Estradiol's salutary effects on keratinocytes following trauma-hemorrhage are mediated by estrogen receptor (ER)-alpha and ER-beta

Although administration of 17beta-estradiol (estrogen) following trauma-hemorrhage attenuates the elevation of cytokine production and mitogen-activated protein kinase (MAPK) activation in epidermal keratinocytes, whether the salutary effects of estrogen are mediated by estrogen receptor (ER)-alpha or ER-beta is not known. To determine which estrogen receptor is the mediator, we subjected C3H/HeN male mice to trauma-hemorrhage (2-cm midline laparotomy and bleeding of the animals to a mean blood pressure of 35 mmHg and maintaining that pressure for 90 min) followed by resuscitation with Ringer's lactate (four times the shed blood volume). At the middle of resuscitation we subcutaneously injected ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), ER-beta agonist diarylpropionitrile (DPN; 5 microg/kg), estrogen (50 microg/kg), or ER antagonist ICI 182,780 (150 microg/kg). Two hours after resuscitation, we isolated keratinocytes, stimulated them with lipopolysaccharide for 24 h (5 microg/mL for maximum cytokine production), and measured the production of interleukin (IL)-6, IL-10, IL-12, and TNF-alpha and the activation of MAPK. Keratinocyte cytokine production markedly increased and MAPK activation occurred following trauma-hemorrhage but were normalized by administration of estrogen, PPT, and DPN. PPT and DPN administration were equally effective in normalizing the inflammatory response of keratinocytes, indicating that both ER-alpha and ER-beta mediate the salutary effects of estrogen on keratinocytes after trauma-hemorrhage.     

Estrogen modulates TNF-alpha-induced inflammatory responses in rat aortic smooth muscle cells through estrogen receptor-beta activation

We have previously shown that 17beta-estradiol (E2) attenuates responses to endoluminal injury of the rat carotid artery, at least in part, by decreasing inflammatory mediator expression and neutrophil infiltration into the injured vessel, with a major effect on the neutrophil-specific chemokine cytokine-induced neutrophil chemoattractant (CINC)-2 beta. Current studies tested the hypothesis that activated rat aortic smooth muscle cells (RASMCs) express these same inflammatory mediators and induce neutrophil migration in vitro and that E2 inhibits these processes by an estrogen receptor (ER)-dependent mechanism. Quiescent RASMCs treated with E2, the ER alpha-selective agonist propyl pyrazole triol (PPT), the ER beta-selective agonist diarylpropiolnitrile (DPN), or vehicle for 24 h were stimulated with tumor necrosis factor (TNF)-alpha and processed for real-time RT-PCR, ELISA, or chemotaxis assays 6 h later. TNF-alpha stimulated and E2 attenuated mRNA expression of inflammatory mediators, including P-selectin, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, monocyte chemoattractant protein (MCP)-1, and CINC-2 beta. DPN dose dependently attenuated TNF-alpha-induced mRNA expression of CINC-2 beta, whereas PPT had no effect. The anti-inflammatory effects of DPN and E2 were blocked by the nonselective ER-inhibitor ICI-182,780. ELISA confirmed the TNF-alpha-induced increase and E2-induced inhibition of CINC-2 beta protein secretion. TNF-alpha treatment of RASMCs produced a twofold increase in neutrophil chemotactic activity of conditioned media; E2 and DPN treatment markedly inhibited this effect. E2 inhibits activated RASMC proinflammatory mediator expression and neutrophil chemotactic activity through an ER beta-dependent mechanism.     

Salutary effects of estrogen receptor-beta agonist on lung injury after trauma-hemorrhage

