Salivary Amylase-Responsive Buccal Tablets Wipe Out Chemotherapy-Rooted Refractory Oral Mucositis.

Oral mucositis (OM) is the most common and refractory complication of cancer chemotherapy and radiotherapy, severely affecting patients' life quality, lowering treatment tolerance, and discouraging patient compliance. Current OM delivery systems mostly affect the comfort of patient use and lead to poor compliance and unsatisfactory effects. Herein, salivary amylases (SAs)-responsive buccal tablets consisting of porous manganese-substituted Prussian blue (PMPB) nanocubes (NCs), anti-inflammatory apremilast (Apr) and starch controller have been engineered. PMPB NCs with large surface area can serve as carriers to load Apr, and their multienzyme-mimicking activity enables them to scavenge reactive oxygen species (ROS), which thus synergize with Apr to mitigate inflammation. More significantly, the starch controller can respond to abundant SAs in the oral cavity and realize the cascade, continuous, and complete drug release after enzymatic decomposition, which not only aids with high tissue affinity to prolong the resistance time but also improves the comfort of use. The preclinical study reveals that contributed by the above actions, such buccal tablets mitigate inflammation, promote endothelium proliferation and migration, and accelerate wound healing for repressing chemotherapy-originated intractable OM with positive oral microenvironment and shorter recovery time, thus holding high potentials in clinical translation.

Porphyromonas gingivalis induced UCHL3 to promote colon cancer progression.

Porphyromonas gingivalis (P. gingivalis), a Gram-negative oral anaerobe, was demonstrated to facilitate colonization and progression in colonic tumor, while the underlying mechanism still remains to be clarified. Here, we identified the proteome profile changed by P. gingivalis infection in HCT116 cells through label-free quantitative proteomics, and found that deubiquitinase UCHL3 was a key protein that response for P. gingivalis infection. By CCK8, colony formation, wound healing assays, and in vivo subcutaneous tumor mouse moudle, we proved that P. gingivalis could promote the proliferation and migration of colon cancer, while the process was inhibited by UCHL3 knock down. Through IP-MS, we identified GNG12 as the UCHL3 interacting protein. The protein level of GNG12 was significantly reduced when knock out UCHL3. Thus we propose that GNG12 is a substrate protein of UCHL3. Furthermore, we demonstrated that overexpression of GNG12 could restore the tumor inhibition effect caused by UCHL3 knock down, and UCHL3-GNG12 axis promote colon cancer progression via the NF-κB signal pathway. Collectively, this study unveiled that P. gingivalis infection up-regulated UCHL3 and stabilized its substrate protein GNG12 to activate the NF-κB signal pathway to promote colon cancer progression. Our study indicate that UCHL3 is a potential biomarker and therapeutic target for colon cancer which infected with P. gingivalis.

Oncolytic Impediment/Promotion Balance Disruption by Sonosensitizer-Free Nanoplatforms Unfreezes Autophagy-Induced Resistance to Sonocatalytic Therapy.

Autophagy as a double-edged sword features an oncolytic impediment/promotion balance, which manipulates tumor progression. From this perspective, a sonosensitizer-free targeting oncolytic nanoplatform (SFTON) consisting of chloroquine (CQ) and porphyrin-structured metal centers (PMCS) was engineered to break this balance for enhancing antitumor activity. Porphyrin structure retention in a ZIF-8-derived hydrophobic carbon skeleton retained high stability and high sonocatalytic activity, and the hydrophobic carbon skeleton capable of adsorbing air provided cavitation nuclei for further elevating sonocatalytic activity. More significantly, the encapsulated CQ as the autophagy inhibitor reprogrammed autophagy, terminated the autophagy-induced self-protection or self-detoxification, and unfroze the resistances to reactive oxygen species (ROS) therapy associated with ROS accumulation and ROS activity. Systematic experiments reveal the action principles and validate that the induced apoptosis and blockaded autophagosome escalation into the autolysosome were two activated pathways to magnify the antitumor sonocatalytic therapy. Contributed by these actions, the SFTON-unlocked oncolytic impediment/promotion balance disruption strategy acquired considerable antitumor outcomes in vivo and in vitro against liver tumor progression, especially after combining with AS1411-mediated active targeting. This impediment/promotion balance disruption enabled by the SFTON can serve as a general method to elevate ROS-based antitumor activity.

