Effect of the lipidosterolic extract of Serenoa repens (Permixon) and its major components on basic fibroblast growth factor-induced proliferation of cultures of human prostate biopsies.

OBJECTIVE: To assess the effect of the lipidosterolic extract of Serenoa repens (LSESr) on in vitro cell proliferation in biopsies of human prostate MATERIAL AND METHODS: Cell proliferation was assessed by incorporation of [3H]thymidine followed by historadiography. RESULTS: Basic fibroblast growth factor (b-FGF) induced a considerable increase in human prostate cell proliferation (from +100 to +250%); the glandular epithelium was mainly affected, minimal labeling being recorded in the other regions of the prostate. Similar results were observed with epidermal growth factor (EGF), although the increase in cell proliferation was not recorded in some cases. Lovastatin, an inhibitor of hydroxymethylglutaryl coenzyme A, antagonized both the basal proliferation and the growth factor-stimulated proliferation of human prostate epithelium (EGF, mean inhibition approximately 80-95%; b-FGF, mean inhibition approximately 40-90%). Geraniol, a precursor of both farnesyl pyrophosphate and geranylgeranyl pyrophosphate, and farnesol, the precursor of farnesyl pyrophosphate, increased cell proliferation only in some prostate specimens, this effect being antagonized by lovastatin. LSESr did not affect basal prostate cell proliferation, with the exception of two prostate specimens in which a significant inhibition of basal proliferation was observed with the highest concentration of LSESr (30 micrograms/ ml). In contrast, LSESr inhibited b-FGF-induced proliferation of human prostate cell cultures; this effect was significant for the highest concentration of LSESr (30 micrograms/ml). In some prostate samples, a similar inhibition was also noted with lower concentrations. Unsaturated fatty acids (UFA), in the range 1-30 ng/ml), did not affect the basal prostate cell proliferation, only a slight increase in cell proliferation was noted in 1 prostate specimen. UFA (1, 10 or 30 micrograms/ml) markedly inhibited the b-FGF-induced cell proliferation down to the basal value. Lupenone, hexacosanol and the unsaponified fraction of LSESr markedly inhibited the b-FGF-induced cell proliferation, whereas a minimal effect on basal cell proliferation was noted. CONCLUSIONS: Despite the large variability in the response of the prostate samples to b-FGF, these results indicate that LSESr and its components affect the proliferative response of prostate cells to b-FGF more than their basal proliferation.

Effect of the lipidic lipidosterolic extract of Serenoa repens (Permixon) on the ionophore A23187-stimulated production of leukotriene B4 (LTB4) from human polymorphonuclear neutrophils.

Although the lipidic extract of Serenoa repens (LESSr, Permixon, Sereprostat) is widely used in patients suffering from benign prostatic hypertrophy (BPH), its mechanism of action is not fully elucidated. It has been demonstrated that infiltration of the prostate by inflammatory cells is one of the aetiologic factors involved in the development of BPH. These inflammatory cell types, such as polymorphonuclear neutrophils (PMNs), produce chemotactic mediators and contribute to the development of the disease. Among the chemotactic factors generated by inflammatory cell types, the derivatives of arachidonic acid have been extensively studied. For instance, leukotriene (LT) B4 is one of the most potent chemotactic factors for PMNs and also exhibits a wide range of biological activities. In order to investigate the potential action of LESSr on arachidonate metabolism, and particularly on the synthesis of LTB4, the effect of this extract on the in vitro synthesis of LT by human PMNs stimulated with the calcium ionophore A23187 was investigated. LESSr significantly inhibits the production of 5-lipoxygenase metabolites (5-HETE, 20-COOH LTB4, LTB4 and 20-OH LTB4) at concentrations as low as 5 microg/ml. Such an effect of LESSr was also observed in the presence of exogenous arachidonic acid (20 microg/ml) and when f-MLP was used as the agonist, suggesting that inhibition of LTB4 production by the extract was unrelated to phospholipase A2 blockade and independent of the stimulating agent. The capability of LESSr to antagonize 5-lipoxygenase metabolites production may contribute, at least partly, to the understanding of its therapeutic activity on the inflammatory component of BPH.

