Panax quinquefolium saponins inhibited immune maturation of human monocyte-derived dendritic cells via blocking nuclear factor-κB pathway

Ethnopharmacological relevance

Panax quinquefolium saponins (PQS), a water-soluble antioxidant extracted from a natural herb, radix panacis quinquefolii (American Ginseng), has yielded encouraging results in the treatment of atherosclerotic diseases. However, the underlying mechanisms remain unclear. Here, we tested the hypothesis that the anti-atherosclerotic effect of PQS might be mediated by suppressing human monocyte-derived dendritic cells (DCs) maturation.

Materials and methods

DCs were derived by incubating purified human monocytes with granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4. DCs were pre-incubated with or without PQS and stimulated by oxidized low density lipoprotein (ox-LDL). Expression of DCs membrane molecules (CD40, CD86, CD1a, HLA-DR) and endocytotic ability were analyzed by FACS, cytokines (IL-12 and TNF-α) were measured by ELISA. Nuclear factor (NF)-κB signaling pathway was determined by Western blotting, and RT-PCR. NF-κB activation was quantified by ELISA.

Results

PQS reduced ox-LDL induced immunophenotypic expressions (CD40, CD1a, CD86, and HLA-DR) and cytokine secretions (IL-12 and TNF-α), and improved endocytotic ability of DCs. These above phenomena were accompanied by decreased protein expression and binding activity of nuclear localized c-Rel subunit.

Conclusions

Our study suggested that PQS inhibited ox-LDL induced immune maturation of DCs in vitro, which might be in part mediated by NF-κB signal transduction pathway.     

Anti-inflammatory activity of Cymbopogon citratus leaves infusion via proteasome and nuclear factor-κB pathway inhibition: Contribution of chlorogenic acid

Ethnopharmacological relevance

Cymbopogon citratus (DC.) Stapf leaves infusion is used in traditional medicine for the treatment of inflammatory conditions, however little is known about their bioactive compounds.

Aim of the study

Investigate the compounds responsible for anti-inflammatory potential of Cymbopogon citratus (Cy) on cytokines production induced by lipopolysaccharide (LPS) in human and mouse macrophages, and the action mechanisms involved.

Materials and methods

An essential oil-free infusion of Cy was prepared and polyphenol-rich fractions (PFs) were obtained from it by column chromatography. Chlorogenic acid (CGA) was identified, by HPLC/PDA/ESI-MSⁿ. The expression of cytokines, namely TNF-α and CCL5, was analyzed by real-time RT-PCR, on LPS-stimulated human macrophages. Activation of nuclear factor (NF)-κB, a master regulator of inflammation, was investigated by western blot and gene reporter assay. Proteasome activity was assessed using a fluorogenic peptide.

Results

Cymbopogon citratus extract and its polyphenols inhibited the cytokine production on human macrophages. This supports the anti-inflammatory activity of Cy polyphenols in physiologically relevant cells. Concerning the effect on the activation of NF-κB pathway, the results pointed to an inhibition of LPS-induced NF-κB activation by Cy and PFs. CGA was identified, by HPLC/PDA/ESI-MSⁿ, as the main phenolic acid of the Cy infusion, and it demonstrated to be, at least in part, responsible by that effect. Additionally, it was verified for the first time that Cy and PFs inhibited the proteasome activity, a complex that controls NF-κB activation, having CGA a strong contribution.

Conclusions

The results evidenced, for the first time, the anti-inflammatory properties of Cymbopogon citratus through proteasome inhibition and, consequently NF-κB pathway and cytokine expression. Additionally, Cy polyphenols, in particular chlorogenic acid, were highlighted as bioactive compounds.     

