The role of CXCL2-mediated crosstalk between tumor cells and macrophages in Fusobacterium nucleatum-promoted oral squamous cell carcinoma progression.

Dysbiosis of the oral microbiota is related to chronic inflammation and carcinogenesis. Fusobacterium nucleatum (Fn), a significant component of the oral microbiota, can perturb the immune system and form an inflammatory microenvironment for promoting the occurrence and progression of oral squamous cell carcinoma (OSCC). However, the underlying mechanisms remain elusive. Here, we investigated the impacts of Fn on OSCC cells and the crosstalk between OSCC cells and macrophages. 16 s rDNA sequencing and fluorescence in situ hybridization verified that Fn was notably enriched in clinical OSCC tissues compared to paracancerous tissues. The conditioned medium co-culture model validated that Fn and macrophages exhibited tumor-promoting properties by facilitating OSCC cell proliferation, migration, and invasion. Besides, Fn and OSCC cells can recruit macrophages and facilitate their M2 polarization. This crosstalk between OSCC cells and macrophages was further enhanced by Fn, thereby amplifying this positive feedback loop between them. The production of CXCL2 in response to Fn stimulation was a significant mediator. Suppression of CXCL2 in OSCC cells weakened Fn's promoting effects on OSCC cell proliferation, migration, macrophage recruitment, and M2 polarization. Conversely, knocking down CXCL2 in macrophages reversed the Fn-induced feedback effect of macrophages on the highly invasive phenotype of OSCC cells. Mechanistically, Fn activated the NF-κB pathway in both OSCC cells and macrophages, leading to the upregulation of CXCL2 expression. In addition, the SCC7 subcutaneous tumor-bearing model in C3H mice also substantiated Fn's ability to enhance tumor progression by facilitating cell proliferation, activating NF-κB signaling, up-regulating CXCL2 expression, and inducing M2 macrophage infiltration. However, these effects were reversed by the CXCL2-CXCR2 inhibitor SB225002. In summary, this study suggests that Fn contributes to OSCC progression by promoting tumor cell proliferation, macrophage recruitment, and M2 polarization. Simultaneously, the enhanced CXCL2-mediated crosstalk between OSCC cells and macrophages plays a vital role in the pro-cancer effect of Fn.

PGRN is involved in macrophage M2 polarization regulation through TNFR2 in periodontitis.

BACKGROUND AND OBJECTIVE: Progranulin (PGRN), a multifunctional growth factor, plays indispensable roles in the regulation of cancer, inflammation, metabolic diseases, and neurodegenerative diseases. Nevertheless, its immune regulatory role in periodontitis is insufficiently understood. This study attempts to explore the regulatory effects of PGRN on macrophage polarization in periodontitis microenvironment. METHODS: Immunohistochemical (IHC) and multiplex immunohistochemical (mIHC) stainings were performed to evaluate the expression of macrophage-related markers and PGRN in gingival samples from periodontally healthy subjects and periodontitis subjects. RAW264.7 cells and bone marrow-derived macrophages (BMDMs) were polarized towards M1 or M2 macrophages by the addition of LPS or IL-4, respectively, and were treated with or without PGRN. Real-time fluorescence quantitative PCR (qRT-PCR), immunofluorescence staining (IF), enzyme-linked immunosorbent assay (ELISA), and flow cytometry were used to determine the expressions of M1 and M2 macrophage-related markers. Co-immunoprecipitation was performed to detect the interaction between PGRN and tumor necrosis factor receptor 2 (TNFR2). Neutralizing antibody was used to block TNFR2 to confirm the role of TNFR2 in PGRN-mediated macrophage polarization. RESULTS: The IHC and mIHC staining of human gingival slices showed a significant accumulation of macrophages in the microenvironment of periodontitis, with increased expressions of both M1 and M2 macrophage markers. Meanwhile, PGRN was widely expressed in the gingival tissue of periodontitis and co-expressed mainly with M2 macrophages. In vitro experiments showed that in RAW264.7 cells and BMDMs, M1 markers (CD86, TNF-α, iNOS, and IL-6) substantially decreased and M2 markers (CD206, IL-10, and Arg-1) significantly increased when PGRN was applied to LPS-stimulated macrophages relatively to LPS stimulation alone. Besides, PGRN synergistically promoted IL-4-induced M2 markers expression, such as CD206, IL-10, and Arg1. In addition, the co-immunoprecipitation result showed the direct interaction of PGRN with TNFR2. mIHC staining further revealed the co-localization of PGRN and TNFR2 on M2 macrophages (CD206+). Blocking TNFR2 inhibited the regulation role of PGRN on macrophage M2 polarization. CONCLUSIONS: In summary, PGRN promotes macrophage M2 polarization through binding to TNFR2 in both pro- and anti-inflammatory periodontal microenvironments.

