Melatonin Enhanced Microglia M2 Polarization in Rat Model of Neuro-inflammation Via Regulating ER Stress/PPARδ/SIRT1 Signaling Axis.

Neuro-inflammation involves distinct alterations of microglial phenotypes, containing nocuous pro-inflammatory M1-phenotype and neuroprotective anti-inflammatory M-phenotype. Currently, there is no effective treatment for modulating such alterations. M1/M2 marker of primary microglia influenced by Melatonin were detected via qPCR. Functional activities were explored by western blotting, luciferase activity, EMSA, and ChIP assay. Structure interaction was assessed by molecular docking and LIGPLOT analysis. ER-stress detection was examined by ultrastructure TEM, calapin activity, and ERSE assay. The functional neurobehavioral evaluations were used for investigation of Melatonin on the neuroinflammation in vivo. Melatonin had targeted on Peroxisome Proliferator Activated Receptor Delta (PPARδ) activity, boosted LPS-stimulated alterations in polarization from the M1 to the M2 phenotype, and thereby inhibited NFκB-IKKß activation in primary microglia. The PPARδ agonist L-165,041 or over-expression of PPARδ plasmid (ov-PPARδ) showed similar results. Molecular docking screening, dynamic simulation approaches, and biological studies of Melatonin showed that the activated site was located at PPARδ (phospho-Thr256-PPARδ). Activated microglia had lowered PPARδ activity as well as the downstream SIRT1 formation via enhancing ER-stress. Melatonin, PPARδ agonist and ov-PPARδ all effectively reversed the above-mentioned effects. Melatonin blocked ER-stress by regulating calapin activity and expression in LPS-activated microglia. Additionally, Melatonin or L-165,041 ameliorated the neurobehavioral deficits in LPS-aggravated neuroinflammatory mice through blocking microglia activities, and also promoted phenotype changes to M2-predominant microglia. Melatonin suppressed neuro-inflammation in vitro and in vivo by tuning microglial activation through the ER-stress-dependent PPARδ/SIRT1 signaling cascade. This treatment strategy is an encouraging pharmacological approach for the remedy of neuro-inflammation associated disorders.

Sirtuin 6 ameliorates arthritis through modulating cyclic AMP-responsive element binding protein/CCN1/cyclooxygenase 2 pathway in osteoblasts.

INTRODUCTION: CCN1 is an immediate-early gene product pivotal for arthritis progression. We have previously shown that sirtuin 6 (SIRT6) inhibited hypoxia-induced CCN1 expression in osteoblasts. Herein we examined the contribution of cyclic AMP-responsive element binding protein (CREB)/CRE to this suppressive action and the influence of CCN1 on cyclooxygenase (COX) 2 synthesis. MATERIALS AND METHODS: MC3T3-E1 murine osteoblasts were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen). Expressions of CCN1, phospho-CREB (Ser133), COX2 and relevant kinases were assessed by Western blot. SIRT6 was overexpressed in cultured osteoblasts and arthritic joints by a lentiviral-based technique. Activities of CCN1 gene promoter constructs were examined by luciferase reporter assay. Interaction between CREB and CCN1 promoter was assessed by chromatin immunoprecipitation (ChIP). Collagen-induced arthritis (CIA) was established in 20 rats to evaluate the effects of SIRT6 therapy on osteoblastic expressions of phospho-CREB, CCN1 and COX2. RESULTS: SIRT6 suppressed hypoxia-enhanced CCN1 expression and CREB phosphorylation. Attenuation of calcium/calmodulin-dependent protein kinase II (CaMKII) may be responsible for SIRT6-induced CREB inhibition. CRE at - 286 bp upstream of the ATG start codon was essential for CCN1 expression under hypoxia and SIRT6 reduced hypoxia-stimulated CREB/CRE interaction. Forced expression of CREB rescued SIRT6-suppressed CCN1 synthesis. CCN1 induced COX2 expression in osteoblasts. In rat CIA, the therapeutic effect of SIRT6 was accompanied by decreases in osteoblastic expressions of phospho-CREB, CCN1 and COX2. CONCLUSION: Our study indicated that the benefits of SIRT6 to inflammatory arthritis and bone resorption are at least partially derived from its modulation of CREB/CCN1/COX2 pathway in osteoblasts.

