Anti-inflammatory effects of methanol extracts of the root of Lilium lancifolium on LPS-stimulated Raw264.7 cells

Aim of the study

Lilium lancifolium is commonly used to treat bronchitis, pneumonia, etc. In this study, we investigated the anti-inflammatory effects of methanol extracts of the root of Lilium lancifolium (LL extracts) in LPS-stimulated Raw264.7 cells.

Material and methods

Levels of NO, PGE₂ and pro-inflammatory cytokines (IL-6 and TNF-α) in the supernatant fraction were determined using sandwich ELISA. Expression of COX-2 and iNOS, phosphorylation of MAPK subgroups (ERK and JNK), and NF-κB activation in extracts were detected via Western blot and immunocytochemistry assays.

Results

The LL extract significantly inhibited NO, PGE₂, IL-6 and TNF-α production in LPS-stimulated cells, and suppressed iNOS and COX-2 expression. A mechanism-based study showed that phosphorylation of ERK1/2 and JNK and translocation of the NF-κB p65 subunit into nuclei were inhibited by the LL extract. Furthermore, interleukin-4 and interleukin-13 production in Con A-induced splenocytes was suppressed.

Conclusion

These results indicate that anti-inflammatory effects of methanol extracts from Lilium lancifolium are due to downregulation of iNOS and COX-2 via suppression of NF-κB activation and nuclear translocation as well as blocking of ERK and JNK signaling in LPS-stimulated Raw264.7 cells.     

A herbal formula containing roots of Salvia miltiorrhiza (Danshen) and Pueraria lobata (Gegen) inhibits inflammatory mediators in LPS-stimulated RAW 264.7 macrophages through inhibition of nuclear factor κB (NFκB) pathway

Ethnopharmacological relevance

The herbal formula DG, containing roots of Salvia miltiorrhiza (Danshen) and Pueraria lobata (Gegen), has long history in treating cardiovascular diseases. It has been shown to be able to reduce intima-media thickening in coronary patients in our previous clinical study. Since intima-media thickening is the hallmark of atherosclerotic disease, the etiology of which is inflammation of the arterial wall, the mechanism underlying the effect of DG may be related to its anti-inflammatory activities.

Aim of study

The present study aims to determine the anti-inflammatory activity of DG and elucidate its underlying mechanisms with regards to its molecular basis of action.

Materials and method

The anti-inflammatory effect of DG was studied by using lipopolysaccharide (LPS)-stimulated activation of nuclear factor κB (NFκB) pathway and subsequent production of inflammatory mediators, including nitric oxide (NO), prostaglandin E₂ (PGE₂), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and macrophage chemotactic protein-1 (MCP-1), in mouse RAW 264.7 macrophages.

Results

The present study demonstrated that DG could suppress the production of NO and PGE₂ through the inhibition of iNOS and COX-2 genes. DG could also inhibit the production of IL-1β, IL-6 and MCP-1, but not TNF-α, through the inhibition of respective mRNA expressions. Further investigations showed the inhibitory effect of DG on activation of IKKα/β and degradation of IκBα, thus preventing nuclear translocation of NFκB. All these results suggested the inhibitory effects of DG on the production of inflammatory mediators through the inhibition of the NFκB pathway.

Conclusions

The inhibitory effects of DG on the production of inflammatory mediators by LPS-stimulated RAW 264.7 macrophages, are accomplished by inhibiting the nuclear translocation of NFκB through inactivating IKKα/β and preventing degradation of IκBα.     

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.     

Wogonoside displays anti-inflammatory effects through modulating inflammatory mediator expression using RAW264.7 cells

Ethnopharmacological relevance

The root of Scutellaria baicalensis Georgi, also called Huangqin in China, is an herbal-based nutraceutical which is usually used in Chinese medicated diet (CMD). As an abundant ingredient in Huangqin, wogonoside is a flavonoid glycoside. The present work investigated the anti-inflammatory activities of wogonoside in lipopolysaccharides (LPS)-induced RAW264.7 cells.

Materials and methods

RAW264.7 cells were used. The inhibition of wogonoside against nitric oxide (NO), prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in LPS-induced RAW264.7 cells were measured. Additionally, the effects of wogonoside on mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), TNF-α and IL-6 were also investigated.

Results and discussion

Wogonoside not only dose-dependently decreased the production of inflammatory mediators including NO and PGE₂ but also inhibited the release of pro-inflammatory cytokines including TNF-α and IL-6 in LPS-induced RAW264.7 cells. Furthermore, wogonoside possessed significantly in vitro inhibitory effects on the gene expression of iNOS, COX2, TNF-α and IL-6.

