Metformin prevents mandibular bone loss in a mouse model of accelerated aging by correcting dysregulated AMPK-mTOR signaling and osteoclast differentiation.

Background: Age-related mandibular osteoporosis frequently causes loose teeth, difficulty eating, and disfiguration in elders. Bmi1-/- mice displaying accelerated skeletal aging represent a useful model for testing interventions against premature jaw bone loss. As an anti-aging agent, metformin may ameliorate molecular dysfunction driving osteoporosis pathogenesis. We explored the mechanisms of mandibular osteopenia in Bmi1-/- mice and prevention by metformin treatment. Methods: Three mouse groups were utilized: wild-type controls, untreated Bmi1-/-, and Bmi1-/- receiving 1 g/kg metformin diet. Mandibular bone phenotype was assessed by X-ray, micro-CT, histology, and immunohistochemistry. AMPK-mTOR pathway analysis, senescence markers, osteoblast and osteoclast gene expression were evaluated in jaw tissue. Osteoclast differentiation capacity and associated signaling molecules were examined in cultured Bmi1-/- bone marrow mononuclear cells ± metformin. Results: Bmi1 loss reduced mandible bone density concomitant with decreased AMPK activity, increased mTOR signaling and cellular senescence in jaw tissue versus wild-type controls. This was accompanied by impaired osteoblast function and upregulated osteoclastogenesis markers. Metformin administration normalized AMPK-mTOR balance, oxidative stress and senescence signaling to significantly improve mandibular bone architecture in Bmi1-/- mice. In culture, metformin attenuated excessive osteoclast differentiation from Bmi1-/- marrow precursors by correcting dysregulated AMPK-mTOR-p53 pathway activity and suppressing novel pro-osteoclastogenic factor Stfa1. Conclusions: Our study newly demonstrates metformin prevents accelerated jaw bone loss in a premature aging murine model by rectifying molecular dysfunction in cellular energy sensors, redox state, senescence and osteoclastogenesis pathways. Targeting such age-associated mechanisms contributing to osteoporosis pathogenesis may help maintain oral health and aesthetics in the growing elderly population. Translational potential: The pronounced mandibular osteopenia exhibited in Bmi1-/- mice represents an accelerated model of jaw bone deterioration observed during human aging. Our finding that metformin preserves mandibular bone integrity in this progeroid model has important clinical implications. As an inexpensive oral medication already widely used to manage diabetes, metformin holds translational promise for mitigating age-related osteoporosis. The mandible is essential for chewing, swallowing, speech and facial structure, but progressively loses bone mass and strength with advancing age, significantly impacting seniors' nutrition, physical function and self-image. Our results suggest metformin's ability to rectify cellular energy imbalance, oxidative stress and osteoclast overactivity may help maintain jaw bone health into old age. Further research is still needed given metformin's multifaceted biology and bone regulation by diverse pathways. However, this preclinical study provides a strong rationale for clinical trials specifically examining mandibular outcomes in elderly subjects receiving standard metformin treatment for diabetes or prediabetes. Determining if metformin supplementation can prevent or delay oral disability and disfigurement from senescent jaw bone loss in the growing aged population represents an important public health priority. In summary, our mechanistic findings in a genetic mouse model indicate metformin merits investigation in rigorous human studies for alleviating morbidity associated with age-related mandibular osteoporosis.

Corrigendum: Identification of hub cuproptosis related genes and immune cell infiltration characteristics in periodontitis.

[This corrects the article DOI: 10.3389/fimmu.2023.1164667.].

Identification of hub cuproptosis related genes and immune cell infiltration characteristics in periodontitis.