Neuronal Death Caused by HMGB1-Evoked via Inflammasomes from Thrombin-Activated Microglia Cells.

Microglial cells are a macrophage-like cell type residing within the CNS. These cells evoke pro-inflammatory responses following thrombin-induced brain damage. Inflammasomes, which are large caspase-1-activating protein complexes, play a critical role in mediating the extracellular release of HMGB1 in activated immune cells. The exact role of inflammasomes in microglia activated by thrombin remains unclear, particularly as it relates to the downstream functions of HMGB1. After receiving microinjections of thrombin, Sprague Dawley rats of 200 to 250 gm were studied in terms of behaviors and immunohistochemical staining. Primary culture of microglia cells and BV-2 cells were used for the assessment of signal pathways. In a water maze test and novel object recognition analysis, microinjections of thrombin impaired rats' short-term and long-term memory, and such detrimental effects were alleviated by injecting anti-HMGB-1 antibodies. After thrombin microinjections, the increased oxidative stress of neurons was aggravated by HMGB1 injections but attenuated by anti-HMGB-1 antibodies. Such responses occurred in parallel with the volume of activated microglia cells, as well as their expressions of HMGB-1, IL-1ß, IL-18, and caspase-I. In primary microglia cells and BV-2 cell lines, thrombin also induced NO release and mRNA expressions of iNOS, IL-1ß, IL-18, and activated caspase-I. HMGB-1 aggravated these responses, which were abolished by anti-HMGB-1 antibodies. In conclusion, thrombin induced microglia activation through triggering inflammasomes to release HMGB1, contributing to neuronal death. Such an action was counteracted by the anti-HMGB-1 antibodies. The refinement of HMGB-1 modulated the neuro-inflammatory response, which was attenuated in thrombin-associated neurodegenerative disorder.

Thrombin-Induced Microglia Activation Modulated through Aryl Hydrocarbon Receptors.

Thrombin is a multifunctional serine protein which is closely related to neurodegenerative disorders. The Aryl hydrocarbon receptor (AhR) is well expressed in microglia cells involving inflammatory disorders of the brain. However, it remains unclear as to how modulation of AhR expression by thrombin is related to the development of neurodegeneration disorders. In this study, we investigated the role of AhR in the development of thrombin-induced neurodegenerative processes, especially those concerning microglia. The primary culture of either wild type or AhR deleted microglia, as well as BV-2 cell lines, was used for an in vitro study. Hippocampal slice culture and animals with either wild type or with AhR deleted were used for the ex vivo and in vivo studies. Simulations of ligand protein docking showed a strong integration between the thrombin and AhR. In thrombin-triggered microglia cells, deleting AhR escalated both the NO release and iNOS expression. Such effects were abolished by the administration of the AhR agonist. In thrombin-activated microglia cells, downregulating AhR increased the following: vascular permeability, pro-inflammatory genetic expression, MMP-9 activity, and the ratio of M1/M2 phenotype. In the in vivo study, thrombin induced the activation of microglia and their volume, thereby contributing to the deterioration of neurobehavior. Deleting AhR furthermore aggravated the response in terms of impaired neurobehavior, increasing brain edema, aggregating microglia, and increasing neuronal death. In conclusion, thrombin caused the activation of microglia through increased vessel permeability, expression of inflammatory response, and phenotype of M1 microglia, as well the MMP activity. Deleting AhR augmented the above detrimental effects. These findings indicate that the modulation of AhR is essential for the regulation of thrombin-induced brain damages and that the AhR agonist may harbor the potentially therapeutic effect in thrombin-induced neurodegenerative disorder.

Metformin Reduces Bone Resorption in Apical Periodontitis Through Regulation of Osteoblast and Osteoclast Differentiation.

