Glycyrrhizic acid ameliorates submandibular gland oxidative stress, autophagy and vascular dysfunction in rat model of type 1 diabetes.

The burden of diabetes mellitus (DM) and associated complications is increasing worldwide, affecting many organ functionalities including submandibular glands (SMG). The present study aims to investigate the potential ameliorative effect of glycyrrhizic acid (GA) on diabetes-induced SMG damage. Experimental evaluation of GA treatment was conducted on a rat model of type I diabetes. Animals were assigned to three groups; control, diabetic and GA treated diabetic groups. After 8 weeks, the SMG was processed for assessment of oxidative stress markers, autophagy related proteins; LC3, Beclin-1 and P62, vascular regulator ET-1, aquaporins (AQPs 1.4 and 5), SIRT1 protein expressions in addition to LC3 and AQP5 mRNA expressions. Also, parenchymal structures of the SMG were examined. GA alleviated the diabetes-induced SMG damage via restoring the SMG levels of oxidative stress markers and ET-1 almost near to the normal levels most probably via regulation of SIRT1, AQPs and accordingly LC-3, P62 and Beclin-1levels. GA could be a promising candidate for the treatment of diabetes-induced SMG damage via regulating oxidative stress, autophagy and angiogenesis.

Smad7 attenuates TGF-ß-mediated aging-related hypofunction of submandibular glands.

Submandibular glands have essential functions in taste, mastication, swallowing, and digestion. Submandibular gland hypofunction is prevalent in the elderly, impairing the patients' quality of life. Current clinical treatment strategies have not decelerated or reversed the pathological process of submandibular gland hypofunction. Therefore, novel restoration strategies should be explored. However, studies on the mechanism of aging-related submandibular gland hypofunction remain very limited. The role of the TGF-ß/Smad pathway in fibrosis has been studied in other organs. Therefore, this study aimed to elucidate the role of TGF-ß/Smad signaling in the aging-related submandibular gland hypofunction. The results showed that Smad7 knockout in mice decreased the salivary flow rate. H&E, Masson trichrome, and immunohistochemistry staining of MCP-1 and α-SMA showed that Smad7 knockout in mice resulted in lymphocytic infiltration, acinar cell atrophy, and interstitial fibrosis. The Western blotting of collagen I and III also confirmed extensive fibrosis. We then found that Smad7 depletion resulted in the TGF-ß-mediated fibrosis via mir-21, mir-29, and np_5318, and NFκB-driven inflammation activation. This study confirmed the inhibitory role of Smad7 in the aging-related submandibular gland hypofunction. Therefore, it provided a promising treatment target for aging-related dysfunction and sialadenitis of submandibular gland.

Dec1 deficiency restores the age-related dysfunctions of submandibular glands.

Age-related organ and tissue-specific cell kinetic and morphological alterations are associated with the incidence of numerous diseases in old age. Salivary dysfunction frequently appears in a wide range of older people and thus is a physiological and biological aspect of aging. The transcription factor Dec1 (differentiated embryo chondrocyte expressed gene 1) is essential for the regulation of cellular senescence. Here, we explored the morphological and physiological abnormalities and the microRNA (miRNA) expression profiles in the submandibular glands (SMGs) of young (3-month-old) and of aged (24-month-old) wild-type (WT) and Dec1KO mice. Hematoxylin-eosin (H-E) staining, Masson's Trichrome staining, immunohistochemistry, immunofluorescence, and quantitative real time PCR were employed. MicroRNA (miRNA) expression profiles were examined using an Agilent system with a Mouse 8x60K array. Immunohistochemical analysis revealed an increased oxidative stress response (8-OHdG), increased expression levels of type I collagen in the fibrotic tissues with substantial amounts of fibroblasts and collagen fibers, the presence of CCl-22-positive lymphocytes infiltrating the SMGs of aged WT mice and a subsequently enhanced expression of fibrosis-associated gene (MMP-2) in the aged SMGs. The water channel protein aquaporin-5 (AQP5) was expressed in the basal cytoplasmic regions of acini in young SMGs but showed a decreased expression in aged SMGs. Myoepithelial cell markers (p63 immunoreactivity and a-SMA immunofluorescence staining) were also decreased in aged SMGs. Quantitative real-time PCR revealed decreased mRNA expression levels of AQP5 and increased mRNA expression levels of Dec1 in aged WT mice. All those characteristics were attenuated in aged Dec1KO mice. There were no apparent differences between young WT and Dec1KO mice. Of the miRNAs analyzed, miR-181c-5p, miR-141-3p, miR-374c-5p and miR-466i-3p are proposed regulatory targets of Dec1 and AQP5 genes that are involved in SMG dysfunction in aged mice. We suggest that a Dec1 deficiency might alleviate the aging-induced hypofunction of SMGs and relevant alterations of Dec1 would be useful to keep SMGs healthy. This study provides clues for determining unique microRNAs concerned with SMG dysfunction. Subsequent activation of such diversely expressed miRNAs be of great value in clarifying the nature of age-related alterations in SMGs.

