• Restorative Dentistry and Endodontics
• /
• 제36권1호
• /
• pp.26-36
• /
• 2011

연구목적: 본 연구는 in vitro 상에서 치수, 치은, 치주인대 세포를 neuropeptide (substance P, Calcitonin gene related peptides (CGRP))및 inflammatory cytokine (TNF-$\alpha$)으로 자극 시 matrix metalloproteinase (MMPs)의 생성 및 발현을 관찰한 것으로 치아와 치아 주변 조직에 염증이 존재할 경우 neuropeptide 및 inflammatory cytokine과 치아 경조직 혹은 치조골의 remodeling에 중요한 역할을 하는 matrix metalloproteinase (MMPs)와의 관계를 규명하고자 하였다. 연구 재료 및 방법: 시편으로는 우식이 없는 건전한 제3대구치(n = 10)를 사용하였으며, 발거 후 즉시 Phosphate buffered saline (PBS)에 보관하고 치아에서 치은과 치주인대 조직을 채취하였다. 치아를 종축으로 절단하고 치수 조직을 채취하여 조각으로 분리한 후 시편을 PBS에서 세 번 세척하였다. Plate에 치수, 치은, 치주인대 시편 조각을 위치시켜 Dulbecco's Modified Eagle Medium (DMEM)을 첨가하여 세포를 배양하였다. 치수, 치은, 치주인대 세포를 culture dish에서 confluence에 도달할 때 까지 배양하여 Fetal Bovine Serum (FBS)가 포함되지 않은 배지로 교환하여, $37^{\circ}C$에서 24시간 동안 배양한 후 Phosphate buffered saline (PBS)로 1회 세척하고 Substance P ($10^{-8}\;M$, $10^{-5}\;M$)가 포함된 배양액과 Mock (배양액만 포함됨)으로 4시간, 24시간동안 자극하였다. CGRP ($10^{-6}\;M$)을 함유한 배양액 및 TNF-$\alpha$(2 ng/mL)를 포함한 배양액으로 각각 24시간동안 세포를 자극하였다. 각각 다른 농도의 TNF-$\alpha$(2 ng/mL, 10 ng/mL, 100 ng/mL)를 포함한 배양액으로 24시간 동안 세포를 자극 한 후 RNase protection assay 및 Enzyme linked immunosorbent assay를 시행하였다. 결과: SP와 CGRP는 치수, 치은, 치주인대 세포의 MMPs발현에 관여 하지 않았다. TNF-$\alpha$로 24시간 자극 시 치수, 치은, 치주인대 세포에서 MMP-1,-12, -13의 발현을 증가시켰다. 반면, TNF-$\alpha$는 치수, 치은, 치주인대 세포들에서 TIMP-3의 발현을 감소시켰다. 서로 다른 농도의 TNF-$\alpha$(2 ng/mL, 10 ng/mL, 100 ng/mL)로 24시간 자극 시 MMP-1과 MMP-13의 발현이 증가하였다. 결론: 치아와 치아 주변 조직에 염증이 존재 시 TNF-$\alpha$가 증가함에 따라 치수, 치은, 치주인대로부터의 치아의 경조직 혹은 치조골의 remodeling에 관여하는 matrix metalloproteinase (MMPs)가 중요한 역할을 하는 것으로 사료된다.

• 한국식품영양과학회지
• /
• 제40권5호
• /
• pp.635-641
• /
• 2011

본 연구는 TNF-${\alpha}$로 자극된 HUVEC에서 녹차씨껍질 EtOAC 추출물이 초기 동맥경화 과정에 중요한 역할을 하는 염증매개인자와 세포부착물질에 미치는 영향을 분석하였다. 녹차씨껍질 EtOAC 추출물의 NO 생성능은 TNF-${\alpha}$만을 처리한 control군에 비해 증가시키는 것을 알 수 있었다. $100{\mu}g$/mL 농도에서는 녹차씨껍질 EtOAC 추출물은 세포독성을 보이지 않았으며 염증매개인자인 TNF-${\alpha}$ 수준 및 세포부착물질인 VCAM-1과 MCP-1의 생성을 억제하였다. 뿐만아니라, 녹차씨껍질 EtOAC 추출물은 총 항산화능은 증가되는 경향을 보였다. 이상의 결과에서, 녹차씨껍질 EtOAC 추출물은 HUVEC에서 TNF-${\alpha}$로 인한 총 항산화능의 수준을 향상시켜 염증생성인자인 TNF-${\alpha}$ 수준 및 세포부착물질인 VCAM-1과 MCP-1의 생성을 억제하여 동맥경화 초기반응을 억제하는데 기여할 것으로 사료된다.

