Ameliorative Effects of Humulus japonicus Extract and Polysaccharide-Rich Extract of Phragmites rhizoma in Rats with Gastrointestinal Dysfunctions Induced by Water Avoidance Stress.

Chronic stress can cause the gastrointestinal disorders characterized by an altered bowel movement and abdominal pain. Studies have shown that Humulus japonicus extract (HJE) has anti-inflammatory and antidiarrheal effects, and Phragmites rhizoma extract (PEP) has antioxidative and antistress effects. The present study aimed to investigate the possible effects of HJE and PEP in rat models with stress-induced gastrointestinal dysfunctions. The rats were exposed to water avoidance stress (WAS, 1 h/day) for 10 days to induce gastrointestinal disorders. We found that WAS significantly increased fecal pellet output during 1 h stress, gastric emptying, colonic contractility, and permeability compared to the normal rats. Pretreatment with HJE and PEP (0.25 and 0.5 mL/kg, both administered separately) improved the increased gastric emptying and colonic contractility induced by electrical field stimulation, acetylcholine, and serotonin and also alleviated the increased colonic permeability. HJE and PEP also increased the claudin-1 and occludin expressions, reduced by WAS. WAS increased the concentration of TNF-α and TBARS and reduced FRAP. HJE and PEP recovered these effects. HJE and PEP improved the gastrointestinal disorders induced by WAS by upregulating the tight junction protein, possibly acting on cholinergic and serotonergic receptors to abolish the colonic hypercontractility and hyperpermeability and degradation of inflammatory cytokines via an antioxidant effect.

Pinus thunbergii Parl. Extracts Reduce Acute Inflammation by Targeting Oxidative Stress.

Pinus thunbergii Parl. (PTP) has traditionally been used for edible and medicinal purposes to treat several disorders, including diabetes and neuralgia. Therefore, this study sought to evaluate the inhibitory effects of PTP leaf ethanol extracts on acute inflammation. Moreover, the reactive oxygen species (ROS) scavenging activity, superoxide dismutase (SOD) activity, lipopolysaccharide (LPS)-induced nitric oxide (NO) generation, and H2O2-induced lipid peroxidation capacity of PTP were assessed in vitro in RAW 264.7 macrophages. Our results suggest that PTP prevents cell damage caused by oxidative free radicals and downregulates the expression of LPS-induced inflammation-associated factors including inducible nitric oxidase synthetase (iNOS), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2). PTP inhibited NO production by 53.5% (P < 0.05) and iNOS expression by 71.5% (P < 0.01) at 100 µg/mL. PTP at 100 µg/mL also inhibited ROS generation by 58.2% (P < 0.01) and SOD activity by 29.3%, as well as COX-2 expression by 83.3% (P < 0.01) and PGE2 expression by 98.6% (P < 0.01). The anti-inflammatory effects of PTP were confirmed in vivo using an arachidonic acid (AA)-induced ear edema mouse model. Ear thickness and myeloperoxidase (MPO) activity were evaluated as indicators of inflammation. PTP inhibited edema formation by 64.5% (P < 0.05) at 1.0 mg/ear. A total of 16 metabolites were identified in PTP extracts and categorized into subgroups, including two phenolic acids (mainly quinic acid), seven flavonoids, five lignans, one sesquiterpenoid, and one long-chain fatty acid. Therefore, our results suggest that PTP possesses anti-inflammatory properties.

Modulation of the TLR4/MyD88/NF-κB Pathway by Humulus japonicus Extract Protects Against Alcohol-Induced Liver Injury in a Rat Model.

