Deok Hyo Yoon

Cordyceps pruinosa extracts induce apoptosis of HeLa cells by a caspase dependent pathway.

AIM OF THE STUDY Cordyceps is a parasitic fungus and has long been used as a traditional Chinese medicine to treat illnesses, promote longevity, increase athletic power, and relieve exhaustion and cancer. In this study, we reveal the mechanisms underlying apoptosis induced by Cordyceps pruinosa butanol fraction (CPBF) in the human cervical adenocarcinoma cell line, HeLa. MATERIALS AND METHODS Proliferation and apoptosis of cells were examined by MTT assay, DNA fragmentation, phosphatidyl serine distribution assay, Western blot analysis, and immunocytochemistry. To determine the association between CPBF related apoptosis and ROS, electron spin resonance (ESR) trapping experiments were used. RESULTS CPBF inhibited proliferation and induced apoptosis in HeLa cells in a dose-dependent manner using a MTT assay, DNA fragmentation, and a phosphatidyl serine distribution assay. Western blot analysis showed that apoptosis in HeLa cells was caspase-3- and -9-dependent. Proteolytic cleavage of PARP and the release of cytochrome c from the mitochondria into the cytosol were significantly increased and the Bcl-2/Bax protein ratio was decreased. Apoptosis induced by CPBF was not prevented by various antioxidants. CONCLUSIONS These results indicate that apoptotic effects of CPBF on HeLa cells are mediated by mitochondria-dependent death-signaling pathway independent of reactive oxygen species, suggesting that CPBF might be effective as an anti-proliferative agent for cancer.

Anti-inflammatory activities and mechanisms of Artemisia asiatica ethanol extract

ETHNOPHARMACOLOGICAL RELEVANCE Artemisia asiatica Nakai (Compositae) is a representative herbal plant used to treat infection and inflammatory diseases. Although Artemisia asiatica is reported to have immunopharmacological activities, the mechanisms of these activities and the effectiveness of Artemisia asiatica preparations in use are not known. MATERIALS AND METHODS To evaluate the anti-inflammatory activities of Artemisia asiatica ethanol extract (Aa-EE), we assayed nitric oxide (NO), tumor necrosis factor (TNF)-α, and prostaglandin E2 (PGE2) in macrophages and measured the extent of tissue injury in a model of gastric ulcer induced in mice by treatment with HCl in EtOH. Putative enzymatic mediators of Aa-EE activities were identified by nuclear fractionation, reporter gene assay, immunoprecipitation, immunoblotting, and kinase assay. Active compound in Aa-EE was identified using HPLC. RESULTS Treatment of RAW264.7 cells and peritoneal macrophages with Aa-EE suppressed the production of NO, PGE2, and TNF-α in response to lipopolysaccharide (LPS) and induced heme oxygenase-1 expression. The Aa-EE also ameliorated symptoms of gastric ulcer in HCl/EtOH-treated mice. These effects were associated with the inhibition of nuclear translocation of nuclear factor (NF)-κB and activator protein (AP)-1, implying that the anti-inflammatory action of the Aa-EE occurred through transcriptional inhibition. The upstream regulatory signals Syk and Src for translocation of NF-κB and TRAF6 for AP-1 were identified as targets of this effect. Analysis of Aa-EE by HPLC revealed the presence of luteolin, known to inhibit NO and PGE2 activity. CONCLUSION The anti-inflammatory activities attributed to Artemisia asiatica Nakai in traditional medicine may be mediated by luteolin through inhibition of Src/Syk/NF-κB and TRAF6/JNK/AP-1 signaling pathways.

In vitro and in vivo anti-inflammatory effect of Rhodomyrtus tomentosa methanol extract.

ETHNOPHARMACOLOGICAL RELEVANCE Rhodomyrtus tomentosa (Aiton) Hassk. is a representative Thai medicinal plant traditionally used in South Asian countries to relieve various inflammatory symptoms. However, no systematic studies on its anti-inflammatory activity and mechanisms have been reported. MATERIALS AND METHODS The effect of the methanol extract from the leaves of this plant (Rt-ME) on the production of inflammatory mediators [nitric oxide (NO) and prostaglandin E2 (PGE2)] and the molecular mechanism of Rt-ME-mediated inhibition, including target enzymes, were studied with RAW264.7, peritoneal macrophage, and HEK293 cells. Additionally, the in vivo anti-inflammatory activity of this extract was evaluated with mouse gastritis and colitis models. RESULTS Rt-ME clearly inhibited the production of NO and PGE2 in lipopolysaccharide (LPS)-activated RAW264.7 cells and peritoneal macrophages in a dose-dependent manner. According to RT-PCR, immunoblotting and immunoprecipitation analyses and a kinase assay with mRNA, whole cell extract, and nucleus lysates from RAW264.7 cells and mice, it was revealed that Rt-ME was capable of suppressing the activation of both nuclear factor (NF)-κB and activator protein (AP)-1 pathways by directly targeting Syk/Src and IRAK1/IRAK4. CONCLUSION Rt-ME could have anti-inflammatory properties by suppressing Syk/Src/NF-kB and IRAK1/IRAK4/AP-1 pathways and will be further developed as a herbal remedy for preventive and/or curative purposes in various inflammatory diseases.

