Effects of Chaihu-Shugan-San on Small Intestinal Interstitial Cells of Cajal in Mice.

Chaihu-Shugan-San (CSS) has been widely used as an alternative treatment for gastrointestinal (GI) diseases in East Asia. Interstitial cells of Cajal (ICCs) are pacemakers in the GI tract. In the present study, we examined the action of CSS on pacemaker potentials in cultured ICCs from the mouse small intestine in vitro and on GI motility in vivo. We used the electrophysiological methods to measure the pacemaker potentials in ICCs. GI motility was investigated by measuring intestinal transit rates (ITR). CSS inhibited the pacemaker potentials in a dose-dependent manner. The capsazepine did not block the effect of CSS. However, the effects of CSS were blocked by glibenclamide. In addition, NG-nitro-L-arginine methyl ester (L-NAME) also blocked the CSS-induced effects. Pretreatment with SQ-22536 or with KT-5720 did not suppress the effects of CSS; however, pretreatment with ODQ or KT-5823 did. Furthermore, CSS significantly suppressed murine ITR enhancement by neostigmine in vivo. These results suggest that CSS exerts inhibitory effects on the pacemaker potentials of ICCs via nitric oxide (NO)/cGMP and ATP-sensitive K+ channel dependent and transient receptor potential vanilloid 1 (TRPV1) channel independent pathways. Accordingly, CSS could provide the basis for the development of new treatments for GI motility dysfunction.

Hemistepsin A alleviates liver fibrosis by inducing apoptosis of activated hepatic stellate cells via inhibition of nuclear factor-κB and Akt.

Hemistepsin A (HsA), isolated from Hemistepta lyrata (Bunge) Bunge, has the ability to ameliorate hepatitis in mice. However, the effects of H. lyrata and HsA on other types of liver disease have not been explored. In this report, we investigated the effects of H. lyrata and HsA on liver fibrosis and the underlying molecular mechanisms in activated hepatic stellate cells (HSCs). Based on cell viability-guided isolation, we found HsA was the major natural product responsible for H. lyrata-mediated cytotoxicity in LX-2 cells. HsA significantly decreased the viability of LX-2 cells and primary activated HSCs, increased the binding of Annexin V, and altered the expression of apoptosis-related proteins, suggesting that HsA induces apoptosis in activated HSCs. HsA reduced the phosphorylation of IKKε and the transactivation of nuclear factor-κB (NF-κB). Moreover, HsA decreased the phosphorylation of Akt and its downstream signaling molecules. Transfection experiments suggested that inhibition of NF-κB or Akt is essential for HsA-induced apoptosis of HSCs. In a CCl4-induced liver fibrosis model, HsA administration significantly decreased ALT and AST activities. Furthermore, HsA attenuated CCl4-mediated collagen deposits and profibrogenic genes expression in hepatic tissue. Thus, HsA may serve as a natural product for managing liver fibrosis through inhibition of NF-κB/Akt-dependent signaling.

The Traditional Medicine Banhasasim-Tang Depolarizes Pacemaker Potentials of Cultured Interstitial Cells of Cajal through M3 Muscarinic and 5-HT3 Receptors in Murine Small Intestine.

BACKGROUND: Banhasasim-tang (BHSST) is a classic herbal formulation in traditional Chinese medicine widely used for gastrointestinal (GI) tract motility disorder. We investigated the effects of BHSST on the pacemaker potentials of cultured interstitial cells of Cajal (ICCs) in small intestine in vitro and its effects on GI motor functions in vivo. METHODS: We isolated ICCs from the small intestines and recorded pacemaker potentials in cultured ICCs with the whole-cell patch-clamp configuration in vitro. Intestinal transit rates (ITR%) were investigated in normal mice and GI motility dysfunction (GMD) mouse models in vivo. RESULTS: BHSST (20-50 mg/mL) depolarized pacemaker potentials and decreased their amplitudes in a concentration-dependent manner. Pretreatment with methoctramine (a muscarinic M2 receptor antagonist) did not inhibit BHSST-induced pacemaker potential depolarization. However, when we applied 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; a muscarinic M3 receptor antagonist), BHSST-induced effects were blocked. Pretreatment with Y25130 (a 5-HT3 receptor antagonist) blocked BHSST-induced effects in ICCs. In addition, when we applied 4-DAMP and Y25130 together, BHSST-induced effects were completely blocked. Pretreatment with Ca2+-free solution or thapsigargin inhibited BHSST-induced effects. Moreover, BHSST blocked both the transient receptor potential melastatin (TRPM) 7 and voltage-sensitive calcium-activated chloride (anoctamin-1, ANO1) channels. In normal mice, ITR% values were significantly increased by BHSST in a dose-dependent manner. The ITR% of GMD mice was significantly reduced relative to those of normal mice, which were significantly reversed by BHSST in a dose-dependent manner. CONCLUSION: These results suggested that BHSST depolarizes the pacemaker potentials of ICCs in a dose-dependent manner through the M3 and 5-HT3 receptors via internal and external Ca2+-dependent and TRPM7- and ANO1-independent pathways in vitro. Moreover, BHSST increased ITR% in vivo in normal mice and GMD mouse models. Taken together, the results of this study showed that BHSST had the potential for development as a prokinetic agent in GI motility function.

