Citri Reticulatae Pericarpium Limits TLR-4-Triggered Inflammatory Response in Raw264.7 Macrophages by Activating RasGRP3.

Inflammation is an important immune response to pathogen invasion, but excessive inflammation leads to tissue injury and even cytokine storm. Therefore, proper response is needed depending on the intensity of the infection. Ras guanine nucleotide releasing protein 3 (RasGRP3) is a regulator of the TLR-mediated response. In low-intensity inflammation, it negatively regulates production of pro-inflammatory cytokines, especially IL-6. Citri Reticulatae Pericarpium, the peel of Citrus reticulata Blanco, is a major medicinal herb in Korean medicine. The present study aims to investigate whether the Citri Reticulatae Pericarpium extract (CRE) has immunomodulatory activity using the Raw264.7 macrophage. Also, we investigated the effect of CRE on RasGRP3 expression. In the present study, CRE reduced IL-6 production in the low-LPS environment (1 ng/mL) and did not in the high-LPS environment (100 ng/mL). The suppression of IL-6 production in the low-LPS environment (1 ng/mL) was abolished after the pretreatment of RasGRP3 siRNA. The reduced RasGRP3 protein content by 100 ng/mL LPS treatment was increased by CRE treatment. Additionally, nobiletin, a major component of CRE showed a suppressive effect on IL-6 production in the low-LPS environment (1 ng/mL). The present results suggest that CRE alleviates inflammatory response via activating RasGRP3 expression in low-intensity inflammation.

Bee Venom Prevents Mucin 5AC Production through Inhibition of AKT and SPDEF Activation in Airway Epithelia Cells.

IL-13 induces mucus metaplasia, which causes airway obstruction in asthma. Bee venom (BV) and its components have shown anti-inflammatory effects in allergic diseases such as atopic dermatitis and asthma. In this study, we investigated the effect of BV on IL-13-induced mucus metaplasia through activation of the signal transducer and activator of transcription (STAT6), and regulation of SAM-pointed domain containing Ets-like factor (SPDEF) and forkhead box A2 (FOXA2) in the airway epithelia cell line A549. In A549 cells, BV (1.0 µg/mL) inhibited IL-13 (10 ng/mL)-induced AKT phosphorylation, increase in SPDEF protein expression, and decrease in FOXA2 protein expression-but not STAT6 phosphorylation. BV also prevented the IL-13-induced increase in mucin 5AC (MUC5AC) mRNA and protein expression. Moreover, we observed that inhibition of phosphoinositide 3 kinase (PI3K)/AKT using LY294002 (50 µM) could reverse the alterations in FOXA2 and MUC5AC expression -by IL-13 and BV. However, LY294002 did not affect IL-13- and BV-induced changes in SPDEF expression. These findings indicate that BV inhibits MUC5AC production through the regulation of SPDEF and FOXA2. The inhibition of MUC5AC production through FOXA2 is mediated via the suppression of PI3K/AKT activation by BV. BV may be helpful in the prevention of mucus metaplasia in asthma.

Effects of egg yolk-derived peptide on osteogenic gene expression and MAPK activation.

The present study investigated the effects of egg yolk-derived peptide (YPEP) on osteogenic activities and MAPK-regulation of osteogenic gene expressions. The effects of YPEP on cell proliferation, alkaline phosphatase activity, collagen synthesis, and mineralization were measured in human osteoblastic MG-63 cells. Activation of MAPKs and downstream transcription factors such as extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK1/2), p38, ELK1, and cJUN were examined using western blot analysis. YPEP dose-dependently increased MG-63 cell proliferation, ALP activity, collagen synthesis, and calcium deposition. YPEP activated ERK1/2, p38, and ELK1 phosphorylation whereas JNK and cJUN were not affected by YPEP. The COL1A1 (collagen, type I, alpha 1), ALPL (alkaline phosphatase), and SPP1 (secreted phosphoprotein 1, osteopontin) gene expressions were increased while BGLAP (osteocalcin) was not affected by YPEP. The ERK1/2 inhibitor (PD98509) blocked the YPEP-induced COL1A1 and ALPL gene expressions as well as ELK1 phosphorylation. The p38 inhibitor (SB203580) blocked YPEP-induced COL1A1 and ALPL gene expressions. SPP1 gene expression was not affected by these MAPK inhibitors. In conclusion, YPEP treatment stimulates the osteogenic differentiation via the MAPK/ELK1 signaling pathway. These results could provide a mechanistic explanation for the bone-strengthening effects of YPEP.