Alcohol induces liver injury related to oxidative stress and inflammatory responses. The purpose of this study was to investigate the hepatoprotective effect of Humulus japonicus extract (HJE) against alcohol-induced liver injury. Furthermore, we investigated the mechanisms of the protective effect of HJE on alcohol-induced liver injury. The pretreatment of HJE decreased the levels of aspartate aminotransferase, alanine aminotransferase, triglyceride, and total cholesterol in the plasma, suppressed the malondialdehyde, myeloperoxidase, and enhanced the activities of superoxide dismutase, glutathione, and catalase. The inhibitory effect of HJE against oxidative stress may be associated with the upregulation of nuclear factor erythroid 2-related factor 2 and its target gene heme oxygenase-1. Moreover, HJE inhibited the pro-inflammatory cytokines (tumor necrosis factor alpha, interleukin-1 beta) by downregulating toll-like receptor 4, myeloid differentiation primary response 88, and nuclear factor kappa B p65. These findings provide evidence for the elucidation of the hepatoprotective mechanisms for HJE.

Polysaccharide-Rich Extract of Phragmites rhizome Attenuates Water Immersion Stress and Forced Swimming Fatigue in Rodent Animal Model.

Our study aimed to investigate the effects of the polysaccharide-rich extract of Phragmites rhizoma (PEP) against water immersion restraint (WIR) stress and forced swimming-induced fatigue. Exposure to WIR stress significantly increased the ulcer index, bleeding score, the weight of the adrenal gland, blood glucose concentrations, total cholesterol, cortisol, and creatine kinase (CK). The weight of the spleen decreased significantly. In addition, myeloperoxidase (MPO) and thiobarbituric acid-reactive substance (TBARS) were significantly upregulated by WIR stress. The antioxidative factors such as glutathione (GSH) and superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in the stomach were decreased by WIR stress. Alterations induced by WIR stress were effectively reversed by pretreatment with PEP. The swimming endurance capacity of mice was significantly prolonged by the oral administration of PEP. Swimming-induced fatigue significantly reduced the body weight; however, the injection of PEP inhibited the decrease of body weight. The PEP-treated group had significantly lower CK levels in plasma, an indicator of muscle damage. These results indicated that PEP has anti-stress and anti-fatigue effects, which are mediated by suppressing the hyperactivation of the hypothalamus-pituitary-adrenal axis, and antagonism of the oxidative damages induced by WIR stress and prolonged swimming times.

Humulus japonicus Extracts Protect Against Lipopolysaccharide/d-Galactosamine-Induced Acute Liver Injury in Rats.

It has been described that Humulus japonicus has potential antituberculosis and anti-inflammatory effects. The antiaging activity of H. japonicus extract (HJE) was also examined not only in yeast but also in human fibroblast cells. We evaluated the protective effect of HJE on hepatotoxicity in this study. We demonstrated the expression of antioxidative enzyme and cytokines in plasma. The vehicle control group received orally administered normal saline. Acute hepatotoxicity rat model was induced by lipopolysaccharide (LPS) (30 µg/kg) and d-galactosamine (D-GalN) (500 mg/kg) by intraperitoneal injection. The positive control group received orally administered silymarin (10 mg/kg). Three HJE groups received 8, 16, and 32 mg/kg. The blood samples were prepared to evaluate malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) level to examine oxidative stress, and hepatic tissue was assayed for myeloperoxidase (MPO). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and tumor necrosis factor-α (TNF-α) activities were also assayed to examine liver cell viability. Pretreatment with HJE decreased levels of AST, ALT, MDA, MPO, and TNF-α and increased levels of SOD and CAT compared with the LPS/D-GalN-treated group. These results suggested that HJE has the potential to reduce oxidative damage and inflammatory reactions in LPS/D-GalN-induced liver injury in the rat model. It can also increase survival rate in LPS/D-GalN-induced hepatotoxicity rat models.

Moisturizing effect of serine-loaded solid lipid nanoparticles and polysaccharide-rich extract of root Phragmites communis incorporated in hydrogel bases.