Rikkunshito Depolarizes Pacemaker Potentials of Cultured Interstitial Cells of Cajal through Ghrelin Receptors in Murine Small Intestine.

BACKGROUND: Rikkunshito has been used to treat gastrointestinal (GI) disorders. The purpose of this study was to investigate the effects of Rikkunshito, a traditional Japanese herbal medicine, on the pacemaker potentials of interstitial cells of Cajal (ICCs) from the small intestines of mice. METHODS: We isolated ICCs from the small intestines of mice, and the whole-cell patch-clamp configuration was used to record the pacemaker potentials in cultured ICCs and membrane currents. RESULTS: Rikkunshito depolarized ICC pacemaker potentials in a dose-dependent manner. Pretreatment with GSK1614343 or (D-Lys3)-growth hormone-releasing peptide-6 inhibited Rikkunshito-induced depolarization of pacemaker potentials. Intracellular GDP-ß-S inhibited Rikkunshito-induced effects. In Ca2+-free solution or in the presence of thapsigargin, Rikkunshito did not depolarize pacemaker potentials. Moreover, in the presence of U-73122 or xestospongin C, Rikkunshito-induced effects were inhibited. However, in the presence of staurosporine, Go6976 or Rottlerin, Rikkunshito depolarized pacemaker potentials. Furthermore, Rikkunshito inhibited both transient receptor potentials melastatin 7 (TRPM7) and Ca2+-activated Cl- channels (ANO1) currents. CONCLUSION: Rikkunshito depolarized pacemaker potentials of ICCs via ghrelin receptor and G protein through internal or external Ca2+-, phospholipase C-, and inositol triphosphate-dependent and protein kinase C-, TRPM7-, and ANO1-independent pathways. The study shows that Rikkunshito may alleviate GI motility disorders through its depolarizing effects on ICCs.

Sipjeondaebo-tang Alleviates Oxidative Stress-Mediated Liver Injury through Activation of the CaMKK2-AMPK Signaling Pathway.

Sipjeondaebo-tang (SDT) is used frequently as a herbal prescription to treat deficiency syndromes in traditional Korean medicine. We investigated the hepatoprotective effects of SDT against oxidative stress and attempted to clarify the underlying molecular mechanisms. SDT pretreatment reduced arachidonic acid (AA) plus iron-mediated cytotoxicity in a concentration-dependent manner and prevented changes in apoptosis-related protein expression. In addition, SDT pretreatment significantly reduced glutathione depletion, hydrogen peroxide production, and mitochondrial dysfunction via treatment with AA plus iron. SDT increased the phosphorylation of AMP-activated protein kinase (AMPK) in accordance with the phosphorylation of Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2). Experiments using an AMPK chemical inhibitor (Compound C) or CaMKK2 chemical inhibitor (STO-609) suggested that the CaMKK2-AMPK signaling pathway contributes to SDT-mediated protection of mitochondria and cells. Moreover, administration of SDT for 4 consecutive days to mice significantly reduced the alanine aminotransferase and aspartate aminotransferase activities induced by carbon tetrachloride, and the numbers of degenerated hepatocytes, infiltrated inflammatory cells, nitrotyrosine-positive cells, and 4-hydroxynonenal-positive cells in liver tissue. Therefore, SDT protects hepatocytes from oxidative stress via CaMKK2-dependent AMPK activation and has the therapeutic potential to prevent or treat oxidative stress-related liver injury.

