Induction of Prostaglandin E₂ by Porphyromonas gingivalis in Human Dental Pulp Cells

Cyclooxygenase-2 (COX-2)-mediated prostaglandin E₂ (PGE₂) plays a key role in development and progression of inflammatory responses and Porphyromonas gingivalis is a common endodontic pathogen. In this study, we investigated induction of COX-2 and PGE₂ by P. gingivalis in human dental pulp cells (HDPCs). P. gingivalis increased expression of COX-2, but not that of COX-1. Increased levels of PGE₂ were released from P. gingivalis-infected HDPCs and this PGE₂ increase was blocked by celecoxib, a selective COX-2 inhibitor. P. gingivalis activated all three types of mitogen-activated protein kinases (MAPKs). P. gingivalis-induced activation of nuclear factor-κB (NF-κB) was demonstrated by the results of phosphorylation of NF-κ B p65 and degradation of inhibitor of κB-α (IκB-α). Pharmacological inhibition of each of the three types of MAPKs and NF-κB substantially attenuated P. gingivalis induced PGE2 production. These results suggest that P. gingivalis should promote endodontic inflammation by stimulating dental pulp cells to produce PGE₂.

Inhibition of Lipopolysaccharide-stimulated Inflammatory Cytokine Production by LY303511 in Human Macrophagic THP-1 Cells

We have previously shown that the specific phosphatidylinositol 3-kinase inhibitor LY294002 (LY29), and its inactive analog LY303511 (LY30), inhibit a monocyte chemoattractant protein-1 (MCP-1) expression in human umbilical vein endothelial cells; these results suggest the potential of LY30 as an anti-inflammatory drug. In this study, we determined the effects of LY30 on the production of various inflammatory cytokines in human macrophagic THP-1 cells which were stimulated with lipopolysaccharide (LPS). LY30 selectively suppressed the mRNA expression of IL-12 p40, TNF-α, and MCP-1 without affecting the expression of IL-1α, IL-6, and IL-8. Inhibition of the production of IL-12 and TNF-α by LY30 was also demonstrated using ELISA assays. In order to elucidate the mechanisms of the action of LY30, we examined the role played by the mitogen-activated protein kinases and the key transcription factors, AP-1 and NF-κB in LPS-stimulated THP-1 cells. The results revealed that LY30 inhibited LPS-induced activation of ERK, but not p38 or JNK. Furthermore, the AP-1 DNA binding activity was suppressed by LY30 based upon the dosage, whereas NF-κB DNA binding was not affected. These results suggest that LY30 selectively inhibits cytokine production in the LPS-stimulated macrophagic THP-1 cells by downregulating the activation of ERK and AP-1.

Clathrin and Lipid Raft-dependent Internalization of Porphyromonas gingivalis in Endothelial Cells

Porphyromonas gingivalis is one of the most important periodontal pathogens and has been to known to invade various types of cells, including endothelial cells. The present study investigated the mechanisms involved in the internalization of P. gingivalis in human umbilical vein endothelial cells (HUVEC). P. gingivalis internalization was reduced by clathrin and lipid raft inhibitors, as well as a siRNA knockdown of caveolin-1, a principal molecule of lipid raft-related caveolae. The internalization was also reduced by perturbation of actin rearrangement, while microtubule polymerization was not required. Furthermore, we found that Src kinases are critical for the internalization of P. gingivalis into HUVEC, while neither Rho family GTPases nor phosphatidylinositol 3-kinase are required. Taken together, this study indicated that P. gingivalis internalization into endothelial cells involves clathrin and lipid rafts and requires actin rearrangement associated with Src kinase activation.

Mechanisms Underlying Enterococcus faecalis-Induced Tumor Necrosis Factor-alpha Production in Macrophages

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-kappaB 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-kappaB, and AP-1.

Mechanisms Underlying Enterococcus faecalis-Induced Tumor Necrosis Factor-alpha Production in Macrophages

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-kappaB 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-kappaB, and AP-1.

