Protecting rat lungs from injury caused by blood flow loss and return by targeting Rev-Erbα

In adherence to the ethical guidelines set forth in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, National Academy Press 1996), meticulous measures were implemented to uphold the welfare of the rats involved in this research. The experimental design obtained approval from the Animal Review Committee of the National Defense Medical Center, Taipei, Taiwan (Permit Number: IACUC-20-216). For the establishment of isolated perfused rat lungs, male Sprague-Dawley rats weighing 350 ± 20 g were subjected to ventilation using a gas mixture composed of 21% O₂ and 5% CO₂ at a rate of 60 breaths per minute. The tidal volume was set at 3 ml, while a positive end-expiratory pressure of 1 cm H₂O was maintained, as previously described [10]. Heparin, at a dosage of 1 U per gram of body weight (BW), was administered via injection into the right ventricle, accompanied by the withdrawal of 10 cc of blood from the same location. Cannulation of the pulmonary artery and left ventricle was performed using silicon tubing, enabling the perfusion of the lungs with a physiological salt solution supplemented with 4% bovine serum albumin. To create a “half-blood” solution, the collected rat blood (10 cc) was mixed with the perfusate prior to recirculation. Throughout the experiment, the isolated lung was placed on an electronic balance to enable the continuous monitoring of real-time changes in lung weight (LW). Additionally, both the pulmonary venous pressure (PVP) and pulmonary arterial pressure (PAP) were constantly recorded using pressure transducers connected to the side arm of the cannula, following established techniques [11].

In accordance with previously established methods, we sought to ascertain the capillary permeability (K_f) by evaluating the augmentation in lung weight induced by an elevation in venous pressure. The quantification of K_f was subsequently expressed in milliliters per minute per centimeter water per 100 g of lung tissue, following the established protocol [12, 13].

The present study employed a randomized experimental design in which rats were assigned to one of eight groups. These included a control group that received dimethyl sulfoxide (DMSO) (n = 6), a drug control group that received 50 mg/kg of SR9009 (50 mg/kg BW, a Rev-Erbα agonist, intraperitoneal injection (i.p.), Sigma-Aldrich, USA), a group subjected to lung IR (IR, n = 6), and three groups that received different doses of SR9009 (12.5 mg, 25 mg, or 50 mg/kg BW) along with lung IR (n = 6 per group), IR + SR8278 (5 mg/kg BW, a Rev-Erbα antagonist), or IR + SR8278 (5 mg/kg BW) + SR9009 (50 mg/kg BW) group. To facilitate intraperitoneal injection, SR9009 was dissolved in 0.5% DMSO and administered 60 min before inducing lung IR. In the IR + SR8278 + SR9009 group, rats were pretreated with SR8278 for 30 min prior to SR9009 injection. The dosages of SR9009 and SR8278 used in this study were based on previous research and preliminary investigations [7, 9] (Supplementary Fig. 1). Lung IR was induced by stopping ventilation and perfusion, leading to ischemia, and then maintaining the lungs in a deflated state for 40 min, followed by 60 min of ventilation and perfusion. In contrast, the control group received perfusate alone for 100 min.

Following the experiment, the right lung was surgically removed at the hilar region. The wet weight of the lung was measured, and the lung weight-to-body weight (LW/BW) ratio was calculated to evaluate the degree of lung edema. Furthermore, a portion of the right upper lung lobe was excised and weighed, and then dried for 48 h at 60 °C to obtain its dry weight. Subsequently, the wet-to-dry (W/D) weight ratio was computed.

Following the experiment, the left lung underwent two rounds of lavage using 2.5 ml of saline to obtain BALF. This fluid was subsequently subjected to centrifugation at 200 × g for 10 min at room temperature. The protein content of the BALF was assessed using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Commercial rat ELISA kits (R&D Systems Inc., Minneapolis, MN, USA) were employed to measure BALF levels of tumor necrosis factor-α (TNF-α), cytokine-induced neutrophil chemoattractant-1 (CINC-1), and interleukin-6 (IL-6). The total cell count in the BALF was determined utilizing a previously described method [11, 13].

Malondialdehyde (MDA) levels were assessed using an established protocol [13]. The measurement involved determining the reaction product of MDA with thiobarbituric acid reactive substances at an absorbance of 532 nm. The resulting values were expressed in nmol/mg protein. To measure the levels of glutathione and hydrogen peroxide (H₂O₂) in the lung tissues, a fluorometric glutathione detection assay kit and a Hydrogen Peroxide Assay Kit from Abcam (Cambridge, MA, USA) were utilized.

Lung tissue homogenate and cell protein lysates, with a protein amount of 30 µg per lane, were subjected to separation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10–12% gel as described in a previous study [13]. Following separation, the proteins were transferred onto polyvinylidene fluoride membranes, following the established protocol. To investigate the presence of specific proteins, the membranes were probed with primary antibodies against β-actin, Rev-Erbα, Bcl-2, NF-κB p65, phospho-NF-κB p65, IκB-α, extracellular signal-regulated kinase( ERK)1/2, phospho-ERK1/2, c-Jun N-terminal kinase (JNK), phospho-JNK, p38, phospho-p38, Protein kinase B (Akt), and phospho-Akt. The obtained results are presented as the relative ratio of the target protein to the reference protein, allowing for the quantification and comparison of protein levels within the samples.