Effects of Gamisoyo-San Decoction, a Traditional Chinese Medicine, on Gastrointestinal Motility.

BACKGROUND: Gamisoyo-San decoction (GSS), a traditional Chinese medicine, has been used to treat various gastrointestinal (GI) symptoms and diseases such as functional dyspepsia. The purpose of this study was to investigate the effect of GSS on GI motility functions in mice. METHODS: Percent intestinal transit rate (ITR%) and gastric emptying (GE) values were measured using Evans Blue and phenol red, respectively, in normal mice and in mice with experimentally induced GI motility dysfunction (GMD). RESULTS: In normal mice, GSS (0.01-1 g/kg) induced higher GE values than non-treated controls. Also, GSS could increase GE in loperamide-induced and cisplatin-induced GE delay models. In addition, GSS increased ITR% in a dose-dependent manner. Loperamide decreased ITR% and GSS recovered this loperamide-induced decrease in ITR%. To examine the effect of GSS on GMD, we used acetic acid (AA)-induced and streptozotocin (STZ)-induced mouse GMD models. The AA mouse model showed a significant decrease in ITR%. However, intragastric treatment with GSS significantly recovered this inhibition. Furthermore, STZ-induced diabetic mice showed a significant reduction in ITR%, which was also significantly inhibited by GSS. CONCLUSION: These results demonstrate that GSS can modulate bowel activity and that it could be used as a gastroprokinetic agent in the treatment of GI motility diseases.

Modulation of Pacemaker Potentials in Murine Small Intestinal Interstitial Cells of Cajal by Gamisoyo-San, a Traditional Chinese Herbal Medicine.

BACKGROUND: The Gamisoyo-san (GSS) has been used for -improving the gastrointestinal (GI) symptoms. The purpose of this study was to investigate the effects of GSS, a traditional Chinese herbal medicine, on the pacemaker potentials of mouse small intestinal interstitial cells of Cajal (ICCs). METHODS: ICCs from the small intestines were dissociated and cultured. Whole-cell patch-clamp configuration was used to record pacemaker potentials and membrane currents. RESULTS: GSS depolarized ICC pacemaker potentials in a dose-dependent manner. Pretreatment with 4-diphenylacetoxypiperidinium iodide completely inhibited GSS-induced pacemaker potential depolarizations. Intracellular GDP-ß-S inhibited GSS-induced effects, and in the presence of U-73122, GSS-induced effects were inhibited. Also, GSS in the presence of a Ca2+-free solution or thapsigargin did not depolarize pacemaker potentials. However, in the presence of calphostin C, GSS slightly depolarized pacemaker potentials. Furthermore, GSS inhibited both transient receptor potential melastatin7 and Ca2+-activated Cl- channel (anoctamin1) currents. CONCLUSION: GSS depolarized pacemaker potentials of ICCs via G protein and muscarinic M3 receptor signaling pathways and through internal or external Ca2+-, phospholipase C-, and protein kinase C-dependent and transient receptor potential melastatin 7-, and anoctamin 1-independent pathways. The study shows that GSS may regulate GI tract motility, suggesting that GSS could be a basis for developing novel prokinetic agents for treating GI motility dysfunctions.

Fucoxanthin, the constituent of Laminaria japonica, triggers AMPK-mediated cytoprotection and autophagy in hepatocytes under oxidative stress.

BACKGROUND: Laminaria japonica has frequently been used as a food supplement and drug in traditional oriental medicine. Among the major active constituents responsible for the bioactivities of L. japonica, fucoxanthin (FX) has been considered as a potential antioxidant. This study was conducted to examine the effects of L. japonica extract (LJE) or FX against oxidative stress on hepatocytes and to elucidate the overall their cellular mechanisms of the effects. METHODS: We constructed an in vitro model with the treatment of arachidonic acid (AA) + iron in HepG2 cells to stimulate the oxidative damage. The cells were pre-treated with LJE or FX for 1 h, and incubated with AA + iron. The effect on oxidative damage and cellular mechanisms of LJE or FX were assessed by cytological examination and several biochemical assays under conditions with or without kinase inhibitiors. RESULTS: LJE or FX pretreatment effectively blocked the pathological changes caused by AA + iron treatment, such as cell death, altered expression of apoptosis-related proteins such as procaspase-3 and poly (ADP-ribose) polymerase, and mitochondria dysfunction. Moreover, FX induced AMPK activation and AMPK inhibitor, compound C, partially reduced the protective effect of FX on mitochondria dysfunction. Consistent with AMPK activation, FX increased the protein levels of autophagic markers (LC3II and beclin-1) and the number of acridine orange stained cells, and decreased the phosphorylation of mTOR and simultaneously increased the phosphorylation of ULK1. And the inhibition of autophagy by 3-methylanine or bafilomycin A1 partially inhibited the protective effect of FX on mitochondria dysfunction. CONCLUSION: These findings suggest that FX have the function of being a hepatic protectant against oxidative damages through the AMPK pathway for the control of autophagy.

