SHED-derived Exosomes Ameliorate Hyposalivation Caused by Sjögren Syndrome via Akt/GSK-3β/Slug-mediated ZO-1 Expression

Abstract

Objective: 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 Sjögren syndrome (SS).Methods: SHED-exos were administered to the submandibular glands (SMGs) of 14-week-old nonobese 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 miRNAs were identified by microarray analysis. Paracellular permeability was evaluated by transepithelial electrical resistance measurement.Results: Local injection of SHED-exos significantly increased saliva secretion and alleviated lymphocytic infiltration in the SMGs of NOD mice. The injected SHED-exos could be taken up by glandular epithelial cells and increased ZO-1 expression. Moreover, SHED-exos increased paracellular permeability, whereas this effect disappeared in ZO-1 knockout cells and reappeared in ZO-1-rescued cells. Furthermore, 180 exosomal miRNAs were identified from SHED-exos, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that the PI3K/Akt pathway might play an important role. SHED-exos treatment induced the downregulation of p-Akt/Akt, p-GSK-3β/GSK-3β, and Slug and the upregulation of ZO-1 in SMGs and SMG-C6 cells. Both the increased ZO-1 expression and paracellular permeability induced by SHED-exos were abolished by IGF1, a PI3K agonist. In addition, 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.

SHED‐exos promote saliva secretion by suppressing p‐ERK1/2‐mediated apoptosis in glandular cells

“…They also expressed the exosome markers CD9, CD63 and heat shock protein 70 (HSP70) (Figure 1c). These results were in keeping with our recent findings (Du et al., 2023). The stimulated saliva flow rate was decreased in 21‐week‐old NOD mice compared with that in 7‐week‐old NOD mice (Figure 1d).…”

“…Previously, we reported that SHEDs protected glandular tissues, thereby avoiding immune damage (Du et al., 2019). Recently, we further proved that SHED‐exos can increase the secretory function of surviving glandular epithelial cells (Du et al., 2023). However, whether SHED‐exos can decrease the cell death of SGECs induced by inflammation and then promote secretion is unknown.…”

“…Exosome‐carried miRNAs play important roles and have attracted much attention (Colombo et al., 2014). Previously, we performed a miRNA microarray of SHED‐exos, and 180 exosomal miRNAs were identified (Du et al., 2023). The original data were uploaded to the Gene Expression Omnibus database (accession number: GSE199497).…”

“…For example, intravenous injection of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, stem cells from human exfoliated deciduous teeth (SHEDs), labial gland‐derived mesenchymal stem cells and conditioned media from dental pulp stem cells exert protective effects on glandular secretory function and alleviate hyposalivation in experimental or clinical SS, mainly through their immunoregulatory effects (Du et al., 2019; Li, Jiang, et al., 2021; Li, Xing, et al., 2021; Matsumura‐Kawashima et al., 2021; Xu et al., 2012). Our recent study also proved that SHED‐derived exosomes (SHED‐exos) can improve the secretory function of surviving glandular epithelial cells and promote salivary secretion in SS mice (Du et al., 2023). However, there is no report on whether cell‐based therapy can decrease cell death in glandular epithelia and thus maintain secretory function.…”

“…For SHED‐exos isolation, SHEDs were cultured in exo‐free medium with exosome‐depleted FBS (Du et al., 2023; Liu et al., 2022; Xie et al., 2022). Then, exosomes were collected from the supernatants and separated by ultracentrifugation and sucrose cushion, according to previous studies (Chen, Fu, et al., 2020; Du et al., 2023; Lamparski et al., 2002; Qu et al., 2009; Yang et al., 2017). Briefly, cell supernatants were centrifuged at 300× g for 10 min, 2000× g for 30 min and 10,000× g for 30 min to remove large vesicles, dead cells and cell debris.…”