Photobiomodulation Recovers the Submandibular Gland in Vismodegib-Treated Rats.

Objective: The submandibular gland (SMG) produces the most saliva, and factors such as aging and chemotherapy can affect its structure and function. However, there are only temporary treatments available for salivary hypofunction. This study aimed to evaluate the effects of photobiomodulation (PBM) on the function of SMG by using a rat animal model and vismodegib, an antagonist of the sonic hedgehog (SHH) pathway. Methods: Vismodegib (10 mg/kg) drug was gavaged orally for 14 days in rats to significantly decrease the SHH signaling proteins [SHH, protein patched homolog 1 (PTCH1), smoothened protein (SMO), glioma-associated oncogene homolog 1 (GLI1)], induce damage in SMG tissue, and affect salivary functional markers AQP5 and Keratin5. After that, in conjunction with vismodegib administration, PBM was performed using an 850 nm high-power light-emitting diode (LED) device treated daily for 6 days at varying total energy densities of 60, 120, and 180 J/cm2 in at least 3 rats per group. The test results were confirmed by Western blot, immunofluorescence staining, and hematoxylin and eosin staining, and the statistics were t-test or one-way analysis of variance (ANOVA) with Tukey's multiple comparisons tests. Results: Significant decreases in the expression of SHH-related proteins (PTCH1, SMO, GLI1, p < 0.05) with damage of SMG ductal cells were observed with vismodegib administration. However, a significant increase in the expression levels of SHH-related proteins (SHH, SMO, GLI1, p < 0.05) and recovery of SMG ductal cells damaged after vismodegib administration were observed for PBM-treated groups. Salivary functional marker AQP5 also showed the same increase or decrease. Conclusions: This study found that vismodegib damages SMG ductal cells and decreases SHH-related proteins and associated salivary functional markers. Also, 850 nm high-power LED recovered the damaged structure of SMG and increased SHH-related proteins and salivary functional markers. The study results suggest that PBM can restore SMG structure and function through SHH signaling.

Effect of acupuncture on regulating IL-17, TNF-ɑ and AQPs in Sjögren's syndrome.

AIM: The aim of the study was to observe the effect of acupuncture on regulating interleukin (IL)-17, tumor necrosis factor (TNF)-ɑ, and aquaporins (AQPs) in Sjögren's syndrome (SS) on patients and on non-obese diabetic (NOD) models. METHODS: Levels of anti-AQP 1, 5, 8, and 9 antibodies, IL-17, and TNF-ɑ in the serum of SS patients were compared prior and following 20 acupuncture treatment visits during 8 weeks. While in murine model, five groups were divided to receive interventions for 4 weeks, including control, model, acupuncture, isoflurane, and hydroxychloroquine. The submaxillofacial gland index, histology, immunohistochemistry of AQP1, 5, salivary flow, together with IL-17, and TNF-ɑ expression in peripheral blood were compared among the groups. RESULTS: Acupuncture reduced IL-17, TNF-ɑ, and immunoglobin A levels, and numeric analog scale of dryness in 14 patients with SS (p < 0.05). The salivary flow was increased, and the water intake decreased in NOD mice receiving acupuncture treatments. IL-17 and TNF-ɑ levels in peripheral serum were down-regulated (p < 0.05) and AQP1, 5 expression in the submandibular glands up-regulated in mice. CONCLUSION: The effect on relieving xerostomia with acupuncture may be achieved by up-regulating the expression of AQP1. AQP5, down-regulating levels of IL-17 and TNF-ɑ, and a decrease in inflammation of glands.

Huoxue Jiedu Recipe represses mitochondrial fission to alleviate submandibular gland inflammation in Sjögren's syndrome.