Ethanol binge drinking during pregnancy and its effects on salivary glands of offspring rats: oxidative stress, morphometric changes and salivary function impairments.

OBJECTIVES: To investigate the biochemical and morphological effects of ethanol (EtOH) binge drinking during pregnancy on parotid glands (PG), submandibular glands (SMG), and saliva of offspring rats. METHODS: Pregnant Wistar rats (n = 8) were exposed to EtOH consumption (3 g/kg/day - 20 % w/v) for three consecutive days. The saliva of 40-day-old offspring rats was collected to determine amylase activity and total protein concentration. PG and SMG were collected to performe oxidative biochemistry, morphometric and immunohistochemistry analyses (Student's t-test, p < .05). RESULTS: EtOH consumption during pregnancy significantly decreased the total protein concentration and decreased amylase activity. In the PG, the EtOH group showed increased lipid peroxidation and decreased antioxidant capacity against peroxyl. In the SMG, the EtOH group showed increased lipid peroxidation and NOx metabolite levels. PG exposed to EtOH showed a decrease of acini, ducts, and total parenchymal area. SMG exposed to EtOH showed an increase in the total stromal area. The expression of CK-19 and Vimentin were found not different between groups. CONCLUSIONS: For the first time, a three-day EtOH binge-drinking protocol during pregnancy is associated with oxidative stress and morphometric alterations in the salivary glands of offspring rats and with the functional reduction of the main salivary enzyme (amylase). CLINICAL RELEVANCE: EtOH consumption during pregnancy altered the morphology and physiology of the salivary glands of offspring rats.

LncRNA and mRNA expression profiles and functional networks of hyposalivation of the submandibular gland in hypertension.

Hyposalivation is a complication of hypertension. However, little is known about the role of long non-coding RNAs (lncRNAs) in salivary glands in hypertension. This study aimed to compare the lncRNA and mRNA expression profiles between spontaneous hypertension rats (SHRs) and Wistar-Kyoto (WKY) rats through microarray analysis and apple bioinformatics methods to analyse their potential roles in hyposalivation. The differentially expressed (DE) lncRNAs and mRNAs were confirmed by quantitative real-time PCR (qRT-PCR). Compared with WKY rats, 225 DE lncRNAs and 473 DE mRNAs were identified in the SMG of SHRs. The pathway analyses of DE mRNAs showed that inflammatory mediator regulation of transient receptor potential channels was involved in hyposalivation in SHRs. Ten DE lncRNAs were chosen for further research. A coding-non-coding gene co-expression (CNC) network and competing endogenous RNA (ceRNA) network analysis revealed that the potential functions of these 10 DE lncRNAs were closely connected with the processes of the immune response. This study showed abundant DE lncRNAs and mRNAs in hypertensive SMGs. Furthermore, our results indicated strong associations between the immune response and hyposalivation and showed the potential of immune-related genes as novel and therapeutic targets for hyposalivation.

Submandibular gland-specific inflammaging-induced hyposalivation in the male senescence-accelerated mouse prone -1 line (SAM-P1).

