Er:YAG Laser Alleviates Inflammaging in Diabetes-Associated Periodontitis via Activation CTBP1-AS2/miR-155/SIRT1 Axis.

Periodontitis is a significant health concern for individuals with diabetes mellitus (DM), characterized by inflammation and periodontium loss. Hyperglycaemia in DM exacerbates susceptibility to periodontitis by inducing inflammaging in the host immune system. The use of erbium-doped yttrium-aluminum-garnet laser (ErL) in periodontitis treatment has gained attention, but its impact on diabetic-associated periodontitis (DP) and underlying mechanisms remain unclear. In this study, we simulated DP by exposing human periodontal ligament fibroblasts (PDLFs) to advanced glycation end products (AGEs) and lipopolysaccharides from P. gingivalis (Pg-LPS). Subsequently, we evaluated the impact of ErL on the cells' wound healing and assessed their inflammaging markers. ErL treatment promoted wound healing and suppressed inflammaging activities, including cell senescence, IL-6 secretion, and p65 phosphorylation. Moreover, the laser-targeted cells were observed to have upregulated expression of CTBP1-AS2, which, when overexpressed, enhanced wound healing ability and repressed inflammaging. Moreover, bioinformatic analysis revealed that CTBP1-AS2 acted as a sponge for miR155 and upregulated SIRT1. In conclusion, ErL demonstrated the ability to improve wound healing and mitigate inflammaging in diabetic periodontal tissue through the CTBP1-AS2/miR-155/SIRT1 axis. Targeting this axis could represent a promising therapeutic approach for preventing periodontitis in individuals with DM.

Galectin-7 promotes proliferation and wound healing capacities in periodontal ligament fibroblasts by activating ERK signaling.

Periodontitis is a progressive inflammation condition and a primary cause of tooth loss in adults. As one of the abundant cell types in the periodontium, periodontal ligament fibroblasts (PDLFs) play an integral role in the maintenance and regeneration of periodontal tissue. Our previous work has shown that the application of Er:YAG laser increased the cell proliferation and migratory capacity of PDLFs via induction of galectin-7. In the present study, we aimed to evaluate if the forced expression of galectin-7 directly affected the cellular phenotypes of PDLFs. Our results showed that the cell proliferation, transwell migration, invasion, and wound healing capacities were all upregulated in PDLFs with the ectopic expression of galectin-7. These results suggest that therapeutic approaches to enhance the expression of galectin-7 in periodontium may accelerate tissue regeneration by recruiting more PDLFs to the injured site.

Magnolol inhibits cancer stemness and IL-6/Stat3 signaling in oral carcinomas.

BACKGROUND/PURPOSE: Cancer stem cells (CSCs) have been known to be implicated in tumorigenesis, metastasis, and drug resistance in oral squamous cell carcinomas (OSCC). In this study, we aimed to investigate whether magnolol, a polyphenolic component derived from Magnolia officinalis, exhibited the anti-CSCs properties. METHODS: The cytotoxicity of magnolol was tested using normal gingival epithelioid SG cells and sphere-forming OSCC-CSCs isolated from SAS, OECM1, and GNM cells. Secondary sphere-forming ability, the proportion of ALDH1 positive cells, Transwell migration, and invasion capacities were examined as well. The chemosensitive effects of magnolol were investigated using MTT, secondary sphere-forming, and invasion assays. RESULTS: Magnolol exerted a higher cytotoxicity of OSCC-CSCs and cancer stemness features, including self-renewal ability, the expression CSC marker, migration, and invasion capacities were all downregulated in magnolol-treated OSCC-CSCs. Moreover, administration of magnolol potentiated the effect of cisplatin, including a decrease in cell viability, self-renewal, and invasion activities. In addition, we observed that the secretion of IL-6 and phosphorylation of Stat3 were decreased in OSCC-CSCs treated with magnolol. CONCLUSION: Our data suggest that magnolol is able to target CSCs and suppress the cancer stemness properties, at least in part, via IL-6/Stat3 signaling. Besides, a dietary supplement of magnolol may function as an adjunct to cisplatin treatment.

