Mouse Submandibular Gland Research Articles

Apigenin alleviates Sjögren’s syndrome-induced salivary gland epithelial cell ferroptosis via ERα signaling-mediated regulation of the ATF3/SLC7A1l axis

BackgroundIn Sjögren’s syndrome (SS)—an autoimmune disease characterized by dry mouth and eyes—salivary gland epithelial cells (SGECs) undergo ferroptosis, which disrupts their integrity and impairs saliva secretion. Apigenin, a phytoestrogen, is known to activate estrogen signalling and alleviate xerostomia in ovariectomized mice; however, its effect on SGEC survival and function in SS remains unclear. We hypothesized that apigenin alleviates SS symptoms and progression by inhibiting ferroptosis in SGECs and aimed to elucidate the underlying mechanism. MethodsApigenin (50 mg/kg) was orally gavaged to non-obese diabetic (NOD)/LtJ female mice (SS model); changes in SS functional indicators were analyzed using mRNA sequencing and bioinformatic analyses of submandibular glands. Interferon-gamma (IFN-γ)-stimulated SGECs were used to model SS in vitro; SGEC activity and aquaporin 5 (AQP5) expression were analyzed. Immunohistochemical staining, transmission electron microscopy, RT-qPCR, western blotting and other methods were used to verify the mechanisms. ResultsApigenin significantly increased salivary secretion and AQP5 expression while inhibiting ferroptosis and immune infiltration in NOD mouse submandibular glands. The oxidative stress gene ATF3 was upregulated and GPX4 was downregulated in NOD mice compared to that in control group (ICR mice); however, apigenin reversed this effect. IFN-γ treatment downregulated AQP5, SLC7A11, and GPX4 expression while promoting ATF3 expression and ferroptosis, which was mitigated by apigenin. ATF3 knockdown increased SLC7A11 and GPX4 expression, inhibiting SS and ferroptosis. Furthermore, apigenin inhibited ferroptosis in SGECs through ESR1 binding to ATF3. ConclusionApigenin alleviates SS by regulating SGEC ferroptosis via the ERα-regulated ATF3/SLC7A11 axis, highlighting its therapeutic potential 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.

Expression of Glucagon-Like Peptide-1 Receptors in the Submandibular Gland of Mice and Its Implications in Type 2 Diabetes.

Introduction Type 2 diabetes mellitus (T2DM) not only affects the pancreas directly involved in glucose metabolism but also impairs salivary gland function. Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that lowers postprandial blood glucose levels by stimulating insulin secretion from the pancreas. Previous studies have revealed the presence of GLP-1 receptors (GLP-1R) in salivary glands. However, the effect of diabetes on salivary gland GLP-1R remains unclear. This study aimed to observe the impact of T2DM on GLP-1R in the submandibular gland (SMG). Materials and methods Twenty-five-week-old mice were randomly divided into four groups (n=5 each): 11w and 13w control groups (CON), and 11w and 13wdiabetes mellitusgroups (DM). After a five-day adaptation period, the DM group mice were subjected to a high-fat diet, while the CON group received a standard diet. The DM group mice were then induced into a state of T2DM by a single low-dose streptozotocin injection at nine weeks of age. Oral glucose tolerance tests (OGTT) were conducted to evaluate mouse glucose tolerance. At 11 and 13 weeks of age, SMG was excised under general anesthesia, and the morphology of SMG was evaluated by hematoxylin-eosin staining, while the distribution and expression of GLP-1R were assessed by immunohistochemical staining. The data obtained were subjected to the Shapiro-Wilk test to confirm normal distribution, the t-test for the OGTTresults, and statistical analysis for other results by one-way analysis of variance. Results Consistent with previous reports, the mice in the DM group showed higher body weight and lower glucose tolerance.Histological analysis revealed an increase in the acinar area and a decrease in the ductal area of the SMG in the DM group. Although there was no significant decrease in the cell count regarding the ductal area, a tendency towardluminal dilation was observed. Interestingly, the expression pattern of GLP-1R was limited to the ductal portion of the SMG, with a decrease in anti-GLP-1R-positive areas observed in the DM group compared to the CON group.While there was no significant difference in anti-GLP-1R-positive areas between the CON11w and CON13w groups, the DM13w group exhibited a significant decrease compared to the DM11w group. These data suggest that diabetes induces both structural changes in the SMG and a reduction in GLP-1R expression, particularly in the ductal regions. Conclusions We found that the expression level of GLP-1R in SMG was decreased in the DM group mice. This data demonstrates the potential relationship between T2DM and GLP-1R expression. Moreover, there was an indication of a temporal decrease in anti-GLP-1R positive areas over time.Thisresult may suggest the involvement of salivary gland GLP-1R in the mechanism of impaired SMG function caused by T2DM, potentially mediated through the decrease in blood GLP-1 levels.