Sjögren's syndrome (SS) is the second most common autoimmune rheumatism. Huoxue Jiedu Recipe (HXJDR) is a kind of traditional Chinese medicine with a variety of pharmacological functions; however, its biological function in SS has not been studied yet. Peripheral blood mononuclear cells (PBMCs) and serum samples were isolated from healthy controls and patients with SS. NOD/Ltj mice were used for developing the SS mouse model. The levels of inflammatory cytokines and NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-related markers as well as dynamin-related protein 1 (Drp1) were determined by ELISA, quantitative real-time PCR, and western blot analysis, respectively. Hematoxylin and eosin and TUNEL staining detected the pathological damage. A transmission electron microscope was used to observe the mitochondrial microstructure. Inflammatory cytokines IL-18, IL-1ß, B-cell activating factor (BAFF), BAFF-receptor (BAFF-R), IL-6, and TNF-α in serum samples and NLRP3 inflammasome-related makers (NLRP3, cysteinyl aspartate-specific proteinase 1 [caspase-1], apoptosis-associated speck-like protein containing a caspase-1 recruitment domain [ASC], IL-1ß) in PBMCs were greatly upregulated in patients with SS. Furthermore, cytoplasmic phosphorylation of Drp1 and mitochondrial Drp1 level were significantly increased in PBMCs, while mitochondrial swelling and fuzzy inner ridge were observed in PBMCs of patients with SS, suggesting increased mitochondrial fission. Compared with control mice, SS mice showed decreased salivary flow rate, increased submandibular gland index, and more severe inflammatory infiltration and damage as well as mitochondrial fission in submandibular gland tissues. After HXJDR administration, these effects were significantly reversed. HXJDR treatment could alleviate the inflammatory infiltration and pathological damage in submandibular glands of SS mice by inhibiting Drp-1-dependent mitochondrial fission.

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.

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.

Protective Effect of Whortleberry Extract on Salivary Gland Damage Induced by Neck Irradiation in Rats.

Radiotherapy is a method of treatment used on malignant head and neck tumors; however, it may lead to adverse effects by influencing other tissues because its effects are not specific to tumor tissues. These adverse effects limit the effectiveness of the treatment and sometimes lead to termination of the treatment. This study aims to histopathologically and biochemically investigate the protective effect of whortleberry against the cellular degeneration and oxidative stress that take place in salivary glands due to radiotherapy. The rats were divided into 6 groups. One group was given radiotherapy only, one group was given radiotherapy and 100 mg/kg of whortleberry, and one group was given radiotherapy and 200 mg/kg of whortleberry. The remaining 3 groups were designated as whortleberry, sham, and control groups. At the end of the study, samples collected were histopathologically and biochemically analyzed. In the group given radiotherapy only, acinar areas were reduced histopathologically, whereas ductal areas increased (P < .01). Oxidative stress increased only in the group given radiotherapy, whereas the oxidative stress levels in the other groups were close to those in the control groups. In conclusion, whortleberry reduces cellular degeneration and oxidative stress that take place in salivary glands due to radiotherapy.

[Effects of estrogen and remifemin on neuronal nitric oxide synthase and transient receptor potential vanilloid 1 in submandibular gland of ovariectomized rats].