Er:YAG laser promotes proliferation and wound healing capacity of human periodontal ligament fibroblasts through Galectin-7 induction.

BACKGROUND/PURPOSE: Among various dental lasers, the erbium-doped yttrium-aluminum-garnet (Er:YAG) laser has great potential for periodontal treatment including soft and hard tissue ablation with minimal thermal side effects under suitable energy densities and it has multiple effects on tissues for wound-healing benefits. In the present study, we sought to reveal the molecular mechanism underlying the impact of Er:YAG laser on PDL fibroblasts. METHODS: Cells were irradiated by a Er:YAG laser with various energy densities (3.6-6.3 J/cm2). MTT assay was used for cell proliferation, and the transwell system was employed for migration and invasion abilities. The wound healing capacity was evaluated by a scratch assay. After confirming these effects, qRT-PCR and western blotting analysis was applied to identify the differentially galectin-7 expression in the irradiated cells. Knockdown experiments were conducted to reveal the functional role of galectin-7 in the modulation of Er:YAG laser-mediated effects. RESULTS: 4.2 J/cm2 was the lowest energy density to induce the optimal cell proliferation, migration and invasion abilities. In the group of upregulated genes, galectin-7 was selected for further examination and its elevation after Er:YAG laser treatment was validated by RT-PCR and Western blot. We demonstrated that silence of galectin-7 abrogated the effects of Er:YAG laser on cell proliferation, migration ad invasion, suggesting the Er:YAG laser promoted these effects through induction of galectin-7. CONCLUSION: These findings indicated that Er:YAG laser may accelerate the regeneration process in periodontal tissues through enhancement of their proliferative and mobile activities. Additionally, the significance of galectin-7 in the Er:YAG laser-elicited benefits was demonstrated.

Magnolol ameliorates the accumulation of reactive oxidative stress and inflammation in diabetic periodontitis.

BACKGROUND/PURPOSE: Periodontal disease and diabetes mellitus (DM) are both chronic inflammatory and highly prevalent diseases. A large amount of evidence suggested that the accumulation of oxidative stress plays a significant role in the deterioration of both diseases. Magnolol has been known to possess anti-inflammatory and anti-oxidant activities in various tissues, but its effects on gingival cells under diabetic conditions have not been fully understood. METHODS: We assessed the generation of reactive oxygen species (ROS), Transwell migration, and wound healing ability in response to the advanced glycation end products (AGEs) stimulation with or without Magnolol treatment. Subsequently, we examined the expression of Nrf2 and HO-1 to ascertain whether Magnolol was able to activate the anti-oxidant signaling. We also measured the secretion of IL-6 and IL-8, and conducted a knockdown experiment to elucidate the effect of Mrf2 on their secretion. RESULTS: The AGEs-induced ROS was dose-dependently downregulated following the Magnolol treatment. Likewise, the reduced Transwell migration and wound healing ability were improved by various concentrations of Magnolol. Results from qRT-PCR indicated that the suppression of Nrf2 and HO-1 following AGEs stimulation was reversed by Magnolol. Also, the AGEs-elicited production of IL-6 and IL-8 was inhibited by Magnolol. Moreover, our results demonstrated that this anti-inflammatory effect was mediated by the upregulation of Nrf2. CONCLUSION: These findings showed that excessive AGEs in the gingiva may lead to the accumulation of ROS and pro-inflammatory cytokines. Supplement of Magnolol may be beneficial to improve the impaired wound healing and inflammation by upregulation of Nrf2 signaling for DM patients with periodontal disease.

miR-200a inhibits proliferation rate in drug-induced gingival overgrowth through targeting ZEB2.

