Effect of wogonin, a plant flavone from Scutellaria radix, on the suppression of cyclooxygenase-2 and the induction of inducible nitric oxide synthase in lipopolysaccharide-treated RAW 264.7 cells

Plant flavonoids show anti-inflammatory activity both in vitro and in vivo. Some flavonoids, such as flavone derivatives, have been reported previously to inhibit nitric oxide (NO) production by suppressing inducible nitric oxide synthase (iNOS) expression. In this investigation, the effects of wogonin, a potent inhibitor of NO production among the flavonoids tested, on cyclooxygenase-2 (COX-2) induction and activity were elucidated further in connection with iNOS, using a mouse macrophage cell line, RAW 264.7. Wogonin inhibited NO and prostaglandin E(2) (PGE(2)) production from lipopolysaccharide-induced RAW cells with IC(50) values of 31 and 0.3 microM, respectively. When added after the induction of iNOS and COX-2, wogonin inhibited the formation of PGE(2) (IC(50) = 0.8 microM), but not the production of NO. Wogonin inhibited COX-2 activity directly (IC(50) = 46 microM) from the homogenate of aspirin-pretreated RAW cells, as determined by measuring [(14)C]PGE(2) formation from [(14)C]arachidonic acid. However, it did not inhibit iNOS or phospholipase A(2) activity. Western blotting showed that wogonin suppressed the induction of both iNOS and COX-2. Prednisolone also suppressed the induction of iNOS and COX-2. Whereas RU-486 (a steroid receptor antagonist) reversed the suppressive activity of prednisolone, it did not affect the suppressive activity of wogonin, suggesting that the suppressive activity of wogonin is not mediated by binding to a steroid receptor. Results from the present study demonstrated that wogonin is a direct COX-2 inhibitor, as well as an inhibitor of iNOS and COX-2 induction. Wogonin may be a potential agent for use in the treatment of inflammatory diseases.     

Neuroprotective effect of wogonin: Potential roles of inflammatory cytokines

Wogonin (5,7-dihydroxy-8-methoxyflavone), an active component originated from the root of Scutellaria baicalensis Georgi, has been reported to possess antioxidant and anti-inflammatory properties. In this study, we investigated the neuroprotective effect of wogonin in a focal cerebral ischemia rat model. Wogonin markedly reduced the infarct volume after 2 h middle cerebral artery occlusion followed by 22 h reperfusion. Wogonin decreased the production of nitric oxide and inflammatory cytokines such as TNF-α and IL-6 in lipopolisaccharide-stimulated microglial cells. While wogonin reduced the activity ofNF-kB, it did not change the activity of mitogen-activated protein kinases family members, p38, ERK and JNK. The lipopolisaccharide-stimulated production of NO and cytokines was significantly blocked by various kinds ofNF-kB inhibitors such asN-acetyl cysteine, pyrrolidinedithiocarbamate and MG-132. The data may indicate that wogonin has neuroprotective effect by preventing the over-activation of microglial cells, possibly by inactivatingNF-kB signaling pathway     

Structure and antiinflammatory activity relationships of wogonin derivatives

A number of wogonin derivatives have been synthesized as congeners of wogonin and evaluated for their inhibitory activities of PGE2 production. Wogonin derivatives modified at the B ring of wogonin were obtained from 2,4-Dihydroxy-3,6-dimethoxyacetophenone (1) via several steps. Most wogonin derivatives exhibited much reduced inhibitory activities against COX-2 catalyzed PGE2 production compared to that of wogonin. Alkylation of 5,7-phenol groups and substitution at the B ring of wogonin generally caused reduction of inhibitory activity.     

Inhibition of prostaglandin E<Subscript>2</Subscript> production by synthetic Wogonin analogs

Effects of wogonin analogs on cyclooxygenase-2 (COX-2) catalyzed prostaglandin E₂ production on lipopolysaccharide (LPS)-induced RAW 264.7 cells were investigated. Wogonin analogs were prepared via several different synthetic pathways. Among wogonin analogs tested, 8-halogeno and 8-nitro analogs showed strong inhibitory activities against COX-2 catalyzed PGE₂ production from LPS-induced RAW 264.7 cells. Effect of wogonin was largely dependent on structural alteration of the 8-methoxy group.     