Protective effect of egg yolk peptide on bone metabolism.

OBJECTIVE: Osteoporosis is a major health problem worldwide, and most current therapy used in osteoporosis treatment acts by either increasing bone formation or decreasing bone resorption. However, the adverse effects of these therapies may preclude their long-term use. We examined the effects of egg yolk water-soluble peptide (YPEP) on bone metabolism as an alternative to current therapeutic agents in ovariectomized (OVX) rats. METHODS: In the first step, the in vitro effects of YPEP on bone loss were determined. The proliferation, collagen content, and alkaline phosphatase activity of preosteoblastic MC3T3-E1 cells and osteoclastogenesis from bone marrow-derived precursor cells were measured. The in vivo experiment confirmed the positive effect of YPEP on bone tissue. Three-month-old female Sprague-Dawley rats were either sham operated or ovariectomized and fed commercial chow diet or 0.1% YPEP-supplemented diet for 3 month. RESULTS: YPEP increased preosteoblastic MC3T3-E1 cell proliferation and alkaline phosphatase activity in a dose-dependent manner. Collagen content was also increased by YPEP treatment. Furthermore, YPEP potently suppressed osteoclastogenesis from bone marrow-derived precursor cells. YPEP (100 µg/mL) abolished the formation of osteoclasts positive for tartrate-resistant acid phosphatase. OVX rats supplemented with YPEP showed an osteoprotective effect, as the bone mineral density and cortical thickness in the tibia were increased compared with the OVX controls. Moreover, histological data indicate that YPEP prevented the cancellous bone loss induced by ovariectomy. None of these protective effects were observed in casein-treated rats. CONCLUSIONS: The present study suggests that YPEP is a promising alternative to current therapeutic agents for the management of osteoporosis.

Anti-inflammatory activity of Motherwort (Leonurus sibiricus L.).

Motherwort (MW), a Korean folk medicine, has been applied to treat inflammatory disease. However, its effect on inflammatory cytokine release from mast cells is not well known. We investigated the anti- inflammatory effect of MW on the secretion of inflammatory cytokine such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 and IL-8 in human mast cell line (HMC-1). MW was treated in vitro before activation of HMC-1 cells with phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187. MW had no cytotoxic effects on HMC-1 cell viability. MW (1 mg/ml) inhibited PMA plus A23187-stimulated gene expression and production of TNF-alpha, IL-6, and IL-8. Stimulation with PMA plus A23187 induced NF-kappaB activation in HMC-1 cells, which was inhibited by MW (1 mg/ml). MW inhibited secretion of TNF-alpha, IL-6, and IL-8 possibly by inhibiting NF-kappaB activation. These results indicate that MW may be helpful in regulating inflammatory diseases.

Flower extract of Panax notoginseng attenuates lipopolysaccharide-induced inflammatory response via blocking of NF-kappaB signaling pathway in murine macrophages.