Neutrophils accentuate renal cold ischemia-reperfusion injury. Dose-dependent protective effect of a platelet-activating factor receptor antagonist.

This study was undertaken to evaluate whether the renal damage induced by cold ischemia-reperfusion was worsened by neutrophils (PMN), and if blockade of platelet-activating factor (PAF) could effectively decrease this injury. After flushing with EuroCollins, 85 kidneys from Sprague-Dawley rats underwent either no cold ischemia or a 4-h cold ischemia, and then were reperfused for 75 min at 37 degrees C and 100 mm Hg in an isolated perfusion circuit. Reperfusion was performed with a Krebs-Henseleit solution containing 4.5% albumin, with and without human PMN (7.5 x 10(5) cells/ml) and with and without addition of a PAF receptor antagonist (BN 52021). Hemodynamic and functional parameters were continuously assessed during reperfusion. At end of the study, PAF production was evaluated. Presence of PMN during reperfusion of nonischemic kidneys produced no alteration of functional parameters or PAF production. After 4-h cold ischemia, the presence of PMN during reperfusion produced a significant worsening of plasma flow rate, glomerular filtration rate and sodium reabsorption in comparison with kidneys reperfused without PMN. Also, higher production of PAF was observed in the kidneys reperfused with PMN than in the kidneys reperfused without PMN. After 4-h cold ischemia, addition of BN 52021 during reperfusion in the presence of PMN significantly increased the plasma flow rate, glomerular filtration rate and sodium reabsorption in comparison with kidneys reperfused without this PAF antagonist. This effect was dose dependent. After 4-h cold ischemia, addition of BN 52021 during reperfusion in the absence of PMN produced no significant effect on functional parameters in comparison with kidneys reperfused without this PAF antagonist. These results indicate that PMN contribute to renal cold ischemia-reperfusion injury evaluated in the isolated perfused kidney. Treatment with a PAF receptor antagonist attenuated this injury in a dose-dependent manner, which suggests that it is mediated by PAF.

Effect of Serenoa repens extract (Permixon) on estradiol/testosterone-induced experimental prostate enlargement in the rat.

The effect of the lipidosterolic extract of Serenoa repens (LSESR) on experimental prostate enlargement was investigated in three groups of rats: shams treated with LSESR (sham rats), castrated animals treated with estradiol and testosterone (castrated rats), castrated animals treated with estradiol/testosterone and treated with LSESR (castrated and treated rats). Following three months of continuous hormonal treatment, the weight of prostates in estradiol/testosterone-treated castrated rats was significantly increased in comparison with sham-operated rats. Such an increase started rapidly, reached a maximum by 30 days and remained at a plateau or slightly declined thereafter. The increase of prostate total weight induced by the hormone treatment was inhibited by administration of LSESR. Indeed, the weight was significantly lower at day 60 and day 90 for the dorsal and lateral regions of the prostate. The weight of the ventral region of the prostate was significantly lower after 30 and 60 days treatment with LSESR. These results demonstrate that administering LSESR to hormone-treated castrated rats inhibits the increase in prostate wet weight. This effect of LSESR may explain the beneficial effect of this extract in human benign prostatic hypertrophy.

Effect of Pygeum africanum extract on A23187-stimulated production of lipoxygenase metabolites from human polymorphonuclear cells.

Pygeum africanum extract has been used for more than 20 years in France in patients suffering from benign prostatic hypertrophy (BPH). The extract displays anti-inflammatory activity and inhibits bladder hyperreactivity during the above conditions. However, the mechanism of action of P. africanum extract has never been clearly resolved. It has been recently demonstrated that infiltration by inflammatory cells may be involved in the development of BPH. Certain of these cell types, such as macrophages, are known to produce chemotactic mediators including leukotrienes, and thus may contribute to the development of the disease. In order to investigate the potential effect of P. africanum extract on arachidonate metabolism, we examined its effect in vitro on leukotriene (LT) synthesis in human polymorphonuclear cells stimulated with the calcium ionophore A23187. Two formulations of the extract were tested, one dissolved in DMSO and one aqueous solution obtained after alkalinization (0.1 N; NaOH/acidification (0.1 N; HCl). Neither formulation had any effect on cell viability which was above 95% in both cases. P. africanum extract dissolved in DMSO significantly inhibited the production of 5-lipoxygenase metabolites (5-HETE, 20-COOH LTB4, LTB4 and 20-OH LTB4) at concentrations as low as 3 micrograms/ml (p < 0.01), while the same extract dissolved in NaOH/HCl only exhibited an inhibitory effect at 10 micrograms/ml (p < 0.01). This difference apparently reflects the greater solubility of the active components in the extract in DMSO. The ability of P. africanum to antagonize 5-lipoxygenase metabolite production may contribute, at least in part, to its therapeutic activity in inflammatory component of BPH.

