Involvement of heme oxygenase-1 in the anti-inflammatory activity of Chrysanthemum boreale Makino extracts on the expression of inducible nitric oxide synthase in RAW264.7 macrophages

Aim of the study

This study is to elucidate the involvement of anti-inflammatory heme oxygenase-1 (HO-1) in the inhibitory activity of a Chrysanthemum boreale Makino (CB) extract on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.

Materials and methods

Cell viability and NO assay were performed. In addition, iNOS expression was detected by Western blotting and real-time PCR. HO-1 expression was also evaluated by Western blotting, and blocking HO-1 activity on NO production was performed.

Results

The CB extract at the highest concentration (100 μg/ml) significantly inhibited NO production by approximately 90% and suppressed iNOS protein expression by approximately 84.8% compared to LPS-stimulated cells. Furthermore, the CB extract (100 μg/ml) inhibited iNOS mRNA expression in a concentration-dependant manner and suppressed iNOS mRNA expression by 94.8%. The CB extract induced the expression of HO-1 in a dose-dependent manner, and blocking HO-1 activity abolished the inhibitory effects of the CB extract. Moreover, the addition of carbon monoxide such as tricarbonyl dichlororuthenium (II) dimmer (RuCO), a byproduct derived from heme degradation, mimicked the inhibitory action of low concentrations of CB extract.

Conclusion

These results suggest that a CB extract has potent anti-inflammatory activity in RAW264.7 macrophages involving the induction of HO-1.     

Achyranthes japonica exhibits anti-inflammatory effect via NF-κB suppression and HO-1 induction in macrophages

Ethnopharmacological relevance

The roots of Achyranthes japonica Nakai have been used in traditional herbal medicine for the treatment of edema and arthritis in Korea.

Aim of the study

In this study, we investigated the molecular mechanism responsible for anti-inflammatory effects of the aqueous extract of A. japonica roots (AJ) in LPS-stimulated macrophages.

Materials and methods

Nitric oxide (NO) production and as inducible nitric oxide synthase (iNOS) expression were examined in TG-elicited peritoneal macrophages and RAW 264.7 cells. Cell viability was monitored by MTT assay. Protein and mRNA expressions were determined by Western blotting and RT-PCR, respectively. The activity of NF-κB and Nrf2 were examined by EMSA, immunocytochemistry or reporter assay.

Results

AJ inhibited LPS-induced NO secretion as well as iNOS expression, without affecting cell viability. Furthermore, AJ suppressed LPS-induced NF-κB activation, degradation of IκB-α, phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. Further study demonstrated that AJ induced heme oxygenase-1 (HO-1) gene expression via nuclear translocation and transactivation of Nrf2. In addition, the inhibitory effects of AJ on iNOS expression were abrogated by small interfering RNA-mediated knock-down of HO-1.

Conclusions

These results suggest that AJ suppresses LPS-induced NO production and iNOS expression in macrophages through the inhibition of IκB/NF-κB and MAPK as well as the Nrf2-mediated HO-1 induction. These findings provide the scientific rationale for anti-inflammatory therapeutic use of A. japonica roots.     

Anti-inflammatory effect of ethyl acetate extract from Cissus quadrangularis Linn may be involved with induction of heme oxygenase-1 and suppression of NF-κB activation

Aim of the study

Cissus quadrangularis (family: Vitaceae) has been widely used in traditional herbal medicine for the treatment of hemorrhoids, gastric ulcers and bone healing. In the present study, we determined the anti-inflammatory activity and the molecular mechanism of the ethyl acetate extract of Cissus quadrangularis stem (CQE) in LPS-stimulated RAW 264.7 macrophage cells.

Materials and methods

The inhibitory effect of CQE on LPS-induced nitric oxide (NO) production was evaluated in conditioned media. Cell viability was monitored by MTT assay. Protein and mRNA expressions were determined by RT-PCR and Western blotting analysis, respectively.

Results

CQE potently inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cells in a dose-dependent manner. The mRNA and protein expressions of inducible nitric oxide synthase (iNOS) were suppressed also by CQE as was p65 NF-κB nuclear translocation. Further study demonstrated that CQE by itself induced heme oxygenase-1 (HO-1) gene expression at the protein and mRNA levels in dose- and time-dependent manner. In addition, the inhibitory effects of CQE on NO production were abrogated by a HO-1 inhibitor, zinc protoporphyrin IX (ZnPP).

Conclusions

Collectively, these results suggest that CQE exerts an anti-inflammatory effect in macrophages, at least in part, through the induction of HO-1 expression. These findings provide the scientific rationale for anti-inflammatory therapeutic use of Cissus quadrangularis stem.     

