Urine metabolomics signatures in reversible cerebral vasoconstriction syndrome.

BACKGROUND: The pathophysiology of reversible cerebral vasoconstriction syndrome is unclear. An unbiased systems-based approach might help to illustrate the metabolite profiling and underlying pathophysiology. METHODS: Urine samples were collected from reversible cerebral vasoconstriction syndrome patients and matched controls recruited in Taipei Veterans General Hospital. 1H-Nuclear magnetic resonance was used to initially explore the metabolic profile, and liquid chromatography tandem mass spectrometry was then used to identify metabolic alterations in reversible cerebral vasoconstriction syndrome. Untargeted metabolite screening was randomly performed on 10 reversible cerebral vasoconstriction syndrome patients and 10 control subjects in the discovery phase. The selected untargeted metabolites were further validated on 47 reversible cerebral vasoconstriction syndrome patients during their ictal stage (with 40 of them having remission samples) and 47 controls in the replication phase. RESULTS AND CONCLUSION: Six metabolites-hippurate, citrate, 1,3,7-trimethyluric acid, ascorbic acid, D-glucurono-6,3-lactone, and D-threo-isocitric acid-with t-test derived p-value < 0.05 and VIP score >1, were identified as potential urine signatures that can well distinguish reversible cerebral vasoconstriction syndrome subjects at ictal stage from controls. Among them, citrate, hippurate, ascorbic acid, and D-glucurono-6,3-lactone were significantly lower, and 1,3,7-trimethyluric acid and D-threo-isocitric acid were higher in reversible cerebral vasoconstriction syndrome patients. Of these, four selected metabolites, citrate, D-glucurono-6,3-lactone, ascorbic acid, and 1,3,7-trimethyluric acid, returned to normal levels in remission. These metabolites are related to pathways associated with free radical scavenging, with the hub molecules being associated with endothelial dysfunction or sympathetic overactivity. Whether these metabolites and their implicated networks play a role in the pathogenesis of reversible cerebral vasoconstriction syndrome remains to be confirmed.

Tetrandrine inhibits human brain glioblastoma multiforme GBM 8401 cancer cell migration and invasion in vitro.

Tetrandrine (TET) has been reported to induce anti-cancer activity in many human cancer cells and also to inhibit cancer cell migration and invasion. However, there are no reports to show TET inhibits cell migration and invasion in human brain glioblastoma multiforme GBM 8401 cells. In this study, we investigated the anti-metastasis effects of TET on GBM 8401 cells in vitro. Under sub-lethal concentrations (from 1, 5 up to 10 µM), TET significantly inhibited cell mobility, migration and invasion of GBM 8401 cells that were assayed by wound healing and Transwell assays. Gelatin zymography assay showed that TET inhibited MMP-2 activity in GBM 8401 cells. Western blotting results indicated that TET inhibited several key metastasis-related proteins, such as p-EGFR(Tyr1068) , SOS-1, GRB2, Ras, p-AKT(Ser473) and p-AKT(Thr308) , NF-κB-p65, Snail, E-cadherin, N-cadherin, NF-κB, MMP-2 and MMP-9 that were significant reduction at 24 and 48 hours treatment by TET. TET reduced MAPK signaling associated proteins such as p-JNK1/2 and p-c-Jun in GBM 8401 cells. The electrophoretic mobility shift (EMSA) assay was used to investigate NF-κB and DNA binding was reduced by TET in a dose-dependently. Based on these findings, we suggested that TET could be used in anti-metastasis of human brain glioblastoma multiforme GBM 8401 cells in the future.

Activation of AMP-activated protein kinase α1 mediates mislocalization of TDP-43 in amyotrophic lateral sclerosis.

