Jiawei Danxuan Koukang Alleviates Arecoline Induced Oral Mucosal Lesions: Network Pharmacology and the Combined Ultra-High Performance Liquid Chromatography (UPLC) and Mass Spectrometry (MS).

Purpose: Arecoline is one of the main toxic components of arecoline to cause oral mucosal lesions or canceration, which seriously affects the survival and life quality of patients. This study analyzed the mechanism of Jiawei Danxuan Koukang (JDK) in alleviating arecoline induced oral mucosal lesions, to provide new insights for the treatment of oral submucosal fibrosis (OSF) or cancerosis. Methods: Metabolomics was applied to analyze the composition of JDK and serum metabolites. The active ingredients of JDK were analyzed by the combined ultra-high performance liquid chromatography and mass spectrometry. The target network of JDK, metabolites and OSF was analyzed by network pharmacology, and molecular docking. Oral mucosal lesions and fibrosis were analyzed by HE and Masson staining. Cell differentiation, proliferation and apoptosis were detected. The expressions of α-SMA, Collagen I, Vimentin, Snail, E-cadherin, AR and NOTCH1 were detected by Western blot. Results: Arecoline induced the gradual atrophy and thinning of rat oral mucosal, collagen accumulation, the increase expressions of fibrosis-related proteins and Th17/Treg ratio. JDK inhibited arecoline-induced oral mucosal lesions and inflammatory infiltration. Arecoline induced changes of serum metabolites in Aminoacyl-tRNA biosynthesis, Alanine, aspartate and glutamate metabolism and Arginine biosynthesis pathways, which were reversed by M-JDK. Quercetin and AR were the active ingredients and key targets of JDK, metabolites and OSF interaction. Arecoline promoted the expression of AR protein, and the proliferation of oral fibroblasts. Quercetin inhibited the effect of arecoline on oral fibroblasts, but was reversed by AR overexpression. Arecoline induced NOTCH1 expression in CAL27 and SCC-25 cells, and promoted cell proliferation, but was reversed by M-JDK or quercetin. Conclusion: JDK improved the arecoline-induced OSF and serum metabolite functional pathway. Quercetin targeted AR protein to improve arecoline-induced OSF. JDK and quercetin inhibited arecoline-induced NOTCH1 protein expression in CAL27 and SCC-25 cells to play an anti-oral cancer role.

Small extracellular vesicles from dental follicle stem cells provide biochemical cues for periodontal tissue regeneration.

BACKGROUND: Treatments based on stem cell-derived small extracellular vesicles (sEVs) have been explored as an alternative to stem cell transplantation-based therapies in periodontal regeneration. Dental follicle stem cells (DFSCs) have shown great potential for regenerative medicine applications. However, it is unclear whether sEVs derived from DFSCs (DFSCs-sEVs) could be used in periodontal regeneration. This study investigates whether DFSCs-sEVs could regenerate damaged periodontal tissue and the potential underlying mechanism. METHODS: DFSCs-sEVs were isolated and identified, and periodontal ligament stem cells (PDLSCs) were cocultured with the isolated sEVs. The effect of DFSCs-sEVs on the biological behaviour of PDLSCs was examined using EdU assay, CCK-8 assay, cell cycle analysis, wound healing, alizarin red staining, qRT-PCR, and western blot analysis. RNA sequencing and functional enrichment analysis were used to detect the signal pathway involved in the effect of DFSCs-sEVs on PDLSCs. PDLSCs were pretreated with ERK1/2 or p38 MAPK inhibitors to investigate the possible involvement of the ERK1/2 and p38 MAPK pathways. Additionally, DFSCs-sEVs were combined with collagen sponges and transplanted into the periodontal defects in SD rats, and then, pathological changes in periodontal tissue were examined using haematoxylin and eosin (HE) staining and micro-CT. RESULTS: PDLSCs could internalize DFSCs-sEVs, thereby enhancing the proliferation assessed using EdU assay, CCK-8 assay and cell cycle analysis. DFSCs-sEVs significantly enhanced the migration of PDLSCs. DFSCs-sEVs promoted osteogenic differentiation of PDLSCs, showing deep Alizarin red staining, upregulated osteogenic genes (RUNX2, BSP, COL1), and upregulated protein expression (RUNX2, BSP, COL1, ALP). We found that p38 MAPK signalling was activated via phosphorylation. Inhibition of this signalling pathway with a specific inhibitor (SB202190) partially weakened the enhanced proliferation. After DFSCs-sEVs transplantation, new periodontal ligament-like structures and bone formation were observed in the damaged periodontal area in rats. Labelled DFSCs-sEVs were observed in the newly formed periodontal ligament and soft tissue of the defect area. CONCLUSIONS: Our study demonstrated that DFSCs-sEVs promoted periodontal tissue regeneration by promoting the proliferation, migration, and osteogenic differentiation of PDLSCs. The effect of DFSCs-sEVs in promoting PDLSCs proliferation may be partially attributed to the activation of p38 MAPK signalling pathway. DFSCs-sEVs provide us with a novel strategy for periodontal regeneration in the future.