Phellinus linteus inhibits inflammatory mediators by suppressing redox-based NF-kappaB and MAPKs activation in lipopolysaccharide-induced RAW 264.7 macrophage

The mushroom Phellinus linteus has been known to exhibit potent biological activity. In contrast to the immuno-potentiating properties of Phellinus linteus, the anti-inflammatory properties of Phellinus linteus have rarely been investigated. Recently, ethanol extract and n-BuOH fractions from Phellinus linteus were deemed most effective in anti-inflammatory activity in RAW 264.7 macrophages. The regulatory mechanisms of Phellinus linteus butanol fractions (PLBF) on the pharmacological and biochemical actions of macrophages involved in inflammation have not been clearly defined yet. In the present study, we tested the role of PLBF on anti-inflammation patterns in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. To investigate the mechanism by which PLBF inhibits NO and PGE2 production as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, we examined the activation of IkappaB and MAPKs in LPS-activated macrophages. PLBF clearly inhibited nuclear translocation of NF-kappaB p65 subunits, which correlated with PLBF's inhibitory effects on IkappaBalpha phosphorylation and degradation. PLBF also suppressed the activation of mitogen-activated protein (MAP) kinases including p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Furthermore, macrophages stimulated with LPS generated ROS via activation of membrane-bound NADPH oxidase, and ROS played an important role in the activation of nuclear factor-kappaB (NF-kappaB) and MAPKs. We demonstrated that PLBF directly blocked intracellular accumulation of reactive oxygen species in RAW 264.7 cells stimulated with LPS much as the NADPH oxidase inhibitors, diphenylene iodonium, and antioxidant pyrrolidine dithiocarbamate did. The suppression of NADPH oxidase also inhibited NO production and iNOS protein expression. Cumulatively, these results suggest that PLBF inhibits the production of NO and PGE2 through the down-regulation of iNOS and COX-2 gene expression via ROS-based NF-kappaB and MAPKs activation. Thus, PLBF may provide a potential therapeutic approach for inflammation-associated disorders.     

Sclareol exhibits anti-inflammatory activity in both lipopolysaccharide-stimulated macrophages and the λ-carrageenan-induced paw edema model

Sclareol (1) is a natural fragrance compound used widely in the cosmetic and food industries. Lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and the λ-carrageenan-induced edema mouse paw model were applied to examine the anti-inflammatory potential of 1 and its possible molecular mechanisms. The experimental results obtained demonstrated that this compound inhibited cell growth, nitric oxide (NO) production, and the expression of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins in LPS-stimulated macrophages. Compound 1 also reduced paw edema, the tissue content of NO, tumor necrosis factor-alpha (TNF-α), malondialdehyde (MDA), iNOS and COX-2 protein expression, and neutrophil infiltration within the tissues after λ-carrageenan stimulation. The present study suggests that the anti-inflammatory mechanisms of 1 might be related to a decrease of inflammatory cytokines and an increase of antioxidant enzyme activity.     

Fermentation improves anti-inflammatory effect of sipjeondaebotang on LPS-stimulated RAW 264.7 cells

Sipjeondaebotang (SJ) has been used as a traditional drug in east-Asian countries. In this study, to provide insight into the biological effects of SJ and SJ fermented by Lactobacillus, we investigated their effects on lipopolysaccharide (LPS)-mediated inflammation in macrophages. The investigation was focused on whether SJ and fermented SJ could inhibit the production of pro-inflammatory mediators such as prostaglandin (PG) E(2) and nitric oxide (NO) as well as the expressions of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB in LPS-stimulated RAW 264.7 cells. We found that SJ modestly inhibited LPS-induced PGE(2), NO and TNF-α production as well as the expressions of COX-2 and iNOS. Interestingly, fermentation significantly increased its inhibitory effect on the expression of all pro-inflammatory mediators. Furthermore, fermented SJ exhibited increased inhibition of p38 MAPK and c-Jun NH(2)-terminal kinase (JNK) MAPK phosphorylation as well as NF-κB p65 translocation by reduced IκBα degradation compared with either untreated controls or unfermented SJ. High performance liquid chromatography (HPLC) analysis showed fermentation by Lactobacillus increases liquiritigenin and cinnamyl alcohol contained in SJ, which are known for their anti-inflammatory activities. Finally, SJ fermented by Lactobacillus exerted potent anti-inflammatory activity by inhibiting MAPK and NF-κB signaling in RAW 264.7 cells.     

