leukotriene-b4 and baicalein

Huang-Lian-Jie-Du-Tang (HLJDT), a famous traditional Chinese prescription with wide anti-inflammatory applications, is an aqueous extract of four herbal materials: Rhizoma coptidis, Radix scutellariae, Cortex phellodendri, and Fructus gardeniae. Its effects on the cyclooxygenase (COX)-2 and 5-lipoxygenase (5-LOX) pathways are thought to be responsible for its anti-inflammatory activity. However, our previous work found that the inhibitory effects of HLJDT act on the 5-LOX pathway but not on the COX pathway. The possibility that HLJDT inhibits COX-2- or 5-LOX-catalyzed eicosanoid generation by downregulating enzyme expression requires further investigation.. To observe the effects of HLJDT and its four major components (baicalin, baicalein, berberine and geniposide) on COX-2- or 5-LOX-catalyzed eicosanoid generation and to distinguish the effects of HLJDT on enzyme activity from those on enzyme expression.. The topical anti-inflammatory activities and inhibition of eicosanoid formation of HLJDT and its components were observed in an arachidonic acid (AA)-induced mouse ear edema model. Macrophage-based systems were established to observe the effects of the drugs on enzyme activity and enzyme expression of COX-2 and 5-LOX. Further experiments were carried out to confirm these effects at the mRNA and protein levels.. Topical treatment of HLJDT significantly inhibited AA-induced mouse ear edema and reduced PGE(2) and LTB(4) release in the edematous ears. Baicalein, geniposide, and berberine also ameliorated the symptoms and suppressed eicosanoid generation with varying efficacies. Cell-based assays showed that HLJDT and baicalein inhibited the PGE(2) levels by decreasing COX-2 enzyme expression without affecting COX-2 enzyme activity in RAW 246.7 murine macrophages. The other experiments on rat peritoneal macrophages indicated that HLJDT and baicalein exerted significant inhibition on LTB(4) production by decreasing 5-LOX enzyme activity. The real-time PCR and western blotting data demonstrated that HLJDT and baicalein reduced COX-2 expression at the mRNA and protein levels, whereas no inhibition on 5-LOX expression was observed.. HLJDT can suppress eicosanoid generation via both the COX and LOX pathways, which definitely contributes to its topical anti-inflammatory activity. We have confirmed that its dual inhibition on the COX and LOX pathways mainly result from the downregulation of COX-2 expression and direct inhibition of 5-LOX activity, respectively. Baicalein worked as a potent active component in most of the tests. These findings about the different inhibitory effects of HLJDT on COX-2 and 5-LOX help to better understand the mechanism of HLJDT and promote safer applications of drug.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Berberine; Cell Line; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Drugs, Chinese Herbal; Edema; Flavanones; Flavonoids; Iridoids; Leukotriene B4; Lipoxygenase Inhibitors; Male; Mice; Mice, Inbred ICR; Rats; Rats, Sprague-Dawley; RNA, Messenger

Neutrophil apoptosis is an important physiological process in the resolution of pulmonary inflammation. Previous studies have shown that eicosapentaenoic acid (EPA; 20:5n-3) increases the rate of apoptosis in a concentration- and time-dependent manner in HL-60 cells. However, it is not known if the EPA-induced apoptosis involves the lipoxygenase (LO) and cyclooxygenase (COX) enzymes or the downstream metabolic products of these enzymes. Thus, the objective of this study was to determine the effects of inhibitors LO and COX enzymes on apoptosis, viability, and necrosis in EPA-treated HL-60 cells.. Cells were incubated with 50 mum EPA in the presence of an enzyme inhibitor (1-10 microm) for 12 h. Compounds were used to inhibit COX 1 and 2 (ibuprofen), 5-, 12-, 15-LO (NDGA), 12-LO (baicalein), 5-LO (AA-861), and 5-LO activating protein (MK-886). Eicosanoid (0.001-1.0 mum) add-back experiments were also conducted; LTB(4) and 5-HETE with 5-LO inhibition and 12-HETE with 12-LO inhibition. Flow cytometry was used to assess apoptosis.. Inhibition of COX 1 and 2 had no effect on apoptosis. Inhibition of 5-LO and 12-LO significantly increased apoptosis in EPA-treated HL-60 cells. Addition of LTB(4) reduced apoptosis to levels significantly lower than in HL-60 cells treated with EPA alone; 5-HETE and 12-HETE also lowered apoptosis to control levels.. These data indicate that inhibition of LO, particularly 5-LO, increased apoptosis in EPA-treated HL-60 cells. Furthermore, this study demonstrated that the products of the LO enzymes, particularly LTB(4), are critical in the regulation of apoptosis in EPA-treated HL-60 cells.