TAR DNA-binding protein-43 (TDP-43) is a nuclear RNA-binding protein involved in many cellular pathways. TDP-43-positive inclusions are a hallmark of amyotrophic lateral sclerosis (ALS). The major clinical presentation of ALS is muscle weakness due to the degeneration of motor neurons. Mislocalization of TDP-43 from the nucleus to the cytoplasm is an early event of ALS. In this study, we demonstrate that cytoplasmic mislocalization of TDP-43 was accompanied by increased activation of AMP-activated protein kinase (AMPK) in motor neurons of ALS patients. The activation of AMPK in a motor neuron cell line (NSC34) or mouse spinal cords induced the mislocalization of TDP-43, recapitulating this characteristic of ALS. Down-regulation of AMPK-α1 or exogenous expression of a dominant-negative AMPK-α1 mutant reduced TDP-43 mislocalization. Suppression of AMPK activity using cAMP-simulating agents rescued the mislocalization of TDP-43 in NSC34 cells and delayed disease progression in TDP-43 transgenic mice. Our findings demonstrate that activation of AMPK-α1 plays a critical role in TDP-43 mislocalization and the development of ALS; thus, AMPK-α1 may be a potential drug target for this devastating disease.

Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation.

In chronic liver diseases, regardless of their etiology, the development of fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. Hepatic stellate cells (HSCs) are the main profibrogenic cells that promote the pathogenesis of liver fibrosis, and so it is important to identify the molecules that regulate HSCs activation and liver fibrosis. Niemann-Pick type C2 (NPC2) protein plays an important role in the regulation of intracellular cholesterol homeostasis by directly binding with free cholesterol. However, the roles of NPC2 in HSCs activation and liver fibrosis have not been explored in detail. Since a high-cholesterol diet exacerbates liver fibrosis progression in both rodents and humans, we propose that the expression of NPC2 affects free cholesterol metabolism and regulates HSCs activation. In this study, we found that NPC2 is decreased in both thioacetamide- and carbon tetrachloride-induced liver fibrosis tissues. In addition, NPC2 is expressed in quiescent HSCs, but its activation status is down-regulated. Knockdown of NPC2 in HSC-T6 cells resulted in marked increases in transforming growth factor-ß1 (TGF-ß1)-induced collagen type 1 α1 (Col1a1), α-smooth muscle actin (α-SMA) expression, and Smad2 phosphorylation. In contrast, NPC2 overexpression decreased TGF-ß1-induced HSCs activation. We further demonstrated that NPC2 deficiency significantly increased the accumulation of free cholesterol in HSCs, increasing Col1a1 and α-SMA expression and activating Smad2, and leading to sensitization of HSCs to TGF-ß1 activation. In contrast, overexpression of NPC2 decreased U18666A-induced free cholesterol accumulation and inhibited the subsequent HSCs activation. In conclusion, our study has demonstrated that NPC2 plays an important role in HSCs activation by regulating the accumulation of free cholesterol. NPC2 overexpression may thus represent a new treatment strategy for liver fibrosis.

Lithospermic acid attenuates 1-methyl-4-phenylpyridine-induced neurotoxicity by blocking neuronal apoptotic and neuroinflammatory pathways.

BACKGROUND: Parkinson's disease is the second most common neurodegenerative disorders after Alzheimer's disease. The main cause of the disease is the massive degeneration of dopaminergic neurons in the substantia nigra. Neuronal apoptosis and neuroinflammation are thought to be the key contributors to the neuronal degeneration. RESULTS: Both CATH.a cells and ICR mice were treated with 1-methyl-4-phenylpyridin (MPP(+)) to induce neurotoxicity in vitro and in vivo. Western blotting and immunohistochemistry were also used to analyse neurotoxicity, neuroinflammation and aberrant neurogenesis in vivo. The experiment in CATH.a cells showed that the treatment of MPP(+) impaired intake of cell membrane and activated caspase system, suggesting that the neurotoxic mechanisms of MPP(+) might include both necrosis and apoptosis. Pretreatment of lithospermic acid might prevent these toxicities. Lithospermic acid possesses specific inhibitory effect on caspase 3. In mitochondria, MPP(+) caused mitochondrial depolarization and induced endoplasmic reticulum stress via increasing expression of chaperone protein, GRP-78. All the effects mentioned above were reduced by lithospermic acid. In animal model, the immunohistochemistry of mice brain sections revealed that MPP(+) decreased the amount of dopaminergic neurons, enhanced microglia activation, promoted astrogliosis in both substantia nigra and hippocampus, and MPP(+) provoked the aberrant neurogenesis in hippocampus. Lithospermic acid significantly attenuates all of these effects induced by MPP(+). CONCLUSIONS: Lithospermic acid is a potential candidate drug for the novel therapeutic intervention on Parkinson's disease.