This study evaluated the moisturizing effect of serine-loaded solid lipid nanoparticles (serine-SLN) and polysaccharide-rich reed (Phragmites communis) root extract (RRE) incorporated in hydrogel bases. The hydrogels with serine-SLN and/or RRE were carefully applied on the volar forearm of human volunteers. Their moisturizing efficacy was evaluated by monitoring conductance values using a skin surface hygrometer. The values of the area under the normalized conductance-time curve (AUCC) were developed and compared as a parameter for the water holding capacity of the skin. Hydrogels with serine-SLN did not significantly moisturize the skin, while hydrogel containing 0.25% RRE produced a significant increase in the moisture content of the skin. However, adding more than 0.25% of RRE into the hydrogel base decreased the moisturizing effect due to the marked reduction of viscosity. Significantly enhanced moisturizing effect was observed with the hydrogel containing 0.25% RRE and 3% serine-SLN, with AUCC increased 2.21 times compared to than blank hydrogel. The results imply that effective delivery of serine into the skin is possible using lipid-based nanocarriers and RRE, which could be a promising strategy to moisturize the skin effectively.

The cytoprotective effect of Rumex Aquaticus Herba extract against hydrogen peroxide-induced oxidative stress in AGS cells.

The Rumex Aquaticus Herba extract containing quercetin-3-ß-D-glucuronopyranoside (ECQ) has been reported to exhibit various pharmacological activities, including anti-inflammatory and anti-oxidative effects. This plant has been traditionally used for the treatment of diarrhea, disinfestation, edema and jaundice, and as an antipyretic drug. The aim of the present study was to investigate the ability of ECQ to protect against oxidative damage and to determine its signaling mechanism in AGS cells. The protein expressions of heme oxygenase-1 (HO-1) and nuclear factor-erythroid 2 related factor 2 (Nrf2) were measured by Western blots. Cell viability was measured by MTT assay. Intracellular reactive oxygen species (ROS) levels were measured using 2',7'-dichlorofluorescein diacetate. Glutathione peroxidase levels were measured using kits. The protein expressions of HO-1 and its upstream mediator, Nrf2, increased after ECQ treatment. The HO-1 inhibitor, ZnPP, repressed the protective effect of ECQ on H2O2-induced cell damage. We found that LY294002, a specific PI3 K/Akt inhibitor, suppressed ECQ-induced HO-1 expression. ECQ significantly attenuated H2O2-induced cytotoxicity and ROS generation. Also, ECQ enhanced the antioxidant enzyme activities of glutathione peroxidase. These results suggest that ECQ exerts a cytoprotective effect against H2O2-induced oxidative stress by upregulation of Nrf2/HO-1 via the PI3 K/Akt pathway.

Ultrasonication processed Panax ginseng berry extract induces apoptosis through an intrinsic apoptosis pathway in HepG2 cells.

Ginseng's major active components, ginsenosides, have been known to show anti-cancer, neuroprotective, and anti-inflammatory activities. Ultrasonication processed Panax ginseng berry extract (UGB) contains various ginsenosides. The components are different from Panax ginseng berry extract (GBE). This study was aimed to investigate the cytotoxic mechanism of UGB in HepG2 cells, human hepatocellular carcinoma cell line. HepG2 cells were treated with UGB (0, 10, 20 µg/ml). Cell growth and cellular apoptosis were evaluated by MTT assay and Annexin V/Pi staining, respectively. Intracellular Reactive oxygen species (ROS) levels were also determined by 2', 7'-dichlorofluorescin diacetate (DCFDA) staining. The expressions of Bax, Bcl-2 and caspase-3, the apoptotic markers, were evaluated by Western Blot. UGB dose-dependently inhibited cell growth and induced apoptotic cell death. Intracellular ROS levels were increased. UGB increased the expression of the cleaved form of caspase-3. Furthermore, UGB induced apoptosis of HepG2 cells through Bax activation and Bcl-2 inhibition. In conclusion, UGB induced apoptosis through an intrinsic pathway in HepG2 cells suggesting that UGB might play a role as a novel substance for anti-cancer effect.