In vitro and in vivo anti-inflammatory activity of Phyllanthus acidus methanolic extract.

ETHNOPHARMACOLOGICAL RELEVANCE Phyllanthus acidus (L.) Skeels (Phyllanthaceae) has traditionally been used to treat gastric trouble, rheumatism, bronchitis, asthma, respiratory disorders, and hepatitis. Despite this widespread use, the pharmacological activities of this plant and their molecular mechanisms are poorly understood. Therefore, we evaluated the immunopharmacological activities of the methanolic extract of the aerial parts of this plant (Pa-ME) and validated its pharmacological targets. MATERIALS AND METHODS Lipopolysaccharide (LPS)-treated macrophages, an HCl/EtOH-induced gastritis model, and an acetic acid-injected capillary permeability mouse model were employed to evaluate the anti-inflammatory activity of Pa-ME. Potentially active anti-inflammatory components of this extract were identified by HPLC. The molecular mechanisms of the anti-inflammatory activity were studied by kinase assays, reporter gene assays, immunoprecipitation analysis, and overexpression of target enzymes. RESULTS Pa-ME suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2) and prevented morphological changes in LPS-treated RAW264.7 cells. Moreover, both HCl/EtOH-induced gastric damage and acetic acid-triggered vascular permeability were restored by orally administered Pa-ME. Furthermore, this extract downregulated the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 and reduced the nuclear levels of NF-κB. Signalling events upstream of NF-κB translocation, such as phosphorylation of Src and Syk and formation of Src/Syk signalling complexes, were also inhibited by Pa-ME. The enzymatic activities of Src and Syk were also suppressed by Pa-ME. Moreover, Src-induced and Syk-induced luciferase activity and p85/Akt phosphorylation were also inhibited by Pa-ME. Of the identified flavonoids, kaempferol and quercetin were revealed as partially active anti-inflammatory components in Pa-ME. CONCLUSION Pa-ME exerts anti-inflammatory activity in vitro and in vivo by suppressing Src, Syk, and their downstream transcription factor, NF-κB.

JAK2-targeted anti-inflammatory effect of a resveratrol derivative 2,4-dihydroxy-N-(4-hydroxyphenyl)benzamide

Chemical derivatization of resveratrol has been widely conducted in an effort to overcome its chemical instability and therapeutic potential. In the present study, we examined the anti-inflammatory effects of resveratrol derivatives containing an amide functionality using in vitro macrophage models that were stimulated by Toll-like receptor (TLR) ligands, and using several animal inflammatory disease models. Of the resveratrol derivatives tested, compound 8 (2,4-dihydroxy-N-(4-hydroxyphenyl)benzamide) most strongly inhibited the production of nitric oxide (NO), tumor necrosis factor (TNF)-α, and prostaglandin E2 (PGE2), as well as the mRNA expression of inducible NO synthase (iNOS), TNF-α, and cyclooxygenase (COX)-2 in lipopolysaccharide (LPS)-activated RAW264.7 cells, differentiated U937 cells, and peritoneal macrophages. The inhibitory activity of compound 8 was apparently mediated by suppressing the phosphorylation of signal transducer and activator of transcription (STAT)-1, STAT-3, STAT-5, and interferon regulatory factor (IRF)-3. The direct target of compound 8 was revealed to be Janus kinase 2 (JAK2) but not TANK-binding kinase (TBK) 1 using the direct kinase assay and analyses of complex formation with these molecules. Additionally, upstream kinase of TBK1 seems to be also inhibited by compound 8. This compound also strongly ameliorated mouse inflammatory symptoms seen in arachidonic acid-induced ear edema, dextran sodium sulfate (DSS)-treated colitis, EtOH/HCl-induced gastritis, collagen type II-triggered arthritis, and acetic acid-induced writhing. Therefore, of the resveratrol derivatives that we tested, compound 8 was determined to have the strongest anti-inflammatory activities in vitro and in vivo and may potentially be developed for use as a novel anti-inflammatory drug.

ERK1- and TBK1-targeted anti-inflammatory activity of an ethanol extract of Dryopteris crassirhizoma.