Modulation of suppressive activity of lipopolysaccharide-induced nitric oxide production by glycosidation of flavonoids

Flavonoids have been demonstrated to exhibit a wide range of biological activities including anti-inflammatory and neuroprotective actions. Although a significant amount of flavonoids has been identified to be present as glycosides in medicinal plants, determinations of the biological activities of flavonoids were mainly carried out with aglycones of flavonoids. Therefore, the exact role of the glycosidation of flavonoid aglycones needs to be established. In an attempt to understand the possible role of glycosidation on the modulation of the biological activities of flavonoids, diverse glycosides of kaempferol, quercetin, and aromadendrin were examined in terms of their anti-inflammatory activity determined with the suppression of lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV2 microglial cells. The results indicated that glycosidation of aglycones attenuated the suppressive activity of aglycones on LPS-induced NO production. Although attenuated, some of glycosides, depending on the position and degree of glycosidation, maintained the inhibitory capability of LPS-induced NO production. These findings suggest that glycosidation of flavonoid aglycones should be considered as an important modulator of the biological activities of flavonoids.     

Anticonvulsant effect of wogonin isolated from Scutellaria baicalensis

In previous studies, we identified sedative effects of Scutellaria baicalensis extracts and found that these extracts or their constituents may also have anticonvulsive effects. Wogonin is a natural product isolated from S. baicalensis, which possesses central nervous system effects such as anxiolytic and neuroprotective activities. In this study, we investigated the effects of wogonin on convulsion related behaviors, such as myorelaxation, motor coordination, and anticonvulsant effects of wogonin on chemical induced seizure and electroshock seizure in mice or rats. The effect of wogonin on membrane potential was also observed. Wogonin was intraperitoneally injected into mice or rats 30 min prior to testing. Animals treated with wogonin did not change locomotor activities as well as endurance times on the rota-rod, which indicates that wogonin did not cause a sedative and myorelaxation effect. Wogonin significantly blocked convulsion induced by pentylenetetrazole and electroshock but not convulsion induced by strychnine. Wogonin also significantly reduced the electrogenic response score, but flumazenil treatment reversed this decrease to the level of the control group. The wogonin treatment increased Cl(-)influx into the intracellular area as dose increased. Flumazenil and bicuculline treatment, however, inhibited the Cl(-) influx induced by wogonin. These results indicate that the anticonvulsive effects produced by wogonin were mediated by the GABAergic neuron.     

Wogonin inhibits excitotoxic and oxidative neuronal damage in primary cultured rat cortical cells

The present study evaluated effects of wogonin (5,7-dihydroxy-8-methoxyflavone) on excitotoxic and oxidative stress-induced neuronal damage in primary cultured rat cortical cells. Wogonin was shown to inhibit the excitotoxicity induced by glutamate or N-methyl-D-aspartic acid, whereas it showed no effects on the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- or kainate-induced toxicity. In addition, wogonin inhibited the oxidative neuronal damage induced by H(2)O(2), xanthine/xanthine oxidase, and by a glutathione depleting agent D,L-buthionine [S,R]-sulfoximine. Furthermore, wogonin dramatically inhibited lipid peroxidation initiated by Fe(2+) and L-ascorbic acid in rat brain homogenates. It also exhibited 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. Taken together, these results demonstrate that wogonin exhibits neuroprotective actions in cultured cortical cells by inhibiting excitotoxicity and various types of oxidative stress-induced damage, and that its antioxidant actions with radical scavenging activity may contribute, at least in part, to the neuroprotective effects.     

