodes a transcription factor expressed in several craniofacial structures of developing mice. The role of Tlx1 in salivary gland development was examined using morphological and immunohistochemical analyses of Tlx1 null mice. Tlx1 is expressed in submandibular and sublingual glands but not parotid glands of neonatal and adult male and female C57Bl/6J (Tlx1+/+ ) mice. TLX1 protein was localized to the nuclei of terminal tubule cells, developing duct cells and mesenchymal cells in neonatal submandibular and sublingual glands, and to nuclei of duct cells and connective tissue cells in adult glands. Occasionally, TLX1 was observed in nuclei of epithelial cells in or adjacent to the acini. Submandibular glands were smaller and sublingual glands were larger in size in mutant mice (Tlx1-/- ) compared to wild-type mice. Differentiation of terminal tubule and proacinar cells of neonatal Tlx1-/- submandibular glands was abnormal; expression of their characteristic products, submandibular gland protein C and parotid secretory protein, respectively, was reduced. At 3 weeks postnatally, terminal tubule cells at the acinar-intercalated duct junction were poorly developed or absent in Tlx1-/- mice. Granular convoluted ducts in adult mutant mice were decreased, and epidermal growth factor and nerve growth factor expression were reduced. Along with normal acinar cell proteins, adult acinar cells of Tlx1-/- mice continued to express neonatal proteins and expressed parotid proteins not normally present in submandibular glands. Sublingual gland mucous acinar and serous demilune cell differentiation were altered. Tlx1 is necessary for proper differentiation of submandibular and sublingual gland acinar cells, and granular convoluted ducts. The mechanism(s) underlying Tlx1 regulation of salivary gland development and differentiation remains unknown.

MANF inhibits Sjögren's syndrome salivary gland epithelial cell apoptosis and antigen expression of Ro52/SSA through endoplasmic reticulum stress/autophagy pathway.

Sjögren's syndrome (SS) is a typical autoimmune disease characterized by lymphocyte infiltration accompanied by the production of Ro52/SSA and La/SSB autoantibodies against whole body ribonucleoprotein particles. The release of type I IFN can induce endoplasmic reticulum stress (ERS) in submandibular gland cells. ERS not only produces a large number of Ro52/SSA antigens and changes their location, but also down-regulates autophagy and increases apoptosis. We collected human submandibular gland tissue samples, established an Experimental Sjögren's syndrome (ESS) mouse model, and used submandibular gland cells to test whether Mesencephalic astrocyte-derived neurotrophic factor (MANF) could reverse ERS-induced autophagy downregulation and reduce apoptosis and Ro52/SSA antigen expression. It was found that MANF could reduce lymphocyte infiltration and the proportion of CD4+ T cell subsets in the salivary glands, reduce the phosphorylation of AKT and mTOR proteins and the expression of ERS-related proteins, and increase the expression of autophagy proteins. We also found that MANF can reduce the expression of Ro52/SSA antigen on the cell membrane and reduce apoptosis. In short, we found that MANF can activate autophagy, inhibit apoptosis and reduce the expression of Ro52/SSA by regulating the AKT/mTOR/LC3B signaling pathway. The above results suggest that MANF may be a protective factor against SS.

Fabrication of polycaprolactone electrospun fibres with retinyl acetate for antioxidant delivery in a ROS-mimicking environment.

