Anti-inflammatory activity of hyperoside through the suppression of nuclear factor-κB activation in mouse peritoneal macrophages

Hyperoside (quercetin-3-O-galactoside) is a flavonoid compound mainly found in the herb plants Hypericum perforatum L and Crataegus pinnatifida. Although hyperoside has a variety of pharmacological effects including anti-viral, anti-oxidative, and anti-apoptotic activities, the anti-inflammatory mechanism of hyperoside in mouse peritoneal macrophages remains unclear. In this study, hyperoside was shown to exert an anti-inflammatory action through suppressed production of tumor necrosis factor, interleukin-6, and nitric oxide in lipopolysaccharide-stimulated mouse peritoneal macrophages. The maximal inhibition rate of tumor necrosis factor-α, interleukin-6, and nitric oxide production by 5 μM hyperoside was 32.31 ± 2.8%, 41.31 ± 3.1%, and 30.31 ± 4.1%, respectively. In addition, hyperoside inhibited nuclear factor-κB activation and IκB-α degradation. The present study suggests that an important molecular mechanism by hyperoside reduces inflammation, which might explain its beneficial effect in the regulation of inflammatory reactions.     

Euonymus alatus extract attenuates LPS-induced NF-κB activation via IKKβ inhibition in RAW 264.7 cells

Aim of the study

The present study was performed to investigate the underlying mechanisms of anti-inflammatory effects with the extract of Euonymus alatus (EEA), and specially focused on nuclear factor κB (NF-κB) signaling pathway by targeting the IκB kinase β (IKKβ).

Materials and methods

The effect of EEA for IKKβ activity was analyzed using an immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. The effect of EEA on lipopolysaccharide (LPS)-induced NF-κB activation in murine macrophage RAW 264.7 cells with western blotting and immunofluorescent staining was evaluated.

Results

IKKβ studies based on IMAP-TR-FRET showed that EEA possesses a potent IKKβ inhibitory activity with IC₅₀ value of 11.83 μg/ml. EEA (10, 30 μg/ml) also attenuated the LPS-induced IκBα phosphorylation/degradation, NF-κB translocation and subsequent NO synthesis in RAW 264.7 cells.

Conclusions

These results suggest that EEA abrogates LPS-induced NF-κB signaling pathway by targeting the IKKβ in RAW 264.7 cells and these properties may provide a molecular basis for understanding the inhibitory effects of EEA on LPS-mediated inflammation.     

Protection of glycyrrhizic acid against AGEs-induced endothelial dysfunction through inhibiting RAGE/NF-κB pathway activation in human umbilical vein endothelial cells

Ethnopharmacological relevance

Licorice (Glycyrrhiza uralensis roots) is used as a traditional medicine for the treatment of diabetes mellitus and its vascular complications. Glycyrrhizic acid (GA, also known as Glycyrrhizin), a triterpenoid saponin glycoside, is considered to be a bioactive component in Licorice and is beneficial to diabetic vascular complications.

Aim of study

The present study was conducted to evaluate the potential protective activities on AGEs-induced endothelial dysfunction, including anti-apoptosis, antioxidant stress and anti-proinflammatory responses, and explore the underlying mechanism.

Materials and methods

Human umbilical vein endothelial cells (HUVECs) were incubated and pre-treated with GA (10⁻⁹–10⁻⁶ M) or RAGE-Ab (5 μg/ml) in the presence or absence of 200 μg/ml AGEs. AO/EB fluorescence staining assay was performed to evaluate anti-apoptosis activity. The superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in cell supernatant were detected by kits while the intracellular reactive oxygen species (ROS) generation was determined by 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) kit. Immunocytochemistry analysis was designed to determine transforming growth factor beta1(TGF-β1) protein expression while immunofluorescence analysis for RAGE and NF-kB. The protein expressions of TGF-β1, RAGE and NF-kB were analyzed by Western blot analysis.