Conclusion

These results suggest that wogonoside may be used as a functional food component for prevention and treatment of inflammation.     

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.     

Biomolecular evidence of anti-inflammatory effects by Clematis mandshurica Ruprecht root extract in rodent cells

Ethnopharmacological relevance

Clematis mandshurica Ruprecht root is widely used in Asia as an analgesic and anti-inflammatory agent. This research investigated the anti-inflammatory effects of Clematis mandshurica Ruprecht root extract (CRE) using RAW 264.7 macrophage cells and carrageenan- (CA-) induced rat paw edema.

Materials and methods

Production of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, nitric oxide (NO) and prostaglandin E₂ (PGE₂) in the culture supernatant, mRNA expression of TNF-α, IL-1β, IL-6, iNOS and COX-2, protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in the extract were assayed. In addition, the effect of CRE on acute inflammation in vivo was observed using CA-induced rat hind paw edema assay. The changes on the histopathology and histomorphometry of hind paw skins—dorsum and ventrum pedis were observed using CA-treated rats.

Results

Treatment with CRE (0.25, 0.5, and 1 mg/mL) resulted in inhibited levels of protein expression of lipopolysaccharide- (LPS-) induced iNOS, COX-2, NF-κB, and MAPKs (ERK, JNK, and p38) as well as production of TNF-α, IL-1β, IL-6, NO, and PGE₂ induced by LPS. Consistent with these results, CRE reduced the LPS-induced expressions of these cytokines, iNOS and COX-2 at the mRNA levels in a dose-dependent manner. In particular, results of the CA-induced rat hind paw edema assay showed an anti-edema effect of CRE. In addition, treatment with CRE resulted in dose-dependent inhibition of CA-induced increases of skin thickness, mast cell degranulation, and infiltrated inflammatory, TNF-α, IL-1β, iNOS, and COX-2-positive cells in both dorsum and ventrum pedis skin, respectively.

Conclusions

These results demonstrate that CRE exhibits anti-inflammatory activities via decreasing production of pro-inflammatory mediators through suppression of the pathways of NF-κB and MAPKs in LPS-induced macrophage cells. In addition, results of the CA-induced rat hind paw edema assay show an anti-edema effect of CRE. Our findings also support the traditional use of CRE in the inflammatory symptoms of rheumatic arthritis and acute icteric hepatitis. Thus, CRE may have therapeutic potential for a variety of inflammation-mediated diseases and may be developed into potent anti-inflammatory drugs.     

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.     

The possible mechanisms of Picrasma quassiodes (D. Don) Benn. in the treatment of colitis induced by 2,4,6-trinitrobenzene sulfonic acid in mice

Ethnopharmacological relevance

Picrasma quassiodes (D. Don) Benn.(PQB) is used in folk medicines for the treatment of colds, upper respiratory infection, acute tonsillitis, acute gastroenteritis, bacillary dysentery and a variety of acute infectious diseases in Asia. Although recent reports indicate that PQB has antibacterial, and anti-inflammatory effects, its effects on colitis and its inhibitory mechanisms have not been previously reported.

Aim of the study

To assess the effects and the mode of action of the extract of Picrasma quassiodes (D. Don) Benn.(PQB) on a model of colitis in mice induced by trinitrobenzene sulfonic acid (TNBS).

Materials and methods

We induced mice colitis using TNBS/ethanol, then different doses of Picrasma quassiodes (D. Don) Benn.(PQB) extract (100, 200 and 400 mg/kg/day) and sulfasalazine (500 mg/kg/day) were administered by gavage for 7 days after the induction of colitis. The mice body weight, colonic wet weight, colonic lengths, myeloperoxidase (MPO) activity, macroscopic and histological colon injury were observed. Pro-inflammatory cytokines such as: tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8) were assayed by enzyme-linked immunoassay. The protein expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in the colons were determined by immunohistochemical analysis.

Results

PQB administration effectively prevented mice diarrhea, decreasing of the body weights, shortening of colon length and increasing of colon wet weight. Macroscopic and histological examinations also indicated that it was protected against colonic edema, ulceration and MPO activity elevation. Furthermore, PQB inhibited the abnormal secretions of pro-inflammatory cytokines, such as TNF-α and IL-8. Additionally, administration of PQB effectively inhibited COX-2 and iNOS protein expression.

