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Background NO (nitric oxide) inhibition could be used for evaluating the drug’s efficacy for NO-mediated inflammatory disorders. The aim of this study was to investigate the influence of L-NAME and melatonin on different NO production levels in RAW 264.7 cell line as an in vitro model for inflammatory diseases. Materials and methods RAW 264.7 macrophage cell line was used to compare the effects of L-NAME and melatonin on basal and Lipopolysaccharide (LPS)-induced iNOS levels. The cells were treated using L-NAME and melatonin for 1 h, afterward incubated with/without LPS for 8 and 24 h. Finally, iNOS mRNA, protein, activity, and nitrite concentrations were evaluated. Results Inhibition rate of nitrite by 1 mM L-NAME compared with LPS control were 78% and 80% during 8 and 24 h, respectively. Real-time PCR showed that in the LPS-treated group, 1 mM L-NAME could result in 14% increase of iNOS-mRNA compared with the control group during 8 h. Dose-dependent activity of iNOS in LPS-induced cells from non-treated to 4 mM L-NAME showed 79% reduction while at the same concentrations of melatonin this decrease was 32% (p-value Conclusion L-NAME showed lower iNOS expression modulating efficacy than melatonin. The result concluded lower potential of the NOS synthetic inhibitors rather than melatonin in the treatment of NO-related disorders.

Background: The purpose of this study was to investigate the anti-inflammatory response of lipopolysaccharide (LPS) activated macrophages (RAW 264.7 murine cell line) to JCE003 which is an extract including Eucommia ulmoides, Juglans regia, Eleutherococcus senticosus, and Zingiber officinale. Methods: An MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed to analyze the survival rate of RAW 264.7 cells. The production of nitric oxide and pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1β, IL-6) in LPS-induced RAW 264.7 cells was measured by enzyme-linked immunosorbent assay. mRNA expression levels of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1β, and IL-6) were analyzed by quantitative polymerase chain reaction analysis. Results: Exposure of LPS-activated RAW 264.7 cells to JCE003 was not cytotoxic up to 400 μg/mL, but cell survival was statistically significantly decreased at 800 μg/mL (p < 0.001). Nitric oxide production was not markedly lowered in LPS-activated RAW 264.7 cells by exposure to JCE003 (10, 50, 100, 200, 400, 800 μl/mL) compared with the Control group. In addition, JCE003 reduced the production of TNF-α in LPS-induced RAW 264.7 cells at 400 μg/mL (p < 0.05), but IFN-γ and TNF-α mRNA expression in LPS-induced RAW 264.7 cells was decreased at 100, 200, and 400 μg/mL JCE003 (p < 0.01). Conclusion: These results suggest that JCE003 inhibited the expression and production of pro-inflammatory cytokines in LPS-activated RAW 264.7 cells. The findings of this study provide basic data for the development of new Korean medicine anti-inflammatory drugs.

Recent evidence has shown that inflammation can contribute to all tumorigenic states. We have investigated the anti-inflammatory effects of a diamine-PEGylated derivative of oleanolic acid (OADP), in vitro and in vivo with inflammation models. In addition, we have determined the sub-cytotoxic concentrations for anti-inflammatory assays of OADP in RAW 264.7 cells. The inflammatory process began with incubation with lipopolysaccharide (LPS). Nitric oxide production levels were also determined, exceeding 75% inhibition of NO for a concentration of 1 µg/mL of OADP. Cell-cycle analysis showed a reversal of the arrest in the G0/G1 phase in LPS-stimulated RAW 264.7 cells. Furthermore, through Western blot analysis, we have determined the probable molecular mechanism activated by OADP, the inhibition of the expression of cytokines such as TNF-α, IL-1β, iNOS, and COX-2, and the blocking of p-IκBα production in LPS-stimulated RAW 264.7 cells. Finally, we have analyzed the anti-inflammatory action of OADP in a mouse acute ear edema, in male BL/6J mice treated with OADP and tetradecanoyl phorbol acetate (TPA). Treatment with OADP induced greater suppression of edema and decreased the ear thickness 14% more than diclofenac. The development of new derivatives such as OADP with powerful anti-inflammatory effects could represent an effective therapeutic strategy against inflammation and tumorigenic processes.