Wogonin inhibits microglial cell migration via suppression of nuclear factor-kappa B activity

Previously, we and others have demonstrated that wogonin, an active component from the root of Scutellaria baicalensis Georgi, has a neuroprotective effect in cerebral ischemic insult. The neuroprotective effect of wogonin may at least in part be due to its anti-inflammatory properties. Microglial cells, well-known residential macrophages in the central nervous system, migrate to the ischemic lesion and play a pivotal role in the development of chronic inflammation. In the present study, we observed that wogonin potently inhibited microglial migration toward a chemokine, monocyte chemoattractant protein-1 (MCP-1). The anti-migratory effect of wogonin was provoked at nanomolar concentrations, at which wogonin did not significantly inhibit the production of cytokines and chemokines. NF-kappaB has previously shown to regulate microglial cell migration, and activation of cAMP-signaling pathway has also been associated with inhibition of microglial cell motility. In the present study, wogonin at low micromolar concentrations completely suppressed the activity of NF-kappaB in MCP-1-stimulated microglia, and NF-kappaB inhibitors such as N-acetyl cysteine and pyrrolidinedithiocarbamate inhibited the MCP-1-induced migration of microglial cells. However, wogonin did not stimulate the production of cAMP in microglial cells. Our results indicate that the anti-inflammatory activity of wogonin is exerted at least in part by suppressing microglial cell motility via inhibition of NF-kappaB activity.     

Wogonin inhibits inducible prostaglandin E(2) production in macrophages

Effects of 5,7-dihydroxy-8-methoxyflavone (wogonin) on cyclooxygenase-2 (COX-2)-mediated prostaglandin E(2) production in macrophages were investigated. Stimulation with lipopolysaccharide (LPS; 1 microg/ml) greatly increased prostaglandin E(2) production in RAW 264.7 murine macrophages. The stimulated prostaglandin E(2) production was abolished in the presence of indomethacin (1 microM) or cycloheximide (2 microM), suggesting that the increased production of prostaglandin E(2) by LPS reflects the inducible synthesis of prostaglandin E(2) by COX-2. Wogonin (0.1-50 microM) concentration-dependently inhibited inducible prostaglandin E(2) production. Wogonin at concentrations as low as 0.5 microM directly attenuated enzymatic activity of COX-2. The protein expression of COX-2 was depressed by wogonin at concentrations of 10 microM and more. These results suggest that wogonin decreases inducible prostaglandin E(2) production in macrophages by inhibiting both COX-2 activity and COX-2 expression. The former action requires much lower doses of wogonin. These wogonin actions may explain, in part, its anti-inflammatory action.     

Inhibition of TPA-induced cyclooxygenase-2 expression and skin inflammation in mice by wogonin, a plant flavone from Scutellaria radix

Wogonin (5,7-dihydroxy-8-methoxyflavone), isolated from Scutellaria radix, was previously reported to inhibit the expression and activity of the enzyme cyclooxygenase-2 in lipopolysaccharide (LPS)-stimulated cells of a mouse macrophage cell line, RAW 264.7. Here, in order to find in vivo effects, inhibition by wogonin of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cyclooxygenase-2 expression and anti-inflammatory activity in vivo were investigated. When applied topically to the dorsal skin of mice, wogonin at doses of 50-200 microg/site/treatment (total of five treatments in 3 days) inhibited cyclooxygenase-2 expression and prostaglandin E2 production induced by multiple treatments with TPA. At 200 microg/site/treatment, wogonin caused a 55.3% reduction of prostaglandin E2 production on the dorsal skin compared with an increased production in the TPA-treated control group. The same compound significantly inhibited mouse ear edema induced by TPA in both preventive (58.1% inhibition) as well as curative treatment (31.3% inhibition) schedules at 200 microg/ear/treatment. Inhibition of neutrophil infiltration was also observed. Therefore, wogonin may be beneficial for cyclooxygenase-2-related skin disorders.     