Although 17beta-estradiol (E2) administration after trauma-hemorrhage attenuates lung injury in male rodents, it is not known whether the salutary effects are mediated via estrogen receptor (ER)-alpha or ER-beta. We hypothesized that the salutary effects of E2 lung are mediated via ER-beta. Male Sprague-Dawley rats underwent trauma-hemorrhage (mean blood pressure 40 mmHg for 90 min, then resuscitation). E2 (50 microg/kg), ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), ER-beta agonist diarylpropiolnitrile (DPN; 5 microg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. At 24 h after trauma-hemorrhage or sham operation, bronchoalveolar fluid (BALF) was collected for protein concentration, LDH activity, and nitrate/nitrite and IL-6 levels. Moreover, lung tissue was used for inducible nitric oxide synthase (iNOS) mRNA/protein expression, nitrate/nitrite and IL-6 levels, and wet/dry weight ratio (n = 6 rats/group). One-way ANOVA and Tukey's test were used for statistical analysis. The results indicated that E2 downregulated lung iNOS expression after trauma-hemorrhage. Protein concentration, LDH activity, and nitrate/nitrite and IL-6 levels in BALF and nitrate/nitrite and IL-6 levels in the lung increased significantly after trauma-hemorrhage; however, administration of DPN but not PPT significantly improved all parameters. Moreover, DPN treatment attenuated trauma-hemorrhage-mediated increase in iNOS mRNA/protein expression in the lung. In contrast, no significant change in the above parameters was observed with PPT. Thus the salutary effects of E2 on attenuation of lung injury are mediated via ER-beta, and ER-beta-induced downregulation of iNOS likely plays a significant role in the DPN-mediated lung protection after trauma-hemorrhage.     

Estrogen prevents intestinal inflammation after trauma-hemorrhage via downregulation of angiotensin II and angiotensin II subtype I receptor

Although angiotensin II (Ang II) plays a key role in development of organ ischemia-reperfusion injury, it remains unclear whether it is involved in development of intestinal injury following trauma-hemorrhage (T-H). Studies have shown that 17beta-estradiol (E2) administration following T-H improves small intestinal blood flow; however, it is unclear whether Ang II plays a role in this E2-mediated salutary effect. Male Sprague-Dawley rats underwent laparotomy and hemorrhagic shock (removal of 60% total blood volume, fluid resuscitation after 90 min). At onset of resuscitation, rats were treated with vehicle, E2, or E2 and estrogen receptor antagonist ICI 182,780 (ICI). A separate group of rats was treated with Ang II subtype I receptor (AT1R) antagonist losartan. At 24 h after T-H, plasma Ang II, IL-6, TNF-alpha, intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-3 levels, myeloperoxidase (MPO) activity, and AT1R expression were determined. T-H significantly increased plasma and intestinal Ang II, IL-6, TNF-alpha levels, intestinal ICAM-1, CINC-1, CINC-3 levels, MPO activity, and AT1R protein compared with shams. E2 treatment following T-H attenuated increased intestinal MPO activity, Ang II level, and AT1R protein expression. ICI administration abolished the salutary effects of E2. In contrast, losartan administration attenuated increased MPO activity without affecting Ang II and AT1R levels. Thus Ang II plays a role in producing small intestine inflammation following T-H, and the salutary effects of E2 on intestinal inflammation are mediated in part by Ang II and AT1R downregulation.     

Tissue compartment-specific role of estrogen receptor subtypes in immune cell cytokine production following trauma-hemorrhage.

Although 17beta-estradiol administration following trauma-hemorrhage attenuates plasma cytokines and alteration in immune cell cytokine production, it is not known whether the salutary effects are mediated via estrogen receptor (ER)-alpha or ER-beta. Accordingly, we examined which ER subtype predominantly mediates the salutary effects of 17beta-estradiol on systemic inflammatory response/immune cell cytokine production in various tissues following trauma-hemorrhage. Male rats underwent trauma-hemorrhage (mean blood pressure: 40 mmHg for 90 min) and fluid resuscitation. The ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), the ER-beta agonist diarylpropionitrile (DPN; 5 microg/kg), 17beta-estradiol (50 microg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation, and various measurements were made 24 h thereafter. 17beta-Estradiol or PPT administration following trauma-hemorrhage prevented the increase in plasma IL-6 and IL-10 levels that were observed in vehicle-treated animals. IL-6 and TNF-alpha production by Kupffer cells increased; however, splenic macrophages (SMPhi), alveolar macrophages (AMPhi), and peripheral blood mononuclear cells (PBMC) had decreased release of these cytokines after trauma-hemorrhage. IL-10 production, however, increased in all macrophage populations. Administration of 17beta-estradiol following trauma-hemorrhage prevented all of these alterations. PPT had the same effects as 17beta-estradiol on IL-6 and TNF-alpha production by Kupffer cells and SMPhi, and DPN had the same effects on AMPhi and PBMC. The same effects as 17beta-estradiol on IL-10 production were observed by PPT on Kupffer cells and DPN on PBMC. Both agonists were equally effective on SMPhi and AMPhi. Thus ER subtypes have tissue compartment-specific roles in mediating the effects of 17beta-estradiol on immune cell functions following trauma-hemorrhage.     

