Phloroglucinol Inhibits Oxidative-Stress-Induced Cytotoxicity in C2C12 Murine Myoblasts through Nrf-2-Mediated Activation of HO-1.

Phloroglucinol is a class of polyphenolic compounds containing aromatic phenyl rings and is known to have various pharmacological activities. Recently, we reported that this compound isolated from Ecklonia cava, a brown alga belonging to the family Laminariaceae, has potent antioxidant activity in human dermal keratinocytes. In this study, we evaluated whether phloroglucinol could protect against hydrogen peroxide (H2O2)-induced oxidative damage in murine-derived C2C12 myoblasts. Our results revealed that phloroglucinol suppressed H2O2-induced cytotoxicity and DNA damage while blocking the production of reactive oxygen species. We also found that phloroglucinol protected cells from the induction of apoptosis associated with mitochondrial impairment caused by H2O2 treatment. Furthermore, phloroglucinol enhanced the phosphorylation of nuclear factor-erythroid-2 related factor 2 (Nrf2) as well as the expression and activity of heme oxygenase-1 (HO-1). However, such anti-apoptotic and cytoprotective effects of phloroglucinol were greatly abolished by the HO-1 inhibitor, suggesting that phloroglucinol could increase the Nrf2-mediated activity of HO-1 to protect C2C12 myoblasts from oxidative stress. Taken together, our results indicate that phloroglucinol has a strong antioxidant activity as an Nrf2 activator and may have therapeutic benefits for oxidative-stress-mediated muscle disease.

Loganin Inhibits Lipopolysaccharide-Induced Inflammation and Oxidative Response through the Activation of the Nrf2/HO-1 Signaling Pathway in RAW264.7 Macrophages.

Inflammation caused by the excessive secretion of inflammatory mediators in abnormally activated macrophages promotes many diseases along with oxidative stress. Loganin, a major iridoid glycoside isolated from Cornus officinalis, has recently been reported to exhibit anti-inflammatory and antioxidant effects, whereas the underlying mechanism has not yet been fully clarified. Therefore, the aim of the present study is to investigate the effect of loganin on inflammation and oxidative stress in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Our results indicated that loganin treatment markedly attenuated the LPS-mediated phagocytic activity and release of nitric oxide (NO) and prostaglandin E2, which was associated with decreased the expression of inducible NO synthase and cyclooxygenase-2. In addition, loganin suppressed the expression and their extracellular secretion of LPS-induced pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-1ß. Furthermore, loganin abolished reactive oxygen species (ROS) generation, and promoted the activation of nuclear factor-E2-related factor 2 (Nrf2) and the expression of heme oxygenase-1 (HO-1) in LPS-stimulated macrophages. However, zinc protoporphyrin, a selective HO-1 inhibitor, reversed the loganin-mediated suppression of pro-inflammatory cytokines in LPS-treated macrophages. In conclusion, our findings suggest that the upregulation of the Nrf2/HO-1 signaling pathway is concerned at least in the protective effect of loganin against LPS-mediated inflammatory and oxidative stress, and that loganin can be a potential functional agent to prevent inflammatory and oxidative damage.

Effect of rosmarinic acid on differentiation and mineralization of MC3T3-E1 osteoblastic cells on titanium surface.

Titanium (Ti) is a widely used biomaterial for dental implants because of its outstanding biocompatibility for hard tissues. Osseointegration, the interaction between implanted biomaterials and living cells in bone, is essential for successful implantation. Rosmarinic acid (RA) is a plant-derived phytochemical with low toxicity and side effects and has various effects that can be applied as a therapeutic substance. The MC3T3-E1 osteoblastic cells on the Ti surface in medium with or without 14 µg/ml RA were used to test RA effects on osteoblast differentiation, cell viability and mineralization during differentiation. RA treatment increased osteoblast differentiation, cell viability and mineralization in MC3T3-E1 osteoblastic cells on Ti surface during differentiation, upregulating Runx-2 and OPG, but downregulating RANKL. This study suggest that RA should be applied as an effective functional and therapeutic substance to enhance osseointegration of osteoblast cells by increasing differentiation, mineralization, and bone formation through the RANKL/RANK/OPG pathway during the differentiation in MC3T3-E1 osteoblastic cells on the Ti surface.

