ABSTRACT
BACKGROUND@#Sjögren's syndrome (SS) is an autoimmune disorder characterized by sicca syndrome and/or systemic manifestations. The treatment is still challenging. This study aimed to explore the therapeutic role and mechanism of exosomes obtained from the supernatant of stem cells derived from human exfoliated deciduous teeth (SHED-exos) in sialadenitis caused by SS.@*METHODS@#SHED-exos were administered to the submandibular glands (SMGs) of 14-week-old non-obese diabetic (NOD) mice, an animal model of the clinical phase of SS, by local injection or intraductal infusion. The saliva flow rate was measured after pilocarpine intraperitoneal injection in 21-week-old NOD mice. Protein expression was examined by western blot analysis. Exosomal microRNA (miRNAs) were identified by microarray analysis. Paracellular permeability was evaluated by transepithelial electrical resistance measurement.@*RESULTS@#SHED-exos were injected into the SMG of NOD mice and increased saliva secretion. The injected SHED-exos were taken up by glandular epithelial cells, and further increased paracellular permeability mediated by zonula occluden-1 (ZO-1). A total of 180 exosomal miRNAs were identified from SHED-exos, and Kyoto Encyclopedia of Genes and Genomes analysis suggested that the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway might play an important role. SHED-exos treatment down-regulated phospho-Akt (p-Akt)/Akt, phospho-glycogen synthase kinase 3β (p-GSK-3β)/GSK-3β, and Slug expressions and up-regulated ZO-1 expression in SMGs and SMG-C6 cells. Both the increased ZO-1 expression and paracellular permeability induced by SHED-exos were abolished by insulin-like growth factor 1, a PI3K agonist. Slug bound to the ZO-1 promoter and suppressed its expression. For safer and more effective clinical application, SHED-exos were intraductally infused into the SMGs of NOD mice, and saliva secretion was increased and accompanied by decreased levels of p-Akt/Akt, p-GSK-3β/GSK-3β, and Slug and increased ZO-1 expression.@*CONCLUSION@#Local application of SHED-exos in SMGs can ameliorate Sjögren syndrome-induced hyposalivation by increasing the paracellular permeability of glandular epithelial cells through Akt/GSK-3β/Slug pathway-mediated ZO-1 expression.
Subject(s)
Mice , Animals , Humans , Sjogren's Syndrome/therapy , Proto-Oncogene Proteins c-akt/metabolism , Tight Junctions/metabolism , Glycogen Synthase Kinase 3 beta , Mice, Inbred NOD , Phosphatidylinositol 3-Kinases/metabolism , Exosomes/metabolism , Xerostomia , Phosphatidylinositol 3-Kinase , MicroRNAs/geneticsABSTRACT
Objective@#To compare the general biological characteristics and the expressions of proteins involved in secretion in stem cells from the pulp of human exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSC).@*Methods@#SHED and DPSC were cultured and collected at passage 4 (P4) and P7. The submandibular gland epithelial and interstitial cells were cultured with tissue culture method. The cell morphology was observed using a phase contrast microscope. Flow cytometry was used to detect stem cell surface markers. Cell counting kit-8 (CCK-8) and IncuCyte ZOOM were used to evaluate cell proliferation. Quantitative real-time PCR (qPCR) was performed to examine the mRNA expressions of proteins involved in fluid and protein secretion.@*Results@#P4 and P7 SHED and DPSC were spindle-shaped. There was no difference in cell morphology among the 4 group cells. P4 and P7 SHED and DPSC expressed CD29, CD44, CD73, and CD90, the mesenchymal stem cell markers, while, CD49f and CD117, the epithelium markers were undetected. There was no difference in cell proliferation among the 4 group cells. Compared with P4 SHED, the expressions of muscarinic cholinergic receptor 1 (MR1), MR3, aquaporin 5 (AQP5), β1-adrenoceptor (β1-AR), α-amylase, and mucin 5B in SHED were not different, while β2-AR expression was decreased (P<0.05). Compared with P4 DPSC, the expressions of MR3, β2-AR, and α-amylase in P7 DPSC were not different, while, the expressions of MR1, AQP5, β1-AR, and mucin 5B were decreased (P<0.05). Compared with primary cultured submandibular gland epithelial cells and gland tissues from a child, the expressions of proteins involved in secretion were all decreased. Compared with submandibular epithelial cells from adults, the expression of AQP5 in P4 DPSC was decreased (P<0.05), while other proteins were not different. The expressions of AQP5, β1-AR, α-amylase and mucin 5B in P7 DPSC were increased (P<0.05), while other proteins were not different. In P4 and P7 DPSC, all the protein expression levels were decreased, compared with those in submandibular gland tissues (P<0.01).@*Conclusions@#Compared with DPSC, SHED have stable growth and the expressions of protein involved fluid and protein secretion are low. Based on its extensive sources and easy separation, SHED can be used as the ideal seed cell for salivary gland tissue engineering and the treatment of salivary gland hypofunction, and the P4 to P7 SHED can be used for experimental study.