Role of estrogen receptor subtypes in estrogen-induced organ-specific vasorelaxation after trauma-hemorrhage

Although endothelin-1 (ET-1)-induced organ hypoperfusion after trauma-hemorrhage is improved by estrogen administration, it remains unclear whether estrogen receptor (ER) subtypes play any role in the attenuation of ET-1-induced vasoconstriction in any specific organ bed. To investigate this, isolated perfusion experiments in the heart, liver, small intestine, kidney, and lung were carried out in sham, at the time of maximum bleedout (MBO; i.e., 5-cm midline incision, with removal of 60% of circulating blood volume over 45 min to maintain a mean blood pressure of 40 mmHg), and 2 h after trauma-hemorrhage and resuscitation (T-H/R). Organ-specific ET-1-induced vasoconstriction was evaluated, and the effects of 17beta-estradiol (E2) and ER-specific agonists propylpyrazole triol (PPT; ERalpha agonist) and diarylpropionitrile (DPN; ERbeta agonist) were determined. ET-1 induced the greatest vasoconstriction in sham animals, with the strongest response in the kidneys, followed by the small intestine and liver. ET-1-induced responses were weakest in the heart and lungs. ET-1-induced vasoconstriction was evident at the time of MBO but was significantly decreased at 2 h after T-H/R. ERbeta plays an important role in cardiac performance, as evidenced by improved heart performance (+dP/dt) in the presence of DPN. DPN also induced a greater effect than PPT in the reduction of ET-1-induced vasoconstriction in the kidneys and lungs. In contrast, PPT attenuated ET-1-induced vasoconstriction in the liver, whereas both DPN and PPT were equally effective in the small intestine. The increased +dP/dt values induced by E2, DPN, or PPT were evident at the time of MBO but were significantly decreased at 2 h after T-H/R. These data indicate that the effects of ET-1 on vasoconstriction and the role of ER subtypes in estrogen-induced vasorelaxation are organ specific and temporally specific after trauma-hemorrhage.     

17beta-Estradiol inhibits keratinocyte-derived chemokine production following trauma-hemorrhage

Neutrophil infiltration is a key step in the development of organ dysfunction following trauma-hemorrhage (T-H). Although we have previously shown that 17beta-estradiol (E2) prevents neutrophil infiltration and organ damage following T-H, the mechanism by which E2 inhibits neutrophil transmigration remains unknown. We hypothesized that E2 prevents neutrophil infiltration via modulation of keratinocyte-derived chemokine (KC), a major attractant for neutrophils. To examine this, male C3H/HeN mice were subjected to T-H or sham operation and thereafter resuscitated with Ringer lactate and E2 (1 mg/kg body wt) or vehicle. Animals were killed 2 h after resuscitation, and Kupffer cells were isolated. Plasma levels and Kupffer cell production capacities of KC, TNF-alpha, and IL-6 were determined by BD Cytometric Bead Arrays; lung mRNA expression of KC was measured with real-time PCR; myeloperoxidase activity assays were performed to determine neutrophil infiltration, and organ damage was assessed by edema formation. Treatment with E2 decreased systemic levels and restored Kupffer cell production of KC, TNF-alpha, and IL-6, as well as KC gene expression and protein in the lung. This was accompanied with a decrease in neutrophil infiltration and edema formation in the lung. These results suggest that E2 prevents lung neutrophil infiltration and organ damage in part by decreasing KC during posttraumatic immune response.     