Results

Pretreatment with GA at a concentration of 10⁻⁸–10⁻⁶ M significantly reduced the AGEs-induced apoptosis in HUVECs. GA significantly increased antioxidant enzyme SOD activity and decreased peroxide degradation product MDA level in a dose-dependent manner. Furthermore, GA also remarkably inhibited the overgeneration of AGEs-induced ROS. Both immunocytochemistry analysis and western blot analysis showed that GA significantly decreased the protein expression of poinflammatory cytokine TGF-β1 in a similar manner which RAGE-Ab did. Additionally, AGEs-induced RAGE and NF-kB protein expressions were down-regulated significantly by the pretreatment with GA or RAGE-Ab.

Conclusion

These findings provide evidences that GA possesses protective activity on AGEs-induced endothelial dysfunction, including anti-apoptosis, anti-inflammation and antioxidant stress, via inhibiting RAGE/NF-kB pathway. GA might be an alternative for the prevention and treatment of diabetic vascular complications in an appropriate dosage.     

Inhibitory effects of salvianolic acid B on CCl4-induced hepatic fibrosis through regulating NF-κB/IκBα signaling

Ethnopharmacological relevance

Hepatic fibrosis, a precursor of liver cirrhosis, is a consequence of severe liver damage that occurs in many patients with chronic liver diseases. Salvianolic acid B (SA-B) is one of water soluble compounds derived from Salvia miltiorrhiza Bunge (Danshen in Chinese) widely used for chronic liver diseases. In this study we investigated the protective effects of SA-B on CCl₄-induced hepatic fibrosis.

Materials and methods

Hepatic fibrosis in rats was induced by carbon tetrachloride (CCl₄). Rats were divided into four groups, including normal controls (N group), model (M group), low SA-B of 10 mg/kg body weight (L group), or high SA-B of 20 mg/kg body weight (H group). After 6 weeks, macroscopic features of the liver and weight ratio of liver to body were measured. Liver fibrosis of the rats was evaluated by HE and Massion staining. Activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) were checked with automated biochemistry analyzer. Serum levels of hyaluronic acid (HA), type IV collagen (IV-C), Laminin (LN) and procollagen III peptide (PIIIP) were detected by radioimmunoassay (RIA). The expression of NF-κB and IκBα was detected by western blotting.

Results

SA-B was shown to reduce CCl₄-induced hepatic fibrosis in rats. The serum levels of ALT, AST, and TBIL were significantly lower in the SA-B treatment groups than in the M group. Compared the M group, the serum levels of HA, LN, IV-C and PIIIP were decreased markedly after treatment with SA-B, especially in the H group. Treatment with SA-B at 10–20 mg/kg (L and N groups, respectively) dose-dependently decreased the expression of NF-κB in the nucleolus and increased the expression levels of NF-κB and IκBα protein in the cytoplasm compared to that of the M group.

Conclusions

This study reveals that SA-B could prevent the progression of liver angiogenesis and alleviate liver fibrosis possibly by regulating the expression of NF-κB and IκBα.     

Er-Miao-San,a traditional herbal formula containing Rhizoma Atractylodis and Cortex Phellodendri inhibits inflammatory mediators in LPS-stimulated RAW264.7 macrophages through inhibition of NF-κB pathway and MAPKs activation

Ethnopharmacological relevance

Er-Miao-San (EMS) is a traditional Chinese herbal formulation that contains combinations of Rhizoma Atractylodis (RA) and Cortex Phellodendri (CP). It exhibits analgesic and anti-inflammatory activities and have been used for the treatment of various “Bi Zheng” for thousand years in China. The aims of the present study were to investigate the anti-inflammatory activities of EMS and elucidate the underlying mechanisms with regard to its molecular basis of action for the best combination.

Materials and methods

The anti-inflammatory effects of EMS were studied by using lipopolysaccharide (LPS)-stimulated activation of nitric oxide (NO) and pro-inflammatory cytokine production in mouse RAW264.7 macrophages. Expression of inducible NO synthase (iNOS), mitogen-activated protein kinases (MAPKs) phosphorylation, p65 phosphorylation, inhibitor-κBα (IκBα) degradation, and NF-κB DNA-binding activity were further investigated.

