Epigallocatechin Gallate Protects against Hypoxia-Induced Inflammation in Microglia via NF-κB Suppression and Nrf-2/HO-1 Activation.

Hypoxia-induced neuroinflammation in stroke, neonatal hypoxic encephalopathy, and other diseases subsequently contributes to neurological damage and neuronal diseases. Microglia are the primary neuroimmune cells that play a crucial role in cerebral inflammation. Epigallocatechin gallate (EGCG) has a protective antioxidant and anti-inflammatory effects against neuroinflammation. However, the effects of EGCG on hypoxia-induced inflammation in microglia and the underlying mechanism remain unclear. In this study, we investigated whether EGCG might have a protective effect against hypoxia injury in microglia by treatment with CoCl2 to establish a hypoxic model of BV2 microglia cells following EGCG pre-treatment. An exposure of cells to CoCl2 caused an increase in inflammatory mediator interleukin (IL)-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 expression, which were significantly ameliorated by EGCG via inhibition of NF-κB pathway. In addition, EGCG attenuated the expression of hypoxia-inducible factor (HIF)-1α and the generation of ROS in hypoxic BV2 cells. Furthermore, the suppression of hypoxia-induced IL-6 production by EGCG was mediated via the inhibition of HIF-1α expression and the suppression of ROS generation in BV2 cells. Notably, EGCG increased the Nrf-2 levels and HO-1 levels in the presence of CoCl2. Additionally, EGCG suppressed hypoxia-induced apoptosis of BV2 microglia with cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-3. In summary, EGCG protects microglia from hypoxia-induced inflammation and oxidative stress via abrogating the NF-κB pathway as well as activating the Nrf-2/HO-1 pathway.

Toll-Like Receptor 5 Promotes the Neurogenesis From Embryonic Stem Cells and Adult Hippocampal Neural Stem Cells in Mice.

Toll-like receptors (TLRs) make a crucial contribution to the innate immune response. TLR5 was expressed in embryoid body derived from mouse embryonic stem cells (mESCs) and ßIII-tubulin-positive cells under all-trans retinoic acid-treated condition. TLR5 was upregulated during neural differentiation from mESCs and augmented the neural differentiation of mESCs via nuclear factor-κB and interleukin 6/CREB pathways. Besides, TLR5 was expressed in SOX2- or doublecortin-positive cells in the subgranular zone of the hippocampal dentate gyrus where adult neurogenesis occurs. TLR5 inhibited the proliferation of adult hippocampal neural stem cells (NSCs) by regulating the cell cycle and facilitated the neural differentiation from the adult hippocampal NSCs via JNK pathway. Also, TLR5 deficiency impaired fear memory performance in mice. Our data suggest that TLR5 is a crucial modulator of neurogenesis from mESCs and adult hippocampal NSCs in mice and represents a new therapeutic target in neurological disorders related to cognitive function.

Inactivation of PI3K/Akt promotes the odontoblastic differentiation and suppresses the stemness with autophagic flux in dental pulp cells.

BACKGROUND/PURPOSE: Autophagy is involved in controlling differentiation of various cell types. The present study aimed to investigate the mechanism related to autophagy in regulating odontogenic differentiation of dental pulp cells. MATERIALS AND METHODS: Human dental pulp cells (HDPCs) were cultured in differentiation inductive medium (DM) and odontoblastic differentiation and mineralization were evaluated by alkaline phosphatase (ALP) staining and Alizarin red S staining, respectively. Tooth cavity preparation was made on the mesial surface of lower first molars in rat. The expression of autophagy-related signal molecules was detected using Western blot analysis and Immunohistochemistry. RESULTS: HDPCs cultured in DM showed increased autophagic flux and declined phosphorylation of phosphoinositide 3-kinases (PI3K), protein kinase B (Akt), and mTOR. Dentin matrix protein-1 (DMP-1) and dentin sialoprotein (DSP), markers of odontoblastic differentiation, were upregulated and autophagic activation showing increased LC3-II and decreased p62 levels was observed during odontogenic differentiation of HDPCs. However, PI3K blocker 3-methyladenine (3MA), lentiviral shLC3 and Akt activator SC79 attenuated the expression of LC3II as well as DMP-1, ALP activity and mineralization enhanced in HDPCs under DM condition. In addition, 3MA, shLC3 and SC79 recovered the expression of pluripotency factor CD146, Oct4 and Nanog downregulated in DM condition. In rat tooth cavity preparation model, the expression of LC3B and DMP-1 was elevated near odontoblast-dentin layer during reparative dentin formation, whereas 3MA significantly reduced the expression of LC3B and DMP-1. CONCLUSION: These findings indicated autophagy promotes the odontogenic differentiation of dental pulp cells modulating stemness via PI3K/Akt inactivation and the repair of pulp.