Androstenediol administration after trauma-hemorrhage attenuates inflammatory response, reduces organ damage, and improves survival following sepsis

Although androstenediol (adiol or 5-androstene-3beta,17beta-diol), a metabolite of dehydroepiandrosterone (DHEA), has protective effects following trauma-hemorrhage (T-H), it remains unknown whether administration of adiol has any salutary effects on the inflammatory response and outcome following a combined insult of T-H and sepsis. Male rats underwent T-H shock [mean arterial pressure (MAP) 40 mmHg for 90 min] followed by resuscitation. Adiol (1 mg/kg body wt) or vehicle was administered at the end of resuscitation. Sepsis was induced by cecal ligation and puncture (CLP) at 20 h after T-H or sham operation. Five hours after CLP, plasma and tissue samples were analyzed for cytokines (IL-6 and IL-10), MPO, neutrophil chemotactic factor (CINC-3), and liver injury (alanine aminotransferase and lactate dehydrogenase). In another group of rats, the gangrenous cecum was removed at 10 h after CLP, the cavity was irrigated with warm saline and closed in layers, and mortality was recorded over 10 days. T-H followed by CLP produced a significant elevation in plasma IL-6 and IL-10 levels, enhanced neutrophil cell activation, and resulted in liver injury. Adiol administration prevented the increase in cytokine production, neutrophil cell activation, and attenuated liver injury. Moreover, rats subjected to the combined insult, receiving vehicle or adiol, had a 50% and 6% mortality, respectively. Since adiol administration suppresses proinflammatory cytokines, reduces liver damage, and decreases mortality after the combined insult of T-H and sepsis, this agent appears to be a novel adjunct to fluid resuscitation for decreasing T-H-induced septic complications and mortality.     

Sexual responses of the male rat medial preoptic area and medial amygdala to estrogen II: site specific effects of selective estrogenic drugs

In the medial preoptic area (MPO) and medial amygdala (MEA), estradiol (E(2)) aromatized from testosterone (T) may act via either estrogen receptor (ER) α or ERβ to mediate mating in male rats. We tested the hypothesis that, in the MPO, ERα exclusively mediates sexual responses to E(2) by monitoring mating in four groups of castrated male rats administered dihydrotestosterone (DHT) subcutaneously and MPO implants delivering either: cholesterol, E(2), propyl pyrazole triol (PPT, ERα-agonist) or diarylpropionitrile (DPN, ER β-agonist); a fifth group of intact males served as DPN toxicity control, receiving DPN MPO implants. In a follow-up study, either 1-methyl-4-phenyl pyridinium (MPP, ERα-antagonist) or blank MPO cannulae were implanted in castrated male rats receiving T subcutaneously, whereas intact MPP toxicity controls received MPP MEA implants. PPT or E(2) MPO implants maintained mating, but cholesterol or DPN MPO implants did not. Moreover, MPP MPO implants interfered with T reinstatement of mating suggesting that, in the MPO, ERα is necessary and sufficient for mating in androgen-maintained male rats and ERβ is not sufficient. Because it is unknown which ER subtype(s) mediate sexual responses of the MEA to E(2), we examined mating following MEA implants of cholesterol, E(2), PPT or DPN in four groups of castrated male rats administered DHT subcutaneously. E(2) MEA implants maintained mounting but mating was significantly decreased in groups receiving PPT, DPN or cholesterol MEA implants suggesting that, unlike the MPO where ERα alone is essential, sexual responses of the MEA to E(2) require simultaneous interactions among multiple ER subtypes.     

17beta-Estradiol modulates vasoconstriction induced by endothelin-1 following trauma-hemorrhage

Although endothelin-1 (ET-1) induces vasoconstriction, it remains unknown whether 17beta-estradiol (E(2)) treatment following trauma-hemorrhage alters these ET-1-induced vasoconstrictive effects. In addition, the role of the specific estrogen receptor (ER) subtypes (ER-alpha and ER-beta) and the endothelium-localized downstream mechanisms of actions of E(2) remain unclear. We hypothesized that E(2) attenuates increased ET-1-induced vasoconstriction following trauma-hemorrhage via an ER-beta-mediated pathway. To study this, aortic rings were isolated from male Sprague-Dawley rats following trauma-hemorrhage with or without E(2) treatment, and alterations in tension were determined in vitro. Dose-response curves to ET-1 were determined, and the vasoactive properties of E(2), propylpyrazole triol (PPT, ER-alpha agonist), and diarylpropionitrile (DPN, ER-beta agonist) were determined. The results showed that trauma-hemorrhage significantly increased ET-1-induced vasoconstriction; however, administration of E(2) normalized ET-1-induced vasoconstriction in trauma-hemorrhage vessels to the sham-operated control level. The ER-beta agonist DPN counteracted ET-1-induced vasoconstriction, whereas the ER-alpha agonist PPT was ineffective. Moreover, the vasorelaxing effects of E(2) were not observed in endothelium-denuded aortic rings or by pretreatment of the rings with a nitric oxide (NO) synthase inhibitor. Cyclooxygenase inhibition with indomethacin had no effect on the action of E(2). Thus, E(2) administration attenuates ET-1-induced vasoconstriction following trauma-hemorrhage via an ER-beta-mediated pathway that is dependent on endothelium-derived NO synthesis.     