AIM OF THE STUDY: The root of Panax notoginseng (PN) is commonly used to treat chronic liver disease with its therapeutic abilities to stop haemorrhage in the circulation, while the PN flower (PN-F) is largely unknown in the biological activities on inflammation and mechanisms of its actions. In this study, the pharmacologic effects of PN-F methanol extract on inflammation were investigated to address potential therapeutic or toxic effects in LPS-stimulated mouse macrophage cells, RAW264.7 cells. MATERIALS AND METHODS: Production of NO, PGE2 and pro-inflammatory cytokines (TNF-alpha and IL-1beta) in supernatant, the expression of iNOS, COX-2 and cytokines, the phosphorylation of MAPK molecules (ERK1/2, JNK and p38 MAPK), and the activation of NF-kappaB in PN-F extract were assayed in LPS-stimulated RAW264.7 cells. RESULTS: PN-F extract significantly inhibited the productions of NO, PGE2, TNF-alpha and IL-1beta on the LPS-stimulated RAW264.7 cells. In addition, PN-F extract suppressed the mRNA and protein expressions of iNOS, COX-2, TNF-alpha and IL-1beta in LPS-stimulated RAW264.7 cells. The molecular mechanism of PN-F extract-mediated attenuation in RAW264.7 cells has close a relationship to suppressing the phosphorylation of MAPK molecules such as ERK1/2, JNK and p38 MAPK, and the translocation of NF-kappaB p65 subunit into nuclear. CONCLUSION: These results indicate that PN-F extract inhibits LPS-induced inflammatory response via the blocking of NF-kappaB signaling pathway in macrophages, and demonstrated that PN-F extract possesses anti-inflammatory properties in vitro.

Inhibitory effects of egg yolk soluble protein on bone resorption.

We determined the effects of yolk water-soluble protein (YSP) on bone resorption. YSP potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven by tumor necrosis factor-alpha (TNF-alpha). YSP (200 microg/ml) abolished the formation of tartarate-resistant acid phosphatase (TRAP)-positive osteoclasts. Furthermore, TNF-alpha induced TRAP activity was greatly inhibited by YSP (100 microg/ml) treatment. Our results suggest that YSP has therapeutic potential for bone-erosive diseases.

Cytotoxic components from the dried rhizomes of Zingiber officinale Roscoe.

Five compounds were isolated from the chloroform-soluble fraction of the methanolic extract of the dried rhizomes of Zingiber officinale (Zingiberaceae) through repeated column chromatography. Their chemical structures were elucidated as 4-, 6-, 8-, and 10-gingerols, and 6-shogaol using spectroscopic analysis. Among the five isolated compounds, 6-shogaol exhibited the most potent cytotoxicity against human A549, SK-OV-3, SK-MEL-2, and HCT15 tumor cells. 6-shogaol inhibited proliferation of the transgenic mouse ovarian cancer cell lines, C1 (genotype: p53(-/-), c-myc, K-ras) and C2 (genotype: p53(-/-), c-myc, Akt), with ED(50) values of 0.58 microM (C1) and 10.7 microM (C2).

Inhibitory effects of Angelicae Gigantis Radix on osteoclast formation.

Angelicae Gigantis Radix (AGR) is one of the most widely used herbal medications. AGR is the dried root of Angelica gigas Nakai (Umbelliferae), which is known as Korean angelica. This study investigated the effects of AGR on osteoclast formation using primary bone marrow cells. TNF-alpha treatment increased tartrate-resistant acid phosphatase (Trap) positive cells and Trap activity in bone marrow cells. However, AGR significantly decreased both TNF-alpha-induced Trap positive cells and Trap activity. RT-PCR analyses revealed that AGR decreased mRNA levels of Trap and matrix metalloproteinase-9 in TNF-alpha-treated bone marrow cells. In addition, AGR decreased TNF-alpha-induced activation of NF-kappaB. These results suggest that AGR has an inhibitory effect on the formation of osteoclasts and its effect is partially related to the NF-kappaB pathway.

Phlomis umbrosa root inhibits mast cell-dependent allergic reactions and inflammatory cytokine secretion.

The effect of an aqueous extract of Phlomis umbrosa Turcz. (Labiatae) root (PUAE) on mast cell-dependent allergic reactions and inflammatory cytokine secretion were investigated. PUAE (0.01-1 g/kg) inhibited compound 48/80-induced systemic allergic reaction. When PUAE was employed in a systemic allergic reaction test, the plasma histamine levels were reduced in a dose-dependent manner. PUAE (0.1 and 1 g/kg) also significantly inhibited the local allergic reaction activated by anti-dinitrophenyl (DNP) IgE. PUAE (0.001-1 mg/mL) dose-dependently inhibited the histamine release from rat peritoneal mast cells activated by compound 48/80 or anti-DNP IgE. PUAE (0.01-1 mg/mL) inhibited the secretion of interleukin (IL)-1beta in phorbol 12-myristate 13-acetate plus calcium ionophore A23187-stimulated human mast cell line (HMC-1) cells. PUAE (1 mg/mL) inhibited the gene expression and production of the main inflammatory cytokine, TNF-alpha, in HMC-1 cells. These results provide evidence that PUAE may be beneficial in the treatment of allergic diseases.