芪麝丸体外抗炎的机制

目的:考察芪麝丸体外对小鼠RAW264.7巨噬细胞炎症模型的抗炎机制.方法:干扰素γ(1×104 U·mL-1)和脂多糖(100μg·L-1)协同诱导小鼠RAW264.7巨噬细胞(1×105/mL)24 h造成炎症模型;Griess反应测定细胞上清液中一氧化氮(NO)产量;Western blot检测诱导型一氧化氮合酶(iNOS)、环氧合酶(COX-2)的蛋白表达及丝裂原活化蛋白激酶(MAPK)信号转导通路的活化情况.结果:芪麝丸(250,500,1000 mg·L-1)呈剂量依赖性抑制细胞上清液中NO含量,且无细胞毒性.较模型组,经芪麝丸500,1000 mg·L-1剂量处理后,iNOS蛋白表达从(1.00±0.06)下调至(0.61±0.07)和(0.02±0.15),(P＜0.01),COX-2蛋白表达从(0.56±0.03)下调至(0.42±0.02),(0.30±0.03),(P＜0.01).胞外信号调节激酶(ERK)磷酸化水平从(1.04±0.04)下调至(0.79±0.06),(0.73 ±0.10),(P＜0.01).p38磷酸化水平从(0.51±0.06)下调至(0.39 ±0.07),(0.29±0.15),(P＜0.05),c-Jun氨基末端激酶(JNK)磷酸化水平从(1.05±0.03)下调至(0.65 ±0.02),(0.66±0.033),(P＜0.01).结论:芪麝丸部分通过抑制MAPK信号转导通路活化过程中关键蛋白胞外信号调节激酶(ERK)、JNK和p38丝裂原活化蛋白激酶(p38)磷酸化,下调iNOS基因和蛋白的表达从而减少NO的产量,同时下调COX-2蛋白表达,而发挥其抗炎效果.     

Effects of hyperin, isoquercitrin and quercetin on lipopolysaccharide-induced nitrite production in rat peritoneal macrophages

The extract of the root of Acanthopanax chiisanensis Nakai is used for the treatment of inflammation. To analyse the action mechanism of this extract, the effect of hyperin (quercetin-3-O-beta-d-galactose) isolated from the ethyl acetate fraction of the root of A. chiisanensis on nitrite production and induction of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS, 1 microg/mL)-stimulated rat peritoneal macrophages were examined. The effect of the structurally related compounds, isoquercitrin (quercetin-3-O-beta-d-glucose) and quercetin (an aglycone of the two compounds) isolated from the extract of the leaves of Vaccinium koreanum Nakai was also examined to compare the effect. It was shown that hyperin inhibited the LPS-induced iNOS expression and nitrite production. Of the three compounds, quercetin showed the most potent inhibitory activity. The phosphorylation of p44/42 mitogen activated protein kinase (MAPK), p38 MAPK and c-Jun N-terminal kinase (JNK) were also inhibited by these compounds. These findings suggested that hyperin in the extract of the root of A. chiisanensis inhibits nitric oxide (NO) production through inhibition of the expression of iNOS by attenuation of p44/p42 MAPK, p38 MAPK and JNK, and thus participates in the antiinflammatory activity of the extract.     

Effects of bee venom on the pro-inflammatory responses in RAW264.7 macrophage cell line

The purpose of this study is to elucidate the molecular mechanism of anti-inflammatory effect of bee venom (BV), which has been used for the treatment of various inflammatory diseases in oriental medicine. With this aim, we examined the effects of BV on the nitric oxide (NO) production by lipopolysaccharide (LPS) or sodium nitroprusside in RAW264.7 macrophages. We further investigated the effects of BV on the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) with RT-PCR in LPS-stimulated RAW264.7 cells. BV suppressed the NO production and decreased the levels of iNOS, COX-2, NF-kappaB and MAPK mRNA in a dose-dependent manner. These results suggest that BV has an anti-inflammatory effect by inhibiting iNOS and COX-2 expression, possibly through suppression of NF-kappaB and MAPK expression.     