Introduction: Periodontitis is an inflammatory disease and its molecular mechanisms is not clear. A recently discovered cell death pathway called cuproptosis, may related to the disease. Methods: The datasets GSE10334 of human periodontitis and control were retrieved from the Gene Expression Omnibus database (GEO) for analysis.Following the use of two machine learning algorithms, least absolute shrinkage and selection operator (LASSO) and support vector machine-recursive feature removal (SVM-RFE) were used to find CRG-based signature. Then the Receiver operating characteristic (ROC) curves was used to evaluate the gene signature's discriminatory ability. The CIBERSORT deconvolution algorithm was used to study the link between hub genes and distinct types of immune cells. Next, the association of the CRGs with immune cells in periodontitis and relevant clusters of cuproptosis were found. The link between various clusters was ascertained by the GSVA and CIBERSORT deconvolution algorithm. Finally, An external dataset (GSE16134) was used to confirm the diagnosis capacity of the identified biomarkers. In addition, clinical samples were examined using qRT-PCR and immunohistochemistry to verifiy the expression of genes related to cuprotosis in periodontitis and the signature may better predict the periodontitis. Results: 15 periodontitis-related DE-CRGs were found,then 11-CRG-based signature was found by using of LASSO and SVM-RFE. ROC curves also supported the value of signature. CIBERSORT results of immune cell signature in periodontitis showed that signature genes is a crucial component of the immune response.The relevant clusters of cuproptosis found that the NFE2L2, SLC31A1, FDX1,LIAS, DLD, DLAT, and DBT showed a highest expression levels in Cluster2 ,while the NLRP3, MTF1, and DLST displayed the lowest level in Cluster 2 but the highest level in Cluster1. The GSVA results also showed that the 11 cuproptosis diagnostic gene may regulate the periodontitis by affecting immune cells. The external dataset (GSE16134) confirm the diagnosis capacity of the identified biomarkers, and clinical samples examined by qRT-PCR and immunohistochemistry also verified that these cuprotosis related signiture genes in periodontitis may better predict the periodontitis. Conclusion: These findings have important implications for the cuproptosis and periodontitis, and highlight further research is needed to better understand the mechanisms underlying this relationship between the cuproptosis and periodontitis.

Role of 1,25-dihydroxyvitamin D in alleviating alveolar bone loss and gingival inflammation in ligature-induced periodontitis.

OBJECTIVES: The goal of this study was to assess if endogenous 1,25(OH)2D deficiency enhanced, whereas exogenous 1,25(OH)2D3 supplementation alleviated alveolar bone loss and gingival inflammation induced by ligature-induced periodontitis. METHODS: A model of ligature-induced experimental periodontitis was generated in wild-type (WT) and Cyp27b1-knockout (KO) mice on a rescue diet (RD), and un-ligated genotype-matched littermates as control, or in WT mice on a normal diet (ND) with vehicle treatment or 1,25(OH)2D3 treatment, and un-ligated WT littermates as control. Alveolar bone mass and turnover, T cell infiltration and inflammatory cytokines in gingival tissues were examined. RESULTS: In WT mice, ligature-induced alveolar bone loss occurred by inhibiting alveolar bone formation. This was characterized by reduction of osteoblast numbers, alkaline phosphatase activity and type I collagen synthesis, as well as by augmentation of osteoclastic alveolar bone resorption and gingival inflammation, including increases of osteoclast numbers, inflammatory positive cells and up-regulation of mRNA expression levels of inflammatory cytokines. Alveolar bone destruction and gingival inflammation were more severe in diet-matched Cyp27b1-KO mice than in WT littermates on RD. Supplementation of exogenous 1,25(OH)2D3 alleviated alveolar bone loss and gingival inflammation in ligated WT mice on ND, but those parameters did not reach levels observed in un-ligated WT ones. CONCLUSIONS: Endogenous 1,25(OH)2D deficiency enhanced, whereas exogenous 1,25(OH)2D3 supplementation alleviated alveolar bone loss and gingival inflammation induced by ligature-induced periodontitis.

Pyrroloquinoline quinone inhibits ligature-induced alveolar bone loss through regulation of redox balance and cell senescence.