Leonurine Promotes the Osteoblast Differentiation of Rat BMSCs by Activation of Autophagy via the PI3K/Akt/mTOR Pathway.

BACKGROUND: Leonurine, a major bioactive component from Herba leonuri, has been shown to exhibit anti-inflammatory and antioxidant effects. The aim of this study was to investigate the effect of leonurine on bone marrow-derived mesenchymal stem cells (BMSCs) as a therapeutic approach for treating osteoporosis. MATERIALS AND METHODS: Rat bone marrow-derived mesenchymal stem cells (rBMSCs) were isolated from 4-weeks-old Sprague-Dawley rats. The cytocompatibility of leonurine on rBMSCs was tested via CCK-8 assays and flow cytometric analyses. The effects of leonurine on rBMSC osteogenic differentiation were analyzed via ALP staining, Alizarin red staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Additionally, autophagy-related markers were examined via qRT-PCR and Western blot analyses of rBMSCs during osteogenic differentiation with leonurine and with or without 3-methyladenine (3-MA) as an autophagic inhibitor. Finally, the PI3K/Akt/mTOR signaling pathway was evaluated during rBMSC osteogenesis. RESULTS: Leonurine at 2-100 µM promoted the proliferation of rBMSCs. ALP and Alizarin red staining results showed that 10 µM leonurine promoted rBMSC osteoblastic differentiation, which was consistent with the qRT-PCR and Western blot results. Compared with those of the control group, the mRNA and protein levels of Atg5, Atg7, and LC3 were upregulated in the rBMSCs upon leonurine treatment. Furthermore, leonurine rescued rBMSC autophagy after inhibition by 3-MA. Additionally, the PI3K/AKT/mTOR pathway was activated in rBMSCs upon leonurine treatment. CONCLUSION: Leonurine promotes the osteoblast differentiation of rBMSCs by activating autophagy, which depends on the PI3K/Akt/mTOR pathway. Our results suggest that leonurine may be a potential treatment for osteoporosis.

Berberine ameliorates the LPS-induced imbalance of osteogenic and adipogenic differentiation in rat bone marrow-derived mesenchymal stem cells.

Lipopolysaccharide (LPS) from oral pathogenic bacteria is an important factor leading to alveolar bone absorption and the implant failure. The present study aimed to evaluate the modulation of berberine hydrochloride (BBR) on the LPS-mediated osteogenesis and adipogenesis imbalance in rat bone marrow-derived mesenchymal stem cells (BMSCs). Cell viability, osteoblastic and adipogenic differentiation levels were measured using the Cell Counting Kit-8 assay, alkaline phosphatase (ALP) staining and content assay, and oil red O staining, respectively. Reverse transcription-quantitative PCR and immunoblotting were used to detect the related gene and protein expression levels. In undifferentiated cells, BBR increased the mRNA expression levels of the osteoblastic genes (Alp, RUNX family transcription factor 2, osteocalcin and secreted phosphoprotein 1) but not the adipogenic genes (fatty acid binding protein 4, Adipsin and peroxisome proliferator-activated receptorγ). LPS-induced osteoblastic gene downregulation, adipogenic gene enhancement and NF-κB activation were reversed by BBR treatment. In osteoblastic differentiated cells, decreased ALP production by LPS treatment was recovered with BBR co-incubation. In adipogenic differentiated cells, LPS-mediated lipid accumulation was decreased by BBR administration. The mRNA expression levels of the pro-inflammatory factors (MCP-1, TNF-α, IL-6 and IL-1ß) were increased by LPS under both adipogenic and osteoblastic conditions, which were effectively ameliorated by BBR. The actions of BBR were attenuated by compound C, suggesting that the role of BBR may be partly due to AMP-activated protein kinase activation. The results demonstrated notable pro-osteogenic and anti-adipogenic actions of BBR in a LPS-stimulated inflammatory environment. This indicated a potential role of BBR for bacterial infected-related peri-implantitis medication.

