Genipin Selectively Inhibits TNF-alpha-activated VCAM-1 But Not ICAM-1 Expression by Upregulation of PPAR-gamma in Human Endothelial Cells

Vascular inflammation process has been suggested to be an important risk factor in the development of atherosclerosis. Recently we reported that induction of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) selectively inhibits vascular cell adhesion molecule-1 (VCAM-1) but not intercellular cell adhesion molecule-1 (ICAM-1) in tumor necrosis factor (TNF)-alpha-activated human umbilical vein endothelial cells (HUVEC). In this study, we investigated whether genipin inhibits expression of cellular adhesion molecules, which is relevant to inflammation. Pretreatment with genipin reduced reactive oxygen species (ROS) production and expression of VCAM-1, but not ICAM-1 in TNF-alpha-activated HUVEC. Genipin dose- and time-dependently increased PPAR-gamma expression and inhibited TNF-alpha-induced phosphorylation of Akt and PKC with different degrees. Finally, genipin prevented TNF-alpha-induced adhesion of U937 monocytic cells to HUVEC. Taken together, these results indicate that upregualtion of PPAR-gamma by genipin selectively inhibits TNF-alpha-induced expression of VCAM-1, in which regulation of Akt and/or PKC play a key role. We concluded that genipin can be used for the treatment of cardiovascular disorders such as atherosclerosis.

Kinetic Changes of COX-2 Expression during Reperfusion Period after Ischemic Preconditioning Play a Role in Protection Against Ischemic Damage in Rat Brain

A brief ischemic insult induces significant protection against subsequent massive ischemic events. The molecular mechanisms known as preconditioning (PC)-induced ischemic tolerance are not completely understood. We investigated whether kinetic changes of cyclooxygenase (COX)-2 during reperfusion time-periods after PC were related to ischemic tolerance. Rats were given PC by occlusion of middle cerebral artery (MCAO) for 10 min and sacrificed after the indicated time-periods of reperfusion (1, 2, 4, 8, 12, 18 or 24 h). In PC-treated rats, focal ischemia was induced by occlusion of MCA for 24 h and brain infarct volume was then studied to determine whether different reperfusion time influenced the damage. We report that the most significant protection against focal ischemia was obtained in rats with 8 h reperfusion after PC. Administration of indomethacin (10 mg/kg, oral) or rofecoxib (5 mg/kg, oral) 48 h prior to PC counteracted the effect of PC. Immunohistochemical analysis showed that COX-2 and HO-1 protein were induced in PC-treated rat brain, which was significantly inhibited by rofecoxib. Taken together, we concluded that the kinetic changes of COX-2 expression during the reperfusion period after PC might be partly responsible for ischemic tolerance.

Induction of Heme Oxygenase-1 by Traditional Herb Mix Extract Improves MKN-74 Cell Survival and Reduces Stomach Bleeding in Rats by Ethanol and Aspirin in vivo

Chinese herb medicines have traditionally been used to treat or alleviate the symptom of various diseases. The rationale for use of certain herbs to certain disorder is now getting unveiled by modern technology. In the present study, we investigated whether herb mix extract (HMX), which is alleged to be useful for gastric ulcer, protects stomach from oxidative stress. Rats were allowed to normal diet with and without HMX (1, 5, 10 mg/kg) for 30 days. To induce gastric ulcer, ethanol (75%, 1.5 ml) or acidified aspirin (100 mg/kg in 0.2 N HCl) was administered by oral route in 24 h-fasted rats and examined the gastric ulceration (bleeding) by measuring the size 1 h after the treatment. Results indicated the area of gastric bleeding was significantly less in HMX fed rats than in normal diet fed ones, and it was dependent on the duration and amount of HMX. To investigate the underlying mechanism by which HMX protects stomach from oxidative stress, expression of enzymes like heme oxygenase (HO), cyclooxygenase (COX), and inducible nitric oxide (iNOS) were investigated in MKN-74 cells, where aspirin or H. pylori was introduced. The results were compared with RAW 264.7 cells to check if there's cell specificities exist. The expression of HO-1 but not COX-2, iNOS was significantly increased by HMX. Furthermore, HO-1 inhibitor, SnPP IX reduced the HO-1 activity and reversed the survival rate in HMX-treated MKN-74 cells. There's no difference between RAW 264.7 cells and MKN-74 cells. We, thus, concluded that HMX is beneficial for protection from oxidative injury, and induction of HO-1 by HMX in gastric cells is, at least, responsible for protection from oxidative stress such as ethanol, aspirin and possibly H. pylori infection.

