Identification of circRNAs expression profiles and functional networks in parotid gland of type 2 diabetes mouse.

BACKGROUND: Circular RNAs (circRNAs) are a novel kind of non-coding RNAs proved to play crucial roles in the development of multiple diabetic complications. However, their expression and function in diabetes mellitus (DM)-impaired salivary glands are unknown. RESULTS: By using microarray technology, 663 upregulated and 999 downregulated circRNAs companied with 813 upregulated and 525 downregulated mRNAs were identified in the parotid glands (PGs) of type2 DM mice under a 2-fold change and P < 0.05 cutoff criteria. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analysis of upregulated mRNAs showed enrichments in immune system process and peroxisome proliferator-activated receptor (PPAR) signaling pathway. Infiltration of inflammatory cells and increased inflammatory cytokines were observed in diabetic PGs. Seven differently expressed circRNAs validated by qRT-PCR were selected for coding-non-coding gene co-expression (CNC) and competing endogenous RNA (ceRNA) networks analysis. PPAR signaling pathway was primarily enriched through analysis of circRNA-mRNA networks. Moreover, the circRNA-miRNA-mRNA networks highlighted an enrichment in the regulation of actin cytoskeleton. CONCLUSION: The inflammatory response is elevated in diabetic PGs. The selected seven distinct circRNAs may attribute to the injury of diabetic PG by modulating inflammatory response through PPAR signaling pathway and actin cytoskeleton in diabetic PGs.

Alteration of tight junctions during botulinum toxin type A-inhibited salivary secretion.

OBJECTIVES: Tight junctions (TJs) are involved in the regulation of salivary secretion via paracellular pathway. Botulinum toxin type A (BTXA) is widely used for the treatment of hypersecretion diseases such as sialorrhea. This study aimed to investigate the role of TJs in BTXA-inhibited secretion of the submandibular gland (SMG). MATERIALS AND METHODS: BTXA was injected into the SMGs of rats, and the same amount of saline was injected as a control. Western blot, real-time PCR, and immunofluorescence staining were used to detect the expression and distribution of TJ proteins. Paracellular permeability was evaluated using the transepithelial electrical resistance (TER) measurements and fluorescent tracer detection in BTXA-stimulated SMG-C6 cells. RESULTS: BTXA injection into the SMGs of rats led to increased expression of claudin (Cldn) -1 and Cldn3. Immunofluorescence staining showed no significant changes in the distribution of TJ proteins. In vitro, BTXA increased the TER values and significantly reduced the permeability of fluorescent tracer, suggesting that BTXA decreased the paracellular permeability. The expression levels of Cldn1, Cldn3, and Cldn4 were upregulated after BTXA treatment. CONCLUSION: The expression of TJ proteins changed in both animal models and SMG-C6 cells after BTXA treatment, which may contribute to the inhibition of salivary secretion.

In Vivo Oral Sentinel Lymph Node Mapping by Near-Infrared Fluorescent Methylene Blue in Rats.

This study aimed to demonstrate the feasibility of near-infrared (NIR) fluorescence imaging using methylene blue (MB) for detecting oral sentinel lymph nodes (SLNs) in rats and compared MB's tracer effects with those of indocyanine green (ICG) in SLN mapping. Different concentrations of MB were injected into the rats' left lingual submucosa to determine the optimal concentration by using a continuous (1 h) MI-1 fluorescence imaging system. To compare the tracer effects of the optimal MB concentration with ICG in oral SLN mapping, MI-1 imaging was continuously monitored for 12 h. The mean signal-to-background ratio (SBR) of the SLNs and SLN fluorescence area fraction were analyzed. SLNs and lymphatic vessels were clearly visible in all rats. The optimal injection dose of MB infected into lingual submucosa for NIR fluorescence imaging was 0.2 mL of 6.68 mM MB. During continuous monitoring for 12 h, the mean SBR of the SLNs was significantly higher in the ICG groups than in the MB groups (p < 0.001). However, the area fraction of SLN fluorescence in the ICG groups increased continuously, owing to strong fluorescent contamination. This study examined the feasibility of detection of draining lymph nodes in the oral cavity of rats using MB NIR fluorescence imaging. MB causes less fluorescent contamination than does ICG, which shows promise for clinical research and application.

High Glucose Reduces the Paracellular Permeability of the Submandibular Gland Epithelium via the MiR-22-3p/Sp1/Claudin Pathway.

