Claudin-10 Decrease in the Submandibular Gland Contributes to Xerostomia.

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

The Role of Endothelial Barrier Function in the Fibrosis of Salivary Gland.

In the salivary glands, fibrosis occurs in many pathological conditions. Endothelial tight junction (TJ)-based barrier function plays a vital role in maintaining the homeostasis of the salivary glands. However, whether endothelial barrier function is changed and involved in the pathogenesis of glandular fibrosis is unknown. Here, by using a mouse model in which the main excretory duct of the submandibular gland (SMG) was ligated to induce inflammation and fibrosis, endothelial barrier function and TJ protein expression and distribution were examined. Both 4-kDa and 70-kDa fluorescence-labeled dextrans permeated more in the 1-, 3-, and 7-d ligated SMGs. Meanwhile, the mRNA level of claudin-5 was increased with an obvious redistribution from apicolateral membranes to lateral membranes and cytoplasm in the fibrotic glands. Notably, the TJ sealer AT1001 significantly attenuated the disrupted endothelial barrier function and thereby ameliorated the glandular fibrosis. Cytokine array detection showed that monocyte chemoattractant protein-1 (MCP-1) was highly enriched in the 3-d ligated SMGs, and MCP-1 directly impaired barrier function, increased claudin-5 expression, induced the relocalization of claudin-5, and activated p-ERK1/2 in cultured human endothelial cells. Furthermore, the upregulation and disorganization of claudin-5 as well as the elevation of MCP-1 and p-ERK1/2 signaling were also confirmed in fibrotic SMGs from patients with chronic sialadenitis and immunoglobulin G4-related sialadenitis. Altogether, our findings revealed that disrupted endothelial barrier function contributed to the progression of glandular fibrosis, and targeting endothelial TJs might be a promising approach to alleviate salivary gland fibrosis-related diseases.

LncRNA MALAT1 affects high glucose-induced endothelial cell proliferation, apoptosis, migration and angiogenesis by regulating the PI3K/Akt signaling pathway.

OBJECTIVE: To investigate the effects of long non-coding ribonucleic acid (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on the high glucose-induced proliferation, apoptosis, migration and angiogenesis of endothelial cells and its potential mechanism. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were divided into 3 groups, including control group (medium with 5.5 mmol/L glucose), high glucose group (HG group, medium with 33.5 mmol/L glucose) and lncRNA MALAT1 knockdown group [HG + MALAT1 small interfering RNA (siRNA) group, medium with 33.5 mmol/L glucose]. Cell Counting Kit-8 (CCK-8) assay was performed to observe the proliferation of HUVECs in each group at different time points. Meanwhile, the wound-healing assay was applied to detect the migratory ability of HUVECs in each group at 0 h and 24 h. The apoptosis rate of each group of cells was measured by means of flow cytometry, and the expression of Bcl-2-associated X protein (Bax) was detected via immunofluorescence at the same time. In addition, the amount of neovascularization in each group of cells was observed through the tube formation assay. Finally, Western blotting was utilized to determine the expression level of proteins in phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in each group of cells. RESULTS: Compared with that in the control group, the expression level of lncRNA MALAT1 in the HG group was elevated markedly (p<0.05). The proliferative capacity of HUVECs in the HG group was increased notably after knocking down lncRNA MALAT1 with siRNA (p<0.05). According to wound-healing assay, the knockdown of lncRNA MALAT1 could prominently reverse the declined HUVECs migratory ability induced by high glucose (p<0.05). Flow cytometry results manifested that the apoptosis level of HUVECs in the HG group was increased markedly, but inhibition on lncRNA MALAT1 could lower the apoptosis level evidently (p<0.05). The results of immunofluorescence showed that the expression of Bax in the HG + MALAT1 siRNA group was remarkably lower than that in the HG group (p<0.05). It was revealed in Western blotting that the knockdown of lncRNA MALAT1 could reverse the inhibition of high glucose on the PI3K/Akt signaling pathway in HUVECs (p<0.05). CONCLUSIONS: Inhibiting lncRNA MALAT1 can promote endothelial cell proliferation, migration and angiogenesis and repress endothelial cell apoptosis simultaneously, whose mechanism may be related to the activation of the PI3K/Akt signaling pathway.

Intrauterine growth retardation leads to the functional change of insulin secretion in the newborn rats.