Cytoprotective effects of fermented oyster extracts against oxidative stress-induced DNA damage and apoptosis through activation of the Nrf2/HO-1 signaling pathway in MC3T3-E1 osteoblasts.

Osteoblast damage by oxidative stress has been recognized as a cause of bone-related disease, including osteoporosis. Recently, we reported that fermented Pacific oyster (Crassostrea gigas) extracts (FO) inhibited osteoclastogenesis and osteoporosis, while promoting osteogenesis. However, since the beneficial potential of FO on osteoblasts is not well known, in the present study, we investigated the cytoprotective effect of FO against oxidative stress in MC3T3-E1 osteoblasts. Our results demonstrated that FO inhibited hydrogen peroxide (H2O2)-induced DNA damage and cytotoxicity through the rescue of mitochondrial function by blocking abnormal ROS accumulation. FO also prevented apoptosis by suppressing loss of mitochondrial membrane potential and cytosolic release of cytochrome c, decreasing the rate of Bax/Bcl-2 expression and reducing the activity of caspase-9 and caspase-3 in H2O2-stimulated MC3T3-E1 osteoblasts, suggesting that FO protected MC3T3-E1 osteoblasts from the induction of caspase dependent- and mitochondria-mediated apoptosis by oxidative stress. In addition, FO markedly promoted the activation of nuclear factor-erythroid-2-related factor 2 (Nrf2), which was associated with the enhanced expression of heme oxygenase-1 (HO-1). However, inhibiting the expression of HO-1 by artificially blocking the expression of Nrf2 using siRNA significantly eliminated the protective effect of FO, indicating that FO activates the Nrf2/HO-1 signaling pathway in MC3T3-E1 osteoblasts to protect against oxidative stress. Based on the present data, FO is thought to be useful as a potential therapeutic agent for the inhibition of oxidative stress in osteoblasts.

Cordycepin induces apoptosis in human bladder cancer T24 cells through ROS-dependent inhibition of the PI3K/Akt signaling pathway.

Cordycepin, a derivative of nucleoside adenosine, is one of the active ingredients extracted from the fungi of genus Cordyceps, which have been used for traditional herbal remedies. In this study, we examined the effect of cordycepin on the proliferation and apoptosis of human bladder cancer T24 cells and its mechanism of action. Cordycepin treatment significantly reduced the cell survival rate of T24 cells in a concentration-dependent manner, which was associated with the induction of apoptosis. Cordycepin activated caspase-8 and -9, which are involved in the initiation of extrinsic and intrinsic apoptosis pathways, respectively, and also increased caspase-3 activity, a typical effect caspase, subsequently leading to poly (ADP-ribose) polymerase cleavage. Additionally, cordycepin increased the Bax/Bcl-2 ratio and truncation of Bid, and destroyed the integrity of mitochondria, which contributed to the cytosolic release of cytochrome c. Moreover, cordycepin effectively inactivated the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, while LY294002, a PI3K/Akt inhibitor, increased the apoptosis-inducing effect of cordycepin. Cordycepin further enhanced the intracellular levels of reactive oxygen species (ROS), while the addition of N-acetyl cysteine (NAC), a ROS inhibitor, significantly diminished cordycepin-induced mitochondrial dysfunction and growth inhibition, and also blocked the inactivation of PI3K/Akt signaling pathway. Furthermore, the presence of NAC significantly attenuated the enhanced apoptotic cell death and reduction of cell viability by treatment with cordycepin and LY294002. Collectively, the data indicate that cordycepin induces apoptosis through the activation of extrinsic and intrinsic apoptosis pathways and the ROS-dependent inactivation of PI3K/Akt signaling in human bladder cancer T24 cells.

Thymosin ß4 is associated with bone sialoprotein expression via ERK and Smad3 signaling pathways in MDPC-23 odontoblastic cells.