Objective: To investigate the effects of estrogen and remifemin on the expression of neuronal nitric oxide synthase (nNOS), transient receptor potential vanilloid 1 (TRPV1), muscarinic acetylcholine receptor, member 1 and 3 (M1 and M3 receptor) and acetylcholinesterase (AChE) in the submandibular gland of rats. Methods: Forty SD female adult rats were divided into SHAM group (sham operation), OVX group (ovarian removal), OVX+E group (ovarian removal + estrogen treatment) and OVX+ICR group (ovarian removal + remifemin treatment), 10 per group. The rats were recovered for 2 weeks after operation. The SHAM group and the OVX group were treated with distilled water, the OVX+E group and the OVX+ICR group were treated with ß-estradiol and remifemin respectively. After 4 weeks, the location and expression of nNOS, TRPV1, M1 and M3 receptors in the submandibular gland were evaluated by immunohistochemistry. The changes of AChE expression in rat submandibular gland were observed by AChE staining. Results: Compared with SHAM group (0.23±0.02, 0.28±0.01, 0.25±0.03, 0.19±0.03), the expression of nNOS, TRPV1, M1 and M3 receptors in OVX group (0.16±0.01, 0.21±0.01, 0.15±0.02, 0.09±0.02) were significantly lower (P<0.05); there were no significant difference between OVX+E group (0.23±0.01, 0.28±0.02, 0.23±0.03, 0.19±0.01) and SHAM group (P>0.05). But compared with OVX group, the expression of nNOS, TRPV1 and M3 receptors in OVX+ICR group were no significantly changed (P>0.05), and only M1 receptor expression (0.22±0.03) was significantly increased (P<0.05). The expression of AChE in OVX group (0.14±0.01) was significantly higher than that in SHAM group (0.10±0.01) (P<0.05). The expression of AChE in OVX+E group (0.15±0.01) was significantly higher than that in SHAM group (P<0.05). The expression of AChE in OVX+ICR group (0.09±0.01) was not significantly different from that in SHAM group (P>0.05). Conclusions: Estrogen can significantly increase the expression of nNOS and TRPV1 in the submandibular gland of rats, suggesting that estrogen may regulate the salivary secretion function of the submandibular gland through nNOS and TRPV1. The mechanism of remifemin is different from that of estrogen, and remifemin does not play a regulatory role by nNOS and TRPV1.

Potential Application of Ixeris dentata in the Prevention and Treatment of Aging-Induced Dry Mouth.

Dry mouth is a common complaint among the elderly population. The aim of this study was to investigate the effect of Ixeris dentata (IXD) extract on aging-induced dry mouth. We used young (two months) and aged (20 months) SD rats in our study. Using water as the vehicle, IXD extract (25, 50, and 100 mg/kg) was given via oral gavage to the young and aged rats for eight weeks. We found that the salivary flow rate relative to the submandibular gland weight was differently influenced by IXD extract treatment. IXD extract augmented the submandibular gland acinar cells, which are depleted during aging. In addition, the decreased salivary alpha-amylase, inositol triphosphate receptor, and aquaporin-5 in the aging rats were upregulated by IXD treatment. Free radical-induced oxidative stress in the aging rats was also alleviated in the IXD-treated group. The formation of high molecular weight complexes of protein disulfide isomerase, decreased expression of an ER chaperone (GRP78), and increased ER stress response (ATF-4, CHOP and p-JNK) in aging rats was regulated with IXD treatment, and eventually increased salivary secretions from the aging submandibular glands. These are the first data to suggest that IXD extract might ameliorate aging-associated oral dryness by regulating the ER environment.

Post-weaning xenohormone intake affects adult rat submandibular gland in a sex-dependent manner.

OBJECTIVES: We previously reported that maternal exposure to genistein and vinclozolin, ingested alone or in combination, affects submandibular salivary glands of rat offspring. Here, we investigated the responsiveness of submandibular gland when such xenohormone exposure occurs later in life. MATERIALS AND METHODS: Chemicals were given orally to male and female Wistar rats (1 mg/kg body weight per day), from weaning to adulthood. Submandibular glands and plasma were collected at postnatal day 100 for histologic and molecular analysis. RESULTS: Whereas no effect was observed in females, increases in granular convoluted tubules area coupled with a modification of salivary secretions were found in male submandibular glands. Genistein and vinclozolin similarly increased the mRNA expression of Cystatin C, Mucin 10, Growth factors, and plasmatic EGF. Negative correlations were found between the expressions of androgen receptor and EGF (-0.34; p < 0.05), TGFα (-0.52; p < 0.01), Mucin 10 (-0.43; p < 0.05), and Cystatin C (-0.42; p < 0.05) as well as between progesterone receptor and EGF (-0.56; p < 0.01). The Spearman correlation test revealed also a positive correlation between salivary EGF-mRNA expression and EGF in plasma (+0.32; p < 0.05). CONCLUSION: Our findings confirm the sex-dependent sensitivity of submandibular salivary glands to dietary xenohormones and underline the influence of the exposure period.