An optoelectronic registration method as applied to PAF-mediated hydrogen peroxide induced arterial thrombosis.

In the present paper, a standardized method for the induction and registration of platelet thrombi in arterioles (500 microns diameter) of small laboratory animals is described in full detail. Using an optoelectronic analogue computer device, different discriminating parameters characteristic for the thrombotic phenomenon are presented. As the topical application of exogenous PAF-acether induces the generation of endogenous PAF-acether according to previous investigations (Bourgain et al. (1985) Prostaglandins 30, 185) it was deemed interesting to investigate the effect of hydrogen peroxide using the described methodology. It was found that the latter substance not only primes the effect of PAF-acether-induced thrombosis, but also can trigger by itself PAF-acether modulated arterial thrombus formation. Experimental evidence is adduced that these thrombotic phenomena can be most efficiently down regulated by specific PAF-acether antagonists.

Inhaled substance P induces activation of alveolar macrophages and increases airway responses in the guinea-pig.

Guinea-pigs pretreated with phosphoramidon or saline were treated with an aerosol of substance P (SP) or saline. 24 h later, the pulmonary inflation pressure (PIP) to substance P or to cumulative doses of acetylcholine or of histamine was recorded. The PIP response to SP itself was significantly enhanced in animals treated with phosphoramidon+SP as compared with phosphoramidon+saline (2.5-fold increase 1 min after the end of the inhalation, P < 0.001). The response to acetylcholine and to histamine was also significantly enhanced in phosphoramidon+substance P-treated as compared with phosphoramidon+saline-treated guinea-pigs (PC200 = 38.9 and 1.6 as compared with 77.6 and 3.9 micrograms/ml, P < 0.01 and P < 0.05 respectively). The production of superoxide anions by alveolar macrophages in response to f-MLP was also enhanced after treatment with phosphoramidon+SP as compared with phosphoramidon+saline (6.4 +/- 0.7 and 3.8 +/- 0.3 cpm, P < 0.001 respectively). In animals treated with saline+SP or saline+saline, the PIP responses and the production of superoxide anion were similar. Altogether these results suggest that SP contributes to the bronchial hyper-responsiveness in asthma and this probably through activation of alveolar macrophages.

Protective effects of somatostatin against gastric damage induced by hemorrhagic shock, stress and PAF in the rat.

Somatostatin is an endogenous cyclic tetradecapeptide which can exert effects on a wide range of gastrointestinal functions, including gastric acid and pepsin secretion, gastric and small intestinal motility, splanchnic blood flow, pancreatic enzyme secretion, intestinal nutrient absorption and gallbladder contractility. Somatostatin has also been shown to reduce the severity of ethanol-induced gastric damage. In this study, we examined the effect of pretreating rats with somatostatin (s.c.) on susceptibility to gastrointestinal damage induced by hemorrhagic shock, stress, platelet-activating factor (PAF), indomethacin or endotoxin. Somatostatin significantly reduced the extent of gastric damage induced by hemorrhagic shock when given at a dose of 20 micrograms/kg or greater (P < 0.05). Somatostatin (20-50 micrograms/kg) also had a dose-dependent protective effect against stress-induced gastric damage. Versus gastric damage induced by intravenous PAF, a dose of 5 micrograms/kg of somatostatin had no effect, while doses of 15-100 micrograms/kg significantly reduced the extent of injury to the stomach. In contrast, somatostatin had no significant effect on gastric or intestinal damage caused by intravenous administration of Salmonella enteritidis endotoxin or by oral administration of indomethacin, despite significantly and dose-dependently (2-10 micrograms/kg) reducing both the volume and titratable acidity of gastric secretion. A protective dose of somatostatin (20 micrograms/kg) had only a small and transient effect on gastric blood flow. The present results demonstrate the effectiveness of somatostatin in protecting the mucosa from injury in a variety of models, and suggest that inhibition of gastric acid secretion is not the sole mechanism underlying these protective effects.