Enhanced immunomodulation and periodontal regeneration efficacy of subgingivally delivered progranulin-loaded hydrogel as an adjunct to non-surgical treatment for Class II furcation involvement in dogs.

AIM: To evaluate the effect of subgingival delivery of progranulin (PGRN)/gelatin methacryloyl (GelMA) complex as an adjunct to scaling and root planing (SRP) on an experimental periodontitis dog model with Class II furcation involvement (FI). MATERIALS AND METHODS: A Class II FI model was established, and the defects were divided into four treatment groups: (a) no treatment (control); (b) SRP; (c) SRP + GelMA; (d) SRP + PGRN/GelMA. Eight weeks after treatment, periodontal parameters were recorded, gingival crevicular fluid and gingival tissue were collected for ELISA and RT-qPCR, respectively, and mandibular tissue blocks were collected for micro computed tomography (micro-CT) scanning and hematoxylin and eosin (H&E) staining. RESULTS: The SRP + PGRN/GelMA group showed significant improvement in all periodontal parameters compared with those in the other groups. The expression of markers related to M1 macrophage and Th17 cell significantly decreased, and the expression of markers related to M2 macrophage and Treg cell significantly increased in the SRP + PGRN/GelMA group compared with those in the other groups. The volume, quality and area of new bone and the length of new cementum in the root furcation defects of the PGRN/GelMA group were significantly increased compared to those in the other groups. CONCLUSIONS: Subgingival delivery of the PGRN/GelMA complex could be a promising non-surgical adjunctive therapy for anti-inflammation, immunomodulation and periodontal regeneration.

Low-dose TNF-α promotes angiogenesis of oral squamous cell carcinoma cells via TNFR2/Akt/mTOR axis.

OBJECTIVES: To assess the role of TNF-α/TNFR2 axis on promoting angiogenesis in oral squamous cell carcinoma (OSCC) cells and uncover the underlying mechanisms. MATERIALS AND METHODS: The expression of TNFR2 and CD31 in OSCC tissues was examined; gene expression relationship between TNF-α/TNFR2 and angiogenic markers or signaling molecules was analyzed; the expression of angiogenic markers, signaling molecules, TNFR1, and TNFR2 in TNF-α-stimulated OSCC cells treated with or without TNFR2 neutralizing antibody (TNFR2 Nab) were assessed; the concentration of angiogenic markers in the supernatant of OSCC cells was detected; conditioned mediums of OSCC cells treated with TNF-α or TNF-α + TNFR2 Nab were applied to human umbilical vein endothelial cells (HUVECs), followed by tube formation and cell migration assays. RESULTS: Significantly elevated expression of TNFR2 and CD31 in OSCC tissues was observed. A positive gene expression correlation was identified between TNF-α/TNFR2 and angiogenic markers or signaling molecules. TNFR2 Nab inhibited the effects of TNF-α on enhancing the expression of angiogenic factors and TNFR2, the phosphorylation of the Akt/mTOR signaling pathway, HUVECs migration, and tube formation. CONCLUSIONS: TNFR2 Nab counteracts the effect of TNF-α on OSCC cells through the TNFR2/Akt/mTOR axis, indicating that blocking TNFR2 might be a promising strategy against cancer.

Hyperlipidemia impairs bone regeneration of closed bone defects and tooth extraction wounds in mice.