Aldehyde dehydrogenase 3A1 activation prevents radiation-induced xerostomia by protecting salivary stem cells from toxic aldehydes.

Xerostomia (dry mouth) is the most common side effect of radiation therapy in patients with head and neck cancer and causes difficulty speaking and swallowing. Since aldehyde dehydrogenase 3A1 (ALDH3A1) is highly expressed in mouse salivary stem/progenitor cells (SSPCs), we sought to determine the role of ALDH3A1 in SSPCs using genetic loss-of-function and pharmacologic gain-of-function studies. Using DarkZone dye to measure intracellular aldehydes, we observed higher aldehyde accumulation in irradiated Aldh3a1-/- adult murine salisphere cells and in situ in whole murine embryonic salivary glands enriched in SSPCs compared with wild-type glands. To identify a safe ALDH3A1 activator for potential clinical testing, we screened a traditional Chinese medicine library and isolated d-limonene, commonly used as a food-flavoring agent, as a single constituent activator. ALDH3A1 activation by d-limonene significantly reduced aldehyde accumulation in SSPCs and whole embryonic glands, increased sphere-forming ability, decreased apoptosis, and improved submandibular gland structure and function in vivo after radiation. A phase 0 study in patients with salivary gland tumors showed effective delivery of d-limonene into human salivary glands following daily oral dosing. Given its safety and bioavailability, d-limonene may be a good clinical candidate for mitigating xerostomia in patients with head and neck cancer receiving radiation therapy.

Effect of Biochanin A versus 17ß estradiol in rat submandibular salivary gland.

The epigenetic nature of development mandates the observation of the effect of any exogenous substance, especially those with estrogenic activities, during critical phases of development. The submandibular gland (SMG) presents as a great model due to extensive postnatal development, and is known to be regulated and affected by hormones as well as growth factors. Herein, we observed postnatal development following low doses of Biochanin A (BCA) and 17ß estradiol (E2) in rats. The pups were randomly divided into four groups: control, BCA, E2, and dimethyl sulfoxide (DMSO), and euthanized at the 6th, 15th, 30th, and 60th postnatal days (PND). SMG morphogenesis was assessed. The nuclear expression of estrogen receptor beta (ERß) was evaluated immunohistochemically; ERß expression was up-regulated by BCA and down-regulated by E2. Similarly, caspase three gene expression, assessed by real time polymerase chain reaction was increased in the BCA group but decreased in the E2 group. A significant decrease in epidermal growth factor gene expression was noted at PND 30. The results presented by this study provide evidence that the effect of a postnatal exposure of the SMG to Biochanin A during development could be linked to sex hormone-dependent disorders.

Effects of Salivary Secretion Stimulation on the Treatment of Chronic Radioactive Iodine-Induced Sialadenitis.

BACKGROUND: The aim of this prospective study was to investigate the effect of salivary stimulation therapy using pilocarpine (a cholinergic agent) on chronic radioactive iodine (RAI)-induced sialadenitis. METHODS: Sixty-one patients with a diagnosis of chronic RAI-induced sialadenitis after thyroidectomy and RAI therapy were enrolled in this prospective study. Patients received salivary stimulation therapy with pilocarpine (5 mg, 3 times daily) over a 3-month period. Subjective symptom scores were assessed using self-reported questionnaires. Salivary flow rates (SFRs) were measured and salivary gland scintigraphy (SGS) was performed to evaluate objective salivary gland functions. RESULTS: After salivary stimulation therapy, subjective symptom scores were significantly improved (p=0.002), but posttreatment unstimulated and stimulated SFRs did not differ significantly from pretreatment values. SGS parameters, that is, uptake ratio (UR), maximum accumulation (MA), Tmin, and maximum secretion (MS) of parotid and submandibular glands were nonsignificantly different after salivary stimulation therapy. CONCLUSION: The study shows that salivary stimulation therapy may reduce the subjective symptoms of RAI-induced chronic sialadenitis but does not significantly induce functional restoration.