It has been demonstrated that oxidative stress is related to periodontitis, and that pyrroloquinoline quinine (PQQ) acts as a powerful antioxidant. This study aimed to explore the effect of PQQ on ligature-induced alveolar bone loss in experimental periodontitis (EP) mice with/without PQQ in the diet. EP mice received a diet supplemented with PQQ for 2 weeks and were compared with sham (control) mice as well as untreated EP mice. Additionally, human periodontal ligament cells (hPDLCs) were treated with PQQ in the presence or absence of lipopolysaccharide (LPS). We found that the bone volume fraction, alkaline phosphatase activity, and the number of antioxidant cells were significantly decreased in EP mice compared with the sham mice, whereas PQQ administration rescued the above effects. In contrast, alveolar bone loss, osteoclast number, cell senescence-associated cells, and cytokines' expression were significantly increased in EP mice compared with the sham mice but were significantly decreased with PQQ supplementation in periodontal tissues. Furthermore, we found that antioxidant enzymes and Bmi-1 protein expression levels were downregulated, whereas the protein expression levels of cell senescence-related proteins including γ-H2AX, IL-6, IL-1ß, p16, and p21 were significantly up-regulated in LPS-induced hPDLCs compared with the control cells. However, PQQ administration partially prevented these changes. These findings suggest that PQQ may alleviate periodontal damage through regulation of the redox balance and cell senescence.

Bioactive Compounds from Abelmoschus manihot L. Alleviate the Progression of Multiple Myeloma in Mouse Model and Improve Bone Marrow Microenvironment.

PURPOSE: Abelmoschus manihot (L.) Medik. (Malvaceae) derived Huangkui capsules (HKC) represent a traditional Chinese medicine that has been widely applied to the clinical therapy of kidney and inflammatory diseases. The present study aimed to determine the potential therapeutic effects and underlying mechanisms of the ingredients on Multiple Myeloma (MM), an incurable disease that exhibits malignant plasma cell clonal expansion in the bone marrow. METHODS: A 5TMM3VT syngeneic MM-prone model was established and treated with HKC. Murine pre-osteoblast MC3T3-E1 and pre-osteoclast Raw264.7 cells were treated with nine flavonoid compounds extracted from the flowers of Abelmoschus manihot. MC3T3-E1 and Raw264.7 cells were then examined by alizarin red staining and tartrate-resistant acid phosphatase activity staining, respectively. The proliferation of two human MM cells (ARP1, H929) was examined by performing an MTT assay following treatment with flavonoid compounds. Additionally, the cell cycle was analyzed via staining and flow cytometry. The differential expressions of certain proteins were detected via Western blotting, transcriptomic RNA-sequencing as well as RT-qPCR. RESULTS: The results revealed that MM-prone animals appeared to be protected following HKC treatment, as evidenced by a prolonged survival rate. Furthermore, four of the nine flavonoid compounds [Hyperin/Hyperoside, HK-2; Cannabiscitrin, HK-3; 3-O-kaempferol-3-O-acetyl-6-O-(p-coumaroyl)-ß-D-glucopyranoside, HK-11; 8-(2''-pyrrolidione-5''-yl)-quercetin, HK-B10] induced the differentiation of murine pre-osteoblast MC3T3-E1 cells. In addition, two compounds [Isomyricitrin, HK-8; quercetin-8-(2''-pyrrolidione-5"-yl)-3'-O-ß-D-glucopyranosid, HK-E3] suppressed osteoclastogenesis in murine Raw264.7 cells. HK-11 directly inhibited MM cells (ARP1 and H929) proliferation and induced G0/G1 cell cycle arrest, which may have involved the suppressing ß-catenin protein, increasing expressions of IL-6 and TNF-α, as well as activating mature TGF-ß1 and some other metabolic pathways. CONCLUSION: These results of the present study indicated that the bio-active ingredients of HKC exerted protective effects on MM mouse survival through promoting osteoblastogenesis and suppressing osteoclastogenesis, thus improving the bone marrow microenvironment to inhibit MM cell proliferation.

TROP2 increases growth and metastasis of human oral squamous cell carcinoma through activation of the PI3K/Akt signaling pathway.