Novel polyprenylated phloroglucinols from Hypericum sampsonii.

Hypericum sampsonii Hance (Clusiaceae) is a folk medicine used in Taiwan to treat blood stasis, relieve swelling, and as an anti-hepatitis drug. Two new polyprenylated phloroglucinol derivatives, hypersampsone R (1) and hypersampsone S (2), and a known prenylated benzophenone, hyperibone K (3) were isolated from the aerial parts of H. sampsonii. Their structures were determined by extensive 1D and 2D NMR, and MS spectral analyses.

Dendrobium nobile Polysaccharide Attenuates Blue Light-Induced Injury in Retinal Cells and In Vivo in Drosophila.

Blue light is the higher-energy region of the visible spectrum. Excessive exposure to blue light is known to induce oxidative stress and is harmful to the eyes. The stems of Dendrobium nobile Lindl. (Orchidaceae), named Jinchaishihu, have long been used in traditional Chinese medicine (TCM) for nourishing yin, clearing heat, and brightening the eyes. The polysaccharide is one of the major components in D. nobile. However, the effect on ocular cells remains unclear. This study aimed to investigate whether the polysaccharide from D. nobile can protect the eyes from blue light-induced injury. A crude (DN-P) and a partially purified polysaccharide (DN-PP) from D. nobile were evaluated for their protective effects on blue light-induced damage in ARPE-19 and 661W cells. The in vivo study investigated the electroretinographic response and the expression of phototransduction-related genes in the retinas of a Drosophila model. The results showed that DN-P and DN-PP could improve blue light-induced damage in ARPE-19 and 661W cells, including cell viability, antioxidant activity, reactive oxygen species (ROS)/superoxide production, and reverse opsin 3 protein expression in a concentration-dependent manner. The in vivo study indicated that DN-P could alleviate eye damage and reverse the expression of phototransduction-related genes, including ninaE, norpA, Gαq, Gß76C, Gγ30A, TRP, and TRPL, in a dose-dependent manner in blue light-exposed Drosophila. In conclusion, this is the first report demonstrating that D. nobile polysaccharide pretreatment can protect retinal cells and retinal photoreceptors from blue light-induced damage. These results provide supporting evidence for the beneficial potential of D. nobile in preventing blue light-induced eye damage and improving eyesight.

Hepatitis C virus core protein stimulates fibrogenesis in hepatic stellate cells involving the obese receptor.

Hepatitis C virus core protein (HCVcp), which is secreted by infected cells, is reported as an immunomodulator in immune cells. However, the effects of HCVcp on hepatic stellate cells (HSCs), the key cells in liver fibrosis, still remain unclear. In this study, we investigated the effects of HCVcp on obese receptor (ObR) related downstream signaling pathways and fibrogenic gene expression in HSCs. LX-2, a human HSC line, was incubated with HCVcp. Inhibitors and short interfering RNAs were used to interrogate the mechanisms of HCVcp action on HSCs. HCVcp (20-100 ng/ml) concentration-dependently stimulated α-smooth muscle actin (α-SMA) protein expression and mRNA expression of α-SMA, procollagen α2(I) and TGF-ß1 genes, with a plateau of 220% of controls at 100 ng/ml. HCVcp induced mRNA and protein expression of ObR. Blocking of Ob-Rb with a neutralizing antibody inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) and AMPKα stimulated by HCVcp. Furthermore, knockdown of Ob-Rb down-regulated HCVcp-induced STAT3, AKT, and AMPKα phosphorylation, and reversed HCVcp-suppressed mRNA expression of matrix metalloproteinase (MMP)-1, peroxisome proliferator-activated receptor (PPAR)γ and sterol regulatory element binding protein-1c (SREBP-1c) genes. AMPKα signaling blockade reversed HCVcp-suppressed SREBP-1c mRNA expression. HCVcp stimulated reactive oxygen species formation and gp91(phox) (a component of NADPH oxidase) protein expression, together with AKT phosphorylation, leading to suppression of PPARγ and SREBP-1c genes. Our results provide a new finding that HCVcp induced ObR-dependent Janus Kinase (JAK) 2-STAT3, AMPKα, and AKT signaling pathways and modulated downstream fibrogenetic gene expression in HSCs.