Thymosin ß4 (Tß4) regulates the expression of molecules associated with dentinogenesis, including bone sialoprotein (BSP). BSP regulates the initiation of mineralization and the direction of dentin growth. However, the association between Tß4 signaling and BSP expression in odontoblasts remains unclear. Therefore, the aim of the present study was to investigate Tß4 mRNA expression in odontoblasts during dentinogenesis and the association between the Tß4 signaling pathway and BSP expression in MDPC­23 odontoblastic cells. Expression and localization of Tß4 mRNA was determined by in situ hybridization during mouse tooth development. The effect of Tß4 signaling on BSP expression was investigated by reverse transcription polymerase chain reaction, western blot analysis, immunofluorescence and a luciferase reporter assay in the presence or absence of specific inhibitors of mitogen activated protein kinase kinase (PD98059) and mothers against decapentaplegic homolog 3 (Smad3; SIS3) in MDPC­23 cells. The expression of Tß4 mRNA in the odontoblast layer was highest at postnatal day 5, known as the advanced bell stage, when odontoblasts actively secrete dentin matrix proteins. Tß4 increased BSP mRNA and protein levels in MDPC­23 cells, but this was inhibited by PD98059 or SIS3 treatment. Tß4 increased levels of phosphorylated (p) extracellular signal­regulated kinase (ERK)1/2, pSmad3, pß­catenin, and runt­related transcription factor 2 (Runx2) protein, but these effects were inhibited by PD98059 or SIS3. Tß4 induced the nuclear translocation of Runx2 and pSmad3, while nuclear translocation of ß­catenin was decreased. Tß4 significantly increased BSP promoter activity, which was decreased by PD98059 or SIS3 treatment. Tß4 induced BSP expression in MDPC­23 cells via ERK and Smad3 signaling pathways, suggesting its role as a signaling molecule in odontoblasts for regulating BSP secretion during dentinogenesis.

Thymosin ß4 Reduces H2O2 Induced Oxidative Stress in MC3T3-E1 Cells on Titanium Surface.

Thymosin ß4 (Tß4) is known to inhibit an inflammatory response and to increase the survival of osteoblasts on titanium (Ti) surfaces. Ti is the most widely used graft material in dentistry; however, an inflammatory response induced following implant placement results in the generation of reactive oxygen species (ROS). The oxidative stress from the production of ROS such as nitric oxide (NO) and hydrogen peroxide (H2O2) can damage surrounding cells, resulting in implant failure by decreasing cell viability. Thus, the aim of this study was to determine the biological effects of Tß4 on the oxidative stress induced to MC3T3-E1 preosteoblasts on the Ti surface. Based on an MTT assay and bromodeoxyuridine immunofluorescence staining, Tß4 was found to increase the proliferation of the H2O2-exposed MC3T3-E1 cells on Ti discs. Reverse transcription-polymerase chain reaction and western blot analyses showed that Tß4 decreased the mRNA and protein expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in H2O2-exposed MC3T3-E1 cells on the Ti discs. Tß4 inhibited the synthesis of intracellular ROS and the secretion of NO and prostaglandin E2 (PGE2) from H2O2-exposed MC3T3-E1 cells on the Ti discs. In conclusion, Tß4 inhibits H2O2-induced iNOS and COX-2 expression with a decrease in ROS, NO, and PGE2 synthesis, which leads to improved cell survival with low cytotoxicity under an oxidative stress condition in MC3T3-E1 cells on the Ti surface. This suggests that Tß4 may be a crucial molecule to reduce oxidative stress-induced cell damage or hypoxia, leading to promoted osseointegration on the Ti surface during implant placement.

Antioxidant and cytoprotective effects of morin against hydrogen peroxide-induced oxidative stress are associated with the induction of Nrf-2­mediated HO-1 expression in V79-4 Chinese hamster lung fibroblasts.

Natural phytochemicals of plant origin, including flavonoids, have been found to be potent antioxidants providing beneficial effects against oxidative stress-related diseases. The present study was carried out to investigate the antioxidant properties of morin, a flavonoid originally isolated from the flowering plants of the Moraceae family. Superoxide dismutase (SOD)­like activity and 2,2'­azino­bis­(3­ethylbenzothiazoline­6­sulfonic acid) (ABTS•+) radical scavenging activity were determined. We also investigated the cytoprotective effects of morin against hydrogen peroxide (H2O2)­induced DNA damage and apoptosis in V79­4 Chinese hamster lung fibroblasts. Our results demonstrated that morin had strong scavenging effects against ABTS•+ radicals with enhanced SOD activity, which varied in a dose-dependent manner. Morin was found to reduce H2O2­induced intracellular reactive oxygen species generation and nuclear DNA damage, and it recovered cell viability damaged by H2O2 via inhibition of mitochondrial dysfunction­mediated apoptosis. Notably, the treatment of V79­4 cells with morin markedly enhanced the expression of heme oxygenase­1 (HO­1) but not quinone oxidoreductase-1, which was associated with the increased expression and phosphorylation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and the downregulation of Kelch­like ECH­associated protein 1 expression. Based on our findings, we conclude that morin effectively ameliorated oxidative stress­induced DNA damage through intrinsic free radical scavenging activity and activation of the Nrf2/HO-1 pathway.