Pistacia chinensis inhibits NO production and upregulates HO-1 induction via PI-3K/Akt pathway in LPS stimulated macrophage cells

Pistacia chinensis has been used for various purposes in China including as an understock for grafting Pistacia vera. However, little attention was given to its health promoting effects. Therefore, in this study, we investigated the effect of Pistacia chinensis methanolic extract (PCME) containing resorcinol class of phenolic lipids on pro-inflammatory mediators and heme oxygenase-1(HO-1) in lipopolysaccharide stimulated RAW264.7 cells. While PCME (2.5-10 μg/ml) inhibited mRNA expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interleukin (IL)-6, it up-regulated HO-1 expression. Likewise, PCME inhibited iNOS protein expression, but not COX-2, and reduced nitric oxide (NO) release. Moreover, Phosphorylated c-Jun N-terminal Kinase (JNK) was attenuated dose-dependently in PCME pre-treated RAW264.7 cells. In addition, PCME up-regulated HO-1 protein expression was diminished by pre-treatment of PI-3K inhibitor. Furthermore, nuclear factor erythroid 2 related factor 2 (Nrf2) repressor was attenuated time-dependently during PCME treatment. Taken together, our study showed (for the first time) that PCME inhibited NO production and up-regulated HO-1 induction via PI-3K/Akt pathway, suggesting the role of Pistacia chinensis as potential sources of anti-inflammatory and antioxidant natural compounds.     

Aloe-emodin from rhubarb (Rheum rhabarbarum) inhibits lipopolysaccharide-induced inflammatory responses in RAW264.7 macrophages

Ethnopharmacological relevance

Rheum rhabarbarum (rhubarb) has long been used for the treatment of inflammation in China and other Asian countries. However, the mechanism underlying the anti-inflammatory activity of this medicinal plant is not fully understood. The present study was designed to investigate the anti-inflammatory effects of anthraquinones, the major constituents in rhubarb, and the molecular mechanism involved in their anti-inflammatory effects.

Materials and methods

RAW264.7 cells were stimulated by lipopolysaccharide (LPS) in the presence or absence of the compounds examined. The proliferation of RAW264.7 cells was assayed by the Alamar-Blue method. The quantity of nitric oxide (NO) was determined by Griess assay. The expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR. Inducible nitric oxide synthase (iNOS), inhibitor of nuclear factor κBα (IκBα), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), and Akt/phosphoinositide 3-kinase (PI3K) protein expression levels were determined by Western blotting.

Results

Aloe-emodin markedly suppressed the production of NO, interleukin-6 (IL-6), and interleukin-1β (IL-1β) in LPS-stimulated RAW264.7 cells with no apparent cytotoxicity. The mRNA expression levels of iNOS, IL-6, and IL-1β genes were also significantly inhibited by aloe-emodin. Western blot analysis showed that aloe-emodin suppressed LPS-induced iNOS protein expression, IκBα degradation, and the phosphorylation of ERK, p38, JNK, and Akt.

Conclusions

These results demonstrate that aloe-emodin is the bioactive component of rhubarb that confers an anti-inflammatory effect through a likely mechanism involving a decrease in pro-inflammatory cytokine production in LPS-induced RAW264.7 macrophages via inhibition of NF-κB, MAPK, and PI3K pathways.     

Mechanism of anti-inflammatory activity of umbelliferone 6-carboxylic acid isolated from Angelica decursiva

Aims of the study

We recently reported the potential antioxidant and anti-inflammatory activities of umbelliferone 6-carboxylic acid (UMC) isolated from the whole plants of Angelica decursiva. In this study, we elucidated the anti-inflammatory mechanisms of UMC in vitro and in vivo.

Methods

The inhibitory effects of UMC on the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), and tumor necrosis factor-α (TNF-α), the expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the activation of nuclear factor kappa B (NF-κB) were evaluated using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The reactive oxygen species (ROS) generation inhibitory activity of UMC was evaluated using t-butyl hydroperoxide (t-BHP)-induced RAW 264.7 cells. Furthermore, the in vivo anti-inflammatory activity of UMC was evaluated using carrageenan induced mouse paw edema model.

Results

UMC dose-dependently inhibited NO and PGE₂ production by down-regulating iNOS and COX-2 protein expression in LPS-stimulated RAW 264.7 macrophages. UMC also suppressed the production of the proinflammatory cytokine TNF-α in LPS stimulated RAW 264.7 cells in a concentration dependent manner. In addition, UMC dose-dependently prevented LPS-induced nuclear translocation of NF-κB in RAW 264.7 macrophages. Furthermore, UMC exhibited the inhibitory activity against t-BHP-induced ROS generation in RAW 264.7 cells with an IC₅₀ value of 705.1 μg/ml. Moreover, UMC inhibited λ-carrageenan induced mouse paw edema by 70.40 and 60.20% at doses of 50 and 25 mg/kg body weight, respectively.

Conclusion

The combined results of this study indicate that UMC is an important anti-inflammatory constituent of A. decursiva and its anti-inflammatory effect was due to its ability to inhibit the production of inflammatory mediators via inhibition of NF-κB activation pathway.