Submandibular Gland Cells Research Articles

Release of secretory immunoglobulin A by submandibular gland via β adrenergic receptor stimulation

ObjectiveSecretory immunoglobulin A (sIgA) is important for mucosal immunity due to the inhibition of pathogen infection. The submandibular gland is known to secrete more sIgA than the parotid and sublingual glands. In this study, we focused on the relationship between the secretion of accumulated intracellular sIgA and β-adrenergic receptor stimulation, and clarified the autonomic regulatory mechanism of sIgA secretion in submandibular gland cells using dispersed gland cells. DesignSprague-Dawley rats (male, 6 weeks old, 200–250 g) were euthanized and their submandibular glands were removed.Dispersed submandibular gland cells placed in Krebs-Ringer-Bicarbonate solution were stimulated by autonomic nerve agonists. The concentration of secreted sIgA was measured using a rat IgA ELISA kit. The results were analysed using ANOVA and Tukey’s test. ResultsCells stimulated with the non-selective β-adrenoreceptor agonist, isoprenaline, secreted significantly more sIgA compared with the unstimulated control. The β2-adrenoreceptor agonist, fenoterol, caused significantly more sIgA secretion than the control, and more sIgA secretion than the β1-adrenoreceptor agonist, xamoterol. sIgA secretion by isoprenaline stimulation was dose dependent. Inhibition of the β receptor by propranolol completely blocked sIgA secretion following isoprenaline stimulation. ConclusionStimulation of β receptors could result in more secretion of intracellularly accumulated sIgA compared with stimulation of other autonomic receptors in the autonomic modulation of mucosal immunity.

CTRP3 promotes TNF-α-induced apoptosis and barrier dysfunction in salivary epithelial cells

BackgroundC1q/tumour necrosis factor-related protein 3 (CTRP3) plays important roles in metabolism and inflammatory responses in various cells and tissues. However, the expression and function of CTRP3 in salivary glands have not been explored. MethodsThe expression and distribution of CTRP3 were detected by western blot, polymerase chain reaction, immunohistochemical and immunofluorescence staining. The effects of CTRP3 on tumour necrosis factor (TNF)-α-induced apoptosis and barrier dysfunction were detected by flow cytometry, western blot, co-immunoprecipitation, and measurement of transepithelial resistance and paracellular tracer flux. ResultsCTRP3 was distributed in both acinar and ductal cells of human submandibular gland (SMG) and was primarily located in the ducts of rat and mouse SMGs. TNF-α increased the apoptotic rate, elevated expression of cleaved caspase 3 and cytochrome C, and reduced B cell lymphoma-2 (Bcl-2) levels in cultured human SMG tissue and SMG-C6 cells, and CTRP3 further enhanced TNF-α-induced apoptosis response. Additionally, CTRP3 aggravated TNF-α-increased paracellular permeability. Mechanistically, CTRP3 promoted TNF-α-enhanced TNF type I receptor (TNFR1) expression, inhibited the expression of cellular Fas-associated death domain (FADD)-like interleukin-1β converting enzyme inhibitory protein (c-FLIP), and increased the recruitment of FADD with receptor-interacting protein kinase 1 and caspase 8. Moreover, CTRP3 was significantly increased in the labial gland of Sjögren's syndrome patients and in the serum and SMG of nonobese diabetic mice. ConclusionsThese findings suggest that the salivary glands are a novel source of CTRP3 synthesis and secretion. CTRP3 might promote TNF-α-induced cell apoptosis through the TNFR1-mediated complex II pathway.

Application of regenerative medicine to salivary gland hypofunction.

Dry mouth results from hypofunction of the salivary glands due to Sjögren's syndrome (SS), various medications, and radiation therapy for head and neck cancer. In severe cases of salivary gland hypofunction, sialagogues are not always effective due to the loss of salivary parenchyma. Therefore, regenerative medicine using stem cell therapy is a promising treatment for severe cases. Stem cells are classified into three groups: tissue stem cells, embryonic stem cells, and induced pluripotent stem cells. Tissue stem cells, such as hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and salivary stem/progenitor cells, could rescue irradiation-induced salivary gland hypofunction. Both HSCs and MSCs can rescue salivary gland hypofunction through soluble factors in a paracrine manner, while salivary stem/progenitor cells can reconstitute the damaged salivary glands. In fact, we clarified that CD133-positive cells in mouse submandibular glands showed stem cell features, which reconstituted the damaged salivary glands. Furthermore, we focused on the challenge of producing functional salivary glands that are three-dimensionally induced from mouse ES cells.