BACKGROUND/PURPOSE: Gingival overgrowth can occur as a result of poor oral hygiene or a side effect of taking certain medications, such as cyclosporine A (CsA). It has been shown that this immunosuppressant drug induces epithelial-to-mesenchymal transition (EMT) in the gingival epithelium but the associated molecular mechanism remains to be elucidated. METHODS: We first assessed the relative expression of microRNA-200a (miR-200a) in response to the CsA treatment using qRT-PCR. Next, luciferase reporter assay was applied to examine whether miR-200a was able to regulate ZEB2 and Western blot was utilized to measure the expression of ZEB2 in normal human gingival fibroblasts (HGFs). To confirm the significance of miR-200a and ZEB2 in the CsA-induced gingival overgrowth, miR-200a inhibitor and shRNA mediated knockdown of ZEB2 were used and cell proliferation in HGFs was assessed by MTT assay. RESULTS: The expression of miR-200a was dose-dependently downregulated following the CsA treatment. Luciferase reporter assay confirmed that ZEB2 was a direct downstream target regulated by miR-200a and ZEB2 was indeed increased after the administration of CsA. We demonstrated that knockdown of ZEB2 hampered the CsA-induced HGFs proliferation and the elevated cell proliferation due to inhibition of miR-200a was reversed by repression of ZEB2. CONCLUSION: Our results showed that insufficient miR-200a in HGFs caused by CsA administration may lead to gingival enlargement mediated by the upregulation of ZEB2. This finding supported that CsA-induced EMT contributed to the adverse effect of using CsA and miR-200a may serve as an upstream target to prevent the overgrowth of the gingiva.

Hinokitiol ablates myofibroblast activation in precancerous oral submucous fibrosis by targeting Snail.

Oral submucous fibrosis (OSF) is a precancerous condition with symptoms of limited mouth opening and areca nut chewing habit has been implicated in its pathogenesis. Hinokitiol, a natural tropolone derived from Chamacyparis taiwanensis, has been reported to improve oral lichen planus and inhibit various cancer cells. Here, we showed that hinokitiol reduced the myofibroblast activities in fBMFs and prevented the arecoline-induced transdifferentiation. Treatment of hinokitiol dose-dependently downregulated the myofibroblast markers as well as various EMT transcriptional factors. In particular, we identified that Snail was able to bind to the E-box in the α-SMA promoter. Our data suggested that exposure of fBMFs to hinokitiol mitigated the hallmarks of myofibroblasts, while overexpression of Snail eliminated the effect of hinokitiol. These findings revealed that the inhibitory effect of hinokitiol on myofibroblasts was mediated by repression of α-SMA via regulation of Snail and showed the anti-fibrotic potential of hinokitiol in the treatment of OSF.

Down-regulation of miR-200b-targeting Slug axis by cyclosporine A in human gingival fibroblasts.

BACKGROUND/PURPOSE: Cyclosporine A (CsA) has been used as an immunosuppressive agent with a side effect of gingival overgrowth. It has been known that CsA-induced epithelial-mesenchymal transition (EMT) in gingiva, but the molecular mechanism has not been fully unveiled. The purpose of the study is to investigate functional roles of microRNAs in gingival overgrowth. METHODS: The effect of CsA on the expression of microRNA-200b (miR-200b) in normal human gingival fibroblasts (HGFs) was determined using qRT-PCR. Luciferase reporter assay and Western blot were utilized to examine the relationship between miR-200b and EMT inducer Slug. Cell proliferation was assessed by MTT assay. RESULTS: CsA was found to downregulate the miR-200b transcript in HGFs in a dose-dependent manner. Luciferase reporter assay confirmed that Slug was a direct target of miR-200b, and the CsA-induced cell proliferation and Slug upregulation were inhibited by overexpression of miR-200b. Additionally, the silence of Slug reversed the increased proliferation of HGFs by miR-200b inhibitor. CONCLUSION: Repression of miR-200b after CsA administration led to an increase in Slug expression. Our results suggested that miR-200b was an upstream effector of the CsA-induced EMT and may act as a therapeutic target for CsA-induced gingival overgrowth.

Upregulation of Slug expression by cyclosporine A contributes to the pathogenesis of gingival overgrowth.