Enhanced serum levels of tumor necrosis factor-α, interleukin-1ß, and -6 in sarcopenia: alleviation through exercise and nutrition intervention.

BACKGROUND: Limited research has been conducted on the post-intervention inflammatory status in sarcopenic patients, despite previous studies revealing elevated pro-inflammatory markers. This study aimed to investigate the potential elevation of specific pro-inflammatory cytokines in sarcopenic patients and evaluate the effects of exercise and nutritional support interventions on these cytokine levels. METHODS: In this post-hoc analysis of a randomized controlled trial (RCT), 57 individuals with sarcopenia from the RCT and 57 non-sarcopenic participants from the same geriatric community cohort that did not participate in the RCT were enrolled. Grip strength and body composition measurements were recorded. Tumor necrotizing factor (TNF)-α, interleukin (IL)-1ß, IL-6, and IL-15 levels were assessed at baseline for both groups and after a 12-week intervention consisting of resistive exercise and supplementation with branched-chain amino acids, calcium, and vitamin D3 in the patients with sarcopenia. RESULTS: The sarcopenic group demonstrated significantly lower body weight, body mass index, grip strength, and skeletal muscle mass index. Moreover, sarcopenic patients exhibited higher levels of TNF-α (p=0.007), IL-1ß (p<0.001), and IL-6 (p<0.001), while no significant difference was observed in IL-15 (p=0.345) between participants with and those without sarcopenia. Following the intervention, the sarcopenic group experienced significant improvements in grip strength and skeletal muscle mass index with a notable reduction in TNF-α (p=0.003), IL-1ß (p=0.012) and IL-6 (p=0.001) levels. CONCLUSIONS: Sarcopenic patients exhibit elevated levels of TNF-α, IL-1ß, and IL-6, which declined after nutrition support and exercise interventions. However, further research is necessary to evaluate the long-term impact of these interventions on cytokine levels.

Protective effects of kaerophyllin against liver fibrogenesis in rats.

BACKGROUND: We previously demonstrated that kaerophyllin, a lignan, isolated from a widely used traditional Chinese herb, Bupleurum scorzonerifolium, leading to the inhibition of hepatic stellate cells (HSCs) activation in vitro. This current study evaluated the in vivo role of kaerophyllin in protecting the liver against injury and fibrogenesis caused by thioacetamide (TAA) in rats and further explored the underlying mechanisms. MATERIALS AND METHODS: Liver fibrosis in Sprague-Dawley rats was induced by intraperitoneal injection of TAA (200 mg/kg) twice per week for 6 weeks. Animals were divided into five groups: vehicle control, TAA control, TAA + low dose kaerophyllin, TAA + high dose kaerophyllin and TAA + curcumin groups. Kaerophyllin (10 or 30 mg/kg) or curcumin (150 mg/mL) was given by gavage twice per day consecutively for 4 weeks starting 2 weeks after TAA injection. Rat HSCs were used to investigate the anti-inflammatory role of kaerophyllin against tumour necrosis factor α (TNF-α) in vitro. Peroxisome proliferator-activated receptor-γ (PPAR-γ) expression was knocked down in rat HSCs using PPAR-γ small interfering RNAs. RESULTS: Kaerophyllin significantly protected liver from injury by reducing serum aspartate transaminase and alanine transaminase levels and by improving the histological architecture and fibrosis score. In addition, kaerophyllin suppressed inflammation by reducing the mRNA of TNF-α, interleukin-1ß (IL-1ß) and monocyte chemoattractant protein-1 (MCP-1) genes. In HSCs, kaerophyllin elevated PPAR-γ activity and reduced TNF-α-stimulated mRNA levels of intracellular adhesion molecule-1 (ICAM-1), MCP-1 and IL-1ß genes, which were reversed by small interfering RNA knockdown of PPAR-γ gene. CONCLUSIONS: Our results demonstrated that kaerophyllin protected the rat liver from TAA-caused injury and fibrogenesis by suppressing hepatic inflammation and inhibiting HSC activation, possibly through upregulation of PPAR-γ expression.

