Proliferation Of RAW264 Research Articles

임실지역 젖소 초유로부터 분리한 TGF-β 함유 유청 단백질의 면역활성

분만 후 3일 이내에 분비되는 임실 지역의 젖소 초유에서 유청 단백질을 효율적으로 분리하고 RAW 264.7 세포의 증식 및 면역활성에 미치는 영향을 조사하였다. 실험에 사용한 초유의 유청 단백질 g당 TGF-<TEX>${\beta}1$</TEX>은 875 pg/mL,TGF-<TEX>${\beta}2$</TEX>는 6,600 pg/mL이었으며 실험에 사용한 초유 유청 단백질 내 총 TGF-<TEX>${\beta}$</TEX>의 양은 7,475 pg/mL이었다. RAW264.7 세포에 대한 초유 유청 단백질 첨가에 따른 세포증식 정도를 알아본 결과 10 mg/mL의 농도까지는 세포성장을 유도하였으나 20 mg/mL 이상의 농도에서는 세포성장을 억제하였다. 이에 RAW 264.7 세포에서의 초유 유청 단백질의 면역관련 실험에 사용할 농도는 최대 10 mg/mL까지로 결정하였다. RAW 264.7 세포에 초유 유청 단백질(0-10 mg/mL)과 LPS(1 <TEX>${\mu}g/mL$</TEX>)를 차례로 반응시키고 NO생성량을 측정한 결과 초유 유청 단백질이 농도 의존적으로 NO의 생성을 억제하였다. 또한 LPS 자극에 의한 염증성 사이토카인(TNF-<TEX>${\alpha}$</TEX>, IL-<TEX>$1{\beta}$</TEX>, IL-6)이 생성되는 과정에서 초유 유청 단백질의 효과를 확인하였다. 그 결과, LPS를 단독으로 첨가했을 때 TNF-<TEX>${\alpha}$</TEX>, IL-<TEX>$1{\beta}$</TEX>, IL-6의 농도가 모두 증가하였으나 초유 유청 단백질을 첨가한 경우에는 염증성 사이토카인의 생성이 농도 의존적으로 감소하는 경향을 보였다. 초유 유청 단백질에 의해 NO를 비롯한 염증성 사이토카인의 생성이 억제되는 것이 heme oxygenase-1의 발현과 관련하는지 알아보고자 초유 유청 단백질을 농도별(0-10 mg/mL)로 처리하여 heme oxygenase-1의 발현여부를 측정한 결과 대조군과 비교하여 3-4배 이상의 발현 증가를 보였다. 이상의 결과들로 미루어보아 LPS로 자극된 RAW 264.7 세포에서 TGF-<TEX>${\beta}$</TEX> 등을 함유한 초유 유청 단백질은 10 mg/mL 이하의 농도에서 농도 의존적으로heme oxygenase-1의 발현을 유도하여 염증성 사이토카인인 TNF-<TEX>${\alpha}$</TEX>, IL-<TEX>$1{\beta}$</TEX>, IL-6 및 NO의 생성을 억제하는 것으로 판단되었다. This experiment was carried out in order to separate bovine colostral whey protein from Imsil province and to test the effect of immunological activity on RAW 264.7 cells. The colostral whey protein contained TGF-<TEX>${\beta}$</TEX> 7, 475 pg/g in total. We first tested the effect of the colostral whey protein on the proliferation of RAW 264.7 cells and it demonstrated cytotoxicity at concentrations greater than 20 mg/mL. Therefore, the immunological activities of colostral whey protein were investigated in maximum concentration of 10 mg/mL on LPS-induced RAW 264.7 cells. Results indicated that colostral whey protein inhibited the LPS-induced nitric oxide (NO) production in a dose-dependent manner. The colostral whey protein also suppressed the productions of proinflammatory cytokines (TNF-<TEX>${\alpha}$</TEX>, IL-<TEX>$1{\beta}$</TEX>, IL-6) in a dose-dependent manner. In addition to the immunological activity, colostral whey protein led to the expression of heme oxygenase-1 (HO-1) in RAW 264.7 cells. In conclusion, colostral whey protein containing TGF-<TEX>${\beta}$</TEX> inhibited the production of NO, TNF-<TEX>${\alpha}$</TEX>, IL-<TEX>$1{\beta}$</TEX>, and IL-6 via expression of HO-1.