Cimetidine-induced androgenic failure causes cell death and changes in actin, EGF and V-ATPase immunoexpression in rat submandibular glands.

Submandibular gland (SMG) is responsive to androgens via androgen receptor (AR). We verified whether cimetidine induces androgenic dysfunction in SMG, and evaluated the structural integrity, cell death and immunoexpression of actin, EGF and V-ATPase in androgen-deficient SMG. Male rats received cimetidine (CMTG) and control animals (CG) received saline. Granular convoluted tubules (GCTs) diameter and number of acinar cell nuclei were evaluated. TUNEL and immunofluorescence reactions for detection of AR, testosterone, actin, EGF and V-ATPase were quantitatively analysed. In CG, testosterone immunolabelling was detected in acinar and ductal cells cytoplasm. AR-immunolabelled nuclei were observed in acinar cells whereas ductal cells showed AR-immunostained cytoplasm, indicating a non-genomic AR action. In CMTG, the weak testosterone and AR immunoexpression confirmed cimetidine-induced androgenic failure. A high cell death index was correlated with decreased number of acinar cells, GCTs diameter and EGF immunoexpression under androgenic dysfunction. Actin immunofluorescence decreased in the SMG cells, but an increased and diffuse cytoplasmic V-ATPase immunolabelling was observed in striated ducts, suggesting a disruption in the actin-dependent V-ATPase recycling due to androgenic failure. Our findings reinforce the androgenic role in the maintenance of SMG histophysiology, and point to a potential clinical use of cimetidine against androgen-dependent glandular tumour cells.

Short-chain fatty acid butyrate induces IL-10-producing B cells by regulating circadian-clock-related genes to ameliorate Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune disease caused by inflammation of the exocrine gland. The pathological hallmark of SS is the infiltration of lymphocytes into the salivary glands. Increased infiltration of T and B cells into salivary glands exacerbates symptoms of SS. Several recent studies have identified the role of gut microbiota in SS. Butyrate, one of the metabolites of the gut microbiota, regulates T cells; however, its effects on B cells and SS remain unknown. This study determined the therapeutic effect of butyrate on regulating B cells in SS. Various concentrations of butyrate were intraperitoneally injected three times per week in NOD/ShiLtJ (NOD) mice, the prototype animal model for SS, and observed for more than 10 weeks. Whole salivary flow rate and the histopathology of salivary glands were investigated. Human submandibular gland (HSG) cells and B cells in mouse spleen were used to confirm the anti-inflammatory and immunomodulatory effects of butyrate. Butyrate increased salivary flow rate in NOD mice and reduced inflammation of salivary gland tissues. It also regulated cell death and the expression of circadian-clock-related genes in HSG cells. Butyrate induced B cell regulation by increasing IL-10-producing B (B10) cells and decreasing IL-17-producing B cells, through the circadian clock genes RAR-related orphan receptor alpha and nuclear receptor subfamily 1 group D member 1. The findings of this study imply that butyrate may ameliorate SS via reciprocal regulation of IL-10- and IL-17-producing B cells.

Erythropoietin Plays a Protective Role in Submandibular Gland Hypofunction Induced by Irradiation