OBJECTIVES: To evaluate the effect of hyperlipidemia on the healing of bone defects. MATERIALS AND METHODS: Apolipoprotein E (ApoE)-deficient mice and wild-type (WT) C57BL/6J mice were fed with an atherogenic high-fat diet (HFD) or a standard chow diet (as control) for 6 weeks. Blood samples were collected to evaluate serum lipid levels. Closed bone defects and open tooth extraction wounds were then created in the mandibles of these animals. One or two weeks after surgery, animals were euthanized. Micro-CT analysis and histomorphometric analysis were conducted to evaluate the healing of bone defects and the alveolar ridge resorption. RESULTS: Bone regeneration of closed bone defects was considerably delayed in the hyperlipidemic Apoe-/- mice and WT mice. No obvious difference was detected in the new bone formation of the tooth extraction wounds. The HFD-fed mice showed more prominent reduction in the lingual alveolar ridge height of the tooth extraction wounds when compared with the control group. CONCLUSIONS: Hyperlipidemia results in delayed bone regeneration in large closed bone defects. Although tooth extraction wounds are small and normally regenerated in a hyperlipidemic microenvironment, the prominent reduction in the alveolar ridge height is also a challenge for future restoration of the dentition.

Characteristics of Microbial Distribution in Different Oral Niches of Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC), one of the most common malignant tumors of the head and neck, is closely associated with the presence of oral microbes. However, the microbiomes of different oral niches in OSCC patients and their association with OSCC have not been adequately characterized. In this study, 305 samples were collected from 65 OSCC patients, including tumor tissue, adjacent normal tissue (paracancerous tissue), cancer surface tissue, anatomically matched contralateral normal mucosa, saliva, and tongue coat. 16S ribosomal DNA (16S rDNA) sequencing was used to compare the microbial composition, distribution, and co-occurrence network of different oral niches. The association between the microbiome and the clinical features of OSCC was also characterized. The oral microbiome of OSCC patients showed a regular ecological distribution. Tumor and paracancerous tissues were more microbially diverse than other oral niches. Cancer surface, contralateral normal mucosa, saliva, and tongue coat showed similar microbial compositions, especially the contralateral normal mucosa and saliva. Periodontitis-associated bacteria of the genera Fusobacterium, Prevotella, Porphyromonas, Campylobacter, and Aggregatibacter, and anaerobic bacteria were enriched in tumor samples. The microbiome was highly correlated with tumor clinicopathological features, with several genera (Lautropia, Asteroleplasma, Parvimonas, Peptostreptococcus, Pyramidobacter, Roseburia, and Propionibacterium) demonstrating a relatively high diagnostic power for OSCC metastasis, potentially providing an indicator for the development of OSCC.

Growth of ZIF-8 Nanoparticles In Situ on Graphene Oxide Nanosheets: A Multifunctional Nanoplatform for Combined Ion-Interference and Photothermal Therapy.

The regulation of intracellular ions' overload to interrupt normal bioprocesses and cause cell death has been developed as an efficient strategy (named as ion-interference therapy/IIT) to treat cancer. In this study, we design a multifunctional nanoplatform (called BSArGO@ZIF-8 NSs) by in situ growth of metal organic framework nanoparticles (ZIF-8 NPs) onto the graphene oxide (GO) surface, subsequently reduced by ascorbic acid and modified by bovine serum albumin. This nanocomplex causes the intracellular overload of Zn2+, an increase of reactive oxygen species (ROS), and exerts a broad-spectrum lethality to different kinds of cancer cells. BSArGO@ZIF-8 NSs can promote cell apoptosis by initiating bim (a pro-apoptotic protein)-mediated mitochondrial apoptotic events, up-regulating PUMA/NOXA expression, and down-regulating the level of Bid/p53AIP1. Meanwhile, Zn2+ excess triggers cellular dysfunction and mitochondria damage by activating the autophagy signaling pathways and disturbing the intracellular environmental homeostasis. Combined with the photothermal effect of reduced GO (rGO), BSArGO@ZIF-8 NSs mediated ion-interference and photothermal combined therapy leads to effective apoptosis and inhibits cell proliferation and angiogenesis, bringing a higher efficacy in tumor suppression in vivo. This designed Zn-based multifunctional nanoplatform will allow promoting further the development of IIT and the corresponding combined cancer therapy strategy.

M1 Macrophages Enhance Survival and Invasion of Oral Squamous Cell Carcinoma by Inducing GDF15-Mediated ErbB2 Phosphorylation.

