Anti-inflammatory effects of magnolol and honokiol are mediated through inhibition of the downstream pathway of MEKK-1 in NF-kappaB activation signaling

Propionibacterium acnes, an anaerobic pathogen, plays an important role in the pathogenesis of acne and seems to initiate the inflammatory process by producing proinflammatory cytokines. In order to demonstrate the anti-inflammatory effects and action mechanisms of magnolol and honokiol, several methods were employed. Through DPPH and SOD activity assays, we found that although both magnolol and honokiol have antioxidant activities, honokiol has relatively stronger antioxidant activities than magnolol {[for DPPH assay, % of DPPH bleaching of magnolol and honokiol (500 microM magnolol: 19.8%; 500 microM honokiol: 67.3%)]; [for SOD assay, SOD activity (200 microM magnolol: 53.4%; 200 microM honokiol: 64.3%)]}. Moreover, the production of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha) induced by P. acnes in THP-1 cells, a human monocytic cell line, was reduced by magnolol and honokiol {[for IL-8 (10 microM magnolol: 42.7% inhibition; 10 microM honokiol: 51.4% inhibition)]; [for TNF-alpha (10 microM magnolol: 20.3% inhibition; 10 microM honokiol: 39.0% inhibition)]}. Cyclooxygenase-2 (Cox-2) activity was also suppressed by them [(15 microM magnolol: 45.8% inhibition), (15 microM honokiol: 66.3% inhibition)]. Using a nuclear factor-kappaB (NF-kappaB) luciferase reporter assay system and Western analysis, we identified that magnolol and honokiol exert their anti-inflammatory effects by inhibiting the NF-kappaB element, which exists in Cox-2, IL-8, and TNF-alpha promoters [(15 microM magnolol: 44.8% inhibition), (15 microM honokiol: 42.3% inhibition)]. Of particular note is that magnolol and honokiol operate downstream of the MEKK-1 molecule. Together with their previously known antibacterial activity against P. acnes and based on these results, we suggest that magnolol and honokiol may be introduced as possible acne-mitigating agents.     

Fibrates suppress chenodeoxycholic acid-induced RANTES expression through inhibition of NF-kappaB activation

We examined agmatine and imidazoline derivatives as putative ligands of trimeric G protein in rat peritoneal mast cells. Agmatine induced a concentration-dependent and pertussis toxin-sensitive secretion of histamine (exocytosis) and arachidonate. Clonidine and idazoxan had no effect. Blockage of Gbetagamma dimers by a specific anti-Gbeta antibody inhibited exocytosis elicited by agmatine and mastoparan. The G protein antagonist [p-Glu(5),D-Trp(7,9,10)]substance P-(5-11) prevented both mastoparan- and agmatine-induced exocytosis when it was allowed to reach its intracellular targets by streptolysin-O permeabilisation. In intact cells, this response was prevented by both the removal of sialic acid residues by neuraminidase and by [D-Pro(4),D-Trp(7,9,10)]substance P-(4-11) acting at the mast cell surface. Exocytosis was restored by permeabilisation of the plasma membrane with streptolysin-O. These results suggest that agmatine might have several molecular targets, exerting its neurotransmitter function at low concentrations (i.e., with high affinity) through membrane receptors and at high concentrations (i.e., with weak affinity) through direct G protein activation.     

21-Methylmelianodiols from Poncirus trifoliata as inhibitors of interleukin-5 bioactivity in Pro-B cells

Interleukin (IL)-5 plays an important role in the progression of allergic inflammation. Here, we have isolated 21alpha-methylmelianodiol and 21beta-methylmelianodiol from Poncirus trifoliata (L.) Raf., a plant of the Rutaceae family, as the inhibitors of IL-5-dependent growth of Y16 pro-B cells by bioassay-guided fractionation. 21alpha-Methylmelianodiol and 21beta-methylmelianodiol inhibited IL-5-dependent growth of Y16 cells in a dose-dependent manner with IC (50) values of 17 microM and 15 microM, respectively. A positive control, tyrphostin AG-490, exhibited an IC (50) value of 23 microM on IL-5 bioactivity. Further, we have documented that 21alpha-methylmelianodiol and 21beta-methylmelianodiol cause G1 arrest of IL-5-induced cell cycle progression of Y16 cells, and also reduce IL-5-dependent survival of the cells by apoptosis. This study could provide a pharmacological potential for P. trifoliata in treatment of IL-5-associated inflammatory disorders.     