Effects of Aqueous Extract of Phyllostachyos Caulis in Taeniam on Longitudinal Bone Growth in Adolescent Rats.

This study examined whether treatment with Phyllostachyos Caulis in Taeniam aqueous extract improves longitudinal bone growth in adolescent male rats. Three-week-old male Sprague-Dawley rats were divided into three groups: a control group, a Phyllostachyos Caulis in Taeniam group (200 mg/kg, p.o.), and a recombinant human growth hormone group (20 µg/kg, s.c.). The total tibial length and the height of each growth plate zone were evaluated by radiography and histomorphometry. The total number of proliferative cells and 5-bromo-2'-deoxyuridine-positive cells were counted after 5-bromo-2'-deoxyuridine staining. Serum total osteocalcin levels were assayed using an enzyme-linked immunosorbent assay. The average total tibial length of the Phyllostachyos Caulis in Taeniam group was significantly longer than that of the control group. The heights of the proliferative and hypertrophic zones in the Phyllostachyos Caulis in Taeniam group were increased, and the ratio of 5-bromo-2'-deoxyuridine-positive to total cells in the proliferative zone was also increased. The serum osteocalcin, growth hormone, and insulin-like growth factor-1 levels were significantly increased in the Phyllostachyos Caulis in Taeniam group compared to the control group. Insulin-like growth factor-1 and insulin-like growth factor-1 receptor were highly expressed in the proliferative and hypertrophic zones in the Phyllostachyos Caulis in Taeniam group. The Phyllostachyos Caulis in Taeniam extract increased bone length, promoted cell proliferation, and activated the growth plate zones, which suggested that the extract could play an important role in longitudinal bone growth. Therefore, the Phyllostachyos Caulis in Taeniam extract might be a good alternative medicine to growth hormone therapy.

The effect of Flos Lonicerae Japonicae extract on gastro-intestinal motility function.

ETHNOPHARMACOLOGICAL RELEVANCE: Flos Lonicerae Japonicae is a well-known herb of traditional Chinese medicine that has been used for heat-clearing, detoxification, anti-inflammation, throat pain and gastro-intestinal (GI) disorder. In order to verify the effect of Flos Lonicerae Japonicae on GI disorder, we investigated the prokinetic effect of GC-7101 on GI motility function. MATERIALS AND METHODS: GC-7101 is the standardized extract of Flos Lonicerae Japonicae. The contractile action of GC-7101 on feline esophageal smooth muscle cell (ESMC) was evaluated by measuring dispersed cell length. The isometric tension study was performed to investigate the effect of GC-7101 on feline lower esophageal sphincther (LES). The prokinetic effect of GC-7101 was investigated by gastric emptying (GE) and gastro-intestinal transit (GIT) in rats. RESULTS: GC-7101 produced concentration-dependent contractions in ESMCs. Pretreatment with 5-HT3 and 5-HT4 receptor blocker (ondansetron and GR113808) inhibited the contractile responses of the GC-7101-induced ESMCs. In isometric tension study, GC-7101 recovered the HCl-induced decreased tone of LES muscle strips. The treatment of GC-7101 enhanced the carbachol-induced contractile responses and the electric field stimulation (EFS)-induced on-contraction. The oral administration of GC-7101 not only significantly accelerated GE and GIT in normal rats but also recovered the delayed GE and GIT, and its effect was more potent than that of conventional prokinetics (e.g., domperidone, a dopamine-receptor antagonist, and mosapride, a 5-HT4-receptor agonist). CONCLUSION: GC-7101 revealed a prokinetic effect through enhancing the contractile responses of ESMCs, tone increases, enhancing the carbarchol- or EFS-induced contractile responses of LES muscle strips, and the acceleration of GE and GIT. We have identified the significant potential of GC-7101 for the development of new prokinetic drugs through this study.

Effect of Rumex Aquaticus Herba Extract Against Helicobacter pylori-Induced Inflammation in Gastric Epithelial Cells.