BACKGROUND/PURPOSE: Gingival overgrowth occurs as a side effect of systemic medication with immunosuppressant cyclosporine A (CsA). Slug, a master regulator of epithelial-mesenchymal transition, is dramatically upregulated in a variety of fibrotic diseases. The aim of this study is to investigate the role of epithelial-mesenchymal transition marker Slug in the pathogenesis of CsA-induced gingival overgrowth. METHODS: Clinically healthy gingiva and CsA-induced gingival overgrowth specimens were analyzed by immunohistochemistry. The effect of CsA on normal human gingival fibroblasts (HGFs) was used to elucidate whether Slug expression could be affected by CsA by real-time reverse transcription-polymerase chain reaction and western blot. Cell proliferation in CsA-treated HGFs with Slug lentiviral-mediated shRNAi knockdown was evaluated by tetrazolium bromide reduction assay. RESULTS: Slug expression was higher in CsA-induced gingival overgrowth specimens than in clinical healthy gingiva (p < 0.05). Slug expression was significantly higher in CsA-induced gingival overgrowth specimens with higher levels of inflammatory infiltrates (p < 0.05). CsA was found to increase Slug transcript and protein expression in HGFs in a dose-dependent manner (p < 0.05). In addition, knockdown of Slug significantly suppressed CsA-induced cell proliferation in HGFs (p < 0.05). CONCLUSION: Taken together, upregulation of Slug in HGFs stimulated by CsA may play an important role in the pathogenesis of CsA-induced gingival overgrowth.

Elevated Snail expression in human gingival fibroblasts by cyclosporine A as the possible pathogenesis for gingival overgrowth.

BACKGROUND/PURPOSE: Cyclosporine A (CsA) is used as an immunosuppressive agent, and its prominent side effect is the induction of gingival overgrowth. Snail is a master regulator of epithelial-mesenchymal transition (EMT). EMT under pathological processes could lead to fibrotic changes. The purpose of this study was to investigate the role of Snail in the pathogenesis of CsA-induced gingival overgrowth. METHODS: The effect of CsA on normal human gingival fibroblasts (HGFs) was used to elucidate whether Snail expression could be induced by CsA by using quantitative real-time reverse transcription-polymerase chain reaction and western blot. The cell proliferation rate in CsA-treated HGFs with Snail lentiviral-mediated short hairpin RNA interference (shRNAi) knockdown was evaluated by tetrazolium bromide reduction assay. RESULTS: CsA increased the Snail transcript and Snail protein expression in HGFs in a dose-dependent manner (p < 0.05). In addition, downregulation of Snail by lentiviral infection significantly reduced CsA-stimulated cell proliferation in HGFs (p < 0.05). CONCLUSION: CsA stimulated Snail expression and cell proliferation in HGFs, while silencing Snail could effectively reverse these phenomena. These results may provide new avenues for the design of novel antifibrotic therapies for CsA-induced gingival overgrowth through targeting Snail.

The modulation of hypoxia-inducible factor-1α/plasminogen activator inhibitor-1 axis in human gingival fibroblasts stimulated with cyclosporine A.

BACKGROUND/PURPOSE: The prominent side effect of the immunosuppressive drug cyclosporine A (CsA) is gingival overgrowth. Hypoxia-inducible factor (HIF)-1α regulates a wide variety of profibrogenic genes, which are closely associated with tissue fibrosis. The aim of this study was to compare HIF-1α expression in normal gingival tissues and CsA-induced gingival overgrowth specimens and further explore the potential mechanisms that may lead to induction of HIF-1α expression. METHODS: Fifteen CsA-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. Western blot was used to investigate the effects of CsA on the expression of HIF-1α in cultured human gingival fibroblasts. The effects of CsA on plasminogen activator inhibitor (PAI)-1 expression were evaluated in environmental hypoxia. RESULTS: HIF-1α staining in gingival tissue was stronger in CsA-induced gingival overgrowth group than normal gingival group (p < 0.05). The expression of HIF-1α was significantly higher in CsA-induced gingival overgrowth specimens with higher levels of inflammatory infiltrates (p = 0.041). CsA was found to upregulate HIF-1α protein in a dose-dependent manner (p < 0.05). Hypoxia increased CsA-induced PAI-1 protein expression than normoxic conditions (p < 0.05). CONCLUSION: These results suggest that HIF-1α expression is significantly upregulated in CsA-induced gingival overgrowth specimens. The activation of HIF-1α may promote fibrogenesis by an increase of PAI-1 expression and a subsequent elevation of extracellular matrix production in gingival tissues.