Growth inhibition and G1 cell cycle arrest mediated by 25-methoxyhispidol A, a novel triterpenoid, isolated from the fruit of Poncirus trifoliata in human hepatocellular carcinoma cells

Poncirus trifoliata (Rutaceae) extracts have been known to possess anti-allergic, anti-inflammatory and antiviral activities. However, other biological activities, especially, the anticancer potential of extracts of P. trifoliata or its constituents, have not been fully investigated yet. In this study, we have evaluated the antiproliferative effects of a novel triterpenoid, 25-methoxyhispidol A, isolated from the fruit of P. trifoliata against SK-HEP-1 human hepatocellular carcinoma cells. Flow cytometric analysis indicated that 25-methoxyhispidol A arrests the cell cycle in the G1 phase at the earlier time and subsequently induces apoptosis of the cancer cells. Further study revealed that the cell cycle arrest in the G1 phase by 25-methoxyhispidol A correlated well with the inhibition of phosphorylation of the retinoblastoma (Rb) protein, and with the down-regulation of cyclin D1 and cyclin-dependent kinase cdk4 and the induction of cdk inhibitor p21 (WAF1/Cip1) protein. These findings suggest the potential of 25-methoxyhispidol A isolated from the fructus of P. trifoliata as an antitumor agent against human hepatocarcinoma cells by arresting the cell cycle and inducing apoptosis.     

Anti-inflammatory effect of torilidis fructus ethanol extract through inhibition of Src

Context: Torilidis fructus, fruits of Torilis japonica Decadolle (Umbelliferae), is a medicinal herb traditionally used as a pesticide, an astrictive, or a medicine for various inflammatory diseases.

Objectives: Due to the lack of pharmacological studies on this herbal medicine, we explored the inhibitory activity of torilidis fructus on the macrophage-mediated inflammatory response using its ethanol extract (Tf-EE).

Material and methods: The Griess assay and prostaglandin (PGE₂) ELISA assay were conducted with Tf-EE (0-75?µg/mL) and LPS (1?µg/mL) treated RAW264.7 cells in cultured media. Tf-EE pretreated RAW264.7 cells were incubated with LPS for 6?h and semi-quantitative PCR was performed. Reporter gene assays, overexpression of target enzymes and immunoblotting were performed on macrophages to determine the molecular targets of Tf-EE.

Results: Tf-EE markedly suppressed the inflammatory response of macrophages, such as lipopolysaccharide (LPS)-induced nitric oxide (NO) and PGE₂ production with IC₅₀ values of 35.66 and 62.47?µg/mL, respectively. It was also found that Tf-EE reduced the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 by 80%. Nuclear translocation and activation of nuclear factor (NF)-κB (p65 and p50) were declined by 60% and 30% respectively, and their regulatory events including the phosphorylation of AKT, IκBα, Src, and the formation of complexes between Src and p-p85 were also recognized to be diminished.

Conclusions: The signalling events managed by Src and p85 complex seemed to be critically involved in Tf-EE-mediated anti-inflammatory response. This might suggest that Tf-EE exhibited anti-inflammatory effects through Src-targeted inhibition of NF-κB.     

Rubiae Radix suppresses the activation of mast cells through the inhibition of Syk kinase for anti-allergic activity

The effect of extracts from various Oriental medicinal herbs on mast-cell-mediated allergic reactions was investigated in this study. Of these extracts, the medicinal herb Rubiae Radix exhibited the most potent activity in the cells, with an IC50 value (concentration necessary to obtain 50% inhibition of the response) of approximately 35 +/- 2.1 microg mL(-1), and its inhibition of compound-48/80-induced systemic anaphylaxis by 48.6 +/- 8.5% at 300 mg kg(-1) in mice. It also inhibited the expression of the pro-inflammatory mediator tumour necrosis factor-alpha (TNF-alpha). As for its mechanism of action, Rubiae Radix suppressed the activating phosphorylation of Syk, a key enzyme in mast-cell signalling processes, and that of Akt in a dose-dependent manner. It also inhibited the MAP kinase ERK1/2, which is critical for the production of inflammatory cytokines in mast cells, as indicated by the suppression of the activating phosphorylation of ERK1/2. These results suggest that Rubiae Radix suppresses the activation of mast cells through the inhibition of Syk for anti-allergic activity.     

Anti-inflammatory potential of Antrodia Camphorata through inhibition of iNOS, COX-2 and cytokines via the NF-kappaB pathway

Antrodia camphorata (A. camphorata), well known in Taiwan as a traditional Chinese medicine, has been shown to exhibit antioxidant and anticancer effects. In the present study, therefore, we have examined the effects of the fermented culture broth of A. camphorata (25-100 microg/ml) in terms of lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production, and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in RAW 264.7 macrophages. Our results indicate concentration-dependent A. camphorata inhibition of LPS-induced NO and PGE2 production, without appreciable cytotoxicity on the RAW 264.7 cells. A. camphorata also attenuates the production of LPS-induced tumor necrosis factor (TNF-alpha) and interleukin (IL)-1beta. Furthermore, A. camphorata blocks the IkappaB-alpha degradation induced by LPS. These results indicate that A. camphorata inhibits LPS induction of cytokine, iNOS and COX-2 expression by blocking NF-kappaB activation. Therefore, we report the first confirmation of the anti-inflammatory potential of this traditionally employed herbal medicine in vitro.     