Activation of estrogen receptor-alpha protects the in vivo rabbit heart from ischemia-reperfusion injury

The estrogen receptor (ER) mediates estrogenic activity in a variety of organs, including those in the reproductive, cardiovascular, immune, and central nervous systems. Experimental studies have demonstrated that 17beta-estradiol (E2) protects the heart from ischemia-reperfusion injury. Two estrogen receptors, ER alpha and ER beta, mediate the actions of estrogen; however, it is not certain which ER mediates the cardioprotective effects of E2. In the present study, the ER-selective agonists 4,4',4'-[4-propyl-(1H)-pyrazole-1,3,5-triyl]tris-phenol (PPT; ER alpha) and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; ER beta) were assessed for their cardioprotective potential in an in vivo rabbit model of ischemia-reperfusion injury. Anesthetized female rabbits were administered PPT (3 mg/kg), DPN (3 mg/kg), E2 (20 microg/rabbit), or vehicle intravenously 30 min before a 30-min occlusion of the left anterior descending coronary artery followed by 4 h of reperfusion. Acute treatment with E2 (17.7 +/- 2.9%; P < 0.001) and PPT (18.1 +/- 2.9%; P < 0.001), but not DPN (45.3 +/- 2.4%) significantly decreased infarct size as a percent of area at risk compared with vehicle (45.3 +/- 2.4%). Coadministration of PPT or E2 with the ER antagonist ICI-182,780 limited the infarct size-sparing effect of the compounds (43.8 +/- 6.6% and 40.6 +/- 5.7% respectively, expressed as a percentage of risk region). PPT reduced the release of cardiac-specific troponin-I and reduced the tissue deposition of the membrane attack complex and C-reactive protein similar to that of E2. The results indicate that activation of ER alpha, but not ER beta, is required for the observed cardioprotective effects of E2.     

Sex specific response of cultured human bone cells to ERα and ERβ specific agonists by modulation of cell proliferation and creatine kinase specific activity

We have previously reported that human cultured bone cells (hObs) respond to estradiol-17β (E2) by stimulating DNA synthesis, creatine kinase BB specific activity (CK) and other parameters sex-specifically. We now investigate the sex specificity of the response of these hObs to estrogen receptor (ER) α and ERβ specific agonists. Real time PCR revealed that all cells express mRNA for both ERs. ERα mRNA but not ERβ mRNA was stimulated by all estrogenic compounds in both pre- and post-menopausal hObs with no effect in male hObs. Cells treated with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ specific agonist) and 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα specific agonist) showed increased DNA synthesis and CK in all female but not male hObs. Raloxifene (Ral), a specific ERα antagonist, inhibited the stimulation of DNA synthesis and CK by E2 or PPT, but not by DPN. DPN and PPT like E2 modulated the expression of both 12 and 15 lipooxygenase (LO) mRNA in both female but not male hObs. 12 and 15 HETE production was modulated only by DPN and PPT in these cells. The LO inhibitor baicaleine inhibited only E2 and PPT but not DPN effects in both female hObs. In conclusion, we provide herein evidence for the separation of age- and sex-dependent mediation via both ERα and ERβ pathways in the effects of estrogens on hObs, with a yet unknown mechanism.     