Autophagy upregulates inflammatory cytokines in gingival tissue of patients with periodontitis and lipopolysaccharide-stimulated human gingival fibroblasts.

BACKGROUND: Periodontitis is an inflammatory disease caused by multiple disease-associated bacterial species in periodontal tissues. Autophagy is known to modulate various inflammation-driven diseases and inflammatory responses, but the role of autophagy related to the pathogenesis of periodontitis is not fully established. We investigated whether autophagic flux regulated the expression of inflammatory cytokines in the gingiva of periodontitis patients and lipopolysaccharide (LPS)-stimulated human gingival fibroblasts (HGFs) and the underlying mechanism. METHODS: The mRNA and protein expression of proinflammatory cytokines was assessed in human gingival tissues collected from patients with periodontitis and HGFs treated with LPS. The expression of signaling molecules related to autophagy was evaluated by immunofluorescence and Western blot analyses. RESULTS: The expression of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and intercellular adhesion molecule-1 (ICAM-1) was increased in the gingival tissues of patients with periodontitis. LC3B-positive cells, a typical autophagic marker, were increased in the gingival tissues of periodontitis patients and LPS-treated HGFs. The conversion ratio of LC3-I to LC3-II was higher in the gingival tissues associated with periodontitis and LPS-treated HGFs compared to the controls. The autophagy inhibitor 3-methyladenine (3MA) significantly abrogated the LPS-sustained inflammatory effect by reducing the expression of IL-6, TNF-α, COX-2, and ICAM-1 in HGFs. The phosphorylation of protein kinase B (AKT) and protein S6K1 (S6), signals involved in the mTOR-dependent mechanism, was decreased in gingiva derived from periodontitis patients and LPS-treated HGFs. CONCLUSIONS: Autophagy augmented the production of inflammatory cytokines by mTOR inactivation via the AKT signaling pathway in the gingival tissues of patients with periodontitis and LPS-stimulated HGFs. These findings would provide a better understanding of the mechanism by which autophagy regulates the inflammatory response associated with periodontal pathogenesis.

Effect of rhBMP-2 applied with a 3D-printed titanium implant on new bone formation in rabbit calvarium.

OBJECTIVE: This study sought to compare the biocompatibility of a three-dimensional (3D)-printed titanium implant with a conventional machined titanium product, as well as the effect of such implant applied with recombinant human Bone Morphogenetic Protein Type 2 (rhBMP-2) for guided bone regeneration. METHODOLOGY: Disk-shaped titanium specimens fabricated either by the conventional machining technique or by the 3D-printing technique were compared by MC3T3-E1 cells cytotoxicity assay. New bone formation was evaluated using a rapid prototype titanium cap applied to the calvaria of 10 rabbits, which were divided into two groups: one including an atelopeptide collagen plug on one side of the cap (group I) and the other including a plug with rhBMP-2 on the other side (group II). At six and 12 weeks after euthanasia, rabbits calvaria underwent morphometric analysis through radiological and histological examination. RESULTS: Through the cytotoxicity assay, we identified a significantly higher number of MC3T3-E1 cells in the 3D-printed specimen when compared to the machined specimen after 48 hours of culture. Moreover, morphometric analysis indicated significantly greater bone formation at week 12 on the side where rhBMP-2 was applied when evaluating the upper portion immediately below the cap. CONCLUSION: The results suggest that 3D-printed titanium implant applied with rhBMP-2 enables new bone formation.

Combined Extract of Leonurus japonicus Houtt, Eclipta prostrata L., and Pueraria lobata Ohwi Improved Hot Flashes and Depression in an Ovariectomized Rat Model of Menopause.