The purposes of this study were to examine the characteristics of Helicobacter pylori and the effect of Rumex Aquaticus Herba extract on the expression of cytokines in H. pylori-infected gastric epithelial cells. Cultured human adenocarcinoma gastric cells (AGS) were infected by H. pylori in RPMI 1640 media. Cell growth was measured by trypan blue assay. Western blot analysis was performed to investigate effect of extract containing Quercetin-3-O-ß-d-glucuronopyranoside (ECQ) on the expression of inflammatory factors and the inhibition on cell growth. Furthermore, we compared the inhibitory effects with various combinations of clarithromycin, amoxicillin, omeprazole, and ECQ. The urease test with Christensen's Urea Agar was performed to identify the urease activity of H. pylori and the effect ECQ has on urease activity. When the cells were exposed to H. pylori, the trypan blue assay revealed a decrease in the rate of cell growth. Western blot analysis showed that H. pylori-infected cells had increased levels of degraded IκB-α and inflammatory factors. Pretreatment with ECQ inhibited interleukin expression induced by H. pylori in a dose-dependent manner. A combination of ECQ and antibiotics inhibited cytokine expression more effectively than other treatments. H. pylori displayed significant urease activity. ECQ did not significantly inhibit urease activity. These data suggest that H. pylori infection has cytotoxic effects against AGS cells, and ECQ may inhibit the production of proinflammatory cytokines in H. pylori-infected AGS cells.

Pharmacological action of DA-9701 on the motility of feline stomach circular smooth muscle.

DA-9701, a new prokinetic agent for the treatment of functional dyspepsia, is formulated with Pharbitis semen and Corydalis tuber. This study wasconducted to determine the pharmacological action of DA-9701 and to identify the receptors involved in DA-9701 -induced contractile responsesin the feline gastric corporal, fundic and antral circular smooth muscle. Concentration-response curve to DA-9701 was established. The tissue trips were exposed to methylsergide, ketanserin, ondansetron, GR 113808, atropine and dopamine before administration of DA-9701. The contractile force was determined before and after administration of drugs by a polygraph.DA-9701 enhanced the spontaneous contractile amplitude of antrum, corpus and fundus. However, it did not change the spontaneous contractile frequency of antrum and corpus, but concentration-dependently reduced that of fundus. In the fundus, DA-9701 -induced tonic contractions were inhibited by dopamine, methylsergide, ketanserine, ondansetron or GR 113808 respectively, but not by atropine, indicating that the contractile responses are mediated by multiple receptors: 5-HT2, 5-HT3, 5-HT4, and dopamine receptors. In the corpus, DA-9701-induced contractions were blocked by atropine, dopamine or GR 113808, but not by methysergide, ketanserin or ondansetron, indicating that they are involved in receptors on both, smooth muscles and neurons: 5-HT4 and dopamine receptors. However, contractile responses to DA-9701 are mainly mediated by dopamine receptors in the antrum. These results suggest that DA-9701 has important roles in gastric accommodation by enhancing tonic activity of fundus, and in gastric emptying and gastrointestinal transit by phasic contractions of corpus and antrum mediated by multiple receptors.

The Effect of Polyphenols Isolated from Cynanchi wilfordii Radix with Anti-inflammatory, Antioxidant, and Anti-bacterial Activity.