Astringinin-mediated attenuation of the hepatic injury following trauma-hemorrhage

Although astringinin administration under adverse circulatory conditions is known to be protective, the mechanism by which astringinin produces the salutary effects remains unknown. We hypothesize that astringinin administration in males following trauma-hemorrhage decreases cytokine production and protects against hepatic injury. Male Sprague-Dawley rats underwent trauma-hemorrhage (mean blood pressure: 40 mmHg for 90 min, then resuscitation). Different doses of astringinin (0.01, 0.03, 0.1, 0.3 mg/kg of body weight) or vehicle were administered intravenously during resuscitation. Concentrations of plasma aspartate aminotransferase (AST) with alanine aminotransferase (ALT) and various hepatic parameters were measured (n = 8 rats/group) at 24 h after resuscitation. One-way ANOVA and Tukey testing were used for statistical analysis. Trauma-hemorrhage significantly increased plasma AST and ALT levels at 24 h postresuscitation; there was a dose-related benefit when astringinin was administered at doses of 0.01 to 0.3 mg/kg. In astringinin-treated (0.3 mg/kg) rats subjected to trauma-hemorrhage, there were significant improvements in liver myeloperoxidase (MPO) activity (237.80 +/- 45.89 vs. 495.95 +/- 70.64 U/mg protein, P < 0.05), interleukin-6 (IL-6) levels (218.54 +/- 34.52 vs. 478.60 +/- 76.21 pg/mg protein, P < 0.05), cytokine-induced neutrophil chemoattractant (CINC)-1 (88.32 +/- 20.33 vs. 200.70 +/- 32.68 pg/mg protein, P < 0.05), CINC-3 (110.83 +/- 26.63 vs. 290.14 +/- 76.82 pg/mg protein, P < 0.05) and intercellular adhesion molecule (ICAM)-1 concentrations (1,868.5 +/- 211.5 vs. 3,645.0 +/- 709.2 pg/mg protein, P < 0.05), as well as in histology. Results show that astringinin significantly attenuates proinflammatory responses and hepatic injury after trauma-hemorrhage. In conclusion, the salutary effects of astringinin administration on attenuation of hepatic injury following trauma-hemorrhage are likely due to reduction of pro-inflammatory mediator levels.     

Protective Effect of Tropisetron on Rodent Hepatic Injury after Trauma-Hemorrhagic Shock through P38 MAPK-Dependent Hemeoxygenase-1 Expression

Tropisetron can decrease inflammatory cell responses and alleviate organ damage caused by trauma-hemorrhage, but the mechanism of these effects remains unknown. The p38 mitogen-activated protein kinase/hemeoxygenase-1 (p38 MAPK/HO-1) pathway exerts anti-inflammatory effects on different tissues. The aim of this study was to investigate whether p38 MAPK/HO-1 plays any role in the tropisetron-mediated attenuation of hepatic injury after trauma-hemorrhage. Male Sprague-Dawley rats underwent trauma-hemorrhage (mean blood pressure maintained at approximately 35–40 mmHg for 90 min), followed by fluid resuscitation. During resuscitation, several treatment regimens were administered: four doses of tropisetron alone (0.1, 0.3, 1, 3 mg/kg body weight), or a single dose of tropisetron (1 mg/kg body weight) with and without a p38 MAPK inhibitor (SB-203580, 2 mg/kg body weight) or HO antagonist (chromium-mesoporphyrin, 2.5 mg/kg body weight). Various parameters were measured, and the animals were sacrificed at 24 h post-resuscitation. The results showed that trauma-hemorrhage increased the following parameters: plasma concentrations of aspartate (AST) and alanine aminotransferases (ALT), hepatic myeloperoxidase (MPO) activity, and levels of cytokine-induced neutrophil chemoattractant-1 and -3 (CINC-1 and CINC-3), intercellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein-1α (MIP-1α). These parameters were significantly improved in the tropisetron-treated rats subjected to trauma-hemorrhage. Tropisetron treatment also increased hepatic p38 MAPK and HO-1 expression compared with vehicle-treated trauma-hemorrhaged rats. Co-administration of SB-203580 or chromium-mesoporphyrin with tropisetron abolished the tropisetron-induced beneficial effects on the above parameters and hepatic injury. These results suggest that the protective effect of tropisetron administration on alleviation of hepatic injury after trauma-hemorrhage is likely mediated through p38 MAPK-dependent HO-1 expression.     