Concurrent expression of Oct4 and Nanog maintains mesenchymal stem-like property of human dental pulp cells.

Human dental pulp stem cells (DPSCs), unique mesenchymal stem cells (MSCs) type, exhibit the characteristics of self-renewal and multi-lineage differentiation capacity. Oct4 and Nanog are pluripotent genes. The aim of this study was to determine the physiological functions of Oct4 and Nanog expression in DPSCs. Herein, we determined the critical role of an Oct4/Nanog axis modulating MSCs properties of DPSCs by lentiviral-mediated co-overexpression or co-knockdown of Oct4/Nanog in DPSCs. MSCs properties including osteogenic/chondrogenic/adipogenic induction differentiation was assayed for expression of osteogenic/chondrogenic/adipogenic markers by quantitative real-time RT-PCR analysis. Initially, we observed that the expression profile of Oct4 and Nanog in dental pulp cells, which exerted properties of MSCs, was significantly up-regulated compared to that of STRO-1-CD146- dental pulp cells. Down-regulation of Oct4 and Nanog co-expression significantly reduced the cell proliferation, osteogenic differentiation capability, STRO-1, CD146, and Alkaline phosphatase (ALP) activity of DPSCs. In contrast, co-overexpression of Oct4 and Nanog enhanced the expression level of STRO-1 and CD146, proliferation rate and osteogenic/chondrogenic/adipogenic induction differentiation capability, and expression of osteogenic/chondrogenic/adipogenic induction differentiation markers. Our results suggest that Oct4-Nanog signaling is a regulatory switch to maintain properties in DPSCs.

Er:YAG laser suppresses pro-inflammatory cytokines expression and inflammasome in human periodontal ligament fibroblasts with Porphyromonas gingivalis-lipopolysaccharide stimulation.

Background/purpose: Periodontitis is an inflammatory condition of the tooth-supporting structures triggered by the host's immune response towards the bacterial deposits around the teeth. It is well acknowledged that pro-inflammatory interleukin (IL)-6, IL-8, MCP-1 as well as the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, are the key modulators in the activation of this response. Erbium-doped yttrium-aluminium-garnet (Er:YAG) laser, a solid-state crystal laser have been commonly used in the treatment of periodontal diseases. However, little is understood about the molecular mechanism of the Er:YAG laser, especially in targeting the host immune response brought on by periodontal pathogens. Hence, the current study focused on the protective effects of Er:YAG laser on periodontitis in-vitro in terms of pro-inflammatory cytokines, chemokines and NLRP3 inflammasome expressions. Materials and methods: Human periodontal ligament fibroblast (PDLFs) were first stimulated with lipopolysaccharides (LPS) from P. gingivalis (Pg-LPS) to simulate periodontitis. Cells were then irradiated with Er:YAG laser of ascending energy densities (3.6-6.3 J/cm2), followed by cell proliferation and wound healing assay. Next, the effects of Er:YAG laser on the expressions of IL-6, IL-8, MCP-1, NLRP3, and cleaved GSDMD were examined. Results: Pg-LPS was found to reduce cell's proliferation rate and wound healing ability in PDLFs and these were rescued by Er:YAG laser irradiation. In addition, LPS stimuli resulted in a marked upregulation in the secretion of IL-6, IL-8 and MCP-1 as well as the mRNA and protein expression of NLRP3 and cleaved-GSDMD protein whereas Er:YAG laser suppressed the elicited phenomena. Conclusion: To our knowledge, this is the first study to look into the laser's implication on the NLRP3 inflammasome in periodontitis models. Our study reveals a crucial role of Er:YAG laser in ameliorating periodontitis in-vitro through the modulation of IL-6, IL-8, MCP-1 and the NLRP3 inflammasome and highlights that the control of the NLRP3 inflammasome may become a potential approach for periodontitis.

Ganoderma Microsporum Immunomodulatory Protein Alleviates Inflammaging and Oxidative Stress in Diabetes-Associated Periodontitis via Nrf2 Signaling Activation: An In Vitro Study.