Knockout of NRAGE promotes autophagy-related gene expression and the periodontitis process in mice.

BACKGROUND AND OBJECTIVE: Neurotrophin receptor-interacting MAGE homologue (NRAGE) plays a crucial role in the regulation of bone metabolism. The present study investigated the regulation role of NRAGE on autophagy activation and periodontitis process during experimental periodontitis. MATERIALS AND METHODS: Six-week-old wild-type (WT) and NRAGE-/- mice were randomly divided into three time points in the periodontitis groups (0, 2, and 4 weeks). Histopathological changes were determined using the tooth mobility, hematoxylin and eosin (H&E) staining, and micro-computed tomography (micro-CT). Osteoclasts activation and number were investigated using tartrate-resistant acid phosphatase (TRAP) staining, immunohistochemistry, and real-time quantitative PCR (RT-PCR). The level of autophagy-related gene expression was measured using immunohistochemistry, immunofluorescence, and RT-PCR. RESULTS: H&E staining and Micro-CT showed that the destruction of the alveolar bone was considerably more severe in the NRAGE-/- group than the WT group after ligation. Tooth mobility in the NRAGE-/- group was obviously higher than that in the WT group. The activation and number of osteoclasts and the level of autophagy-related gene expression in NRAGE-/- group were significantly higher than that in WT group. CONCLUSIONS: The present study showed that knockout of NRAGE induced autophagy-related gene expression and accelerated the process of periodontitis disease via increasing the activity and differentiation of osteoclast.

Chromium-catalysed efficient N-formylation of amines with a recyclable polyoxometalate-supported green catalyst.

A simple and efficient protocol for the formylation of amines with formic acid, catalyzed by a polyoxometalate-based chromium catalyst, is described. Notably, this method shows excellent activity and chemoselectivity for the formylation of primary amines; diamines have also been successfully employed. Importantly, the chromium catalyst is potentially non-toxic, environmentally benign and safer than the widely used high valence chromium catalysts such as CrO3 and K2Cr2O7. The catalyst can be recycled several times with a negligible impact on activity. Finally, a plausible mechanism is provided based on the observation of intermediate and control experiments.

LINC01315 Impairs microRNA-211-Dependent DLG3 Downregulation to Inhibit the Development of Oral Squamous Cell Carcinoma.

Recent studies have revealed that long non-coding RNAs (lncRNAs) involve in the progression of oral squamous cell carcinoma (OSCC). These lncRNAs have emerged as biomarkers or therapeutic targets for OSCC. We here aimed to investigate the role of lncRNA LINC01315 in OSCC and the related mechanisms. LINC01315 and DLG3 were determined to be poorly expressed while microRNA-211 (miR-211) was highly expressed in OSCC tissues and cells using RT-qPCR and western blot analysis. Based on the results obtained from dual-luciferase reporter gene, RIP, and FISH assays, LINC01315 was found to upregulate DLG3 expression by competitively binding to miR-211. Upon altering the expression of LINC01315, and/or miR-211 in OSCC cells with shRNA, mimic, or an inhibitor, we assessed their effects on OSCC cell proliferation, migration, invasion, and apoptosis. LINC01315 knockdown enhanced OSCC cell proliferation, migration and invasion, but dampened their apoptosis, all of which could be reversed by miR-211 inhibition. Elevation of DLG3, a target gene of miR-211, activated the Hippo signaling pathway, whereby suppressing OSCC progression in vitro. Finally, their roles in tumor growth were validated in vivo. These findings suggest that LINC01315 elevates DLG3 expression by competitively binding to miR-211, thereby suppressing OSCC progression.

Profiling risk factors of micro-invasive carcinoma within oral potentially malignant disorders: a cross-sectional study.