Conclusions

These results suggest that PQB has an anti-inflammatory effect on TNBS-induced colitis due to the down-regulations of the productions and expressions of inflammatory mediators, and that it may be a potential inflammatory bowel disease (IBD) drug candidate.     

Bioassay-guided isolation of anti-inflammatory components from the root of Rehmannia glutinosa and its underlying mechanism via inhibition of iNOS pathway

Ethnopharmacological relevance

The root of Rehmannia glutinosa (RR) is commonly used to reduce inflammation in various traditional Chinese herbal formulae; however, little is known regarding its active component(s).Aim of study: The objective of the present study was to examine the active component(s) responsible for the anti-inflammatory activity of RR via anti-nitric oxide production assay-guided fractionation; and the underlying anti-inflammatory mechanism of action of such component(s) was further investigated.

Materials and methods

Anti-nitric oxide (NO) activities with lipopolysaccharides (LPS)-stimulated RAW264.7 murine macrophages was used as screening platform. Gene, protein and inflammatory mediators' expression were also studied using real-time PCR, western blotting and ELISA, respectively.

Results

Using anti-NO assay-guided fractionation, sub-fraction C3 (from 31.25 to 62.5 μg/ml, p=0.001 to 0.01) possessed 100-fold more potent anti-inflammatory effect than that of the aqueous extract of RR. Characterization of C3 showed that the anti-inflammatory effect could be partly due to the presence of rehmapicrogenin, which could significantly inhibit NO production (p<0.001). C3 was further demonstrated in blocking inflammation by inhibiting gene (p<0.001) and protein expression of inducible NO synthase (iNOS) dose-dependently. Besides, C3 also significantly inhibited the production of prostaglandin E₂ (p<0.001 to 0.01), IL-6 (p<0.001 to 0.05) and COX-2 (p<0.05).

Conclusions

Rehmapicrogenin was, for the first time, shown to possess nitric oxide inhibitory activities. Bioassay-guided fractionation demonstrated that rehmapicrogenin-containing subfraction C3 exhibited potent anti-inflammatory effect by inhibiting iNOS, COX-2 and IL-6, while rehmapicrogenin was only partially responsible for the anti-inflammatory effect of RR.     

Atropa acuminata Royle Ex Lindl. blunts production of pro-inflammatory mediators eicosanoids., leukotrienes,cytokines in vitro and in vivo models of acute inflammatory responses

Ethnopharmacological relevance

Atropa acuminata Royle Ex Lindl. has been widely used in folk medicine for several inflammatory disorders such as arthritis, asthma, conjunctivitis, encephalitis, pancreatitis, peritonitis, acute infections and neuroinflammatory disorders.

Aim of the study

Our aim was to evaluate Atropa acuminata for its anti-inflammatory properties and to delineate its possible mechanism of action on the modulation of the inflammatory mediators.

Materials and methods

We investigated the inhibitory action of ethanolic extract of Atropa acuminata (AAEE) on production of NO, TNF-α and IL-1β in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and also assayed it for COX 1/2 and 5-LOX inhibitory activities. Next AAEE was tested in acute inflammatory animal models., carragenean induced rat paw edema, carragenean induce pleurisy in rats and vascular permeability in mice and the effects on NO, PGE₂ and LTB₄ production in the pleural fluid and paw exudates were evaluated. In addition the effects on leukocyte migration and exudation and vascular permeability were also observed.

Results

Our findings summarized novel anti-inflammatory mechanisms for Atropa acuminata based on dual in vitro cyclooxygenase 1/2/ and 5-Lipoxygenase inhibitory activities and also significant downregulation of nitric oxide (NO) and pro-inflammatory cytokin (TNF-α and Il-1 β) release in LPS-stimulated RAW 246.7 macrophage cell line. In acute inflammatory models in vivo (carragenean induced edema, carragenean induced pleurisy in rats and vascular permeability in mice), AAEE exhibited an extensive diverse mechanism for anti-inflammatory properties. This was indicated on the basis of dose dependent suppression of multi targeted inflammatory mediators., NO, TNF-α and IL-1β, eicosanoids., PGE₂ and leukotrienes., LTB₄ along with significantly decreased leucocyte migration, exudation and decreased vascular permeability. These effects were more potent and prolonged than traditional NSAIDS, thereby indicating fewer side effects. AAEE was found to be safe for long term administration, as confirmed by the results of acute toxicity studies and MTT assay. The complex mode of action of the herbs was attributed possibly due to the high polyphenolic, flavanol and flavonoid content present in the extracts as observed by means of quantitative screening for phytochemicals.