The winemaking process produces a huge number of pomaces that generally are used for energy purposes. Further valuable applications such as health-promoting properties are still under investigation. The seeds of the white berries of Mantonico and Pecorello cv. were extracted in a Soxhlet apparatus, using n-hexane and chloroform as solvents. Extracts were characterized by NMR and GC-MS analyses. They were assayed in vitro as wound healing and anti-inflammatory agents in HaCaT and RAW 264.7 cell lines, respectively. n-hexane Mantonico extract resulted in the most interesting wound healing sample, while n-hexane Pecorello, containing a good number of carotenoids, resulted in a good anti-inflammatory candidate. These preliminary findings underlined the benefit of grape seed extracts valorization due to their health-promoting properties.

Current pharmacological therapies for the management of chronic articular diseases are far from being satisfactory, so new strategies need to be investigated. We tested the intra-articular pain relieving properties of a system of molecules from a characterized Centella asiatica extract (14G1862) in a rat model of osteoarthritis induced by monoiodoacetate (MIA). 14G1862 (0.2–2 mg mL−1) was intra-articularly (i.a.) injected 7 days after MIA, behavioural and histological evaluations were performed 14, 30 and 60 days after treatments. Moreover, the effect of 14G1862 on nitrate production and iNOS expression in RAW 264.7 macrophages stimulated with LPS was assessed. In vitro, 14G1862 treatment attenuated LPS-induced NO production and iNOS expression in a comparable manner to celecoxib. In vivo, 14G1862 significantly reduced mechanical allodynia and hyperalgesia, spontaneous pain and motor alterations starting on day 14 up to day 60. The efficacy was higher or comparable to that evoked by triamcinolone acetonide (100 μg i.a.) used as reference drug. Histological evaluation highlighted the improvement of several morphological parameters in MIA + 14G1862-treated animals with particularly benefic effects on joint space and fibrin deposition. In conclusion, i.a. treatment with Centella asiatica is a candidate to be a novel effective approach for osteoarthritis therapy.

Neglected diseases are becoming more prevalent due to globalization. This has inspired active research in the development of new drugs for the treatment of parasitic diseases such as Chagas disease.With the aim of finding new trypanocidal agents, we report the in vitro evaluation of a new series of 3-amidocoumarins with or without hydroxyl substituents at position 4 of the coumarin ring.Electrochemical and biological assays were performed in order to assess the antioxidant and trypanocidal potential of these compounds and to better understand the mechanisms involved in their activity.Most of the studied compounds showed high trypanocidal activity against both epimastigote and trypomastigote forms, with IC50 values in the low micromolar range. Some of them have greater activity and selectivity than the reference compound, nifurtimox.Compound 2 is the most active of this series, being also non-cytotoxic against murine RAW 264.7 macrophages. Electrochemical and radical scavenging experiments were carried out, providing new information about the profile of the best derivatives, and the potential therapeutic application of the new 3-amidocoumarins.

Aim: Confirm the use of Plectanthus spp. plants in traditional medicine, particularly as anti-inflammatory and anti-infective agents. Materials & Methods: Compounds previously isolated from Plectranthus spp. were studied for their anti-inflammatory activity using the SNAP assay and RAW 264.7 cells, by the quantification of nitric oxide. An halimane diterpene and its derivatives were tested in infected macrophages with M. tuberculosis H37Rv, using CFU counts assay, at their minimum inhibitory concentration values. Results: The isolated compounds tested at noncytotoxic concentrations, did not reveal nitric oxide scavenging in the S-nitroso-N-acetylpenicillamine and the cellular assays. On the other hand, promising results were obtained regarding one semisynthetic halimane derivative (11R*,13E)-halima-5,13-diene-11,15-diol), previously prepared (2.1 × 105 CFU/mL), with an effect similar to the antitubercular drugs ethambutol (2.0 × 105 CFU/mL) and isoniazid (1.2 × 105 CFU/mL). Conclusion: The present report demonstrates the relevance of Plectranthus spp. in medicinal chemistry drug development for TB and other infective respiratory complaints. Also, this work suggests that further studies involving other inflammatory mediators are needed to validate the anti-inflammatory use of these medicinal plants.

The aim of this study was to determine whether exposure to radiation from single or multiple radio-frequency (RF) signals at 900 and 2450 MHz would induce effects in the RAW 264.7 cell line.Cell cultures were exposed to single or combined RF for 4, 24, 48, or 72 h in a GTEM electromagnetic test chamber. At the end of the radiation exposure time, viability and cell growth were analyzed by flow cytometry, nitric oxide (NO) production was measured by colorimetry, the expression of HSP70 and TNF-α was ascertained by qPCR, and the phagocytic activity was observed by microscopy.NO production increased after 48 h exposure at 2450 MHz, compared with controls. The group subjected to the combined interaction of two RFs showed an increase of HSP70 after 48 h exposure and a significant increase of NO and TNF-α after 72 h. The phagocytic activity of macrophages decreased in all groups as exposure time increased.Our results indicated a decrease in phagocytic activity and an increase in inflammatory, cytoprotective, and cytotoxic responses in macrophages after continuous and combined exposure of multiple RF signals. Multiple RF interact in everyday life, the immune response in humans is unknown.