Periodontitis, characterized by inflammation and loss of periodontal tissue, is a significant health complication for individuals with diabetes mellitus (DM). Buildup of advanced glycation end-products (AGEs) in DM poses an increased risk of periodontitis via inflammaging. Ganoderma immunomodulatory protein (GMI) shows promise in suppressing inflammaging by mitigating oxidative stress and inflammation via Nrf2 modulation. However, its specific protective effects are not fully understood. Thus, this study aimed to investigate GMI's anti-inflammaging properties and its underlying mechanism in diabetic-associated periodontitis (DP). We first simulated DP by culturing human gingival fibroblasts (HGFs) with AGEs and lipopolysaccharides from P. gingivalis (LPS). We then evaluated the impact of GMI on cell proliferation, migration and wound healing. Additionally, we assessed GMI's effects on the components of inflammaging such as reactive oxygen species (ROS) formation, cellular senescence expression, IL-6 and IL-8 secretions, and NF-κB phosphorylation. Next, we explored whether GMI's anti-inflammaging effects are mediated through the Nrf2 pathway by evaluating Nrf2 and HO-1, followed by the assessment of IL-6 and IL-8 post-Nrf2 knockdown. Our findings revealed that GMI treatment suppressed ROS production, cell senescence, IL-6 and IL-8 and NF-κB phosphorylation. Furthermore, GMI upregulated Nrf2/HO-1 expression and its protective effects were reversed when Nrf2 was knocked down. In conclusion, GMI exerts its anti-inflammaging effect via the modulation of the Nrf2/NF-κB signaling axis in DP in vitro, highlighting its potential as an effective adjunct treatment for diabetes-related periodontitis.

Resveratrol attenuates advanced glycation end product-induced senescence and inflammation in human gingival fibroblasts.

Background/purpose: The accumulation of advanced glycation end products (AGEs) lead to a series of immune responses such as: increased oxidative stress and inflammation which contribute to the development of diabetic complications and periodontal disease. Resveratrol is a natural compound that has anti-oxidant and anti-inflammatory effects. Studies have found that diabetes-induced periodontitis is mainly caused by oxidative stress, aging and increased inflammation. In view of resveratrol has been proposed to have the ability in anti-oxidant and anti-inflammation in a variety of tissues. However, the role of resveratrol in diabetic periodontitis remains to be investigated. In this study, we aimed to investigate the role of resveratrol in preventing and treating diabetic periodontitis. Materials and methods: First, cell proliferation was measured in AGEs-treated human gingival fibroblast with or without resveratrol. We examined the reactive oxygen species (ROS) generation, senescence-associated beta-galactosidase (SA-ß-gal) and senescence marker p16 in human gingival fibroblasts (HGFs) stimulated with AGEs with or without the treatment of resveratrol. To determine whether resveratrol has the potential to regulate inflammaging which is mediated via the NF-κB signaling pathway and, the expression of p65 and p-IκB were also investigated. Furthermore, the concentration of interleukin (IL)-6 and IL-8 were also measured in AGEs-stimulated HGFs treated with or without resveratrol. Results: ROS generation, cell senescence, and the secretion of IL-6 and IL-8 were significantly upregulated following the treatment of AGEs. However, the administration of resveratrol suppresses the generation of IL-6 and IL-8 and cell senescence via inhibiting NF-κB signaling pathway. Our results revealed that resveratrol inhibits inflammaging by downregulating NF-κB signaling pathway. Conclusion: According to our findings, AGEs increase senescence and the production of proinflammatory cytokines in the gingiva, while the administration of resveratrol impedes inflammaging via suppressing NF-κB signaling pathway.

Quercetin ameliorates advanced glycation end product-induced wound healing impairment and inflammaging in human gingival fibroblasts.