Y-27632 enables long-term expansion of mouse submandibular gland epithelial cells via inactivation of TGF-β1/CTGF/p38 and ROCK2/JNK signaling pathway

ObjectivesThis study aimed to investigate the effects of Y-27632 on the long-term maintainence of mouse submandibular epithelial cells (SG-Epis) in vitro and to elucidate the underlying mechanisms. MethodsThe role of the Rho-associated kinase (ROCK) inhibitor Y-27632 in maintaining SG-Epis and its underlying mechanisms were evaluated by examining the in vitro expansion of mouse SG-Epis. Changes in key cellular characteristics, such as proliferation, long-term expansion, and mRNA and protein expression, were assessed in the presence or absence of Y-27632. ResultsTreatment with Y-27632 significantly enhanced the proliferative potential of SG-Epis, preserving Krt8 and Krt14 expression over 17 passages. In the absence of Y-27632, SG-Epis lost their epithelial morphology. However, Y-27632 treatment maintained the epithelial morphology and downregulated mRNA levels of Tgf-β1, Ctgf, and Rock2. Treatment with TGF-β1 indicated that TGF-β/CTGF/p38 signaling is responsible for the maintenance of SG-Epis, while RNA interference studies revealed that ROCK2/c-Jun N-terminal kinase (JNK) signaling is also crucial for SG-Epis proliferation and maintenance. ConclusionsThe TGF-β1/CTGF/p38 and ROCK2/JNK signaling pathways are responsible for SG-Epis proliferation, and Y-27632 treatment effectively inactivates these pathways, enabling long-term in vitro maintenance of SG-Epis. The culture method utilizing Y-27632 provides an effective approach for the in vitro expansion of SG-Epis.

Upregulation of Amy1 in the salivary glands of mice exposed to a lunar gravity environment using the multiple artificial gravity research system.

Introduction: Space is a unique environment characterized by isolation from community life and exposure to circadian misalignment, microgravity, and space radiation. These multiple differences from those experienced on the earth may cause systemic and local tissue stress. Autonomic nerves, including sympathetic and parasympathetic nerves, regulate functions in multiple organs. Saliva is secreted from the salivary gland, which is regulated by autonomic nerves, and plays several important roles in the oral cavity and digestive processes. The balance of the autonomic nervous system in the seromucous glands, such as the submandibular glands, precisely controls serous and mucous saliva. Psychological stress, radiation damage, and other triggers can cause an imbalance in salivary secretion systems. A previous study reported that amylase is a stress marker in behavioral medicine and space flight crews; however, the detailed mechanisms underlying amylase regulation in the space environment are still unknown. Methods: In this study, we aimed to elucidate how lunar gravity (1/6g) changes mRNA expression patterns in the salivary gland. Using a multiple artificial gravity research system during space flight in the International Space Station, we studied the effects of two different gravitational levels, lunar and Earth gravity, on the submandibular glands of mice. All mice survived, returned to Earth from space, and their submandibular glands were collected 2days after landing. Results: We found that lunar gravity induced the expression of the salivary amylase gene Amy1; however, no increase in Aqp5 and Ano1, which regulate water secretion, was observed. In addition, genes involved in the exocrine system, such as vesicle-associated membrane protein 8 (Vamp8) and small G proteins, including Rap1 and Rab families, were upregulated under lunar gravity. Conclusion: These results imply that lunar gravity upregulates salivary amylase secretion via Rap/Rab signaling and exocytosis via Vamp8. Our study highlights Amy1 as a potential candidate marker for stress regulation in salivary glands in the lunar gravity environment.