Flavonoids from Scutellaria barbata inhibit activation of tumor-associated macrophages by blocking the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-κB signaling pathway

OBJECTIVE: To determine the efficacy of Scutellaria barbata flavonoids and polysaccharides on Ishikawa endometrial carcinoma cells co-cultured with U937 macrophages.METHODS: The presence of CD163 and CD206 was determined by flow cytometry. Thiazolyl Blue Tetrazolium Bromide assays were used to assess the proliferation effect of tumor-associated macrophages(TAMs) on Ishikawa cells. The secretion of interleukin(IL)-10 in the co-culture conditioned media was examined using an enzyme-linked immunosorbent assay. The protein expression levels of Toll-like receptor 4(TLR4), myeloid differentiation factor 88(MyD88) and nuclear factor(NF)-κB p65 were detected by Western blot. The mRNA expression levels of TLR4 and MyD88 were analyzed by real-time polymerase chain reaction(PCR). The expression levels of IL-12, IL-1β and tumor necrosis factor-α(TNF-α) were evaluated with real-time PCR.RESULTS: Compared with the U937 control group,the expression levels of CD163 and CD206 in the TAM group were higher(P 0.05). TAMs co-cultured with Ishikawa cells for 24 or 48 h showed higher proliferation rates(P 0.05). The expression levels of IL-12 decreased than compared with those in the U937 untreated group(P 0.05) and those of the Scutellaria barbata flavonoids group(P 0.05). The expression levels of CD206, CD163, IL-10, IL-1β and TNF-α, NF-κB p65 and TLR4/MyD88 in the TAMs control group were greater than those in the U937 untreated group(P 0.05) and those of the Scutellaria barbata flavonoids group(P 0.05).CONCLUSION: Scutellaria barbata flavonoids may inhibit TAM activation by blocking the TLR4/MyD88/NF-κB signaling pathway.     

Fuyuan Decoction inhibits nitric oxide production via inactivation of nuclear factor-κB in SW1353 chondrosarcoma cells

Ethnopharmacological relevance

Fuyuan Decoction (FYD) is an empirical formula of treating Bi Zheng in traditional Chinese medicine (TCM). Despite the fact that the efficiency of FYD on treating osteoarthritis has been verified in clinic, the underlying mechanisms are not totally understood. This study was to investigate the effects and mechanisms of FYD on nitric oxide (NO) production and nuclear factor (NF)-κB activation in interleukin (IL)-1β-stimulated chondrocytes.

Materials and methods

SW1353 human chondrosarcoma cells were pretreated with various concentrations of FYD-containing serum (FYD-CS), and then were stimulated by IL-1β. Amounts of NO were determined by Griess reaction assay. Inducible NO synthase (iNOS) expression, inhibitor-κBα (IκBα) degradation and nuclear translocation of p65 protein were determined by Western blot assay. DNA binding activity of NF-κB was determined by ELISA assay using Trans AM^™ kit for p65.

Results

10% and 20% (v/v) FYD-CS significantly decreased NO production in a concentration-dependent manner (p<0.05 or p<0.01) as compared to control in IL-1β-induced SW1353 cells. Besides, 10% and 20% FYD-CS also significantly reduced iNOS protein expression by about 60% and 70% (both p<0.01), respectively. Furthermore, 10% and 20% FYD-CS markedly decreased IκBα degradation by about 45% and 26% (p<0.01 or p<0.05), lessened P65 content in the nucleus by about 28% and 60% (both p<0.01), and repressed DNA binding activity of P65 by about 30% and 45% (both p<0.01) in IL-1β-induced SW1353 cells.

Conclusions

These findings suggested that FYD could inhibit NO production and iNOS expression in IL-1β-induced chondrocytes through suppressing NF-κB activation.     

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.     

Effect of warming yang and supplementing kidney moxibustion therapy on the NF-κB signaling pathway of vascular dementia rats

Objective

To observe the effect of warming yang and supplementing kidney moxibustion therapy on the behavioristics and the expression levels of hippocampal IL-1β, TNF-α and NF-κB related genes and proteins of vascular dementia (VD) rats, and to explore the mechanism of warming yang and supplementing kidney moxibustion therapy in inhibiting VD inflammatory response. MethodsSeventy-eight SD rats, except 10 rats as sham operation group (group A), were established into VD models by applying the ischemia reperfusion method in bilateral common carotid arteries. 30 models were successful and were randomly divided into model group (group B), moxibustion group (group C) and western medication group (group D), with 10 rats in each group. After modeling, suspended moxibustion was conducted at “Dàzhuī (大椎GV 14)”, “Mìngmén (命门GV 4)” and “Guānyuán (关元CV 4)” of the rats in group C for 15?min/time. The intervention was performed for once a day, and intervention for 4 consecutive weeks was needed. Intragastric administration with nimodipine (2?mg?kg^?1?d^?1) was carried out in the rats in group D for 4 consecutive weeks. Morris water maze test was adopted for behavioral test in the rats in each group. HE staining was conducted in order to observe the pathological changes. RT-qPCR method and Western blot method were used for detecting the expression levels of hippocampal IL-1β, TNF-α and NF-κB related genes and proteins.