Results

The present study demonstrated that EMS could suppress the production of NO in LPS-stimulated RAW264.7 macrophages. However, CP and RA did not have significant inhibitory effect on them. EMS also inhibited the production of tumor necrosis factor-alpha, interleukin-1 beta and macrophage chemotactic protein-1. Further investigations showed EMS could suppress iNOs expression and p38 phosphorylation. EMS significantly decreased the content of IκBα, reduced the level of phosphorylated p65 and suppressed the NF-κB DNA-binding activity. All these results suggested the inhibitory effects of EMS on the production of inflammatory mediators through the inhibition of the NF-κB pathway.

Conclusions

Our results indicated that EMS inhibited inflammatory events and iNOS expression in LPS-stimulated RAW264.7 cells through the inactivation of the MAPK and NF-κB pathway. This study gives scientific evidence validating the use of EMS in treatment of patients with “Bi Zheng” in clinical practice in traditional Chinese medicine.     

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.     

Carthamus tinctorius L. prevents LPS-induced TNFα signaling activation and cell apoptosis through JNK1/2–NFκB pathway inhibition in H9c2 cardiomyoblast cells

Aim of the study

Severe and potentially fatal hypotension and cardiac contractile dysfunction are common symptoms in patients with sepsis. In our previous study, we found that estradiol and estrogen-receptor α have cardio-protective effects in myocardial cells exposed to LPS. Estradiol supplementation has been shown to induce breast and cervical cancers. Flos Carthami, the flower of Carthamus tinctorius L. (Compositae) is an important traditional Chinese medicine used for the treatment of heart disease and inflammation, and therefore might be a potential alternative to Estradiol in the prevention of heart damage. This study investigated the effect of Flos Carthami (FC_EtOH) ethanolic extract on LPS-induced apoptosis in H9c2 cardiomyoblast cells.

Materials and methods

H9c2 cells induced apoptosis with LPS administration (1 μg/mL). H9c2 cells were divided into five groups: Control, LPS (1 μg/mL), and three FC_EtOH (31.25, 62.5,and 125 μg/mL). We detected apoptosis using MTT, LDH, TUNEL assay. JC-1 staining and Western blot were used to detect pro-apoptosis proteins, anti-apoptosis proteins, MAPK proteins (JNK, ERK, and P38), and NFκB expression.

Results

FC_EtOH (62.5 μg/mL) inhibited LPS-induced apoptosis by suppressing JNK1/2 activity, which resulted in the reduction of both IκB degradation and NFκB activation. In addition, FC_EtOH led to the activation of anti-apoptotic proteins, Bcl-2 and Bcl-xL, the stabilization of the mitochondria membrane and the down-regulation of extrinsic and intrinsic pro-apoptotic proteins, such as TNFα, active caspase-8, t-Bid, Bax, active caspases-9, and -3.

Conclusions

Carthamus tinctorius L. possesses the ability to suppress JNK activity and inhibit LPS-induced TNFα activation and apoptosis in H9c2 cardiomyoblast cells. Carthamus tinctorius L could potentially serve as a cardio-protective agent against LPS-induced apoptosis.     

Inhibitory effects of casticin on migration of eosinophil and expression of chemokines and adhesion molecules in A549 lung epithelial cells via NF-κB inactivation

Ethnopharmacological relevance

The fruits of Vitex rotundifolia L. have long been used for the treatment of inflammation of the respiratory tract in East Asia.

Aim

To determine if casticin, one of the constituents of Vitex rotundifolia L., has anti-allergic and anti-inflammatory effects in asthma.