Roles of Orphan Nuclear Receptor Small Heterodimer Partner in Bone Development: Microcomputed Tomographic Analysis of Bone Microarchitecture in SHP Knockout Mice / 대한골다공증학회지

OBJECTIVES: Orphan nuclear receptor small heterodimer partner (SHP) is involved in osteoblastic differentiation. This study was undertaken to demonstrate a role of SHP in in vivo bone development using microcomputed tomographic (microCT) analysis of SHP knockout (KO) mice. MATERIAL & METHODS: Tibia bones were harvested from 1-, 4-, 8- and 20-week-old wild type (WT) and SHP KO mice. The microarchitecture of tibial bone was analyzed using a microCT (Skyscan 1172; Skyscan, Kontich, Belgium). Samples were scanned at a resolution of 17 microm (isotropic). The X-ray was operated with 50 kV, 200 microA of energy, 1.2 sec of exposure time, and a 0.5 mm thick aluminum filter. Projections were acquired over an angular range of 180degrees. For quantification of the bone mineral density (BMD), the microCT was calibrated using 2 standard phantoms with densities of 0.25 and 0.75 g/cm3. The image slices were reconstructed and analyzed using CT analyzer software (CTan, Skyscan). RESULTS: The CT values of tibial trabecular bone were significantly decreased in SHP KO compared to WT at 20-week-old mice determined by microCT; (bone volume / tissue volume [BV/TV, 40%], BMD [80%], and trabecular number [Tb.N, 50%]). However, the CT values were not significantly different between WT and SHP KO in cortical bone. Furthermore, the qualitative indices of trabecular bone such as the structure model index (SMI) and the polar moment inertia (PMI) did not differ between WT and SHP KO mice. CONCLUSION: These microCT results supports that SHP may act as a positive regulator of trabecular bone formation.

Reduction of Spinal Cyclooxygenase-2 with Transcutaneous Electrical Nerve Stimulation and Cold Therapy in Rats of Carrageenan-induced Inflammatory Muscle Pain / 대한해부학회지

The purpose of this study was to investigate the effects of transcutaneous electrical nerve stimulation (TENS) and cold therapy on cyclooxygenase-2 (COX-2) expression in lumbar spinal cord and on secondary hyperalgesia produced by muscle pain. Muscle pain was experimentally induced by injection of 2% carrageenan into gastrocnemius muscle of Sprague-Dawley rats. For assessment of secondary hyperalgesia, the rats were tested for paw withdrawal latency (PWL) and tail flick latency (TFL) to heat stimulus. COX-2 expression in lumbar cord was examined using RT-PCR and immunohistochemistry. Secondary hyperalgesia to heat stimulus was significantly reduced in the both TENS- and cold-treatment groups, compared to the control group. The COX-2 mRNA levels were down-regulated in the lumbar spinal cord in the both TENS- and cold-treatment groups. In addition, COX-2 immunoreactivity was decreased in the dorsal horn of the lumbar spinal cord in the both TENS- and cold-treatment groups. These results suggested that decreased COX-2 expression in the lumbar spinal cord of the subjects receiving TENS treatment and cold therapy might be an important factor for reducing secondary hyperalgesia produced by muscle pain.

Altered Atrial Natriuretic Peptide System in the Kidney of Bilateral Obstructive Uropathic Rats / 대한신장학회잡지