In accordance with previously published methods, the immunohistochemical staining protocol for myeloperoxidase (MPO) was employed [14]. Lung tissue sections, measuring 4 mm in thickness, were obtained from paraffin blocks. To eliminate peroxidase activity, the sections were subjected to dewaxing and treated with 3% H₂O₂ and 100% methanol for a duration of 15 min. For MPO staining, a primary anti-MPO rabbit antibody (diluted at 1:100, obtained from Thermo Fisher Scientific, Rockford, IL, USA) was applied to the sections, followed by three washes with phosphate-buffered saline (PBS) for 5 min each. Subsequently, a rat-specific horseradish peroxidase polymer anti-rabbit antibody (manufactured by Nichirei Corporation, Tokyo, Japan) was added and allowed to incubate for 30 min. The sections were then washed thrice with PBS. To visualize the MPO staining, a horseradish peroxidase substrate was applied for a duration of 3 min. Hematoxylin was used as a counterstain for the sections. All staining tests were performed in triplicate to ensure consistency and reliability of the results.

To examine the tissue architecture and detect any pathological alterations, hematoxylin and eosin staining were applied on lung tissue sections that were embedded in paraffin. The amount of polymorphonuclear neutrophils present in the interstitium was measured by counting cells in 10 high-power field views (×400) chosen randomly and then averaging them. This was done by two laboratory technicians who were unaware of the slide’s source. Furthermore, a semi-quantitative grading was carried out on the hematoxylin and eosin sections to evaluate the extent of pathological lesions, as previously described [15] .

The TUNEL assay was performed on 5 mm-thick paraffin-embedded lung tissue sections using the FragELTM DNA Fragmentation Detection Kit and Fluorescent-TdT Enzyme (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s instructions. TUNEL-positive nuclei were detected through fluorescence microscopy.

The MLE-12 cells were cultured in DMEM/F-12 medium, which was supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (10 mg/mL). The cells were maintained at a temperature of 37 °C and a CO₂ concentration of 5% in a humidified air environment, following previously established protocols [12, 16]. To induce hypoxia-reoxygenation (HR), the MLE-12 cells were subjected to 2 h of hypoxia (1% O₂, 5% CO₂, and 94% N₂) at 37 °C, followed by 1 h of reoxygenation (5% CO₂ and 95% room air). Prior to HR induction, the cells were treated with different substances: either a control vehicle, SR9009 (10 µM), or Rev-Erbα siRNA (25 nM). The dosage of SR9009 and Rev-Erbα siRNA was determined through reference to a prior study [17, 18] and analysis of Western blot results (Supplementary Fig. 2). Transfection of Rev-Erbα siRNA was accomplished by incubating the cells with DharmaFECT™ 1 siRNA transfection reagent (Dharmacon Inc. Chicago, IL, USA) for 24 h. As negative controls, MLE-12 cells treated with a non-targeting control siRNA were used. After 24 h, the culture medium was replaced with the appropriate medium. At 48 h from the initiation of Rev-Erbα siRNA treatment, the MLE-12 cells were exposed to 2 h of hypoxia followed by 1 h of reoxygenation. Protein extraction was conducted by lysing the cells, and Western blot analysis was performed. Additionally, the supernatant was collected and subjected to keratinocyte chemoattractant (KC) analysis using a mouse KC/CXCL1 ELISA kit (R&D, Inc.). All experiments were conducted in triplicate.

The data obtained from the experiments were subjected to statistical analysis using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). The results are expressed as means accompanied by standard deviations. To assess the differences between the groups, a one-way analysis of variance (ANOVA) was performed, followed by post-hoc Bonferroni tests for conducting multiple comparisons. Lung weight gain and pulmonary artery pressure (PAP) were compared among the groups using a repeated measures two-way ANOVA, followed by post-hoc Bonferroni tests. The threshold for determining statistical significance was set at a p-value of less than 0.05.

In comparison to the control group treated with a vehicle, the induction of IR injury resulted in a notable increase in lung weight gain, vascular filtration coefficient (K_f), ratio of lung weight to body weight (LW/BW), ratio of wet to dry lung weight (W/D weight), and protein concentrations in the BALF. In addition, the administration of SR8278 exhibited a trend towards exacerbating IR damage, although this trend did not reach statistical significance (not shown). Conversely, rats that received pretreatment with SR9009 showed a significant reduction in IR-induced lung edema in a dose-dependent manner, as demonstrated in Supplementary Fig. 1A-F. Notably, the protective effects of SR9009 on IR-induced lung edema were significantly diminished when the SR8278 was added, as shown in Fig. 1A-E.