Recently, Cynanchi wilfordii Radix has gained wide use in Asian countries as a functional food effective for relieving fatigue, osteoporosis, and constipation, particularly in menopausal disorders. However, its anti-inflammatory and anti-microbial activities have not been explored in detail to date. The anti-inflammatory, antioxidant, and anti-bacterial properties of the Cynanchi wilfordii Radix extracts obtained with water, methanol, ethanol, and acetone were compared. All 4 polyphenol-containing extracts exhibited anti-inflammatory and antioxidant effects. The ethanol extract was found to elicit the most potent reduction of nitric oxide (NO), prostaglandin E2 (PGE2), and cytokine (IL-1ß, IL-6, IL-10, and TNF-α) levels, as well as inhibit the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a concentration-dependent manner. The evaluation of antioxidant activity also revealed the ethanol extract to have the highest free radical scavenging activity, measured as 85.3±0.4%, which is equivalent to 99.9% of the activity of α -tocopherol. In the assessment of anti-bacterial activity, only ethanol extract was found to inhibit the growth of the Bacillus species Bacillus cereus and Bacillus anthracis. These results show that polyphenols of Cynanchi wilfordii Radix have anti-inflammatory, antioxidant, and anti-bacterial properties that can be exploited and further improved for use as a supplementary functional food, in cosmetics, and for pharmaceutical purposes.

The role of phosphatidylcholine and deoxycholic acid in inflammation.

AIMS: Phosphatidylcholine with deoxycholic acid (PC/DA) is widely used to reduce localized fat deposits with mild adverse effects. We previously demonstrated that PC induces lipolysis with mild PMN infiltration, while DA induces adipose tissue damage. Therefore, the aim of this study was to extend our understanding of the pro-inflammatory responses of PC, DA, and PC/DA. MAIN METHODS: We evaluated the level of edema and polymononuclear (PMN) infiltration by histopathological examination. Myeloperoxidase (MPO) activity was analyzed using an MPO activity assay kit. Levels of inflammatory cytokines (IL-1ß and IL-6) and PGE2 were measured by ELISA. KEY FINDINGS: A low and high dose of PC failed to induce an inflammatory response, whereas DA led to an intense inflammatory response in a dose dependent manner. Combined PC/DA treatment resulted in a mild inflammatory response that was notably less severe than higher DA. Together, these results demonstrated that DA plays a role in inflammation caused by combined PC/DA. Histopathological examination and measurement of MPO activity indicated that DA was the primary cause of edema and PMN infiltration. Further, increased levels of cytokines (IL-1ß and IL-6) and PGE2 demonstrated that DA might directly induce inflammation, whereas PC alone has no effect on inflammation. SIGNIFICANCE: These results indicate that DA rather than PC is responsible for inflammation, and that PC may not aggravate inflammatory responses induced by DA. Thus, the results of this study suggest that the adverse effects of PC/DA during localized fat treatment may be solely due to DA.

The effect of DA-9701 on 5-hydroxytryptamine-induced contraction of feline esophageal smooth muscle cells.

Serotonin, or 5-hydroxytryptamine (5-HT), is a monoamine neurotransmitter found in blood platelets, the gastrointestinal (GI) tract, and the central nervous system (CNS) of animals and humans. The signaling pathways of 5-hydroxytryptamine (5-HT)-induced contractions in cat esophageal smooth muscle cell (ESMC)s have been identified, but the downstream components of the 5-HT signaling pathway remain unclear. DA-9701 is the standardized extract of the Pharbitis nil Choisy seed (Pharbitidis Semen, Convolvulaceae) and the root of Corydalis yahusuo W.T. Wang (Corydalis Tuber, Papaveraceae). DA-9701 is known to have strong gastroprokinetic effects and a good safety profile. In this study, we investigated the 5-HT signaling pathway at the G-protein level, and we explored the mechanisms by which DA-9701 induces smooth muscle contraction. Freshly isolated smooth muscle cells were harvested from the feline esophagus, and cells were permeabilized to measure their length. 5-HT produced esophageal smooth muscle contractions in a dose-dependent manner. Furthermore, 5-HT produced a relatively long-acting contraction. 5-HT binds to the 5-HT2, 5-HT3 and 5-HT4 receptors to induce smooth muscle contraction in feline ESMCs. These receptors, which are located in esophageal smooth muscle, are coupled to Gαq, Gαo and Gαs. These G proteins activate PLC, which leads to Ca2+/calmodulin-dependent MLCK activation, resulting in MLC20 phosphorylation and cell contraction. Conversely, DA-9701 inhibits 5-HT-induced contraction by inhibiting MLC20 phosphorylation.