Effect of Thymosin beta4 on the Differentiation and Mineralization of MC3T3-E1 Cell on a Titanium Surface.

Osteoblasts are responsible for the synthesis of bone matrix through the secretion of collagenous and non-collagenous proteins with mineralization. Thymosin beta4 (Tbeta4) is an actin-sequestering peptide that is involved in the regulation of cell proliferation, differentiation and motility. A recent study reported that the inhibition of Tbeta4 mRNA synthesis strongly decreases the level of gene expression of bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP), osteocalcin (OCN), osteonectin (ON) and collagen type I (Col I) with mineralization during differentiation in odontoblasts. Titanium (Ti) is used commonly as an implant material for dental implants, which have strong mechanical potential and good biocompatibility with bone. This study examined whether Tbeta4 can be a potential molecule for promoting the differentiation and mineralization of MC3T3-E1 cells on a Ti surface. Tbeta4 increased the viability of MC3T3-E1 cells during differentiation on Ti discs compared to that of the control. The expression of Tbeta4 mRNA and protein in the Tbeta4-treated MC3T3-E1 cells was higher than the control during differentiation on the Ti discs. In addition, Tbeta4 increased the formation of mineralization nodules and the mRNA expression of alkaline phosphatase (ALP), DSPP, dentin matrix protein1 (DMP1), BSP and Col I compared to that of the control in MC3T3-E1 cells during differentiation on Ti discs. From the results, Tbeta4 increased the viability and promoted the differentiation and mineralization of MC3T3-E1 cells on Ti discs. This highlights the potential use of Tbeta4 for increasing osseointegration through osteoblast differentiation and mineralization on Ti discs.

Sanguinarine Induces Apoptosis of Human Oral Squamous Cell Carcinoma KB Cells via Inactivation of the PI3K/Akt Signaling Pathway.

Preclinical Research Sanguinarine, an alkaloid isolated from the root of Sanguinaria canadensis and other plants of the Papaveraceae family, selectively induces apoptotic cell death in a variety of human cancer cells, but its mechanism of action requires further elaboration. The present study investigated the pro-apoptotic effects of sanguinarine in human oral squamous cell carcinoma KB cells. Sanguinarine treatment increased DR5/TRAILR2 (death receptor 5/TRAIL receptor 2) expression and enhanced the activation of caspase-8 and cleavage of its substrate, Bid. Sanguinarine also induced the mitochondrial translocation of pro-apoptotic Bax, mitochondrial dysfunction, cytochrome c release to the cytosol, and activation of caspase-9 and -3. However, a pan-caspase inhibitor, z-VAD-fmk, reversed the growth inhibition and apoptosis induced by sanguinarine. Sanguinarine also suppressed the phosphorylation of phosphoinositide 3-kinase (PI3K) and Akt in KB cells, while co-treatment of cells with sanguinarine and a PI3K inhibitor revealed synergistic apoptotic effects. However, pharmacological inhibition of AMP-activated protein kinase and mitogen-activated protein kinases did not reduce or enhance sanguinarine-induced growth inhibition and apoptosis. Collectively, these findings indicate that the pro-apoptotic effects of sanguinarine in KB cells may be regulated by a caspase-dependent cascade via activation of both intrinsic and extrinsic signaling pathways and inactivation of PI3K/Akt signaling. Drug Dev Res 77 : 227-240, 2016. © 2016 Wiley Periodicals, Inc.

Secretory leukocyte protease inhibitor promotes differentiation and mineralization of MC3T3-E1 preosteoblasts on a titanium surface.