INTRODUCTION: We have previously demonstrated that auxiliary metformin therapy promotes healing of apical periodontitis. Here we aimed to investigate the effects of metformin on osteoblast differentiation and osteoclast formation in cultured cells and rat apical periodontitis. METHODS: Murine pre-osteoblasts MC3T3-E1 and macrophages RAW264.7 were cultured under hypoxia (2% oxygen) or normoxia (21% oxygen) and stimulated with receptor activator of nuclear factor-κB ligand (RANKL) when indicated. Metformin was added to the cultures to evaluate its anti-hypoxic effects. Expressions of osteoblast differentiation regulator runt-related transcription factor 2 (RUNX2), RANKL, and osteoclast marker tartrate-resistant acid phosphatase (TRAP) were assessed by Western blot. Apical periodontitis was induced in mandibular first molars of 10 Sprague-Dawley rats. Root canal therapy with or without metformin supplement was performed. Periapical bone resorption was measured by micro-computed tomography. Immunohistochemistry was used to examine RUNX2, RANKL, and TRAP expressions. RESULTS: Hypoxia suppressed RUNX2 expression and enhanced RANKL synthesis in pre-osteoblasts. TRAP production increased in macrophages after hypoxia and/or RANKL stimulation. Metformin reversed hypoxia-induced RUNX2 suppression and RANKL synthesis in pre-osteoblasts. Metformin also inhibited hypoxia and RANKL-enhanced TRAP synthesis in macrophages. Intracanal metformin diminished bone loss in rat apical periodontitis. Comparing with vehicle control, cells lining bone surfaces in metformin-treated lesions had significantly stronger expression of RUNX2 and decreased synthesis of RANKL and TRAP. CONCLUSIONS: Alleviation of bone resorption by intracanal metformin was associated with enhanced osteoblast differentiation and diminished osteoclast formation in rat apical periodontitis. Our results endorsed the role of metformin as an effective medicament for inflammatory bone diseases.

Endodontic malpractice litigations in the United States from 2000 to 2021.

Background/purpose: Little is known regarding the outcomes and distinguishing characteristics of lawsuits related to endodontic procedures. This study used a verdict-based data from United States of America to analyze the factors associated with endodontic malpractice lawsuits and mitigate the risk of litigation. Materials and methods: The LexisNexis legal database was used to search for endodontic malpractice cases from January 1, 2000 to December 31, 2021 using the terms "medical malpractice" and (I) "endodontist" (II) "endodontics" (III) "root canal" (IV) "dental pulp." Each case was reviewed for reported medical characteristics and litigation outcomes. Results: A total of 650 cases were initially identified, and 97 cases were included in the final analysis. Eighty-four (86.6%) of the 97 defendants were general practitioners; 42 cases favored the plaintiff, 53 (54.6%) favored the defendant, 1 was partial win/loss, and 1 was settled. The annual case mean was 4.41 ± 2.17 (Mean ± SD). The major allegations favored for the patients involving paresthesia, root perforation, rubber dam not use, wrong tooth therapy, and infections. Plaintiffs who claimed with post-procedural reasons had a significantly higher winning rate than non-post-procedural reasons (P < 0.05). Conclusion: In the present study, 54.6% of endodontic litigation favored the dentists in the US. The authors recommend that general practitioners refer complicated cases to endodontists and treat carefully to avoid paresthesia, canal perforation and infections. Clinicians should always diagnose and treat correctly, shared decision making with the patient, use rubber dam routinely, and timely management to prevent malpractice claims.

Oral microbiome and serological analyses on association of Alzheimer's disease and periodontitis.

OBJECTIVE: To investigate the association between Alzheimer's disease (AD) and periodontitis in the aspects of periodontal status, serological markers, and oral microbiome. MATERIALS AND METHODS: Twenty AD and 20 healthy subjects were enrolled in this age- and gender-matched case-control study. Clinical periodontal parameters and serum biomarkers, including amyloid ß42 (Aß42 ), Tau, phosphorylated Tau (pTau), triglyceride, pro-inflammatory cytokines, and anti-Porphyromonas gingivalis lipopolysaccharide (LPS) antibody were examined. The saliva samples were analyzed for oral microbiome composition. RESULTS: Alzheimer's disease patients with Clinical Dementia Rating (CDR) ≥1 exhibited significantly more clinical attachment loss (CAL) than those with lower CDR. The levels of serum Tau protein, hsCRP and anti-P. gingivalis LPS antibody were markedly elevated in the AD group compared with the control group. Serum pTau protein level was positively correlated with anti-P. gingivalis LPS antibody titer. Moreover, the increased abundances of Capnocytophaga sp ora clone DZ074, Eubacterium infirmum, Prevotella buccae, and Selenomonas artemidis were detected in the AD group. Interestingly, serum levels of Aß42, pTau, and anti-P. gingivalis LPS antibody were strongly related to the gene upregulation in human pathogen septicemia. CONCLUSIONS: Our study suggested the association of periodontal infection and oral microbiome with AD. Further large-scale studies with longitudinal follow-up are warranted.