Aging has pronounced effects on mammalian tissues and cells, but the impacts of aging on salivary gland function are relatively unknown. This study aims to evaluate the effects of aging on submandibular gland (SMG) and parotid gland (PG) functions in the male senescence-accelerated mouse. In vivo analysis at the systemic level revealed that salivary secretion induced by pilocarpine, a muscarinic agonist, from the SMG was significantly decreased in aged mice, whereas salivary secretion from the PG was not affected. To evaluate organ-level function, the SMG was perfused with the muscarinic agonists carbachol and calcium ionophore A23187 ex vivo to induce salivary secretion, and decreased saliva production was also observed in the aged SMG. Histological analysis revealed the presence of CD4-positive lymphocytes infiltrating the aged SMG. Furthermore, real-time PCR revealed that the aged SMG exhibited accelerated cell aging, increased levels of the inflammatory cytokine interleukin-6, and decreased mRNA levels of the water channel protein aquaporin-5 (AQP5). In summary, these results demonstrate that SMG function in aged mice was diminished, and that cell senescence, chronic inflammation, and the decreased gene expression of AQP5 are the likely causes of hyposalivation in the SMG of aged mice.

Switching to normal diet reverses kwashiorkor-induced salivary impairments via increased nitric oxide level and expression of aquaporin 5 in the submandibular glands of male Wistar rats.

Kwashiorkor, a form of malnutrition, has been shown to cause impaired salivary secretion. However, there is dearth of information on the mechanism that underlies this complication. Also, whether returning to normal diet after kwashiorkor will reverse these complications or not is yet to be discerned. Thus, this study aimed at assessing the mechanisms that underlie kwashiorkor-induced salivary impairments and to evaluate the effects of switching back to normal-diet on kwashiorkor-induced salivary impairments. Weaning rats were randomly divided into 3 groups (control group, kwashiorkor group (KG), re-fed kwashiorkor group (RKG)) of 7 rats each. The control group had standard rat chow while the KG and RKG were fed 2% protein diet for 6 weeks to induce kwashiorkor. The RKG had their diet changed to standard rat-chow for another 6 weeks. Blood and stimulated saliva samples were collected for the analysis of total protein, electrolytes, amylase, immunoglobulin A (IgA) secretion rate, leptin, and ghrelin. Tissue total protein, nitric oxide level, expressions of Na+/K+-ATPase, muscarinic (M3) receptor, and aquaporin 5 in the submandibular glands were also determined. Data were presented as means ± SEM and compared using ANOVA with Tukey's post hoc test. RKG showed improved salivary function evidenced by reduced salivary lag-time and potassium and increased flow rate, sodium, amylase, IgA secretion rate, leptin, submandibular nitric oxide level, and aquaporin 5 expression compared with KG. This study for the first time demonstrated that kwashiorkor caused significant reduction in salivary secretion through reduction of nitric oxide level and aquaporin 5 expression in submandibular salivary glands. Normal-diet re-feeding after kwashiorkor returned salivary secretion to normal.

An experimental chronic obstructive sialadenitis model by partial ligation of the submandibular duct characterised by sialography, histology, and transmission electron microscopy.

Extensive investigation into the mechanism of chronic obstructive sialadenitis (COS) calls for a reliable model that mimics the onset and progression of the disease. Duct obstruction is closely correlated with COS, and partial duct obstruction is usually observed in COS. Partial ligation of ducts or vessels is a valid treatment to mimic pathological processes in certain animal studies. In this study, we developed a novel animal model of COS by incomplete ligation of Wharton's duct and clarified the corresponding morphological alterations in the submandibular gland. Partial ligation of Wharton's duct in Japanese white rabbits resulted in a gradually progressive COS model as demonstrated by sialographic, gross, microscopic, and ultrastructural changes in acinar and ductal degenerations over 8 weeks. The model is a feasible option for investigating the pathogenesis of COS and evaluating the efficacy and safety of novel treatments of COS.

Differential immunohistochemical expression of type I collagen and matrix metalloproteinase 2 among major salivary glands of young and geriatric mice.