TGF-β1 maintains the developmental potential of embryonic submandibular gland epithelia separated with mesenchyme

ObjectiveThe objective of this study was to investigate the impact of transforming growth factor β1 (TGF-β1) on epithelial development using an ex vivo model of submandibular gland (SMG) epithelial-mesenchymal separation. Materials and methodsThe ex vivo model was established by separating E13 mouse SMG epithelia and mesenchyme, culturing them independently for 24 h, recombining them, and observing branching morphogenesis. Microarray analysis was performed to evaluate the transcriptome of epithelia treated with and without 1 ng/ml TGF-β1. Differential gene expression, pathway enrichment, and protein-protein interaction networks were analyzed. Quantitative real-time polymerase chain reaction, Western blot, and immunofluorescence were employed to validate the mRNA and protein levels. ResultsRecombined SMGs using separated epithelia and mesenchyme that were cultured for 24 h showed a significant inhibition of epithelial development compared to SMGs recombined immediately after separation. The level of TGF-β1 decreased in the SMG epithelia after epithelia-mesenchyme separation. Epithelia that were separated from mesenchyme for 24 h and pretreated with 1 ng/ml TGF-β1 continued to develop after recombination with mesenchyme, while epithelia without 1 ng/ml TGF-β1 treatment did not. Microarray analysis suggested pathway enrichment related to epithelial development and an upregulation of Sox2 in the 1 ng/ml TGF-β1-treated epithelia. Further experiments validated the phosphorylation of SMAD2 and SMAD3, upregulation of SOX2 and genes associated with epithelial development, including Prol1, Dcpp1, Bhlha15, Smgc, and Bpifa2. Additionally, 1 ng/ml TGF-β1 inhibited epithelial apoptosis by improving the BCL2/BAX ratio and reducing cleaved caspase 3. ConclusionsThe addition of 1 ng/ml TGF-β1 maintained the developmental potential of embryonic SMG epithelia separated from mesenchyme for 24 h. This suggests that 1 ng/ml TGF-β1 may partially compensate for the role of mesenchyme during the separation phase, although its compensation is limited in extent.

Establishment and characterization of cMYB-expressing human salivary adenoid cystic carcinoma cell lines (UM-HACC-14, UM-HACC-6) and matching patient-derived xenograft model (UM-PDX-HACC-14)

Limited availability of authentic human adenoid cystic carcinoma (ACC) cell lines has hindered progress in understanding mechanisms underpinning the biology of this disease and the development of safe and effective therapies. Surgical human ACC specimens (UM-HACC-6, UM-HACC-14) were dissociated into single cell suspensions and cultured in fibronectin-coated flasks. Alternatively, tumor fragments were transplanted subcutaneously into female immunodeficient (SCID) mice to establish patient-derived xenograft tumors (PDX; UM-PDX-HACC-14). Both ACC cell lines showed continuous growth in monolayers for over 100 passages. Total RNA-Seq, RT-PCR, and FISH analysis revealed that both are MYB-NFIB fusion negative. Western blots revealed passage-dependent expression of E-Cadherin, PCNA, p63, phospho-c-MYB, and NFIB. Both, UM-HACC-14 and UM-HACC-6 cells exhibited tumorigenic potential when injected orthotopically into mouse submandibular glands. UM-HACC-14, patient-matching UM-PDX-HACC-14, and the UM-HACC-6 cell line are new, authenticated preclinical models of ACC that are well suited for mechanistic and developmental therapeutics studies.

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.

ΔNp63 regulates Sfrp1 expression to direct salivary gland branching morphogenesis.

Branching morphogenesis is a complex process shared by many organs including the lungs, kidney, prostate, as well as several exocrine organs including the salivary, mammary and lacrimal glands. This critical developmental program ensures the expansion of an organ's surface area thereby maximizing processes of cellular secretion or absorption. It is guided by reciprocal signaling from the epithelial and mesenchymal cells. While signaling pathways driving salivary gland branching morphogenesis have been relatively well-studied, our understanding of the underlying transcriptional regulatory mechanisms directing this program, is limited. Here, we performed in vivo and ex vivo studies of the embryonic mouse submandibular gland to determine the function of the transcription factor ΔNp63, in directing branching morphogenesis. Our studies show that loss of ΔNp63 results in alterations in the differentiation program of the ductal cells which is accompanied by a dramatic reduction in branching morphogenesis that is mediated by dysregulation of WNT signaling. We show that ΔNp63 modulates WNT signaling to promote branching morphogenesis by directly regulating Sfrp1 expression. Collectively, our findings have revealed a novel role for ΔNp63 in the regulation of this critical process and offers a better understanding of the transcriptional networks involved in branching morphogenesis.

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.