Most malignant neoplasms of the oral cavity are oral squamous cell carcinoma (OSCC), which is a type of highly malignant tumor with a propensity for forming distant metastases. Trophoblast cell surface antigen 2 (TROP2) is a transmembrane protein that is overexpressed in several types of tumor cells, although its role and regulatory mechanism in OSCC have not been determined. The aim of the present study was to examine the effects of TROP2 in human OSCC cell lines. The present study demonstrated that TROP2 protein expression was upregulated in OSCC cell lines. Transfection of short hairpin RNA (shRNA) targeting TROP2 (sh­TROP2) reduced cell proliferation, migration and invasion of OSCC cell lines, whereas overexpression of TROP2 increased proliferation, migration and invasion. sh­TROP2 transfection in OSCC cell lines inhibited tumor growth in OSCC mouse models. Furthermore, TROP2 expression activated the phosphoinositide 3­kinase (PI3K)/Akt signaling pathway in human OSCC cells. These results suggest that TROP2 induces cell growth, migration and invasion through activation of the PI3K/Akt signaling pathway in OSCC cells.

POM121 is identified as a novel prognostic marker of oral squamous cell carcinoma.

Background: The aim of this study was to confirm the role of nuclear pore membrane protein 121(POM121) in oral squamous cell carcinoma and to explore the underlying mechanism. Methods: POM121mRNA and protein expressions were evaluated in OSCC tissues and normal oral tissues by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. The relationship between POM121 expression and clinical characteristics was analyzed. Bioinformatics analysis was performed to explore the possible mechanisms how POM121 affected OSCC. Results: We confirmed that POM121 mRNA expression in OSCC tissues was significantly higher than that in non-tumorous tissues, as was POM121 protein expression. POM121 expression was associated with distant metastasis and TNM stage. Multivariate analysis confirmed POM121 expression as an independent prognostic factor for OSCC patients. OSCC patients with high POM121 expression had a worse overall survival (OS) compared with patients with low POM121 expression. Bioinformatics analysis indicated POM121 may regulate OSCC through hedgehog and /or p53 signaling pathway. Conclusion: Targeting of POM121 expression levels could provide new diagnostic and therapeutic strategies for OSCC patients.

1,25-dihydroxyvitamin D deficiency accelerates alveolar bone loss independent of aging and extracellular calcium and phosphorus.

BACKGROUND: Vitamin D is critical for bone homeostasis and immunomodulation. We therefore assessed whether 1,25-dihydroxyvitamin D (1,25(OH)2 D) deficiency in mice with targeted deletion of the gene encoding 25-hydroxyvitamin D-1α-hydroxylase (1α(OH)ase [1αOH)ase-/- mice]) results in alveolar bone loss and periodontal inflammation in vivo. METHODS: Ten-week-old and 12-month-old 1α(OH)ase-/- mice and wild-type littermates were fed a normal diet or a rescue diet, and the phenotype of the periodontium was then analyzed using microcomputed tomography, histology, immunohistochemistry, and real-time Reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Alveolar bone loss was increased and maxillary bone mineral density (BMD), osteoblast numbers, and the number of osterix-positive cells were decreased significantly in 1α(OH)ase-/- mice compared with wild-type mice. Although aging from 10 weeks to 12 months accentuated these changes, and a rescue diet reduced them, the alterations in the 1α(OH)ase-/- mice exceeded the effects of aging and diet change. Nuclear factor kappa light-chain-enhancer of activated B cells (NF-кB) p65 and CD3 positive cells, and the gene expression levels of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, matrix metalloproteinase (MMP)-3 and -8 were all increased significantly in periodontal tissues of 1α(OH)ase-/- mice compared with wild-type mice. Aging from 10 weeks to 12 months also accentuated these changes, and a rescue diet reduced them, however, the alterations in the 1α(OH)ase-/- mice exceeded the effects of aging and diet change. CONCLUSION: 1,25(OH)2 D deficiency in the 1α(OH)ase-/- mice accelerated alveolar bone loss by inhibiting osteoblastic bone formation and enhancing periodontal tissue degeneration in a calcium- and phosphorus- as well as an age-independent manner.

Comparison of gene expression profiles between dental pulp and periodontal ligament tissues in humans.