Epimedium koreanum Ameliorates Oxidative Stress-Mediated Liver Injury by Activating Nuclear Factor Erythroid 2-Related Factor 2.

Oxidative stress induced by reactive oxygen species is the main cause of various liver diseases. This study investigated the hepatoprotective effect of Epimedium koreanum Nakai water extract (EKE) against arachidonic acid (AA)[Formula: see text][Formula: see text][Formula: see text]iron-mediated cytotoxicity in HepG2 cells and carbon tetrachloride (CCl4-)-mediated acute liver injury in mice. Pretreatment with EKE (30 and 100[Formula: see text][Formula: see text]g/mL) significantly inhibited AA[Formula: see text][Formula: see text][Formula: see text]iron-mediated cytotoxicity in HepG2 cells by preventing changes in the expression of cleaved caspase-3 and poly(ADP-ribose) polymerase. EKE attenuated hydrogen peroxide production, glutathione depletion, and mitochondrial membrane dysfunction. EKE also increased the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), transactivated anti-oxidant response element harboring luciferase activity, and induced the expression of anti-oxidant genes. Furthermore, the cytoprotective effect of EKE against AA[Formula: see text][Formula: see text][Formula: see text]iron was blocked in Nrf2 knockout cells. Ultra-performance liquid chromatography analysis showed that EKE contained icariin, icaritin, and quercetin; icaritin and quercetin were both found to protect HepG2 cells from AA[Formula: see text][Formula: see text][Formula: see text]iron via Nrf2 activation. In a CCl4-induced mouse model of liver injury, pretreatment with EKE (300[Formula: see text]mg/kg) for four consecutive days ameliorated CCl4-mediated increases in serum aspartate aminotransferase activity, histological activity index, hepatic parenchyma degeneration, and inflammatory cell infiltration. EKE also decreased the number of nitrotyrosine-, 4-hydroxynonenal-, cleaved caspase-3-, and cleaved poly(ADP-ribose) polymerase-positive cells in hepatic tissues. These results suggest EKE is a promising candidate for the prevention or treatment of oxidative stress-related liver diseases via Nrf2 activation.

The physico-chemical alteration of lovastatin and enhanced antioxidant effect of Bacillus subtilis fermented-red yeast rice product.

Red yeast rice product (RYP) has been used as a food supplement because of its lipid lowering, and in food additives as a natural colorant. Lovastatin of RYP is a hypolipidemic commercial drug. To enhance the beneficial effects of RYP, we performed a bioconversion with Bacillus subtilis. This B. subtilis-fermentation process of RYP increased the ratio of the active open-hydroxyl acid form and the prodrug lactone form of lovastatin, which is a potent cholesterol synthesis inhibitor. 3(2H)-benzofuranone was newly produced in the fermented red yeast rice product (FRYP) as analyzed by GC-MS. FRYP increased the free radical scavenging activity compared with RYP. FRYP blocked xanthine oxidase (XO)-induced oxidative cytotoxicity and inhibited the H2O2-induced intracellular ROS in cells. This is the first study to illustrate that B. subtilis-fermented FRYP is useful for facilitating the alteration in the physico-chemical property of lovastatin and enhancing antioxidant activity, which may have greater pharmacological activity.

Cheongsangbangpung-tang ameliorated the acute inflammatory response via the inhibition of NF-κB activation and MAPK phosphorylation.

