High-Throughput Combined Analysis of Saliva Microbiota and Metabolomic Profile in Chinese Periodontitis Patients: A Pilot Study.

The onset and progression of periodontitis involves complicated interactions between the dysbiotic oral microbiota and disrupted host immune-inflammatory response, which can be mirrored by the changes in salivary metabolites profile. This pilot study sought to examine the saliva microbiome and metabolome in the Chinese population by the combined approach of 16s rRNA sequencing and high-throughput targeted metabolomics to discover potential cues for host-microbe metabolic interactions. Unstimulated whole saliva samples were collected from eighteen Stage III and IV periodontitis patients and thirteen healthy subjects. Full-mouth periodontal parameters were recorded. The taxonomic composition of microbiota was obtained by 16s rRNA sequencing, and the metabolites were identified and measured by ultra-high performance liquid chromatography and mass spectrometry-based metabolomic analysis. The oral microbiota composition displayed marked changes where the abundance of 93 microbial taxa differed significantly between the periodontitis and healthy group. Targeted metabolomics identified 103 differential metabolites between the patients and healthy individuals. Functional enrichment analysis demonstrated the upregulation of protein digestion and absorption, histidine metabolism, and nicotinate and nicotinamide metabolism pathways in the dysbiotic microbiota, while the ferroptosis, tryptophan metabolism, glutathione metabolism, and carbon metabolism pathways were upregulated in the patients. Correlation analysis confirmed positive relationships between the clinical parameters, pathogen abundances, and disease-related metabolite levels. The integral analysis of the saliva microbiome and metabolome yielded an accurate presentation of the dysbiotic oral microbiome and functional alterations in host-microbe metabolism. The microbial and metabolic profiling of the saliva could be a potential tool in the diagnosis, prognosis evaluation, and pathogenesis study of periodontitis.

Interleukin-22 Inhibits Apoptosis of Gingival Epithelial Cells Through TGF-ß Signaling Pathway During Periodontitis.

Periodontitis is a chronic inflammatory disease characterized by the destruction of tooth-supporting tissues. The gingival epithelium is the first barrier of periodontal tissue against oral pathogens and harmful substances. The structure and function of epithelial lining are essential for maintaining the integrity of the epithelial barrier. Abnormal apoptosis can lead to the decrease of functional keratinocytes and break homeostasis in gingival epithelium. Interleukin-22 is a cytokine that plays an important role in epithelial homeostasis in intestinal epithelium, inducing proliferation and inhibiting apoptosis, but its role in gingival epithelium is poorly understood. In this study, we investigated the effect of interleukin-22 on apoptosis of gingival epithelial cells during periodontitis. Interleukin-22 topical injection and Il22 gene knockout were performed in experimental periodontitis mice. Human gingival epithelial cells were co-cultured with Porphyromonas gingivalis with interleukin-22 treatment. We found that interleukin-22 inhibited apoptosis of gingival epithelial cells during periodontitis in vivo and in vitro, decreasing Bax expression and increasing Bcl-xL expression. As for the underlying mechanisms, we found that interleukin-22 reduced the expression of TGF-ß receptor type II and inhibited the phosphorylation of Smad2 in gingival epithelial cells during periodontitis. Blockage of TGF-ß receptors attenuated apoptosis induced by Porphyromonas gingivalis and increased Bcl-xL expression stimulated by interleukin-22. These results confirmed the inhibitory effect of interleukin-22 on apoptosis of gingival epithelial cells and revealed the involvement of TGF-ß signaling pathway in gingival epithelial cell apoptosis during periodontitis.

An Appearance Data-Driven Model Visualizes Cell State and Predicts Mesenchymal Stem Cell Regenerative Capacity.

Mesenchymal stem cells (MSCs) are widely used in treating various diseases. However, lack of a reliable evaluation approach to characterize the potency of MSCs has dampened their clinical applications. Here, a function-oriented mathematical model is established to evaluate and predict the regenerative capacity (RC) of MSCs. Processed by exhaustive testing, the model excavates four optimal fitted indices, including nucleus roundness, nucleus/cytoplasm ratio, side-scatter height, and ERK1/2 from the given index combinations. Notably, three of them except ERK1/2 are cell appearance-associated features. The predictive power of the model is validated via screening experiments of these indices by predicting the RC of newly enrolled and chemical inhibitor-treated MSCs. Further RNA-sequencing analysis reveals that cell appearance-based indices may serve as major indicators to visualize the results of integration-weighted signals in and out of cells and reflect MSC stemness. In general, this study proposes an appearance data-driven predictive model for the RC and stemness of MSCs.