Anti-inflammatory effects of catechols in lipopolysaccharide-stimulated microglia cells: inhibition of microglial neurotoxicity

Microglial activation plays a pivotal role in the pathogenesis of neurodegenerative diseases by producing various proinflammatory cytokines and nitric oxide (NO). In the present study, the anti-inflammatory and subsequent neuroprotective effects of catechol and its derivatives including 3-methylcatechol, 4-methylcatechol, and 4-tert-butylcatechol were investigated in microglia and neuroblastoma cells in culture. The four catechol compounds showed anti-inflammatory effects with different potency. The catechols significantly decreased lipopolysaccharide (LPS)-induced NO and tumor necrosis factor (TNF)-alpha production in BV-2 microglia cells. The catechols also inhibited the expression of inducible nitric oxide synthase (iNOS) and TNF-alpha at mRNA or protein levels in the LPS-stimulated BV-2 cells. In addition, the catechols inhibited LPS-induced nuclear translocation of p65 subunit of nuclear factor (NF)-kappaB, IkappaB degradation, and phosphorylation of p38 mitogen-activated protein kinase (MAPK) in BV-2 cells. Moreover, the catechols attenuated the cytotoxicity of LPS-stimulated BV-2 microglia toward co-cultured rat B35 neuroblastoma cells. The catechols, however, did not protect B35 cells against H(2)O(2) toxicity, indicating that the compounds exerted the neuroprotective effect by inhibiting the inflammatory activation of microglia in the co-culture. The anti-inflammatory and neuroprotective properties of the catechols in cultured microglia and neuroblastoma cells suggest a therapeutic potential of these compounds for the treatment of neurodegenerative diseases that are associated with an excessive microglial activation.     

天然产物汉黄芩素的研究进展

天然产物汉黄芩素(wogonin)具有广泛的生物活性,如抗氧化、抗炎、神经保护、抗肿瘤和抗病毒活性等,目前引起了国内外学者的广泛关注。本文综述了近年来国内外关于汉黄芩素的植物提取、人工合成、生物活性及其作用机制方面的研究进展。     

Wogonin, baicalin, and baicalein inhibition of inducible nitric oxide synthase and cyclooxygenase-2 gene expressions induced by nitric oxide synthase inhibitors and lipopolysaccharide

We previously reported that oroxylin A, a polyphenolic compound, was a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In the present study, three oroxylin A structurally related polyphenols isolated from the Chinese herb Huang Qui, namely baicalin, baicalein, and wogonin, were examined for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. The results indicated that these three polyphenolic compounds inhibited LPS-induced NO production in a concentration-dependent manner without a notable cytotoxic effect on these cells. The decrease in NO production was in parallel with the inhibition by these polyphenolic compounds of LPS-induced iNOS gene expression. However, these three compounds did not directly affect iNOS enzyme activity. In addition, wogonin, but not baicalin or baicalein, inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity. Furthermore, N-nitro-L-arginine (NLA) and N-nitro-L-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS (but not COX-2) protein expression, which was inhibited by these three polyphenolic compounds. Wogonin, but not baicalin or baicalein, similarly inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cells. These results indicated that co-treatment with NOS inhibitors and polyphenolic compounds such as wogonin effectively blocks acute production of NO and, at the same time, inhibits expression of iNOS and COX-2 genes.     

Inhibitory effects of wogonin on the invasion of human breast carcinoma cells by downregulating the expression and activity of matrix metalloproteinase-9

Wogonin, a naturally occurring monoflavonoid extracted from Scutellariae radix, has been shown to possess tumor therapeutic potential in vitro and in vivo. However, the effects of wogonin on tumor cells invasion remains poorly understood. In this study, we performed in vitro experiments to investigate the anti-invasive and anti-metastatic activity of wogonin in MDA-MB-231 human breast carcinoma cells. Wogonin caused a concentration-dependent suppression of cell migration, adhesion and invasion. The mechanism revealed that wogonin significantly inhibited the expression and activity of both endogenous and phorbol-12-myristate-13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) potentially associating with the suppression of translocation of protein kinase C (PKC) δ and phosphorylation of extracellular signal-regulated kinase (ERK1/2). These results suggested that wogonin could inhibit the invasion of tumor cells by downregulating the expression and activity of MMP-9, the possible targets may be PKCδ and ERK1/2.     