Effect of Gamiseunggal-Tang on immediate type allergic reaction in mice.

BACKGROUND & OBJECTIVE: The herbal formulation, Gamiseunggal-Tang (G-Tang) has long been used for various allergic diseases. The mechanism of its action is largely unknown. We carried out this study to determine the effect of G-Tang on the mast cell-mediated anaphylactic reactions in vivo and in vitro murine models. METHODS: In this study, the effects of G-Tang on the mast cell-mediated anaphylactic reactions were examined by using the ear swelling, histamine assay, and ELISA method in murine model. RESULTS: Anal administration of G-Tang showed dose-dependent inhibitory activity on the compound 48/80-induced ear swelling response (P<0.05) and histamine release (P<0.01). G-Tang (0.001-0.1 g/kg) significantly inhibited passive cutaneous anaphylaxis (P<0.05) in mice. The production of tumour necrosis factor-alpha (TNF-alpha) was also significantly inhibited (about 47.4%, at 0.1 mg/ml, P<0.01) by treatment of G-tang in anti-dinitrophenyl IgE antibodystimulated mast cells. INTERPRETATION & CONCLUSION: Findings of our study showed that G-Tang inhibited immediate type allergic reaction in a murine model and may be beneficial in the treatment of allergic inflammatory diseases.

Egg yolk soluble protein stimulates the proliferation and differentiation of osteoblastic MC3T3-E1 cells.

We determined the effects of yolk water-soluble protein (YSP) on bone formation in pre-osteoblastic MC3T3-E1 cells. YSP (50-5,000 microg/ml) increased cell proliferation and collagen content. Alkaline phosphatase (ALP) activity was also increased by YSP treatment. After enhancement of ALP activity, significant augmentation of calcification was observed. These results suggest that YSP is a promising agent for the prevention and treatment of bone loss.

Cytotoxic isoquinoline alkaloids from the aerial parts of Corydalis incisa.

Three known isoquinoline alkaloids were isolated from the chloroform-soluble fraction of the methanolic extract of the aerial parts of Corydalis incisa (Papaveraceae) through repeated column chromatography. Their chemical structures were elucidated as corynoline (1), corynoloxine (2) and 6-oxocorynoline (3) using spectroscopic analysis. Compounds 1-3 exhibited cytotoxicity against human A549, SK-OV-3, SK-MEL-2 and HCT15 tumor cells.

Protective effects of a novel herbal decoction on focal cerebral ischemia in a rodent model.

BACKGROUND: Herbs have been used to treat stroke and coma patient in traditional Korean medicine (TKM). The novel decoction, Guhpoongchungsimhwan (GCH), was developed on the basis of clinical data and TKM theory. METHODS: We examined the neuroprotective effect of GCH on cerebral ischemia. The middle cerebral artery occlusion (MCAO) model was used to produce cerebral ischemia in Sprague-Dawley rats. Subjects were treated with GCH (50 or 200 mg/kg) or vehicle alone (controls) 0 and 2 hours after MCAO. The functional status was tested 24 hours after MCAO by neurological examination (clinical score) and by series of motor function tasks (foot placement and parallel bar crossing). RESULTS: The infarct volume was determined by 2,3,5-triphenyltetrazolium chloride staining 24 hours after surgery, and the expression of cyclooxygenase-2 was determined by immunohistochemistry. The clinical score of the GCH-treated group (200 mg/kg) was significantly lower than that of the control group (p<0.05), indicating fewer neurological deficits. The impairment of motor functions induced by MCAO was significantly reduced by the administration of GCH (p<0.05). The infarct volume was significantly smaller in the GCH-treated group (203.1 +/- 40.2 mm(3), p<0.05), as compared to the control group (377.8 +/- 32.6 mm(3)). The level of motor function in the GCH-treated group was associated with reduced infarct volume. In the analysis of immunohistochemistry, GCH treatment markedly inhibited the ischemia-induced expression of PTGS2 (prostaglandin-endoperoxidase synthase 2) or cyclooxygenase 2 (COX2), which plays an important role in ischemic neuronal cell death. CONCLUSION: The results showed that GCH reduced the infarct size and the functional deficits in MCAO rats.

