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Acute Effects of Estradiol on Lung Inflammation Due to Intestinal Ischemic Insult in Male Rats

Breithaupt-Faloppa, Ana Cristina*; Thais Fantozzi, Evelyn; Romero, Daniel Cancelli; Rodrigues, Adriana da Silva; de Sousa, Paulo Thales Rocha*; Lino dos Santos Franco, Adriana; Oliveira-Filho, Ricardo Martins; Boris Vargaftig, Bernardo; Tavares de Lima, Wothan

doi: 10.1097/SHK.0000000000000092
Basic Science Aspects

ABSTRACT: Intestinal ischemia and reperfusion (intestinal I/R) causes acute lung inflammation that is characterized by leukocyte migration, increased lung microvascular permeability, and, in severe forms, noncardiogenic pulmonary edema and acute respiratory distress syndrome. Female sex hormones interfere with immune response, and experimental and clinical evidence shows that females are more resistant than males to organ injury caused by gut trauma. To reduce the lung inflammation caused by intestinal I/R, we have acutely treated male rats with estradiol. Intestinal I/R was performed by the clamping (45 min) of the superior mesenteric artery (SMA), followed by 2 h of intestinal reperfusion (unclamping SMA). Groups of rats received 17β estradiol (E2, 280 µg/kg, i.v., single dose) 30 min after the SMA occlusion (ischemia period) or 1 h after the unclamping of SMA (reperfusion period). Leukocytes influx into the lung and microvascular leakage were assessed by lung myeloperoxidase activity and Evans blue dye extravasation, respectively. The lung expression of adhesion molecules (intercellular adhesion molecule 1, platelet endothelial cell adhesion molecule 1, and vascular cell adhesion molecule [VCAM]) was evaluated by immunohistochemistry. Interleukin 1β (IL-1β), IL-10, and NOx concentrations were quantified in supernatants of cultured lung tissue. We have found that intestinal I/R increased the lung myeloperoxidase activity and Evans blue dye extravasation, which were reduced by treatment of rats with E2. Intestinal I/R increased ICAM-1 expression only, and it was decreased by E2 treatment. However, E2 treatment reduced the basal expression of platelet endothelial cell adhesion molecule 1. E2 treatment during intestinal ischemia was effective to reduce the levels of IL-10 and IL-1β in explant supernatant, but only IL-10 levels were reduced by E2 at reperfusion phase. The treatment with E2 did not affect NOx concentration. Taken together, our data suggest that estradiol modulates the lung inflammatory response induced by lung injury, likely by acute effects. Thus, acute estradiol treatment could be considered as a potential therapeutic agent in ischemic events.

*Laboratory of Cardiovascular Surgery and Physiopathology of Circulation (LIM-11), Heart Institute (InCor), Medicine School, Department of Pharmacology, Institute of Biomedical Sciences, and Department of Clinical and Toxicological Analyses, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil

Received 12 Aug 2013; first review completed 3 Sep 2013; accepted in final form 7 Nov 2013

Address reprint requests to Wothan Tavares de Lima, PhD, Laboratório de Fisiopatologia da Inflamação Experimental, Instituto de Ciências Biomédicas, University of São Paulo, Av Prof Lineu Prestes, 1524, 05508-900 São Paulo, Brazil. E-mail:

This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP: 09/54823-2) and Conselho Nacional de Pesquisa (CNPq). W.T.L. is fellow researcher of CNPq.

Intestinal ischemia and reperfusion (intestinal I/R) accounts for organ injury in a wide range of insults, such as organ transplantation, hemorrhagic shock, and major surgery. Intestinal I/R causes severe local and remote hemodynamic changes, systemic inflammation, increase in vascular permeability, endothelial cell activation, and an imbalance between vasodilator and vasoconstrictor factors. As a consequence of intestinal I/R, circulating leukocytes, basically neutrophils, become activated and migrate into the lungs. Activated neutrophils release a wide range of inflammatory mediators that contribute to the induction of acute lung injury (ALI), which can evolve into a more dangerous condition known as acute respiratory distress syndrome (ARDS) (1–4). Acute respiratory distress syndrome is a clinically relevant lung disorder characterized by a significant mortality rate. Recent studies have contributed to the understanding of the role of inflammatory pathways in the pathogenesis of ARDS (5, 6). In this context, even though new therapies have shifted ARDS mortality from 70% to 40% (7), new therapies providing a better control of lung homeostasis could represent an additional therapeutic advance. Experimental evidence shows that the immune system is affected by female sex hormones (8–10). In fact, a favorable outcome after hemorrhagic shock is influenced by female sex hormones (9–11). Frink et al. (12) investigating the role of female sex hormones on polytraumatized patients confirmed that women present lower levels of systemic inflammatory cytokines and are therefore more resistant to multiple organ failure. Interestingly, a prospective study showed that women younger than 50 years tolerated shock-trauma better than did men of the same age (13). Notwithstanding clinical and experimental studies report sex specificity in some immune responses (14, 15), there are no consistent evaluations regarding the influence of female hormones on inflammatory conditions, notably in the lungs after gut trauma.