Effect of the nifedipine-atenolol association on arterial myocyte migration and proliferation.

The in vitro effect of nifedipine and atenolol, either alone or in combination, on the proliferation and migration of rat aortic smooth muscle cells was investigated. Nifedipine inhibited the replication of arterial myocytes in concentrations ranging between 10 and 100 microM. The inhibition, evaluated as cell number, was dose- and time-dependent with an IC50 of 39 and 34 microM after 48 and 72 h, respectively; the cell doubling time increased with drug concentrations up to 118 h versus 28 h for controls. Atenolol alone failed to reduce arterial myocyte proliferation, and did not influence the effect of nifedipine on cell proliferation. Nifedipine and atenolol alone inhibited in a dose-dependent manner rat aortic myocytes migration induced by fibrinogen as chemotactic agent. When the combination nifedipine-atenolol was investigated, an additive inhibitory effect on cell migration was observed. These results provide in vitro support for a potential effect of this drug association on early steps of atherogenesis.

Beta 2-adrenoceptor stimulation augments the IL-4-induced CD23 expression and release and the expression of differentiation markers (CD14, CD18) by the human monocytic cell line, U 937.

The effect of beta 2-adrenoceptor agonists and interleukin-4 (IL-4) on the CD23 expression on, and release from, the human promonocytic cell line, U 937, was investigated. As assessed by flow cytometry, incubation of U 937 cells in the presence of salbutamol, fenoterol or IL-4 induced a concentration- and time-dependent increase in CD23 expression, that was maximal after 48 hr and followed by a decrease thereafter. In addition, salbutamol potentiated the effect of IL-4, the optimal concentration of the drug being a function of the concentration of this cytokine. This synergy between IL-4 and beta 2-adrenoceptor agonists was also observed for the release of the soluble form of CD23. The effect on CD23 expression of salbutamol and fenoterol, but not of IL-4, was blocked in the presence of D,L-propranolol (1 microM) or butoxamine (1 microM). The alpha-adrenoceptor agonist, norepinephrine (1 microM), was ineffective in inducing CD23 expression or potentiating the one evoked by IL-4. Salbutamol down-regulated the expression of Fc gamma RI (CD64) and Fc gamma RII (CD32) whereas IL-4 was ineffective. Only when added together at the onset of the culture did salbutamol and IL-4 induce, after 48 hr, the expression of the monocyte marker, CD14. The expression of CD18 was up-regulated in response to salbutamol either alone or in combination with IL-4, this cytokine alone being inefficient. These data suggest that IL-4 and beta 2-adrenoceptor agonists induce differentiation of U 937 cells into monocyte-like cells.

[Immunomodulatory and anti-oxidant effects of bovine lactoferrin in man]. / Effets immunomodulateurs et anti-oxydants de la lactoferrine bovine chez l'homme.

Lf presented differential effects in the induction of normal human monocyte activation. Lf inhibits the free radical generation by normal human monocytes in response to PMA. This effect on free radical generation is linked to the nature of the divalent cationic metal that substitutes the Apo-Lf. Indeed, the substitution of Fe2+ by another cationic transition metal (Cu2+, Zn2+, Pt2+ or Au2+) modifies the anti-oxidant effect of Lf. Among the different substituted Lf the Lf saturated with Cu2+ is the more potent inhibitor of free radical generation by normal human monocytes. In addition to its anti-oxidant effect, Lf stimulates, in combination with LPS, the production of cytokines (IL-1 beta, TNF-alpha and IL-6) and of prostacyclin (PGI2) by normal human monocytes. This potentiating effect is independent of the cation that substitutes the Lf, since the Apo-Lf is able to induce such a potentiating effect. In addition, whereas Lf does not modulate the phenotype of normal human monocytes stimulated or not with IL-4 it does enhance the IL-4-induced release of the soluble form of CD23 by these cells.