BACKGROUND: Cheongsangbangpung-tang (CBT) is a traditional herbal formula used in Eastern Asia to treat heat-related diseases and swellings in the skin. The present study was conducted to evaluate the anti-inflammatory effects of cheongsangbangpung-tang extract (CBTE) both in vitro and in vivo. METHODS: The in vitro effects of CBTE on the lipopolysaccharide (LPS)-induced production of inflammation-related proteins were examined in RAW 264.7 cells. The levels of nitric oxide (NO) were measured with the Griess reagent. Inflammatory cytokines and prostaglandin E2 (PGE2) were detected using the enzyme-linked immunosorbent assay (ELISA) method. Inflammation-related proteins were detected by Western blot. The effect of CBTE on acute inflammation in vivo was evaluated using carrageenan (CA)-induced paw oedema. To evaluate the anti-inflammatory effect, paw oedema volume, thickness of the dorsum and ventrum pedis skin, number of infiltrated inflammatory cells, and number of COX-2-, iNOS-immunoreactive cells were measured. RESULTS: In an in vitro study, CBTE inhibited the production of NO and PGE2 and also decreased the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) activity, interleukin (IL)-1ß, IL-6 and tumuor necrosis factor-α. In LPS-activated macrophages, nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) signalling is a pivotal pathway in the inflammatory process. These plausible molecular mechanisms increased the phosphorylation of I-κBα, while the activation of NF-κB and the phosphorylation of MAPK by LPS were blocked by CBTE treatment. In our in vivo study, a CA-induced acute oedematous paw inflammation rat model was used to evaluate the anti-inflammatory effect of CBTE. CBTE significantly reduced the increases in paw swelling, skin thicknesses, infiltrated inflammatory cells and iNOS-, COX-2 positive cells induced by CA injection. CONCLUSIONS: Based on these results, CBTE should favourably inhibit the acute inflammatory response through modulation of NF-κB activation and MAPK phosphorylation. Furthermore, the inhibition of CBTE in rat paw oedema induced by CA is considered to be clear evidence that CBTE may be a useful source to treat inflammation.

Liqustri lucidi Fructus inhibits hepatic injury and functions as an antioxidant by activation of AMP-activated protein kinase in vivo and in vitro.

Medicinal herbs are used to treat or prevent various diseases, and function to regulate protective mechanisms as nutraceuticals. Fructus Ligustri lucidi is the fruit of Ligustrum lucidum and has been used for its tonic effects on the liver. This study was designed to examine the effects of Fructus Ligustri lucidi water extract (FLL) against severe oxidative stress and mitochondrial impairment in vivo and in vitro and to elucidate its cellular mechanisms of action. Treatment of HepG2 cells with arachidonic acid (AA) + iron successfully induced oxidative stress and apoptosis, as indicated by depletion of glutathione, formation of ROS, decreses in mitochondrial membrane potential (Δψm), and altered expression of apoptosis-related proteins, such as procaspase-3 and Bcl-xL. FLL treatment significantly blocked these pathological changes and the mitochondrial dysfunction caused by AA + iron, which were similar with the effect of aminoimidazole-carboxamide-ß-d-ribofuranoside (AICAR). Moreover, FLL induced the activation of AMP-activated protein kinase (AMPK), which was mediated by its upstream kinase LKB1. Inhibition or activation of AMPK revealed the role of AMPK in cellular protection conferred by FLL in LKB1-deficient cells. In mice, oral administration of 100 mg/kg FLL activated AMPK in the liver, and protected against oxidative stress and liver injury induced by CCl4 injection. Among the components of FLL, chlorogenic acid was found to be responsible for the protection of hepatocytes against AA + iron-induced cellular damage. Overall, our results confirmed that FLL has the ability to protect hepatocytes against oxidative injury through regulation of the AMPK signaling pathway.

Anti-inflammatory Steroid from Phragmitis rhizoma Modulates LPS-Mediated Signaling Through Inhibition of NF-κB Pathway.

Lipopolysaccharides (LPS) strongly stimulate immune cells, and unabated activation of immune system by LPS may lead to an exacerbation of sickness and depression. In this study, stigmasta-3,5-dien-7-one (ST) was isolated from Phragmitis rhizoma as a negative regulator of LPS-induced inflammation in macrophages. ST effectively reduced nitric oxide (NO), prostaglandin E2, and pro-inflammatory cytokine levels, which were markedly raised by LPS treatment. In addition, ST blocked the nuclear factor-kappa B (NF-κB) signaling pathway via down-regulation of phospho-p38 mitogen-activated protein kinase and phosphorylation and degradation of the inhibitor of NF-κB α. To our knowledge, this is the first study showing anti-inflammatory activities of ST isolated from Phragmitis rhizoma.