• The Korean Journal of Physiology and Pharmacology
• /
• 제18권3호
• /
• pp.233-239
• /
• 2014

The present study was designed to investigate the effect Hypericum Perforatum (HP), on behavioral changes, corticosterone, TNF-${\alpha}$ levels and tryptophan metabolism and disposition in bilateral ovariectomized rats compared to $17{\alpha}$-ethinylestradiol. Behavioral analysis by measuring immobility time in forced swimming test and open field test, serum and hippocampal corticosterone and TNF-${\alpha}$ along with hippocampal kynurenine/tryptophan ratio were determined in mature ovariectomized rats treated orally either by HP at three different doses 125, 250, and 500 mg/kg/day or by $17{\alpha}$-ethinylestradiol $30{\mu}g/kg/day$ for 30 days. Ovariectomized rats showed significant increase in immobility time in the forced swimming test. Along with elevation in serum and hippocampal TNF-${\alpha}$ and corticosterone levels associated with significant increase in hippocampal kynurenine/tryptophan ratio. Immobility time in the forced swimming test was decreased in rats treated by different doses of HP in a dose dependent manner and $17{\alpha}$-ethinylestradiol with no concomitant changes in the open field test. Only Rats treated with HP exhibited significant decrease in the elevated serum and hippocampal TNF-${\alpha}$ and corticosterone, which couldn't explain the associated insignificant effect on hippocampaus kynurenine/tryptophan ratio in comparison to ovariectomized untreated rats. It is concluded that increased tryptophan metabolism toward kynurenine secondary to elevated corticosterone and TNF-${\alpha}$ might be one of the pathohphysiological mechanisms that could explain depression like state observed in this rat model. Further, the observed attenuating effect of HP on TNF-${\alpha}$ and corticosterone could contribute in its antidepressant effect in this animal model by other ways than their effects on tryptophan-kynurenine metabolism pathway.

• International Journal of Oral Biology
• /
• 제35권2호
• /
• pp.43-49
• /
• 2010

Enterococcus faecalis, a gram-positive bacterium, has been implicated in endodontic infections, particularly in chronic apical periodontitis. Proinflammatory cytokines, including tumor necrosis factor-$\alpha$ (TNF-$\alpha$), are involved in the pathogenesis of these apical lesions. E. faecalis has been reported to stimulate macrophages to produce TNF-$\alpha$. The present study investigated the mechanisms involved in TNF-$\alpha$ production by a murine macrophage cell line, RAW 264.7 in response to exposure to E. faecalis. Both live and heat-killed E. faecalis induced high levels of gene expression and protein release of TNF-$\alpha$. Treatment of RAW 264.7 cells with cytochalasin D, an inhibitor of endocytosis, prevented the mRNA up-regulation of TNF-$\alpha$ by E. faecalis. In addition, antioxidant treatment reduced TNF-$\alpha$ production to baseline levels. Inhibition of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase also significantly attenuated E. faecalis-induced TNF-$\alpha$ expression by RAW 264.7 cells. Furthermore, activation of NF-${\kappa}B$ and AP-1 in RAW 264.7 cells was also stimulated by E. faecalis. These results suggest that the phagocytic uptake of bacteria is necessary for the induction of TNF-$\alpha$ in E. faecalis-stimulated macrophages, and that the underlying intracellular signaling pathways involve reactive oxygen species, ERK, p38 MAP kinase, NF-${\kappa}B$, and AP-1.

• 대한한방내과학회지
• /
• 제22권1호
• /
• pp.79-85
• /
• 2001

Object : Hepatitis Treatment-tang No.1 has been used for the treatment of Liver disease and Jaundice. Long-term EtOH exposure leads to immunoregulatory and detoxification impairment. This study aimed to determine the relationship between TNF-${\alpha}$ production and expression, and EtOH-induced cytotoxicity on Hep G2 cells. Method : Cells were incubated with EtOH in the presence or absence of HT. The cells were tested after 24 hours and, again, after 48 hours. Cytoviability and TNF-${\alpha}$ release were analyzed by MTT assay and enzyme linked immunosorbent assay (ELISA), respectively. After 24 hours of EtOH exposure, the cytoviability decreased, and the release of TNF-${\alpha}$ was increased. Increased amounts of TNF-${\alpha}$ contribute to EtOH-induced cytotoxicity. The Anti-TNF-${\alpha}$ antibody almost abolished it. Interestingly, EtOH-induced cytotoxicity and TNF-${\alpha}$ production were inhibited by HT. Moreover, when HT was used in combination with the anti-TNF-${\alpha}$ antibody, there was a marked inhibition of EtOH-induced cytotoxicity. Results : These results suggest that HT may prevent the cytotoxicity through partial inhibition of the TNF-${\alpha}$ secretion.