Topics: Apoptosis; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Cyclooxygenase Inhibitors; Eicosapentaenoic Acid; Enzyme Inhibitors; Fatty Acids, Unsaturated; Flavanones; Guaiacol; HL-60 Cells; Humans; Hydroxyeicosatetraenoic Acids; Ibuprofen; Indoles; Leukotriene B4; Lignans; Lipoxygenase Inhibitors; Neutrophils; Respiratory Distress Syndrome

This study investigated role of spinal lipoxygenase metabolites in induction of hyperalgesia and development of opioid analgesic tolerance. In the rat, nociception was measured using formalin and tail-flick tests. Intrathecal administration of leukotriene receptor agonist (LTB4) augmented the second phase of the formalin response and marginally increased sensitivity to acute thermal stimulation in the tail-flick test, responses suppressed by 6-(6-(3R-hydroxy-1E,5Z-undecadien-1-yl)-2-pyridinyl)-1,5S-hexanediol (U75302), a leukotriene BLT receptor antagonist. Treatment with 15-hydroxyperoxyeicosatetranoic acid (HPETE) increased phase II formalin activity, but had no effect on tail-flick responses. 12-HPETE failed to produce an effect in either nociceptive test. In the second part of this study, chronic spinal morphine for 5 days produced progressive decline in morphine antinociception and loss in analgesic potency. These effects were attenuated by co-administration of morphine with selective and nonselective lipoxygenase inhibitors. These results suggest involvement of lipoxygenase metabolites in both pain modulation and induction of opioid tolerance at the spinal level.

Topics: Analgesics, Opioid; Animals; Benzoquinones; Dose-Response Relationship, Drug; Drug Tolerance; Flavanones; Formaldehyde; Hindlimb; Hyperalgesia; Injections, Spinal; Leukotriene B4; Leukotrienes; Lipid Peroxides; Lipoxygenase; Lipoxygenase Inhibitors; Male; Masoprocol; Morphine; Pain; Pain Measurement; Rats; Rats, Sprague-Dawley; Spinal Cord; Time Factors

Diets high in fat are associated with an increased risk of prostate cancer, although the molecular mechanism is still unknown. We have previously reported that arachidonic acid, an omega-6 fatty acid common in the Western diet, stimulates proliferation of prostate cancer cells through production of the 5-lipoxygenase metabolite, 5-HETE (5-hydroxyeicosatetraenoic acid). We now show that 5-HETE is also a potent survival factor for human prostate cancer cells. These cells constitutively produce 5-HETE in serum-free medium with no added stimulus. Exogenous arachidonate markedly increases the production of 5-HETE. Inhibition of 5-lipoxygenase by MK886 completely blocks 5-HETE production and induces massive apoptosis in both hormone-responsive (LNCaP) and -nonresponsive (PC3) human prostate cancer cells. This cell death is very rapid: cells treated with MK886 showed mitochondrial permeability transition between 30 and 60 min, externalization of phosphatidylserine within 2 hr, and degradation of DNA to nucleosomal subunits beginning within 2-4 hr posttreatment. Cell death was effectively blocked by the thiol antioxidant, N-acetyl-L-cysteine, but not by androgen, a powerful survival factor for prostate cancer cells. Apoptosis was specific for 5-lipoxygenase-programmed cell death was not observed with inhibitors of 12-lipoxygenase, cyclooxygenase, or cytochrome P450 pathways of arachidonic acid metabolism. Exogenous 5-HETE protects these cells from apoptosis induced by 5-lipoxygenase inhibitors, confirming a critical role of 5-lipoxygenase activity in the survival of these cells. These findings provide a possible molecular mechanism by which dietary fat may influence the progression of prostate cancer.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Antineoplastic Agents; Apoptosis; Cell Membrane; Dietary Fats; Drug Combinations; Drugs, Chinese Herbal; Flavanones; Flavonoids; Glycyrrhiza; Humans; Hydroxyeicosatetraenoic Acids; Ibuprofen; Indoles; Leukotriene B4; Lipoxygenase Inhibitors; Male; Mitochondria; Models, Biological; Nucleosomes; Oxidative Stress; Paeonia; Permeability; Phosphatidylserines; Prostatic Neoplasms; Tumor Cells, Cultured