Materials and methods

The in vitro anti-inflammatory activity of casticin was studied in A549 human type II-like epithelial lung cells using an eotaxin inhibition assay. Additionally, its effects on eotaxin, regulated on activation normal T cell expressed and secreted (RANTES), vascular cell adhesion molecule (VCAM)-1, and inter-cellular adhesion molecule (ICAM)-1 expression were investigated by real time-polymerase chain reaction (real time-PCR). The inhibition of nuclear factor κB (NF-κB) activity in the presence of casticin was determined by analyzing confocal microscopy images of fluorescence immunocytochemical analysis while the suppression of inhibitory κB (IκB)-α phosphorylation was studied using Western blot analysis. Finally, the inhibitory effect of casticin on eosinophil migration toward prestimulated A549 cell media was measured using the human eosinophilic leukemia cell line.

Results and discussion

Casticin significantly suppressed eotaxin production in cytokine activated A549 lung epithelial cells. Casticin also suppressed the mRNA expression levels of eotaxin, RANTES, VCAM-1, and ICAM-1, which subsequently contributed to the inhibition of eosinophil migration. Furthermore, casticin inhibited IκB-α phosphorylation and nuclear translocation of p65 in A549 cells.

Conclusion

Casiticin inhibited the eosinophil migration and activity of chemokines and adhesion molecules involved in the inflammatory process of asthma by suppressing the NF-κB pathway. These results suggest that casticin has the potential for use in the treatment of allergic asthma.     

Flower extract of Panax notoginseng attenuates lipopolysaccharide-induced inflammatory response via blocking of NF-κB signaling pathway in murine macrophages

Aim of the study

The root of Panax notoginseng (PN) is commonly used to treat chronic liver disease with its therapeutic abilities to stop haemorrhage in the circulation, while the PN flower (PN-F) is largely unknown in the biological activities on inflammation and mechanisms of its actions. In this study, the pharmacologic effects of PN-F methanol extract on inflammation were investigated to address potential therapeutic or toxic effects in LPS-stimulated mouse macrophage cells, RAW264.7 cells.

Materials and methods

Production of NO, PGE₂ and pro-inflammatory cytokines (TNF-α and IL-1β) in supernatant, the expression of iNOS, COX-2 and cytokines, the phosphorylation of MAPK moleduces (ERK1/2, JNK and p38 MAPK), and the activation of NF-κB in PN-F extract were assayed in LPS-stimulated RAW264.7 cells.

Results

PN-F extract significantly inhibited the productions of NO, PGE₂, TNF-α and IL-1β on the LPS-stimulated RAW264.7 cells. In addition, PN-F extract suppressed the mRNA and protein expressions of iNOS, COX-2, TNF-α and IL-1β in LPS-stimulated RAW264.7 cells. The molecular mechanism of PN-F extract-mediated attenuation in RAW264.7 cells has close a relationship to suppressing the phosphorylation of MAPK molecules such as ERK1/2, JNK and p38 MAPK, and the translocation of NF-κB p65 subunit into nuclear.

Conclusion

These results indicate that PN-F extract inhibits LPS-induced inflammatory response via the blocking of NF-κB signaling pathway in macrophages, and demonstrated that PN-F extract possesses anti-inflammatory properties in vitro.     

Lagerstroemia speciosa extract inhibit TNF-induced activation of nuclear factor-κB in rat cardiomyocyte H9c2 cells

Aim of the study

Lagerstroemia speciosa has been used as a folk medicine among people with diabetes in the Philippines. It is known to exhibit antidiabetic, antiobesity, and glucose transport activities through mechanisms not well defined. Diabetes leads to cardiomyocyte hypertrophy in association with an upregulation of vasoactive factors and activation of nuclear factor (NF)-κB and activating protein-1. We therefore investigated the effect of Lagerstroemia speciosa on the activation of NF-κB as a key mediator of cardiomyocyte hypertrophy, in rat cardiomyocyte H9c2 cells.

Materials and methods

Water extract of Lagerstroemia speciosa (Lythraceae family) was prepared. H9c2 cells were used for treatment of Lagerstroemia speciosa extract with/without tumor necrosis factor (TNF). To examine NF-κB's activation, we performed an electrophoretic mobility shift assay (EMSA).