OBJECTIVE: This study aimed to demonstrate the immunohistochemical changes associated with MMP-2 and type 1 collagen separately for the first time in the major salivary glands (the parotid, submaxillary, and sublingual glands) that occur with aging in mice. MATERIAL AND METHODS: Fourteen Balb/c white mice (50-80 g) were used in this study. The animals were divided into two equal groups. Group I consisted of young animals (2-month-old) (n=7) and Group II consisted of older animals (18-month-old) (n=7). After routine histological follow-ups, Hematoxylin-eosin (H&E), Masson's Trichrome staining and immunohistochemical staining was performed for type I collagen and MMP-2. RESULTS: We observed that there were age-related decreases in the number of acinar cells, increase in eosinophilic zymogen granules in cells, collagen accumulation in fibrotic areas and dilatation in interlobular ducts. Also, while type I collagen and MMP-2 immunoreactivity were moderate in the salivary glands of the young mice, they were high in the salivary glands of the old mice (p=0.001). In the H-score assessment, MMP-2 immunoreactivity was lower at a significant level in young mice than in old mice (p=0.001). CONCLUSIONS: This study showed that anatomical, physiological and morphological abnormalities occur in all three major salivary glands as a natural consequence of aging.

Retroductal Nanoparticle Injection to the Murine Submandibular Gland.

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Efficacy and Non Invasive Treatment of Sialorrhea in the Goldenhar Syndrome.

BACKGROUND AND OBJECTIVE: Goldenhar syndrome (ocular-auricular-vertebral syndrome), a rare congenital condition arising from defects in the first and second brachial arches, consists in clinical variety of features ranging from facial abnormalities, ear-eye abnormalities, vertebral defects and congenital heart problems and severe obstructive sleep apnea. Due to craniofacial abnormalities, patients presents mechanical obstructive phenomena and sialorrhea that cause prone position, language's fastening, use of nasopharyngeal cannulas and tracheal intubation. METHODS: In this article, we report a case of a 16 years old child affected by Goldenhar syndrome and sialorrhea to demonstrate improvement of the daily patient management, through inoculations of botulinum toxin type A. Due to severe sialorrhea which caused tracheobronchial daily aspirations, the caregivers used an external aspirators. RESULTS: In the first infiltration (August 2016) the parotid and submandibular glands bilaterally were inoculated with incobotulinum toxin type A (Xeomin®, Merz Pharma) with dosages of 5 UI for each of them, for a total of 20 UI without clinical efficacy (no quantitative and qualitative saliva reducing during 3 months). In the second (November 2016) and third (February 2017) infiltrations each parotid and each submandibular glands were injected with a (dosage of 7 UI and 5 UI respectively (total of 24 UI of incobotulinumtoxin A) with important clinical results (saliva production and tracheo-bronchial aspirations reduced). CONCLUSION: Therefore, botulinum toxin type A could be a good and non invasive treatment of sialorrhea in Goldenhar syndrome to improve oral hygiene and daily patient management.

Dietary nitrate protects submandibular gland from hyposalivation in ovariectomized rats via suppressing cell apoptosis.

Xerostomia, a major oral symptom of menopause, is a subjective feeling of dry mouth associated with oral pain and difficulties in deglutition and speech, which significantly reduces patient's quality of life. Dietary nitrate, which can be converted to nitric oxide, has multiple physiological functions in the body, including antioxidant activity and vasodilatation; however, its protective effect against xerostomia remains poorly understood. The present study aimed to evaluate the effects of dietary nitrate on estrogen deficiency-induced xerostomia. We established an ovariectomized (OVX) rat model, which included five groups: sham-operated, OVX, OVX + 0.4 mM nitrate, OVX + 2 mM nitrate, and OVX + 4 mM nitrate (n = 6). After ovariectomy, animals in the nitrate treatment groups received appropriate amounts of sodium nitrate dissolved in distilled water for 3 months. The results showed that nitrate treatment reduced body weight and water intake, and increased serum nitrate and nitrite levels. Furthermore, nitrate uptake increased saliva secretion as evidenced by saliva flow rates and aquaporin 5 expression, and alleviated histological lesions as evidenced by reduction of the fibrotic area and cell atrophy in the salivary glands. Although protective effects of nitrate against estrogen deficiency-induced xerostomia were observed at all doses, treatment with 2 mM nitrate was more effective than that with 0.4 mM and 4 mM nitrate. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase-3 expression analyses showed that nitrate also protected cells from apoptosis, possibly through upregulation of Cu-Zn superoxide dismutase (Cu-Zn SOD) known to inhibit oxidative stress-related apoptosis. Our findings indicate that nitrate could improve functional activity of the salivary glands in OVX rats by suppressing apoptosis and upregulating Cu-Zn SOD expression, suggesting that dietary nitrate may potentially prevent hyposalivation in menopausal women.