ETHNOPHARMACOLOGICAL RELEVANCE Dryopteris crassirhizoma Nakai (Aspiadaceae) has been traditionally used as an herbal medicine for treating various inflammatory and infectious diseases such as tapeworm infestation, colds, and viral diseases. However, no systematic studies on the anti-inflammatory actions of Dryopteris crassirhizoma and its inhibitory mechanisms have been reported. We therefore aimed at exploring the anti-inflammatory effects of 95% ethanol extracts (Dc-EE) of this plant. MATERIALS AND METHODS The anti-inflammatory effect of Dc-EE on the production of inflammatory mediators in RAW264.7 cells and HCl/EtOH-induced gastritis was examined. Inhibitory mechanisms were also evaluated by exploring activation of transcription factors, their upstream signalling, and target enzyme activities. Finally, the active components from this extract were also identified using HPLC system. RESULTS Dc-EE diminished the production of nitric oxide (NO) and prostaglandin (PG)E(2) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells in a dose-dependent manner. Dc-EE also downregulated the levels of mRNA expression of pro-inflammatory genes such as inducible NO synthase (iNOS), cyclooxygenase (COX)-2, and TNF-α by inhibiting the activation of activator protein (AP-1) and IRF3. Indeed, the extract strongly blocked the activities of their upstream kinases ERK1 and TBK1. This extract also strongly ameliorated gastritis symptoms stimulated by HCl/EtOH in mice. According to HPLC fingerprinting, resveratrol, quercetin, and kampferol were identified from Dc-EE. CONCLUSION Dc-EE displays strong anti-inflammatory activity by suppressing ERK/AP-1 and TBK1/IRF3 pathways, which contribute to its major ethno-pharmacological role as an anti-inflammatory and anti-infectious disease remedy.

In vivo and in vitro anti-inflammatory activities of Persicaria chinensis methanolic extract targeting Src/Syk/NF-κB

ETHNOPHARMACOLOGIC RELEVANCE Persicaria chinensis L. (Polygonaceae) [also synonym as Polygonum chimnense L.] has been used as Chinese traditional medicine to treat ulcer, eczema, stomach ache, and various inflammatory skin diseases. Due to no molecular pharmacological evidence of this anti-inflammatory herbal plant, we investigated the inhibitory mechanisms and target proteins contributing to the anti-inflammatory responses of the plant by using its methanolic extract (Pc-ME). MATERIALS AND METHODS We used lipopolysaccharide (LPS)-treated macrophages and a murine HCl/EtOH-induced gastritis model to evaluate the anti-inflammatory activity of Pc-ME. HPLC analysis was employed to identify potential active components of this extract. Molecular approaches including kinase assays, reporter gene assays, immunoprecipitation analysis, and overexpression of target enzymes were used to confirm target enzymes. RESULTS Pc-ME inhibited LPS-induced nitric oxide and prostaglandin E2 release by RAW264.7 macrophages and ameliorated HCl/EtOH-induced gastric ulcers in mice. The nuclear translocation of NF-κB (p65 and p50) was suppressed by Pc-ME. Phosphorylation of Src and Syk, their kinase activities, and formation of the signaling complex of these proteins were repressed by Pc-ME. Phosphorylation of p85 and Akt induced by Src or Syk overexpression was blocked by Pc-ME. In the mouse gastritis model, orally administered Pc-ME suppressed the increased phosphorylation of IκBα, Αkt, Src, and Syk. Caffeic acid, kaempferol, and quercetin, identified as major anti-inflammatory components of Pc-ME by HPLC, displayed strong nitric oxide inhibitory activity in LPS-treated macrophages. CONCLUSION Pc-ME might play a pivotal ethnopharmacologic role as an anti-inflammatory herbal medicine by targeting Syk and Src kinases and their downstream transcription factor NF-κB.

Methanol extract of Evodia lepta displays Syk/Src-targeted anti-inflammatory activity.

ETHNOPHARMACOLOGICAL RELEVANCE Evodia lepta (Spreng.) Merr., in the Rutaceae family, is a medicinal plant traditionally used to treat inflammatory symptoms such as in meningitis and hepatitis. However, no study has systematically investigated its anti-inflammatory activities including its molecular mechanism. MATERIALS AND METHODS The effects of a methanol extract from the roots Evodia lepta (El-ME) were evaluated using lipopolysaccharide (LPS)-treated RAW264.7 cells producing nitric oxide (NO) and prostaglandin E2 (PGE2), and an HCl/ethanol-induced mouse gastritis model. Target molecules were identified by analyzing the activation of transcription factors and their upstream kinases. RESULTS El-ME reduced the production of NO and PGE2 from LPS-activated RAW264.7 cells in a dose-dependent manner. El-ME also ameliorated the gastritis symptoms of EtOH/HCl-treated mice. The extract suppressed production of mRNA for the inducible NO synthase (iNOS) and cyclooxygenase (COX)-2; the nuclear translocation of nuclear factor (NF)-κB; the phosphorylation of upstream kinases that activate NF-κB; and the kinase activities of Syk and Src. CONCLUSION The anti-inflammatory effects of El-ME might be due to its suppression of Syk/Src and NF-κB. Considering the in vitro and in vivo efficacy of El-ME, Evodia lepta could be developed into an anti-inflammatory herbal remedy.