Experimental data showed that estradiol exerts a protective effect on the intestinal microcirculation in a model of sepsis. These data reinforced the role of sex on the control of inflammation and interaction of leukocyte with endothelium (16). Estrogens can affect the functional activity of endothelial cells, which actively recruit immune and inflammatory cells to lymphoid and peripheral tissues through the expression of adhesion molecules and chemokines (17). Interestingly, estrogen restores both immune and other organ functions in males and ovariectomized females following trauma-hemorrhagic shock (18). Similarly, Yu et al. (19) demonstrated that intestinal injury after trauma-hemorrhagic shock is positively modulated by estradiol. In addition, the low levels of circulating male sex hormones in female animals are also considered to contribute to the divergent sex profile of immune responses observed after injury and blood loss (20).

In the present study, we have considered that estradiol could exert protective effects on the lung after intestinal I/R. Our aim was to investigate whether estradiol exerts its effects at the time of intestinal ischemia or during the phase of intestinal reperfusion. To this purpose, estradiol was given to rats after 30 min of intestinal ischemia or 1 h after the intestinal perfusion reestablishment. In this context, we quantified leukocyte migration into the lung and lung microvascular permeability. In parallel, to clarify some putative mechanisms underlying the effects of estradiol, we also quantified the expression of vascular adhesion molecules, notably intercellular adhesion molecule 1 (ICAM-1), platelet endothelial cell adhesion molecule 1 (PECAM-1), and vascular cell adhesion molecule (VCAM).

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Male Wistar rats (6–8 weeks) from our departmental animal facilities were used. The animals were kept in a temperature- and humidity-controlled environment in a 12-h light-dark cycle, in groups of five rats per cage, and they were allowed free access to water and diet. All animal experiments were approved by the local Animal Care Committee.

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Intestinal I/R rat model

The rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 µg/kg, i.p.). The abdomen was opened, and the superior mesentery artery was clamped during 45 min (ischemia period). The vascular clamp was released, and the intestinal perfusion reestablished (reperfusion period, 2 h). At the end of the reperfusion period, the animals were killed under deep anesthesia by exsanguination via the abdominal aorta. Sham-operated rats were used as controls and a group of nonmanipulated rats was added to obtain normal values of the variables studied (basal).

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Lung myeloperoxidase activity after intestinal I/R

Neutrophil presence in the lung was indirectly determined using the myeloperoxidase (MPO) activity assay. To this purpose, samples of lung tissue were obtained from rats killed after the intestinal reperfusion period. The lungs were perfused via pulmonary artery with phosphate-buffered saline (pH 7.0) containing 5 IU/mL heparin. The samples were processed as described elsewhere (21). Briefly, samples of lung homogenates (10 µL) were incubated for 15 min with H2O2 and O-dianisidine (Sigma, St Louis, Mo). The reaction of MPO activity was stopped by the addition of NaN3 (1%). Absorbance was determined at 450 nm using a microplate reader (Bio-Tek Instruments, Winooski, Vt), and the data were expressed as MPO activity per milligram of tissue.