Paeoniae radix reduces PDGF-stimulated hepatic stellate cell migration.

Hepatic stellate cells (HSCs) play a key role in the pathogenesis of liver fibrosis. In chronic liver injury, HSCs undergo transdifferentiation to an activated myofibroblastic phenotype and migrate to injured areas in response to chemotactic factors, producing extracellular matrix proteins such as collagen type I to repair the damage as well as overexpression of α-smooth muscle actin (α-SMA). Paeoniae Radix, the root of Paeonia lactiflora Pall, was investigated for PDGF-BB-induced HSC chemotaxis. Rat HSCs and LX-2, a human HSC cell line, were used for the in vitro experiments. Cell migration was analyzed by wound-healing and transwell assays. An ELISA and a Sircol collagen assay kit were used to detect the expressions of α-SMA and of collagen, respectively. Phosphorylations of mitogen-activated protein kinases, including ERK 1/2, p38, and JNK, were evaluated with immunoblotting. Results indicated that PDGF-BB increased migration as well as α-SMA and collagen expression in HSCs. Paeoniae Radix extracts and its active components, paeonol and 1,2,3,4,6-penta- O-galloyl- ß-D-glucose (PGG), inhibited PDGF-BB-induced HSC migration and α-SMA and collagen expressions in a concentration-dependent manner. The inhibitory effects were associated with downregulation of PDGF receptor- α, ERK, p38, and JNK activation. Both paeonol and PGG participate in HSC migration, but via differential mechanisms.

The Components of Flemingia macrophylla Attenuate Amyloid ß-Protein Accumulation by Regulating Amyloid ß-Protein Metabolic Pathway.

Flemingia macrophylla (Leguminosae) is a popular traditional remedy used in Taiwan as anti-inflammatory, promoting blood circulation and antidiabetes agent. Recent study also suggested its neuroprotective activity against Alzheimer's disease. Therefore, the effects of F. macrophylla on Aß production and degradation were studied. The effect of F. macrophylla on Aß metabolism was detected using the cultured mouse neuroblastoma cells N2a transfected with human Swedish mutant APP (swAPP-N2a cells). The effects on Aß degradation were evaluated on a cell-free system. An ELISA assay was applied to detect the level of Aß1-40 and Aß1-42. Western blots assay was employed to measure the levels of soluble amyloid precursor protein and insulin degrading enzyme (IDE). Three fractions of F. macrophylla modified Aß accumulation by both inhibiting ß-secretase and activating IDE. Three flavonoids modified Aß accumulation by activating IDE. The activated IDE pool by the flavonoids was distinctly regulated by bacitracin (an IDE inhibitor). Furthermore, flavonoid 94-18-13 also modulates Aß accumulation by enhancing IDE expression. In conclusion, the components of F. macrophylla possess the potential for developing new therapeutic drugs for Alzheimer's disease.

Effects of armepavine against hepatic fibrosis induced by thioacetamide in rats.