Panax ginseng increases hypoxia-induced down-regulated cellular response related genes in human neuroblastoma cells, SK-N-MC.

BACKGROUND: Many studies have suggested that hypoxia plays a crucial role in the pathogenesis of various neurological disorders. To determine protective effect of Panax ginseng (PG) on hypoxia (0.1% O(2))-induced cell death in human neuroblastoma cells SK-N-MC, we profiled the gene expression among hypoxia, PG-treated hypoxia and normoxia groups. METHODS: To determine protective effect on hypoxia-induced cytotoxicity of PG, we performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. We compared the gene expression profiles among hypoxia, PG-treated hypoxia (100 mug/ml, 6 hours) and normoxia groups using 8K human cDNA microarray analysis. Additionally, in order to identify differentially expressed genes between hypoxia and PG-treated hypoxia groups, hierarchical clustering of genes was also performed. RESULTS: MTT assay showed that PG protected hypoxia-induced cell death. In cDNA microarray analysis, hypoxia remarkably down-regulated IGF-II mRNA-binding protein 3 (IMP-3), integrin alpha 2 (ITGA2), syndecan binding protein (SDCBP), insulin-like growth factor binding protein 3 (IGBP3) and M-phase phosphoprotein 10 (MPHOSPH10), belonging to category of cellular physiologic response (global M<-3.5). In cluster analysis, 1428 genes exhibited differential expression levels between hypoxia and PG-treated hypoxia groups. Of them, the expressions of 11 genes were increased more than two-fold by PG treatment compared to those in hypoxia group. Particularly, of 11 genes, the expression levels of cellular physiologic response related genes such as MPHOSPH10, IMP-3 and SDCBP, which markedly down-regulated by hypoxia, are increased more than four-fold by PG treatment, compared to hypoxia group. CONCLUSION: In summary, hypoxia induced down-regulation of cellular physiologic response related genes in human neuroblastoma cells, SK-N-MC, and PG ameliorated the hypoxia-induced down-regulation of such genes. These results indicate possible usage of PG in hypoxia-induced neuronal injury including ischemia, trauma and degenerative diseases.

Effects of ephedrae herba on melanogenesis and gene expression profiles using cDNA microarray in B16 melanocytes.

Ephedrae Herba (EH) is obtained by drying the stems of Ephedra sinica Stapf (family Ephedraceae). EH has been used clinically to treat colds and to reduce edema in skin. The effects of EH on melanogenesis were studied in B16 murine melanocytes. The tyrosinase activity and melanin content were measured after incubation with EH. Both reverse transcription-polymerase chain reaction and cDNA microarray analysis were used to study the mechanism of EH action. EH decreased tyrosinase activity and melanin content in a dose-dependent manner. EH decreased microphthalmia-associated transcription factor (MITF) and tyrosinase gene expression. These effects were controlled by MITF-mediated regulation of tyrosinase gene expression. EH also altered the expression of about 100 other genes. These results suggest that EH may be used clinically to treat freckles and liver spots.

Anticancer effects of oligomeric proanthocyanidins on human colorectal cancer cell line, SNU-C4.