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Lung microvascular leakage

The Evans blue (EB) dye extravasation technique was used to evaluate the lung vascular permeability. Briefly, rats were injected with EB dye (20 µg/kg, i.v.; Sigma), 20 min before the end of intestinal reperfusion period (2 h). After the animals were killed, the lungs were perfused by pulmonary artery as described above. Two samples of the lung parenchyma were taken and weighed. One fragment of lung tissue was incubated in formamide (4 mL/g wet weight) at room temperature for 24 h. The other fragments were dried in an oven at 60°C for at least 48 h, and the corresponding dry/wet tissue weight ratio was then calculated to avoid undervaluation of changes of permeability due to edema formation. The concentration of EB dye extracted to formamide was determined spectrophotometrically at 620 nm (Bio-Tek Instruments) using a standard curve (0.3–100 µg/mL of the dye in formamide). The amount of extravasated dye was expressed as micrograms per gram of dry tissue weight.

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Measurement of lactate dehydrogenase

Lactate dehydrogenase (LDH) activity in serum was determined from samples using commercially available kits (Labtest Diagnóstica, Lagoa Sonata, Brazil) and expressed as units per liter. Basal values were obtained using samples from nonmanipulated rats. Absorbance was determined at 340 nm in a microplate reader (Bio-Tek Instruments).

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Lung tissue culture (explant)

Lung tissue culture was performed according to Proust et al. (22). Briefly, samples of lung parenchyma were taken, and each sample was cut in small pieces (2 × 2 mm), which were then incubated in 24-well plastic plates (four pieces per well) with 1 mL of Dulbecco modified Eagle media (DMEM) for 24 h at 37°C in a humidified atmosphere with 5% CO2. After the incubation, the media was removed and stored in aliquots at −80°C. The tissue pieces were dried in an oven at 60°C and subsequently weighed.

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Determination of total nitric oxide

Total nitric oxide (NOx) was determined by measuring the formation of the stable oxidation products of NO, namely, nitrite (NO2) and nitrate (NO3). Nitric oxide in oxygen-containing solutions is chemically unstable and undergoes rapid oxidation to NO2. Various tissue components catalyze this oxidation and promote further oxidation of NO2 to NO3. Therefore, we measured both NO2 and NO3 to accurately determine the level of total NO.

NO3 in lung explant media was first reduced to NO2 through the incubation of the samples with nitrate reductase (0.15 U/mL). Thereafter, the concentrations of NO2 in the samples were determined by Griess reagent reaction. The control values consisted of data from sample media obtained of basal group of rats. The nitrite levels were obtained using a standard curve of NaNO2 (5–60 µM) at optical density (at 540 nm) using a microplate reader (Bio-Tek Instruments).

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Quantification of cytokines

Interleukin 10 (IL-10) and IL-1β were quantified in media aliquots from lungs explants using Duo Set commercial kit (R&D Systems, Minneapolis, Minn). The data were obtained by absorbance (450 nm) in a microplate reader (Bio-Tek Instruments). The results are expressed as picograms per milligram of dry weight (lung tissue).

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Immunohistochemical analyses of lung tissue

After the animals were killed, the left lobe of the lung was expanded by intratracheal injection of OCT media (optimal cutting temperature, 5 mL). The lung was immediately frozen in nitrogen-hexane solution. Cryosections (8 µm) were fixed in cold acetone during 10 min. Nonspecific sites were then blocked by incubation at room temperature with Tris-buffered saline/Tween 20 containing bovine serum albumin (0.5%) during 15 min. The sections were incubated overnight (in a humidified box, 4°C) with the selected primary antibodies. The sections were then incubated during 15 min with H2O2 (2%) solution to block the endogenous peroxidase. Subsequently, the sections were incubated for 2 h at 37°C, with the correspondent peroxidase-conjugated secondary antibody. The sections were rinsed and stained with chromogen (AEC [3-amino-9-ethylcarbazole]). AEC-stained objects in vessel walls were identified after a threshold determination, and the number of objects per area was quantified using an image analyzer (NIS-elements; Nikon, Tokyo, Japan). The background reaction was determined in lung sections incubated in the absence of primary antibody (negative control). Primary and secondary antibodies (all purified as immunoglobulin G) were as follows: (primary antibodies) anti-CD31 (PECAM-1) and anti-VCAM (Millipore, Billerica, Mass) and anti-ICAM (Cedarlene, Burlington, Ontario, Canada); secondary antibodies: horseradish peroxidase–conjugated goat anti–mouse (Chemicon International, Temecula, Calif).

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Pharmacological treatments

Groups of rats were treated 30 min after clipping the superior mesenteric artery (SMA, ischemia period) or 1 h after the removal of the clamp (intestinal reperfusion period) with a single intravenous injection of 17β-estradiol (280 µg/kg) dissolved in sterile saline.