Immunohistochemical Localization of YAP and TAZ in Mouse Submandibular Gland Morphogenesis

Immunohistochemical Localization of YAP and TAZ in Mouse Submandibular Gland Morphogenesis

TARGET ONCOGENIC RECEPTORS IN TUMOURS, FROM ITS INITIAL CLINICAL BREAKTHROUGHS TO CURRENT CLINICAL STANDARD THERAPY

Epidermal growth factor (EGF) is a polypeptide hormone originally isolated from mouse submaxillary gland, which is a potent mitogen for a wide variety of culture cells of both epithelial and mesenchymal origin, and its important role in the proliferation, differentiation, and survival of neural and glial precursor cells. The physiological effects of EGF are mediated by an EGF receptor with tyrosine-specific protein kinase activity. The traditionally accepted view is that normal epidermal growth factor receptor (EGFR) is no tumorigenic, whereas mutated EGFR such as an oncogenic receptor EGFRvIII is oncogenic. In recent, it has been implicated that EGF could be beneficial for burn, wound healing, diabetic foot ulcer, and show an attractive perspective. In addition, cosmetic containing EGF would be effective in improving the plasticity of skin, helps in anti-aging and whitening, and controls the amount of erythema and sebum in the skin. In our team we have successfully prepared a series of 350 bottles of Shampoo liquid and 26 bottles of recombinant human EGF (rhEGF) spray, and 4 bottles of EGF-Silvadence ointment. The initial results indicated that prepared rhEGF is safe and available in clinical use. On the other hand, progress on the interaction of EGF with its altered oncogenic receptor signaling through its downstream molecules such Ras/Raf/MAPK (see figure in the full text, George Zhu,1991) and/or PI3k/akt pathway in the development of some cancers such as A431 human epidermoid carcinoma cells, brain glioblastoma, breast, pancreas and lung cancers. In addition to a series of gefitinib, erlotinib, osimertinib and the CIMA vax-EGF vaccine, an antioncogenic receptor antibody based fusion protein [for example Cetuximab-based IL-10 fusion protein, CmAb-(IL10)2] provide a potential strategy to improve cancer immunotherapy. Peer Review History: Received 1 December 2023; Revised 26 January 2024; Accepted 2 March; Available online 15 March 2024 Academic Editor: Dr. Tamer Elhabibi, Suez Canal University, Egypt, tamer_hassan@pharm.suez.edu.eg Average Peer review marks at initial stage: 6.0/10 Average Peer review marks at publication stage: 8.0/10 Reviewers: Dr. Bilge Ahsen KARA, Ankara Gazi Mustafa Kemal Hospital, Turkey, ahsndkyc@gmail.com Dr. Asia Selman Abdullah, University of Basrah, Iraq, asia_abdullah65@yahoo.com

Ser252Trp mutation in fibroblast growth factor receptor 2 promotes branching morphogenesis in mouse salivary glands

ObjectivesThe purpose of this study was to perform morphological and immunohistochemical (IHC) analysis of the submandibular glands (SMGs) in early development in Apert syndrome model mice (Ap mice). MethodsACTB-Cre homozygous mice were mated with fibroblast growth factor receptor 2 (Fgfr2+/Neo-S252W) mice; ACTB-Cre heterozygous mice (ACTB-Cre mice) at embryonic day (E) 13.5 served as the control group, and Fgfr2+/S252W mice (Ap mice) served as the experimental group. Hematoxylin and eosin (H&E) staining was performed on SMGs; Total SMG area and epithelial area were determined, and the epithelial occupancy ratio was calculated. Immunostaining was performed to assess the localization of FGF signaling-related proteins. Next, bromodeoxyuridine (BrdU)-positive cells were evaluated to assess cell proliferation. Finally, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to assess apoptosis in SMGs. ResultsThe epithelial occupancy ratio was significantly higher in SMGs of Ap mice compared with that in SMGs of controls. FGF7 and bone morphogenetic protein 4 (BMP4) exhibited different localizations in SMGs of Ap mice compared with SMGs of controls. Cell proliferation was higher in SMGs of Ap mice compared with that of controls; however, apoptosis did not different significantly between the two groups. ConclusionOur results suggest that enhanced FGF signaling conferred by missense mutations in FGFR2 promotes branching morphogenesis in SMGs of Ap mice.

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.