Protective effect of extract from Rumex aquaticus herba on ethanol-induced gastric damage in rats.

BACKGROUND/AIMS: In this study, we investigated the gastroprotective effect of extract including quercetin-3-O-ß-D-glucuronopyranoside (EIQ) from Rumex aquaticus herba against the ethanol-induced gastric damage in rats. METHODS: The rats were divided into eight groups composed of a non-ethanol group, only EIQ (10 mg/kg) group, groups with absolute ethanol after pretreatment with various doses of EIQ (1, 3 and 10 mg/kg), rebamipide (10 mg/kg), stillen (40 mg/kg) and a control receiving only absolute ethanol. Ethanol-induced gastric lesions, lipid peroxidation, neutrophil infiltration and glutathione level were measured. Superoxide dismutase (SOD) and catalase activity were assessed by an assay kit. Protein expression of SOD, catalase or hemoxygenase-1 (HO-1) was assessed by western blotting analysis. RESULTS AND CONCLUSION: In the absolute ethanol treated group, gastric lesion and malondialdehyde levels were significantly increased with enhanced myeloperoxidase activity. Administration of EIQ 1 h prior to ethanol treatment significantly inhibited the formation of gastric lesions and the elevation of the malondialdehyde levels with myeloperoxidase activity. In addition, pretreatment with EIQ significantly increased the level of glutathione, and elevated the activity and protein expression of radical scavenging enzymes, such as SOD, catalase and HO-1. EIQ may exert anti-inflammatory and anti-oxidative effects against ethanol-induced gastric injury through the reduction of lipid peroxidation, myeloperoxidase activity and free radicals.

HMC05 attenuates vascular contraction through inhibition of RhoA/Rho-kinase signaling pathway.

AIM OF THE STUDY: HMC05, an extract from eight different herbal mixtures, has been developed to treat cardiovascular disease. This extract has a vasorelaxant and anti-atherosclerotic action. We hypothesized that HMC05 attenuates vascular contraction through inhibition of the RhoA/Rho-kinase signaling pathway. MATERIALS AND METHODS: Rat aortic ring preparations were mounted in organ baths and subjected to contraction and relaxation. Phosphorylation of 20 kDa myosin light chains (MLC(20)) and myosin phosphatase targeting subunit 1 (MYPT1) were examined by immunoblot. We also measured the amount of GTP RhoA as a marker for RhoA activation. RESULTS: In endothelium-denuded aortic ring preparations, HMC05 relaxed vascular contraction induced by 6.0 mM NaF, 100 nM phenylephrine, 30 nM thromboxane A(2) agonist U46619 or 1.0 µM protein kinase C (PKC) activator phorbol-12,13-dibutyrate (PDBu) in a decreasing order. HMC05 relaxed aortic ring preparations precontracted with sodium fluoride (NaF) whether endothelium was intact or denuded. Pre-incubation with HMC05 for 30 min dose-dependently inhibited the NaF-induced contractile response. In vascular strips, HMC05 decreased the phosphorylation level of both MLC(20) and MYPT1(Thr855) induced by 6.0 mM NaF. Furthermore, HMC05 decreased the amount of GTP RhoA activated by NaF. CONCLUSIONS: HMC05 attenuates vascular contraction through inhibition of the RhoA/Rho-kinase signaling pathway. HMC05 may be useful for the treatment and/or prevention of cardiovascular diseases associated with activation of RhoA/Rho-kinase signaling pathway.

Quercetin-3-O-beta-d-glucuronopyranoside (QGC)-induced HO-1 expression through ERK and PI3K activation in cultured feline esophageal epithelial cells.