The traditional herbal medicine, Ge-Gen-Tang, inhibits pacemaker potentials by nitric oxide/cGMP dependent ATP-sensitive K(+) channels in cultured interstitial cells of Cajal from mouse small intestine.

ETHNOPHARMACOLOGICAL RELEVANCE: Ge-Gen-Tang (GGT) is a traditional Chinese medicinal formula composed of Puerariae radix (Pueraria lobata Ohwi), Ephedrae Herba (Ephedra sinica Stapf), Cinnamomi Ramulus (Cinnamomum cassia Blume), Paeoniae Radix (Paeonia lactiflora Pallas), Glycyrrhizae Radix preparata (Glycyrrhiza uralensis Fischer), Zingiberis Rhizoma (Zingiber officinale Roscoe), and Zizyphi Fructus (Ziziphus jujuba Mill. var. inermis Rehder) and is widely used to ameoliorate the symptoms of gastrointestinal (GI) disorders related to diarrhea and intestinal mucosal immunity and for anti-cold, antipyretic and analgesic in Eastern Asia. AIM OF THE STUDY: Interstitial cells of Cajal (ICCs) are pacemaker cells in the GI tract that generate rhythmic oscillations in membrane potentials known as slow waves. We investigated the effects of GGT on pacemaker potentials in cultured ICCs from the mouse small intestine, and sought to identify the receptors and the action mechanisms involved. MATERIALS AND METHODS: Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues. All experiments on ICCs were performed on within 12h after culture. A whole-cell patch-clamp configuration was used to record potentials (current clamp) from cultured ICCs. Intracellular Ca(2+) ([Ca(2+)]i) increase was studied in cultured ICCs using fura-2AM. All of the experiments were performed at 30-32°C. RESULTS: Under the current clamping mode, GGT decreased the amplitude and frequency of pacemaker potentials; however, these effects were blocked by intracellular GDPßS, a G-protein inhibitor, and glibenclamide, a specific ATP-sensitive K(+) channels blocker. Prazosin (α1-adrenoceptor antagonist) and butoxamine (ß2-adrenoceptor antagonist) did not block the GGT-induced effects, whereas atenolol (ß1-adrenoceptor antagonist) blocked the GGT-induced effects. Also, yohimbine (α2-adrenoceptor antagonist) partially blocked the GGT-induced effects. Pretreatment with SQ-22536, an adenylate cyclase inhibitor, did not block the GGT-induced effects, whereas pretreatment with ODQ, a guanylate cyclase inhibitor, or L-NAME, an inhibitor of nitric oxide (NO) synthase, did. Additionally, [Ca(2+)]i analysis showed that GGT decreased [Ca(2+)]i. CONCLUSION: These results suggest that GGT inhibits pacemaker potentials in ICCs in a G protein-, cGMP- and NO-dependent manner through stimulation of α2 and ß1-adrenoceptors.