This study aims to explore the radioprotective effects of recombinant human erythropoietin (rhEPO) on rats' submandibular gland hypofunction induced by irradiation (IR). Thirty rats were divided into 3 groups: 1) control group, 2) IR group, and 3) IR+rhEPO group. The IR group and IR+rhEPO group received a single dose of 15Grays (Gy) (0.98Gy/min), plus, the IR+rhEPO group also received subcutaneous administration of rhEPO at a dose of 3,000 IU/kg body weight 3days before irradiation and then repeated every 24hours for the first 2 weeks after irradiation. Immunohistochemistry analysis to erythropoietin receptor was performed to detect the levels of erythropoietin receptor in submandibular glands with or without radiation. Ninetydays after irradiation, the salivary flow rates were assessed, and the submandibular gland of every rat was subjected to hematoxylin and eosin staining and immunohistochemical staining with antiaquaporin 5 and anti-proliferating cell nuclear antigen antibodies. Apoptosis was examined by the terminal deoxynucleotidyl transferase biotin-dUDP nick end-labeling assay. In addition, to examine the protective role of rhEPO on human submandibular gland cells, the apoptotic and proliferation rate of cells under a radiation dose of 8Gy was detected. One-way analysis of variance was carried out to analyze the results of each group, and the P value was set at 0.05. Erythropoietin receptor was expressed in the submandibular glands at a low level under normal conditions but upregulated after irradiation. rhEPO administration remarkably alleviated gland atrophy, increased salivary flow rates with upregulation of aquaporin-5 compared with the IR group. In addition, fewer apoptotic cells and more proliferative cells were observed in the IR+rhEPO group compared with the IR group, both invivo and invitro. rhEPO administration may be a useful countermeasure to mitigate submandibular gland hypofunction after therapeutic radiation exposure.

Cell Sheets Restore Secretory Function in Wounded Mouse Submandibular Glands.

Thermoresponsive cell culture plates release cells as confluent living sheets in response to small changes in temperature, with recovered cell sheets retaining functional extracellular matrix proteins and tight junctions, both of which indicate formation of intact and functional tissue. Our recent studies demonstrated that cell sheets are highly effective in promoting mouse submandibular gland (SMG) cell differentiation and recovering tissue integrity. However, these studies were performed only at early time points and extension of the observation period is needed to investigate duration of the cell sheets. Thus, the goal of this study was to demonstrate that treatment of wounded mouse SMG with cell sheets is capable of increasing salivary epithelial integrity over extended time periods. The results indicate that cell sheets promote tissue organization as early as eight days after transplantation and that these effects endure through Day 20. Furthermore, cell sheet transplantation in wounded SMG induces a significant time-dependent enhancement of cell polarization, differentiation and ion transporter expression. Finally, this treatment restored saliva quantity to pre-wounding levels at both eight and twenty days post-surgery and significantly improved saliva quality at twenty days post-surgery. These data indicate that cell sheets engineered with thermoresponsive cell culture plates are useful for salivary gland regeneration and provide evidence for the long-term stability of cell sheets, thereby offering a potential new therapeutic strategy for treating hyposalivation.

Proteomics-based analysis indicating α-enolase as a potential biomarker in primary Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease. Its etiology is not well understood. Salivary glands are the main target organ in pSS, investigating the changes of salivary protein in pSS patients may not only be a valuable way of identifying new biomarkers/antigens for pSS, but also of revealing the pathogenesis underlying this autoimmune disease. In the present study, we aimed to investigate new biomarkers and explore their potential role in pSS. In this study, α-enolase (ENO1) was found to be overexpressed in pSS by 1D gel electrophoresis/mass spectrometry. The finding was verified by Western blots, immunohistochemistry (IHC), and polymerase chain reaction (PCR) results in both saliva and labial salivary glands. The expression level of immunoglobulin G (IgG) antibody to ENO1 was then tested by enzyme-linked immunosorbent assay (ELISA). ENO1 autoantibody was found to be overexpressed in pSS compared with healthy controls. The effects of ENO1 overexpression on rat submandibular gland cell line SMG-C6 was investigated in vitro. The expressions of proteins related to saliva secretion and immunomodulatory were upregulated in ENO1 overexpressed SMG-C6 cells. Both ENO1 and anti-ENO1 autoantibody are overexpressed in pSS patients. Nevertheless, their potential role in the pathogenesis of pSS warrants further study.