YS 49, a Synthetic Isoquinoline Alkaloid, Protects Sheep Pulmonary Artery Endothelial Cells from tert-butylhydroperoxide-mediated Cytotoxicity

Endothelium, particularly pulmonary endothelium, is predisposed to injury by reactive oxygen species (ROS) and their derivatives. Heme oxygenase (HO) has been demonstrated to provide cytoprotective effects in models of oxidant-induced cellular and tissue injuries. In the present study, we investigated the effects of YS 49 against oxidant [tert-butylhydroperoxide (TBH) ]-induced injury using cultured sheep pulmonary artery endothelial cells (SPAECs). The viability of SPAECs was determined by quantifying reduction of a fluorogenic indicator Alamar blue. We found that TBH decreased cell viability in a time- and concentration-dependent manner. YS 49 concentration- and time-dependently increased HO-1 induction on SPAECs. As expected, YS 49 significantly decreased the TBH-induced cellular injury. In the presence of zinc protophorphyrin, HO-1 inhibitor, effect of YS 49 was significantly inhibited, indicating that HO-1 plays a protective role for YS 49. Furthermore, YS 49 showed free radical scavenging activity as evidenced by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and inhibition of lipid peroxidation. However, YS 49 did not inhibit apoptosis induced by lipopolysaccharide (LPS) in SPAECs. Taken together, HO-1 induction along with strong antioxidant action of YS 49 may be responsible for inhibition of TBH-induced injury in SPAECs.

Inhibition of Inducible Nitric Oxide Synthase Expression by YS 49, a Synthetic Isoquinoline Alkaloid, in ROS 17/2.8 Cells Activated with TNF-alpha, IFN-gamma and LPS

Nitric oxide (NO) has been suggested to act as a mediator of cytokine-induced effects of turn over of bone. Activation of the inducible nitric oxide synthase (iNOS) by inflammation has been related with apoptotic cell death in osteoblast. YS 49, a synthetic isoquinoline alkaloid, inhibits NO production in macrophages activated with cytokines. In the present study, we investigated the molecular mechanism of YS 49 to inhibit iNOS expression in ROS 17/2.8 cells, which were activated with combined treatment of inflammatory cytokines (TNF-alpha, IFN-gamma) and lipopolysaccharide (LPS). Results indicated that YS 49 concentration-dependently reduced iNOS mRNA and protein expression, as evidenced by Northern and Western blot analysis, respectively. The underlying mechanism by which YS 49 suppressed iNOS expression was not to affect iNOS mRNA stability but to inhibit activation and translocation of NF-kappaB by preventing the degradation of its inhibitory protein IkappaBalpha. As expected, YS 49 prevented NO-induced apoptotic cell death by sodium nitroprusside. Taken together, it is concluded that YS 49 inhibits iNOS expression by interfering with degradation of phosphorylated inhibitory kappaBalpha (p-IkappaBalpha). These actions may be beneficial for the treatment of inflammation of the joint, such as rheumatoid arthritis.

Consistent and Specific Suppression of Mucin Release from Cultured Hamster Tracheal Surface Epithelial Cells by Poly-L-Lysine

Poly-L-lysine (PLL) was reported to suppress mucin release from airway goblet cells during 30 min treatment period. In this study, we investigated whether PLL consistently suppresses mucin release from cultured airway goblet cells during 24 h after 30 min treatment and also specifically suppresses the release of mucin without any effects on the other releasable glycoproteins. Confluent primary hamster tracheal surface epithelial (HTSE) cells were metabolically radiolabeled with 3H-glucosamine for 24 h and chased for 30 min in the presence of varying concentrations of PLL to assess the effects on 3H-mucin release and on the total elution profile of the treated culture medium. The total mucin content during 24 h after 30 min treatment of PLL was assesed to investigate the consistency of effects. PLL did not affect the release of the other releasable glycoproteins whose molecular weights were less than mucin, and decreased the total mucin content during 24 h after 30 min treatment. We conclude that PLL can specifically suppress mucin release from cultured airway goblet cells and the suppression on mucin release is consistent. This finding suggests that PLL might be used as a specific airway mucin-regulating agent by directly acting on airway mucin-secreting cells.

Caffeic Acid Phenethyl Ester Inhibits the PKC-Induced IL-6 Gene Expression in the Synoviocytes of Rheumatoid Arthritis Patients

To gain insight on the role of pro-inflammatory cytokines in the pathogenesis and treatment of rheumatoid arthritis (RA), the phorbol 12-myristate 13-acetate (PMA) -induced IL-6 gene expression and the effect of caffeic acid phenethyl ester (CAPE) on the PMA-induced IL-6 gene expression were investigated in human fibroblast-like synoviocytes (FLSs). Synovial tissue samples were obtained from rheumatoid arthritis patients, and FLSs were isolated. The cells were stimulated with PMA (100 nM) for 6 hrs to induce IL-6 gene. The cells were pretreated with CAPE (20, 50, 100 microM) prior to PMA treatment. PMA increased IL-6 RNA expression, binding activities of transcription factors (NF-kappaB, AP-1) to IL-6 promoter, and IL-6 promoter activity. However, CAPE inhibited PMA-induced IL-6 mRNA expression in dose-dependent manner, and also inhibited the increased binding activities of transcription factors to IL-6 promoter and IL-6 promoter activity. These results suggest that CAPE might regulate PKC-mediated IL-6 expression and inflammatory reactions in RA.