M2 macrophages are generally recognized to have a protumor role, while the effect of M1 macrophages in cancer is controversial. Here, the in vitro and in vivo effects of conditioned medium from M1 macrophages (M1-CM) on oral squamous cell carcinoma (OSCC) cells and a potential mechanism were studied. CCK-8, colony formation, EdU labeling, xenograft growth, and Transwell assays were utilized to observe cell survival/proliferation and migration/invasion, respectively, in OSCC cell lines treated with basic medium (BM) and M1-CM. The ErbB2 phosphorylation inhibitor (CI-1033) and GDF15 knockout cell lines were used to appraise the role of ErbB2 and GDF15 in mediating the effects of M1-CM. Compared with BM, M1-CM significantly enhanced the survival/proliferation of SCC25 cells. The migration/invasion of SCC25 and CAL27 cells also increased. Mechanically, M1-CM promoted GDF15 expression and increased the phosphorylation of ErbB2, AKT, and ErK. CI-1033 significantly declined the M1-CM-induced activation of p-AKT and p-ErK and its protumor effects. M1-CM stimulated enhancement of p-ErbB2 expression was significantly decreased in cells with GDF15 gene knockout vs without. In xenograft, M1-CM pretreatment significantly promoted the carcinogenic potential of OSCC cells. Our results demonstrate that M1 macrophages induce the proliferation, migration, invasion, and xenograft development of OSCC cells. Mechanistically, this protumor effect of M1 macrophages is partly associated with inducing GDF15-mediated ErbB2 phosphorylation.

Carboxymethyl chitin or chitosan for osteoinduction effect on the human periodontal ligament stem cells.

Human periodontal ligament stem cells (hPDLSCs) are seeding cells for tissue-engineered treatment of alveolar bone regeneration. To elucidate carboxymethyl chitosan (CMCTS) and carboxymethyl chitin (CMCT) effect on osteogenic differentiation, hPDLSCs were isolated and treated with CMCTS or CMCT. Cell viability and multiplication capacity were measured. The expression of classic osteogenic related molecules, including Alkaline Phosphatase (ALP), Phosphoprotein 1 (OPN), RUNX family transcription factor 2 (Runx2) and Osteocalcin (OCN), were determined. Mineralization levels were detected by Alizarin Red staining. Results showed that both CMCTS and CMCT treatment had the maximal promoting ability for hPDLSCs viability below the concentration of 100 µg/mL, while CMCTS improved hPDLSCs mineralization significantly. CMCTS induced multiple-factor high expression, including ALP, Runx2, OPN and OCN, whereas slightly osteoinductive bioactivity of CMCT was mainly due to ALP. Therefore, CMCTS had a more significant advantage for osteoinductive differentiation of hPDLSCs than CMCT, which may be a promising material for periodontal regeneration.

Alveolar ridge preservation with fibroblast growth factor-2 modified acellular dermal matrix membrane and a bovine-derived xenograft: An experimental in vivo study.

OBJECTIVE: To investigate the effect of a bone substitute material combined with fibroblast growth factor-2 (FGF-2) loaded barrier membrane on the preservation of alveolar ridge after tooth extraction. MATERIAL AND METHODS: Four dogs were included. Six extraction sockets of each animal received the 3 treatments and were randomly divided into three groups. Group A: negative control; Group B: bovine xenografts + membrane; and Group C: bovine xenografts + FGF-2-loaded membrane. CBCT and histological analysis were performed to evaluate changes in the width and height of alveolar ridges and extraction socket bone healing 8 weeks post-extraction. RESULTS: CBCT showed that the alveolar bone in Group A was significantly thinner than that in Group B and Group C at 1 and 3 mm apically from the alveolar crest. The alveolar width at 1 mm in Group C (60.99 ± 15.36%) was significantly thicker than that in Group B (39.75 ± 30.18%). Histomorphmetrical measurements showed that the buccal alveolar width at 1 mm was significantly thicker in Groups B and C than in Group A. Additionally, buccal bone height and lingual bone width at 1 mm in Group C (87.06 ± 10.34%, 89.09 ± 10.56%) were significantly greater than in Group A (53.48 ± 23.94%, 82.72 ± 12.59%). CONCLUSION: The present findings indicate that application of bovine bone combined with barrier membrane with or without FGF-2 over tooth sockets can effectively reduce ridge absorption, especially in terms of ridge width and FGF-2 modified membrane seems to improve the outcomes obtained with membrane alone.

Alteration of salivary microbiome in periodontitis with or without type-2 diabetes mellitus and metformin treatment.