In Vitro Biocompatibility of Endodontic Sealers Incorporating Antibacterial Nanoparticles

The main cause of endodontic disease is bacteria. Disinfection is presently achieved by cleaning the root canal system prior to obturation. Following setting, root canal filling is devoid of any antibacterial effect. Endodontic sealers with antimicrobial properties yet biocompatible may enhance root canal therapy. For this purpose, quaternized polyethylenimine nanoparticles which are antibacterial polymers, biocompatible, nonvolatile, and stable may be used. The aim of the present study was to examine the impact of added QPEI on the cytotoxicity of AH Plus, Epiphany, and GuttaFlow endodontic sealers. The effect of these sealers on the proliferation of RAW 264.7 macrophage and L‐929 fibroblast cell lines and on the production of TNFα from macrophages was examined. Cell vitality was evaluated using a colorimetric XTT assay. The presence of cytokines was determined by two‐site ELISA. Results show that QPEI at 1% concentration does not impair the basic properties of the examined sealers in both macrophages and fibroblast cell lines. Incorporation of 1% QPEI into the sealers did not impair their biocompatibility. QPEI is a potential clinical candidate to improve antibacterial activity of sealers without increasing cytotoxicity.

The effect of hydrophilic titanium surface modification on macrophage inflammatory cytokine gene expression

Chemical modification of microrough titanium dental implants to produce a hydrophilic surface with increased wettability and improved surface energy has been demonstrated clinically to achieve superior bone wound healing and osseointegration compared to that achieved with a microrough titanium surface alone. As the recruitment of the necessary osseoinductive precursors involved in bone wound healing and osseointegration to the wound site is facilitated by the action of cytokines, this study sought to determine the in vitro effect of hydrophilic surface modification on the expression of pro-inflammatory cytokines from adherent macrophages. The surface topography and composition of the titanium surfaces was characterized by scanning electron microscopy and X-ray photoelectron spectroscopy. Macrophage attachment and proliferation was assessed using an MTT assay. The expression of 84 pro-inflammatory cytokines and chemokines by adherent RAW 264.7 cells, a murine leukaemic monocyte cell line, was assessed by PCR array after 24 h culture on either smooth polished, sand-blasted acid-etched (SLA) or hydrophilic-modified SLA (SLActive) titanium surfaces. Following 24 h culture on titanium, surface microroughness activated pro-inflammatory cytokine gene transcription in RAW 264.7 cells. Although there was no significant difference in the degree of cellular attachment or proliferation of RAW 264.7 cells to the different titanium surfaces, by 24 h the hydrophilic surface elicited a gene expression profile with significant down-regulation of the key pro-inflammatory cytokines Tnfα, IL-1α, IL-1β and the chemokine Ccl-2. Down-regulation of the expression of pro-inflammatory cytokine genes may thus modulate the inflammatory response and may facilitate the enhanced bone wound healing and osseointegration observed clinically using implants with a microrough hydrophilic surface.

Inflammatory cytokine response to titanium chemical composition and nanoscale calcium phosphate surface modification

Nanoscale surface modification of titanium dental implants with calcium phosphate (CaP) has been shown to achieve superior bone wound healing and osseointegration compared with smooth or microrough titanium surfaces alone. As bone healing has been shown to be influenced by the action of cytokines, this study examined whether changes in cytokine gene expression from RAW 264.7 cells cultured on commercially pure and titanium alloy (Ti–6Al–4V) microrough or nanoscale crystalline CaP-modified surfaces, may influence downstream events in bone wound healing and osseointegration. Whilst no significant difference in the attachment or proliferation of RAW 264.7 cells was observed, the nanoscale CaP-modified surface elicited a gene expression profile with marked down-regulation of a number of pro-inflammatory cytokines and chemokines. Inflammatory cytokine gene expression was further influenced by chemical composition, with lower levels of pro-inflammatory markers noted following exposure of the macrophage-like cells to titanium alloy (Ti–6Al–4V) compared with the commercially pure titanium surface. Down-regulation of pro-inflammatory cytokine gene expression (confirmed at the protein level for TNFα and CCL5), may thus facilitate the enhanced bone wound healing and osseointegration observed clinically with nanoscale calcium phosphate-modified implant surfaces.