Long-Term Maintenance of Acinar Cells in Human Submandibular Glands After Radiation Therapy

In a combined retrospective and prospective study, human salivary glands were investigated after radiation treatment for head and neck cancers. The aim was to assess acinar cell loss and morphologic changes after radiation therapy and to determine whether irradiated salivary glands have regenerative potential. Irradiated human submandibular and parotid salivary glands were collected from 16 patients at a range of time intervals after completion of radiation therapy (RT). Control samples were collected from 14 patients who had not received radiation treatments. Tissue sections were analyzed using immunohistochemistry to stain for molecular markers. Human submandibular and parotid glands isolated less than 1 year after RT showed a near complete loss of acinar cells. However, acinar units expressing functional secretory markers were observed in all samples isolated at later intervals after RT. Significantly lower acinar cell numbers and increased fibrosis were found in glands treated with combined radiation and chemotherapy, in comparison to glands treated with RT alone. Irradiated samples showed increased staining for duct cell keratin markers, as well as many cells coexpressing acinar- and duct cell-specific markers, in comparison to nonirradiated control samples. After RT, acinar cell clusters are maintained in human submandibular glands for years. The surviving acinar cells retain proliferative potential, although significant regeneration does not occur. Persistent DNA damage, increased fibrosis, and altered cell identity suggest mechanisms that may impair regeneration.

Cordycepin-loaded Nanoparticles from Cassava Starch Promote the Proliferation of Submandibular Gland Cells and Inhibit the Growth of Oral Squamous Carcinoma Cells

This study examined associations between the effect of treatment with nano-cassava starch that contained cordycepin (CS) extract, targeting human submandibular gland cells (HSGs), and human oral squamous carcinoma cells (HSC-4). Cassava starch nanoparticles (CSNPs) were prepared by either physical or acid treatment. These nanoparticles were then loaded with either CS or cordyceps medium and then treated with HSG or HSC-4 cells in different concentrations of CS and nanoparticles. Moreover, the protein secretion, reactive oxygen species (ROS) activity and the expression of salivary-specific genes, antioxidant gene were determined after treatment. CSNPs can enhance the activity of CS at low concentrations. Cordycepin-loaded cassava starch nanoparticles (CCSNPs) increased HSG proliferation, protein secretion, and the expression of salivary-specific genes, AMY and AQP5. Besides, CCSNPs also protected and scavenged of ROS via the stimulation of the antioxidant genes in HSGs, indicating the protective roles of CS to HSGs. On the other hand, CCSNPs inhibited the growth of HSC-4 cells by stimulating ROS generation and reducing protein secretion. This finding suggested that CCSNPs presented the dual actions against HSGs and human oral squamous carcinoma cells, and the encapsulation of CS with cassava nanoparticles enhanced the activity of CS.

An In Vitro Barrier Model of the Human Submandibular Salivary Gland Epithelium Based on a Single Cell Clone of Cell Line HTB-41: Establishment and Application for Biomarker Transport Studies.

The blood–saliva barrier (BSB) consists of the sum of the epithelial cell layers of the oral mucosa and salivary glands. In vitro models of the BSB are inevitable to investigate and understand the transport of salivary biomarkers from blood to saliva. Up to now, standardized, cell line-based models of the epithelium of the submandibular salivary gland are still missing for this purpose. Therefore, we established epithelial barrier models of the submandibular gland derived from human cell line HTB-41 (A-253). Single clone isolation resulted in five different clones (B2, B4, B9, D3, and F11). Clones were compared to the parental cell line HTB-41 using measurements of the transepithelial electrical resistance (TEER), paracellular marker permeability assays and analysis of marker expression for acinar, ductal, and myoepithelial cells. Two clones (B9, D3) were characterized to be of acinar origin, one clone (F11) to be of myoepithelial origin and one isolation (B4) derived from two cells, to be presumably a mixture of acinar and ductal origin. Clone B2, presumably of ductal origin, showed a significantly higher paracellular barrier compared to other clones and parental HTB-41. The distinct molecular identity of clone B2 was confirmed by immunofluorescent staining, qPCR, and flow cytometry. Experiments with ferritin, a biomarker for iron storage, demonstrated the applicability of the selected model based on clone B2 for transport studies. In conclusion, five different clones originating from the submandibular gland cell line HTB-41 were successfully characterized and established as epithelial barrier models. Studies with the model based on the tightest clone B2 confirmed its suitability for transport studies in biomarker research.

Mechanism of cAMP-PKA Signaling Pathway Mediated by Shaoyao Gancao Decoction () on Regulation of Aquaporin 5 and Muscarinic Receptor 3 Levels in Sjögren's Syndrome.