Mechanism involved in Danshen-induced fluid secretion in salivary glands.

AIM: Danshen's capability to induce salivary fluid secretion and its mechanisms were studied to determine if it could improve xerostomia. METHODS: Submandibular glands were isolated from male Wistar rats under systemic anesthesia with pentobarbital sodium. The artery was cannulated and vascularly perfused at a constant rate. The excretory duct was also cannulated and the secreted saliva was weighed in a cup on an electronic balance. The weight of the accumulated saliva was measured every 3 s and the salivary flow rate was calculated. In addition, the arterio-venous difference in the partial oxygen pressure was measured as an indicator of oxygen consumption. In order to assess the mechanism involved in Danshen-induced fluid secretion, either ouabain (an inhibitor of Na(+)/K(+) ATPase) or bumetanide (an inhibitor of NKCC1) was additionally applied during the Danshen stimulation. In order to examine the involvement of the main membrane receptors, atropine was added to block the M3 muscarinic receptors, or phentolamine was added to block the α1 adrenergic receptors. In order to examine the requirement for extracellular Ca(2+), Danshen was applied during the perfusion with nominal Ca(2+) free solution. RESULTS: Although Danshen induced salivary fluid secretion, 88.7 ± 12.8 µL/g-min, n = 9, (the highest value around 20 min from start of DS perfusion was significantly high vs 32.5 ± 5.3 µL/g-min by carbamylcholine, P = 0.00093 by t-test) in the submandibular glands, the time course of that secretion differed from that induced by carbamylcholine. There was a latency associated with the fluid secretion induced by Danshen, followed by a gradual increase in the secretion to its highest value, which was in turn followed by a slow decline to a near zero level. The application of either ouabain or bumetanide inhibited the fluid secretion by 85% or 93%, and suppressed the oxygen consumption by 49% or 66%, respectively. These results indicated that Danshen activates Na(+)/K(+) ATPase and NKCC1 to maintain Cl(-) release and K(+) release for fluid secretion. Neither atropine or phentolamine inhibited the fluid secretion induced by Danshen (263% ± 63% vs 309% ± 45%, 227% ± 63% vs 309% ± 45%, P = 0.899, 0.626 > 0.05 respectively, by ANOVA). Accordingly, Danshen does not bind with M3 or α1 receptors. These characteristics suggested that the mechanism involved in DS-induced salivary fluid secretion could be different from that induced by carbamylcholine. Carbamylcholine activates the M3 receptor to release inositol trisphosphate (IP3) and quickly releases Ca(2+) from the calcium stores. The elevation of [Ca(2+)]i induces chloride release and quick osmosis, resulting in an onset of fluid secretion. An increase in [Ca(2+)]i is essential for the activation of the luminal Cl(-) and basolateral K(+) channels. The nominal removal of extracellular Ca(2+) totally abolished the fluid secretion induced by Danshen (1.8 ± 0.8 µL/g-min vs 101.9 ± 17.2 µL/g-min, P = 0.00023 < 0.01, by t-test), suggesting the involvement of Ca(2+) in the activation of these channels. Therefore, IP3-store Ca(2+) release signalling may not be involved in the secretion induced by Danshen, but rather, there may be a distinct signalling process. CONCLUSION: The present findings suggest that Danshen can be used in the treatment of xerostomia, to avoid the systemic side effects associated with muscarinic drugs.

[Effects of Crude Atractylodis Rhizoma and Processed Atractylodis Rhizoma on AQP1 - AQP5 and Hemorheology in Healthy Rats].