Isoliquiritigenin inhibits microglia-mediated neuroinflammation in models of Parkinson's disease via JNK/AKT/NFκB signaling pathway

Isoliquiritigenin (ISL) is a flavonoid with numerous pharmacological properties, including anti-inflammation, yet its role in Parkinson's disease (PD) with microglia-mediated neuroinflammation remains unknown. In this study, the effects of ISL on inhibiting microglia-mediated neuroinflammation in PD were evaluated in the 1-methyl-4-phenylpyridinium (MPTP)-induced mouse model of PD and in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Our results showed that ISL prevented behavioral deficits and excessive microglial activation in MPTP-treated mice. Moreover, ISL was found to prevent the elevation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and mitigate the phosphorylation of c-Jun N-terminal protein kinase (JNK), protein kinase B (AKT), nuclear factor kappa light-chain enhancer of activated B cells (NFκB), and inhibitor of NFκB protein ɑ (IκBɑ) in the substantia nigra and striatum of MPTP-treated mice and LPS-stimulated BV-2 cells. Meanwhile, in LPS-stimulated BV-2 cells, ISL inhibited the production of inflammatory mediators such as interleukin (IL)-1β, IL-6 and tumor necrosis factor alpha (TNF-α). In addition, the agonist of JNK partly abolished the inhibitory effects of ISL in LPS-treated BV-2 cells. Our results demonstrated that ISL inhibits microglia-mediated neuroinflammation in PD models probably through deactivating JNK/AKT/NFκB signaling pathways. The novel findings suggest the therapeutic potential of ISL for microglia-mediated neuroinflammation in PD.     

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.     

Modified yukmijihwangtang suppresses the production of proinflammatory cytokines in the intravesical hydrochloric acid-induced cystitis rat model via the NF-κB pathway

Yukmijihwangtang (YM), a boiled extract of medicinal plants, has been prescribed for patients with kidney dysfunction in Korea; however, the mechanism underlying its therapeutic effects has not been fully elucidated. This study was conducted to evaluate the beneficial effects on bladder function by using modified YM (M-YM), which included Ulmi radicis cortex in addition to the six traditional medicinal plants in YM. Bladder irritation of the rats was caused by intravesical instillation of HCl. The animals were divided into six groups: sham group, cystitis-injury group with no treatment, cystitis-injury group with prednisolone treatment (5 mg/kg), and cystitis-injury with M-YM treatment (100, 200 or 500 mg/kg groups). Whole bladders were collected at day eight after injury. Samples were analyzed by histological and immunological examinations. An in vitro study was performed to determine whether M-YM extracts inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production and IκB phosphorylation in a human uroepithelial cell line of T24 cells. Administration of M-YM notably improved bladder histological changes, and suppressed IL-6/TNF α production and IκB phosphorylation in a rat model of chronic cystitis. M-YM also inhibited LPS-induced NO production and IκB phosphorylation in T24 cells. This study suggests that administration of M-YM might be an applicable therapeutic traditional medicine for the treatment of interstitial cystitis.     

Anti-inflammatory effect of Sanshuibaihu decoction may be associated with nuclear factor-κB and p38 MAPKα in collagen-induced arthritis in rat