We aimed to explore the effects of type-2 diabetes mellitus (T2DM) and hypoglycemic therapy on the salivary microbiome in periodontitis patients and identify the potential salivary micro-biomarker for the early warning of T2DM. Saliva samples were collected from healthy individuals (Health), periodontitis patients (P), T2DM patients, periodontitis patients with T2DM (DAP), and DAP patients treated with Metformin (Met). Samples were determined by16S rRNA gene sequencing. 29 phyla, 322 genera, and 333 species of salivary microbiome were annotated. Compared to the Health group, the P and DAP group showed a significantly higher diversity of saliva microbiota, while the T2DM and Met group had no significant difference in microbial abundance but showed a trend of increasing diversity. Other than well-known periodontitis-inducing pathogens, the proportion of Prevotella copri, Alloprevotella rava, and Ralstonia pickettii, etc. were also significantly increased in periodontitis patients with or without T2DM. After effective glycemic control, the abundance of Prevotella copri, Alloprevotella rava, Ralstonia pickettii, etc. decreased in periodontitis patients with companion T2DM. The accuracies of the classification models in differentiating Health-vs.-P, DAP-vs.-P, and T2DM-vs.-P were 100%, 96.3%, and 98.1%, respectively. Hypoglycemic therapy could reconstruct the saliva microbiota and hence improve the localized conditions of diabetes patients with periodontitis.

Progranulin inhibits LPS-induced macrophage M1 polarization via NF-кB and MAPK pathways.

BACKGROUND: Macrophage M1 polarization plays a pivotal role in inflammatory diseases. Progranulin (PGRN) has potential anti-inflammation action, however, the effect of PGRN on macrophage M1 polarization has been poorly studied. Our study aimed to investigate the effect of PGRN on lipopolysaccharide (LPS)-induced macrophage M1 polarization and clarify the underlying mechanisms. METHODS: RAW264.7 cells were polarized to M1 macrophage by LPS with or without recombinant PGRN (rPGRN) and tumor necrosis factor alpha antibody (anti-TNF-α). A cell counting kit-8 assay (CCK-8), flow cytometry, Quantitative Real-Time PCR assay (q-PCR), Western blot assay and enzyme-linked immunosorbent assay (ELISA) were used to determine the effect of different treatments on cell proliferation, expression of surface phenotype marker and expressions and secretion of inflammatory cytokines. The activation of NF-κB/mitogen-activated protein kinase (MAPK) pathways and the nuclear translocation of NF-κB p65 were detected by Western blot and immunofluorescence respectively. THP-1 and primary bone marrow-derived monocytes (BMDMs) were also used to demonstrate effect of PGRN on expressions and secretion of inflammatory cytokines induced by LPS. RESULTS: In RAW264.7 cells, rPGRN at concentrations below 80 ng/ml significantly promoted cell proliferation in dose dependent fashion. rPGRN significantly inhibited LPS-induced change of phenotype (CD86/CD206 ratio) and function (tumor necrosis factor (TNF-α) and inducible nitric oxide synthase (iNOS) expressions). LPS-stimulated secretion of TNF-α and activated phosphorylation of IKKα/ß, IкBα, p65, JNK and p38 and the nucleus translocation of NF-кB p65 were also significantly downregulated by rPGRN. In addition, recombinant TNF-α (rTNF-α) significantly boosted TNF-α and iNOS expression vs the control group. Moreover, anti-TNF-α significantly inhibited LPS-induced TNF-α and iNOS expression. In THP-1 and BMDM cells, reversing effect of rPGRN on LPS-enhanced expressions of TNF-α and iNOS and secretion of TNF-α was further demonstrated. CONCLUSIONS: PGRN down-regulates LPS-induced macrophage M1 polarization in phenotype and function via NF-κB/MAPK signaling pathways.

EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation.