BACKGROUND: Although being associated with an elevated plasma atrial natriuretic peptide(ANP), its precise role in the postobstructive diuresis has not been fully understood. Evidence has been provided suggesting that the locally-synthesized ANP in the kidney contributes to the regulation of urinary sodium excretion. The present study was aimed to investigate whether an altered regulation of local ANP system is involved in the postobstructive diuresis. METHODS: Male Sprague-Dawley rats were used. Both proximal ureters were ligated for 48 hours, after which the kidneys were taken without releasing the ligature, being designated as bilateral ureteral obstruction(BUO) group; or the ligature was released and 4 or 24 hr later, urinary data were collected, being designated as BUR-4 or BUR-24, respectively. Sham operated rats were used as control. Plasma ANP levels were determined by radioimmunoassay. The expression of ANP and natriuretic peptide receptor(NPR)-A mRNAs was determined by reverse transcription-polymerase chain reaction(RT-PCR). To further examine whether the altered renal ANP system, if any, was associated with an altered biological effects of guanylyl cyclase, ANP-stimulated cGMP accumulation was determined in membrane preparations of the glomeruli and papillae by radioimmunoassay. RESULTS: The plasma ANP level was increased in BUO group compared with that in the control(260.5+/-32.5 vs. 133.3+/-23.5pg/mL, p<0.05), decreased in BUR-4 group(3.6+/-0.5 vs. 143.5+/-42.8pg/mL, p<0.01), while not significantly different in BUR-24 group. In BUR-4. the urinary flow rate increased compared with that in the control(1598+/-370 vs. 215+/-34 microL/hr, p<0.01), along with increases of FENa(11.5+/-4.1 vs. 0.25+/-0.02%, p<0.05) and UNaV (153.7+/-23.7 vs. 36.5+/-9.3microEq/hr, p<0.01). In BUR-24, the urinary parameters were normalized. Renal tissue expression of ANP mRNA was increased in BUO as well as in BUR-4, while not changed in BUR-24. NPR-A mRNA expression was decreased in the kidney of BUO. The ANP-stimulated accumulation of cGMP in the isolated glomeruli and papillae in BUO was significantly reduced. The guanylyl cyclase activities were partly recovered in BUR-4 and completely in BUR-24. CONCLUSION: An enhanced local activity of ANP in the kidney may be causally related to the postobstructive diuresis.

Decreased Formation of Nitric Oxide in Rats Treated with FK506 / 대한신장학회잡지

The present study was aimed at investigating whether FK506 alters the regulation of nitric oxide(NO) system. Male Sprague-Dawley rats were treated with FK506(1 mg/kg/day, i.m.) for 3 weeks. Control group was without treatment of FK506. Plasma levels and urinary excretion of NO metabolites(nitrite/nitrate, NOx) were measured. The protein expression of NO synthases(NOS) and tissue contents of NOx were determined in the kidney and thoracic aorta. The aorta was also examined of its changes in isometric tension in responses to acetylcholine and sodium nitroprusside. The arterial pressure did not significantly differ between FK506-treated and control groups. Plasma NOx levels remained unaltered, while urinary NOx excretion was significantly decreased in FK 506-treated group. Tissue contents of NOx were significantly decreased, although the expression of ecNOS and iNOS proteins was significantly altered neither in the kidney nor in the aorta. Acetylcholine-induced relaxation of the isolated aortic ring was significantly attenuated, whereas sodium nitroprusside-induced relaxation was not significantly affected. These results suggest that FK506 decreases the tissue contents of NO, without significantly affecting the expression of NOS.

Decreased nitric oxide synthesis in rats with chronic renal failure

The present study was aimed at investigating whether an altered role of nitric oxide (NO) is involved in chronic renal failure (CRF). Rats were subjected to 5/6 nephrectomy and kept for 6 weeks to induce CRF. On the experimental day, after measurement of arterial pressure under anesthesia, the arterial blood was collected, and thoracic aorta and kidney were rapidly taken. NO metabolites (NOx) were determined in the plasma, urine, aorta and kidney. The expression of NO synthase (NOS) isozymes was determined in the kidney and aorta by Western blot analysis. The expression of NOS mRNA in the glomeruli was also determined by RT-PCR. There were significant increases in arterial pressure and serum creatinine levels in CRF. Urine NOx levels were decreased in CRF, whereas plasma NOx levels were not altered. Aorta and kidney tissue NOx levels were also decreased in CRF. The expression of endothelial constitutive (ec) and inducible (i) isoforms of NOS proteins was decreased in the kidney and aorta in CRF. Accordingly, the expression of ecNOS and iNOS mRNA was decreased in the glomeruli in CRF. In conclusion, NO synthesis is decreased in the kidney and vasculature of CRF rats.