Conclusion

Our study provides scientific evidence to support the traditional anti-inflammatory uses of Atropa acuminata and is probably due to inhibitory effects on multiple inflammatory mediators which indicates a promising potential for the development of a strong anti-inflammatory agent from this plant.     

In vitro anti-inflammatory effects of arctigenin,a lignan from Arctium lappa L., through inhibition on iNOS pathway

Ethnopharmacological relevance

Arctigenin, a bioactive constituent from dried seeds of Arctium lappa L. (Compositae) which has been widely used as a Traditional Chinese Medicine for dispelling wind and heat included in Chinese Pharmacophere, was found to exhibit anti-inflammatory activities but its molecular mechanism remains unknown yet.

Aim of the study

To investigate the anti-inflammatory mechanism of arctigenin.

Materials and methods

Cultured macrophage RAW 264.7 cells and THP-1 cells were used for the experiments. Griess assay was used to evaluate the inhibitory effect of arctigenin on the overproduction of nitric oxide (NO). ELISA was used to determine the level of pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). The inhibitory effect on the enzymatic activity of cyclooxygenase-2 (COX-2) was tested by colorimetric method. Western blot was used to detect the expression of inducible nitric oxide synthase (iNOS) and COX-2.

Results

Arctigenin suppressed lipopolysaccharide (LPS)-stimulated NO production and pro-inflammatory cytokines secretion, including TNF-α and IL-6 in a dose-dependent manner. Arctigenin also strongly inhibited the expression of iNOS and iNOS enzymatic activity, whereas the expression of COX-2 and COX-2 enzymatic activity were not affected by arctigenin.

Conclusions

These results indicated that potent inhibition on NO, TNF-α and IL-6, but not COX-2 expression and COX-2 activity, might constitute the anti-inflammatory mechanism of arctigenin. Arctigenin suppressed the overproduction of NO through down-regulation of iNOS expression and iNOS enzymatic activity in LPS-stimulated macrophage.     

Anti-inflammatory and antitumoural effects of Uncaria guianensis bark

Ethnopharmacological relevance

Uncaria guianensis (Aublet) Gmell (Rubiaceae) is a medicinal plant from the jungles of South and Central America, used to treat cancer, arthritis, diabetes, and inflammation. Evaluate the anti-inflammatory and anti-tumor effects of Uncaria guianensis preparations.

Materials and methods

Bio-guided fractionation of a hydroethanolic extract of Uncaria guianensis was performed, evaluating the fractions and subfractions for their effect on inflammatory mediators, tumour necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and prostaglandin E₂ (PGE₂) by ELISA and nitric oxide (NO) by the Griess reaction in cultured supernatant from RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). The expression of cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS) and inhibitor of κB (IκB) were investigated in RAW 264.7 macrophages by flow cytometry. The activity of NF-κB in HeLa cells transfected with a luciferase reporter system was determined. The effect of Uncaria guianensis on the inflammatory response in vivo was assessed in BALB/c mice stimulated with LPS, on rat paw oedema induced by carrageenan, and on tumour growth and lung metastasis in BALB/c mice inoculated with 4T1 mammary tumour cells. Immune cell infiltrates and inflammatory mediators were evaluated in the tumour by immunohistochemistry.

Results

Sub-fraction Ug AIV inhibited, to varying degrees, NO, TNF-α, IL-6 and PGE₂ production by macrophages in vitro (30 μg/ml) and in the serum of LPS-challenged mice (5 mg/kg). Macrophage expression of Cox-2 was inhibited (35%), IκB degradation was completely inhibited and NF-κB activation was inhibited (70%) by Ug AIV at 30 μg/ml. Ug AIV decreased paw oedema by 86% (5 mg/kg) and serum NO and TNF-α by 45% and 65% respectively. Ug AIV reduced 4T1 mammary tumour growth by 91% on day 33 post-inoculation as well as the levels of serum NO, IL-6 and TNF-α in the same animals. Ug AIV decreased the number of tumour-infiltrating T lymphocytes, macrophages and neutrophils as well as the number of cells positive for COX-2, iNOS, IL-6, TNF-α and p65.

Conclusions

As Ug AIV was not cytotoxic for tumour cells or macrophages, its anti-tumour effect may be due to a reduction in pro-tumoural inflammatory processes in the tumour microenvironment, possibly mediated through NF-κB.