OBJECTIVE: To study the different effects of crude Atractylodis Rhizoma and processed Atractylodis Rhizoma in healthy rats, in order to prove the traditional theory that the crude Atractylodis Rhizoma has dry effects and the dry effects can be weaken by processing. METHODS: Health rats had been orally administered with pure water, crude Atractylodis Rhizoma, processed Atractylodis Rhizoma and atropine. The concentration of AQP1 and AQP5 in submaxillary gland were measured by ELISA. Their index of submaxillary gland, hemorheology and moisture content of intestine were also measured. RESULTS: There were obvious differences of concentration of AQP1 and AQP5 in submaxillary gland, index of submaxillary gland, hemorheology and moisture content of intestine between the rats which had been orally administered crude Atractylodis Rhizoma and the rats administered processed Rhizoma Atractylodes. CONCLUSIONS: The dry effects of Atractylodis Rhizoma works on rats' moisture content of intestine, index of submaxillary gland and hemorheology. The dry effects can be weaken by processing.

Role of androgen and estrogen receptors for the action of dehydroepiandrosterone (DHEA).

Dehydroepiandrosterone (DHEA) is an abundant steroid hormone, and its mechanism of action is yet to be determined. The aim of this study was to elucidate the importance of androgen receptors (ARs) and estrogen receptors (ERs) for DHEA function. Orchidectomized C57BL/6 mice were treated with DHEA, DHT, 17ß-estradiol-3-benzoate (E2), or vehicle. Orchidectomized AR-deficient (ARKO) mice and wild-type (WT) littermates were treated with DHEA or vehicle for 2.5 weeks. At termination, bone mineral density (BMD) was evaluated, thymus and seminal vesicles were weighted, and submandibular glands (SMGs) were histologically examined. To evaluate the in vivo ER activation of the classical estrogen signaling pathway, estrogen response element reporter mice were treated with DHEA, DHT, E2, or vehicle, and a reporter gene was investigated in different sex steroid-sensitive organs after 24 hours. DHEA treatment increased trabecular BMD and thymic atrophy in both WT and ARKO mice. In WT mice, DHEA induced enlargement of glands in the SMGs, whereas this effect was absent in ARKO mice. Furthermore, DHEA was able to induce activation of classical estrogen signaling in bone, thymus, and seminal vesicles but not in the SMGs. In summary, the DHEA effects on trabecular BMD and thymus do not require signaling via AR and DHEA can activate the classical estrogen signaling in these organs. In contrast, DHEA induction of gland size in the SMGs is dependent on AR and does not involve classical estrogen signaling. Thus, both ERs and ARs are involved in mediating the effects of DHEA in an organ-dependent manner.

Salivary gland hypofunction in tyrosylprotein sulfotransferase-2 knockout mice is due to primary hypothyroidism.

BACKGROUND: Protein-tyrosine sulfation is a post-translational modification of an unknown number of secreted and membrane proteins mediated by two known Golgi tyrosylprotein sulfotransferases (TPST-1 and TPST-2). We reported that Tpst2-/- mice have mild-moderate primary hypothyroidism, whereas Tpst1-/- mice are euthyroid. While using magnetic resonance imaging (MRI) to look at the thyroid gland we noticed that the salivary glands in Tpst2-/- mice appeared smaller than in wild type mice. This prompted a detailed analysis to compare salivary gland structure and function in wild type, Tpst1-/-, and Tpst2 -/- mice. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative MRI imaging documented that salivary glands in Tpst2-/- females were (≈) 30% smaller than wild type or Tpst1-/- mice and that the granular convoluted tubules in Tpst2-/- submandibular glands were less prominent and were almost completely devoid of exocrine secretory granules compared to glands from wild type or Tpst1-/- mice. In addition, pilocarpine-induced salivary flow and salivary α-amylase activity in Tpst2-/- mice of both sexes was substantially lower than in wild type and Tpst1-/- mice. Anti-sulfotyrosine Western blots of salivary gland extracts and saliva showed no differences between wild type, Tpst1-/-, and Tpst2-/- mice, suggesting that the salivary gland hypofunction is due to factor(s) extrinsic to the salivary glands. Finally, we found that all indicators of hypothyroidism (serum T4, body weight) and salivary gland hypofunction (salivary flow, salivary α-amylase activity, histological changes) were restored to normal or near normal by thyroid hormone supplementation. CONCLUSIONS/SIGNIFICANCE: Our findings conclusively demonstrate that low body weight and salivary gland hypofunction in Tpst2-/- mice is due solely to primary hypothyroidism.