Effect of cefadroxil on antigen-induced bronchial hyperresponsiveness and eosinophil accumulation in lung from sensitized guinea pigs.

The effect of a semi-synthetic cephalosporin, Cefadroxil, on antigen-induced bronchial hyperresponsiveness and eosinophil accumulation in lungs from sensitized guinea pigs was investigated and compared to the effects of Cetirizine and Ketotifen. When aerosol-sensitized guinea pigs were pretreated 1 h before the antigen challenge with Cefadroxil (100 mg/kg i.p.) a partial but significant inhibition of the bronchial hyperresponsiveness to aerosolized acetylcholine chloride was observed. Furthermore, the treatment of guinea pigs (115 mg/kg, per os) 24 and 1 h before ovalbumin challenge also significantly reduced bronchial hyperresponsiveness. In contrast, no significant inhibition was noted when the guinea pigs were treated by a single dose of Cefadroxil (115 mg/kg per os) 1 h before challenge. Pretreatment of the guinea pigs with Cetirizine (1 mg/kg per os) or Ketotifen (0.1 mg/kg per os) completely inhibited the antigen-induced bronchial hyperresponsiveness. Cefadroxil (100 mg/kg i.p.) slightly inhibited the accumulation of eosinophils in the peribronchial area induced by antigen challenge. In contrast, no significant reduction was noted when the guinea pigs were treated per os with Cefadroxil (115 mg/kg), Cetirizine (1 or 10 mg/kg) or Ketotifen (0.1 mg/kg). These results show that Cefadroxil is effective in reducing antigen-induced bronchial hyperresponsiveness, an effect independent of a reduction in the pulmonary inflammation, namely eosinophil accumulation in lung.

Effect of platelet-activating factor on tumor necrosis factor-induced superoxide generation from human neutrophils. Possible involvement of G proteins.

The effect of platelet-activating factor (PAF) and of two specific PAF antagonists on tumor necrosis factor (TNF) induced superoxide production by human polymorphonuclear neutrophils (PMN) was examined. PAF alone (0.1 pM to 0.1 nM) failed to evoke superoxide production; however, when PAF was added for 10 min to cells upon prior incubation with 10 ng/mL TNF for 50 min, superoxide production was significantly enhanced as compared to that induced by TNF alone. Maximum amplification (+30%) was obtained with 10 pM PAF; however, the effect was completely abolished by two structurally unrelated PAF antagonists, BN 52021 and BN 52111. The antagonists also decreased by 25% the superoxide production elicited solely by TNF, implicating the involvement of endogenous PAF in this process. Pretreatment of the PMN with either pertussis or cholera toxin attenuated the PAF amplified superoxide production in TNF stimulated cells, suggesting that G proteins sensitive to these toxins may be involved in the mechanisms controlling amplification.

Effect of iloprost on reperfusion-induced arrhythmias and myocardial ion shifts in isolated rat hearts.

Isolated hearts excised from normotensive (NT) and spontaneously hypertensive (SH) rats subjected to transient normothermic global ischemia were used to study the effect of chronic treatment with iloprost on reperfusion-induced arrhythmias and myocardial ion shifts. After 30 min of ischemia, iloprost given s.c. in doses of 10, 50, 100 and 200 micrograms/kg per day for 14 days reduced the incidence of reperfusion-induced ventricular fibrillation (VF) in isolated hearts from the control value of 91 to 83, 75, 50 (P less than 0.05) and 25% (P less than 0.01) respectively, in NT rats. In the SH groups, the incidence of VF was also reduced from 100 to 75, 58, 33 (P less than 0.01) and 17% (P less than 0.001), respectively, with 10, 50, 100 and 200 micrograms/kg per day of iloprost. A similar reduction was observed in the incidence of reperfusion-induced ventricular tachycardia (VT). Ischemia and reperfusion caused significant changes in myocardial ion contents, i.e. an increase in Na+ and Ca2+ and a decrease in K+ and Mg2+ concentrations. The myocardial water content was also increased in parallel to the Na+ gain. The effect of iloprost given s.c. in doses of 50 and 200 micrograms/kg per day for 14 days was also measured on myocardial ion contents after 15- or 30-min ischemia and 30-min ischemia plus 10-min reperfusion. The higher iloprost dose significantly reduced the myocardial Na+, Ca2+ and water gains and the loss of K+ induced by ischemia and reperfusion in the NT and SH groups, while the decrease in Mg2+ content was alleviated only in SH rats. The results suggest that long-term iloprost treatment reduces the incidence of reperfusion-induced VF and VT by preventing Na+, Ca2+ and water accumulation as well as K+ and Mg2+ loss from myocardial tissue.