AIM: Oligomeric proanthocyanidins (OPC), natural polyphenolic compounds found in plants, are known to have antioxidant and anti-cancer effects. We investigated whether the anti-cancer effects of the OPC are induced by apoptosis on human colorectal cancer cell line, SNU-C4. METHODS: Colorectal cancer cell line, SNU-C4 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. The cytotoxic effect of OPC was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylt-etrazolium bromide (MTT) assay. To find out the apoptotic cell death, 4, 6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, reverse transcription-polymerase chain reaction (RT-PCR), and caspase-3 enzyme assay were performed. RESULTS: In this study, cytotoxic effect of OPC on SNU-C4 cells appeared in a dose-dependent manner. OPC treatment (100 microg/mL) revealed typical morphological apoptotic features. Additionally OPC treatment (100 microg/mL) increased level of BAX and CASPASE-3, and decreased level of BCL-2 mRNA expression. Caspase-3 enzyme activity was also significantly increased by treatment of OPC (100 microg/mL) compared with control. CONCLUSION: These data indicate that OPC caused cell death by apoptosis through caspase pathways on human colorectal cancer cell line, SNU-C4.

Citrus Reticulata blanco induces apoptosis in human gastric cancer cells SNU-668.

Citrus fruits have been known to reduce the proliferation of many cancer cells. The antiproliferative effects of Citrus reticulata Blanco (CR) extract, the immature tangerine peel, on human gastric cancer cell line SNU-668 were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, 4,6-diamidineo-2-phenylindole staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, reverse transcription-polymerase chain reaction expressions of BCL-2, BAX and CASP-3 genes, caspase-3 activity, and immunocytochemistry of caspase-3. From the results of the morphological and biochemical assays, CR (50 microg/ml) increased the apoptosis of human gastric cancer cells with typical apoptotic characteristics, including morphological changes of chromatin condensation and apoptotic body formation. CR (50 microg/ml) reduced the expression of BCL-2, whereas the expression of BAX and CASP-3 was increased compared with the control group. Furthermore, caspase-3 activity and caspase-3 protein expression in the CR-treated group was significantly increased compared with that in control group. These results suggest that CR may induce the apoptosis through the caspase-3 pathway in human gastric cancer cells.

Citri Reticulatae Viride Pericarpium extract induced apoptosis in SNU-C4, human colon cancer cells.

Citri Reticulatae Viride Pericarpium (CR) has been used traditionally in Korea to promote the Liver Qi activity and the function of digestive system. We investigated whether the immature peels of Citrus reticulata Blanco (Rutaceae) induced cell-death on SNU-C4, human colon cancer cells. Cytotoxicity of CR was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cell death was identified as apoptosis using 4,6-diamidineo-2-phenylindole (DAPI) staining and terminal deoxy-nucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. The expression of pro-apoptotic gene, Bax, was increased and the expression of anti-apoptotic gene, Bcl-2, was decreased by CR-treatment. The expression and activity of major apoptotic gene, caspase-3 was significantly increased by CR-treatment. Considering the above results, CR could induce the apoptosis on SNU-C4, human colon cancer cells via Bax-related caspase-3 activation. And it might provide the experimental data for the future clinical use of CR on colon cancer.

Susceptibility for ischemic stroke in Korean population is associated with polymorphisms of the interleukin-1 receptor antagonist and tumor necrosis factor-alpha genes, but not the interleukin-1beta gene.

Enhanced release of proinflammatory cytokines may contribute to the pathogenesis of ischemic stroke. Interleukin-1 receptor antagonist (IL-1Ra) is an anti-inflammatory cytokine, and tumor necrosis factor (TNF)-alpha and IL-1beta are proinflammatory cytokine. To determine the role of cytokines in genetic susceptibility to ischemic stroke, we genotyped ischemic stroke patients (n = 152) and the healthy control subjects (n = 165) for IL-1Ra, TNF-alpha and IL-1beta polymorphism by polymerase chain reaction-restriction fragment length polymorphism methods. The analysis shown the association of IL1RN*1, IL1RN*2 allele (IL1RN*1, OR=0.44, P = 0.0206 IL1RN*2, OR=2.90, P = 0.0141) and TNF1, TNF2 allele (TNF1, OR=2.16, P = 0.0225; TNF2, OR=2.16, P = 0.0225) to ischemic stroke. However, the genetic polymorphism of IL-1beta was not associated with ischemic stroke. Our results suggest that IL-1Ra and TNF-alpha gene polymorphism is associated with the susceptibility to ischemic stroke.