Menopause leads to ovarian hormone loss, which causes symptoms such as weight gain, hot flashes, and depression. Exploring nutraceuticals is important for treating menopausal symptoms that extensively impact women's quality of life. We hypothesized that a combination of Leonurus japonicus Houtt, Eclipta prostrata L., and Pueraria lobata Ohwi (LEPE) would alleviate menopausal symptoms in an ovariectomized menopausal rat model. Bilateral ovariectomy was performed and animals were assigned to five groups: (1) Sham, (2) Vehicle, (-) Control, (3) LEPE (100 mg/kg bw), (4) LEPE (200 mg/kg bw), and (5) Estradiol (3 µg/kg bw). LEPE was orally administered daily for 12 weeks. LEPE supplementation did not affect growth performance (body weight and feed intake) or body composition (lean mass and fat in tissue). LEPE did not cause deviations in aspartate aminotransferase, alanine aminotransferase, estradiol, and follicle-stimulating hormone levels, indicating no hepatotoxicity or endocrine disturbance. LEPE decreased type I collagen (CTX-1) but did not affect bone mineral density or osteocalcin. LEPE decreased tail temperature and increased rectal temperature, improving menopause-related vasomotor symptoms. Furthermore, LEPE ameliorated depression-related behavior, including in forced swimming and tail suspension tests. Thus, LEPE may improve menopausal symptoms by enhancing vasomotor symptoms and depression in an ovariectomized rat menopause model.

SLPI in periodontal Ligament is not sleepy during biophysical force-induced tooth movement.

AIM: We aimed to identify a key molecule that maintains periodontal tissue homeostasis during biophysical force-induced tooth movement (BTM) by orchestrating alveolar bone (AB) remodelling. MATERIALS AND METHODS: Differential display-PCR was performed to identify key molecules for BTM in rats. To investigate the localization and expression of the identified molecules, immunofluorescence, real-time RT-PCR and Western blotting were performed in rats and human periodontal ligament (PDL) cells. Functional test and micro-CT analysis were performed to examine the in vivo effects of the identified molecules on BTM. RESULTS: Secretory leucocyte peptidase inhibitor (SLPI) in the PDL was revealed as a key molecule for BTM-induced AB remodelling. SLPI was enhanced in the PDL under both compression and tension, and downregulated by an adenyl cyclases inhibitor. SLPI induced osteoblastogenic genes including runt-related transcription factor 2 (Runx2) and synergistically augmented tension-induced Runx2 expression. SLPI augmented mineralization in PDL cells. SLPI induced osteoclastogenic genes including receptor activator of nuclear factor kappa-Β ligand (RANKL) and synergistically augmented the compression-induced RANKL and macrophage colony-stimulating factor (MCSF) expression. Finally, the in vivo SLPI application into the AB significantly augmented BTM. CONCLUSIONS: SLPI or its inhibitors might serve as a biological target molecule for therapeutic interventions to modulate BTM.

Mineral trioxide aggregate-induced AMPK activation stimulates odontoblastic differentiation of human dental pulp cells.

AIM: To investigate the role of autophagy in MTA-induced odontoblastic differentiation of human dental pulp cells (HDPCs). METHODOLOGY: In MTA-treated HDPCs, odontoblastic differentiation was assessed based on expression levels of dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP1), alkaline phosphatase activity (ALP) activity by ALP staining and the formation of mineralized nodule by Alizarin red S staining. Expression of microtubule-associated protein 1A/1B-light chain3 (LC3), adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signalling molecules and autophagy-related genes was analysed by Western blot analysis and Acridine orange staining was used to detect autophagic lysosome. For in vivo experiments, tooth cavity preparation models on rat molars were established and the expression of proteins-related odontogenesis and autophagy markers was observed by Immunohistochemistry and Western blot analysis. Kruskal-Wallis with Dunn's multiple comparison was used for statistical analysis. RESULTS: Mineral trioxide aggregate (MTA) promoted odontoblastic differentiation of HDPCs, accompanied by autophagy induction, including formation of autophagic lysosome and cleavage of LC3 to LC3II (P < 0.05). Conversely, inhibition of autophagy through 3MA significantly attenuated the expression level of DSPP (P < 0.05) and DMP1 (P < 0.05) as well as formation of mineralized nodules (P < 0.05), indicating the functional significance of autophagy in MTA-induced odontoblastic differentiation. Also, MTA increased the activity of AMPK (P < 0.01), whereas inhibition of AMPK by compound C downregulated DSPP (P < 0.01) and DMP1 (P < 0.05), but increased the phosphorylation of mTOR (P < 0.05), p70S6 (P < 0.01) and Unc-51-like kinases 1 (ULK1) (ser757) (P < 0.01), explaining the involvement of AMPK pathway in MTA-induced odontoblast differentiation. In vivo study, MTA treatment after tooth cavity preparation on rat molars upregulated DMP-1 and DSPP as well as autophagy-related proteins LC3II and p62, and enhanced the phosphorylation of AMPK. CONCLUSION: MTA induced odontoblastic differentiation and mineralization by modulating autophagy with AMPK activation in HDPCs. Autophagy regulation is a new insight on regenerative endodontic therapy using MTA treatment.