Current pharmacological therapies for the management of chronic articular diseases are far from being satisfactory, so new strategies need to be investigated. We tested the intra-articular pain relieving properties of a system of molecules from a characterized Centella asiatica extract (14G1862) in a rat model of osteoarthritis induced by monoiodoacetate (MIA). 14G1862 (0.2&ndash, 2 mg mL&minus, 1) was intra-articularly (i.a.) injected 7 days after MIA, behavioural and histological evaluations were performed 14, 30 and 60 days after treatments. Moreover, the effect of 14G1862 on nitrate production and iNOS expression in RAW 264.7 macrophages stimulated with LPS was assessed. In vitro, 14G1862 treatment attenuated LPS-induced NO production and iNOS expression in a comparable manner to celecoxib. In vivo, 14G1862 significantly reduced mechanical allodynia and hyperalgesia, spontaneous pain and motor alterations starting on day 14 up to day 60. The efficacy was higher or comparable to that evoked by triamcinolone acetonide (100 &mu, g i.a.) used as reference drug. Histological evaluation highlighted the improvement of several morphological parameters in MIA + 14G1862-treated animals with particularly benefic effects on joint space and fibrin deposition. In conclusion, i.a. treatment with Centella asiatica is a candidate to be a novel effective approach for osteoarthritis therapy.

The winemaking process produces a huge number of pomaces that generally are used for energy purposes. Further valuable applications such as health-promoting properties are still under investigation. The seeds of the white berries of Mantonico and Pecorello cv. were extracted in a Soxhlet apparatus, using n-hexane and chloroform as solvents. Extracts were characterized by NMR and GC-MS analyses. They were assayed in vitro as wound healing and anti-inflammatory agents in HaCaT and RAW 264.7 cell lines, respectively. n-hexane Mantonico extract resulted in the most interesting wound healing sample, while n-hexane Pecorello, containing a good number of carotenoids, resulted in a good anti-inflammatory candidate. These preliminary findings underlined the benefit of grape seed extracts valorization due to their health-promoting properties.

Atherosclerosis is an inflammatory disease characterized by the accumulation of macrophages in the vessel wall. Macrophages depend on their polarization to exert either pro-inflammatory or anti-inflammatory effects. Macrophages of the anti-inflammatory phenotype express high levels of CD163, a scavenger receptor for the hemoglobin-haptoglobin complex. CD163 can also bind to the pro-inflammatory cytokine TWEAK. Using ApoE-deficient or ApoE/CD163 double-deficient mice we aim to investigate the involvement of CD163 in atherosclerosis development and its capacity to neutralize the TWEAK actions. ApoE/CD163 double-deficient mice displayed a more unstable plaque phenotype characterized by an increased lipid and macrophage content, plaque size, and pro-inflammatory cytokine expression. In vitro experiments demonstrated that the absence of CD163 in M2-type macrophages-induced foam cell formation through upregulation of CD36 expression. Moreover, exogenous TWEAK administration increased atherosclerotic lesion size, lipids, and macrophages content in ApoE-/-/CD163-/- compared with ApoE-/-/CD163+/+ mice. Treatment with recombinant CD163 was able to neutralize the proatherogenic effects of TWEAK in ApoE/CD163 double-deficient mice. Recombinant CD163 abolished the pro-inflammatory actions of TWEAK on vascular smooth muscle cells, decreasing NF-kB activation, cytokines and metalloproteinases expression, and macrophages migration. In conclusion, CD163-expressing macrophages serve as a protective mechanism to prevent the deleterious effects of TWEAK on atherosclerotic plaque development and progression. © 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology

