Radicicol Inhibits iNOS Expression in Cytokine-Stimulated Pancreatic Beta Cells

Here, we show that radicicol, a fungal antibiotic, resulted in marked inhibition of inducible nitric oxide synthase (iNOS) transcription by the pancreatic beta cell line MIN6N8a in response to cytokine mixture (CM: TNF-alpha, IFN-gamma, and IL-1beta). Treatment of MIN6N8a cells with radicicol inhibited CM-stimulated activation of NF-kappaB/Rel, which plays a critical role in iNOS transcription, in a dose-related manner. Nitrite production in the presence of PD98059, a specific inhibitor of the extracellular signal-regulated protein kinase-1 and 2 (ERK1/2) pathway, was dramatically diminished, suggesting that the ERK1/2 pathway is involved in CM-induced iNOS expression. In contrast, SB203580, a specific inhibitor of p38, had no effect on nitrite generation. Collectively, this series of experiments indicates that radicicol inhibits iNOS gene expression by blocking ERK1/2 signaling. Due to the critical role that NO release plays in mediating destruction of pancreatic beta cells, the inhibitory effects of radicicol on iNOS expression suggest that radicicol may represent a useful anti-diabetic activity.

Silibinin Inhibits LPS-Induced Macrophage Activation by Blocking p38 MAPK in RAW 264.7 Cells

We demonstrate herein that silibinin, a polyphenolic flavonoid compound isolated from milk thistle (Silybum marianum), inhibits LPS-induced activation of macrophages and production of nitric oxide (NO) in RAW 264.7 cells. Western blot analysis showed silibinin inhibits iNOS gene expression. RT-PCR showed that silibinin inhibits iNOS, TNF-alpha, and IL1beta. We also showed that silibinin strongly inhibits p38 MAPK phosphorylation, whereas the ERK1/2 and JNK pathways are not inhibited. The p38 MAPK inhibitor abrogated the LPS-induced nitrite production, whereas the MEK-1 inhibitor did not affect the nitrite production. A molecular modeling study proposed a binding pose for silibinin targeting the ATP binding site of p38 MAPK (1OUK). Collectively, this series of experiments indicates that silibinin inhibits macrophage activation by blocking p38 MAPK signaling.

Inhibition of p65 Nuclear Translocation by KIOM-79 / 대한해부학회지

We demonstrate that KIOM-79, combined extracts isolated from Magnolia officinalis, Pueraria lobata, Glycyrrhiza uralensis, and Euphorbia pekinensis, inhibits LPS-induced expression of iNOS gene in RAW 264.7 cells. Treatment of RAW 264.7 cells with KIOM-79 inhibited LPS-stimulated nitric oxide production in a doserelated manner. Immunohisto-chemical staining of iNOS and RT-PCR analysis showed that the decrease of NO was due to the inhibition of iNOS gene expression. Immunostaining of p65 and EMSA showed that KIOM-79 inhibited NF-kappa/Rel nuclear translocation and DNA binding, respectively. Collectively, this series of experiments indicates that KIOM inhibits iNOS gene expression by blocking NF-kappa/Rel. Due to the critical role that NO release plays in mediating inflammatory responses, the inhibitory effects of KIOM-79 on iNOS suggest that KIOM-79 may represent a useful anti-inflammatory agent.

The Expression of P53 and Phosphorylation of H2AX in Germ Cells of Varicocele Rats / 대한비뇨기과학회지

PURPOSE: To explore the expressions of P53 and phosphorylation-H2AX in varicocele-induced rat testes. MATERIALS AND METHODS: A total of 16 adult male Sprague-Dawley rats underwent an operation; 12 underwent an experimental varicocele and 4, as controls, were sham-operated. Groups of 4 varicocele-induced rats underwent a left orchiectomy after 2 or 3 weeks, or both orchiectomies after 4 weeks. The sham-operated rats underwent both orchiectomies after 4 weeks. Sections of both testes from each animal were studied. The changes in the expressions of P53 and phosphorylation of H2AX were determined using immunohistochemistry and western blot. RESULTS: Immunohistochemical staining of the left testes in the varicocele- induced rats showed that the expressions of P53 and phosphorylation of H2AX had not begun 2 weeks postoperatively, but remarkable results were observed after 3 and 4 weeks. Both testes of the varicocele-induced rats showed the expressions of P53 and phosphorylation of H2AX after 4 weeks, with the left testes being more distinctive in immunohistochemical staining compared to the right. Western blot of the left testes in the varicocele- induced rats also showed unclear expressions of P53 and gamma-H2AX after 2 weeks. Considerable distinction was seen after 3 and 4 weeks compared to the control group. CONCLUSIONS: Our results suggest that experimental varicocele is associated with increased sperm DNA damage. These changes may be related to abnormal spermatogenesis.