Biomolecular evidence of anti-inflammatory effects by Clematis mandshurica Ruprecht root extract in rodent cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Clematis mandshurica Ruprecht root is widely used in Asia as an analgesic and anti-inflammatory agent. This research investigated the anti-inflammatory effects of Clematis mandshurica Ruprecht root extract (CRE) using RAW 264.7 macrophage cells and carrageenan- (CA-) induced rat paw edema. MATERIALS AND METHODS: Production of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, nitric oxide (NO) and prostaglandin E2 (PGE2) in the culture supernatant, mRNA expression of TNF-α, IL-1ß, IL-6, iNOS and COX-2, protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in the extract were assayed. In addition, the effect of CRE on acute inflammation in vivo was observed using CA-induced rat hind paw edema assay. The changes on the histopathology and histomorphometry of hind paw skins-dorsum and ventrum pedis were observed using CA-treated rats. RESULTS: Treatment with CRE (0.25, 0.5, and 1 mg/mL) resulted in inhibited levels of protein expression of lipopolysaccharide- (LPS-) induced iNOS, COX-2, NF-κB, and MAPKs (ERK, JNK, and p38) as well as production of TNF-α, IL-1ß, IL-6, NO, and PGE2 induced by LPS. Consistent with these results, CRE reduced the LPS-induced expressions of these cytokines, iNOS and COX-2 at the mRNA levels in a dose-dependent manner. In particular, results of the CA-induced rat hind paw edema assay showed an anti-edema effect of CRE. In addition, treatment with CRE resulted in dose-dependent inhibition of CA-induced increases of skin thickness, mast cell degranulation, and infiltrated inflammatory, TNF-α, IL-1ß, iNOS, and COX-2-positive cells in both dorsum and ventrum pedis skin, respectively. CONCLUSIONS: These results demonstrate that CRE exhibits anti-inflammatory activities via decreasing production of pro-inflammatory mediators through suppression of the pathways of NF-κB and MAPKs in LPS-induced macrophage cells. In addition, results of the CA-induced rat hind paw edema assay show an anti-edema effect of CRE. Our findings also support the traditional use of CRE in the inflammatory symptoms of rheumatic arthritis and acute icteric hepatitis. Thus, CRE may have therapeutic potential for a variety of inflammation-mediated diseases and may be developed into potent anti-inflammatory drugs.

O-Methylated flavonol isorhamnetin prevents acute inflammation through blocking of NF-κB activation.

Here, we isolated isorhamnetin, a natural 3'-O-methylated flavonoid, from water dropwort (Oenanthe javanica, Umbelliferae) and investigated its ability to protect against acute inflammation in vivo and in vitro. To induce paw swelling, the hind paw of each rat was injected with a carrageenan 1h after vehicle or isorhamnetin treatment. In vitro effect and mechanism studies were performed in lipopolysaccharide (LPS)-activated macrophages. Administration of isorhamnetin markedly inhibited the swelling volume and the thickness of hind paws. Moreover, isorhamnetin significantly reduced inflammatory cell infiltration and pro-inflammatory gene expression in rats. Isorhamnetin pretreatment inhibited inducible nitric oxide synthase (iNOS) expression and NO release in LPS-stimulated cells. Activation of nuclear factor-kappa B (NF-κB) and activating protein-1 (AP-1) is the key step in the iNOS gene induction. Isorhamnetin specifically inhibited NF-κB luciferase activity, but not AP-1. Pretreatment with isorhamnetin suppressed NF-κB nuclear translocation in accordance with decreased phosphorylation and degradation of inhibitory-κB. Consistently, TNF-α, IL-1ß and IL-6 expression, representative NF-κB target genes, were almost completely prohibited by isorhamnetin. Furthermore, isorhamnetin inhibited LPS-induced JNK and AKT/IKKα/ß phosphorylation. Our results suggest that isorhamnetin inhibited JNK, and AKT/IKKα/ß activation, leading to NF-κB inactivation, which might contribute to the inhibition of the acute inflammatory response.

Roots of Erigeron annuus Attenuate Acute Inflammation as Mediated with the Inhibition of NF- κ B-Associated Nitric Oxide and Prostaglandin E2 production.

Erigeron annuus is a naturalized plant belonging to Compositae (asteraceae) family, which is called the annual fleabane, and commonly found at meadows and roadside. This study investigated the anti-inflammatory effects of the extract of E. annuus roots (EER), as assessed by the paw edema formation and histological analysis in rat, and the productions of nitric oxide (NO), prostaglandin E2 (PGE2), and pro-inflammatory cytokines in Raw264.7 murine macrophages. Carrageenan treatment promoted infiltration of inflammatory cells and caused swelling in the hind paw. Oral administrations of EER (0.3 g/kg and 1 g/kg) attenuated acute inflammation similar to the result using dexamethasone (1 mg/kg). Treatment of macrophages with lipopolysaccharide (LPS) simulated inflammatory condition: LPS significantly increased the productions of NO, PGE2, and proinflammatory cytokines. EER suppressed activation of macrophages, preventing the induction of iNOS and COX-2 protein expressions. LPS treatment induced phosphorylation of I- κ B α and increased the level of nuclear NF- κ B protein, both of which were suppressed by concomitant treatment of EER. In conclusion, EER ameliorated acute inflammation in rats, and the induction of NO, PGE2, and proinflammatory cytokines in Raw264.7 cells. EER's effects may be associated with its inhibition of NF- κ B activation, suggesting its effect on inflammatory diseases.