Effect of polyunsaturated fatty acids on the reactive oxygen and nitrogen species production by raw 264.7 macrophages

Polyunsaturated fatty acids (PUFAs) can affect various functions of the immune system including inflammatory responses. An oxidative burst of phagocytes accompanied by reactive oxygen species (ROS) and reactive nitrogen species (RNS) formation is one of the phagocyte functions that could be modulated by PUFAs. To investigate the effects of omega-3 (alpha-linolenic, docosahexaenoic, eicosapentaenoic) and omega-6 (arachidonic, linoleic) PUFAs on lipopolysaccharide (LPS)-stimulated ROS and RNS production by the murine macrophage cell line RAW 264.7. Murine peritoneal macrophages RAW 264.7 were stimulated with LPS (0.1 microg/ml) and treated with 0.1-100 microM omega-3 or omega-6 PUFAs for either 8 (ROS production) or 20 h (RNS production). The cytotoxicity of PUFAs was evaluated by an ATP (adenosine triphosphate) test after both 8 and 20 h of treatment with PUFAs. Changes in ROS production by LPS-treated macrophages subsequently activated with phorbol myristate acetate (PMA) or opsonized zymosan particles (OZP) were determined by luminol-enhanced chemiluminescence, whilst the production of RNS was determined as the concentration of nitrites in cell supernatants (Griess reaction). Changes in inducible nitric oxide synthase (iNOS) expression were evaluated by Western blot analysis. The antioxidant properties of PUFAs were tested by TRAP (total peroxyl radical-trapping antioxidant parameter) assay. All PUFAs in 100 microM concentration except eicosapentaenoic acid decreased ROS production. The effect was most significant when docosahexaenoic acid was used. Arachidonic acid decreased PMA-activated ROS production even in 1 and 10 microM concentrations. On the other hand, 10 and 100 microM eicosapentaenoic acid potentiated ROS production. As concerns RNS production, all the fatty acids that were tested in a concentration of 100 microM decreased iNOS expression and nitrite accumulation. Fatty acids had no significant effect on the viability and proliferation of RAW 264.7 cells. The TRAP assay confirmed that none of the tested PUFAs exerted any significant antioxidant properties. High concentrations of PUFAs of both omega-3 and omega-6 groups can inhibit ROS and RNS formation by stimulated macrophages. The expression of iNOS can also be inhibited. This effect, together with the absence of antioxidant activity and cytotoxic properties, indicates that PUFAs can participate in the regulation of enzymes responsible for reactive species production.

Effects of a traditional Chinese herbal preparation on osteoblasts and osteoclasts

BackgroundBone formation and resorption is a balanced and continuous process. When osteoclastic bone resorption exceeds osteoblastic bone formation, bone density decreases, which can lead to osteoporosis. Er-Zhi-Wan (EZW), a famous traditional Chinese formulation, has been developed as a restorative formula for hundreds of years, which contains two herbs viz. Herba Ecliptae and Fructus Ligustri Lucidi. EZW is widely used to prevent and treat various kidney diseases for its actions of nourishing the kidney yin and strengthening tendon and bone. The objective of current study was to investigate the effects of EZW on proliferation and differentiation of osteoblasts and osteoclasts in vitro using a serum pharmacological method. MethodsThe rats were orally administered EZW (0.45, 1.8 and 7.2gkg−1) for total seven doses and twice a day, and then the different concentrations of EZW-containing serum were prepared. The proliferation of primary cultural osteoblasts, UMR106 and RAW264.7 cells and differentiation of osteoclasts were determined after these cells were treated with different concentrations of EZW-containing serum for a period of time. ResultsThe serum from rats treated with EZW for 4 days did not facilitate proliferation of primary cultural osteoblasts and UMR106 cells, but evidently inhibited both proliferation of RAW264.7 cells and differentiation of osteoclasts from RAW264.7 cells induced by receptor activator of nuclear factor κB ligand (RANK-L) and macrophage-colony stimulating factor (M-CSF). ConclusionAntiosteoporotic activity of EZW is carried out mainly via restraint of osteoclastic bone resorption, which is in accordance with the traditional Chinese medicine theory on nourishing the kidney yin. Therefore EZW has favorable potency to develop a new anti-osteoporotic agent in clinic.

Mechanisms Involved in Enhancement of Osteoclast Formation and Function by Low Molecular Weight Hyaluronic Acid

Hyaluronic acid (HA) is a component of the extracellular matrix that has been shown to play an important role in bone formation, resorption, and mineralization both in vivo and in vitro. We examined the effects of HA at several molecular weights on osteoclast formation and function induced by RANKL (receptor activator of NF-kappa B ligand) in a mouse monocyte cell line (RAW 264.7). HA at M(r) < 8,000 (low molecular weight HA (LMW-HA)) enhanced tartrate-resistant acid phosphatase-positive multinucleated cell formation and tartrate-resistant acid phosphatase activity induced by RANKL in a dose-dependent manner, whereas HA at M(r) > 900,000 (high molecular weight HA (HMW-HA)) showed no effect on osteoclast differentiation. LMW-HA enhanced pit formation induced by RAW 264.7 cells, whereas HMW-HA did not, and LMW-HA stimulated the expression of RANK (receptor activator of NF-kappa B) protein in RAW 264.7 cells. In addition, we found that LMW-HA enhanced the levels of c-Src protein and phosphorylation of ERKs and p38 MAPK in RAW 264.7 cells stimulated with RANKL, whereas the p38 MAPK inhibitor SB203580 inhibited RANKL-induced osteoclast differentiation. This enhancement of c-Src and RANK proteins induced by LMW-HA was inhibited by CD44 function-blocking monoclonal antibody. These results indicate that LMW-HA plays an important role in osteoclast differentiation and function through the interaction of RANKL and RANK.