Acute activation of ER alpha decreases food intake, meal size, and body weight in ovariectomized rats

Estradiol exerts many of its actions by coupling with two nuclear estrogen receptor (ER) proteins, ER alpha, and ER beta. While the acute, anorexigenic effect of estradiol appears to involve such a mechanism, the relative contributions of ERalpha and ERbeta are equivocal. To address this problem, food intake was monitored in ovariectomized (OVX) rats following acute administration of a selective ER alpha agonist (4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol, PPT; dose range = 0-200 microg), a selective ER beta agonist (2,3-bis(4-hydroxyphenyl)-propionitrile, DPN; dose range = 0-600 microg), and a physiological (4 microg) dose of estradiol benzoate (EB). While PPT-treated rats displayed dose-dependent decreases in daily food intake and body weight, neither of these measures was influenced by any dose of DPN. In addition, DPN failed to modulate the anorexigenic effect of PPT when the two ER agonists were coadministered. Meal pattern analysis revealed that the anorexigenic effect of 75 microg PPT (a dose of PPT that produced a similar decrease in daily food intake as 4 microg EB) was mediated by a decrease in meal size, not meal number. Thus, PPT, like EB and endogenous estradiol, decreases food intake by selectively affecting the controls of meal size. The finding that acute administration of 75 microg PPT failed to induce a conditioned taste aversion suggests that the anorexigenic effect of this dose of PPT is not secondary to malaise. Taken together, our findings demonstrate that selective activation of ER alpha decreases food intake, body weight, and meal size in the ovariectomized rat.     

The role of MIP-1 alpha in the development of systemic inflammatory response and organ injury following trauma hemorrhage

Although MIP-1alpha is an important chemokine in the recruitment of inflammatory cells, it remains unknown whether MIP-1alpha plays any role in the development of systemic inflammatory response following trauma-hemorrhage (T-H). C57BL/6J wild type (WT) and MIP-1alpha-deficient (KO) mice were used either as control, subjected to sham operation (cannulation or laparotomy only or cannulation plus laparotomy) or T-H (midline laparotomy, mean blood pressure 35 +/- 5 mmHg for 90 min, followed by resuscitation) and sacrificed 2 h thereafter. A marked increase in serum alpha-glutathione transferase, TNF-alpha, IL-6, IL-10, MCP-1, and MIP-1alpha and Kupffer cell cytokine production was observed in WT T-H mice compared with shams or control. In addition lung and liver tissue edema and neutrophil infiltration (myeloperoxidase (MPO) content) was also increased following T-H in WT animals. These inflammatory markers were markedly attenuated in the MIP-1alpha KO mice following T-H. Furthermore, compared with 2 h, MPO activities at 24 and 48 h after T-H declined steadily in both WT and KO mice. However, normalization of MPO activities to sham levels within 24 h was seen in KO mice but not in WT mice. Thus, MIP-1alpha plays an important role in mediating the acute inflammatory response following T-H. In the absence of MIP-1alpha, acute inflammatory responses were attenuated; rapidly recovered and less remote organ injury was noted following T-H. Thus, interventions that reduce MIP-1alpha levels following T-H should be useful in decreasing the deleterious inflammatory consequence of trauma.     

Estrogen rescues heart failure through estrogen receptor Beta activation

Background

Recently, we showed that exogenous treatment with estrogen (E2) rescues pre-existing advanced heart failure (HF) in mice. Since most of the biological actions of E2 are mediated through the classical estrogen receptors alpha (ERα) and/or beta (ERβ), and both these receptors are present in the heart, we examined the role of ERα and ERβ in the rescue action of E2 against HF.

Methods

Severe HF was induced in male mice by transverse aortic constriction-induced pressure overload. Once the ejection fraction (EF) reached ~?35%, mice were treated with selective agonists for ERα (PPT, 850 μg/kg/day), ERβ (DPN, 850 μg/kg/day), or E2 (30 μg/kg/day) together with an ERβ-antagonist (PHTPP, 850 μg/kg/day) for 10 days.

Results

EF of HF mice was significantly improved to 45.3?±?2.1% with diarylpropionitrile (DPN) treatment, but not with PPT (31.1?±?2.3%). E2 failed to rescue HF in the presence of PHTPP, as there was no significant improvement in the EF at the end of the 10-day treatment (32.5?±?5.2%). The improvement of heart function in HF mice treated with ERβ agonist DPN was also associated with reduced cardiac fibrosis and increased cardiac angiogenesis, while the ERα agonist PPT had no significant effect on either cardiac fibrosis or angiogenesis. Furthermore, DPN improved hemodynamic parameters in HF mice, whereas PPT had no significant effect.

Conclusions

E2 treatment rescues pre-existing severe HF mainly through ERβ. Rescue of HF by ERβ activation is also associated with stimulation of cardiac angiogenesis, suppression of fibrosis, and restoration of hemodynamic parameters.