The aim of this study was to investigate if armepavine (Arm, C19H23O3N) could exert inhibitory effects against hepatic fibrosis in rats. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with tumour necrosis factor-α (TNF-α) to evaluate the inhibitory effects of Arm. Rats were injected with thioacetamide (TAA; 300 mg/kg, intraperitoneally) thrice a week for 4 weeks to induce hepatic fibrosis, with Arm (3 or 10 mg/kg) given by gavage twice a day. Liver sections were taken for western blotting, fibrosis scoring and immunofluorescence staining. Arm (1-10 µm) concentration-dependently attenuated TNF-α-stimulated: (i) protein expressions of α-smooth muscle actin (α-SMA), collagen type I and angiopoietin-1; (ii) H2O2 production; and (iii) NF-κB, JunD and C/EBPß (cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT)/enhancer binding protein-ß (EBPß)) nuclear translocations in HSC-T6 cells. In vivo Arm treatment significantly reduced plasma aspartate transaminase and alanine transaminase levels, hepatic α-SMA expression and collagen contents, and fibrosis scores of TAA-injected rats. Moreover, Arm treatment decreased α-SMA- and NF-κB-positive cells in immunohistochemical staining, and mRNA expression levels of IL-6, TGF-ß1, TIMP-1, col1α2, iNOS and ICAM-1 genes, but up-regulated the metallothionein gene in the livers of TAA-injected rats. Our results indicated that Arm exerted both in vitro and in vivo antifibrotic effects in rats, with inhibition of NF-κB, JunD and C/EBPß pathways.

Dendrobium nobile protects retinal cells from UV-induced oxidative stress damage via Nrf2/HO-1 and MAPK pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Excessive UV irradiation and ROS exposure are the main contributors of ocular pathologies. Pseudobulb of Dendrobium nobile Lindl. is one of the sources of Shihu and has long been used in traditional Chinese medicine as a tonic to nourish stomach, replenish body fluid, antipyretic and anti-inflammation. AIM OF STUDY: This study aimed to investigate whether D. nobile could protect ocular cells against oxidative stress damage. MATERIALS AND METHODS: Retinal-related cell lines, ARPE-19 and RGC-5 cells, were pretreated with D. nobile extracts before H2O2- and UV-treatment. Cell viability and the oxidative stress were monitored by sulforhodamine B (SRB) and SOD1 and CAT assay kits, respectively. The oxidative stress related proteins were measured by Western blotting. RESULTS: Under activity-guided fractionation, a sesquiterpene-enriched fraction (DN-2) and a major component (1) could ameliorate H2O2- and UV-induced cytotoxicity and SOD1 and CAT activity, but not dendrobine, the chemical marker of D. nobile. Western blotting showed both DN-2 and compound 1 protected ARPE-19 cells against UV-induced oxidative stress damage by regulating MAPK and Nrf2/HO-1 signaling. CONCLUSION: Our results suggest D. nobile extract protects retinal pigment epithelia cells from UV- and oxidative stress-damage, which may have a beneficial effect on eye diseases.

Metabolomic and proteomic characterization of sng and pain phenotypes in fibromyalgia.

BACKGROUND: Fibromyalgia (FM) is characterized by chronic widespread pain. Its pathophysiological mechanisms remain poorly understood, and effective diagnosis and treatments are lacking. This study aimed to identify significantly changed biosignatures in FM and propose a novel classification for FM based on pain and soreness (sng) symptoms. METHODS: Urine and serum samples from 30 FM patients and 25 controls underwent metabolomic and proteomic profiling. RESULTS: Compared with controls, FM patients showed significant differential expression of three metabolites in urine and five metabolites and eight proteins in serum. Of them, DETP, 4-guanidinobutanoic acid, SM(d18:1/18:0), PC(20:1(11Z)/18:0), S100A7, SERPINB3, galectin-7 and LYVE1 were first reported as potential biomarkers for FM. Furthermore, lactate, 2-methylmaleate and cotinine in urine and lactate, SM(d18:1/25:1), SM(d18:1/26:1) and prostaglandin D2 (PGD2) and PCYOX1, ITIH4, PFN1, LRG1, C8G, C8A, CP, CDH5 and DBH in serum could differentiate pain- (PG) and sng-dominant groups (SG). Lactate, 2-methylmaleate, cotinine, PCYOX1, ITIH4, PFN1 and DBH have a higher level in SG. SM(d18:1/25:1), SM(d18:1/26:1), PGD2, LRG1, C8G, C8A, CP and CDH5 in SG are lower than PG. The omics results indicated disordered free radical scavenging, and lipid and amino acid metabolism networks and resulting NF-κB-dependent cytokine generation in FM. Lactate level was altered simultaneously in urine and serum and significantly higher in sng-dominant patients than others. CONCLUSIONS: In this study, we identified potential biomarkers from FM patients. The selected biomarkers could discriminate sng and pain phenotypes in FM patients. These results could help elucidate the underlying pathological mechanisms for more effective diagnosis and therapy for FM.