Osteoarthritis (OA), a degenerative joint disorder, has been reported as the most common cause of disability worldwide. The production of inflammatory cytokines is the main factor in OA. Previous studies have been reported that obeticholic acid (OCA) and OCA derivatives inhibited the release of proinflammatory cytokines in acute liver failure, but they have not been studied in the progression of OA. In our study, we screened our small synthetic library of OCA derivatives and found T-2054 had anti-inflammatory properties. Meanwhile, the proliferation of RAW 264.7 cells and ATDC5 cells were not affected by T-2054. T-2054 treatment significantly relieved the release of NO, as well as mRNA and protein expression levels of inflammatory cytokines (IL-6, IL-8 and TNF-α) in LPS-induced RAW 264.7 cells. Moreover, T-2054 promoted extracellular matrix (ECM) synthesis in TNF-α-treated ATDC5 chondrocytes. Moreover, T-2054 could relieve the infiltration of inflammatory cells and degeneration of the cartilage matrix and decrease the levels of serum IL-6, IL-8 and TNF-α in DMM-induced C57BL/6 mice models. At the same time, T-2054 showed no obvious toxicity to mice. Mechanistically, T-2054 decreased the extent of p-p65 expression in LPS-induced RAW 264.7 cells and TNF-α-treated ATDC5 chondrocytes. In summary, we showed for the first time that T-2054 effectively reduced the release of inflammatory mediators, as well as promoted extracellular matrix (ECM) synthesis via the NF-κB-signaling pathway. Our findings support the potential use of T-2054 as an effective therapeutic agent for the treatment of OA.

Extracellular bacterial ribonucleases such as binase from Bacillus pumilus possess cytotoxic activity against tumor cells with a potential for clinical application. Moreover, they may induce activation of tumor-derived macrophages either into the M1-phenotype with well-documented functions in the regulation of the antitumor immune response or into M2-macrophages that may stimulate tumor growth, metastasis, and angiogenesis. In this study, binase or endogenous RNase1 (but not RNA or short oligonucleotides) stimulated the expression of activated NF-κB p65 subunit in macrophages. Since no changes in MyD88 and TRIF adaptor protein expression were observed, toll-like receptors may not be involved in RNase-related NF-κB pathway activation. In addition, short exposure (0.5 hr) to binase induced the release of cytokines such as IL-6, МСР-1, or TNF-α (but not IL-4 and IL-10), indicative for the polarization into antitumor M1-macrophages. Thus, we revealed increased expression of activated NF-κB p65 subunit in macrophages upon stimulation by binase and RNase1, but not RNA or short oligonucleotides.

Ethnopharmacological importance Decoction and infusion prepared from aerial parts of Lavandula stoechas L. (L. stoechas) have been traditionally used as remedy against several components of metabolic syndrome (MetS) and associated disorders including type II diabetes and cardiovascular diseases by Anatolian people. Aim of the study The aim is to elucidate the potential ameliorative effects of L. stoechas aqueous extracts on insulin resistance and inflammation models through multitarget in vitro approaches and also to elucidate mechanism of action by analyzing transcriptional and metabolic responses. Materials and methods An aqueous extract was prepared and fractionated to give rise to ethyl acetate (EE) and butanol (BE) extracts. The anti-insulin resistance effects of BE and EE were evaluated on palmitate induced insulin resistance model of H4IIE, C2C12 and 3T3L1 cells by using several metabolic parameters. Specifically, whole genome transcriptome analysis was performed by using microarray over 55.000 genes in control, insulin resistant and EE (25 µg/mL) treated insulin resistant H4IIE cells. Anti-inflammatory effects of both extracts were analyzed in LPS-stimulated RAW264.7 macrophages. Results Both EE and BE at low doses (25–50 µg/mL) significantly decreased hepatic gluconeogenesis in H4IIE cell line by suppressing the expression of PEPCK and G6Pase. In C2C12 myotubes, both extracts increased the insulin stimulated glucose uptake more effectively than metformin. Both extracts decreased the isoproterenol induced lipolysis in 3T3L1 cell line. Moreover, they also effectively increased the expression of lipoprotein lipase protein level in insulin resistant myotubes at low doses. EE increased the protein level of PPARγ and stimulated the activation AKT in insulin resistant H4IIE and C2C12 cell lines. The results obtained from biochemical assays, mRNA/protein studies and whole genome transcriptome analyses were found to be complementary and provided support for the hypothesis that EE might be biologically active against insulin resistance and act through the inhibition of liver gluconeogenesis and AKT activation. Besides, LPS induced inflammation in RAW264.7 macrophages was mainly inhibited by EE through suppression of iNOS/NO signaling, IL1β and COX-2 genes. HPLC-TOF/MS analysis of EE of L. stoechas mainly resulted in caffeic acid, apigenin, luteolin, rosmarinic acid and its methyl ester, 4-hydroxybenzoic acid, vanillic acid, ferrulic acid and salicylic acid. Conclusion Data suggest that EE of L. stoechas contains phytochemicals that can be effective in the treatment/prevention of insulin resistance and inflammation. These results validate the traditional use of L. stoechas in Anatolia against several metabolic disorders including metabolic syndrome.