Background: Increased cancer rates denote that one in two people will be diagnosed with cancer in their lifetime. Over 60% of cancer patients receive radiotherapy, either as a stand-alone treatment or in combination with other treatments such as chemotherapy and surgery. Whilst radiotherapy is effective in destroying cancer cells, it also causes subsequent damage to healthy cells and surrounding tissue due to alterations in the tumor microenvironment and an increase in reactive oxygen species (ROS). This can cause extensive damage that impairs tissue function, and the likelihood of tissue regeneration and restoration of function is significantly reduced as new healthy cells cannot survive in the damaged environment. In the treatment of head and neck cancers, radiotherapy can cause salivary gland dysfunction. This significantly impairs the patient's quality of life and there is currently no cure, only palliative treatment options. Tissue engineering approaches are used to mimic the microenvironment of the tissue and can mediate the damaged microenvironment via bioactive compounds, to support the delivery, survival, and proliferation of new, healthy cells into the damaged environment. Methods: In this study, retinyl acetate, a derivative of vitamin A, was successfully incorporated into electrospun polycaprolactone fibres. Results: SEM images and characterization analyses showed that all scaffolds produced had similar characteristics, including fiber morphology and scaffold wettability. The vitamin scaffolds were shown to exert an antioxidant effect through scavenging activity of both DPPH and hydroxyl radicals in vitro. Critically, the antioxidant scaffolds supported the growth of human submandibular gland cells and significantly upregulated the expression of GPx1, an antioxidant enzyme, when cultured under both normal conditions and under a simulated oxidative stress environment. Discussion: These results suggest that incorporation of retinyl acetate into electrospun fibres has may mediate the damaged microenvironment post cancer radiation therapy.

TGF-β1 Triggers Salivary Hypofunction via Attenuating Protein Secretion and AQP5 Expression in Human Submandibular Gland Cells.

Aging-related salivary gland degeneration usually causes poor oral health. Periductal fibrosis frequently occurs in the submandibular gland of the elderly. Transforming growth factor β1 (TGF-β1) is the primary driving factor for fibrosis, which exhibits an increase in the fibrotic submandibular gland tissue. This study aimed to investigate the effects of TGF-β1 on the human submandibular gland (HSG) cell secretory function and its influences on aquaporin 5 (AQP5) expressions and distribution. We found that TGF-β1 reduces the protein secretion amount of HSG and leads to the abundance alteration of 151 secretory proteins. Data are available via ProteomeXchange with the identifier PXD043185. The majority of HSG secretory proteins (84.11%) could be matched to the human saliva proteome. Meanwhile, TGF-β1 enhances the expression of COL4A2, COL5A1, COL7A1, COL1A1, COL2A1, and α-SMA, hinting that TGF-β1 possesses the potential to drive HSG fibrosis-related events. Besides, TGF-β1 also attenuates the AQP5 expression and its membrane distribution in HSGs. The percentage for TGF-β1-induced AQP5 reduction (52.28%) is much greater than that of the TGF-β1-induced secretory protein concentration reduction (16.53%). Taken together, we concluded that TGF-β1 triggers salivary hypofunction via attenuating protein secretion and AQP5 expression in HSGs, which may be associated with TGF-β1-driven fibrosis events in HSGs.

Nigella sativa oil mitigates xerostomia and preserves salivary function in radiotherapy-treated mice.

This study aimed to assess if Nigella sativa oil (NSO), a health supplement containing thymoquinone as a major component, can act as a protective agent in salivary gland stem cells following radiotherapy (RT) damage. Forty, 10-week-old, male C3H/HeJ mice were randomized to four experimental groups: sham RT + H2O gavage (control) (N = 4); 15 Gy RT + H2O gavage (N = 12); sham RT + NSO gavage (N = 12); and 15 Gy RT + NSO gavage (N = 12). Weight changes, saliva production, and salivary gland histopathologic staining were recorded for each group over the course of the experiment. All mice in the sham RT + H2O gavage and sham RT + NSO gavage groups demonstrated 100% 60-day survival. RT + H2O compared to RT + NSO gavaged mice were significantly underweight by an average of 6.4 g (p < .001). Salivary output showed significant decline in RT + H2O gavaged mice at days 3 and 16, whereas salivary output in RT + NSO during these same time periods was comparable to the control. At day 60, all mice that survived recovered salivary function regardless of their treatment arm. Salivary specimens from the RT + NSO gavage group demonstrated early signs of recovery of Kr 5+ salivary gland stem cells in both submandibular and sublingual glands at day 16 with complete recovery by day 60, marked by strong histopathologic staining, whereas the RT + H2O gavage group did not recover as effectively. NSO may help preserve salivary function in mice treated with RT and may mitigate xerostomia by accelerating the recovery of salivary gland stem cells. Not applicable.