正交试验优选枸橘的提取工艺

目的：对枸橘的提取工艺进行优选。方法：以乙醇浓度、提取时间、溶剂倍数为考察因素,枸橘苷、欧前胡素的转移率为评价指标,运用正交试验优化提取的最佳工艺条件,同时用方差分析对其进行评价。结果：枸橘最佳提取工艺条件为70%乙醇溶液提取两次,第1次6倍量,提取60 min,第2次4倍量,提取45 min。结论：优选出的提取工艺简单、科学、合理,为枸橘的开发利用奠定基础。     

Humic acid suppresses the LPS-induced expression of cell-surface adhesion proteins through the inhibition of NF-kappaB activation

Humic acid (HA), a potential toxin when penetrating the drinking well water of blackfoot disease-endemic areas in Taiwan, has been implicated as one of the etiological factors of this disease. In this study, we investigated the effects of HA on the expression of human vascular endothelial-leukocyte adhesion molecules and the activation of nuclear factor kappa B (NF-kappaB) in cultured human umbilical vein endothelial cells (HUVECs). The expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin was monitored by flow cytometry. Pretreatment of HUVECs with HA inhibited the lipopolysaccharide (LPS)-induced expression of these three adhesion molecules in a dose- and time-dependent manner. Since NF-kappaB can regulate the expression of these adhesion molecules, NF-kappaB activation was assessed by electrophoretic mobility shift assay (EMSA). Our results reveal that the activation of NF-kappaB by LPS is suppressed by HA in a dose- and time-dependent manner. Furthermore, HA reduces NF-kappaB binding to DNA slightly, but completely inhibits the degradation of IkappaBalpha at a concentration of 100 microg/ml. Thus, all our data demonstrate that HA can inhibit the LPS-induced expression of adhesion molecules through the inhibition of NF-kappaB activation. HA may also suppress the immune or inflammatory reaction of HUVECs responsible for endotoxin, which could be one possible explanation for the causes of the infection and inflammation observed for patients with blackfoot disease. Our results also suggest that immune or inflammatory disturbance occurs for patients with blackfoot disease and that NF-kappaB may be a critical molecule in the pathogenesis of this disease.     

Anti-inflammatory effect of novel andrographolide derivatives through inhibition of NO and PGE2 production

Andrographolide (1) is a major diterpene lactone exhibiting anti-inflammatory effects and is found in the plant Andrographis paniculata (Burm. f) Nees, which is widely used in Traditional Chinese Medicine. Synthesis of more effective drugs from andrographolide is very interesting and can prove to be highly useful. In this study, we investigated the anti-inflammatory effects of andrographolide and its derivatives (compounds 2–6) through dimethylbenzene-induced ear edema in mice. Substances under study were administrated intragastrically and the structure–activity relationship was analyzed. Results showed that compounds 5 and 6 significantly inhibited ear edema compared with compound 1 (p < 0.05), indicating that the introduction of p-Chlorobenzylidene to C-15 of compound 2 enhances the anti-inflammatory effect. Moreover, compound 6 exhibited the strongest anti-inflammatory effect against ear edema in mice (79.4%; 1.35 mmol/kg, ig) and paw edema in rats (50.4%; 0.90 mmol/kg, ig). In addition, compound 6 significantly (p < 0.05) inhibited granuloma formation and reduced the increase in vascular permeability induced by peritoneal injection of 0.6% acetic acid solution in mice. Findings indicate that compound 6 exerts its enhanced anti-inflammatory effects by decreasing serum iNOS activity, NO production, and PGE₂ production.     

Apomorphine-induced inhibition of histamine release in rat peritoneal mast cells.

The apomorphine-induced inhibition of histamine release in rat peritoneal mast cells was studied by means of secretagogues stimulating different pathways of mast cell activation. Apomorphine inhibited the mast cell response to all releasing agents (lysophosphatidylserine plus nerve growth factor, compound 48/80, substance P, ATP, tetradecanoylphorbolacetate, melittin). The IC50 ranged from 4 microM to 24 microM at concentrations of secretagogues releasing 30-50% of mast cell histamine. However, the potency of the drug decreased at higher secretagogue concentrations. Mast cells, pretreated with apomorphine and washed, released little histamine upon stimulation. The secretory response could be partially restored on increasing the concentration of secretagogues. The results suggest that apomorphine affects a regulatory step controlling the terminal sequence of mast cell secretory activity. As indicated by the reduced potency of the drug, the control by the apomorphine-sensitive reaction loses efficiency under conditions of massive histamine release.     