Autophagy-Related Protein MAP1LC3C Plays a Crucial Role in Odontogenic Differentiation of Human Dental Pulp Cells.

BACKGROUND: Autophagy plays important roles in odontogenic differentiation of dental pulp cells (DPCs) in the developmental stage of tooth bud. Few studies have reported the role of autophagy during reparative dentin formation process. The objective of this study was to discover gene expression pattern correlated to autophagy and their role during odontogenic differentiation process in DPCs. METHODS: After tooth cavities were prepared on the mesial surface of lower first molar crown of rats. Odontogenic differentiation and reparative dentin formation were assessed based on detection of morphology change with hematoxylin and eosin staining. RESULTS: After tooth cavities were prepared on the mesial surface of lower first molar crown of rats, odontogenic differentiation and reparative dentin formation were assessed based on detection of morphology change with hematoxylin and eosin staining and dentin sialophosphoprotein (DSPP), whereas autophagy inhibitor 3-methyladenine (3MA) reversed. Results of quantitative polymerized chain reaction array of autophagosome formation related genes revealed that GABARAPL2 was prominently upregulated while expression of other ATG8 family members were moderately increased after tooth cavity preparation. In addition, human DPCs incubated in differentiation medium predominantly upregulated MAP1LC3C, which selectively decreased by 3MA but not by autophagy enhancer trehalose. Knock-down of MAP1LC3C using shRNA resulted in strong downregulation of dentin matrix protein 1 and DSPP as well-known odontogenic marker compared to knock-down of MAP1LC3B during odontogenic differentiation process of human DPCs. CONCLUSION: Our results suggest that MAP1LC3C plays a crucial role in odontogenic differentiation of human DPCs via regulating autophagic flux.

Effect of rhBMP-2 applied with a 3D-printed titanium implant on new bone formation in rabbit calvarium

Abstract Objective This study sought to compare the biocompatibility of a three-dimensional (3D)-printed titanium implant with a conventional machined titanium product, as well as the effect of such implant applied with recombinant human Bone Morphogenetic Protein Type 2 (rhBMP-2) for guided bone regeneration. Methodology Disk-shaped titanium specimens fabricated either by the conventional machining technique or by the 3D-printing technique were compared by MC3T3-E1 cells cytotoxicity assay. New bone formation was evaluated using a rapid prototype titanium cap applied to the calvaria of 10 rabbits, which were divided into two groups: one including an atelopeptide collagen plug on one side of the cap (group I) and the other including a plug with rhBMP-2 on the other side (group II). At six and 12 weeks after euthanasia, rabbits calvaria underwent morphometric analysis through radiological and histological examination. Results Through the cytotoxicity assay, we identified a significantly higher number of MC3T3-E1 cells in the 3D-printed specimen when compared to the machined specimen after 48 hours of culture. Moreover, morphometric analysis indicated significantly greater bone formation at week 12 on the side where rhBMP-2 was applied when evaluating the upper portion immediately below the cap. Conclusion The results suggest that 3D-printed titanium implant applied with rhBMP-2 enables new bone formation.

APE1 Promotes Pancreatic Cancer Proliferation through GFRα1/Src/ERK Axis-Cascade Signaling in Response to GDNF.