Differentiation by LPS and IFN-γ of expression of adenosine receptors in macrophage cell lines RAW264 and J774

Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) are external and internal activator of macrophages, respectively. During infection, adenosine has physiological effects at the site of inflammation mainly through four subtypes of adenosine receptors including A1, A2a, A2b, and A3. Moreover, A2a adenosine receptor is a critical part of the physiological negative feedback mechanism for limitation and termination of tissue-specific and systemic inflammatory responses. It was useful and meaningful to gain information about interaction with LPS, which generates the inflammation, and IFN-γ, which is a major activation factor for macrophages, and adenosine receptors, which terminate the inflammation. The aim of this study is to evaluate the abilities of LPS and IFN-γ on the expression of adenosine receptors in mouse macrophage cell lines RAW264 and J774. LPS increased the proliferation in RAW264, but not J774. IFN-γ didn’t alter the proliferation in RAW264 nor J774. LPS significantly potentiated the expression of all adenosine receptors in J774, but not in RAW, whereas IFN-γ markedly potentiated the expression of all adenosine receptors in RAW, but not in J774. These results suggest that LPS and IFN-γ may differently affect the expression of adenosine receptors in macrophage cell lines RAW264 and J774.

Knockdown of glucocorticoid receptor expression by RNA interference promotes cell proliferation in murine macrophage RAW264.7 cells

It is well documented that glucocorticoids (GC) promote arrest in the G1-S transition of the cell cycle in many cell types, resulting in a decrease in proliferation. However, the relationship between glucocorticoid receptor (GR) and the cell-cycle regulation remains unclear. Suppression of GR is important for exploring GR dependent processes. This study applied RNA interference targeting GR to the murine macrophage RAW264.7 cells. Transient transfection of the GR-siRNA expression vector reduced GR synthesis as measured on mRNA and protein level by RT-PCR and Western blot. GR-siRNA also depressesed GR transcriptional activity. A cell line [RAW-(GR−)] stably transfected with GR-siRNA expression vector was then established, the decreased GR level in this cell line was confirmed by Western blot. MTT assay showed RAW-(GR−) cells grew faster than control cells, which indicated that knockdown of GR promoted cell proliferation in macrophages. Further studies showed decreased p27 expression, increased PKC-α expression and enhanced basal and LPS-induced NF-κB activity in RAW-(GR−) cells as compared to the RAW-control cells. In contrast, virtually no change in p21, ERK1/2 and p38 expression was detected. In conclusion, these results indicate that GR itself is an inhibitor of cell proliferation in RAW264.7 cell line. This effect may be associated with the decreased expression of p27, the increased expression of PKC-α, and the activation of NF-κB. As all the experiments are carried out in GC free or serum-free medium, this study also shows the possibility for GR to have some constitutive functions, which are independent on GC activation.

PHOSPHATIDYLINOSITOL 3-KINASE REGULATES PROLIFERATION OF RAW 264.7 MACROPHAGES

Phosphatidylinositol 3-kinase (PI3-kinase) is an enzyme that acts as a direct biochemical link between a novel phosphatidylinositol pathway and a number of proteins containing intrinsic or associated kinase activities. Here we demonstrate that wortmannin, PI3-kinase inhibitor, decreases the proliferation of RAW 264.7 macrophages and that another structurally unrelated inhibitor of PI3-kinase, LY294002, also inhibits the proliferation. These results indicate a possible involvement of PI3-kinase in RAW 264.7 macrophages growth regulation. Wortmannin stimulation of RAW 264.7 macrophages is followed by sustained expression of the mRNA of c-fos and a transient expression of the mRNA of c-jun. We also show that the wortmannin and LY294002 induce a cell cycle arrest in asynchronously growing cells leading to an inhibition of cell proliferation after 12 h of treatment. In addition, wortmannin or LY294002 inhibited the phorbol 12-myristate 13-acetate-induced macrophages proliferation potently. These results suggest that PI3-kinase plays an important role in growth regulation of RAW 264.7 macrophages and that protein kinase C is a down stream effector of PI3-kinase.