Endocannabinoids mediate hyposalivation induced by inflammogens in the submandibular glands and hypothalamus.

OBJECTIVE: The aim of this study was to investigate the factors that could participate on salivary glands hypofunction during inflammation and the participation of endocannabinoids in hyposalivation induced by the presence of inflammogens in the submandibular gland (SMG) or in the brain. DESIGN: Salivary secretion was assessed in the presence of inflammogens and/or the cannabinoid receptor antagonist AM251 in the SMG or in the brain of rats. At the end of the experiments, some systemic and glandular inflammatory markers were measured and histopathological analysis was performed. RESULTS: The inhibitory effect observed 1h after lipopolysaccharide (LPS, 50µg/50µl) injection into the SMG (ig) was completely prevented by the injection of AM251 (5µg/50µl) by the same route (P<0.05). The LPS (ig)-induced increase in PGE2 content was not altered by AM251 (ig), while the glandular production of TNFα induced by the endotoxin (P<0.001) was partially blocked by it. Also, LPS injection produced no significant changes in the wet weight of the SMG neither damage to lipid membranes of its cells, nor significant microscopic changes in them, after hispopathological analysis, compared to controls. Finally, TNFα (100ng/5µl) injected intracerebro-ventricularly (icv) inhibited methacholine-induced salivary secretion evaluated 30min after (P<0.01), but the previous injection of AM251 (500ng/5µl, icv) prevented completely that effect. CONCLUSION: We conclude that endocannabinoids mediate the hyposialia induced by inflammogens in the SMG and in the brain. The hypofunction would be due to changes on signalling pathway produced by inflammatory compounds since anatomical changes were not observed.

[Effect of prescriptions replenishing vital essence, tonifying Qi and activating blood on TNF-alpha, IL-1beta expressions in serum and submaxillary gland of NOD mice with Sjogren's syndrome].

OBJECTIVE: To observe effect of prescriptions replenishing vital essence, tonifying Qi and activating blood on expression of tumor necrosis factor-alpha (TNF-alpha) and interleukin-IP (IL-1beta) in serum and submaxillary gland of non-obese diabetic (NOD) mice with Sjogren's syndrome. METHOD: Thirty-two NOD mice were divided into four groups at random: the model group, the traditional Chinese medicine (TCM) group, the hydroxychloroquine group, the TCM and western medicine (WM) group, with 8 mice in each group. Eight Balb/C mice were taken as the normal normal control group. The TCM group was orally administered with 0.4 mL decoction replenishing vital essence, tonifying Qi and activating blood (100 g x kg(-1)) everyday; the hydroxychloroquine group were given 0.4 mL hydroxychloroquine (60 mg x kg(-1)) everyday; the TCM WM group were given 0.4 mL decoction, replenishing vital essence tonifying Qi and activating blood (50 g x kg(-1)) and hydroxychloroquine (60 mg x kg(-1)) everyday. Mice were sacrificed after eight weeks, and their arterial blood and tissues of submaxillary gland were collected. The levels of TNF-alpha, IL-1beta in serum were detected by ELISA. Expressions of TNF-alpha, IL-1beta protein in submaxillary gland were detected by immunohisto-chemistry. RESULT: Compared with other groups, TNF-alpha, IL-1beta in serum and submaxillary gland in the model group were higher (P < 0.05). The normal group showed lower serum TNF-alpha level than other groups (P < 0.05), but without statistical significance compared with the TCM group. IL-1beta in serum in the TCM group and the TCM WM group were lower than that of the hydroxychloroquine group (P < 0.05), but without statistical significance compared with the normal group. TNF-alpha protein expression in the TCM group and the TCM WM group showed no significant difference compared with the normal group, whereas the TCM WM group were notably lower than that of the hydroxychloroquine group (P < 0.05). IL-1beta expression in the TCM WM group showed no significant difference compared with the normal group. CONCLUSION: The decoction replenishing vital essence, tonifying Qi and activating blood can decrease the levels of TNF-alpha, IL-1beta in serum and submaxillary gland of NOD mice with Sjogren's syndrome. It may improve pathological damage of submaxillary gland by regulating Th1/Th2 cell factors, in order to achieve the therapeutic effect on SS.