Targeting histone deacetylase-3 blocked epithelial-mesenchymal plasticity and metastatic dissemination in gastric cancer.

BACKGROUND AND PURPOSE: Histone deacetylase (HDAC) inhibitors (HDIs) can modulate the epithelial-mesenchymal transition (EMT) progression and inhibit the migration and invasion of cancer cells. Emerging as a novel class of anti-cancer drugs, HDIs are attracted much attention in the field of drug discovery. This study aimed to discern the underlying mechanisms of Honokiol in preventing the metastatic dissemination of gastric cancer cells by inhibiting HDAC3 activity/expression. EXPERIMENTAL APPROACH: Clinical pathological analysis was performed to determine the relationship between HDAC3 and tumor progression. The effects of Honokiol on pharmacological characterization, functional, transcriptional activities, organelle structure changes, and molecular signaling were analyzed using binding assays, differential scanning calorimetry, luciferase reporter assay, HDAC3 activity, ER stress response element activity, transmission electron microscopy, immune-blotting, and Wnt/ß-catenin activity assays. The in vivo effects of Honokiol on peritoneal dissemination were determined by a mouse model and detected by PET/CT tomography. KEY RESULTS: HDAC3 over-expression was correlated with poor prognosis. Honokiol significantly abolished HDAC3 activity (Y298) via inhibition of NFκBp65/CEBPß signaling, which could be reversed by the over-expression of plasmids of NFκBp65/CEBPß. Treatments with 4-phenylbutyric acid (a chemical chaperone) and calpain-2 gene silencing inhibited Honokiol-inhibited NFκBp65/CEBPß activation. Honokiol increased ER stress markers and inhibited EMT-associated epithelial markers, but decreased Wnt/ß-catenin activity. Suppression of HDAC3 by both Honokiol and HDAC3 gene silencing decreased cell migration and invasion in vitro and metastasis in vivo. CONCLUSIONS AND IMPLICATIONS: Honokiol acts by suppressing HDAC3-mediated EMT and metastatic signaling. By prohibiting HDAC3, metastatic dissemination of gastric cancer may be blocked. Conceptual model showing the working hypothesis on the interaction among Honokiol, HDAC3, and ER stress in the peritoneal dissemination of gastric cancer. Honokiol targeting HDAC3 by ER stress cascade and mitigating the peritoneal spread of gastric cancer. Honokiol-induced ER stress-activated calpain activity targeted HDAC3 and blocked Tyr298 phosphorylation, subsequently blocked cooperating with EMT transcription factors and cancer progression. The present study provides evidence to demonstrate that HDAC3 is a positive regulator of EMT and metastatic growth of gastric cancer cells. The findings here imply that overexpressed HDAC3 is a potential therapeutic target for honokiol to reverse EMT and prevent gastric cancer migration, invasion, and metastatic dissemination. • Honokiol significantly abolished HDAC3 activity on catalytic tyrosine 298 residue site. In addition, Honokiol-induced ER stress markedly inhibited HDAC3 expression via inhibition of NFκBp65/CEBPß signaling. • HDAC3, which is a positive regulator of metastatic gastric cancer cell growth, can be significantly inhibited by Honokiol. • Opportunities for HDAC3 inhibition may be a potential therapeutic target for preventing gastric cancer metastatic dissemination.

Aryl hydrocarbon receptor deficiency augments dysregulated microangiogenesis and diabetic retinopathy.