There are anatomical and functional differences between human dental pulp (DP) and periodontal ligament (PDL). However, the molecular biological differences and function of these tissues are poorly understood. In the present study, we employed a cDNA microarray array to screen for differentially expressed genes (DEGs) between human DP and PDL tissues, and used the online software WebGestalt to perform the functional analysis of the DEGs. In addition, the STRING database and KEGG pathway analysis were applied for interaction network and pathway analysis of the DEGs. DP and PDL samples were obtained from permanent premolars (n=16) extracted for orthodontic purposes. The results of the microarray assay were confirmed by RT-qPCR. The DEGs were found to be significantly associated with the extracellular matrix and focal adhesion. A total of 10 genes were selected to confirm the results. The mRNA levels of integrin alpha 4 (ITGA4), integrin alpha 8 (ITGA8), neurexin 1 (NRXN1) and contactin 1 (CNTN1) were significantly higher in the DP than in the PDL tissues. However, the levels of collagen type XI alpha 1 (COL11A1), aggrecan (ACAN), collagen type VI alpha 1 (COL6A1), chondroadherin (CHAD), laminin gamma 2 (LAMC2) and laminin alpha 3 (LAMA3) were higher in the PDL than in the DP samples. The gene expression profiles provide novel insight into the characterization of DP and PDL tissues, and contribute to our understanding of the potential molecular mechanisms of dental tissue mineralization and regeneration.

Cranial base characteristics in anteroposterior malocclusions: A meta-analysis.

OBJECTIVE: To investigate cranial base characteristics in malocclusions with sagittal discrepancies. MATERIALS AND METHODS: An electronic search was performed in PubMed, Embase, Web of Science, and the Cochrane Library. A fixed- or random-effect model was applied to calculate weighted mean difference with 95% confidence intervals (CIs) according to statistical heterogeneity. Outcome measures were anterior, posterior, and total cranial base length and cranial base angle. Sensitivity analysis and publication bias were conducted. RESULTS: Twenty studies that together included 1121 Class I, 1051 Class II, and 730 Class III cases qualified for the final analysis. Class III malocclusion demonstrated significantly reduced anterior (95% CI: -1.74, -0.53; P < .001 vs Class I; 95% CI: -3.30, -2.09; P < .001 vs Class II) and total cranial base length (95% CI: -3.33, -1.36; P < .001 vs Class I; 95% CI: -7.38, -4.05; P < .001 vs Class II). Further, Class II patients showed significantly greater anterior and total cranial base length than did Class I patients (95% CI: 0.51, 1.87; P < .001 for SN; 95% CI: 2.20, 3.30; P < .001 for NBa). Cranial base angle was significantly smaller in Class III than in Class I (95% CI: -3.14, -0.93; P < .001 for NSBa; 95% CI: -2.73, -0.68; P  =  .001 for NSAr) and Class II malocclusions (95% CI: -5.73, -1.06; P  =  .004 for NSBa; 95% CI: -6.11, -1.92; P < .001 for NSAr) and greater in Class II than in Class I malocclusions (95% CI: 1.38, 2.38; P < .001 for NSBa). CONCLUSIONS: This meta-analysis showed that anterior and total cranial base length and cranial base angle were significantly smaller in Class III malocclusion than in Class I and Class II malocclusions, and that they were greater in Class II subjects compared to controls.

[Comparison between J-hook and micro-implant anchorage in the treatment of patients with bimaxillary protrusion].

OBJECTIVE: To compare the difference between J-hook and micro-implant anchorage in the treatment of patient with bimaxillary protrusion. METHODS: Thirty patients with bimaxillary protrusion were divided into two groups (J-hook and micro-implant groups) and treated with MBT appliance. Four first premolars were extracted in all patients. Cephalometric analyses were carried out before and after treatment. RESULTS: In J-hook group and micro-implant group,computerized cephalometric analysis revealed that before treatment U6C-PP was (12.4 +/- 0.2) mm and (12.5 +/- 0.1) mm, respectively,and after treatment U6C-PP was (12.6 +/- 0.1) mm and (12.8 +/- 0.1) mm,respectively. The difference between J-hook group and microimplant group was significant (P < 0.01). The other differences of cephalometric analyses between J-hook group and micro-implant group was not significant. CONCLUSIONS: Both J-hook and micro-implant could provide adequate anchorage in the treatment of patients with bimaxillary protrusion.