In vitro and in vivo inhibitory activities of rutin,wogonin, and quercetin on lipopolysaccharide-induced nitric oxide and prostaglandin E(2) production

Flavonoids are widely distributed in plants, but their biological functions are still unclear. In the present study, in vitro and in vivo experiments were performed to demonstrate the inhibitory activities of rutin, wogonin, and quercetin on lipopolysaccharide-induced nitric oxide (NO) and prostaglandin E(2) production in RAW 264.7 macrophages, primary peritoneal macrophages, and Balb/c mice, respectively. In vitro results showed that wogonin and quercetin dose-dependently suppressed lipopolysaccharide-induced NO production in RAW 264.7 macrophages and primary peritoneal macrophages without a notable cytotoxic effect on either cell types associated with a decrease in inducible nitric oxide synthase (iNOS) protein expression in both cells. Rutin, at 80 microM only, had a slight but obvious inhibitory effect on lipopolysaccharide-induced NO production in primary peritoneal macrophages. Both wogonin and quercetin attenuated lipopolysaccharide-induced prostaglandin E(2) production in vitro. Intravenous injection of lipopolysaccharide (10 mg/kg, i.v.) resulted in a time-dependent induction of NO production in serum, and pretreatment with the L-arginine analog N-nitro-L-arginine methyl ester (L-NAME) blocked this induction. Intravenous pretreatment of Balb/c mice with rutin, wogonin or quercetin for 1 h followed by lipopolysaccharide treatment significantly inhibited lipopolysaccharide-induced NO production, but no inhibition of prostaglandin E(2) production was found. A decrease in iNOS protein, but not cyclooxygenase-2 protein, was detected in liver and lung specimens of lipopolysaccharide-treated Balb/c mice in the presence of rutin, wogonin or quercetin. In conclusion, data obtained both in vitro and in vivo suggest that wogonin and quercetin exert inhibitory activity on lipopolysaccharide-induced NO production through suppression of iNOS expression.     

Inhibition of contact dermatitis in animal models and suppression of proinflammatory gene expression by topically applied Flavonoid,Wogonin

Wogonin (5,7-dihydroxy-8-methoxyflavone) is a down-regulator of cyclooxygenase-2 and inducible nitric oxide synthase expression, contributing to anti-inflammatory activity in vivo. For further characterization of modulatory activity on proinflammatory gene expression in vivo, the effect of wogonin was examined in this experiment using animal models of skin inflammation. By topical application, wogonin inhibited an edematic response as well as proinflammatory gene expression against contact dermatitis in mice. Wogonin inhibited ear edema (19.4-22.6%) at doses of 50-200 microg/ear and down-regulated interleukin-1beta induction (23.1%) at 200 microg/ear in phenol-induced simple irritation. Wogonin (2x50-2x200 microg/ear) also inhibited edematic response (51.2-43.9%) and down-regulated proinflammatory gene expression of cyclooxygenase-2, interleukin-1beta, interferon-gamma, intercellular adhesion molecule-1 and inducible nitric oxide synthase with some different sensitivity against picryl chloride-induced delayed hypersensitivity reaction. All these results clearly demonstrate that wogonin is a down-regulator of proinflammatory gene expression in animal models of skin inflammation. Therefore, wogonin may have potential for a new anti-inflammatory agent against skin inflammation.     

Effects of naturally occurring flavonoids on nitric oxide production in the macrophage cell line RAW 264.7 and their structure-activity relationships.