Pancreatic cancer is the worst exocrine gastrointestinal cancer leading to the highest mortality. Recent studies reported that aberrant expression of apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is involved in uncontrolled cell growth. However, the molecular mechanism of APE1 biological role remains unrevealed in pancreatic cancer progression. Here, we demonstrate that APE1 accelerates pancreatic cancer cell proliferation through glial cell line-derived neurotrophic factor (GDNF)/glial factor receptor α1 (GFRα1)/Src/ERK axis-cascade signaling. The proliferation of endogenous APE1 expressed-MIA PaCa-2, a human pancreatic carcinoma cell line, was increased by treatment with GDNF, a ligand of GFRα1. Either of downregulated APE1 or GFRα1 expression using small interference RNA (siRNA) inhibited GDNF-induced cancer cell proliferation. The MEK-1 inhibitor PD98059 decreased GDNF-induced MIA PaCa-2 cell proliferation. Src inactivation by either its siRNA or Src inhibitor decreased ERK-phosphorylation in response to GDNF in MIA PaCa-2 cells. Overexpression of GFRα1 in APE1-deficient MIA PaCa-2 cells activated the phosphorylation of Src and ERK. The expression of both APE1 and GFRα1 was gradually increased as progressing pancreatic cancer grades. Our results highlight a critical role for APE1 in GDNF-induced pancreatic cancer cell proliferation through APE1/GFRα1/Src/ERK axis-cascade signaling and provide evidence for future potential therapeutic drug targets for the treatment of pancreatic cancer.

Combination of mineral trioxide aggregate and propolis promotes odontoblastic differentiation of human dental pulp stem cells through ERK signaling pathway.

The aim of this study is to investigate combined effects of mineral trioxide aggregate (MTA) and propolis on odontoblastic differentiation of human dental pulp stem cells (DPSCs) and to find a signaling pathway involved. Combination of MTA and propolis significantly up-regulated the expression of DSPP and DMP1, and facilitated a mineral nodule formation (p < 0.05). Treatments with MTA, propolis or combined increased the phosphorylation of extracellular signal-regulated kinases (ERK), one of mitogen-activated protein kinases signaling cascades during odontogenic differentiation of DPSCs (p < 0.05), and U0126, an inhibitor of ERK, decreased calcium deposits (p < 0.05). Combination of MTA and propolis promotes odontogenic differentiation and mineralization of DPSCs through ERK pathway.

Transcriptome analysis to identify long non coding RNA (lncRNA) and characterize their functional role in back fat tissue of pig.

Long non coding RNAs (lncRNA) have been previously found to be involved in important cellular activities like epigenetics, implantation, cell growth etc. in pigs. However, comprehensive analysis of lncRNA in back fat tissues at different developmental stages in pigs is still lacking. In this study we conducted transcriptome analysis in the back fat tissue of a F1 crossbred Korean Native Pig (KNP) × Yorkshire Pig to identify lncRNA. We investigated their role in 16 pigs at two different growth stages; stage 1 (10 weeks, n = 8) and stage 2 (26 weeks, n = 8). After quality assessment of sequencing reads, we got a total of 1,641,165 assembled transcripts out of eight paired end read from each stage. Among them, 6808 lncRNA transcripts were identified by filtering on the basis of multiple parameters like read length ≥ 200 nucleotides, exon numbers ≥2, FPKM ≥0.5, coding potential score < 0 etc. PFAM and RFAM were used to filter out all possible protein coding genes and housekeeping RNAs respectively. A total of 103 lncRNAs and 1057 mRNAs were found to be differentially expressed (DE) between the two stages (|log2FC| > 2, q < 0.05). We also identified 306 genes located around 100 kb upstream and 234 genes downstream around these DE lncRNA transcripts. The expression of top eleven DE lncRNAs (COL4A6, LY7S, MYH2, OXCT1, SMPDL3A, TMEM182, TTC36, RFOOOO4, RFOOO15, RFOOO45, CADM2) had been validating by qRT-PCR. Pathway and GO terms analysis showed that, positive regulation of biosynthetic process, Wnt signaling pathway, cellular protein modification process, and positive regulation of nitrogen compound were differentially enriched. Our results suggested that, KEGG pathways such as protein digestion and absorption, Arrhythmogenic right ventricular cardiomyopathy (ARVC) to be significantly enriched in both DE lncRNAs as well as DE mRNAs and involved in back fat tissues development. It also suggests that, identified lncRNAs are involved in regulation of important adipose tissues development pathways.