Tight junctions (TJs) play an important role in water, ion, and solute transport through the paracellular pathway of epithelial cells; however, their role in diabetes-induced salivary gland dysfunction remains unknown. Here, we found that the TJ proteins claudin-1 and claudin-3 were significantly increased in the submandibular glands (SMGs) of db/db mice and high glucose (HG)-treated human SMGs. HG decreased paracellular permeability and increased claudin-1 and claudin-3 expression in SMG-C6 cells. Knockdown of claudin-1 or claudin-3 reversed the HG-induced decrease in paracellular permeability. MiR-22-3p was significantly downregulated in diabetic SMGs and HG-treated SMG-C6 cells. A miR-22-3p mimic suppressed claudin-1 and claudin-3 expression and abolished the HG-induced increases in claudin-1 and claudin-3 levels in SMG-C6 cells, whereas a miR-22-3p inhibitor produced the opposite effects. Specificity protein-1 (Sp1) was enhanced in diabetic SMGs and HG-treated SMG-C6 cells, which promoted claudin-1 and claudin-3 transcription through binding to the corresponding promoters. A luciferase reporter assay confirmed that miR-22-3p repressed Sp1 by directly targeting the Sp1 mRNA 3'-untranslated region (3'-UTR). Consistently, the miR-22-3p mimic suppressed, whereas the miR-22-3p inhibitor enhanced, the effects of HG on Sp1 expression. Taken together, our results demonstrate a new regulatory pathway through which HG decreases the paracellular permeability of SMG cells by inhibiting miR-22-3p/Sp1-mediated claudin-1 and claudin-3 expression.

MicroRNA-mRNA expression profiles and functional network after injection of botulinum toxin type A into submandibular glands.

Botulinum toxin type A (BTXA) is effective for the treatment of sialorrhea. MicroRNAs (miRNAs) have significant functions in salivary diseases, but the role of miRNAs during BTXA-inhibited salivary secretion is not yet clear. A total of 19 differentially expressed (DE) miRNAs and 1072 DE mRNAs were identified following BTXA injected into submandibular glands of rats (n = 4) through miRNA sequencing and microarray analysis. Bioinformatic analysis identified that several pathways may be associated with the inhibition of salivary secretion, such as the MAPK signalling pathway, tight junctions, and cytokine-cytokine receptor interaction. We predicted the target genes of DE miRNAs and established the miRNA-mRNA interaction network. The intersection of DE mRNAs and target genes of DE miRNAs was performed and seven mRNAs were obtained: Egr2, Paqr9, Zkscan1, Usp6n, Cyb561a3, Zfhx4, and Clic5. These findings explore the mechanism of BTXA in inhibiting salivary secretion and probably will provide new ideas for clinical application.

Aberrantly expressed lncRNAs and mRNAs after botulinum toxin type A inhibiting salivary secretion.

OBJECTIVE: In this study, we sought to determine the expression profiles of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) and construct functional networks to analyze their potential roles following botulinum toxin type A (BTXA)-mediated inhibition of salivary secretion. METHODS: The submandibular gland of rats in the BTXA and control groups was injected with BTXA and saline, respectively. Microarray analysis was used to identify the differentially expressed lncRNAs and mRNAs. Gene ontology and pathway analysis were performed to examine the biological functions. Functional networks, including lncRNA-mRNA co-expression and competing endogenous RNA (ceRNA) networks, were constructed to reveal the interaction between the coding and non-coding genes. RESULTS: Microarray analysis revealed that 254 lncRNAs and 631 mRNAs were differentially expressed between the BTXA and control groups. Bioinformatic analysis revealed that most of the mRNAs were closely related to transmembrane transporter activity. lncRNA-mRNA co-expression and ceRNA networks were constructed, and several critical mRNA-lncRNA axes and key microRNAs related to salivary secretion were identified. CONCLUSIONS: Our study identified differentially expressed lncRNAs and mRNAs through microarray analysis and explored the interactions between the coding and non-coding genes through bioinformatic analysis. These findings provide new insights into the mechanism of BTXA-mediated inhibition of salivary secretion.

Disruption of tight junctions contributes to hyposalivation of salivary glands in a mouse model of type 2 diabetes.

Tight junction (TJ) plays an important role in regulating paracellular fluid transport in salivary glands; however, little is known about the involvement of TJs in diabetes salivary glands. This study aimed to investigate the alterations of TJs and their possible contribution in diabetes-induced hyposalivation. Here, we observed that the morphologies of submandibular glands (SMGs) were impaired, characterized by enlarged acini accumulation with giant secretory granules, which were significantly reduced in atrophic ducts in SMGs of db/db mice, a spontaneous model of type-2 diabetes. However, the secretory granules were increased and scattered in the acini of diabetes parotid glands (PGs). Other ultrastructural damages including swollen mitochondria, expansive endoplasmic reticulum, and autophagosomes were observed in the diabetes group. The levels of TJ proteins including claudin-1 (Cldn1) and claudin-3 (Cldn3) were increased, whereas those of claudin-4 (Cldn4), occludin (Ocln), and zonula occludens-1 (ZO-1) were decreased in SMGs of db/db mice. Higher Cldn1 and Cldn3 and lower claudin-10 (Cldn10) and Ocln levels were observed in PGs of diabetes mice. Taken together, the structures of SMGs and PGs were impaired in diabetes mice, and the disruption of TJ integrity in both SMGs and PGs may contribute to diabetes-induced hyposalivation.