Diabetic retinopathy (DR) is a pathophysiologic vasculopathic process with obscure mechanisms and limited effective therapeutic strategies. Aryl hydrocarbon receptor (AhR) is an important regulator of xenobiotic metabolism and an environmental sensor. The aim of the present study was to investigate the role of AhR in the development of DR and elucidate the molecular mechanism of its downregulation. DR was evaluated in diabetes-induced retinal injury in wild type and AhR knockout (AhR-/-) mice. Retinal expression of AhR was determined in human donor and mice eyes by immunofluorescence since AhR activity was examined in diabetes. AhR knockout (AhRKO) mice were used to induce diabetes with streptozotocin, high-fat diet, or genetic double knockout with diabetes spontaneous mutation (Leprdb) (DKO; AhR-/-×Leprdb/db) for investigating structural, functional, and metabolic abnormalities in vascular and epithelial retina. Structural molecular docking simulation was used to survey the pharmacologic AhR agonists targeting phosphorylated AhR (Tyr245). Compared to diabetic control mice, diabetic AhRKO mice had aggravated alterations in retinal vasculature that amplified hallmark features of DR like vasopermeability, vascular leakage, inflammation, blood-retinal barrier breakdown, capillary degeneration, and neovascularization. AhR agonists effectively inhibited inflammasome formation and promoted AhR activity in human retinal microvascular endothelial cells and pigment epithelial cells. AhR activity and protein expression was downregulated, resulting in a decrease in DNA promoter binding site of pigment epithelium-derived factor (PEDF) by gene regulation in transcriptional cascade. This was reversed by AhR agonists. Our study identified a novel of DR model that target the protective AhR/PEDF axis can potentially maintain retinal vascular homeostasis, providing opportunities to delay the development of DR.

ATMIN enhances invasion by altering PARP1 in MSS colorectal cancer.

Genomic instability is a key cancer indicator. It results from defects in the DNA damage response (DDR) and increased replication stress. Herein, we examined how ataxia-telangiectasia mutated interactor (ATMIN), a DDR pathway involved in mismatch repair-proficient (microsatellite stability [MSS]), acts in colorectal carcinoma (CRC). Firstly, ATMIN mRNA expression was detected in CRC specimens with MSS characteristics, and the effects of ectopic ATMIN expression and ATMIN knockdown on invasion abilities were gauged in MSS cell lines. To understand the molecular mechanism, co-immunoprecipitation analyses in vitro were employed. Interestingly, ATMIN expression was positively correlated with advanced stages (P < .001), lymph node metastases (P = .002), and deeper invasion (P = .037) in MSS tumors; and significantly changed the cell motility in vitro. In the high-throughput analysis, ATMIN was found to act on the Wnt signaling pathway via PARP1. PAPR1 inhibition, in turn, significantly decreased invasion abilities resulting from ATMIN overexpression in cancer cell. Taken together, ATMIN, which alters the Wnt signaling pathway regulating CRC progression, plays as a crucial prognostic factor in MSS tumors.

ATMIN Suppresses Metastasis by Altering the WNT-Signaling Pathway via PARP1 in MSI-High Colorectal Cancer.

BACKGROUND: Constant DNA damage occurs in cells, and the cells are programmed to respond constitutively. This study explored the roles of ataxia-telangiectasia mutated interactor (ATMIN), one of the impaired pathways involving the DNA damage response (DDR) in mismatch repair-deficient [microsatellite instability (MSI)-high] colorectal carcinoma (CRC). METHODS: Expression of ATMIN messenger RNA (mRNA) was detected in CRC specimens with microsatellite instability (MSI) characteristics. The effects of ectopic ATMIN expression and ATMIN knockdown on invasion abilities were evaluated in MSI-high cell lines, and liver metastasis ability was investigated in vivo. Protein-protein interactions were assessed by coimmunoprecipitation analyses in vitro. RESULTS: Decreased ATMIN expression was positively correlated with advanced stage of disease (P < 0.05), lymph node metastases (P < 0.05), and deeper invasion (P < 0.05) in MSI-high tumors. Transient or stable ATMIN knockdown significantly increased cell motility. Moreover, in the high-throughput microarray and gene set enrichment analysis, ATMIN was shown to act on the Wnt-signaling pathway via PARP1. This cascade influences ß-catenin/transcription factor 4 (TCF4) binding affinity in MSI-high tumors, and PARP1 inhibition significantly decreased the number of metastases from ATMIN knockdown cancer cells. CONCLUSIONS: The results not only indicated the critical role of ATMIN, but also shed new light on PARP1 inhibitors, providing a basis for further clinical trials of MSI-high CRC.

The possible role of sirtuin 5 in the pathogenesis of apical periodontitis.