Dynamic laser speckle (DLS) technique or biospeckle is a noninvasive approach that can sensitively monitor the dynamics of a biological material such as the metabolism of cells. In turn, stimulation of cell activity is widely used in microbiology: it can be done by means of many agents, such as lipopolysaccharide (LPS), which is the most common component within the cell wall of Gram-negative bacteria, and it has been extensively used in in vitro models studying inflammation. The main challenge is to monitor the change of behavior inside the cells during their stimulation. This work tested the viability of the BSL to monitor the change in the metabolism of RAW cells when LPS is applied. The results show different answers of cells to distinct concentrations of LPS, and the inflammatory process promoted by the LPS could be best distinguished from the RAW cells alone in a concentration of 10 ng/mL. This proved the ability of the DLS to monitor the agent stimulus.

Introduction: Adverse left ventricle remodeling can be measured as a reduction in ejection fraction after myocardial infarction. Left ventricle remodeling leads to congestive heart failure and is a main determinant of mortality and morbidity after myocardial infarction. Berberine is an isoquinoline alkaloid extracted from barberry that has anti-inflammatory and anti-oxidant activities. Pretreatment with long-term administration of high doses of berberine has shown beneficial effects in experimental diabetes and cardiac ischemia reperfusion injury. However, the poor solubility and the short half-life in the circulation have impeded the clinical use of berberine. Purpose: To examine whether encapsulation of berberine into long-circulating liposomes could improve its therapeutic availability and efficacy to protect cardiac function in vivo. Methods: Berberine was loaded into liposomes at a concentration of 0.3 mg/ml. Lipopolysaccharide (LPS) activated mouse macrophages RAW 264.7 were treated with free berberine or liposome-encapsulated berberine (Lipo-Berb) and analyzed for cell viability, reactive oxygen species production and cytokine secretion. C57BL/6J male mice (10-12 week old) subjected to myocardial infarction (MI) via permanent ligation of the left anterior descending artery were blindly selected for intravenous injection of empty liposomes, free berberine or lipo-Berb (1.5 mg/kg). Three doses were administered at the onset of MI, and at 3 and 6 days after MI. Ejection fraction was assessed by echocardiography at baseline, 7 and 28 days after MI. Results: Free berberine improved the viability of LPS-insulted macrophages, reduced production of reactive oxygen species and inhibited the secretion of inflammatory mediators including IL-6 and TNFα. As expected these protective effects of berberine in vitro were diminished upon encapsulation into liposomes. In vivo, however, the liposome-encapsulated berberine significantly preserved ejection fraction after 28 days of MI while free berberine did not show any preservation of ejection fraction (29.5±1.9 for lipo-Berb, 18.2±3.2 for free berberine and 18.0±3.1 for empty liposomes; n=6-10; p

Introduction: Adverse left ventricle remodeling can be measured as a reduction in ejection fraction after myocardial infarction. Left ventricle remodeling leads to congestive heart failure and is a main determinant of mortality and morbidity after myocardial infarction. Berberine is an isoquinoline alkaloid extracted from barberry that has anti-inflammatory and anti-oxidant activities. Pretreatment with long-term administration of high doses of berberine has shown beneficial effects in experimental diabetes and cardiac ischemia reperfusion injury. However, the poor solubility and the short half-life in the circulation have impeded the clinical use of berberine. Purpose: To examine whether encapsulation of berberine into long-circulating liposomes could improve its therapeutic availability and efficacy to protect cardiac function in vivo. Methods: Berberine was loaded into liposomes at a concentration of 0.3 mg/ml. Lipopolysaccharide (LPS) activated mouse macrophages RAW 264.7 were treated with free berberine or liposome-encapsulated berberine (Lipo-Berb) and analyzed for cell viability, reactive oxygen species production and cytokine secretion. C57BL/6J male mice (10-12 week old) subjected to myocardial infarction (MI) via permanent ligation of the left anterior descending artery were blindly selected for intravenous injection of empty liposomes, free berberine or lipo-Berb (1.5 mg/kg). Three doses were administered at the onset of MI, and at 3 and 6 days after MI. Ejection fraction was assessed by echocardiography at baseline, 7 and 28 days after MI. Results: Free berberine improved the viability of LPS-insulted macrophages, reduced production of reactive oxygen species and inhibited the secretion of inflammatory mediators including IL-6 and TNFα. As expected these protective effects of berberine in vitro were diminished upon encapsulation into liposomes. In vivo, however, the liposome-encapsulated berberine significantly preserved ejection fraction after 28 days of MI while free berberine did not show any preservation of ejection fraction (29.5±1.9 for lipo-Berb, 18.2±3.2 for free berberine and 18.0±3.1 for empty liposomes; n=6-10; p