Submandibular gland and caries susceptibility in the obese Zucker rat.

BACKGROUND: Obesity is a prevalent disorder characterized as marked insulin resistance and low grade inflammation. We tested the hypothesis that obesity upregulates inflammatory markers in the submandibular gland in association with derangements of its architecture and pre-disposition to caries in obese Zucker rats (OZR). We also examined the potential impact of chromium picolinate (Cr(Pic)3), a nutritional supplement suggested to improve glycemic control, on the aforementioned parameters. DESIGN: Male OZR were treated with diets lacking and containing 5 or 10 mg/kg chromium (as Cr(Pic)3) from 6 weeks to about 6 months of age; lean Zucker rats (LZR) served as controls. Thereafter, glycemic status, salivary tissue architecture, and the levels of several inflammatory markers were determined in association with caries susceptibility. RESULTS: OZR showed reduced insulin sensitivity, increased ratio of phospho-nuclear factor-kappa B (NF-κB) to total NF-κB, and increased intercellular adhesion molecule-1 level but similar histological features compared to LZR. Importantly, compared to LZR, OZR displayed rampant caries and a tendency for reduced dentin mineral density. Treatment of OZR with Cr(Pic)3 attenuated upregulation of these proinflammatory indicators in association with reduced severity of caries without improving insulin sensitivity. CONCLUSIONS: Obesity promotes proinflammatory changes within the submandibular gland, without affecting glandular architecture, in association with rampant caries; Cr(Pic)3 treatment provided some protective effects.

Effects of Chinese herbs on salivary fluid secretion by isolated and perfused rat submandibular glands.

AIM: To determine whether Chinese herbs (CHs) relieve xerostomia (dry mouth) by increasing salivary secretion. METHODS: The submandibular glands of Wistar rats were surgically isolated and perfused arterially with buffered salt solution. After control perfusion, recording started 5 min prior to the start of stimulation. After fluid secretion was induced by 0.2 mumol/L carbamylcholine (CCh) in the perfusate for 10 min, Chinese herb (CH) was added in the perfusion for 5 min. CCh was then overloaded at 0.2 mumol/L in the perfusion for 20 min. The volume of salivary fluid secretion was recorded by a computer-controlled balance system. RESULTS: Saliva secretion formed an initial ephemeral peak at 30 s followed by a gradual increase to a sustained level. CH alone induced no or little saliva in all types of CH selected. During perfusion with CH, overloading of CCh promoted fluid secretion in 15 of 20 CHs. This promotion was classified into four patterns, which were eventually related to the categories of CH: Overall sustained phase was continuously raised (Yin-nourishing, fluid production-promoting and heat-clearing agents); The sustained secretion rose to reach a maximum then decreased (Qi-enhancing agent); Sustained secretion rose to reach the highest maximum and was then sustained with a slight decline (swelling-reducing, phlegm-resolving and pus-expelling agents); Stimulation of salivary secretion without any added stimulants. Addition of CCh raised the fluid secretion to reach the highest maximum then sharply decreased to a lower sustained level (blood activating agent). CONCLUSION: The present findings lead to the conclusion that various CHs have different promotional effects directly on the salivary gland.