Our previous study showed the inhibitory effect of Qing-Fei-Tang (Q.F.T.) and baicalein on the leukotriene (LT)B4 synthesis of human alveolar macrophages. It has recently been demonstrated that LTs support various cell growth, and basophil and its precursor numbers increase in atopic patients. Therefore, we examined the effect of anti-allergic drugs, including Q.F.T., Xiao-Qing-Long-Tang (X.Q.L.T.), Chai-Pu-Tang (C.P.T.), baicalein and ketotifen which have been used for treatment of bronchial asthma, on human basophil growth in vitro using cord blood mononuclear cells as a basophil precursor source and conditioned medium of T cell leukemia cell line Mo as a growth factor. Two-week cultured basophil numbers identified by alcian blue-safranin staining and those histamine contents assayed fluorometrically were inhibited by Q.F.T. (1.0 mg/ml), X.Q.L.T. (0.01-1.0 mg/ml), C.P.T. (0.01-1.0 mg/ml), baicalein (1-100 microM) or ketotifen (1-100 microM) in a dose-dependent manner while low dose (0.01-0.1 mg/ml) of Q.F.T. showed an enhancing effect on the basophil growth and the histamine content. However, LTB4 or LTC4 failed in restoring the basophil growth reduced by 1 mg/ml of C.P.T. or 100 microM of ketotifen. These results suggest that anti-allergic drugs may modulate basophil growth and differentiation in vitro and/or in vivo and therefore be useful and reasonable for controlling allergic diseases including bronchial asthma.

Topics: Basophils; Dose-Response Relationship, Drug; Drug Interactions; Drugs, Chinese Herbal; Flavanones; Flavonoids; Histamine; Histamine H1 Antagonists; Humans; Ketotifen; Leukotriene B4; SRS-A

A traditional Chinese remedy, Qing-Fei-Tang (Seihai-to, T90), has been used for treatment of chronic respiratory diseases with long-lasting cough and sputum, e.g. chronic bronchitis. We examined the effect of T90 and its main component flavonoid, baicalein, on the lucigenin-dependent chemiluminescence (CL) and leukotriene B4 (LTB4) synthesis of human alveolar macrophages (AM). AM were obtained by bronchoalveolar lavage from patients with various respiratory diseases, including sarcoidosis, idiopathic pulmonary fibrosis, bronchial asthma, chronic bronchitis and lung cancer. CL were observed by stimulating 1 x 10(5) AM with phorbol myristate acetate in the presence of lucigenin. LTB4 were generated by incubating 1 x 10(6)/ml AM with Ca ionophore A23187 for 30 min and determined by reverse phase high performance liquid chromatography and radioimmunoassay. T90 (0.2-2.0 mg/ml) and baicalein (0.1-100 microM) inhibited both CL and LTB4 production of AM in a dose-dependent fashion. These inhibitory effects were not due to cytotoxic effects of the procedure because neither 2 mg/ml T90 nor 100 M baicalein affected the viability of AM nor lactate dehydrogenase release from AM. These results suggest that T90 exerts its effect on inflammatory lung diseases through the anti-inflammatory action, i.e. inhibiting the oxidative and arachidonate metabolism of local inflammatory lung cells.

Topics: Acridines; Cells, Cultured; Drugs, Chinese Herbal; Flavanones; Flavonoids; Humans; Leukotriene B4; Luminescent Measurements; Macrophages; Pulmonary Alveoli; Radioimmunoassay