Inhibitory effects of traditional herbal formula pyungwi-san on inflammatory response in vitro and in vivo.

Pyungwi-san (PWS) is a traditional basic herbal formula. We investigated the effects of PWS on induction of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), pro-inflammatory cytokines (interleukin-6 (IL-6) and tumor necrosis factor- α (TNF- α )) and nuclear factor-kappa B (NF- κ B) as well as mitogen-activated protein kinases (MAPKs) in lipopolysaccharide-(LPS-) induced Raw 264.7 cells and on paw edema in rats. Treatment with PWS (0.5, 0.75, and 1 mg/mL) resulted in inhibited levels of expression of LPS-induced COX-2, iNOS, NF- κ B, and MAPKs as well as production of prostaglandin E2 (PGE2), nitric oxide (NO), IL-6, and TNF- α induced by LPS. Our results demonstrate that PWS possesses anti-inflammatory activities via decreasing production of pro-inflammatory mediators through suppression of the signaling pathways of NF- κ B and MAPKs in LPS-induced macrophage cells. More importantly, results of the carrageenan-(CA-) induced paw edema demonstrate an anti-edema effect of PWS. In addition, it is considered that PWS also inhibits the acute edematous inflammations through suppression of mast cell degranulations and inflammatory mediators, including COX-2, iNOS and TNF- α . Thus, our findings may provide scientific evidence to explain the anti-inflammatory properties of PWS in vitro and in vivo.

Bojesodok-eum, a Herbal Prescription, Ameliorates Acute Inflammation in Association with the Inhibition of NF-κB-Mediated Nitric Oxide and ProInflammatory Cytokine Production.

Bojesodok-eum (BSE) is a herbal prescription consisting of Coptidis Rhizoma and Scutellariae Radix as main components. This paper investigated the effects of BSE on the induction of nitric oxide (NO), prostaglandin E(2) (PGE(2)), and proinflammatory cytokines that are caused by lipopolysaccharide (LPS) in murine macrophage cell line and on the paw edema formation in animals. Administration of BSE (0.3 g/kg and 1 g/kg) in rats significantly inhibited carrageenan-induced paw edema formation, as did dexamethasone, an anti-inflammatory positive control drug. In cell model, treatment of BSE decreased the production of NO and PGE(2) in RAW264.7 cells stimulated by LPS. BSE also inhibited the expression of iNOS and COX-2 protein as well as COX activity in a concentration-dependent manner. Consistently, BSE suppressed the ability of LPS to produce TNF-α, interleukin-1ß, and interleukin-6. LPS treatment induced nuclear NF-κB level and I-κBα phosphorylation, which were inhibited subsequent treatment of BSE, suggesting its repression of LPS-inducible NF-κB activation. BSE abrogated the induction of NO, PGE(2), and proinflammatory cytokines, as well as iNOS and COX-2 protein expression in RAW264.7 cells stimulated by LPS as mediated with NF-κB inhibition.

Anti-inflammatory effects of bangpungtongsung-san, a traditional herbal prescription.

Bangpungtongsung-san (BPTS), a traditional oriental herbal prescription, is widely used for expelling wind, draining heat, and providing general improvement to the immune system. In this study, we investigated the effects of BPTS on induction of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), proinflammatory cytokines, nuclear factor-kappa B (NF-κB), and mitogen-activated protein kinases (MAPKs) in lipopolysaccharide- (LPS- ) stimulated Raw 264.7 cells, and on paw edema in rats. At concentrations of 0.5, 0.75, and 1 mg/mL, treatment with BPTS inhibited levels of expression of LPS-induced NF-κB and MAPKs (ERK, JNK, and p38) as well as production of proinflammatory mediators, such as nitric oxide (NO), prostaglandin E(2) (PGE(2)), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) by LPS. These results suggest that BPTS may exert anti-inflammatory effects via reduction of proinflammatory mediators, including NO, PGE(2), TNF-α, and IL-6 through suppression of the signaling pathways of NF-κB and MAPKs in LPS-induced macrophages. In addition, using the carrageenan-induced paw edema assay, an antiedema effect of BPTS was observed in rats. These findings may provide scientific evidence validating the use of BPTS in treatment of patients with heat syndrome in Korean oriental medicine.