Differential immunohistochemical expression of type I collagen and matrix metalloproteinase 2 among major salivary glands of young and geriatric mice

Abstract Objective This study aimed to demonstrate the immunohistochemical changes associated with MMP-2 and type 1 collagen separately for the first time in the major salivary glands (the parotid, submaxillary, and sublingual glands) that occur with aging in mice. Material and Methods Fourteen Balb/c white mice (50-80 g) were used in this study. The animals were divided into two equal groups. Group I consisted of young animals (2-month-old) (n=7) and Group II consisted of older animals (18-month-old) (n=7). After routine histological follow-ups, Hematoxylin-eosin (H&E), Masson's Trichrome staining and immunohistochemical staining was performed for type I collagen and MMP-2. Results We observed that there were age-related decreases in the number of acinar cells, increase in eosinophilic zymogen granules in cells, collagen accumulation in fibrotic areas and dilatation in interlobular ducts. Also, while type I collagen and MMP-2 immunoreactivity were moderate in the salivary glands of the young mice, they were high in the salivary glands of the old mice (p=0.001). In the H-score assessment, MMP-2 immunoreactivity was lower at a significant level in young mice than in old mice (p=0.001). Conclusions This study showed that anatomical, physiological and morphological abnormalities occur in all three major salivary glands as a natural consequence of aging.

Botulinum Toxin Therapy for Nonmotor Aspects of Parkinson's Disease.

The medical treatment of Parkinson's disease (PD) is symptomatic both in terms of motor and nonmotor aspects. The nonmotor symptoms therapy should be taken into account as many of them negatively influence the quality of life and are treatable. Botulinum neurotoxin (BoNT) offers effective treatment for drooling and bladder dysfunctions and alternative treatment for constipation and pain related to parkinsonism. BoNT for drooling is probably the best-documented therapy for nonmotor symptoms by clinical trials. Ultrasound-guided injections into parotid and submandibular glands can be recommended as the first line treatment, especially when anticholinergic oral medications are not indicated due to the risk of confusion, cognitive decline, or psychosis. BoNT therapy of constipation and neurogenic bladder is not so well evidenced and the latter indication is based on positive effects in clinical trials in multiple sclerosis and posttraumatic spine injuries. BoNT injections into the salivary glands or bladder are effective and safe with relatively low risk of complications. Authors reviewed the literature on the use of BoNT for all nonmotor aspects of PD, showed their position in guidelines and systematic reviews and gave the practical remarks on injection techniques and dosing.

Clinical significance of hypoechoic submandibular gland lesions in type 1 autoimmune pancreatitis.

AIM: To assess the role of ultrasonography of submandibular glands (SGs) in the diagnosis of type 1 autoimmune pancreatitis (AIP). METHODS: Thirty-seven patients who were definitively diagnosed with type 1 AIP according to the international consensus diagnostic criteria (ICDC) for AIP at our institution between December 1990 and April 2016 were retrospectively reviewed. Findings by physical examination, ultrasonography, and scintigraphy of SGs were analyzed to reach a diagnosis based on the ICDC for AIP. The efficacy of corticosteroid treatment in the resolution of hypoechoic lesions in SGs was also evaluated by assessment with ultrasonography before and after treatment in 18 cases. RESULTS: The sensitivity of multiple hypoechoic lesions in SGs by ultrasonography for the diagnosis of sialadenitis in type 1 AIP (84%) was higher than that of physical examination (46%), scintigraphy (28%), and SGs thickness (49%). Ultrasonographic evidence of hypoechoic lesions in SGs improved the definitive diagnosis of sialadenitis and type 1 AIP by the ICDC criteria in 11 (30%) and 2 (5.4%) cases, respectively. Multiple hypoechoic lesions in SGs were resolved or disappear by corticosteroid administration in 14 of 16 cases with hypoechoic lesions in SGs, whereas the ultrasonographic findings in the remaining 2 cases with hypoechoic lesions in SGs and the 2 cases with homogenous SG parenchyma remained unchanged after corticosteroid administration. CONCLUSION: SG ultrasonography to detect multiple hypoechoic lesions might be useful for type 1 AIP diagnosis by improving diagnostic accuracy together with the ICDC sialadenitis criteria.