Claudin-10 Decrease in the Submandibular Gland Contributes to Xerostomia

Tight junction proteins play a crucial role in paracellular transport in salivary gland epithelia. It is clear that severe xerostomia in patients with HELIX syndrome is caused by mutations in the claudin-10 gene. However, little is known about the expression pattern and role of claudin-10 in saliva secretion in physical and disease conditions. In the present study, we found that only claudin-10b transcript was expressed in human and mouse submandibular gland (SMG) tissues, and claudin-10 protein was dominantly distributed at the apicolateral membranes of acini in human, rat, and mouse SMGs. Overexpression of claudin-10 significantly reduced transepithelial electrical resistance and increased paracellular transport of dextran and Na+ in SMG-C6 cells. In C57BL/6 mice, pilocarpine stimulation promoted secretion and cation concentration in saliva in a dose-dependent increase. Assembly of claudin-10 to the most apicolateral portions in acini of SMGs was observed in the lower pilocarpine (1 mg/kg)–treated group, and this phenomenon was much obvious in the higher pilocarpine (10 mg/kg)–treated group. Furthermore, 7-, 14-, and 21-wk-old nonobese diabetic (NOD) and BALB/c mice were used to mimic the progression of hyposalivation in Sjögren syndrome. Intensity of claudin-10 protein was obviously lower in SMGs of 14- and 21-wk-old NOD mice compared with that of age-matched BALB/c mice. In the cultured mouse SMG tissues, interferon-γ (IFN-γ) downregulated claudin-10 expression. In claudin-10–overexpressed SMG-C6 cells, paracellular permeability was decreased. Furthermore, IFN-γ stimulation increased p-STAT1 level, whereas pretreatment with JAK/STAT1 antagonist significantly alleviated the IFN-γ–induced claudin-10 downregulation. These results indicate that claudin-10 functions as a pore-forming component in acinar epithelia of SMGs, assembly of claudin-10 is required for saliva secretion, and downregulation of claudin-10 induces hyposecretion. These findings may provide new clues to novel therapeutic targets on hyposalivation.

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging.

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration. The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

Gut microbiota combined with fecal metabolomics reveals the effects of FuFang Runzaoling on the microbial and metabolic profiles in NOD mouse model of Sjögren’s syndrome

ObjectiveSjögren’s syndrome (SS) is an inflammatory autoimmune disease characterized by high levels of chronic lymphocyte infiltration. Differences and dysfunction in the gut microbiota and metabolites may be closely related to the pathogenesis of SS. The purpose of this study was to reveal the relationship between the gut microbiota and metabolome in NOD mice as a model of SS and the role of FuFang Runzaoling (FRZ), which is a clinically effective in treating SS.MethodsNOD mice were gavaged with FRZ for 10 weeks. The ingested volume of drinking water, submandibular gland index, pathologic changes of the submandibular glands, and serum cytokines interleukin (IL)-6, IL-10, IL-17 A, and tumor necrosis factor-alpha (TNF-α) were determined. The roles of FRZ on gut microbiota and fecal metabolites were explored by 16 S rRNA gene sequencing and liquid chromatography-mass spectrometry (LC-MC), respectively. The correlation between them was determined by Pearson correlation analysis.ResultsCompared with the model group, the drinking water volume of NOD mice treated with FRZ increased and the submandibular gland index decreased. FRZ effectively ameliorated lymphocyte infiltration in the small submandibular glands in mice. Serum levels of IL-6, TNF-α, and IL-17 A decreased, and IL-10 increased. The Firmicutes/Bacteroidetes ratio in the FRZ treatment group was higher. FRZ significantly downregulated the relative abundance of the family Bacteroidaceae and genus Bacteroides, and significantly upregulated the relative abundance of genus Lachnospiraceae_UCG-001. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) revealed the significant change in fecal metabolites after FRZ treatment. Based on criteria of OPLS-DA variable influence on projection > 1, P < 0.05, and fragmentation score > 50, a total of 109 metabolites in the FRZ-H group were differentially regulated (47 downregulated and 62 upregulated) compared to their expressions in the model group. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed enriched metabolic of sphingolipid metabolism, retrograde endocannabinoid signaling, GABAergic synapse, necroptosis, arginine biosynthesis, and metabolism of histidine, alanine, aspartate, and glutamate. Correlation analysis between gut microbiota and fecal metabolites suggested that the enriched bacteria were related to many key metabolites.ConclusionsTaken together, we found FRZ could reduce the inflammatory responses in NOD mice by regulating the gut microbiota, fecal metabolites, and their correlation to emerge a therapeutic effect on mice with SS. This will lay the foundation for the further studies and applications of FRZ, and the use of gut microbiotas as drug targets to treat SS.Graphical

Major depression-related factor NEGR1 controls salivary secretion in mouse submandibular glands