Background/purpose: Diabetes mellitus (DM) and periodontal disease are both prevalent and chronic inflammatory disorders that have significant health impact. Many studies have pointed out that advanced glycation end-products (AGEs) in DM induces inflammaging, which is a pre-aging and hyperinflammatory condition, and it has been linked to a greater likelihood in developing periodontitis. Inflammaging in DM has been shown to be driven by AGEs-induced cell senescence, inflammatory cytokines, and oxidative stress, resulting in the degradation of periodontium. Quercetin has shown abilities to decrease inflammation and oxidative stress in a variety of tissues, however, the effect in diabetic periodontitis remains uncertain. Thus, the aim of this study was to investigate its impacts on inflammaging in diabetic periodontitis. Materials and methods: We examined cell proliferation in human gingival fibroblasts (HGF), wound healing, IL-6 and IL-8 secretions, cellular senescence expression, and the formation of reactive oxygen species (ROS) in response to AGE stimulation with and without Quercetin intervention. Following that, we looked into NF-κß activity to see if Quercetin mediate its effects via this pro-inflammatory signaling. Results: Quercetin at 20 µM and below did not have any impact on HGFs' cell proliferation rate. Quercetin intervention improved the AGEs-impaired wound healing, in addition to the attenuation of AGEs-induced ROS in a dose-dependent pattern. Moreover, Quercetin therapy dose-dependently inhibited AGEs-induced cell senescence activity along with its senescence associated secretion phenotype (SASP) secretions such as IL-6 and IL-8. Western blot analysis indicated that Quercetin was able to reverse the phosphorylation of p65 and Iκß in AGEs-stimulated HGFs, demonstrating it can modulate NF-κß pathway. Conclusion: Accumulation of AGEs can elicit inflammaging in HGFs, as seen by increased pro-inflammatory cytokines, cell senescence expression and oxidative stress. The results proposed that Quercetin is able to ameliorate inflammaging in diabetic periodontitis and improve wound healing via the suppression of NF-κß pathway and hence, may be a promising approach for treatment of diabetes-associated periodontitis.

Enhancement of cancer stem-like and epithelial-mesenchymal transdifferentiation property in oral epithelial cells with long-term nicotine exposure: reversal by targeting SNAIL.

Cigarette smoking is one of the major risk factors in the development and further progression of tumorigenesis, including oral squamous cell carcinoma (OSCC). Recent studies suggest that interplay cancer stem-like cells (CSCs) and epithelial-mesenchymal transdifferentiation (EMT) properties are responsible for the tumor maintenance and metastasis in OSCC. The aim of the present study was to investigate the effects of long-term exposure with nicotine, a major component in cigarette, on CSCs and EMT characteristics. The possible reversal regulators were further explored in nicotine-induced CSCs and EMT properties in human oral epithelial (OE) cells. Long-term exposure with nicotine was demonstrated to up-regulate ALDH1 population in normal gingival and primary OSCC OE cells dose-dependently. Moreover, long-term nicotine treatment was found to enhance the self-renewal sphere-forming ability and stemness gene signatures expression and EMT regulators in OE cells. The migration/cell invasiveness/anchorage independent growth and in vivo tumor growth by nude mice xenotransplantation assay was enhanced in long-term nicotine-stimulated OE cells. Knockdown of Snail in long-term nicotine-treated OE cells was found to reduce their CSCs properties. Therapeutic delivery of Si-Snail significantly blocked the xenograft tumorigenesis of long-term nicotine-treated OSCC cells and largely significantly improved the recipient survival. The present study demonstrated that the enrichment of CSCs coupled EMT property in oral epithelial cells induced by nicotine is critical for the development of OSCC tumorigenesis. Targeting Snail might offer a new strategy for the treatment of OSCC patients with smoking habit.

Bromelain inhibits the inflammation and senescence effect in diabetic periodontitis: A preliminary in vitro study.