To investigate the observed variation in glucose tolerance and insulin secretion in intrauterine growth retarded newborn rats and to explore the mechanism of the variations, Sprague-Dawley pregnant rats were allocated into two groups: a control group and an intrauterine energy restricted group. The intrauterine growth retardation (IUGR) in the rats was induced by 50% calorie restriction in pregnant rats from gestational day 15 until term as compared to the control group. The pancreata of control and IUGR newborn rats were dissected respectively. RT-PCR was used to study the mRNA level related to insulin synthesis and exocytosis. Intraperitoneal glucose tolerance tests were done to study the function of the pancreatic islet. We found that birth weight and pancreas mass of IUGR newborn rats were significantly lower than those of controls. Although no significant differences were observed in mRNA level of insulin and PDX-1, the expression of genes related to insulin exocytosis such as munc13-1, vamp-2, syntaxin1a, rab3a were reduced significantly in IUGR newborn rats. IUGR animals were glucose-intolerant. The observed blood insulin level and insulin secretion response to glucose challenge were both found to be at reduced level in IUGR newborn rats as compared with the normal control group rats. With these findings, we hypothesize that IUGR can induce changes in glucose homeostasis due to, at least in part, a reduced function of insulin exocytosis in newborn rats.

Comparison of M22-based ELISA and human-TSH-receptor-based luminescence assay for the measurement of thyrotropin receptor antibodies in patients with thyroid diseases.

Previously, a new procedure for measuring serum TSH receptor autoantibodies (TRAb) was reported in which the autoantibodies inhibit binding of a human monoclonal thyroid stimulating antibody M22 to TSHR-coated ELISA plate wells (TRAb ELISA). The aim of the present study was to evaluate the clinical performance of this assay in comparison to the second generation TRAb assay (TRAb LIA) based on the recombinant human TSH-receptor and chemiluminescence technology (TRAb LIA). Among the 158 patients, 84 patients suffered from Graves' disease (GD), 34 patients had Hashimoto's thyroiditis (HT), and 40 patients had euthyroid nodular thyroid disease (NTD) without signs of autoimmunity. TRAb measurements were performed according to the manufacturer's instructions. Out of 84 GD patients, 80 (95.2%) were TRAb positive as detected by the TRAb LIA. One GD patient had TRAb values within the grey zone (1.0-1.5 IU/l). All patients with HT and NTD were negative except in 6 (8.1%) cases whose TRAb values were within the grey zone. On the basis of the recommended cutoff value (TRAb 1.0 IU/l), the TRAb ELISA found 78 of 84 (92.9%) GD patients to be TRAb positive. None of the patients with HT, but two cases (5.0%) with NTD were TRAb positive. The diagnostic sensitivity of the TRAb LIA and TRAb ELISA assays was 95.2 and 92.9%, while the specificity was 100% and 97.3%, respectively. There was a close correlation (r=0.968, p<0.0001) between both assays in 84 patients with GD. Additionally, the between-run imprecision close to the cutoff limit was assessed. The calculated between-run coefficient of variation (CV) of the TRAb ELISA was 28.2% at the recommended cutoff value of 1.0 IU/l. Due to the evaluated imprecision data we propose a higher cutoff value correlating with a between-run CV of 20% (functional assay sensitivity). Our results indicate that due to a worse imprecision the TRAb ELISA has a slightly lower sensitivity and specificity compared to the TRAb LIA assay. These findings suggest that the M22 monoclonal antibody-based TRAb ELISA is not as reliable as other second generation TRAb assays in the diagnosis of Graves' diseases.

Reference data and predictive diagnostic models for calcaneus bone mineral density measured with single-energy X-ray absorptiometry in 7428 Chinese.

Calcaneus bone mineral density (BMD) of 7428 Chinese (4126 women, 3302 men; aged 22-94 years) was measured using single-energy X-ray absorptiometry (SXA). A reference range of calcaneus BMD values for healthy Chinese men and women was established and the usefulness of this method for screening and diagnosis in osteoporosis was evaluated. The peak BMD occurred at 20-24 years old and peak BMD in women was significantly lower than in men. BMD loss in the calcaneus started at the age of 35 years for women, and at 63 years in men. BMD loss rate was 1.2%/year for women and 0.56%/year for men after 50 years. The young normal reference for calcaneus BMD was 442.1+/-69.6 mg/cm2 for men and 388.3+/-61.7 mg/cm2 for women calculated from the mean BMD value of subjects whose age ranged from 20 to 49 years. The accumulated BMD loss in the calcaneus is similar to that of Ward's triangle. Multiple linear regression showed that both age and weight were important factors. The incidence of osteoporosis in older men and women (> or = 60 years) is 6.6% and 32.1% respectively. We conclude that calcaneus BMD measurement is useful and sensitive for the screening and diagnosis of osteoporosis. A predictive diagnostic model for osteoporosis based on the calcaneus was constructed using multiple linear regression and the WHO criteria for diagnosing osteoporosis can be applied to calcaneus BMD.