iNOS Induction by Polysaccharide Isolated from Astragalus membranaceus / 체질인류학회지

Astragalus membranaceus is used as a natural herbal medicine in East Asia for preventing carcinogenesis and reducing side effects induced by chemotherapy in cancer patients. Although the mechanism of anti-tumor activity is not known, the polysaccharides may potentiate the host defense mechanism through the activation of immune system. The objective of this study is to investigate the mechanism by which APS activates macrophages. To analyze macrophage activation and iNOS gene expression, we performed nitrite generation assay, immunohistochemistry, and RT-PCR. In the present study we show that a polysaccharide isolated from the Astragalus membranaceus (Astragalus Polysaccharide, APS) significantly induces nitric oxide (NO). Immunohistochemical staining of inducible NO synthase (iNOS) showed that the increase of NO was due to the induction of iNOS production. To further study the mechanism responsible for the induction of iNOS, we investigated the effect of APS on the iNOS mRNA expression. RT-PCR analysis showed that APS produced significant induction of iNOS gene expression. In conclusion, we demonstrate that a polysaccharide isolated from Astragalus membranaceus stimulates macrophages to generate NO through the activation of iNOS gene expression.

Immunohistochemical Study on Platelet-Derived Growth Factor alpha-Receptor (PDGF -alpha R) in Developing Canine CNS / 대한해부학회지

Platelet-derived growth factor (PDGF) was initially described for its mitogenic activity on smooth muscle cells, fibroblast, and glial cells. The biological activities of PDGF include stimulation of mitogenesis, differentiation, wound healing, inflammation, and tumor formation. The localization of platelet-derived growth factor-alpha Receptor (PDGF-alpha R) in central nervous system was commonly restricted to oligodendrocyte progenitors during late embryonic and postnatal development. However, several studies recently demonstrated that postnatal neurons could also synthesize PDGF-alpha R in rodents. In the present study, to analyze the distributional pattern of PDGF-alpha R during postnatal development of the canine CNS, we used immunohistochemical method on sections of canine brain tissue. We found that neurons of various CNS regions, including cerebral cortex, striatum, diencephalon, nuclei of brain stem, cerebellum, spinal cord, exhibited the immunoreactivity to PDGF-alpha R as early as postnatal day 0. Generally PDGF-alpha R immunoreactivity was well localized in the dendrites and axons of neuron during the postnatal day 14 and postnatal day 28, and then showed diminished pattern. But neuronal immunoreactivity to PDGF-alpha R were maintained postnatal 6 month. These results suggest that the localization of PDGF-alpha R in postnatal developing neurons supports the several roles of PDGF for neurons including maturation and survival.

Plasticity of Interstitial Cells of Cajal (ICC) and Intestinal Motility in Murine Small Bowel Obstruction / 체질인류학회지

Interstitial cells of Cajal (ICC) are the pacemakers in gastrointestinal slow wave, and also transduce signal inputs from the enteric nervous system to smooth muscle. The abnormal motility corresponded to a lack or decreasing of ICC and a disruption of electrical slow waves. So we developed partial obstruction model in murine small intestine, and found that ICC and electrical slow wave were absent or decreased oral to the occlusion site in previous study. In an additional series of experiments, we examined the ability of tissue regenerate the ICC phenotype and normal electrical slow waves after surgical treatment to relieve the mechanical obstruction, and the animals were allowed to recover for 1~2 months. Removal of the obstruction led to the normal gross appearance and the redevelopment of ICC and recovery of slow wave activity within 30 days. These data demonstrate the plasticity of ICC networks in response to partial obstruction, and suggest that adult tissue retain the ability to regenerate functional ICC. This model may be useful for estimating molecular factors responsible for the regulation of the ICC phenotype. More work is needed to find out the factors in ICC for the therapy of intestinal motility disorders.