Background/purpose: Diabetes mellitus (DM) is a chronic metabolic disorder that affects millions of people worldwide. A growing evidence suggests that hyperglycemia in DM causes a pre-aging and pro-inflammatory condition known as inflammaging, which increases periodontitis susceptibility. Bromelain has been demonstrated to have anti-inflammatory and anti-aging properties in variety of tissues, but its effects on diabetic periodontitis remain unclear. Thus, the aim of this study is to investigate the its Bromelain's impact in diabetic periodontitis in terms of inflammation and senescence activity. Materials and methods: We assessed the wound healing capacity, production of pro-inflammatory cytokines Interleukin (IL)-6 and IL-8 and senescence marker p16 in human gingival fibroblasts (HGFs) in response to Advanced glycation end-products (AGEs) stimulant, with or without Bromelain treatment. The expression of p65, p-ERK, and p-p38 were also examined to elucidate whether Bromelain's anti-inflammaging activity is mediated through NF-κB and MAPK/ERK signaling pathway. Results: Bromelain concentrations ranging from 2.5 to 20 g/mL had no adverse effect on HGF cell proliferation. Bromelain improved wound healing in HGFs with AGEs stimulation. In addition, Bromelain suppressed the production of pro-inflammatory cytokines IL-6 and IL-8 in HGFs elicited by AGEs. Meanwhile, Bromelain treatment also inhibited the senescence activity and expression of p16 in AGEs-stimulated HGFs. Western blot analysis indicated that the upregulation of p-ERK, p-p38 and p65 induced by AGEs were inhibited by Bromelain in HGFs. Conclusion: These data suggest that excessive AGEs in the gingiva may lead to the accumulation of pro-inflammatory cytokines and marked senescence activity. Bromelain application may be helpful in enhancing wound healing by suppressing inflammaging via downregulation of NF-κB and MAPK/ERK signaling pathways in DM individuals with periodontal disease.

Anti-inflammaging effects of vitamin D in human gingival fibroblasts with advanced glycation end product stimulation.

Background/purpose: :Both periodontal disease and diabetes mellitus (DM) are long-term inflammatory disorders that are highly prevalent and have a significant health impact. Inflammaging, a state of pre-aging and hyperinflammatory state has been acknowledged for its role in DM patients to have heightened risk of periodontitis. Numerous evidences revealed that inflammaging contributed by cell senescence, acceleration of inflammation and oxidative stress participates in the destruction of periodontium in DM. Abilities of vitamin D in suppressing inflammation and oxidative stress have been revealed in a range of tissues, however in DM's gingival cells, the effect remain undefined. Materials and methods: : Under the stimulation of advanced glycation end-products (AGEs), we assessed the cell proliferation in human gingival fibroblast (HGF), IL-6 and IL-8 secretions, cellular senescence expression and generation of reactive oxygen species (ROS) with or without vitamin D intervention. Following that, we examined the expression of Nrf2 and HO-1 to see if vitamin D was able to modulate the anti-oxidant signaling. A knockdown experiment was then conducted to proof the participation of Nrf2 on the secretion of pro-inflammatory IL-6 and IL-8. Results: : Following the treatment of vitamin D, AGEs-elicited IL-6 and IL-8 production and cell senescence were dose-dependently repressed. Moreover, vitamin D attenuated AGEs-induced ROS in a dose-dependent pattern. Results from qRT-PCR demonstrated vitamin D reversed the suppression of Nrf2 and HO-1 induced by AGEs. Our findings revealed that the anti-inflammatory and anti-oxidant effect in vitamin D was mediated via the upregulation of Nrf2 expression. Conclusion: : These data showed that high levels of AGEs in the gingiva lead to inflammaging reflected by increased pro-inflammatory cytokines, cell senescence expression and oxidative stress. Vitamin D supplementation can reduce oxidative stress and inflammation via the upregulation of Nrf2 signaling and hence, may be a potential approach for treatment of diabetes-associated periodontitis.

Emerging Role of MicroRNA-200 Family in Dentistry.

MicroRNAs (miRNAs) are endogenous non-coding RNAs ~22 nucleotides in length, which have been shown to participate in various biological processes. As one of the most researched miRNAs, the miR-200 family has been found to regulate several factors that are associated with the epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs) behavior. In this review, we briefly summarize the background of the miR-200 family and their implication in various dental diseases. We focus on the expression changes, biological functions, and clinical significance of the miR-200 family in oral cancer; periodontitis; oral potentially malignant disorder; gingival overgrowth; and other periodontal diseases. Additionally, we discuss the use of the miR-200 family as molecular biomarkers for diagnosis, prognostic, and therapeutic application.