Flavonoids affect the inflammatory process of the mammalian system and possess anti-inflammatory as well as immunomodulatory activities in vitro and in vivo. Since nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is one of the inflammatory mediators, the effects of various naturally occurring flavonoids on NO production in LPS-activated RAW 264.7 cells were evaluated in vitro. Flavonoids such as apigenin, wogonin, luteolin, tectorigenin, and quercetin inhibited NO production, as measured by nitrite formation at 10-100 microM. The most active among 26 flavonoid derivatives tested were apigenin, wogonin, and luteolin, having IC50 values of 23, 17, and 27 microM, respectively, while AMT, a synthetic selective iNOS inhibitor, had an IC50 value of 0.09 microM. In contrast, flavanones, such as naringenin, and flavonoid glycosides, such as apiin, did not demonstrate significant inhibition up to 100 microM. These results clearly indicated that a C-2,3 double bond might be important, and that the potency of inhibition depended upon the substitution patterns of the flavonoid molecules. The inhibitory activity of flavonoids was not due to direct inhibition of iNOS enzyme activity because they did not reasonably inhibit iNOS activity, as measured by [3H]citrulline formation from [3H]arginine, up to 100 microM. In contrast, wogonin and luteolin concentration-dependently reduced iNOS enzyme expression, when measured by western blotting, at 10-100 microM. All these results clearly demonstrated that certain flavonoids inhibit NO production in lipopolysaccharide-activated RAW 264.7 cells, and their inhibitory activity might be due to reduction of iNOS enzyme expression.     

Neuroprotective effect of curcumin is mainly mediated by blockade of microglial cell activation

Curcumin, the major yellow pigment in turmeric (Curcuma longa), is a well-documented naturally-occurring anti-oxidant with numerous pharmacological activities such as anti-inflammatory, anti-carcinogenic and anti-bacterial effects. In this study, curcumin's neuroprotective effect was carefully examined using a coculture system, based on reports that curcumin-containing plants are neuroprotective. Coculturing neuronal cells and activated microglial cells enhanced dopamine-induced neuronal cell death from 30% up to 50%. However, curcumin did not protect dopamine-directed neuronal cell death and sodium nitroprosside (SNP)-induced NO generation, but only blocked activated microglial cell-mediated neuronal cell damage under inflammatory conditions. Indeed, curcumin blocked the production of pro-inflammatory and cytotoxic mediators such as NO, TNF-alpha, IL-1alpha, and IL-6 produced from Abeta(25-35)/IFN-gamma- and LPS-stimulated microglia, in a dose-dependent manner. Therefore, our results suggest that curcumin-mediated neuroprotective effects may be mostly due to its anti-inflammatory effects.     

Relaxant effect induced by wogonin from Scutellaria baicalensis on rat isolated uterine smooth muscle

Context: Wogonin is a flavone derivative isolated from Scutellaria baicalensis Georgi (Labiatae) root, which is a traditional Chinese drug used as an anti-inflammatory and for management of dysmenorrhea.

Objective: The effect of wogonin on the uterus has not yet been examined. We investigated the relaxant effects of wogonin on contractile activity of isolated uterine strips of rats.

Materials and methods: The effect of wogonin on spontaneous uterine contraction, and uterine contraction induced by agonists, K⁺-depolarization and oxytocin in Ca²⁺-free solution was observed. To clarify the type of potassium channel, we tested the effects of 4-aminopyridine, tetraethylammonium and glibenclamide.

Results: Wogonin reduced the contractile amplitude of uterine strip smooth muscle of rats in a dose-dependent manner. The concentration of wogonin for reducing the contraction amplitude by 50% (IC₅₀) on spontaneous contractions was 60.5 μM. Wogonin also inhibited the contraction induced by three agonists (oxytocin, prostaglandin F_2α and acetylcholine). For the uterine strips pretreated with oxytocin in Ca²⁺-free solution or K⁺-depolarization, wogonin showed relaxant effect on the induced uterine contractions. In addition, whereas the inhibitive effect of wogonin on the contraction of uterine smooth muscle in rats could be partly blocked by 4-aminopyridine and tetraethylammonium, it was not influenced by glibenclamide.

Discussion and conclusion: Wogonin significantly inhibited the contraction of rat uterine smooth muscle probably through the inhibition of the inflow of extracellular calcium into cells via cell membrane, and intracellular release of calcium ions. In addition, the relaxant effect induced by wogonin might be due in part to the opening of voltage-dependent and large conductance Ca²⁺-activated K⁺ channels.