• The Korean Journal of Physiology and Pharmacology
• /
• 제9권6호
• /
• pp.315-322
• /
• 2005

In this study, the authors investigated whether death of vascular smooth muscle cell (VSMC) had a pathological pertinence. Conditioned media obtained from rat aorta smooth muscle cell (SMC) that were induced death by expressing FADD in the absence of tetracycline (FADD-SMC) triggered death of normal SMC. DNA fragmentation and caspase-3 activation were observed in dying SMC by conditioned media. FADD-SMC showed transcriptional activation of tumor necrosis factor $(TNF)-{\alpha}$. Conditioned medium contained $TNF-{\alpha}$, indicating secretion of the cytokine from dying FADD-SMC. It was investigated if secreted $TNF-{\alpha}$ was functional. Conditioned medium activated ERK and p38 MAPK pathways and induced MMP-9 expression, whereas depletion of the cytokine with its soluble receptor (sTNFR) remarkably inhibited induction of MMP-9 by conditioned medium. These findings suggest that $TNF-{\alpha}$ in conditioned medium seems to be active. Then, contribution of $TNF-{\alpha}$ on death-inducing activity of conditioned medium was examined. Depletion of $TNF-{\alpha}$ with soluble $TNF-{\alpha}$ receptor decreased the death activity of conditioned medium by 35%, suggesting that $TNF-{\alpha}$ play a partial role in the death activity. Boiling of medium almost completely abolished the death-inducing activity, suggesting that other heat labile death inducing proteins existed in conditioned medium. Taken together, these results indicate that SMC undergoing death could contribute to inflammation by expressing inflammatory cytokines and pathological complications by inducing death of neighboring cells.

• Journal of Microbiology and Biotechnology
• /
• 제16권2호
• /
• pp.219-225
• /
• 2006

The rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay have been used to detect endotoxins present in vaccines. Currently, the rabbit pyrogen test is used to detect endotoxins in hepatitis B (HB) vaccines, even though the HB surface protein, which is the active ingredient, is overexpressed in and purified from eukaryotic cells that lack these endotoxins. Although the LAL clot assay is sensitive and reliable and can be used to replace the rabbit pyrogen test, its reaction is limited by the lack of responsiveness to the Gram-positive bacterial components. Furthermore, aluminum hydroxide in the HB vaccine can interfere with the LAL assay. In contrast, macrophages can detect the endotoxin as well as other pyrogens, and secrete $TNF-{\alpha}$. Therefore, this study was undertaken to examine the possibility of replacing the animal tests with a more efficient $TNF-{\alpha}$ secretion assay. With this in mind, we determined if aluminum hydroxide in the HB vaccines affects the $TNF-{\alpha}$ secretion assay. HB vaccines and the HB protein solutions spiked with lipopolysaccharide (LPS) produced the same level of dose-dependent $TNF{\alpha}$ secretion and temperature increase in rabbits, indicating that aluminum hydroxide in the HB vaccine does not interfere with the pyrogenic response in rabbits, nor does it interfere with $TNF-{\alpha}$ secretion. In addition, the $TNF-{\alpha}$ assay was found to be more sensitive than the LAL assay, and correlated well with the pyrogen test and the LAL assay. These results suggest that the $TNF-{\alpha}$ assay in RAW264.7 cells is a good substitute for the current pyrogen assays that are used for detecting LPS in HB vaccines as well as in other vaccines containing aluminum.