Diclofenac inhibits IFN-gamma plus lipopolysaccharide-induced iNOS gene expression via suppression of NF-kappaB activation in RAW 264.7 macrophages

Diclofenac, a phenylacetic acid derivative, is a widely used non-steroidal anti-inflammatory drug (NSAID) to provide effective relief of inflammation and pain. Nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been implicated as a mediator of inflammation. We examined the inhibitory effects of diclofenac on the induction of iNOS in RAW 264.7 macrophages which were activated with lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). Treatment of RAW 264.7 cells with diclofenac and other NSAIDs (aspirin and indomethacin) significantly inhibited NO production and iNOS protein expression induced by LPS plus IFN-gamma. Also, diclofenac but not aspirin and indomethacin, inhibited iNOS mRNA expression and nuclear factor-kappa B (NF-kappaB) binding activity concentration-dependently. Furthermore, transfection of RAW 264.7 cells with iNOS promoter linked to a CAT reporter gene revealed that only diclofenac inhibited the iNOS promoter activity induced by LPS plus IFN-gamma through the NF-kappaB sites of iNOS promoter. Taken together, these suggest that diclofenac may exert its anti-inflammatory effect by inhibiting iNOS gene expression at the transcriptional level through suppression of NF-kappaB activation.

Role of NF- kappaB binding sites in the regulation of inducible nitric oxide synthase by tyrosine kinase

In macrophages, lipopolysaccharide (LPS) alone or in combination with interferon- gamma (IFN- gamma) has been shown to release a nitric oxide (NO) through the increase of the transcription of the inducible nitric oxide synthase (iNOS) gene. To investigate the exact intracellular signaling pathway of the regulation of iNOS gene transcription by LPS plus IFN- gamma, the effects of protein tyrosine kinase (PTK) inhibitor and protein kinase C (PKC) inhibitors on NO production, iNOS mRNA expression, nuclear factor- kappaB (NF- kappaB) binding activity and the promoter activity of iNOS gene containing two NF- kappaB sites have been examined in a mouse macrophage RAW 264.7 cells. LPS or IFN- gamma, stimulated NO production, and their effect was enhanced synergistically by mixture of LPS and IFN- gamma. The PTK inhibitor such as tyrphostin reduced LPS plus IFN- gamma-induced NO production, iNOS mRNA expression and NF- kappaB binding activity. In contrast, PKC inhibitors such as H-7, Ro-318220 and staurosporine did not show any effect on them. In addition, transfection of RAW 264.7 cells with iNOS promoter linked to a CAT reporter gene revealed that tyrphostin inhibited the iNOS promoter activity through the NF- kappaB binding site, whereas PKC inhibitors did not. Taken together, these suggest that PTK, but not PKC pathway, is involved in the regulation of the iNOS gene transcription through the NF- kappaB sites of iNOS promoter in RAW 264.7 macrophages by LPS plus IFN- gamma.

(3H)Ryanodine binding sites of SR vesicles of the chicken pectoral muscle

To investigate the properties of ryanodine binding sites of the bird skeletal SR vesicles, SDS PAGE, purification of RyR, and (3H)ryanodine binding study were carried out in the SR vesicles prepared from the chicken pectoral muscle. The chicken SR vesicles have two high molecular weight (HMW) protein bands as in eel SR vesicles on SDS PAGE. The HMW bands on SDS PAGE were found in the (3H) ryanodine peak fraction (Fr3-5) obtained from the purification step of the ryanodine receptor protein. Bmax and KD of the chicken (3H)ryanodine binding sites were 12.52 pmol/mg protein and 14.53 nM, respectively. Specific (3H)ryanodine binding was almost maximal at 50~100 micrometer Ca2+, but was not increased by 5 mM AMP and not inhibited by high Ca2+. Binding was significantly inhibited by 20~100 micrometer ruthenium red and 1 mM tetracaine, but slightly inhibited by Mg2+. From the above results, it is suggested that chicken SR vesicles have the ryanodine binding sites to which the binding of ryanodine is almost maximal at 50~10 micrometer Ca2+, is significantly inhibited by ruthenium red and tetracaine, slightly inhibited by Mg2+, but not affected by AMP and not inhibited by high Ca2+.

Effect of Intraventricular Atropine on the Heart Rate of the Rabbit / 대한마취과학회지

The effects of intraventricular atropine on the heart rate was investigated in the rabbit. Intraventricular administration of atropine in a dose of 10, 30, 100, or 300 ug produced dose dependant bradycardia. Atropine (100 ug) induced bradycardia was abolished by bilateral vagotomy or intravenous atropine, and inhibited by intravenous propranolol but not by intravenous Regitine. Intraventricular Ecolid or regitine pretreatment diminished the bradycardia induced by intraventricular atropine. From the above results, it is suggested that a central adrenergic mechanism as well as vagal activity plays an important role in the intraventricular atropine-induced bradycardia.