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.     

Cytotoxic and apoptotic activity of Cheilanthes farinosa (Forsk.) Kaulf. against human hepatoma,Hep3B cells

Aim of the study

Cheilanthes farinosa (Forsk.) Kaulf., family: Adianthaceae, is a fern of immense medicinal properties used in ethno-medicine. The Gaddis tribe of Himachal Pradesh, India, has been using this fern to treat liver damage. Aim of the current study was to determine the apoptosis inducing and cytotoxic activity, if any, of this fern towards hepatic cancer cells.

Materials and methods

Water extract of the plant was used in the study. MTT assay was performed in hepatocellular carcinoma cell line, Hep3B as well as murine macrophage cell line, RAW264.7 to analyze the cytotoxic activity of the plant. Further, the apoptosis inducing action of water extract of the plant was evaluated using comet assay, DNA fragmentation analysis, DAPI staining of chromatin and Annexin V-FITC staining.

Results

This plant was found to produce considerable cytotoxicity in hepatoma cell line, Hep3B without inducing substantial damage to non-cancerous cell line RAW264.7. In addition, this plant was found to induce apoptosis in Hep3B cells. This was substantiated by comet assay, DNA fragmentation analysis, DAPI staining of chromatin and Annexin V-FITC staining for detecting early stage of apoptosis.

Conclusions

This investigation shows that the water extract of Cheilanthes farinosa has antiproliferative and apoptotic activity in human liver cancer cells and is not deleterious towards non-cancerous macrophage cell line.     

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.     

Ethanol extract of Alismatis Rhizoma reduces acute lung inflammation by suppressing NF-κB and activating Nrf2

Ethnopharmacological relevance

The tuber of Alisma orientale Juzepzuk, a medicinal herb that has been used for the treatment of various disorders in Korea, has an anti-inflammatory effect. Here, we investigated a possible underlying mechanism and a protective effect on acute lung injury (ALI).

Materials and methods

Alisma orientale tuber was extracted in 80% ethanol and dried. The powder of the ethanol extract of Alisma orientale tuber (EEAO) was dissolved in PBS. The effect of EEAO on NF-κB and Nrf2 activities was analyzed with RAW 264.7 cells. The effect of EEAO on lung inflammation was determined by histologic and molecular biological analyses of the lung tissue of C57BL/6 mice that were gavaged once a day with 0.3 or 1.2 g/kg of EEAO for 14 days, prior to an intranasal administration of LPS (0.01 g/kg) for inducing ALI.

Results

EEAO pre-treatment of RAW 264.7 cells suppressed NF-κB activity and the expression of its dependent genes including COX-2, IL-1β and iNOS. Similar treatment enhanced Nrf2 activity and the expression of Nrf2-regulated genes including NQO-1, HO-1 and GCLC. LPS instillation induced acute neutrophilic lung inflammation, which was significantly suppressed by pre-treatment with EEAO. Analysis of the lungs revealed that EEAO pre-treatment induced the expression of Nrf2-regulated genes, with concomitant down-regulation of inflammatory gene expression.

Conclusions

EEAO attenuated lung inflammation in LPS-induced ALI mice, which was associated with differential regulation of NF-κB and Nrf2 activities. We suggest that EEAO can be developed as a potential therapeutics for the treatment of ALI.     

Chemopreventive action of Lygodium flexuosum extract in human hepatoma PLC/PRF/5 and Hep 3B cells

Ethnopharmacological relevance

Lygodium flexuosum (Lygodiaceae), a medicinal fern used in Indian traditional medicine against liver disorders.

Aim of the study

The rationale of the study was to examine whether the n-hexane extract from plant Lygodium flexuosum affects apoptosis on human hepatoma PLC/PRF/5 and Hep 3B cells.

Materials and methods

Chemopreventive activity of the Lygodium flexuosum extract was determined by MTT assay, annexin-V FITC binding to phosphatidyl serine and cleavage of PARP. Subdiploid condition of cells treated with Lygodium flexuosum was analyzed by flow cytometry. Further, used transiently transfected NF-κB reporter in PLC/PRF/5 cells to evaluate the inhibitive effect of Lygodium flexuosum extract.