BACKGROUND: Low concentrations of tumor necrosis factor-alpha (TNF-α) and its receptor TNFR2 are both reported to promote osteogenic differentiation of osteoblast precursor cells. Moreover, low concentrations of TNF-α up-regulate the expression of EphB4. However, the molecular mechanisms underlying TNF-α-induced osteogenic differentiation and the roles of TNFR2 and EphB4 have not been fully elucidated. RESULTS: The ALP activity, as well as the mRNA and protein levels of RUNX2, BSP, EphB4 and TNFR2, was significantly elevated in MC3T3-E1 murine osteoblast precursor cells when stimulated with 0.5 ng/ml TNF-α. After TNFR2 was inhibited by gene knockdown with lentivirus-mediated shRNA interference or by a neutralizing antibody against TNFR2, the pro-osteogenic effect of TNF-α was partly reversed, while the up-regulation of EphB4 by TNF-α remained unchanged. With EphB4 forward signaling suppressed by a potent inhibitor of EphB4 auto-phosphorylation, NVP-BHG712, TNF-α-enhanced expressions of TNFR2, BSP and Runx2 were significantly decreased. Further investigation into the signaling pathways revealed that TNF-α significantly increased levels of p-JNK, p-ERK and p-p38. However, only the p-ERK level was significantly inhibited in TNFR2-knockdown cells. In addition, the ERK pathway inhibitor, U0126 (10 µM), significantly reversed the positive effect of TNF-α on the protein levels of RUNX2 and BSP. CONCLUSIONS: The EphB4, TNFR2 and ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation.

Conditioned medium derived from FGF-2-modified GMSCs enhances migration and angiogenesis of human umbilical vein endothelial cells.

BACKGROUND: Angiogenesis plays an important role in tissue repair and regeneration, and conditioned medium (CM) derived from mesenchymal stem cells (MSC-CM) possesses pro-angiogenesis. Nevertheless, the profile and concentration of growth factors in MSC-CM remain to be optimized. Fibroblast growth factor-2 (FGF-2) has been proven to be an effective angiogenic factor. Thus, the aim of this study was to verify whether FGF-2 gene overexpression optimized CM from human gingival mesenchymal stem cells (hGMSCs) and whether such optimized CM possessed more favorable pro-angiogenesis effect. METHODS: First, FGF-2 gene-modified hGMSCs were constructed using lentiviral transfection technology (LV-FGF-2+-hGMSCs) and the concentration of angiogenesis-related factors in LV-FGF-2+-hGMSC-CM was determined by ELISA. Then, human umbilical vein endothelial cells (HUVECs) were co-cultured for 3 days with LV-FGF-2+-hGMSC-CM, and the expression level of placenta growth factor (PLGF), stem cell factor (SCF), vascular endothelial growth factor receptor 2 (VEGFR2) in HUVECs were determined by qRT-PCR, western blot, and cellular immunofluorescence techniques. The migration assay using transwell and in vitro tube formation experiments on matrigel matrix was conducted to determine the chemotaxis and angiogenesis enhanced by LV-FGF-2+-hGMSC-CM. Finally, NOD-SCID mice were injected with matrigel mixed LV-FGF-2+-hGMSC-CM, and the plug sections were analyzed by immunohistochemistry staining with anti-human CD31 antibody. RESULTS: LV-FGF-2+-hGMSC-CM contained significantly more FGF-2, vascular endothelial growth factor A (VEGF-A), and transforming growth factor ß (TGF-ß) than hGMSC-CM. HUVECs pretreated with LV-FGF-2+-hGMSC-CM expressed significantly more PLGF, SCF, and VEGFR2 at gene and protein level than hGMSC-CM pretreated HUVECs. Compared with hGMSC-CM, LV-FGF-2+-hGMSC-CM presented significantly stronger chemotaxis to HUVECs and significantly strengthened HUVECs mediated in vitro tube formation ability. In vivo, LV-FGF-2+-hGMSC-CM also possessed stronger promoting angiogenesis ability than hGMSC-CM. CONCLUSIONS: Overexpression of FGF-2 gene promotes hGMSCs paracrine of angiogenesis-related growth factors, thereby obtaining an optimized conditioned medium for angiogenesis promotion.

Curcumin suppresses the proliferation and tumorigenicity of Cal27 by modulating cancer-associated fibroblasts of TSCC.