OBJECTIVES: To investigate the clinicopathological profile and risk factors of micro-invasive carcinoma within oral potentially malignant disorders (OPMD). METHODS: Micro-invasive carcinomas were identified in a large prospective series of OPMD patients (n = 810) from eastern China. Logistic regression was applied to evaluate odds ratios (OR) with 95% confidence interval (CI) for indicative of malignant risk in general OPMD. RESULTS: Leukoplakia (41.4%), lichen planus (28.0%), and lichenoid lesion (23.7%) were the most 3 clinical subtypes of OPMD. A total of 62 (7.7%) micro-invasive carcinomas within OPMD were identified, and 96.8% of micro-invasive carcinoma was found within leukoplakia and erythroplakia. Multivariate regression analysis revealed that the risk of malignant change within OPMD located on lateral/ventral tongue (OR, 15.1; 95% CI, 1.85-122.8; P = 0.011) was higher than other sites. The risk of malignant change within non-homogenous type (OR, 103.3; 95% CI, 13.39-796.7; P < 0.001) was strikingly higher than other subtypes of OPMD, respectively. Intriguingly, the risk of micro-invasive carcinoma diagnosed in current smoker (OR, 3.96; 95% CI, 1.31-12.02; P = 0.015) was higher than non-smoker. CONCLUSION: This large-scale cross-sectional study elucidated the clinical factors and risk assessment of micro-invasive carcinoma within OPMD. CLINICAL RELEVANCE: Non-homogenous lesions located on lateral/ventral tongue might be monitored at closer intervals, and the need for rigorous management to detect malignant changes.

Expression of autophagy­associated proteins in rat dental irreversible pulpitis.

Autophagy serves an important role in numerous diseases, as well as in infection and inflammation. Irreversible pulpitis (IP) is one of the most common inflammatory endodontic diseases, and autophagy has been reported to regulate IP in vitro. However, the level of autophagy in the IP pathogenic process in vivo remains unknown. The aim of the current study was, thus, to investigate the levels of autophagy­associated proteins in rats with IP in vivo. A rat dental IP model was successfully constructed, and five different time points (0, 1, 3, 5 and 7 days) were investigated. The levels of the autophagy­related 5 (ATG5), ATG7, light chain 3 (LC3) and Beclin­1 proteins exhibited a time­dependent increase in rats with IP, whereas the levels of mammalian target of rapamycin and p62/sequestosome 1 were decreased. In addition, the levels of ATG proteins were specifically increased in odontoblasts and microvascular endothelial cells in pulpitis tissue. Based on these findings, autophagy may serve an important role in IP, and the present study data provide a new insight into the IP pathogenesis and treatment.

lncRNA PLAC2 activated by H3K27 acetylation promotes cell proliferation and invasion via the activation of Wnt/ß­catenin pathway in oral squamous cell carcinoma.

As a new group of important effector molecules involved in multiple cancer types, including breast cancer, lung cancer and oral squamous cell carcinoma, long noncoding RNAs (lncRNAs) have attracted considerable attention recently. However, the underlying cause that induces the dysregulated lncRNAs in cancer remains poorly understood. In the present study, the regulatory model of the lncRNA placenta­specific protein 2 (PLAC2) upregulation in oral squamous cell carcinoma (OSCC) was investigated and its biological functions in OSCC malignant progression was identified. A reverse transcription­quantitative polymerase chain reaction assay identified that PLAC2 is upregulated in OSCC cell lines and primary tissue samples. Furthermore, bioinformatic analysis followed by chromatin immunoprecipitation verified an enriched histone H3 on lysine 27 (H3K27) acetylation (H3K27ac) at the promoter region of the PLAC2 gene. Knockdown of cAMP­response element binding protein­binding protein (CBP) significantly reduced the enrichment level of H3K27ac, and thereby induced a decreased expression of PLAC2. Functionally, overexpression of PLAC2 promotes OSCC cell proliferation, migration and invasion, whereas knockdown of PLAC2 exerted an opposite effect. Furthermore, the Wnt/ß­catenin signaling pathway was activated by PLAC2 and mediated the PLAC2­induced malignant progress of OSCC. In conclusion, the present results indicated that lncRNA PLAC2 is transcriptionally activated by H3K27ac modification at the promoter region in OSCC, and promotes cell growth and metastasis via activating Wnt/ß­catenin signaling pathway. Therefore, PLAC2 may serve as a promising biomarker for OSCC prognosis and therapy.

miR-23a-3p suppresses cell proliferation in oral squamous cell carcinomas by targeting FGF2 and correlates with a better prognosis: miR-23a-3p inhibits OSCC growth by targeting FGF2.