First-in-man mesenchymal stem cells for radiation-induced xerostomia (MESRIX): study protocol for a randomized controlled trial.

BACKGROUND: Salivary gland hypofunction and xerostomia are major complications following radiotherapy for head and neck cancer and may lead to debilitating oral disorders and impaired quality of life. Currently, only symptomatic treatment is available. However, mesenchymal stem cell (MSC) therapy has shown promising results in preclinical studies. Objectives are to assess safety and efficacy in a first-in-man trial on adipose-derived MSC therapy (ASC) for radiation-induced xerostomia. METHODS: This is a single-center, phase I/II, randomized, placebo-controlled, double-blinded clinical trial. A total of 30 patients are randomized in a 1:1 ratio to receive ultrasound-guided, administered ASC or placebo to the submandibular glands. The primary outcome is change in unstimulated whole salivary flow rate. The secondary outcomes are safety, efficacy, change in quality of life, qualitative and quantitative measurements of saliva, as well as submandibular gland size, vascularization, fibrosis, and secretory tissue evaluation based on contrast-induced magnetic resonance imaging (MRI) and core-needle samples. The assessments are performed at baseline (1 month prior to treatment) and 1 and 4 months following investigational intervention. DISCUSSION: The trial is the first attempt to evaluate the safety and efficacy of adipose-derived MSCs (ASCs) in patients with radiation-induced xerostomia. The results may provide evidence for the effectiveness of ASC in patients with salivary gland hypofunction and xerostomia and deliver valuable information for the design of subsequent trials. TRIAL REGISTRATION: EudraCT, Identifier: 2014-004349-29. Registered on 1 April 2015. ClinicalTrials.gov, Identifier: NCT02513238 . First received on 2 July 2015. The trial is prospectively registered.

Nicotinamide Phosphoribosyltransferase Upregulation by Phenylephrine Reduces Radiation Injury in Submandibular Gland.

PURPOSE: Radiation therapy for head and neck cancer commonly leads to radiation sialadenitis. Emerging evidence has indicated that phenylephrine pretreatment reduces radiosensitivity in the salivary gland; however, the underlying cytoprotective mechanism remains unclear. Nicotinamide phosphoribosyltransferase (NAMPT) is not only a key enzyme for the nicotinamide adenine dinucleotide salvage pathway, but also a cytokine participating in cell survival, metabolism, and longevity, with a broad effect on cellular functions in physiology and pathology. However, the regulatory events of NAMPT in response to the irradiated salivary gland are unknown. METHODS AND MATERIALS: The cell viability of primary cultured submandibular gland cells was determined using the PrestoBlue assay. NAMPT expression was measured using reverse transcriptase polymerase chain reaction and Western blotting in vitro and in vivo. Silent information regulator 1 (SIRT1) and phosphorylated Akt protein levels were examined by Western blotting. The cellular locations of NAMPT and SIRT1 were detected by immunohistochemistry. NAMPT promoter activity was assessed using the luciferase reporter gene assay. RESULTS: NAMPT was mainly distributed in the cytoplasm of granular convoluted tubule cells and ductal cells in normal submandibular glands. mRNA and protein expression of NAMPT was downregulated after radiation but upregulated with phenylephrine pretreatment both in vivo and in vitro. Moreover, the protein expression of phosphorylated Akt and SIRT1 was decreased in irradiated glands, and phenylephrine pretreatment restored the expression of both. SIRT1 was mainly located in the cell nucleus and cytoplasm in the normal submandibular gland. Phenylephrine dramatically enhanced the expression of SIRT1, which was significantly reduced by radiation. Furthermore, phenylephrine induced a marked increase of NAMPT promoter activity. CONCLUSIONS: These findings reveal the regulatory mechanisms of NAMPT expression, which help to understand the mechanism of the cytoprotective role of phenylephrine on irradiated tissues.