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Statistical analysis

Data are represented as mean ± SEM. Comparisons between groups were made by analysis of variance followed by Student-Newman-Keuls post hoc test using the GraphPad Prism software (version 5.0, La Jolla, Calif). P < 0.05 was considered significant.

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ALI induced by intestinal I/R (effects of estradiol treatment)

Lung MPO activity (Fig. 1A) was increased by intestinal I/R in comparison to control groups. Treatment of rats with estradiol at the time of intestinal ischemia (30 min) or at the first hour of intestinal reperfusion was effective in reducing the MPO activity. However, the MPO activity after estradiol treatment at 1 h of intestinal reperfusion was lower when compared with treatment at the ischemia phase.

Intestinal I/R significantly increased the extravasation of EB dye in the lung in comparison to controls. Both protocols of treatment with E2 significantly reduced the dye extravasation (Fig. 1B), but the magnitude of this reduction was higher when the steroid was given at the ischemia phase.

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Effects of in vivo treatment with estradiol on the levels of cytokine in the lung explants

The concentrations of IL-10 and IL-1β were quantified in aliquots of cultured lung explants. Figure 2 shows that the levels of these mediators were significantly higher in samples obtained from intestinal I/R groups than in sham-operated animals. Estradiol treatment at the time of intestinal ischemia was effective in reducing the levels of IL-10 and IL-1β in the explant media. On the other hand, only IL-10 levels were reduced when estradiol treatment was carried out 1 h after unclamping of SMA.

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Effect of in vivo treatment with estradiol on the levels of NOx

As observed in Figure 3, the levels of NOx in the supernatant of lung explants collected after intestinal I/R were significantly increased when compared with sham-operated group. The estradiol treatment given after 30 min of intestinal ischemia or 1 h after clamp removal did no alter NOx levels.

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LDH activity after intestinal I/R

Serum levels of LDH can be considered as a systemic parameter of tissue injury. Figure 4 shows that after the intestinal I/R an increased serum activity of LDH was observed in comparison to sham-operated group.

The treatment of rats with estradiol at the 30th min of ischemia reduced the activity of LDH in serum, a result that was not observed when the hormone was administered at the first hour of intestinal reperfusion period.

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Effects of estradiol treatment on the expression of adhesion molecules in lung vessels

Figure 5 shows the expression of ICAM-I (Fig. 5A), PECAM-I (Fig. 5B), and VCAM (Fig. 5C) in lung vessels induced by intestinal I/R. In the figure, we show that intestinal I/R augmented the expression of ICAM-I in comparison to sham-operated group. Both protocols of estradiol treatment were effective in attenuating the increased expression of ICAM-I. In contrast, neither intestinal I/R nor estradiol treatment altered PECAM-I expression. On the other hand, whereas intestinal I/R did not modify the basal expression of VCAM, the treatment of rats with estradiol was effective in decreasing the basal expression of VCAM (Fig. 6).

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In this study, we show that estradiol attenuates the lung inflammation induced by intestinal I/R in male rats. This is an interesting finding because ALI accounts for respiratory dysfunction and systemic inflammation and can evolve to a multiple organ failure condition. Efforts to better understand the underlying mechanisms of intestinal I/R are important, and in this context, a possible beneficial role of estradiol on the lung injury after ischemic events could open a valuable clinical prospect.

A number of studies indicate that female sex hormones are linked to immune regulation, and in this scenario, an increased number of studies on the role of estradiol as modulator of lung health or inflammatory response have been published in the last 10 years (8–10). Clinical and experimental data reveal that estradiol is also implicated in protecting the function of the female immune system (23). In general terms, an ischemic event in a given organ or structure followed by reperfusion can trigger an inflammatory network. In this context, I/R accounts for an unbalance between proinflammatory and anti-inflammatory mediators, neutrophil and endothelial cell activation, and increased microvascular permeability.