Sanshuibaihu decoction (SSBH) is an anti-arthritic Chinese herbal formula which has been used in the treatment of rheumatoid arthritis (RA) for many years. We herein aimed to confirm its anti-arthritic effect and explore the potential mechanism of action on collagen-induced arthritis (CIA) in rats. CIA was induced by immunizing 50 female Wistar rats with bovine type II collagen. 13 days following the immunization rats with CIA were treated with SSBH (50 mg/kg), leflunomide (LEF) (10 mg/kg) and physiological saline for 30 days, and rats without CIA were left untreated. After the treatment, paw edema was obviously improved in SSBH-treated rats, with the significant difference of arthritis score (F = 6.032, P = 0.006) observed between the three treated groups. In pathological observation, SSBH-treated rats showed a significant improvement of inflammatory infiltration, synovial hyperplasia, cartilage and bone destruction and joint fusion. After the treatment of SSBH, radiological score of knee (t = 11.504, P = 0.000) and ankle joints (t = 9.250, P = 0.000) was decreased significantly. In situ hybridization on joint tissue section indicated only slight synovial hyperblastosis and expression of NF-κB in SSBH-treated rats. Image analysis indicated a significant difference of means of integrated optical density (MIOD) (F = 3.956, P = 0.040) and means of stained area (MSA) (F = 3.867, P = 0.032) of NF-κB between the three treated groups. MIOD and MSA of SSBH-treated group were significantly lower vs control. Enzyme linked immunosorbent assay (ELISA) showed a significant difference (F = 10.167, P = 0.000) of the amount of p-p38 MAPKα in the three treated groups. The detected amount of p-p38 MAPKα in SSBH-treated group was significantly lower vs control. These results show SSBH has an inhibiting effect on CIA, which may be associated with NF-κB and p38 MAPKα.     

The protective effect of smilax glabra extract on advanced glycation end products-induced endothelial dysfunction in HUVECs via RAGE-ERK1/2-NF-κB pathway

Ethnopharmacological relevance

Smilax glabra Roxb. (SGR) is a traditional Chinese herb that has been used in folk for the treatment of diabetic vascular complications. Advanced glycation end products (AGEs)-induced endothelial dysfunction has been thought to be a major cause of diabetic vascular complications. The present study was conducted to investigate the protective effect of SGR extract on AGEs-induced endothelial dysfunction and its underlying mechanisms.

Materials and methods

Human umbilical vein endothelial cells (HUVECs) were exposed to 200 μg/ml AGEs to induce endothelial dysfunction. Acridine orange/ethidium bromide (AO/EB) fluorescence assay and Annexin-V/PI double-staining were performed to determine endothelium apoptosis. Dihydroethidium (DHE) fluorescence probe, SOD and MDA kits were used to evaluate oxidative stress. The effect of SGR extract on AGEs-induced TGF-beta1 expression was determined by immunocytochemistry and western blotting. Attenuations of SGR extract on receptor for AGEs (RAGE) expression, extracellular regulated protein kinases (ERK1/2) activation and NF-κB phosphorylation were determined by immunofluorescence assay and western blotting. The blockade assays for RAGE and ERK1/2 were carried out using a specific RAGE-antibody (RAGE-Ab) or a selective ERK1/2 inhibitor PD98059 in immunofluorescence assay.

Results

The pretreatment of SGR extract (0.125, 0.25 and 0.5 mg crude drug/ml) significantly attenuated AGEs-induced endothelium apoptosis, and down-regulated TGF-beta1 protein expression in HUVECs. It was also well shown that SGR extract could down-regulate dose-dependently ROS over-generation, MDA content, TGF-beta1 expression, ERK1/2 and NF-κB activation whereas increase significantly SOD activity. Furthermore, the AGEs-induced ERK1/2 activation could be attenuated by the blockade of RAGE-Ab (5 μg/ml) while the NF-κB activation was ameliorated by ERK1/2 inhibitor PD98059 (10 μM).

Conclusion

These results indicated that SGR extract could attenuate AGEs-induced endothelial dysfunction via RAGE-ERK1/2-NF-κB pathways. Our findings suggest that SGR extract may be beneficial for attenuating endothelial dysfunction in diabetic vascular complications.     

Acupotomy inhibits aberrant formation of subchondral bone through regulating osteoprotegerin/receptor activator of nuclear factor-κB ligand pathway in rabbits with knee osteoarthritis induced by modified Videman method

OBJECTIVE: To investigate the effects of acupotomy on inhibiting abnormal formation of subchondral bone in rabbits with knee osteoarthritis(KOA). METHODS: A total of 24 New Zealand rabbits were randomly divided into four groups of 6 rabbits each [control, model, electroacupuncture(EA) and acupotomy]. Eighteen KOA model rabbits were established using a modified Videman method. Rabbits in EA and acupotomy groups received the intervention for 3 weeks. Then, the cartilage and subchondral bone unit w...     

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.