Syk and Src are major pharmacological targets of a Cerbera manghas methanol extract with kaempferol-based anti-inflammatory activity

ETHNOPHARMACOLOGICAL RELEVANCE Cerbera manghas L. (Apocynaceae), a semi-mangrove medicinal plant distributed throughout tropical and subtropical countries, is traditionally known to possess analgesic, anti-inflammatory, anti-convulsant, cardiotonic, and hypotensive activity. In vitro and in vivo anti-inflammatory activities of a methanol extract of the leaves of Cerbera manghas and the underlying molecular mechanisms were investigated to validate the ethnopharmacological use of this plant. MATERIALS AND METHODS The effect of Cerbera manghas methanol extract (Cm-ME) on the production of inflammatory mediators and the induction of HCl/EtOH-treated gastritis was explored using macrophages, HEK293 cells, and ICR mice. The molecular targets of this extract and potential active components in Cm-ME were also investigated. RESULTS Cm-ME inhibited the production of nitric oxide (NO) in lipopolysaccharide (LPS)-treated RAW264.7 cells and peritoneal macrophages in a dose-dependent manner. This extract also suppressed the expression of NO synthase (iNOS) and cyclooxygenase (COX)-2. NF-κB-mediated enhancement of luciferase activity, nuclear translocation of p50 and p65, and phosphorylation of IκBα were markedly reduced by Cm-ME treatment. Direct enzyme assays, reporter gene assays, and immunoprecipitation analysis of kinases revealed Syk and Src as immunopharmacological targets of Cm-ME. Moreover, this extract strongly ameliorated the gastric symptoms induced by HCl/EtOH treatment of mice. Finally, HPLC analysis and pharmacological tests identified kaempferol as an active component of the extract with Src/Syk inhibitory activities. CONCLUSION Inhibition of Syk/Src and the NF-κB pathway by kaempferol could play a key role in the anti-inflammatory pharmacological action of Cerbera manghas.

AP-1 pathway-targeted inhibition of inflammatory responses in LPS-treated macrophages and EtOH/HCl-treated stomach by Archidendron clypearia methanol extract

ETHNOPHARMACOLOGICAL RELEVANCE Archidendron clypearia Jack. (Fabaceae) is a representative ethnomedicinal herbal plant prescribed for various inflammatory diseases such as pharyngolaryngitis and tonsillitis. However, the pharmacology behind this plants anti-inflammatory properties has not been fully understood. Therefore, in this study, the anti-inflammatory mechanism of a 95% methanol extract (Ac-ME) was explored. MATERIALS AND METHODS The anti-inflammatory mechanism of Ac-ME on the AP-1 activation pathway, which plays a critical role in the production of prostaglandin (PG)E2 in RAW264.7 cells and peritoneal macrophages and in induction of acute gastritis caused by HCl/EtOH, was investigated using immunoblotting, immunoprecipitation analyses, and reporter gene activity assays. In particular, enzyme assays and HPLC analysis were employed to identify direct target enzymes of Ac-ME and to detect active chemical components from the plant extract. RESULTS Ac-ME clearly reduced the nuclear levels of total and phospho-forms of c-Jun, FRA-1, and ATF-2. Consequently, this extract suppressed both the production of PGE2 in lipopolysaccharide (LPS)-activated RAW264.7 and peritoneal macrophage cells and PGE2-dependent induction of gastritis lesion in stomach under EtOH/HCl exposure. Analysis of AP-1 upstream signalling revealed that the AP-1 activation pathway consisting of IRAK1, TRAF6, TAK1, MKK3/6, and p38 was predominantly inhibited by Ac-ME. Similarly, this extract directly blocked the enzyme activity of IRAK1, indicating that this enzyme is an inhibitory target of Ac-ME and is involved in the suppression of the AP-1 pathway. HPLC analysis showed that quercetin, which inhibits PGE2 production, is an active component in Ac-ME. CONCLUSION Ac-ME is an ethnomedicinal remedy with an IRAK1/p38/AP-1-targeted inhibitory property. Since AP-1 is a major inflammation-inducing transcription factor, the therapeutic potential of Ac-ME in other AP-1-mediated inflammatory symptoms will be further tested.