Electrostatic Charge-Mediated Apoptotic Vesicle Biodistribution Attenuates Sepsis by Switching Neutrophil NETosis to Apoptosis.

Mesenchymal stem cell (MSC) therapy can attenuate organ damage and reduce mortality in sepsis; however, the detailed mechanism is not fully elucidated. In this study, it is shown that MSC-derived apoptotic vesicles (apoVs) can ameliorate multiple organ dysfunction and improve survival in septic mice. Mechanistically, it is found that tail vein-infused apoVs mainly accumulate in the bone marrow of septic mice via electrostatic charge interactions with positively charged neutrophil extracellular traps (NETs). Moreover, apoVs switch neutrophils NETosis to apoptosis via the apoV-Fas ligand (FasL)-activated Fas pathway. In summary, these findings uncover a previously unknown role of apoVs in sepsis treatment and an electrostatic charge-directed target therapeutic mechanism, suggesting that cell death is associated with disease development and therapy.

Curcumin reduces apoptosis and promotes osteogenesis of human periodontal ligament stem cells under oxidative stress in vitro and in vivo.

AIMS: Human periodontal ligament stem cells (hPDLSCs) tether the teeth to the surrounding bone and are considered as major functional stem cells responsible for regeneration of the alveolar bone and periodontal ligament tissue. However, the outcome of stem cell regenerative therapy is affected by the survival rate and their differentiation potential of transplanted cells. This is primarily because of local oxidative stress and chronic inflammation at the transplantation site. Therefore, our study aimed to explore whether a natural antioxidant, curcumin could increase the tissue regeneration ability of transplanted hPDLSCs. MAIN METHODS: A hydrogen peroxide environment and a rat cranial bone defect model were built to mimic the oxidative stress conditions in vitro and in vivo, respectively. We evaluated the effect of curcumin on oxidative status, apoptosis, mitochondrial function and osteogenic differentiation of H2O2-stimulated hPDLSCs in vitro. We also measured the effect of curcumin on cell viability and bone repair ability of transplanted hPDLSCs in vivo. KEY FINDINGS: Our data showed that curcumin enhanced cell proliferation, reduced the reactive oxygen species (ROS) levels and apoptosis, maintained the standard mitochondrial structure and function, and promoted osteogenic differentiation of H2O2-stimulated hPDLSCs. The extracellular regulated protein kinases 1/2 (Erk1/2) signaling pathway was determined to be involved in the osteogenic differentiation of the H2O2-stimulated hPDLSCs. Moreover, curcumin enhanced the viability and the bone repair ability of hPDLSCs in vivo. SIGNIFICANCE: Curcumin reduced apoptosis and promoted osteogenesis of the hPDLSCs under oxidative stress, and might therefore have a potential clinical use with respect to tissue regeneration.

Potential Research Tool of Stem Cells from Human Exfoliated Deciduous Teeth: Lentiviral Bmi-1 Immortalization with EGFP Marker.

Stem cells from human exfoliated deciduous teeth (SHED) are a favourable source for tissue engineering, for its great proliferative capacity and the ease of collection. However, the transplantation of stem cells and the study of stem cell-based tissue engineering require massive stem cells. After long-term expansion, stem cells face many challenges, including limited lifespan, senescence, and loss of stemness. Therefore, a cell line capable of overcoming those problems should be built. In this study, we generated a Bmi-1-immortalized SHED cell line with an enhanced green fluorescent protein (EGFP) marker (SHED-Bmi1-EGFP) using lentiviral transduction. We compared this cell line with the original SHED for cell morphology under a microscope. The expression of Bmi-1 was detected with Western blot. Replicative lifespan determination and colony-forming efficiency assessment were using to assay proliferation capability. Senescence-associated ß-galactosidase assay was performed to assay the senescence level of cells. Moreover, multipotency, karyotype, and tumour formation in nude mice of SHED and SHED-Bmi1-EGFP were also tested. Our results confirmed that Bmi-1 immortalization did not affect the main features of SHED. SHED-Bmi1-EGFP could be passaged for a long time and stably expressed EGFP. SHED-Bmi1-EGFP at a late passage showed low activity of ß-galactosidase and similar multilineage differentiation as SHED at an early passage. The immortalized cells had no potential tumourigenicity ability in vivo. Moreover, we provided some suggestions for potential applications of the immortalized SHED cell line with the EGFP marker. Thus, the immortalized cell line we built can be used as a functional tool in the lab for long-term studies of SHED and stem cell-based regeneration.