Mineralized bone matrix constituted with collagenous and non-collagenous proteins was synthesized by osteoblasts differentiated from mesenchymal stem cells. Secretory leukocyte protease inhibitor (SLPI), a serine protease inhibitor, promotes cell migration and proliferation, and suppresses the inflammatory response. Recent studies reported that SLPI regulates the formation of dentin and mineralization by odontoblasts and increases the adhesion and viability of preosteoblasts on a titanium (Ti) surface. Ti and its alloys are widely used implant materials in artificial joints and dental implants owing to their biocompatibility with bone. Therefore, this study aimed to examine whether SLPI can be an effective molecule in promoting differentiation and mineralization of osteoblasts on a Ti surface. In order to investigate the effects of SLPI on osteoblasts, an MTT assay, PCR, western blotting and Alizarin Red S staining were performed. The results demonstrated that SLPI increased the viability of osteoblasts during differentiation on Ti discs compared with that of the control. The expression levels of SLPI mRNA and protein were higher than that of the control after treatment of osteoblasts with SLPI on Ti discs during differentiation. SLPI increased the formation of mineralized nodules and mRNA expression of alkaline phosphatase, dentin sialophosphoprotein, dentin matrix protein 1, bone sialoprotein, and collagen I in osteoblasts on Ti discs compared with that of the control. In conclusion, SLPI increases the viability and promotes the differentiation and mineralization of osteoblasts on Ti surfaces, suggesting that SLPI is an effective molecule for achieving successful osseointegration between osteoblasts and a Ti surface.

660 nm Red LED Induces Secretory Leukocyte Protease Inhibitor (SLPI) in Lipopolysaccharide-Stimulated RAW264.7 Cell.

SLPI acts as a modulator of the innate immune responses of macrophages, neutrophils and odontoblasts, and LPS-inducible anti-inflammatory cytokine to suppress the production of pro-inflammatory products by macrophages. Many studies have revealed the effects of light emitting diodes (LEDs) on the tissue repair and inflammatory responses. Although the anti-inflammatory mechanisms of irradiation with LEDs in gingival fibroblasts are known, the effects of 660 nm red LEDs on the inflammation remain unclear. Moreover, there is no report regarding the molecular mechanism for the relationship between SLPI and biological effects of LEDs. The effects of 660 nm red LEDs on inflammation with SLPI were investigated by examining the effects of 660 nm LED on the SLPI expression of RAW264.7 cells after LPS stimulation. This paper reports that the 660 nm red LED induced SLPI expression or reduced the LPS response, and inhibited NF-κB activation directly, leading to the suppression of pro-inflammatory cytokines, such as TNF-α and IL-1ß, suggesting that it might be a useful wavelength LED for inflammation therapy.

The Effect of Thymosin ß4 for Osteoblast Adhesion on Titanium Surface.

Titanium (Ti) is the most widely used implant material in dentistry and orthopedics but the release of metal ions from Ti implants results in increased bone resorption by enhancing the production of inflammatory cytokines from the macrophages and facilitating osteoclast differentiation. Thymosin ß4 (Tß4) has several biological activities, such as promoting wound healing, angiogenesis, cell proliferation and migration in mammalian cells. This study examined the role of Tß4 in osteoblasts via focal adhesions (FAs) and ERK1/2 signaling related to cell adhesion and proliferation for cell survival on the Ti surface. As a result, cell adhesion and proliferation increased in the Tß4 treated cells (Tß4/MC3T3-E1) but was significantly lower in the Tß4 knock-down cells by Tß4-siRNA (si-Tß4/MC3T3-E1) than that of the untreated cells. The levels of FAK phosphorylation, paxillin expression, and paxillin localization were higher in the Tß4/IMC3T3-E1 cells than that of the untreated cells but lower in the si-Tß4/MC3T3-E1 cells. In addition, the levels of cell proliferation, Grb2 and Ras protein expression and phosphorylation of ERK1/2 were higher in the Tß4/MC3T3-E1 cells than in the untreated cells but lower in the si-Tß4/IMC3T3-E1 cells. These results suggest that Tß4 might be a good nanomolecule that promotes osteoblast survival by facilitating adhesion and proliferation on the Ti surface.

Function of Secretory Leukocyte Protease Inhibitor (SLPI) in Odontoblast During Mouse Tooth Development.