To investigate the mechanism of cAMP-PKA signaling pathway mediated by Chinese medicine formula Shaoyao Gancao Decoction (, SGD) on the regulation of aquaporin 5 (AQP5) and muscarinic receptor 3 (M3R) levels in Sjögren's syndrome (SS). Of the 30 mice, 5 were randomly selected as control, and others were used for creating SS model. After successful modeling, mice were randomly divided into 5 groups (n=5 per group) and intragastrically administered with saline (8 mL/kg), pilocarpine (1.4 mg/kg), or low, medium and high doses SGD (0.14, 0.21, 0.35 g/kg Radix paeoniae with 0.01 g/kg Radix glycyrrhizae, respectively) for 6 weeks. Human labial gland acinar cells were treated with pilocarpine or varying doses of SGD with saline as the placebo. Hematoxylin and eosin staining was used to observe the histopathological changes of the submandibular glands of mice. The serum levels of anti-SS antigen A (SS-A), anti-SS antigen B (SS-B), M3R, and α-fodrin in submandibular glands of mice were measured by enzyme-linked immunosorbent assay. Immunofluorescence staining was used to observe the spatial localization of AQP5 and M3R in acinar cells. Reverse transcriptase polymerase chain reaction and Western blot were used to detect the expressions of PKA, cAMP, Epac1, AQP5, M3R, nuclear factor kappa-B (NF-κB), and tumor necrosis factor (TNF)-α in submandibular gland tissues and cells of each group. Compared to normal mice, body weight, 5-min salivary secretion, 30-min secretion of tears and breakup time of tear film of model mice decreased at 1-6 weeks after immunization (all P<0.05), whereas water intake increased (all P<0.05). In the model group, glands of the submandibular glands showed atrophy, accompanied by acini of different sizes, decreased numbers and loose arrangement, with catheter dilatation and different degrees of lymphocyte infiltration. Conditions of mice in SGD groups were improved. The positive expression of AQP5 and M3R were higher in the acinar cells treated with all doses SGD compared to the normal group; serum levels of SS-A, SS-B, and α-fodrin were lower, and that of M3R was higher in all doses SGD treated animals than the model or pilocarpine treated ones (all P<0.05). Compared to the model and pilocarpine groups, the mRNA and protein levels of NF-κB and TNF-α were lower in mice or cells treated with medium or high-dose SGD (all P<0.05), while those of PKA, Epac1, AQP5 and M3R were higher (all P<0.05). SGD can improve symptoms of SS by regulating the cAMP-PKA signaling pathway and increasing AQP5 and M3R levels.

Characterization of antiapoptotic microRNAs in primary Sjögren's syndrome.

During the development of primary Sjögren's syndrome (pSS), aberrant expression of autoantigen is a hallmark event. To explore the regulation of autoantigen tripartite motif containing 21 (Ro/SSA, TRIM21), microRNA profiling was performed in our previous study. In which, two TRIM21-targeting microRNAs were identified, namely miR-1207-5p and miR-4695-3p. To further pursue their roles in the development of pSS, assays were performed with cultured human submandibular gland (HSG) cells, and salivary gland tissues. Results showed that transfection of miR-1207-5p or miR-4695-3p mimics down-regulated not only the expression of TRIM21, but also the levels of pro-apoptotic genes B cell lymphoma 2 associated X (BAX), Caspase 9 (CASP-9) and Caspase 8 (CASP-8). This finally led to antiapoptotic phenotypes in HSG cells. Consistent with the antiapoptotic activity, transfection of microRNA inhibitors up-regulated the expression of TRIM21 and led to a pro-apoptotic phenotype. These therefore propose miR-1207-5p and miR-4695-3p as two antiapoptotic microRNAs functioning through apoptosis pathway. Supporting this speculation, assays performed with salivary gland tissues revealed down-regulation of miR-1207-5p and miR-4695-3p, as well as up-regulation of TRIM21 and pro-apoptotic CASP-8 gene in pSS samples. SIGNIFICANCE OF THE STUDY: For pSS patients, apoptosis of acinar and ductal epithelial cells has been proposed to be a potential mechanism that impairs the secretion of salivary glands. In our study, two autoantigen-targeting microRNAs were characterized as antiapoptotic microRNAs functioning through apoptosis pathway, which may be potential targets for the treatment of pSS.