Kaerophyllin inhibits hepatic stellate cell activation by apoptotic bodies from hepatocytes.

BACKGROUND: Hepatic stellate cells (HSCs), the key cell type for hepatic fibrosis, become activated and profibrogenic in the presence of hepatocyte apoptotic bodies (ABs). Bupleurum scorzonerifolium (BS), a widely used traditional Chinese herb for liver diseases, was fractionated, and the inhibitory effects of BS extracts on AB-induced HSC migration were screened. The activity-guided fractionation led to a lignan, kaerophyllin. In this study, the anti-fibrotic effects of kaerophyllin were studied in the presence of ABs. METHODS: LX-2 cells phagocytosing ultraviolet (UV)-induced HepG2 ABs were investigated by confocal microscopy and flow cytometry. AB-induced HSC activation was evaluated by immunoblotting and real-time PCR analyses. HSC migration was measured by wound-healing assays. RESULTS: HepG2 ABs induced LX-2 activation, with the production of collagen I and α-smooth muscle actin, upregulated profibrogenic gene transcriptions and increased NF-κB activity, cell migration and phagocytosis. Kaerophyllin from BS antagonized AB-induced HSC migration and activation. CONCLUSIONS: Kaerophyllin inhibited AB-induced LX-2 activation and migration with downregulation of Akt/ERK phosphorylations and NF-κB activity. Our study suggests a novel platform for screening anti-fibrotic compounds with ABs.

Differential inhibitory effects of salvianolic acids on activation of rat hepatic stellate cells by platelet-derived growth factor.

Platelet-derived growth factor (PDGF) induces cell proliferation together with oxidative stress. The present study investigated the effects of salvianolic acid A (Sal A) and B (Sal B) on the PDGF-induced signaling cascades in hepatic stellate cells (HSCs). HSC-T6, a rat hepatic stellate cell line, was stimulated with PDGF (10 ng/mL). The inhibitory effects of Sal A and B on oxidative stress-related signaling pathways were assessed in vitro. The protein levels were measured by Western blotting. FACS analysis was applied to detect the thioredoxin (Trx) level. Sal A and B showed different inhibitory abilities on the PDGF-related pathway. Sal A inhibited 70-kDa ribosomal S6 kinase (p70(s6k)) and associated proteins. Sal B attenuated PDGF-induced c-jun-N-terminal kinase (JNK), p38, and PKC- δ phosphorylations. Both Sal A and B diminished the activation of PKD, Trx, heme-oxygenase (HO)-1, and Nrf2. Taken together, our results showed that Sal A and B attenuated PDGF-induced ROS formation in HSCs, possibly through different signaling pathways.

S-Petasin, the Main Sesquiterpene of Petasites formosanus, Inhibits Phosphodiesterase Activity and Suppresses Ovalbumin-Induced Airway Hyperresponsiveness.