Water-Soluble Arginyl-Diosgenin Analog Attenuates Hippocampal Neurogenesis Impairment Through Blocking Microglial Activation Underlying NF-κB and JNK MAPK Signaling in Adult Mice Challenged by LPS.

Microglia-mediated neuroinflammatory responses are well known to inhibit neurogenesis in the dentate gyrus (DG) of the adult hippocampus, and growing evidence indicates that therapeutic intervention to suppress microglial activation could be an effective strategy for restoring the impaired neurogenesis and memory performance. In the present study, we investigated the effects of water-soluble arginyl-diosgenin analog (Arg-DG) on the adult hippocampal neurogenesis using a central LPS-induced inflammatory mice model, along with the fundamental mechanisms in vivo and in vitro using LPS-stimulated microglial BV2 cells. Arg-DG (0.6 mg/kg) attenuates LPS-impaired neurogenesis by ameliorating the proliferation and differentiation of neural stem cells (NSCs), and prolonging their survival. The impaired neurogenesis in the hippocampal DG triggered the cognitive function, and that treatment of Arg-DG led to the recovery of cognitive decline. Arg-DG also suppressed the production of LPS-induced pro-inflammatory cytokines in hippocampal DG by blocking microglial activation. In in vitro study, Arg-DG inhibited the production of nitric oxide (NO), nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) expression, and prostaglandin D2 production (PGD2), as well as the pro-inflammatory cytokines, such as interleukin (IL)-6, IL-1ß, and tumor necrosis factor alpha (TNF-α). The anti-inflammatory effect of Arg-DG was regulated by NF-κB and MAPK JNK signaling both in vivo, and in LPS-stimulated microglial BV2 cells. Taken together, these results suggest that Arg-DG might have the potential to treat various neurodegenerative disorders resulting from microglia-mediated neuroinflammation.

A synthetic diosgenin primary amine derivative attenuates LPS-stimulated inflammation via inhibition of NF-κB and JNK MAPK signaling in microglial BV2 cells.

Diosgenin, a precursor of steroid hormones in plants, is known to exhibit diverse pharmacological activities including anti-inflammatory properties. In this study, (3ß, 25R)­spirost­5­en­3­oxyl (2­((2((2­aminoethyl)amino)ethyl)amino)ethyl) carbamate (DGP), a new synthetic diosgenin derivative incorporating primary amine was used to investigate its anti-inflammatory effects and underlying mechanisms of action in lipopolysaccharide (LPS)-stimulated microglial BV2 cells. Pretreatment with DGP resulted in significant inhibition of nitric oxide (NO) synthesis, and down-regulation of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated microglial BV2 cells. In addition, DGP decreased the production of reactive oxygen species (ROS) and pro-inflammatory cytokines such as interleukin (IL)-6, IL-1ß, and tumor necrosis factor alpha (TNF-α). The inhibitory effects of DGP on these inflammatory mediators in LPS-stimulated microglial BV2 cells were regulated by NF-κB signaling through blocking p65 nuclear translocation and NF-κB p65/DNA binding activity. DGP also blocked the phosphorylation of c-Jun amino-terminal kinase (JNK), but not p38 kinase or extracellular signal-regulated kinases (ERK). The NF-κB inhibitor JSH-23 and JNK-specific inhibitor SP600125 significantly decreased NO production and IL-6 release in LPS-stimulated BV2 cells, respectively. The overall results demonstrate that DGP has anti-inflammatory effects on LPS-stimulated BV2 cells via inhibition of NF-κB and JNK activation, suggesting that DGP is a potential prophylactic agent in various neurodegenerative disorders.

Toll-like receptor 2 promotes neurogenesis from the dentate gyrus after photothrombotic cerebral ischemia in mice.