Introduction: Adverse left ventricle remodeling can be measured as a reduction in ejection fraction after myocardial infarction. Left ventricle remodeling leads to congestive heart failure and is a main determinant of mortality and morbidity after myocardial infarction. Berberine is an isoquinoline alkaloid extracted from barberry that has anti-inflammatory and anti-oxidant activities. Pretreatment with long-term administration of high doses of berberine has shown beneficial effects in experimental diabetes and cardiac ischemia reperfusion injury. However, the poor solubility and the short half-life in the circulation have impeded the clinical use of berberine. Purpose: To examine whether encapsulation of berberine into long-circulating liposomes could improve its therapeutic availability and efficacy to protect cardiac function in vivo. Methods: Berberine was loaded into liposomes at a concentration of 0.3 mg/ml. Lipopolysaccharide (LPS) activated mouse macrophages RAW 264.7 were treated with free berberine or liposome-encapsulated berberine (Lipo-Berb) and analyzed for cell viability, reactive oxygen species production and cytokine secretion. C57BL/6J male mice (10-12 week old) subjected to myocardial infarction (MI) via permanent ligation of the left anterior descending artery were blindly selected for intravenous injection of empty liposomes, free berberine or lipo-Berb (1.5 mg/kg). Three doses were administered at the onset of MI, and at 3 and 6 days after MI. Ejection fraction was assessed by echocardiography at baseline, 7 and 28 days after MI. Results: Free berberine improved the viability of LPS-insulted macrophages, reduced production of reactive oxygen species and inhibited the secretion of inflammatory mediators including IL-6 and TNFα. As expected these protective effects of berberine in vitro were diminished upon encapsulation into liposomes. In vivo, however, the liposome-encapsulated berberine significantly preserved ejection fraction after 28 days of MI while free berberine did not show any preservation of ejection fraction (29.5±1.9 for lipo-Berb, 18.2±3.2 for free berberine and 18.0±3.1 for empty liposomes; n=6-10; p

Melilotus suaveolens Ledebis a traditional medicinal plant for treating inflammation-related disease. This explores the inner anti-inflammatory mechanism ofn-butanol extract fromM. suaveolens Ledeb. Inflammatory cellular model was established by lipopolysaccharide intervention on RAW264.7 cell line. Levels of secreted cytokines TNF-α, IL-1β, IL-6, NO and IL-10 in supernatant, mRNA expression of TNF-α, COX-2, iNOS and HO-1, protein expression of COX-2 and HO-1, activation of NF-κB and ingredients in the extract were assayed by ELISA, real time quantitative PCR, western blot, immunocytochemical test and HPLC fingerprint test, respectively. As a result, the extract could not only markedly reduce the production of pro-inflammatory mediators to different extents by blocking NF-κB activation but also promote the release of anti-inflammatory mediator HO-1 significantly. Each 1 g extract contained 0.023531 mg coumarin and another two high polar ingredients, probably saponins. It can be concluded that the extract has similar effects on antagonizing pro-inflammatory mediators and cytokines like Dexamethasone, and has effects on promoting the production of anti-inflammatory mediators.

Multinuclear osteoclasts are derived from CD11b-positive mononuclear cells in bone marrow and in circulation. FACS sorting experiments showed impaired osteoclastogenesis in RAW264.7 cells with low CD11b expression. Neutralizing antibodies and siRNA against CD11b inhibited osteoclastogenesis induced by RANKL. Although primary cultured mouse bone marrow macrophages expressed CD11a and CD11b, osteoclastogenesis induced by M-CSF and RANKL was inhibited in the presence of anti-CD11b or anti-CD18 but not anti-CD11a antibodies. Furthermore, anti-CD11b antibodies inhibited NFATc1 expression induced by M-CSF and RANKL in BMMs. These findings suggest, at least partly, an important role of CD11b in osteoclastogenesis.