Degenerative Change of Cerebellar Purkinje Cells by Harmaline Treatment / 체질인류학회지

The indole alkaloid harmaline has been to cause tremor and ataxia, and produce cerebellar neurotoxicity in rat. Degeneration of Purkinje cell alligned in narrow parasagittal bands result from excitation of inferior olivary nucleus in harmaline-treated rats. The objective of this study was to investigate the hypothesis that excitation of climbing fiberinduced by harmaline mediates Purkinje cell injury or degeneration. For this purpose, the inferior olive of rats was chemically ablated by using 3-acetyl pyridine, a neurotoxic chemical, and cerebellar damage followed by administration of harmaline was analyzed using immunohistochemical markers for neurons, glial cells. The results demonstrated that a subset of Purkinje cell in the vermis and paravermis degenerated after harmaline treatment, but harmaline produced little or no Purkinje cell degeneration after inferior olivary ablation. These results suggested that harmalineinduced activation of inferior olivary neurons may lead to release of glutamate from climbing fiber synaptic terminal distributed over the Purkinje cells, and may lead to cytotoxic degeneration of Purkinje cells.

Postnatal Development of Parvalbumin and Calbindin D-28k Immunoreactivities in the Canine Anterior Cingulate Cortex: Transient Expression in Layer V Pyramidal Cells / 대한해부학회지

We have examined the ontogeny of parvalbumin and calbindin D-28k immunoreactivities in the canine anterior cingulate cortex from the day of birth (P0) through P180. At P7, parvalbumin immunoreactivity appears firstly in layer VI multipolar cells. The parvalbumin immunoreactivity in GABAergic interneurons appears to follow an 'inside-out' gradient of radial mergence and reaches an adult-like pattern by the end of the 6th postnatal month. Immunoreactivity is limited mainly to developing nonpyramidal cells, whereas pyramid-like parvalbumin immunoreactive cells are transiently observed in layer V from the P14 to the P90. The developmental pattern of calbindin D-28k immunoreactivity differs from that of parvalbumin immunoreactivity. Calbindin D-28k immunoreactivity develops firstly in layer V pyramidal cells from P0, which continues through the third postnatal month. Calbindin D-28k immunoreactive interneurons are located in the infragranular layers and white matter at P0 and increase in both the supragranular and infragranular layers by P14. This is followed by an adult-like pattern at the P180. These data suggested that parvalbumin and calbindin D-28k may play a role in protecting immature neurons from intracelluar calcium influx during postnatal development.

The Morphological Study of the Protein Gene Product (PGP) 9.5-like Immunoreactive Enteric Nervous System in Murine Ileum: Close Relation to Interstitial Cells of Cajal (ICC) in Confocal Laser Scanning Microscopy / 대한해부학회지

This study was performed to investigate the morphology of the enteric nervous system and interstitial cells of Cajal (ICC) in the murine ileum. The PGP9.5-like immunoreactive (PGP9.5-LI) neurons and the c-Kit-like immunoreactive (c-Kit-LI) ICCs were stained by indirect immunofluorescence method and were observed under the confocal laser scanning microscopy. According to three dimensional reconstruction study, it was found that the PGP9.5-LI neurons and the c-Kit-LI ICCs were widely distributed in the intestinal wall : (1) In circular muscle layer, PGP9.5-LI nerve fibers were paralell to circular muscle layer. (2) In the myenteric plexus, the PGP9.5-LI nerve were closely apposed to the adjacent PGP9.5-LI nerve, constituting the networks. (3) In double-labeling immunohistochemistry using anti-PGP9.5 and anti-c-Kit antibodies, the c-Kit-LI networks encircled around the neural strands. The characteristic arrangement of the PGP9.5-LI enteric nervous system and the ICC containing c-Kit-positive cells provide a morphological basis upon the mechanism regulating gastro-intestinal motility and the pathogenesis of gatro-intestinal disorders.

Change of Interstitial Cells of Cajal (ICC) and Intestinal Motility in Murine Small Bowel Obstruction / 대한해부학회지

Interstitial cells of Cajal (ICC) are the pacemakers in gastrointestinal slow wave, and also transduce signal inputs from the enteric nervous system to smooth muscle. The abnormal motility corresponded to a lack or decreasing of ICC and a disruption of electrical slow waves. So we developed partial obstruction model in murine small intestine and investigated changes in the ICC networks and electrical activity in the obstructed bowel using c-kit immunohistochemistry and intracelluar electrophysiological techniques. Two weeks following the onset of a partial obstruction, the small intestine increased in diameter and muscular hypertrophy was developed oral to the obstruction site. ICC were absent or only weak at 1 ~25 mm oral to the occlusion site, and this disruption was accompanied by the loss of electrical slow wave. ICC networks and slow waves were normal appearance aboral to the clip. In conclusion, The present results showed that partial intestinal obstruction induced the loss of ICC networks and slow waves. These result will provide a valuable aid for understanding pathogenesis of intestinal motility disorder, and this model may be an important tool for evaluating genetic or molecular factor for the therapeutic opportunities of motility disorder in human.