Secretory leuckocyte protease inhibitor (SLPI) is thought as a regulating protein on the synthesis and degradation of matrix proteins. But there was no report of expression and function of SLPI on the tooth development, especially on the odontoblasts. As observed by in-situ hybridization and immunohistochemical analysis, SLPI was expressed in odontoblasts and predentin on post-natal day 4 (PN4). On PN10, SLPI was observed under the dentin and apical region including odontoblasts processes. Further, on PN15, expression of SLPI was the same pattern compared to PN10. SLPI was expressed under layer of the odontoblasts and in odontoblasts on PN20. Matrix metalloproteinase-2 (MMP-2) and -9 levels in SLPI/MDPC-23 cells were higher than that of the MDPC-23 cells. The gene expression of SLPI, bone sialoprotein (BSP), osteocalcin (OCN), osteonectin (ON), and collagen type I (Col I) was higher in SLPI/MDPC-23 than that of MDPC-23 cells and the expression of dentin sialophosphoprotein (DSPP) was lower in SLPI/MDPC-23. Taken together, our results suggest that SLPI may be a MMP-2 and -9 regulating molecule in odontoblasts during dentin matrix formation and acts as a signaling molecule for dentin matrix related proteins during odontoblasts differentiation and mineralization.

Secretory Leukocyte Protease Inhibitor (SLPI) Increases Focal Adhesion in MC3T3 Osteoblast on Titanium Surface.

An appropriate interaction between implanted materials and the surrounding tissue is essential for successful implantation. Titanium (Ti) and some of its alloys have been used in dentistry and orthopedics as a substitutive material for hard tissue, such as teeth or natural bone. Nevertheless, metal ions released from titanium and alloy implants have adverse biological effects on biological tissues or cells. Secretory leukocyte protease inhibitor (SLPI) promotes cell migration, proliferation and wound healing. FAK and ERK1/2 signaling regulate cell adhesion and proliferation for cell survival. This study evaluated the potential of SLPI as a molecule to increase the cell adhesion on the Ti surface. Compared with the untreated cells, SLPI increased the adhesion of MC3T3-E1 cells to Ti discs, formation of actin stress fibers, paxillin expression and the phosphorylation of FAK. Moreover, SLPI enhanced the level of Grb2 and Ras expression and ERK1/2 phosphorylation in the MC3T3-E1 cells on Ti discs. These results suggest that SLPI can increase the interaction between the implanted Ti material and surrounding bone in orthodontic and dental surgery, making an effective nanomolecule for successful implantation.

Dynamin II involves in cell migration and actin formation of NIH3T3 cells.

It was previously reported that in Ras transformed NIH3T3 cells, dynamin II acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration. However, these results do not provide sufficient evidence of a relationship between dynamin II and the Ras signal transduction pathway leading to membrane ruffling and cell migration. The results showed that a dynamin II association with myosin II as a signaling molecule is involved in NIH3T3 cell migration through the Ras/PI3K signaling pathway, and is associated with the p85 subunit of PI3K. Confocal microscopy also revealed co-localization between dynamin II and paxillin after PDGF stimulation. In addition, immunofluorescence results showed that dynamin II was colocalized with the actin filament. After stimulating the NIH3T3 cells with PDGF and treating them with an actin inhibitor, such as Cytochalasin D, it was observed that dynamin II with the myosin II complex inhibited binding to the actin. Therefore, dynamin II is localized in focal adhesion when cell migration is triggered and binds to the actin filament component, suggesting that it is a good candidate nanomolecule to regulate the cell attachment and migration to the materials such as implants etc.

Expression of thymosin ß4 in odontoblasts during mouse tooth development.

Thymosin ß4 (Tß4) is expressed in developing tissue, where it stimulates cell differentiation and migration. Further, Tß4 is expressed during molar development in mice, but the expression and function of Tß4 in odontoblasts during mammalian tooth development have not yet been reported. Therefore, this study examined the expression and function of Tß4 in differentiating odontoblasts during tooth development. As observed by immunohistochemistry, Tß4 was expressed in the oral epithelium and inside cells of the tooth bud on embryonic day 15 (E15). Further, on E17, Tß4 was expressed strongly in the dental lamina and oral epithelium, but only expressed in part of the cells in the outer and inner dental epithelium. Tß4 was strongly expressed in the entire cytoplasm of odontoblasts on postnatal day 1 (PN1) and expressed intensively in the apical area of odontoblasts on PN4. Further, expression of Tß4 was increased gradually in odontoblasts from PN1 to PN21. In an odontoblast cell line, MDPC-23, expression of Tß4 mRNA and protein was increased strongly on day 4 and gradually decreased from day 14. The gene expression of dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP), osteocalcin (OCN), osteonectin (ON), and collagen type I, related with mineralization, was significantly decreased in si-Tß4/MDPC-23 during differentiation compared to that in MDPC-23 cells. Taken together, our results suggest that Tß4 may be involved in oral epithelial cell proliferation at the initial stage of tooth development and regulates the expression and secretion of proteins during odontoblast differentiation.