Cancer-associated fibroblasts (CAFs) are "activated" fibroblasts in the tumor microenvironment (TME) and play a vital role in all steps of cancer development. Increasing evidence focusing on the function of CAFs suggests that CAFs are candidate therapeutic targets and that drugs targeting the modification of CAFs would suppress tumor progression and be beneficial to tumor treatment and prevention. In the present study, we found that curcumin reversed the phenotype of CAFs to that of peri-tumor fibroblast (PTF)-like cells by downregulating the expression of α-SMA (a special marker for CAFs) and inhibiting the secretion of pro-carcinogenic cytokines, including transforming growth factor-ß1 (TGF-ß1), matrix metalloproteinases 2 (MMP2), and stromal cell-derived factor-1 (SDF-1). We further demonstrated that the conditioned medium (CM) derived from CAFs promoted the proliferation of Cal27, and this effect was confirmed by the xenograft model. More importantly, we found that curcumin blocked the CAF-mediated enhancement of Cal27 proliferation in vitro and in vivo. In conclusion, our data suggest that curcumin reverses cell phenotype from CAF to PTF-like cells and suppresses the CAF-mediated proliferation and tumorigenicity of Cal27 by inhibiting TSCC CAFs.

Enhancement of periodontal tissue regeneration by conditioned media from gingiva-derived or periodontal ligament-derived mesenchymal stem cells: a comparative study in rats.

BACKGROUND: Evidence has demonstrated conditioned medium (CM) from periodontal ligament stem cells (PDLSCs) improved periodontal regeneration. Gingival mesenchymal stem cells (GMSCs) have been considered an alternative strategy for regenerative medicine. To determine whether GMSC-CM could promote periodontal wound healing, we compared the effects of GMSC-CM and PDLSC-CM on periodontal regeneration and the underlying mechanisms in rat periodontal defects. METHODS: Cell-free CMs were collected from PDLSCs, GMSCs, and gingival fibroblasts (GFs) using ultracentrifugation (100-fold concentration). Periodontal defects were created on the buccal side of the first molar in the left mandible of 90 rats by a surgical method. Collagen membranes loaded with concentrated CMs (α-MEM, GF-CM, GMSC-CM, PDLSC-CM) were transplanted into periodontal defects. After 1, 2, and 4 weeks, the animals were sacrificed and specimens including the first molar and the surrounding tissues were separated and decalcified. Hematoxylin-eosin and Masson's trichrome staining were performed to evaluate periodontal regeneration. Immunohistochemical staining for tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-10 was conducted to analyze inflammation. Immunohistochemistry of BSP-II and Runx2 was performed to analyze osteoblast differentiation. RESULTS: Histological analysis showed the amount of newly formed periodontal tissue was significantly higher in both the GMSC-CM and PDLSC-CM groups than in the other groups, with no significant difference between these two groups. At 1 and 2 weeks, the expression levels of TNF-α and IL-1ß were significantly lower in the GMSC-CM and PDLSC-CM groups than in the other three groups, while there was no significant difference between these two groups. IL-10 expression was significantly higher in the GMSC-CM group than in the PDLSC-CM group and the other three groups. At 1, 2, and 4 weeks, BSP-II and Runx2 expressions were significantly higher in the GMSC-CM and PDLSC-CM groups than in the other three groups, with no significant difference between the two groups. CONCLUSIONS: Our results demonstrate that GMSC-CM transplantation can significantly promote periodontal regeneration in rats and achieve the same effect as PDLSC-CM. The mechanism of periodontal regeneration may involve the regulation of inflammatory factors and the promotion of osteogenic differentiation of bone progenitor cells in the wound region by CMs from MSCs.

Proanthocyanidins Promote Osteogenic Differentiation of Human Periodontal Ligament Fibroblasts in Inflammatory Environment Via Suppressing NF-κB Signal Pathway.

Proanthocyanidins (PA) have been proven to suppress inflammation and promote osteogenic differentiation. However, whether PA could promote osteogenic differentiation of human periodontal ligament fibroblasts (HPDLFs) in inflammatory environment is unclear. Here, HPDLFs were stimulated by tumor necrosis factor-α (TNF-α), PA, or their combination, and osteogenic differentiation- and mineralization-associated markers were detected by quantitative real-time polymerase chain reaction (qRT-PCR), alizarin red S staining, and alkaline phosphatase (ALP) activity assay. The result showed that PA significantly upregulated expression of osteogenesis-related genes and proteins and ALP activity in HPDLFs compared with the control in non-inflammatory environment. Moreover, PA significantly reversed inhibition of osteogenesis-related gene and protein expression, ALP activity, and mineralization caused by TNF-α. The underlying mechanism was that PA could regulate osteogenesis of HPDLFs via suppressing nuclear factor-kappa beta (NF-κB) signal pathway. These findings suggest that PA may contribute to bone generation in inflammatory microenvironment via suppressing NF-κB signal pathway. Thus, PA may be a potential treatment agent for periodontal bone regeneration.