     

Estradiol improves cardiac and hepatic function after trauma-hemorrhage: role of enhanced heat shock protein expression

Although studies indicate that 17beta-estradiol administration after trauma-hemorrhage (T-H) improves cardiac and hepatic functions, the underlying mechanisms remain unclear. Because the induction of heat shock proteins (HSPs) can protect cardiac and hepatic functions, we hypothesized that these proteins contribute to the salutary effects of estradiol after T-H. To test this hypothesis, male Sprague-Dawley rats ( approximately 300 g) underwent laparotomy and hemorrhagic shock (35-40 mmHg for approximately 90 min) followed by resuscitation with four times the shed blood volume in the form of Ringer lactate. 17beta-estradiol (1 mg/kg body wt) was administered at the end of the resuscitation. Five hours after T-H and resuscitation there was a significant decrease in cardiac output, positive and negative maximal rate of left ventricular pressure. Liver function as determined by bile production and indocyanine green clearance was also compromised after T-H and resuscitation. This was accompanied by an increase in plasma alanine aminotransferase (ALT) levels and liver perfusate lactic dehydrogenase levels. Furthermore, circulating levels of TNF-alpha, IL-6, and IL-10 were also increased. In addition to decreased cardiac and hepatic function, there was an increase in cardiac HSP32 expression and a reduction in HSP60 expression after T-H. In the liver, HSP32 and HSP70 were increased after T-H. There was no change in heart HSP70 and liver HSP60 after T-H and resuscitation. Estradiol administration at the end of T-H and resuscitation increased heart/liver HSPs expression, ameliorated the impairment of heart/liver functions, and significantly prevented the increase in plasma levels of ALT, TNF-alpha, and IL-6. The ability of estradiol to induce HSPs expression in the heart and the liver suggests that HSPs, in part, mediate the salutary effects of 17beta-estradiol on organ functions after T-H.     

Selective estrogen receptor-alpha and estrogen receptor-beta agonists rapidly decrease pulmonary artery vasoconstriction by a nitric oxide-dependent mechanism

Both endogenous and exogenous estrogen decrease pulmonary artery (PA) vasoconstriction. Whether these effects are mediated via estrogen receptor (ER)-alpha or ER-beta, and whether the contribution of ERs is stimulus-dependent, remains unknown. We hypothesized that administration of the selective ER-alpha agonist propylpyrazole triol (PPT) and/or the selective ER-beta agonist diarylpropiolnitrile (DPN) rapidly decreases PA vasoconstriction induced by pharmacologic and hypoxic stimuli via a nitric oxide (NO)-dependent mechanism. PA rings (n = 3-10/group) from adult male Sprague-Dawley rats were suspended in physiologic organ baths. Force displacement was measured. Vasoconstrictor responses to phenylephrine (10(-8)M - 10(-5)M) and hypoxia (Po(2) 35-45 mmHg) were determined. Endothelium-dependent and -independent vasorelaxation were measured by generating dose-response curves to acetylcholine (10(-8)M - 10(-4)M) and sodium nitroprusside (10(-9)M - 10(-5)M). PPT or DPN (10(-9)M - 5 x 10(-5)M) were added to the organ bath in the presence and absence of the NO-synthase inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) (10(-4)M). Selective ER-alpha activation (PPT, 5 x 10(-5)M) rapidly (<20 min) decreased phenylephrine-induced vasoconstriction. This effect, as well as PPT's effects on endothelium-dependent vasorelaxation, were neutralized by l-NAME. In contrast, selective ER-beta activation (DPN, 5 x 10(-5)M) rapidly decreased phase II of hypoxic pulmonary vasoconstriction (HPV). l-NAME eliminated this phenomenon. Lower PPT or DPN concentrations were less effective. We conclude that both ER-alpha and ER-beta decrease PA vasoconstriction. The immediate onset of effect suggests a nongenomic mechanism. The contribution of specific ERs appears to be stimulus specific, with ER-alpha primarily modulating phenylephrine-induced vasoconstriction, and ER-beta inhibiting HPV. NO inhibition eliminates these effects, suggesting a central role for NO in mediating the pulmonary vascular effects of both ER-alpha and ER-beta.