The subgranular zone (SGZ) of hippocampal dentate gyrus (HDG) is a primary site of adult neurogenesis. Toll-like receptors (TLRs), are involved in neural system development of Drosophila and innate immune response of mammals. TLR2 is expressed abundantly in neurogenic niches such as adult mammalian hippocampus. It regulates adult hippocampal neurogenesis. However, the role of TLR2 in adult neurogenesis is not well studied in global or focal cerebral ischemia. Therefore, this study aimed to investigate the role of TLR2 in adult neurogenesis after photochemically induced cerebral ischemia. At 7 days after photothrombotic ischemic injury, the number of bromodeoxyuridine (BrdU)-positive cells was increased in both TLR2 knock-out (KO) mice and wild-type (WT) mice. However, the increment rate of BrdU-positive cells was lower in TLR2 KO mice compared to that in WT mice. The number of doublecortin (DCX) and neuronal nuclei (NeuN)-positive cells in HDG was decreased after photothrombotic ischemia in TLR2 KO mice compared to that in WT mice. The survival rate of cells in HDG was decreased in TLR2 KO mice compared to that in WT mice. In contrast, the number of cleaved-caspase 3 (apoptotic marker) and the number of GFAP (glia marker)/BrdU double-positive cells in TLR2 KO mice were higher than that in WT mice. These results suggest that TLR2 can promote adult neurogenesis from neural stem cell of hippocampal dentate gyrus through increasing proliferation, differentiation, and survival from neural stem cells after ischemic injury of the brain.

The role of Hedgehog signaling in cementoblast differentiation.

OBJECTIVE: It has been well known that Hedgehog (Hh) signaling plays an important role in bone development, however, its function in cementogenesis has not yet been reported. This study was intended to elucidate the role of Hh signaling in cementoblast differentiation. DESIGN: Expression changes of various Hh signaling components and levels of skeletogenic markers (alkaline phosphatase, osteocalcin, osteopontin) and osteogenic transcription factors (RUNX2, Osterix) by Hh signaling modulators during OCCM-30 cementoblast differentiation were determined by quantitative real-time reverse transcriptase polymerase chain reaction. To investigate effects of Hh signaling modulators on the mineralization of cementoblast, alkaline phosphatase and alizarin red S staining were used. Then, the interaction between Hh and BMP signaling during cementoblast differentiation was evaluated using co-treatment of BMP7 and Hh signaling modulators. RESULTS: We observed the consistent expression of Hh signaling molecules in the OCCM-30, which were up-regulated during cementoblast differentiation. We also found that the treatment of cells with Purmo, an Hh activator, enhanced cementoblast differentiation by increasing the mRNA expression of skeletogenic markers and osteogenic transcription factors, as well as increasing alkaline phosphate activity and mineralization capability. On the contrary, an Hh antagonist, like Cyclo, effectively inhibited cementoblast differentiation. Furthermore, BMP7 promoted cementoblast differentiation through crosstalk with the Hh signaling. CONCLUSION: These results suggest that Hh signaling is involved in cementoblast differentiation, and Hh signaling molecules may therefore represent new therapeutic targets in periodontal treatment and regeneration.

Leptin Induces Odontogenic Differentiation and Angiogenesis in Human Dental Pulp Cells via Activation of the Mitogen-activated Protein Kinase Signaling Pathway.

INTRODUCTION: Up-regulation of odontogenic differentiation, dentin formation, and angiogenesis in dental pulp are key factors in vital pulp therapy. The aim of this study was to investigate whether leptin could promote odontogenic differentiation and angiogenesis in human dental pulp cells (hDPCs). In addition, the involvement of the intracellular signaling pathway in these effects was determined. METHODS: The viability of hDPCs treated with leptin was examined using the water soluble tetrazolium salt-1 assay. Real-time polymerase chain reaction was performed to determine messenger RNA (mRNA) expression levels of odontogenic and angiogenic markers. Western blot analysis was used to measure odontogenic and angiogenic protein expression levels and assess mitogen-activated protein kinase (MAPK) pathway involvement. Alkaline phosphatase (ALP) and alizarin red staining were used to evaluate expression levels of ALP and calcified nodule formation after treatment with leptin and/or the presence of MAPK inhibitors. RESULTS: All concentrations of leptin used in this study did not significantly affect the viability of hDPCs. However, mRNA and protein levels of odontogenic and angiogenic markers, ALP activity, and calcified nodule formation were significantly increased in the leptin-treated group compared with those in the control group. Leptin enhanced phosphorylation of extracellular signal-related kinases, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases within 5 minutes after treatment. However, leptin-induced dentin sialophosphoprotein and vascular endothelial growth factor protein expression and mineralization were appreciably blocked by the presence of MAPK inhibitors. CONCLUSIONS: Leptin can induce angiogenesis, odontogenic differentiation, and mineralization in hDPCs via activating the MAPK signaling pathway.