Anti-inflammatory effect of berkeleyacetal C through the inhibition of interleukin-1 receptor-associated kinase-4 activity

Berkeleyacetal C (BAC) isolated from Penicillium sp. which had isolated from a soil sample collected in Fukushima, inhibited NO production and induction of iNOS protein in RAW264.7 cells stimulated by the Toll-like receptor (TLR) 2 ligand, peptidoglycan (PGN) or TLR4 ligand, lipopolysaccharide (LPS). The other inflammatory mediator production by these stimulators was also suppressed by BAC in a concentration-dependent manner. BAC inhibited LPS- or PGN-activated nuclear translocation of nuclear factor (NF)-κB and MyD88-dependent signaling molecules. However, it showed no effect on LPS-induced nuclear translocation of interferon regulatory factor (IRF)-3, a MyD88-independent signaling molecule. To clarify the mechanistic basis for BAC ability to inhibit translocation of NF-κB and activated MyD88-dependent signaling molecules, we examined interleukin-1 receptor-associated kinase (IRAK)-4, existing to the most upstream on MyD88-dependent signaling molecules, in vitro kinase assay. BAC suppressed IRAK-4 kinase activity in a concentration-dependent manner. These findings suggest that BAC inhibits LPS- and PGN- induced NO production and iNOS expression by decreasing the level of the translocating of NF-κB in nuclear through inhibiting the kinase activity of IRAK-4 in inflammatory cells.     

Anti-inflammatory effect of propolis through inhibition of nitric oxide production on carrageenin-induced mouse paw edema

The anti-inflammatory effect of propolis was compared with that of diclofenac, a non-steroidal anti-inflammatory drug, and L-N(G)-nitro arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, using carrageenin-induced mouse paw edema. When administered 10 min prior to carrageenin injection, propolis (1 : 1000, 1 : 100, p.o.), diclofenac (12.5, 50 mg/kg, p.o.) and L-NAME (10, 100 mg/kg, s.c.) showed a significant anti-inflammatory effect. The anti-inflammatory effects of propolis and L-NAME were significantly inhibited by L-arginine, a precursor of nitric oxide, but not by D-arginine. In contrast, the anti-inflammatory effect produced by diclofenac was not inhibited by either D-arginine or L-arginine. These results indicate that the anti-inflammatory effect of propolis on mouse paw edema acts via the inhibition of nitric oxide production, similar to that of L-NAME but not diclofenac.     

Anti-Helicobacter pylori activity of the metabolites of poncirin from Poncirus trifoliata by human intestinal bacteria.

Poncirin was isolated from water extract of the fruits of Poncirus trifoliata and metabolized by human intestinal bacteria. The inhibitory effect of poncirin and its metabolites by these bacteria on the growth of Helicobacter pylori (HP) was investigated. Among them, ponciretin (5,7-dihydroxy-4'-methoxyflavanone), the main metabolite most potently inhibited the growth of HP, with a minimum inhibitory concentration (MIC) of 10-20 microg/ml. However, poncirin and its metabolites except ponciretin did not inhibit the growth of HP, nor did they inhibit HP urease.     

Motorcycle exhaust particles induce IL-8 production through NF-kappaB activation in human airway epithelial cells

Motorcycle exhaust particles (MEP) are among the major air pollutants, especially in urban area of Taiwan. In our previous study, data showed that MEP induce proinflammatory and proallergic response profiles in BALB/c mice. Effects of MEP on interleukin (IL)-8 production in A549 human airway epithelial cells were further investigated in this study. It was found that MEP enhanced IL-8 protein and mRNA expression in human epithelial cells. Pretreatment with an NF-kappaB inhibitor (1 mM PDTC), extracellular signal-regulated kinase (ERK) inhibitor (50 microM PD98059), JNK inhibitor (25 microM SP600125), p38 inhibitor (2 microM SB203580), and three antioxidants (500 U/ml superoxide dismutase [SOD], 50 microM vitamin E, 10 mMN-acetylcysteine [NAC]) attenuated the MEP-induced increase in IL-8 production. Through further, direct detection of nuclear factor (NF)-kappaB activation in epithelial cells using immunoblotting of nuclear p65 and NF-kappaB reporter assay, data showed that MEP induced nuclear translocation of p65 and enhancement of NF-kappaB luciferase gene expression. MEP also induced activation of ERK, JNK, and p38 signaling pathways and produced an increase of oxidative stress in A549 cells. By using mitogen-activated protein kinase (MAPK) inhibitors and antioxidant, it was demonstrated that ERK inhibitor, JNK inhibitor, and antioxidants but not p38 inhibitor attenuated the MEP-induced increase in NF-kappaB reporter activity. In conclusion, evidence shows that filter-trapped particles emitted from unleaded gasoline-fueled, two-stroke motorcycle engines induce an increase in IL-8 production by activation of NF-kappaB in human airway epithelial cells.