Delayed healing and induction of secretory leukocyte protease inhibitor in polycystic ovary syndrome rat skin wounds.

Secretory leukocyte protease inhibitor (SLPI) and estrogen promote wound healing through a decrease in the excessive inflammatory response, accelerating re-epithelialization and increasing the amount of collagen deposition. The excessive administration of estradiol valerate (EV) using hormonal therapy decreases the concentration of estrogen abruptly and induces the polycystic ovary syndrome (PCOS). In this study, the PCOS rat skin wound area was wider than that of the normal groups and the rate of keratinocyte migration in PCOS was lower than the normal group. The numbers of inflammatory cells and macrophages recruited in the PCOS group were larger than that of the normal group. More collagen was deposited in the healing area of the normal group than in the PCOS group. The level of SLPI expression was higher in the PCOS group than the normal group after wounding, with the exception of the epithelium. On the other hand, mRNA and protein expression levels of transforming growth factor-ß1 (TGF-ß1) were lower in the PCOS group than in the normal group. Matrix metalloproteinase-2 (MMP-2) and MMP-9 levels in the PCOS group were significantly lower than that of the normal group. Therefore, increased SLPI in PCOS skin wounds may help prevent an excessive inflammatory response and aberrant collagen deposition but not are sufficient to accelerate PCOS skin wound healing, suggesting that SLPI may act as a local rather than a systemic modulating molecule in PCOS rat skin wounds.

Expression of dynamin II in odontoblast during mouse tooth development.

Odontoblasts secrete a collagen-based matrix and release numerous membrane-bound matrix vesicles, which are involved in dentin formation during tooth development. Dynamin II is a GTPase protein that contributes a variety of vesicular budding events, such as endocytotic membrane fission, caveolae internalization and protein trafficking in the Golgi apparatus. However, the expression and function of dynamin II in odontoblasts has not been reported. Therefore, this study examined the expression and possible role of dynamin II in odontoblasts during tooth development and mineralization. The levels of mRNA and protein expression in MDPC23 cells were significantly high at the early stages of differentiation and then decreased gradually thereafter. Immunohistochemistry showed that dynamin II was not expressed near the region of the odontoblasts at embryonic day 17 (E17) and E21. However, dynamin II was expressed strongly in the odontoblast layer at postnatal day 1 (PN1) and decreased gradually at PN3 and PN5. In addition, at PN15 in the functional stage, the dynamin II protein was also expressed in the odontoblast process as well as adjacent to the nuclear region. In conclusion, dynamin II may be involved in the transport of vesicles containing collageneous and non-collageneous proteins for dentin formation in odontoblast, suggesting that it is a good nanomolecule as a candidate to regulate the secretion of collagen on the bone and other nano material.

Induction of secretory leukocyte protease inhibitor (SLPI) in estradiol valerate (EV) induced polycystic ovary.

The excessive administration of estradiol valerate induces polycystic ovary syndrome by formation of follicular cysts. Secretory leukocyte protease inhibitor (SLPI) promotes wound healing by decreasing the excessive inflammatory response, stimulating keratinocyte proliferation and increasing collagen deposition through the inhibition of protease activity. In this study, SLPI expression was high in the ovarian stroma, corpus luteum, unilaminar primary follicle, multilaminar primary follicle and granulose layer of the antral follicle in polycystic ovary (PCO) compared to the normal ovary. SLPI was expressed strongly in the theca around the cyst in PCO compared to the mature follicle in the normal ovary. The levels of SLPI mRNA and protein expression were higher in PCO than in the normal ovary, and the level of MMP-2 expression was lower in PCO. These results showed that the formation of a cyst was initiated from a multilaminar primary follicle and SLPI expression was increased depending on the morphological changes in the follicle and ovarian stroma. Therefore, an increase in SLPI may be related to the suppression of tissue disruption, and act as a protease inhibitor in PCO, suggesting that SLPI increases independently of the estrogen concentration in pathological tissues.