Autophagy induction plays a protective role against hypoxic stress in human dental pulp cells.

Human dental pulp exposed to hypoxic conditions induces cell death accompanied by autophagy. However, the role of hypoxia-induced autophagy in human dental pulp cells (HDPCs) is unclear. The present study aimed to investigate the role of autophagy in hypoxia-induced apoptosis of HDPCs. Cobalt chloride (CoCl2 ) treated HDPCs, to mimic hypoxic conditions, decreased cell viability. Also, apoptosis-related signal molecules, cleaved caspase-3 and PARP levels, were enhanced in CoCl2 -treated HDPCs. HDPCs exposed to CoCl2 also promoted autophagy, showing upregulated p62 and microtubule-associated protein 1 light chain 3 (LC3)-II levels, typical autophagic markers, and increased acidic autophagolysosomal vacuoles. Autophagy inhibition by 3 methyladenine (3MA) or RNA interference of LC3B resulted in increased levels of cleaved PARP and caspase-3, and the release of cytochrome c from mitochondria into cytosol in the CoCl2 -treated HDPCs. However, autophagy activation by rapamycin enhanced the p62 and LC3-II levels, whereas it reduced PARP and caspase-3 cleavage induced by CoCl2. These results revealed that CoCl2 -activated autophagy showed survival effects against CoCl2 -induced apoptosis in the HDPCs. CoCl2 upregulated HIF-1α and decreased the phosphorylation of mTOR/p70S6K. HIF-1α inhibitor, YC-1 decreased p62 and LC3-II levels, whereas it augmented PARP and caspase-3 cleavage in response to CoCl2 . Also, YC-1 enhanced the phosphorylation of mTOR and p70S6K suppressed by CoCl2 , demonstrating that CoCl2 -induced autophagy via mTOR/p70S6K is mediated by HIF-1α. Taken together, these finding suggest that CoCl2 -induced autophagy mediated by the mTOR/p70S6K pathway plays a protective role against hypoxic stress in HDPCs.

Effect of Platelet-rich Fibrin on Odontoblastic Differentiation in Human Dental Pulp Cells Exposed to Lipopolysaccharide.

INTRODUCTION: Platelet-rich fibrin (PRF), as an autologous fibrin matrix, is known to contain platelets, leukocytes, and growth factors to control inflammation and to facilitate the healing process. The purpose of this study was to investigate the effects of PRF on odontoblastic differentiation in human dental pulp cells (HDPCs) treated with lipopolysaccharide (LPS). METHODS: Gene expression of inflammatory cytokines and adhesion molecules on the HDPCs cultured with or without LPS and PRF extract (PRFe) were evaluated by reverse-transcription polymerase chain reaction and Western blot analysis. In addition, odontoblastic differentiation was determined by measuring alkaline phosphatase (ALP) activity using ALP staining, the expression of odontogenesis-related genes, and the extent of mineralization using alizarin red S staining. RESULTS: Treatment with PRFe significantly attenuated the LPS-stimulated expression of interleukin (IL)-1ß, IL-6, and IL-8 in HDPCs. In addition, PRFe inhibited the up-regulation of vascular cell adhesion molecule 1 and the production of intracellular adhesion molecule 1 in HDPCs exposed to LPS. Expression of dentin sialophosphoprotein and dentin matrix acidic phosphoprotein 1, ALP activity, and mineralization were enhanced by PRFe in LPS-treated HDPCs. CONCLUSIONS: These results suggest that PRF has effects associated not only with inhibition of inflammation in HDPCs exposed to LPS but also stimulation of odontoblastic differentiation.