Upon induction of IR, there was an initial rapid rise in pulmonary artery pressure (PAP), which later declined after reperfusion, as indicated in Fig. 1F. Even after 60 min of reperfusion, the PAP in the IR-exposed lungs remained significantly higher compared to the baseline and the control group, where PAP remained stable during the 100-minute observation period. Administration of SR9009 effectively mitigated the IR-induced changes in PAP, displaying a significant suppression of PAP alterations (p < 0.05; Fig. 1F). However, the addition of SR8278 significantly counteracted the protective effects of SR9009 on preventing the elevation of PAP.

The levels of TNF-α, CINC-1, IL-6, and total cell counts in BALF showed a significant increase in the IR group compared to the control group (p < 0.05; Fig. 2). In contrast, rats pretreated with SR9009 exhibited a significant reduction in TNF-α, CINC-1, IL-6 levels, and total cell count in BALF induced by IR injury, when compared to rats subjected to IR injury alone (Fig. 2). However, the protective effects of SR9009 on the IR-induced increase were significantly blocked by the addition of SR8278 (Fig. 2).

The Western blot analysis revealed a notable decrease in the expression levels of Rev-Erbα proteins in the lung tissue of the IR group when compared to the control groups (p < 0.05; Fig. 3). However, treatment with SR9009 significantly increased the expression of Rev-Erbα protein compared to the IR group (p < 0.05; Fig. 3). Notably, the protective effects of SR9009 on the IR-induced increase in Rev-Erbα protein expression were significantly blocked by the addition of SR8278.

Compared to the control group, the IR group exhibited a significant rise in the number of MPO-positive cells, as well as increased levels of MDA and H₂O₂, along with a reduction in glutathione level in lung tissue (p < 0.05; Fig. 4A–D). Conversely, the administration of SR9009 significantly attenuated these IR-induced effects. However, the addition of SR8278 blocked the protective effects of SR9009.

The histological assessment exhibited a significant increase in interstitial thickening and cellular infiltration in the IR group in comparison to the control group (Fig. 5A). Nonetheless, the administration of SR9009 substantially decreased these histological changes, neutrophil infiltration (Fig. 5B), and lung injury scores (Fig. 5C) in the IR group. Moreover, the addition of the SR8278 significantly blocked the effect of SR9009.

The number of TUNEL-positive cells (Fig. 6A) and the protein levels of cleaved caspase-3 (Fig. 6B) were significantly higher, and the protein level of Bcl-2 was significantly lower (Fig. 6C) in the lungs of the IR group compared to the control group. But SR9009 treatment significantly attenuated the severity of these apoptosis-related changes in lungs exposed to IR. However, the addition of the SR8278 significantly blocked the effect of SR9009.

Figure 7 A and C show a significant increase in the levels of NF-κB p65 in the nucleolus and Akt phosphorylation in the IR group. Conversely, Fig. 7B demonstrates a significant decrease in the cytoplasmic level of IκB-α compared to the control group. Treatment with SR9009 resulted in a significant rise in IκB-α levels while reducing NF-κB p65 levels and Akt phosphorylation. However, the addition of SR8278 effectively blocked the effects of SR9009.

Activation of the mitogen-activated protein kinase (MAPK) pathway, including phosphorylation of ERK, JNK, and p38, was significantly increased in lung tissue due to IR. However, the administration of SR9009 successfully decreased the activation of all three MAPKs induced by IR, as shown in Fig. 8A-C. Notably, the addition of SR8278 reversed the effect of SR9009.

In MLE-12 cells from the HR group, Western blot analysis revealed a significant decrease in the expression levels of Rev-Erbα proteins compared to the control groups (p < 0.05; Fig. 9A). However, treatment with SR9009 resulted in a notable increase in Rev-Erbα protein expression compared to the HR group (p < 0.05; Fig. 9A). Furthermore, SR9009 demonstrated a reduction in the elevated levels of KC and phospho-NF-κB p65 protein induced by HR in MLE-12 cells. Additionally, SR9009 attenuated the HR-induced decrease in IκB-α protein expression. To investigate the role of Rev-Erbα protein in the beneficial effects of SR9009, siRNA was used to silence the Rev-Erbα gene in MLE-12 cells. The introduction of Rev-Erbα siRNA effectively blocked the beneficial effects of SR9009, providing further evidence of the involvement of Rev-Erbα protein (Fig. 9B-D).

Below is the link to the electronic supplementary material.

Additional file 1: Supplementary Figure S1. The effect of varying doses of SR9009 on pulmonary edema. Lung weight gain (A); vascular filtration coefficient (K_f)(B); lung weight/body weight (LW/BW) (C); wet/dry (W/D) weight ratio (D); protein concentration in bronchoalveolar lavage fluid (BALF)(E); and pulmonary artery pressure (F) increased significantly in the ischemia-reperfusion (IR) group. The increase in these parameters was significantly attenuated by treatment with SR9009 in dose-dependent manner. Data are expressed as mean ± SD (n = 6 per group). *p < 0.05, **p < 0.01, ***p < 0.001 compared to control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared to IR group

Additional file 2: Supplementary Fig. 2. Rev-Erbα siRNA was transfected into MLE-12 cells at different concentrations, and the expression of Rev-Erbα and β-actin (used as a loading control) was examined through immunoblotting.