Pathogenic role of plasmacytoid dendritic cells in the development and onset of Sjögren’s syndrome-like exocrinopathy in non-obese diabetic mice

Abstract Plasmacytoid dendritic cells (pDCs), which are the most potent producers of type I IFNs, have been demonstrated to be critically involved in the induction of multiple autoimmune diseases. In Sjögren’s syndrome (SS), the presence of pDCs and activated type I IFN pathways has been shown in the salivary glands of patients, and the disease severity closely correlates with type I IFN activity. However, the particular role of pDCs in the pathogenesis of SS remains unknown. To investigate whether pDCs play a crucial role in the development and onset of SS, we administrated a pDC-depleting anti-BST2 antibody to female non-obese diabetic (NOD) mice, a spontaneous model of SS during the early stage of SS development, from 4 to 7 weeks of age. Mice were analyzed at 10 weeks of age around the clinical onset of the disease. Anti-BST2 treated mice had significantly higher salivary flow rate than IgG-treated control group, indicating the remission of SS-like xerostomia by pDC depletion. Meanwhile, anti-BST2 treatment inhibited leukocyte infiltration of submandibular glands (SMGs), as demonstrated by H&amp;E staining. Consistent with these results, flow cytometric analysis provided evidence that administration of anti-BST2 not only lowered the frequency of total leukocytes, CD4 T cells, CD8 T cells, B cells and pDCs, but also reduced the proportion of CD44+CD4 T cells, CD44+CD8 T cells and CD86+B cells among the total SMG cells of NOD mice. These changes were accompanied by downregulation of CD40, CD40L, Lt-α, Lt-β, TNF-α, IFN-γ, CXCL9, CXCL11 and NOS2, as determined by RT2 Profiler PCR array, suggesting an attenuation of SMG inflammation. Taken together, pDC depletion at the early stage of disease development hinders the pathogenesis of SS-like exocrinopathy.

Sex-mediated elevation of the specialized pro-resolving lipid mediator levels in a Sjögren's syndrome mouse model.

Our previous results showed that the specialized pro-resolving mediator (SPM) Resolvin D1 (RvD1) promotes resolution of inflammation in salivary glands in non-obese diabetic (NOD)/ShiLtJ, a mouse model for Sjögren's syndrome (SS). Additionally, mice lacking the RvD1 receptor ALX/FPR2 show defective innate and adaptive immune responses in salivary glands. Particularly, ALX/FPR2 KO mice exhibit exacerbated inflammation in their salivary glands in response to systemic LPS treatment. Moreover, female ALX/FPR2 KO mice show increased autoantibody production and loss of salivary gland function with age. Together, these studies suggest that an underlying SPM dysregulation could be contributing to SS progression. Therefore, we investigated whether SPM production is altered in NOD/ShiLtJ using metabololipidomics and enzyme-linked immunosorbent assay (ELISA). Our results demonstrate that SPM levels were broadly elevated in plasma collected from NOD/ShiLtJ female mice after disease onset, whereas these drastic changes did not occur in male mice. Moreover, gene expression of enzymes involved in SPM biosynthesis were altered in submandibular glands (SMG) from NOD/ShiLtJ female mice after disease onset, with 5-LOX and 12/15-LOX being downregulated and upregulated, respectively. Despite this dysregulation, the abundances of the SPM products of these enzymes (ie, RvD1 and RvD2) were unaltered in freshly isolated SMG cells suggesting that other cell populations (eg, lymphocytes) may be responsible for the overabundance of SPMs that we observed. The elevation of SPMs noted here appeared to be sex mediated, meaning that it was observed only in one sex (females). Given that SS primarily affects females (roughly 90% of diagnosed cases), these results may provide some insights into the mechanisms underlying the observed sexual dimorphism.

Exposure to Maghemite Nanoparticles Induces Epigenetic Alterations in Human Submandibular Gland Cells.