Oral squamous cell carcinomas (OSCCs) are one of the most ubiquitous malignancies the world over, and are accompanied by a high mortality. microRNAs (miRNAs) have increasingly garnered attention with regards to the roles they play in initiation and progression of various kinds of cancers, including OSCC. It has been reported, that miR-23a-3p promotes the development of tumors for prostate cancer, gastric cancer and gliomas. The functions of miR-23a-3p in OSCC however, remain unclear. In this study, fibroblast growth factor 2 (FGF2) is revealed as a direct target of miR-23a-3p, based on luciferase assays and immunoblotting. The expression of miR-23a-3p and FGF2 were found to be significantly downregulated and upregulated in OSCC tissues respectively. This indicates a reverse correlation between miR-23a-3p and FGF2 levels. Using in vitro approaches we ascertained that miR-23a-3p might contribute to the inhibition of growth and inhibition through increasing apoptosis in OSCC cells; while an inhibitor of miR-23a-3p could reverse this effect. Examination of a clinical cohort of OSCC patients suggested that reduced expression of miR-23a-3p is correlated with more advanced cancerous stage and poorer differentiation of OSCC cell. Additionally, a survival analysis and the Cox-hazard regression model showed that higher levels of miR-23a-3p can be used reliably for prognosis of OSCC patients. This study indicates that miR-23a-3p might suppress tumor proliferation, invasion and promote apoptosis of OSCC by targeting FGF2. miR-23a-3p has the potential to be used as prognostic indicator, and could be exploited as a therapeutic reagent for OSCC in the future.

Animal Models Used for Testing Hydrogels in Cartilage Regeneration.

Local cartilage or osteochondral lesions are painful and harmful. Besides pain and limited function of joints, cartilage defect is considered as one of the leading extrinsic risk factors for osteoarthritis (OA). Thus, clinicians and scientists have paid great attention to regenerative therapeutic methods for the early treatment of cartilaginous defects. Regenerative medicine, showing great hope for regenerating cartilage tissue, relies on the combination of biodegradable scaffolds and particular biological factors, such as growth factors, genetic cues. Among all biomaterials, hydrogels have become a promising type of scaffolds for simultaneous cell growth and drug delivery in cartilage tissue engineering. A wide range of animal models have been applied in testing repair with hydrogels in cartilage defects. This review summarized the current animal models used to test hydrogels technologies for the regeneration of cartilage. Advantages and disadvantages in the establishment of the cartilage defect animal models among different species were emphasized, as well as the feasibility of replication of diseases in animals.

Role of protein delta homolog 1 in the proliferation and differentiation of ameloblasts.

Protein delta homolog 1 (DLK1) regulates the odontoblastic differentiation of human dental pulp stem cells. It was hypothesized that DLK1 may exert regulatory effects on epithelial­mesenchymal interactions in tooth development. The present study investigated the expression of DLK1 during the development of mouse enamel and its role in the proliferation and differentiation of ameloblast­lineage cells (ALCs). DLK1 expression was upregulated in ameloblasts in the first mandibular molar during the entire process of enamel development. The mRNA and protein levels of DLK1 were significantly upregulated following ameloblastic induction in ALCs. In addition, overexpression of DLK1 promoted the proliferation of ALCs, inhibited ameloblastic differentiation, upregulated the expression of amelogenin and enamelin, and downregulated the expression of odontogenic ameloblast­associated protein and kallikrein 4. The results of the present study suggested that DLK1 may be a potent regulator of ameloblast proliferation and differentiation, and may regulate enamel formation during tooth development.

The effect of delta-like 1 homologue on the proliferation and odontoblastic differentiation in human dental pulp stem cells.