Decreased nitric oxide synthesis in rats with chronic renal failure

The present study was aimed at investigating whether an altered role of nitric oxide (NO) is involved in chronic renal failure (CRF). Rats were subjected to 5/6 nephrectomy and kept for 6 weeks to induce CRF. On the experimental day, after measurement of arterial pressure under anesthesia, the arterial blood was collected, and thoracic aorta and kidney were rapidly taken. NO metabolites (NOx) were determined in the plasma, urine, aorta and kidney. The expression of NO synthase (NOS) isozymes was determined in the kidney and aorta by Western blot analysis. The expression of NOS mRNA in the glomeruli was also determined by RT-PCR. There were significant increases in arterial pressure and serum creatinine levels in CRF. Urine NOx levels were decreased in CRF, whereas plasma NOx levels were not altered. Aorta and kidney tissue NOx levels were also decreased in CRF. The expression of endothelial constitutive (ec) and inducible (i) isoforms of NOS proteins was decreased in the kidney and aorta in CRF. Accordingly, the expression of ecNOS and iNOS mRNA was decreased in the glomeruli in CRF. In conclusion, NO synthesis is decreased in the kidney and vasculature of CRF rats.

Increased expression of nitric oxide synthase coincides with reversal of renovascular hypertension

The present study was aimed at investigating whether there are changes in the expression of nitric oxide synthase (NOS) in relation with the unclipping-induced fall of blood pressure in two-kidney, one clip (2K1C) hypertension. Male Sprague-Dawley rats were made 2K1C hypertensive by clipping the left renal artery for four weeks. Sham-clipped rats served as control. The expression of endothelial constitutive (ec) NOS proteins and tissue levels of NO metabolites were determined in the kidney. Systolic blood pressure was significantly increased in clipped rats compared with that in the control. The development of hypertension was associated with decreases in the expression of ecNOS proteins and tissue levels of NO metabolites in the clipped kidney. The blood pressure at twenty-four hours after removal of the renal arterial clip fell to the control level. Accordingly, in the unclipped kidney, the expression of ecNOS proteins and tissue contents of NO metabolites were increased to the control level. The contralateral kidney was not affected by the development or reversal of hypertension. It is suggested that an enhanced expression of ecNOS in the unclipped kidney is an important component in the reversal of renovascular hypertension.

The Effect of Rotating Shift Work(2 days intervals) on the changes of 17-OHCS and Free Cortisol / 성인간호학회지

In this study, 20 students in C university were selected by control group and 14 shift work nurses working in C university hospital by test group It took part in case of day shift(8AM-4PM), evening shift(4PM-12MN), and night shift(12MN-8AM) to know a urinary 17-OHCS and free cortisol changes which works in a rapidly rotating shift work system. The working team were an rest period of a day and an oberservation of an week. It obtained an urine specimen before and after work shift in 2nd day. Test group and control group of shift work compared to change of levels of urinary 17-OHCS and free cortisol of urine collected from nurses of day shift, evening shift, and night shift. The data was analyzed by t-test, paired t-test. The results are as follows. 1. Compare 8AM with 4PM in day shift. Control group increased 4.1mg at 8AM, 4.2mg at 4PM in a case of 17-OHCS(p=.84) and also test group increased a little 3.5mg at start time of work, 3.6mg at stop time(p=.97). In a case of free cortisol control group decreased 3.8microgram at 8AM, 2.4microgram at 4PM(p=.12) and test group decreased 8.3microgramat start time of work, 3.2microgram at stop time(p=.22). 2. Compare 4PM with 12MN in evening shift. Control group decreased 4.2mg at 4PM, 2.9mg at 12MN in a case 17-OHCS(p=.54), but test group increased 1.7mgat start time of work, 3.4mg at stop time(p=.07). In a case of free cortisol control group decreased 2.4microgram at 4PM, 1.9microgram AT 12MN(p=.23) and also test group decreased a little 2.6microgram at start time of work, 1microgram at stop time(p=.43). 3. Compared 12MN with 8AM in night shift. Control group increased 3.9mg at 12MN, 4.1mg at 8AM in a case of 17-OHCS(p=.79) and also test group increased 6.3mg at start time of work, 8.4mg at stop time(p=.16). In a case of free cortisol control group increased 1.9microgram at 12MN, 3.8microgram at 8AM(p=.08) and test group increased 4.4microgram at start time of work, 11.6microgram at stop time(p=.04). As a result of this study reveals that health level of nurses is decreased in a rotating shift work(2 days internal). As mentioned above, it was confirmed that the rotating shift work had influence on the health of the nurse. For it, I present the objective base data to measure the level healthy in order to extend the understanding of the physical aspects of the nurses. Moreover, it is considered that one can make use of it as an objective base data with a view to the rational management for the nursing administration.