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction.

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)(1), and lay out a procedure for local gland irradiation.

Interleukin-17 Impairs Salivary Tight Junction Integrity in Sjögren's Syndrome.

Sjögren's syndrome (SS) is an inflammatory autoimmune disease that causes secretory dysfunction of the salivary glands. It has been reported that proinflammatory cytokine interleukin-17 (IL-17) was elevated and tight junction (TJ) integrity disrupted in minor salivary glands from SS patients. However, whether the elevated IL-17 in SS affects TJ integrity and thereby alters the function of salivary gland is unknown. Here, by using nonobese diabetic (NOD) mice as SS model, we found that the stimulated salivary flow rate was significantly decreased in NOD mice. Lymphocyte infiltration was mainly observed in submandibular glands (SMGs), but not parotid glands (PGs), of NOD mice. IL-17 was significantly increased and mainly located in lymphocytic-infiltrating regions in SMGs but not detectable in PGs of NOD mice. Meanwhile, the epithelial barrier function was disrupted, as evidenced by an increased paracellular tracer clearance and an enlarged acinar TJ width in SMGs of NOD mice. Furthermore, claudin-1 and -3 were elevated especially at the basolateral membranes, whereas claudin-4, occludin, and zonula occludens-1 (ZO-1) were reduced in SMGs of NOD mice. Moreover, occludin and ZO-1 were dispersed into cytoplasm in SMGs of NOD mice. However, no change in the expression and distribution of TJ proteins was found in PGs. In vitro, IL-17 significantly decreased the levels and apical staining of claudin-4 and ZO-1 proteins in the cultured SMG tissues, as well as claudin-1, occludin, and ZO-1 in PG tissues. Moreover, IL-17 activated the phosphorylation of IκBα and p65 in SMG cells, whereas pretreatment with NF-κB inhibitor pyrrolidine dithiocarbamate suppressed the IL-17-induced downregulation of claudin-4 and ZO-1 in SMG tissues. Taken together, these findings indicate that IL-17 derived from infiltrating lymphocyte impairs the integrity of TJ barrier through NF-κB signaling pathway, and thus might contribute to salivary gland dysfunction in SS.

Interferon-gamma increased epithelial barrier function via upregulating claudin-7 expression in human submandibular gland duct epithelium.

Tight junctions (TJs) are necessary for salivary gland function and may serve as indicators of salivary gland epithelial dysfunction. IgG4-related disease (IgG4-RD) is a newly recognized fibro-inflammatory condition which disrupts the TJ associated epithelial barrier. The salivary glands are one of the most frequently involved organs in IgG4-RD, however, changes of the TJ associated epithelial barrier in salivary gland duct epithelium is poorly understood. Here, we investigated the regulation and function of TJs in human submandibular gland ductal epithelial cells (HSDECs) in normal and IgG4-RD. We examined submandibular gland (SMG) tissue from eight control individuals and 22 patients with IgG4-RD and established an HSDEC culture system. Immunohistochemistry, immunocytochemistry, western blotting, and measurement of transepithelial electrical resistance (TER) were performed. Claudin-4, claudin-7, occludin, and JAM-A were expressed at the apical side of the duct epithelium in submandibular gland (SMG) tissue and at the cell borders in HSDECs of normal and IgG4-RD. The expression and distribution of TJs in SMG tissue were not different in control individuals and patients with IgG4-RD in vivo and in vitro. Although interferon-gamma (IFNγ) generally disrupts the integrity and function of TJs, as manifested by decreased epithelial barrier function, IFNγ markedly increased the epithelial barrier function of HSDECs via upregulation of claudin-7 expression in HSDECs from patients with IgG4-RD. This is the first report showing an IFNγ-dependent increase in epithelial barrier function in the salivary gland duct epithelium. Our results provide insights into the functional significance of TJs in salivary gland duct epithelium in physiological and pathological conditions, including IgG4-RD.