leukotriene-b4 and Hemolysis

Silsosangami is a dried decoctum of a mixture of seven Korean herbal medicine, which is consisted of seven herbs (indicated as concentrations) of Typhae Pollen, Pteropi Faeces, Paeoniae Radicis rubra, Cnidii Rhizoma, Persicae Semen, Carthami Flos and Curcumae Tuber. In the present study, the effects of Silsosangami water extract (SSG) on hemolysis in human blood were studied. Using an in vitro system, only Curcumae Tuber, Persicae Semen and Paeoniae Radicis rubra had the strongest effects on hemolysis; Typhae Pollen and Pteropi Faeces had the slight effects; and Cnidii Rhizoma and Carthami Flos had no effect. On the other hand, the SSG inhibited neutrophil functions, including degranulation, superoxide generation, and leukotriene B4 production, without any effect on 5-lipoxygenase activity. This SSG reduced nitric oxide (NO) and prostaglanin E2 (PGE2) production in mouse peritoneal macrophages stimulated with lipopolysaccharide, without the influence on the activity of inducible NO synthase (iNOS), cyclooxygenase COX-2 and COX-1 being observed. SSG significantly reduced mouse paw oedema induced by carrageenan. Western blot analysis showed that SSG reduced the expression of iNOS and COX-2. These results suggested that SSG might be used as a novel antithrombotic therapeutic agents in post-myocardial infarction and also, indicated that SSG exerts anti-inflammatory effects related to the inhibition of neutrophil functions and of NO and PGE2 production, which could be due to a decreased expression of iNOS and COX-2.

Topics: Animals; Anti-Inflammatory Agents; Carrageenan; Cyclooxygenase 2; Dinoprostone; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Edema; Erythrocytes; Gene Expression; Hemolysis; Herbal Medicine; Humans; Korea; Leukotriene B4; Macrophages, Peritoneal; Medicine, East Asian Traditional; Membrane Proteins; Mice; Neutrophils; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Pancreatic Elastase; Phytotherapy; Plant Extracts; Prostaglandin-Endoperoxide Synthases; Thromboxane B2

Pseudomonas aeruginosa phospholipase C (PLC) is a critical component in the pathogenesis of severe P. aeruginosa infections. However, P. aeruginosa can produce a hemolytic (PLC-H) as well as a nonhemolytic (PLC-N) variant, both having a MW of about 77 kD. In the past, studies did not distinguish between both types of PLC with regard to the induction of inflammatory mediators from human cells.. We compared the ability of P. aeruginosa PLC-H and PLC-N to generate leukotriene B4 (by HPLC) and oxygen (O2-) metabolites (luminol-enhanced chemiluminescence), and to release beta-glucuronidase and histamine (fluorophotometry from human granulocytes. Therefore, human neutrophilic granulocytes (PMN; 1 x 10(7)) or human peripheral blood mononuclear cells (5 x 10(6)) were treated with purified P. aeruginosa PLC-H (up to 10 units) as well as culture supernatants (cutoff: MW > 50,000) of P. aeruginosa PAOl capable of producing both PLC-H and PLC-N, and PAOl mutant strains deficient in the production of either or both phospholipases. Controls were PLC-H from Clostridium perfringens and PLC-N from Bacillus cereus.. PLC-H-containing P. aeruginosa culture supernatant, purified P. aeruginosa PLC-H as well as PLC-H from P. perfringens activated human leukocytes for a significant (p < 0.05) increase in inflammatory mediator release. In this regard, purified PLC-H (10 units) from P. aeruginosa activated human PMN for a significant increase in the generation of oxygen metabolites (30 +/- 5.4 x 10(3) cpm) and in leukotriene B4 (6.1 +/- 2.0 ng), in the release of beta-glucuronidase (15.8 +/- 1.1%) and of histamine (25.8 +/- 6.2%) as compared to the corresponding control values (3 +/- 1 x 10(3) cpm; 0.2 +/- 0.1 ng; 5.1 +/- 1.0%, 5.1 +/- 1.5%). Culture supernatants containing no PLC or only PLC-N, as well as PLC-N from B. cereus, failed to activate or only slightly stimulated human granulocytes for inflammatory mediator release.. The data thus provide evidence that P. aeruginosa PLC-H can be a potent inducer of inflammatory mediator release, at least in vitro. Our results therefore contribute to the understanding of the pathophysiological role of P. aeruginosa PLCs.

Topics: Bacillus cereus; Clostridium perfringens; Glucuronidase; Granulocytes; Hemolysis; Histamine Release; Humans; In Vitro Techniques; Inflammation Mediators; Leukotriene B4; Luminescent Measurements; Molecular Weight; Pseudomonas aeruginosa; Pseudomonas Infections; Reactive Oxygen Species; Type C Phospholipases; Virulence

The generation of the 5-lipoxygenase product, leukotriene B4 (LTB4) by human mononuclear phagocytes (monocytes) following incubation with 25 different uropathogenic strains of Escherichia coli correlated with the haemolytic activity of the strains (r = 0.572, P less than 0.01). LTB4 generation by human neutrophils (PMN), however, was unrelated to this haemolytic potential (r = 0.164). In contrast, both prelabelled monocytes and PMN were stimulated by haemolytic strains of E. coli and by haemolytic culture supernatants to release significant amounts of [3H]arachidonic acid. There was a significant correlation between haemolytic activity and [3H]arachidonic acid release generated by individual strains from monocytes (r = 0.804, P less than 0.001) and PMN (r = 0.888, P less than 0.001). In addition, nonhaemolytic strains but not their culture supernatants were capable of causing slow release of both [3H]arachidonic acid and LTB4 from PMN and mononuclear cells. These results suggest that both the possession of haemolytic activity, and the direct interaction of bacteria with the leukocyte surface are mechanisms by which uropathogenic strains of E. coli may cause the release and metabolism of arachidonic acid. In addition, there was synergistic augmentation by nonhaemolytic bacteria of the PMN LTB4 response to haemolytic culture supernatants or to low doses of the calcium ionophore A23187. These results support an ionophore-like mechanism for the activation of the cell by haemolysin. LTB4 generation by PMN incubated with haemolytic supernatants was also augmented by particulate zymosan in a manner dependent on the dose of zymosan, suggesting that the direct interaction of E. coli with PMN may involve an activation mechanism similar to that for zymosan. These results demonstrate differing responses of peripheral mononuclear cells and PMN from the same donors to identical strains of E. coli and suggest that the generation of the potent chemotactic agent LTB4 in response to E. coli infection in vivo need not depend solely on the elaboration of cytotoxic haemolysins by individual strains.

Topics: Adenosine Triphosphate; Arachidonic Acid; Arachidonic Acids; Escherichia coli; Hemolysis; Humans; L-Lactate Dehydrogenase; Leukotriene B4; Lymphocyte Activation; Monocytes; Neutrophils