OBJECTIVES: We investigated the relation between expression of sirtuin 5 (SIRT5) in osteoblastic cells and progression of apical periodontitis. The role of SIRT5 in hypoxia-induced reactive oxygen species (ROS) formation and osteoblast apoptosis was also examined. MATERIALS AND METHODS: Progression of rat apical periodontitis was monitored by conventional radiography and microcomputed tomography. SIRT5 and oxidative stress biomarker 8-OHdG in bone-lining cells were assessed by immunohistochemistry. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was used to demonstrate apoptosis. In primary human osteoblasts cultured under hypoxia, Western blot was used to analyze SIRT5 expression and cleavage of pro-caspase 3 and poly(ADP-ribose) polymerase (PARP). SIRT5 was overexpressed through lentiviral technique. ROS formation and mitochondrial membrane potential changes were assessed by MitoSOX-Red and JC-1 fluorescence, respectively. Immunofluorescence microscope was used to evaluate mitochondrial release of cytochrome c. RESULTS: In rat apical periodontitis, disease progression was accompanied by decreased expression of SIRT5, increased oxidative stress, and enhanced apoptosis in bone-lining cells. SIRT5 was suppressed in cultured osteoblasts under hypoxia. SIRT5 overexpression ameliorated hypoxia-enhanced ROS formation, mitochondrial depolarization, cytochrome c leakage, activation of caspase-3, and PARP fragmentation. CONCLUSIONS: SIRT5 is able to alleviate hypoxia-enhanced osteoblast apoptosis. SIRT5 augmentation may have therapeutic potential for apical periodontitis.

Role and Mechanism of LIF in Oral Squamous Cell Carcinoma Progression.

Background: Metastasis is a severe problem in patients with oral squamous cell carcinoma (OSCC), which is the fifth most common cancer worldwide. Leukemia inhibitory factor (LIF) has been studied in different cancers, while the role of LIF in OSCC remains unclear. Methods: LIF expression was detected in 100 OSCC samples by immunohistochemistry. Effects of LIF on cell motility were evaluated in OSCC cell lines. High-throughput microarray analysis was also conducted. The correlation between LIF and the downstream effector was analyzed by real-time quantitative reverse transcription PCR. Results: Patients with OSCC who had lymph node metastasis or advanced cancer stages showed high LIF expression. OSCC patients with higher LIF expression, advanced stage, large tumor size, or lymph node metastasis had significantly shorter overall survival. LIF regulated cancer cell motilities through outside-in signaling. The inhibin beta A subunit (INHBA) gene was identified as a crucial downstream effector of LIF-promoted OSCC progression and restored migration and invasion abilities in LIF knockdown transfectants. Conclusion: LIF enhances regional lymphatic spread, thus leading to an advanced cancer stage. Regulation of LIF downstream molecules such as INHBA inhibits the invasion or migration ability of cancer cells. Thus, LIF can be a potential target in preventing cancer metastasis and spread.

Intracanal Metformin Promotes Healing of Apical Periodontitis via Suppressing Inducible Nitric Oxide Synthase Expression and Monocyte Recruitment.

INTRODUCTION: We have previously shown that intracanal metformin ameliorates apical periodontitis, partially by modulation of osteoblast apoptosis. The action of metformin on other cell types pertinent to the development of apical periodontitis needs to be examined. In the present study, we aimed to analyze whether its effects on the expression of inducible nitric oxide synthase (iNOS) and monocyte recruitment contribute to the therapeutic effect on apical periodontitis. METHODS: Lipopolysaccharide (LPS)-induced expression of iNOS in a human monocytic cell line, Mono-Mac-6, was assessed by Western blot. The amount of nitrite in culture medium was assessed to quantify nitric oxide (NO) production. C-C motif chemokine ligand-2 (CCL-2) synthesis was measured by enzyme-linked immunosorbent assay. Experimental apical periodontitis in rats was treated with root canal debridement with or without intracanal metformin medication. Lesion progression was assessed by conventional radiography and micro-computed tomographic imaging. Cellular expression of iNOS and the number of monocytes/macrophages were assessed by immunohistochemistry. RESULTS: Metformin suppressed LPS-induced iNOS and NO production by monocytes. More importantly, metformin inhibited LPS-enhanced CCL-2 synthesis through modulation of the iNOS/NO pathway. Intracanal metformin reduced bone resorption associated with apical periodontitis and suppressed iNOS expression and monocyte recruitment. CONCLUSIONS: Our results confirmed the therapeutic efficacy of intracanal metformin for apical periodontitis. Suppression of monocyte recruitment through modulation of iNOS expression and NO production is an important mechanism underlying the beneficial effect of metformin.