Even though nanotechnology has revolutionized the biomedical research, a plethora of studies debate the nanoparticles safety. In order to contribute to these studies, we evaluated the cytotoxic and epigenetic effects of maghemite nanoparticles covered with citric acid on human submandibular gland cells. Objective: This work objective was to evaluate the cytotoxic effects and epigenetic alterations induced in human salivary gland cells after treatment with maghemite nanoparticles covered with citric acid. Methods: For that, human submandibular gland cells were cultured and treated with nanoparticles for 24 or 48 hours. To assess cytotoxicity we used lactate dehydrogenase, a general oxidative stress indicator assay and microscopy. Epigenetic status was detected by colorimetric assays and the results were confirmed by quantitative polymerase chain reaction. Results: No cytotoxic effects were detected on cells exposed to up 3.0 mgFemL-1 for 48 hours, although cytoplasmic vacuoles formation was detected by light microscopy analyses. An increased generation of reactive oxygen species in cells exposed to nanoparticles was evidenced and iron clusters accumulated in the cytoplasm of treated cells. Global DNA methylation and histones H3 and H4 acetylation were also altered in response to nanoparticles exposure, thus suggesting a reprogramming of the epigenome. Transcripts accumulation analyses showed that genes related to iron metabolism and oxidative stress were upregulated, while the gene related to epigenetic reprogramming presented reduced transcript accumulation after treatment. Conclusion: We concluded that maghemite nanoparticles covered with citric acid exposure provoked several biological responses without impairment of human submandibular gland cells viability. This is the first report on the epigenetic effects of maghemite nanoparticles on this cell lineage.

Cytotoxic Tph-like cells are involved in persistent tissue damage in IgG4-related disease

Objectives The aim of this study was to determine pathological features of T peripheral helper (Tph)-like (PD-1+CXCR5−CD4+ T) cells in IgG4-related disease (IgG4-RD). Methods Tph-like cells in the blood and submandibular glands (SMGs) from IgG4-RD patients were analyzed by flow cytometry. Correlations between level of a Tph-like cell subset and clinical parameters of IgG4-RD were investigated. The cytotoxic capacity of Tph-like cells was also examined. Expression profiles of a molecule related to a Tph-like cell subset in IgG4-RD SMGs were assessed by immunohistochemistry. Results Tph-like cells from IgG4-RD patients highly expressed a fractalkine receptor, CX3CR1. Percentages of circulating CX3CR1+ Tph-like cells were significantly correlated with clinical parameters including IgG4-RD Responder Index, number of involved organs, and serum level of soluble IL-2 receptor. CX3CR1+ Tph-like cells abundantly possessed cytotoxic T lymphocyte-related molecules such as granzyme A, perforin, and G protein-coupled receptor 56. Functional assays revealed their cytotoxic potential against vascular endothelial cells and ductal epithelial cells. Immunohistochemistry showed that fractalkine was markedly expressed in vascular endothelial cells and ductal epithelial cells in IgG4-RD SMGs. Conclusion CX3CR1+ Tph-like cells are thought to contribute to persistent tissue injury in IgG4-RD and are a potential clinical marker and/or therapeutic target for inhibiting progression of IgG4-RD.

Characterization of the Active Components of Aurantii fructus Causing Dry Mouth by Analysis of Aquaporin 5 Expression

Aurantii fructus, the dried immature fruit of Citrus aurantium L., Rutaceae, or its cultivated variants, has been used in traditional Chinese medicine for thousands of years. Unprocessed Aurantii fructus can trigger various side effects, collectively known as “zao”. Dry mouth is the obvious symptom. Fengyan pieces, a processed product of Aurantii fructus, can relieve dry mouth but does not affect its efficacy. In order to analyze this mechanism, the effect on the gastrointestinal tract was characterized by measuring the levels of gastrin, a vasoactive intestinal peptide, in rat serum. Chemical analysis of Aurantii fructus pieces (raw product) and Fengyan pieces was performed by ultra-performance liquid chromatography-mass spectrometry (quadrupole time-of-flight) in combination with chemometrics methods to dereplicate the characteristic components. Aquaporin 5 expression in human submandibular gland cells was measured to validate the effect of the identified components. The results showed that serum gastrin content was different between the different dose groups, and Fengyan pieces showed lower levels of serum vasoactive intestinal peptide than Aurantii fructus pieces. 3,5,6,7,8,3′,4′-Heptamethoxyflavone was the distinctive component, and it has most effectively downregulated aquaporin 5 expression in the submandibular glands of mice. It also reduced aquaporin 5 mRNA and protein expression in human submandibular gland cells. Collectively, these results suggest that processing did not affect the efficacy of Fengyan pieces, but did relieve dry mouth and that 3,5,6,7,8,3′,4′-heptamethoxyflavone was the Aurantii fructus component responsible for dry mouth. This study provides a new scientific basis for reduction of the dry mouth effects of Aurantii fructus from processing.