Effect of matrix metalloproteinase 8 inhibitor and chlorhexidine on the cytotoxicity, oxidative stress and cytokine level of MDPC-23.

OBJECTIVE: This study investigated the effect of matrix metalloproteinase-8 inhibitor I (MMP8-I) and chlorhexidine (CHX) on the viability, oxidative stress and cytokine secretion of MDPC-23 under short-term (30min) and long-term (3 days) culture. METHODS: MDPC-23 were treated with MMP8-I or CHX for 30min, 1day, 2days and 3days to detect the proliferation by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. In the following assays, MDPC-23 treated with 0.0003% CHX were referred to CHX group, treated with 8µM MMP8-I were MMP8-I group. Cells without additional treatment were regarded as control group. The cell cycle, reactive oxygen species (ROS) level, and apoptosis were assessed by flow cytometry. The cytokine level was quantified by enzyme-linked immunosorbent assay (ELISA). RESULTS: In 30min, CHX at concentrations higher than 0.0003% dilution inhibited cell proliferation when compared to the control group. MMP8-I (0.1-500µM) showed no obvious cytotoxicity to MDPC-23, and MMP8-I (1000µM) inhibited cell proliferation. In 3 days, CHX (0.0003%) significantly inhibited cell growth, while MMP8-I (8µM) had no cytotoxicity. In the CHX group, the S phase population was decreased, and cellular ROS were increased in 3 days. In the MMP8-I group, the change of S phase population and cellular ROS was not significant compared with the control group. Cell apoptosis was not elevated in the MMP8-I group, while the apoptosis rate was increased in the CHX group both in 30min and 3 days. In 30min, CHX treatment significantly increased the secretion of interleukin (IL)-1ß and IL-8, but slightly increased the secretion of IL-10, while MMP8-I caused no change in cytokines. In 3 days, CHX treatment significantly increased the secretion of IL-1ß, IL-6, and IL-8, and inhibited the secretion of IL-10. MMP8-I treatment caused the increase of IL-6. SIGNIFICANCE: Compared with CHX, MMP8-I at low concentration did not result in cytotoxicity, oxidative stress, or the disorder of immune response.

Substance P attenuates hyperoxia­induced lung injury in neonatal rats.

The aim of the study was to investigate the effects of substance P (SP) in hyperoxia­induced lung injury in newborn rats and to elucidate its protective mechanism of action via the sonic hedgehog (SHH) signaling pathway. Twelve­hour­old neonatal Sprague­Dawley rats were randomly divided into one of four groups: air, hyperoxia, air + SP and hyperoxia + SP. In a separate set of experiments, the neonatal rat pups were exposed to 21 or 95% O2 for 14 days with or without intraperitoneal administration of rat SP. The animals were sacrificed at 3, 7 and 14 days, respectively, of hyperoxia exposure. Lung pathology and grade of lung tissue injury were examined by light microscopy. Oxidative stress was evaluated by malondialdehyde (MDA) and antioxidant activity was measured by superoxide dismutase (SOD) in tissue homogenates. The expression of SHH mRNA and protein were detected by quantitative polymerase chain reaction (qPCR) and western blot analysis, respectively. In the hyperoxia group, marked characteristics of acute lung injury (ALI) were observed. Compared with the simple hyperoxia treatment, the lung damage was significantly ameliorated following the addition of SP. Furthermore, the levels of MDA were decreased and SOD was significantly increased following the addition of SP. SP stimulation may result in activation of the SHH signaling pathway and the expression of SHH markedly increased following treatment with SP. The present study demonstrated that SP protected against the hyperoxia­induced lung damage by attenuating oxidative stress, elevating the antioxidant activities and upregulating the signaling pathway of SHH.