Results

Lygodium flexuosum extract inhibited the cell viability and induced apoptosis in hepatoma cells in a concentration dependent manner as evidenced by apoptotic changes such as flipping of phosphatidyl serine, cleavage of PARP. Cell cycle analysis showed the subG1 apoptotic population in cells treated with higher concentrations of the extract. When activated with exogenous TNF-α in transfected hepatoma cells it was observed that NF-κB dependent gene expression was inhibited by treatment with Lygodium flexuosum extract in PLC/PRF/5 cells dose-dependently.

Conclusions

This investigation suggests that the Lygodium flexuosum extract has antiproliferative and apoptotic activity in both cancer cells and has inhibitive role in TNF-α induced NF-κB activation in PLC/PRF/5 cells confirms the potential of the extract as a chemopreventive agent.     

Evaluating the anti-inflammatory potential of Tectaria cicutaria L. rhizome extract in vitro as well as in vivo

Ethnopharmacological relevance

The rhizome of Tectaria cicutaria has been used in the folklore system of Indian traditional medicine (Ayurveda) for the treatment of various disorders such as rheumatic pain, chest complaints, burns, sprain, poisonous bites, tonsilitis, toothache, gum complaints, cuts and wounds. The present work has for the first time tried to elucidate the anti-inflammatory potential of aqueous extract of Tectaria cicutaria rhizome (TCR_aq) in vitro as well as in vivo.

Materials and methods

Anti-inflammatory potential of TCR_aq was analyzed in vivo in carrageenan induced rat paw edema model. Serum antioxidant status in TCR_aq-treated as well as untreated control rodents was measured by oxygen radical absorbance capacity (ORAC) assay. In vitro experiments for analyzing the anti-inflammatory potential of TCR_aq were performed on murine macrophage cell line, RAW 264.7. Analysis of nitric oxide release in RAW 264.7 cells was done by Griess reaction. RT-PCR and western blotting experiment was performed to analyze the expression of iNOS. Expression of COX-2 and NFκB proteins was evaluated by western blotting.

Results

TCR_aq significantly reduced the paw volume in Sprague-Dawley rats at a dose of 200 mg/kg body weight, which was comparable with the standard diclofenac treatment. The rats treated with TCR_aq showed a significant increase in the serum antioxidant levels compared to the untreated control animals. TCR_aq was able to reduce the nitric oxide (NO) levels in RAW 264.7 cells that had been stimulated with lipopolysaccharide (LPS). This was accompanied by a corresponding decrease in iNOS expression at mRNA and protein level. Interestingly, TCR_aq was found to decrease the expression of COX-2 as well as the nuclear translocation of NFκB in RAW 264.7 cells.

Conclusion

Our study signifies the anti-inflammatory potential of Tectaria cicutaria and scientifically validates its traditional use in inflammatory conditions.     

Anti-inflammatory effects of ethyl acetate fraction from Melilotus suaveolens Ledeb on LPS-stimulated RAW 264.7 cells

Aim of the study

This paper aimed to elucidate the anti-inflammatory effects of EtOAc fraction prepared from Melilotus suaveolens Ledeb ethanol extract with a cellular model of LPS-stimulated RAW 264.7 cell.

Materials and methods

Some key pro-inflammatory cytokines and mediators including IL-1β, IL-6, NO, iNOS, COX-2 and TNF-α, two important anti-inflammatory cytokines and mediators IL-10 and HO-1, I-κB and NF-κB were studied by sandwich ELISA, real-time PCR, western blot analysis and immunocytochemistry. At last a HPLC fingerprint was taken to evaluate the fraction.

Results

The EtOAc fraction could significantly inhibit the production of IL-1β, IL-6, NO, TNF-α, COX-2 in LPS-stimulated cell than that of single LPS-stimulated cell (p < 0.01 or p < 0.05), and the extract could increase the production of IL-10 and HO-1 than that of single LPS intervention cell (p < 0.01 or p < 0.05). Meanwhile, the extract also could inhibit the production of NF-κB compared to single LPS-stimulated cell. All the results showed that the extract had a good anti-inflammatory effect on LPS-stimulated RAW264.7 cell.