Metformin Ameliorates Periapical Lesions through Suppression of Hypoxia-induced Apoptosis of Osteoblasts.

INTRODUCTION: Intramuscular injection of metformin has been shown to inhibit the progression of periapical lesions in rats by decreasing the number of receptor activator of nuclear factor-κß ligand- and tartrate-resistant acid phosphatase-positive cells. In this study, we investigated the effect of metformin on hypoxia-induced apoptosis of osteoblasts and the therapeutic activity of intracanal metformin in induced periapical lesions in rats. METHODS: The influence of metformin on hypoxia-induced mitochondrial superoxide production in human osteoblasts was examined by using MitoSOX (Invitrogen, Carlsbad, CA) fluorescence dye signaling. The release of cytochrome c from mitochondria and the cleavage of procaspase-9 and poly(adenosine diphosphate-ribose) polymerase were evaluated by Western blot analysis. Apoptotic cell fraction was assessed by DNA content flow cytometry. In a rat model of induced periapical lesions, the effect of intracanal metformin on disease progression was appraised by 2-dimensional radiography and micro-computed tomographic imaging. Oxidative lesions and apoptotic activity of osteoblasts in vivo were estimated, respectively, by 8-hydroxy-2'-deoxyguanosine staining and terminal deoxynucleotidyl transferase dUTP nick end labeling. RESULTS: Metformin inhibited hypoxia-enhanced mitochondrial superoxide production in osteoblasts. Metformin suppressed hypoxia-induced cytochrome c release from mitochondria and the cleavage of procaspase-9 and poly(adenosine diphosphate-ribose) polymerase. Metformin repressed hypoxia-augmented apoptotic cell fraction. In a rat model, intracanal metformin diminished the size of periapical lesions and the oxidative damage and apoptotic activity in osteoblasts. CONCLUSIONS: Hypoxia increased oxidative stress in osteoblasts and enhanced cell death through activation of the mitochondrial pathway of apoptosis. Metformin attenuated the oxidative and cytotoxic action of hypoxia. The therapeutic effect of metformin on periapical lesions is partially caused by its antioxidative activity.

Connective tissue growth factor decreases mitochondrial metabolism through ubiquitin-mediated degradation of mitochondrial transcription factor A in oral squamous cell carcinoma.

BACKGROUND/PURPOSE: Deregulation of metabolic pathways is one of the hallmarks of cancer progression. Connective tissue growth factor (CTGF/CCN2) acts as a tumor suppressor in oral squamous cell carcinoma (OSCC). However, the role of CTGF in modulating cancer metabolism is still unclear. METHODS: OSCC cells stably overexpressing CTGF (SAS/CTGF) and shRNA against CTGF (TW2.6/shCTGF) were established. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were examined by the Seahorse XF24 analyzer. The expression of CTGF and mitochondrial biogenesis related genes was measured by real-time polymerase chain reaction or Western blot analysis. RESULTS: CTGF decreased OCR, ECAR, adenosine triphosphate (ATP) generation, mitochondrial DNA (mtDNA), and mitochondrial transcription factor A (mtTFA) protein expression in OSCC cells. Overexpression of mtTFA restored CTGF-decreased OCR, ECAR, mtDNA copy number, migration and invasion of SAS/CTGF cells. Immunoprecipitation assay showed a higher level of ubiquitinated mtTFA protein after CTGF treatment. MG132, an inhibitor of proteasomal degradation, reversed the effect of CTGF on mtTFA protein expression in SAS cells. CONCLUSION: CTGF can decrease glycolysis, mitochondrial oxidative phosphorylation, ATP generation, and mtDNA copy number by increasing mtTFA protein degradation through ubiquitin proteasome pathway and in turn reduces migration and invasion of OSCC cells. Therefore, CTGF may be developed as a potential additive therapeutic drug for oral cancer in the near future.

Differential protective effects of connective tissue growth factor against Aß neurotoxicity on neurons and glia.