Cordycepin attenuates Salivary Hypofunction through the Prevention of Oxidative Stress in Human Submandibular Gland Cells.

Xerostomia (dry mouth) is a significant age-related condition. Meanwhile, cordycepin, the natural therapeutic agent, has demonstrated an anti-aging effect. Therefore, the present study aimed to investigate the preventive effects of cordycepin on secretory function in an in vitro model of hydrogen peroxide (H2O2)-induced salivary hypofunction. After being exposed to H2O2, human submandibular gland (HSG) cells were treated with various concentrations of cordycepin (6.25-50 µM) for 24, 48, and 72h. To evaluate cell proliferation and reactive oxygen species (ROS) generation, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and 2, 7'-dichlorodihydrofluorescein diacetate assays were performed. The amylase activity was kinetically measured by 2-chloro-p-nitrophenol linked with maltotrioside. The expression of salivary, antioxidant and apoptotic markers at mRNA and protein levels were performed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence analysis, respectively. We demonstrated that cordycepin (6.25-25 µM) contributed to significant increases in expression of the salivary marker genes, alpha-amylase 1 (AMY1A) and aquaporin-5 (AQP5), and in amylase secretion without changes in cell viability. Under oxidative stress, HSG cells showed remarkable dysfunction. Cordycepin rescued the protective effects partially by decreasing ROS generation and restoring the expression of the salivary proteins, AMY and AQP5 via anti-oxidant and anti-apoptotic activity. In addition, the amount of amylase that was secreted from HSG cells cultured in cordycepin was increased. In conclusion, cordycepin demonstrated a protective effect on H2O2-induced HSG cells by decreasing ROS generation and upregulating the salivary function markers, AMY1A and AQP5, at both the transcriptional and translational levels.

Disruption of tight junction structure contributes to secretory dysfunction in IgG4-related sialadenitis.

IgG4-related sialadenitis (IgG4-RS) is a chronic fibro-inflammatory disease characterized by swelling of salivary glands and varying degrees of xerostomia. Tight junctions (TJs) play an essential role in maintaining secretory function by regulating the paracellular flow of ions and water. However, whether TJs are altered and contribute to the hyposecretion in IgG4-RS is not fully understood. Here, a total of 399 differentially expressed proteins were identified in IgG4-RS submandibular glands (SMGs) and enriched in the regulation of actin cytoskeleton and the salivary secretion. Real-time PCR results showed that the mRNA levels of claudin-3, -4, -6, -7, -8, -10, -12, occludin, and ZO-1 were significantly lower, whereas claudin-1 and -5 were higher in IgG4-RS SMGs. Immunohistochemical and immunofluorescence staining revealed that claudin-1, -3, -4, occludin, and ZO-1 were mainly distributed at apicolateral membranes in acini and ducts of SMGs from controls, whereas claudin-1 protein intensity at apicolateral membrane was elevated, while the staining of claudin-3, -4, and ZO-1 were reduced in IgG4-RS SMGs. Occludin was dispersed into cytoplasm of acini and ducts in SMGs of patients. Among them, claudin-3 and ZO-1 protein levels were positively correlated with saliva flow rate. Furthermore, the decreased fluorescence intensity of F-actin at peri-apicolateral membranes and the loss of ZO-1 staining at the same location were observed in acinar and ductal cells of IgG4-RS SMGs, which might be responsible for disorganization of TJ complex. Taken together, these findings indicate that the integrity of TJ complex of SMGs is impaired and might contribute to hyposalivation of IgG4-RS patients.