• 미생물학회지
• /
• 제52권3호
• /
• pp.380-382
• /
• 2016

청국장은 대두발효식품으로 발효 중 생성된 다양한 펩타이드가 들어 있다. 이들 펩타이드가 포함된 청국장추출물을 세포에 처리하면 세포 신호전달에 영향을 미친다. 인간 유방암 MDA-MB-231 세포에 청국장추출물을 처리해 주었을 때 세포의 성장은 농도의존적으로 억제되었다. 청국장추출물이 유방암세포의 증식을 억제하였고 암과 염증이 관련이 있으므로, 청국장추출물이 염증 유전자의 하나인 $TNF{\alpha}$ 발현의 억제 여부를 알아 보았다. 인간유방암 MDA-MB 231 세포에 청국장 추출물을 처리하면 $TNF{\alpha}$ 발현은 억제되었다. $TNF{\alpha}$ 저해제는 자가면역질환 치료제로 개발되어 있다. 본 청국장추출물도 $TNF{\alpha}$ 억제 효과가 있으므로, 이들 자가면역 질환의 치료제로 개발될 수 있을 것이다.

• The Korean Journal of Physiology and Pharmacology
• /
• 제20권4호
• /
• pp.347-355
• /
• 2016

Cryptotanshinone (CPT) is a natural compound isolated from traditional Chinese medicine Salvia miltiorrhiza Bunge. In the present study, the regulatory effect and potential mechanisms of CPT on tumor necrosis factor alpha ($TNF-{\alpha}$) induced lectin-like receptor for oxidized low density lipoprotein (LOX-1) were investigated. Human umbilical vein endothelial cells (HUVECs) were cultured and the effect of $TNF-{\alpha}$ on LOX-1 expression at mRNA and protein levels was determined by Real-time PCR and Western blotting respectively. The formation of intracellular ROS was determined with fluorescence probe $CM-DCFH_2-DA$. The endothelial ox-LDL uptake was evaluated with DiI-ox-LDL. The effect of CPT on LOX-1 expression was also evaluated with SD rats. $TNF-{\alpha}$ induced LOX-1 expression in a dose- and time- dependent manner in endothelial cells. $TNF-{\alpha}$ induced ROS formation, phosphorylation of $NF-{\kappa}B$ p65 and ERK, and LOX-1 expression, which were suppressed by rotenone, DPI, NAC, and CPT. $NF-{\kappa}B$ inhibitor BAY11-7082 and ERK inhibitor PD98059 inhibited $TNF-{\alpha}-induced$ LOX-1 expression. CPT and NAC suppressed $TNF-{\alpha}-induced$ LOX-1 expression and phosphorylation of $NF-{\kappa}B$ p65 and ERK in rat aorta. These data suggested that $TNF-{\alpha}$ induced LOX-1 expression via ROS activated $NF-{\kappa}B/ERK$ pathway, which could be inhibited by CPT. This study provides new insights for the anti-atherosclerotic effect of CPT.

• The Korean Journal of Physiology
• /
• 제30권1호
• /
• pp.77-83
• /
• 1996

Platelet-activating factor(PAF) is a phospholipid mediator of pulmonary inflammation, and immunologic reaction. In this study, the role of PAF on tumor necrosis factor$(TNF_{-{\alpha}})$ production by rat alveolar macrophages(AM) was examined. When PAF $(10^{-12}{\sim}10{-16}\;M)$ alone was added to AM culture, $(TNF_{-{\alpha}})$ production was not significantly increased above the resting level. In contrast, the combined addition of PAF $(10^{-6}\;M)$ and muramyl dipeptide(MDP) $(1.0\;{\mu}g\ml)$ to AM cultures markedly enhanced $(TNF_{-{\alpha}})$ production with 8.2 fold increase compared with AM culture in resting state. This potentiative effect was 313% above the sum of the separate effects of PAF and MDP. To characterize MDP effects on $(TNF_{-{\alpha}})$ production, the dose-response of AM cultured with various concentrations of MDP was tested. High level of MDP $(10\;{\mu}g\ml)$ could not significantly enhance the potentiation effect on $(TNF_{-{\alpha}})$ production compared with AM cultures with low level of MDP $(0.1\;{\mu}g\ml)$, i.e. 112.5% vs 107.8%, respectively when $10^{-10}$ M of PAF was simultaneously added to the cell culture. These data support that the potentiation of TNF. g production in AM culture is mediated by PAF rather than MDf It was also evaluated whether the similar result was obtained in silica, respirable toxic particle-treated AM culture. $(TNF_{-{\alpha}})$ production was also significantly enhanced in the PAF $(10^{-6}\;M)$ and silica $(50\;{\mu}g\ml)$-added cell cultures with 4.7 fold above the value of silica alone-stimulated cells. These results indicate that PAF can potentiate $(TNF_{-{\alpha}})$ production by MDP-or silica- stimulated AM and suggest that PAF may play a potent role in lung inflammation and disease associated with microbe and occupational dust exposures.