Progranulin promotes osteogenic differentiation of human periodontal ligament stem cells via tumor necrosis factor receptors to inhibit TNF-α sensitized NF-kB and activate ERK/JNK signaling.

OBJECTIVE: To investigate the molecular mechanism of Progranulin (PGRN) in promoting osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in inflammatory environment. BACKGROUND: Progranulin is an antagonist of tumor necrosis factor (TNF) receptors (TNFRs) and is known to promote inflammatory periodontal bone defect regeneration. METHODS: TNFR1- and TNFR2-silenced hPDLSCs designed as hPDLSCs-sh-TNFR1 and hPDLSCs-sh-TNFR2 were cultured with osteoinductive medium containing TNF-α and (or) PGRN. Immunofluorescence, quantitative real-time PCR, and western blot were used to, respectively, detect expressions of TNFR1\TNFR2 and osteogenic differentiation markers as well as phosphorylation level in NF-κB\MAPK-related pathways. RESULTS: Immunofluorescence and real-time PCR showed that TNFR1 and TNFR2 positively expressed in hPDLSCs. TNF-α stimulation could significantly decrease the expressions of ALP and RUNX2 in hPDLSCs, whereas PGRN treatment could significantly enhance their expressions, and reverse TNF-α-mediated expression suppression of ALP and RUNX2 in hPDLSCs. In hPDLSCs-sh-TNFR1, TNF-α mediated osteogenic inhibition decreased, but both TNF-α + PGRN and alone PGRN significantly promoted expression of ALP and RUNX2. PGRN significantly enhanced expression of P-ERK1/2 and P-JNK, while corresponding inhibitors eliminated PGRN-stimulated osteogenic differentiation. In hPDLSCs-sh-TNFR2, no significant difference existed in osteogenic markers and P-JNK expression between the PGRN group and the control group. However, PGRN still activated P-ERK1/2 expression. Besides, PGRN antagonized TNF-α-enhanced NF-κB P65 expression. CONCLUSION: Progranulin promotes osteogenic differentiation of hPDLSCs via TNFR1 to inhibit TNF-α-sensitized NF-κB and via TNFR2 to activate JNK signaling. The mechanism by which PGRN activates ERK signaling remains to be explored.

Salivary chromogranin A and myeloid-related protein-8/14 from periodontitis patients with type 2 diabetes.

OBJECTIVES: The bidirectional relationship between diabetes mellitus and chronic periodontitis is well known from clinical trials. Periodontitis in diabetic patients is characterized by severe inflammation and tissue destruction. The purpose of this study was to investigate the levels of chromogranin A (CgA), a stress marker, and myeloid-related protein (MRP)-8/14, an inflammatory marker, in saliva from patients with periodontitis and diabetes mellitus, and to investigate the relationship between CgA and MRP-8/14 in all individuals and in the three groups separately. METHOD AND MATERIALS: Stimulated saliva was collected from 20 diabetic patients with chronic periodontitis, 16 patients with chronic periodontitis, and 21 healthy individuals. Salivary CgA and MRP-8/14 were determined with enzyme-linked immunosorbent assay. Salivary CgA and MRP-8/14 levels were assessed in the saliva of diabetic periodontitis and periodontitis patients, and the relationship with periodontal disease severity was investigated. RESULTS: CgA values in saliva samples from chronic periodontitis patients and diabetic patients with chronic periodontitis were significantly higher than those of the control group. MRP-8/14 values in saliva from chronic periodontitis patients and diabetic patients with chronic periodontitis was significantly higher than that in the control group. Salivary CgA level was positively correlated to MRP-8/14 in all individuals, but there was no significant correlation within the chronic periodontitis patient group, diabetic patients with chronic periodontitis group, and the healthy patient group. No significant correlation between salivary CgA/MRP-8/14 and clinical parameters of periodontitis was found in the three groups. CONCLUSIONS: The results suggest that salivary CgA and MRP-8/14 could be related to the pathogenesis of periodontitis and diabetes. CgA concentration in saliva was positively associated with increased MRP-8/14 in all individuals.