Impaired clearance of amyloid-ß peptide (Aß) leads to abnormal extracellular accumulation of this neurotoxic protein that drives neurodegeneration in sporadic Alzheimer's disease (AD). Connective tissue growth factor (CTGF/CCN2) expression is elevated in plaque-surrounding astrocytes in AD patients. However, the role of CTGF in AD pathogenesis remains unclear. Here we characterized the neuroprotective activity of CTGF. We found that CTGF facilitated Aß uptake and subsequent degradation within primary glia and neuroblastoma cells. CTGF enhanced extracellular Aß degradation via membrane-bound matrix metalloproteinase-14 (MMP14) in glia and extracellular MMP13 in neurons. In the brain of a Drosophila AD model, glial-expression of CTGF reduced Aß deposits, improved locomotor function, and rescued memory deficits. Neuroprotective potential of CTGF against Aß42-induced photoreceptor degeneration was disrupted through silencing MMPs. Therefore, CTGF may represent a node for potential AD therapeutics as it intervenes in glia-neuron communication via specific MMPs to alleviate Aß neurotoxicity in the central nervous system.

Ataxia-telangiectasia mutated interactor regulates head and neck cancer metastasis via KRas expression.

OBJECTIVES: Relapse is the most serious problem affecting the morbidity and mortality rates of patients with head and neck squamous cell carcinoma (HNSCC). Although HNSCC has been studied for several decades, the exact mechanism of cancer recurrence remains unclear. MATERIALS AND METHODS: ataxia-telangiectasia mutated interactor (ATMIN) messenger RNA(mRNA) expression was detected in HNSCC samples by quantitative RT-PCR, and was analyzed with patients' clinical outcomes by Kaplan-Meier analyses. The ectopic ATMIN expression or ATMIN silencing on invasion ability was evaluated in HNSCC cell lines. Lymph node metastasis ability was investigated by buccal orthotopic implantation in vivo. All statistical tests were two-sided. RESULTS: ATMIN mRNA expression was positively correlated with patients' clinical outcomes. ATMIN blockage reduced invasion, migration, and metastasis abilities both in vitro and in vivo. Evidence from a buccal orthotopic implantation mice model showed that silenced ATMIN expression prolongs mice survival and reduced lymph node metastasis. In high-throughput microarray and bioinformative analyses, KRas was identified as a crucial downstream effector in ATMIN-mediated HNSCC metastasis and was positively associated with patients' clinical stages and ATMIN mRNA expression. CONCLUSIONS: The role of ATMIN and its regulatory mechanisms in HNSCC progression are reported for the first time. The study results improve our understanding of the ATMIN-KRas axis leading to HNSCC migration or invasion and metastasis and facilitates the identification of possible therapy targets of downstream genes for designing effective therapeutic strategies in personalized medicine.

Identifying N-linked glycan moiety and motifs in the cysteine-rich domain critical for N-glycosylation and intracellular trafficking of SR-AI and MARCO.

BACKGROUND: The accumulation of soluble oligomeric amyloid-ß peptide (oAß) proceeding the formation of senile plaques contributes to synaptic and memory deficits in Alzheimer's disease. Our previous studies have indentified scavenger receptor A (SR-A), especially SR-A type I (SR-AI), as prominent scavenger receptors on mediating oAß clearance by microglia while glycan moiety and scavenger receptor cysteine-rich (SRCR) domain may play the critical role. Macrophage receptor with collagenous structure (MARCO), another member of class A superfamily with a highly conserved SRCR domain, may also play the similar role on oAß internalization. However, the role of N-glycosylation and SRCR domain of SR-AI and MARCO on oAß internalization remains unclear. RESULT: We found that oAß internalization was diminished in the cells expressing SR-AI harboring mutations of dual N-glycosylation sites (i.e. N120Q-N143Q and N143Q-N184Q) while they were normally surface targeted. Normal oAß internalization was observed in 10 SR-AI-SRCR and 4 MARCO-SRCR surface targeted mutants. Alternatively, the SRCR mutants at ß-sheet and α-helix and on disulfide bone formation obstructed receptor's N-glycosylation and surface targeting. CONCLUSION: Our study reveals that N-glycan moiety is more critical than SRCR domain for SR-A-mediated oAß internalization.