Salivary gland cells, which secrete water in response to neuronal stimulation, are closely connected to other neurons. Transcriptomic studies show that salivary glands also express some proteins responsible for neuronal function. However, the physiological functions of these common neuro-exocrine factors in salivary glands are largely unknown. Here, we studied the function of Neuronal growth regulator 1 (NEGR1) in the salivary gland cells. NEGR1 was also expressed in mouse and human salivary glands. The structure of salivary glands of Negr1 knockout (KO) mice was normal. Negr1 KO mice showed tempered carbachol- or thapsigargin-induced intracellular Ca2+ increases and store-operated Ca2+ entry. Of interest, the activity of the large-conductance Ca2+-activated K+ channel (BK channel) was increased, whereas Ca2+-activated Cl- channel ANO1 channel activity was not altered in Negr1 KO mice. Pilocarpine- and carbachol-induced salivation was decreased in Negr1 KO mice. These results suggest that NEGR1 influence salivary secretion though the muscarinic Ca2+ signaling.

SHED-derived exosomes ameliorate hyposalivation caused by Sjögren's syndrome via Akt/GSK-3β/Slug-mediated ZO-1 expression.

Sjögren's syndrome (SS) is an autoimmune disorder characterized by sicca syndrome and/or systemic manifestations. The treatment is still challenging. This study aimed to explore the therapeutic role and mechanism of exosomes obtained from the supernatant of stem cells derived from human exfoliated deciduous teeth (SHED-exos) in sialadenitis caused by SS. SHED-exos were administered to the submandibular glands (SMGs) of 14-week-old non-obese diabetic (NOD) mice, an animal model of the clinical phase of SS, by local injection or intraductal infusion. The saliva flow rate was measured after pilocarpine intraperitoneal injection in 21-week-old NOD mice. Protein expression was examined by western blot analysis. Exosomal microRNA (miRNAs) were identified by microarray analysis. Paracellular permeability was evaluated by transepithelial electrical resistance measurement. SHED-exos were injected into the SMG of NOD mice and increased saliva secretion. The injected SHED-exos were taken up by glandular epithelial cells, and further increased paracellular permeability mediated by zonula occluden-1 (ZO-1). A total of 180 exosomal miRNAs were identified from SHED-exos, and Kyoto Encyclopedia of Genes and Genomes analysis suggested that the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway might play an important role. SHED-exos treatment down-regulated phospho-Akt (p-Akt)/Akt, phospho-glycogen synthase kinase 3β (p-GSK-3β)/GSK-3β, and Slug expressions and up-regulated ZO-1 expression in SMGs and SMG-C6 cells. Both the increased ZO-1 expression and paracellular permeability induced by SHED-exos were abolished by insulin-like growth factor 1, a PI3K agonist. Slug bound to the ZO-1 promoter and suppressed its expression. For safer and more effective clinical application, SHED-exos were intraductally infused into the SMGs of NOD mice, and saliva secretion was increased and accompanied by decreased levels of p-Akt/Akt, p-GSK-3β/GSK-3β, and Slug and increased ZO-1 expression. Local application of SHED-exos in SMGs can ameliorate Sjögren syndrome-induced hyposalivation by increasing the paracellular permeability of glandular epithelial cells through Akt/GSK-3β/Slug pathway-mediated ZO-1 expression.

Examination of age-related changes in the submandibular glands of male mice.

Salivary gland hypofunction adversely affects the oral environment and daily life by causing dry mouth (xerostomia). Senescence-related atrophy of salivary gland tissues is one cause of xerostomia, and it is particularly common among the elderly. However, the underlying mechanism is poorly understood, and no treatment has been established. Therefore, we examined age-related changes in senescence-associated secretory phenotype (SASP) factors, which regulate stemness and cellular senescence, in mouse submandibular glands. We analyzed the submandibular glands of 6-week-old (young group, n = 6) and 82-week-old mice (aged group, n = 6). We performed salivary flow rate measurements, histological analysis including immunohistochemistry, and quantitative real-time PCR. The salivary flow rate was significantly lower in the aged group than in the young group. In addition, immunostaining and quantitative real-time PCR illustrated that aquaporin-5 and α-amylase expressions were significantly decreased in aged mice, indicating salivary gland hypofunction. c-Kit and cytokeratin 5 expressions were also significantly decreased in this group, suggesting that the regenerative abilities of the submandibular glands were reduced because of decreased stem and progenitor cell counts. Furthermore, the levels of p16INK4a and p21 (the senescence markers) and TGF-β1 and IL-6 (SASP factors) were significantly increased in mice, suggesting that senescence had been promoted. The decreased numbers of stem and progenitor cells and increased levels of SASP factors might be associated with age-related changes in mouse submandibular glands. These results might facilitate the development of treatments for senescence-related submandibular gland hypofunction.