Based on the salutary effects of estradiol on female rodents subjected to gut trauma (24), in this study, we focused our attention on the effects of estradiol given to male rats at the ischemia or reperfusion phases. More importantly, because reperfusion is also associated to tissue injury (4), our present data suggest that estradiol can exert a therapeutic effect, being in line with reports indicating that this hormone plays an important role in inflammation. Indeed, 17β-estradiol plays a protective role after I/R of heart (25), brain (26), and liver (27). Furthermore, 17β-estradiol can also improve cell and organ functions after trauma and hemorrhagic shock (10, 28, 29). The gut of female rats is more resistant to the harmful effects of a hemorrhagic shock than the gut of male rats, reinforcing the role of female sex hormones on these events (30). Overall, our data identified that estradiol might mediate the magnitude of lung inflammation caused by intestinal I/R.

Although being nonspecific, the elevation of LDH activity in the serum is a highly sensitive marker of tissue damage (31). Our results revealed augmented levels of LDH in serum, thus confirming tissue damage caused by intestinal I/R. Interestingly, estradiol treatment reduced LDH levels; such an effect was observed only when it was given at the time of intestinal ischemia. We have also showed that estradiol treatment reduced the MPO activity and EB dye extravasation in lungs after intestinal I/R. Noteworthy, these effects were observed when estradiol was given at the time of ischemia and at the intestinal reperfusion period. Notwithstanding intestinal perfusion was reestablished, estradiol was effective to reduce the ALI. Therefore, our data support the view that estradiol could represent a putative alternative to control the progress of inflammatory events triggered by the reperfusion of an ischemic insult.

Cell trafficking among compartments is mediated by adhesion molecules. In this context, our data showing that intestinal I/R increases the expression adhesion molecule ICAM-1 are in line with experimental evidence showing increased expression of ICAM-1 and increased neutrophil lung influx after intestinal I/R (32). Our hypothesis was that estradiol could mediate the reduction of the MPO activity by interfering with the expression of adhesion molecules. Indeed, our data revealed that estradiol reduced the expression of ICAM-1. Interestingly, estradiol treatment at the time of ischemia blunted the ICAM-1 expression. Moreover, when given at 1 h of the intestinal reperfusion, estradiol was effective to reduce the ICAM-1 expression. In contrast, PECAM-1 and VCAM expression were not affected by intestinal I/R. Taking these results into account, we hypothesize that upon ischemic and reperfusion events, estradiol could be considered as a pharmacological tool to modulate ALI.

Recently, we have shown that intestinal I/R induces lung of male rodents to generate inflammatory mediators (33). Here, we confirm this ability and demonstrate that lung generation of IL-1β and IL-10 is negatively mediated by estradiol. Interestingly, we identified a selective effect of estradiol on IL-1β release by the lung. Indeed, estradiol treatment appears to be effective in this regard only when occurring during the ischemic period. This selectivity of estradiol treatment diverges in what IL-10 is concerned, because the hormone was effective to reduce IL-10 generation irrespective of the time of ischemia or reperfusion. Thus, we suggest that once the inflammatory response was decreased (by IL-1β), the production of anti-inflammatory IL-10 is reduced as part of an endogenous control of inflammatory response. Our data allowed us to infer that estradiol acts through distinct pathways evoked by intestinal I/R. In fact, estradiol mediates the cell traffic to the lung and the lung release of IL-1β and IL-10. In summary, as a result of estradiol effects, the lung inflammatory response induced by intestinal I/R might be mitigated.

One important aspect of estradiol effects is its ability to mediate NO generation (34). It has been suggested that neutrophil recruitment and the increased microvascular permeability observed after intestinal I/R might be mediated by NO (35). In addition, estradiol modulates the NO generation by lung of female rats submitted to intestinal I/R (24). Thus, we investigated whether the observed generation of NO by the lung explant after intestinal I/R could be modulated by estradiol in male rats. Our present data rejected this hypothesis, because both schedules of treatments with estradiol (at the time of ischemia and 1 h after intestinal reperfusion) did not alter the values of NO generated by lung tissue. Taken together, our results suggest a sex difference in the regulatory mechanisms of estradiol on the lung inflammatory response caused by intestinal I/R.

We showed that estradiol was effective in reducing lung inflammation even after 1 h of the reestablishment of intestinal perfusion. Thus, our data could indicate that the effects of estradiol are important in the modulation of an already established inflammatory response. It is noteworthy that the estradiol effects observed in this study may be associated to its acute effects and could be considered a potential therapeutic benefit.

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Intestinal ischemia and reperfusion; lung inflammation; estradiol; rat; neutrophils

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