Morphology of the c-Kit-Immunoreactive Interstitial Cells of Cajal (ICC) in the Mouse Intestine / 대한해부학회지

Interstitial cells of Cajal (ICC) are the pacemalkers in gastrointestinal muscles, and these cells also mediate or transduce inputs from the enteric nervoius system. Immunolabelling of interstitial cells of ICC in intestinal wall is recently developed by using specific marker, anti-c-kit antibody. Immunohistochemistry was done for c-Kit-positive ICC network in attempt to provide a morphological basis for the mechanism regulating gastro-intestinal movement. Cryosection and whole-mount preparations of mouse ileum and colon were immunolabelled using the anti-c-Kit. Immunolabelled specimens were observed under a confocal laser scanning microscopy. According to three dimensional reconstruction study, it was found that the c-Kit-positive cells were widely distributed in the intestinal wall: (1) circular muscle layer, (2) myenteric plexus, (3) deep muscular plexus in ileum, (4) submucosal plexus and longitudinal muscle layer in colon. The characteristic profiles of ICC containing c-Kit-positive cells provide a morphological basis upon the mechanism regulating gastro-intestinal motility.

Immunohistochemical Study on Platelet-Derived Growth Factor alpha-Receptor (PDGF-alphaR) in the Canine Cerebellum During Postnatal Development / 대한해부학회지

The biological activities of PDGF include stimulation of mitogenesis, chemotaxis, and differentiation. In nervous system, previous studies have shown that PDGF has an important role in the generation of cells of a glial lineage. However, several studies demonstrated that mature and immature neurons could also synthesize PDGF-alphaR. In the present study, to analyze the distributional pattern of PDGF-alphaR during postnatal development of the canine cerebellum, we used immunohistochemistry. We found that neurons of cerebellum, including Purkinje cells and granules cells, showed immunoreactivity to PDGF-alphaR (IRPDGF-alphaR) as early as postnatal day 0. Whereas IRPDGF-alphaR immunoreactivity in the Purkinje cells were maintained at all postnatal ages. Our data support that PDGF may have the important roles during development and survival of neurons.

Influence of Total Ginseng Saponin on Contractile Responses of Vasoconstrictors in the Isolated Rat Aorta

BACKGROUND: It has been known that Ginseng extract causes the hypotensive action while it rather produces the hypertensive action. Some studies have suggested that Ginseng extract causes a biphasic response on blood pressure, namely, transient fall followed by prolonged elevation. It has been also shown that administration of Korean Red Ginseng powder has no effect on blood pressure in normotensive and hypertensive rats. The present study was designed to examine the effect of total Ginseng saponin on contractile responses of vasoconstrictors in the rat aorta and to establish the mechanism of its action. METHODS: The ring segment of aorta was mounted in a muscle bath filled with oxygenated Krebs solution for the measurement of isometric tension. After the equilibration period, under the presence of total Ginseng saponin, isometric tension induced by some vasoconstrictors were observed and compared to the control responses. The data were expressed as % of the control tension. RESULTS: Phenylephrine (an adrenergic alpha1-receptor agonist) and high potassium (a membrane depolarizing agent) caused greatly contractile responses in the rat aorta, respectively. However, in the presence of total ginseng saponin (600 g/ml), the contractile responses of phenylephrine (10(-6) and 10(-5) M) and high potassium (3.5 x 10(-2) and 5.6 x 10(-2) M) were markedly potentiated whereas prostglandin F2alpha(5 x 10(-6) M)-induced contractile responses was not affected. The contractile responses induced by phenylephrine (10(-5) M) and high potassium (3.5 x 10(-2) M) even under the presence of total ginseng saponin (600 g/ml) were greatly inhibited by the pretreatment of nicardipine (10(-6) M), a calcium channel blocker. CONCLUSION: Taken together, these experimental results suggest that total ginseng saponin can enhance the contractile responses evoked by stimulation of adrenergic alpha1-receptor and the membrane depolarization in the isolated rat aortic strips, which seems to be associated to calcium influx.