Results

The activation of NF-κB by TNF was completely blocked by a Lagerstroemia speciosa extract in a dose- and time-dependent manner in H9c2 cells.

Conclusion

Overall, our results indicate that Lagerstroemia speciosa can inhibit DNA-binding of NF-κB. This may explain its possible inhibition of diabetes-induced caridomyocyte hypertrophy.     

Chrysanthemum indicum Linné extract inhibits the inflammatory response by suppressing NF-κB and MAPKs activation in lipopolysaccharide-induced RAW 264.7 macrophages

Aims of study

Although the flowers of Chrysanthemum indicum Linné (Asteraceae) have long been used in traditional Korean and Chinese medicine to treat inflammatory diseases, the underlying mechanism(s) by which these effects are induced remains to be defined. We investigated the effects of a 70% ethanolic extract of C. indicum (CIE) on the activities of cellular signaling molecules that mediate inflammatory responses.

Materials and methods

Production of NO, PGE₂, TNF-α, and IL-1β by ELISA, mRNA and protein expression of iNOS and COX-2, phosphorylation of MAPKs, and activation of NF-κB by RT-PCR and Western blotting were examined in LPS-induced RAW 264.7 macrophages.

Results

The CIE strongly inhibited NO, PGE₂, TNF-α, and IL-1β production, and also significantly inhibited mRNA and protein expression of iNOS and COX-2 in LPS-induced RAW 264.7 macrophages, in a dose-dependent manner. Furthermore, the CIE clearly suppressed nuclear translocation of NF-κB p65 subunits, which correlated with an inhibitory effect on IκBα phosphorylation. The CIE also attenuated the activation of ERK1/2 and JNK in a dose-dependent manner.

Conclusion

Our results suggest that the anti-inflammatory properties of CIE might result from the inhibition of inflammatory mediators, such as NO, PGE₂, TNF-α, and IL-1β, via suppression of MAPKs and NF-κB-dependent pathways.     

Lipid-soluble extracts from Salvia miltiorrhiza inhibit production of LPS-induced inflammatory mediators via NF-κB modulation in RAW 264.7 cells and perform antiinflammatory effects in vivo

Salvia miltiorrhiza, a traditional Chinese herbal medicine, is used to treat various inflammatory diseases. In the present study, the antiinflammatory effects of S. miltiorrhiza lipid-soluble extracts (SMLE) were demonstrated in vitro and in vivo, along with its underlying mechanism of action. SMLE significantly inhibited the production of NO, TNF-α, IL-1β and IL-6 in lipopolysaccharides (LPS)-stimulated RAW 264.7 cells. SMLE also inhibited the LPS-induced degradation of IκB-α in the cytoplasm and the translocation of p65 to the nucleus in RAW 264.7 cells. In addition, SMLE inhibited the production of intracellular reactive oxygen species (ROS) and the surface expression of CD14 induced by LPS. In animal models, intraperitoneal administration of SMLE increased the survival rate of endotoxemia and sepsis in mice. The topical administration of SMLE significantly inhibited ear edema induced by PMA. It was found that SMLE inhibits the LPS-induced gene and protein expression of iNOS, TNF-α, IL-1β and IL-6 in macrophages by blocking NF-κB activation, and these effects are mediated, at least in part, through the inhibition of intracellular ROS generation and the surface expression of CD14. The results suggest a possible therapeutic application of SMLE in inflammatory diseases and provide scientific evidence in support of the traditional Chinese medical practice of treatment with S. miltiorrhiza.     