S-Petasin is the main sesquiterpene of Petasites formosanus, a traditional folk medicine used to treat hypertension, tumors and asthma in Taiwan. The aim of the present study was to investigate its inhibitory effects on phosphodiesterase (PDE) 1-5, and on ovalbumin (OVA)-induced airway hyperresponsiveness (AHR) in a murine model of allergic asthma. S-Petasin concentration-dependently inhibited PDE3 and PDE4 activities with 50% inhibitory concentrations (IC(50)) of 25.5, and 17.5 µM, respectively. According to the Lineweaver-Burk analysis, S-petasin competitively inhibited PDE3 and PDE4 activities with respective dissociation constants for inhibitor binding (K(i)) of 25.3 and 18.1 µM, respectively. Both IC(50) and K(i) values for PDE3 were significantly greater than those for PDE4. S-Petasin (10-30 µmol/kg, administered subcutaneously (s.c.)) dose-dependently and significantly attenuated the enhanced pause (P(enh)) value induced by methacholine (MCh) in sensitized and challenged mice. It also significantly suppressed the increases in total inflammatory cells, lymphocytes, neutrophils, eosinophils and levels of cytokines, including interleukin (IL)-2, IL-4 and IL-5, tumor necrosis factor (TNF)-α and interferon (IFN)-γ in bronchoalveolar lavage fluid (BALF) of these mice. In addition, S-petasin (10-30 µmol/kg, s.c.) dose-dependently and significantly attenuated total and OVA-specific immunoglobulin E (IgE) levels in the serum and BALF, and enhanced the IgG(2a) level in serum of these mice. The PDE4(H) value of S-petasin was >300 µM; therefore, its PDE4(H)/PDE4(L) value was calculated to be >17. In conclusion, the present results for S-petasin at least partially explain why Petasites formosanus is used as a folk medicine to treat asthma in Taiwan.

Anti-inflammatory effects and chemical study of a flavonoid-enriched fraction from adlay bran.

Anti-inflammation-guided fractionation and purification were used to evaluate the bioactivity and components of adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) bran. Results showed that the fraction with high phenolic and flavonoid contents from the ethanol extracts of adlay bran suppressed LPS-stimulated IL-6 and TNF-α secretions in a concentration-dependent manner in RAW 264.7 cells and murine peritoneal macrophages. Fifteen compounds, including a novel aurone derivative, two chromones, one dihydrochalcone, one chalcone, four flavanones, five flavones and one isoflavone, were isolated from the active fraction. The structure of the new compound was elucidated by spectroscopic methods, including 1D and 2D NMR and MS. All of the isolates are reported for the first time from adlay except naringenin. LC/MS was also provided as an analytical platform. Our results suggest that flavonoids in adlay bran, partially at least, contribute to its anti-inflammatory effect. Thus, adlay bran may be beneficial to the health of consumers.

Inhibition of endothelial adhesion molecule expression by Monascus purpureus-fermented rice metabolites, monacolin K, ankaflavin, and monascin.

BACKGROUND: Inflammation is an independent risk factor of cardiovascular diseases and is associated with endothelial dysfunction. Monascus purpureus-fermented rice, containing naturally occurring statins and various pigments, has lipid-modulating, anti-inflammatory and antioxidative effects. RESULTS: The effects of monacolin K, ankaflavin and monascin, as metabolites from Monascus-fermented rice, on the expression of cell adhesion molecules (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecular-1 (VCAM-1) and E-selectin) by tumor necrosis factor (TNF)-α-treated human aortic endothelial cells (HAECs) were investigated. Supplement of HAECs with these Monascus-fermented rice metabolites significantly suppressed cellular binding between the human monocytic cells U937 and TNF-α-stimulated HAECs. Immunoblot analysis showed that Monascus-fermented rice metabolites significantly attenuated TNF-α-induced VCAM-1 and E-selectin but not ICAM-1 protein expression. Gel shift assays showed that Monascus-fermented rice metabolites treatment reduced TNF-α-activated transcription factor nuclear factor (NF)-κB. Furthermore, Monascus-fermented rice metabolites also attenuated reactive oxygen species (ROS) generation in vitro and in TNF-α-treated HAECs. Supplement with an ROS scavenger N-acetylcysteine gave similar results as compared with Monascus-fermented rice metabolites. CONCLUSION: Monascus-fermented rice metabolites reduced TNF-α-stimulated endothelial adhesiveness as well as downregulating intracellular ROS formation, NF-κB activation, and VCAM-1/E-selectin expression in HAECs, supporting the notion that the various metabolites from Monascus-fermented rice might have potential implications in clinical atherosclerosis disease.