Tumor necrosis factor alpha 'primes' the platelet-activating factor-induced superoxide production by human neutrophils: possible involvement of G proteins.

It has recently been demonstrated that very low concentrations of platelet-activating factor (PAF) and various cytokines can prime human neutrophils (PMN) to respond in an enhanced manner to subsequent agonistic stimulation. We were interested to ascertain whether superoxide generation by human PMN could be amplified by PAF following initial stimulation with tumor necrosis factor (TNF) and the effect of cholera and pertussis toxin on this process. PAF alone (0.1 pM-0.1 nM) failed to evoke any superoxide production; however, when PAF was added for 10 min to cells previously incubated for 50 min with 10 ng/ml TNF, superoxide production was significantly enhanced relative to that induced by TNF alone. Maximum amplification (+30%) was obtained with 10(-12) M PAF, this effect being completely abolished by BN 52021 and BN 52111, two structurally unrelated PAF antagonists. The PAF antagonists also decreased by 25% the superoxide production elicited solely by TNF, indicating that TNF-induced superoxide generation is partially mediated by a mechanism involving endogenous PAF. Pretreatment of the PMN with pertussis or cholera toxin reduced the amplification of superoxide production induced by PAF in TNF-stimulated PMN, implicating pertussis and cholera toxin-sensitive G-proteins in the amplification process.

Platelet-activating factor and cytokine interactions in shock.

Many types of shock are characterized by profound hemodynamic alterations and depression of immune processes. Among the various mediators implicated in shock conditions, there is much evidence to suggest that, together with various cytokines, the inflammatory and chemotactic autacoid, platelet-activating factor (PAF), plays an important role. Studies on several animal models have shown that infusion of PAF mimicks the shock state, that markedly increased levels of PAF are produced in shock and that PAF antagonists afford significant protection against diverse forms of shock. The precise mechanism by which PAF antagonists protect against shock remains unclear; however, it is becoming apparent that in traumatic states a complex interaction occurs between PAF and cytokines, which leads to the acute phase reaction and circulatory collapse. We propose that PAF antagonists may be effective in counteracting shock because of their antiprotease activity and their ability to inhibit deleterious PAF/cytokine auto-generated feedback processes.

PAF/cytokine auto-generated feedback networks in microvascular immune injury: consequences in shock, ischemia and graft rejection.

The catastrophe theory evolved by Thom and Zeeman proposes a mathematical definition for the abrupt or 'catastrophic' changes that can suddenly occur in normally well-ordered and smooth-running systems. We have integrated this theory with our own PAF/cytokine feedback network hypothesis to explain the control and dysfunction of the inflammatory response. This process involves the activation of cells and factors such as proteases, and is coordinated by mediators such as PAF, cytokines and growth factors, minute amounts of which can prime cells to respond in an enhanced manner to subsequent agonistic stimuli. PAF and certain cytokines also possess the unique property of being able to induce the release of each other and their own generation in vivo. This 'singularity' may enable a self-generating feedback network to become established. The priming ability of these mediators indicates the extreme sensitivity of the inflammatory process and importance of a homeostatic equilibrium between the vectors involved in the priming and feedback processes and internal suppressive mechanisms. In pathological conditions, one can consider the phenomenon of PAF and cytokine autogeneration as a 'fold' in the feedback network and an expression of the singularity characteristic of the catastrophe hypothesis. This may lead to systemic toxicity and microcirculatory collapse, a characteristic feature of shock, sepsis, asthma, ischemia and graft rejection. A combination of drugs antagonizing the various feedback components may inhibit this catastrophic process and thus provide more successful therapy of these conditions.