Triptolide down-regulates COX-2 expression and PGE2 release by suppressing the activity of NF-κB and MAP kinases in lipopolysaccharide-treated PC12 cells

As an active compound extracted from the Chinese herb Tripterygium wilfordii, triptolide (TP) was demonstrated to have potent antiinflammatory and immunosuppressive properties in previous studies. Recently, it has been shown that TP prevented the loss of dopaminergic neurons in the substantia nigra of rats in a model of Parkinson's disease, but little is known about the precise neuroprotective mechanism of TP. This study was designed to elucidate whether the neuroprotective effect of TP is partially based on its direct inhibition of inflammatory molecules by investigating the effects of TP on the expression of cyclooxygenase (COX)‐2 and prostaglandin E2 (PGE2) related to the nuclear factor (NF)‐κB pathway in lipopolysaccharide (LPS)‐stimulated PC12 cells. The activation of related upstream molecules such as NF‐κB, P38, extracellular signal‐regulated kinase (ERK)1/2, and beta‐alanyl‐alpha‐ketoglutarate transaminase (AKT), in PC12 cells were investigated by real time polymerase chain reaction (PCR), western blotting and enzyme‐linked immunosorbent assay (ELISA). Our results showed that TP directly inhibited the expression of both mRNA and protein of COX‐2 (p < 0.01), decreased PGE2 production (p < 0.01) in a dose‐dependent manner, down‐regulated NF‐κB activity (p < 0.01), and significantly inhibited the phosphorylation of p38, ERK1/2 (p42/p44) and AKT in PC12 cells after LPS challenge. This suggests that the neuroprotective effects of TP may be partially mediated by direct inhibition of the expression of COX‐2, activation of NF‐κB, and phosphorylation of p38, ERK1/2 (p42/p44) and AKT proteins of neuronal cells. Copyright © 2011 John Wiley & Sons, Ltd.     

Terminalia chebula extract acts as a potential NF-κB inhibitor in human lymphoblastic T cells

Terminalia chebula (TC) is native to southern Asia to southwestern China and is used in traditional medicine for the treatment of human ailments including malignant tumors and diabetes. This plant also has antibacterial and immunomodulatory properties. Nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) is responsible for the expression of numerous genes involved in cell survival, proliferation, angiogenesis, inflammation, invasion and metastasis, among other processes. This study aims to assess the NF-κB inhibitory effect of TC extract in human lymphoblastic T (Jurkat) cells. The effects of TC extract were investigated using the FRET-based Gene Blazer technique in transfected Jurkat-NF-κB-RE-bla cells. The concentration of TC extract required for NF-κB inhibition was determined by a cell proliferation assay. Treatment with TC extract (50 μg/mL) inhibited NF-κB activity and protected against IκBα degradation and strongly suppressed IκBα phosphorylation in Jurkat-NF-κB-RE-bla cells. This treatment might be crucial for inhibiting NF-κB translocation and activation. In addition, the TC extract downregulated certain NF-κB regulated genes, including IL-8 and MCP-1, in Jurkat-NF-κB-RE-bla cells. Moreover, gallic acid was identified from the TC extract demonstrating its ability to inhibit NF-κB activity in Jurkat-NF-κB-RE-bla cells. Further studies to identify the role of gallic acid in NF-κB inhibition may uncover the crucial antiinflammatory and antitumor properties of the TC extract.     

Diarylheptanoid hirsutenoxime inhibits toll-like receptor 4-mediated NF-κB activation regulated by Akt pathway in keratinocytes

Microbial products, including lipopolysaccharides, may be involved in the pathogenesis of inflammatory skin diseases. We examined the effect of hirsutenoxime on the Toll-like receptor 4-mediated activation of Akt and nuclear factor (NF)-κB in lipopolysaccharide-stimulated keratinocytes. Hirsutenoxime, a cell signaling Akt inhibitor, and Bay 11-7085, an inhibitor of NF-κB activation, attenuated the lipopolysaccharide-induced expression of Toll-like receptor 4, activation of NF-κB and Akt, and the production of chemokines and reactive oxygen/nitrogen species. Hirsutenoxime may reduce the Toll-like receptor 4 expression-mediated NF-κB activation, which is regulated by the Akt pathway in keratinocytes exposed to lipopolysaccharides. This effect may reduce the skin inflammatory response.