Conclusions

Taken together, the anti-inflammatory actions of M. suaveolens Ledeb EtOAc fraction might be due to the down-regulation of IL-1β, IL-6, NO, TNF-α and COX-2 via the suppression of NF-κB activation, and another pathway was up regulating the production of IL-10 and HO-1. Meanwhile, the EtOAc fraction might be further studied to isolate the active anti-inflammatory ingredients besides coumarin.     

Kinsenoside, a high yielding constituent from Anoectochilus formosanus, inhibits carbon tetrachloride induced Kupffer cells mediated liver damage

Aim

In the present study, we have evaluated the hepatoprotective ability of kinsenoside, a major component of Anoectochilus formosanus, in vitro and in vivo.

Materials and methods

The inhibitory action of kinsenoside on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells and Kupffer cells were investigated. Mice hepatic injury was produced by CCl₄ twice a week for 3 weeks. Mice in the three CCl₄ group were treated daily with water and kinsenoside throughout the experimental period.

Results

In LPS-stimulated macrophage RAW 264.7 cells and Kupffer cells, kinsenoside inhibited nitric oxide (NO) production and also blocked LPS-induced inducible NO synthase expression. Furthermore, kinsenoside inhibited the NF-κB activation induced by LPS, and this is associated with the abrogation of IκBα degradation, with subsequent decreases in nuclear p65 and p50 protein levels. Moreover, the phosphorylations of p38, ERK and JNK in LPS-stimulated RAW 264.7 cells were suppressed by kinsenoside. In the in vivo study, kinsenoside significantly protected the liver from injury, by reducing the activities of plasma aminotransferase, and by improving the histological architecture of the liver. kinsenoside inhibited Kupffer cell activation by reducing the CD 14 mRNA and protein expressions.

Conclusion

These results indicate that kinsenoside alleviates CCl₄-induced liver injury, and this protection is probably due to the suppression of Kupffer cell activation.     

Methylene chloride fraction of the leaves of Thuja orientalis inhibits in vitro inflammatory biomarkers by blocking NF-κB and p38 MAPK signaling and protects mice from lethal endotoxemia

Aim of the study

Thuja orientalis (TO) has been a recognized herbal medicine across Northeast Asian countries for thousands of years and used for the treatment of various inflammatory diseases through as yet undefined mechanisms. In this study, we set out to determine whether the anti-inflammatory effects of this plant are mediated to suppress mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) activation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.

Materials and methods

RAW 264.7 cells were pretreated with the methylene chloride fraction of TO (MTO) and stimulated with LPS. Nitric oxide (NO) release was determined by the accumulation of nitrite in the culture supernatants and tumor necrosis factor-α (TNF-α) and IL-6 secretion were determined by immunoenzymatic assay. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression were evaluated via RT-PCR and Western blotting. NF-κB activation was also evaluated by reporter gene assay and electrophoretic mobility shift assay (EMSA). In addition, the protective effect of MTO was evaluated by use of the LPS-induced endotoxin shock model in mice.

Results

We found that MTO significantly suppressed LPS-stimulated NO and IL-6 production without affecting cell viability. MTO inhibited the expression of LPS-induced iNOS and COX-2 protein and their mRNA expression. Also, TNF-α and IL-6 secretion were decreased by MTO in both PMA and ionomycin-stimulated splenocytes. As a result, MTO inhibited pro-inflammatory cytokines such as TNF-α and IL-6, which is hypothesized as being due to the suppression of LPS-induced p38 MAPK and NF-κB activation. Moreover, MTO improved the survival rate during lethal endotoxemia by inhibiting the production of TNF-α in an animal model and our LC-MS analysis showed that a major component of MTO was pinusolide.

Conclusions

We demonstrate here the evidence that the methylene chloride fraction of Thuja orientalis (MTO) potentially inhibits the biomarkers related to inflammation in vitro and in vivo, and might be provided as a potential candidate for the treatment of inflammatory diseases.