Heme oxygenase-1 (HO-1) is one of the antioxidant enzymes which help protect against cellular damage. The present study examined the ability of Quercetin-3-O-beta-D-glucuronopyranoside (QGC), flavonoid glucoside extracted from Rumex Aquaticus Herba, to induce expression of HO-1 and analyzed its signaling mechanism in cultured feline esophageal epithelial cells (EEC). Culture of the esophageal epithelial cells from cat was prepared. The data suggested that QGC could result in enhanced antioxidant enzyme defense system via HO-1 expression and Nrf2 translocation involving both the ERK and PI3K-Akt pathways as well as partly PKC pathways in EEC.

Inhibitory effect of genistein on agonist-induced modulation of vascular contractility.

The present study was undertaken to determine whether treatment with genistein, the plant-derived estrogen-like compound influences agonist-induced vascular smooth muscle contraction and, if so, to investigate related mechanisms. The measurement of isometric contractions using a computerized data acquisition system was combined with molecular experiments. Genistein completely inhibited KCl-, phorbol ester-, phenylephrine-, fluoride- and thromboxane A(2)-induced contractions. An inactive analogue, daidzein, completely inhibited only fluoride-induced contraction regardless of endothelial function, suggesting some difference between the mechanisms of RhoA/Rho-kinase activators such as fluoride and thromboxane A(2). Furthermore, genistein and daidzein each significantly decreased phosphorylation of MYPT1 at Thr855 had been induced by a thromboxane A(2) mimetic. Interestingly, iberiotoxin, a blocker of large-conductance calcium-activated potassium channels, did not inhibit the relaxation response to genistein or daidzein in denuded aortic rings precontracted with fluoride. In conclusion, genistein or daidzein elicit similar relaxing responses in fluoride-induced contractions, regardless of tyrosine kinase inhibition or endothelial function, and the relaxation caused by genistein or daidzein was not antagonized by large conductance K(Ca)-channel inhibitors in the denuded muscle. This suggests that the RhoA/Rho-kinase pathway rather than K(+)-channels are involved in the genistein-induced vasodilation. In addition, based on molecular and physiological results, only one vasoconstrictor fluoride seems to be a full RhoA/Rho-kinase activator; the others are partial activators.

The protective effect of eupatilin on indomethacin-induced cell damage in cultured feline ileal smooth muscle cells: involvement of HO-1 and ERK.

Chronic users of non-steroidal anti-inflammatory drugs frequently develop ulcerative lesions in their intestines. The purpose of the present study was to investigate whether eupatilin, an active ingredient derived from Artemisia plants, prevents this side effect in vitro. Extracts of the whole herb of Artemisia asiatica Nakai have been used in oriental medicine for the treatment of inflammation. As measured by the MTT assay, the treatment of cultured feline ileal smooth muscle cells (ISMCs) with 2.5mM indomethacin for 2h decreased the cell viability to 43%. Pretreatment with eupatilin resulted in dose-dependent inhibition on indomethacin-induced cell damage. This cytoprotective effect of eupatilin required concentrations of more than 150 microM and incubation periods of longer than 16 h. Pretreatment of ISMC with cycloheximide, an inhibitor of protein synthesis, attenuated the cytoprotective effect of eupatilin, suggesting that eupatilin induces proteins that are responsible for the cytoprotection. Heme oxygenase-1 (HO-1), which is known as a cytoprotective enzyme due to its anti-inflammatory actions, is a candidate protein since ZnPP, an HO-1 inhibitor, repressed the protective effect of eupatilin on indomethacin-induced cell damage in a concentration-dependent manner. Western blot analysis revealed that eupatilin-mediated HO-1 induction occurred in a concentration- and time-dependent manner. We also found that PD98059, a MEK (MAPK/ERK kinase) inhibitor, attenuated the eupatilin-induced HO-1 expression and nuclear translocation of transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). Taken together, the data imply that eupatilin protects ISMC from cell damage caused by indomethacin, and that its cytoprotective action could be attributed to eupatilin-mediated HO-1 induction via ERK and Nrf2 signaling in ISMC.