INTRODUCTION: This study aimed to investigate the functions of delta-like homologue 1 (DLK1) in the proliferation and differentiation of human dental pulp stem cells (hDPSCs). METHODS: Immunohistochemical analysis was used to determine the expression of alkaline phosphatase (ALP), dentin sialophosphoprotein (DSPP), DLK1, NOTCH1 and p-ERK1/2 in the mouse first maxillary molar. Recombinant lentivirus was constructed to overexpress DLK1 stably in hDPSCs. The cell viability and proliferation of hDPSCs were examined by CCK8 and EdU incorporation assay respectively. The odontoblastic differentiation of hDPSCs was determined by detection of ALPase activity assay, ALP and alizarin red staining and the expression of mineralization-related genes including ALP, DSPP and dental matrix protein. The mRNA and protein levels of DLK1 and p-ERK1/2 protein expression were detected. ERK inhibitor was used to test the differentiation effect of DLK1 on hDPSCs. RESULTS: Delta-like homologue 1 was highly expressed on the odontoblasts and dental pulp cells on the first maxillary molar; the expression of p-ERK1/2 is similar with the DLK1 in the same process. The expression level of DLK1 increased significantly after the odontoblastic induction of hDPSCs. DLK1 overexpression increased the proliferation ability of hDPSCs and inhibited odontoblastic differentiation of hDPSCs. The protein level of p-ERK1/2 significantly increased in hDPSCs/dlk1-oe group. ERK signalling pathway inhibitor reversed the odontoblastic differentiation effects of DLK1 on hDPSCs. CONCLUSIONS: The proliferation of hDPSCs was promoted after DLK1 overexpression. DLK1 inhibited the odontoblastic differentiation of hDPSCs, which maybe through ERK signalling pathway.

The Effect of NRAGE on cell cycle and apoptosis of human dental pulp cells and MDPC-23.

OBJECTIVES: Neurotrophin receptor-mediated melanoma antigen-encoding gene homology (NRAGE) is an important regulator of proliferation, cell cycle arrest and apoptosis. Our previous study showed that NRAGE is an important regulator of proliferation and odontogenic differentiation of mouse dental pulp cells. This study aimed to investigate the effects of NRAGE on the cell cycle and apoptosis on human dental pulp cells (hDPCs) and MDPC-23. MATERIALS AND METHODS: Cells were infected by recombinant lentivirus to stably knockdown the expression of NRAGE, then the biological effects of NRAGE on the MDPC-23 was detected. The cell cycle distributions and apoptosis of hDPCs and MCPC-23 were performed by flow cytometric analysis. Simultaneously, the cell cycle and apoptosis were also detected after cells treated with IKK inhibitor. RESULTS: The mRNA and protein levels of NRAGE decreased significantly after infected by recombinant lentivirus. Knockdown of NRAGE inhibited the apoptosis in hDPCs and MCPC-23. Knockdown of NRAGE show significantly G0G1 arrest in hDPCs, while no significantly difference in MDPC-23. Meanwhile, Knockdown of NRAGE activated the NF-κB signaling pathway. After treated with IKK inhibitor, the effect of NRAGE knockdown on apoptosis was reversed in both hDPCs and MDPC-23. CONCLUSION: NRAGE is a potent regulator for cell cycle and apoptosis of hDPCs. Knockdown of NRAGE inhibited apoptosis of hDPCs and MDPC-23 through the NF-κB signaling pathway.

Effect of storage temperature on the viability of human periodontal ligament fibroblasts.

BACKGROUND: The viability of periodontal ligament fibroblasts (PDLF) can affect the long-term prognosis of replanted avulsed teeth. When immediate replantation of an avulsed tooth is not possible, the cells should be incubated in a physiological storage medium instantly to maintain their biological activity. The ability of different storage media to preserve PDLF viability has been previously evaluated. However, few studies have showed the effect of temperature on the viability of PDLF cultured with various storage media in vitro. MATERIAL AND METHODS: This study was designed to measure PDLF activity by CCK-8 assay to compare the effectiveness at 4, 22 (room temperature), and 37°C under various storage media. RESULTS: Statistical analysis demonstrated that tap water, saline, and saliva decreased cell viability as the storage temperature increased. But the temperature played only a minor role on cell viability when cells were incubated in Hank's balanced salt solution (HBSS), Dubelco's modified Eagle's medium (DMEM), or milk. CONCLUSIONS: Within the parameters of this study, it seems that room temperature is adequate for storing the avulsed teeth in HBSS, DMEM, or milk in the extra-alveolar period.