α7nAChR Agonist Activates the Nrf2/HO-1 Signaling Pathway to Reduce Acute Lung Injury Induced by Endotoxic Shock


 Objective: Alpha 7 nicotinic acetylcholine receptors (α7nAChRs) can inhibit the activation of macrophages and the production of pro-inflammatory cytokines and exert inhibitory effects on systemic and local inflammatory responses. The objective of this study was to observe the protective effect of α7nAChR agonist against acute lung injury (ALI) caused by endotoxic shock, and to explore the regulatory mechanism of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. Methods: A total of 40 Sprague-Dawley rats were randomly divided into sham operation group (Sham group, n=10), endotoxic shock-induced ALI model group (ALI group, n=10), ALI + α7nAChR agonist (PHA568487, 3134, Tocris Bioscience, USA) group (PHA group, n=10) and ALI + α7nAChR agonist + Nrf2 inhibitor (ML385, HY-100523, MCE, USA) group (ML group, n=10). The rats received a tail vein injection of LPS to initiate ALI. Six hrs after injection, arterial blood was analyzed for blood gases and lung wet weight/dry weight (W/D) was determined. Lung histopathology was determined by H&E staining and apoptosis quantified by TUNEL. Levels of intercellular adhesion molecule-1 (ICAM-1), TNF-α, IL-1β, malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD), choline acetyltransferase (ChAT) and acetylcholine esterase (AchE) in bronchoalveolar lavage fluid were measured via ELISA. Western blotting revealed levels of nuclear factor kappa-B (NF-κB), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), phosphatidylin-ositol-3-kinase (PI3K), protein kinase B (Akt), phosphorylated Akt (p-Akt), HO-1, Nrf2, thioredoxin reductase 1 (Trx-1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Results: It was found that α7nAChR agonist increased the partial pressure of oxygen (PaO2) and pH, reduced the partial pressure of carbon dioxide (PaCO2) and W/D ratio, alleviated pulmonary edema and oxidative stress injury, and suppressed inflammatory responses. At the same time, it activated PI3K to phosphorylate Akt, inhibited cell apoptosis, and protected the lung tissues of ALI rats. Moreover, α7nAChR agonist facilitated nuclear translocation of Nrf-2 and up-regulated HO-1 and Trx-1 expression. Nrf-2 activity was required for the protective effect of α7nAChR . Conclusion: α7nAChR agonist can improve endotoxic shock-induced ALI by activating the cholinergic anti-inflammatory pathway and the Nrf2/HO-1 signaling pathway.

Results: It was found that α7nAChR agonist increased the partial pressure of oxygen (PaO 2 ) and pH, reduced the partial pressure of carbon dioxide (PaCO 2 ) and W/D ratio, alleviated pulmonary edema and oxidative stress injury, and suppressed in ammatory responses. At the same time, it activated PI3K to phosphorylate Akt, inhibited cell apoptosis, and protected the lung tissues of ALI rats. Moreover, α7nAChR agonist facilitated nuclear translocation of Nrf-2 and up-regulated HO-1 and Trx-1 expression. Nrf-2 activity was required for the protective effect of α7nAChR .
Conclusion: α7nAChR agonist can improve endotoxic shock-induced ALI by activating the cholinergic anti-in ammatory pathway and the Nrf2/HO-1 signaling pathway.

Background
Endotoxin-induced acute lung injury (ALI) is a common complication occurring after septic shock or systemic in ammatory responses, with high morbidity and mortality rates [1]. It is pathologically manifested as increased permeability of pulmonary capillary endothelium and alveolar epithelium, which leads to non-cardiogenic pulmonary edema and ventilation/perfusion mismatch, thus causing acute respiratory distress syndrome (ARDS) [2,3]. ALI/ARDS is more serious and complicated than the original disease. In the development process of ALI/ARDS, the lung tissues suffer from the activation and aggregation of in ammatory cells and oxidative free radical injury, and the synthesis and excessive release of in ammatory mediators can lead to uncontrolled in ammatory responses, immune dysfunction, pulmonary microcirculation disturbance, etc [4][5][6]. Therefore, determining the mechanisms of in ammatory pathogenesis will be clinically signi cant for improving the prognosis of ALI/ARDS and reducing its mortality rate. Alpha 7 nicotinic acetylcholine receptors (α7nAChRs) play a vital role in the regulation of the body's peripheral in ammation. Receptor activation inhibits macrophage activation through intracellular signal transduction, thus suppressing the production of the pro-in ammatory cytokines, TNF and ILs, and exerts inhibitory effects on local and systemic in ammatory responses in the body [7]. In mouse models of Escherichia coli-induced pneumonia, applying an α7nAChR agonist can reduce the level of the chemokine monocyte chemotactic protein-2 (MIP-2) in alveolar lavage uid, the extravascular exudation of neutrophils, and the mortality rate of these rats [8]. Moreover, acetylcholine additionis able to remarkably reduce the release of various pro-in ammatory cytokines by LPS-stimulated human macrophages cultured in vitro., but this effect disappears after the knockout of α7nAChRs in rats. The above ndings suggest that α7nAChR agonists may play an anti-in ammatory role in ALI, whose mechanism is correlated with anti-oxidative stress and in ammatory stimulation.
Studies have revealed that nuclear factor erythroid 2-related factor 2 (Nrf2) dissociates from the Nrf2/kelch-like ECH-associated protein 1 (Keapl) complex under oxidative stress, which facilitates the nuclear translocation of Nrf2 and activates antioxidant response element (ARE)-mediated protein kinase pathways such as protein kinase C (PKC), phosphatidyl-inositol 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) pathways [9]. The target genes regulated by Nrf2 include stress genes and antioxidant genes, mainly including catalase (CAT), superoxide dismutase (SOD) and heme oxygenase-1 (HO-1). Among them, HO-1 is considered to promote adaptive cytoprotective responses against oxidative stress and in ammatory stimulation [10]. It can alleviate endotoxic shockinduced lung injury through resisting in ammation and oxidative stress and inhibiting cytokine release.

Preparation of endotoxic shock model
The rat model of endotoxic shock was established according to Martin A's method [11]. Rats were anesthetized by intraperitoneal injection of 2% pentobarbital sodium (3 mg/kg) and placed on a xed rack. Next, the ALI model was established by tail vein injection of LPS (Escherichia coli O55: B5, Sigma, USA). Reduction of MAP by 50% represents successful modeling. Sham group received a 200 μL tail vein injection of the vehicle, 10% dimethyl sulfoxide (DMSO). At 30 min after modeling, the PHA group received a tail vein injection of 0.4 mg/kg PHA568487, while MLA group underwent tail vein injection of 0.4 mg/kg PHA568487 and 6 mg/kg ML385.

Sample collection and detection
Six hours after LPS injection, rats were anesthetized with 2% pentobarbital sodium and xed on the operating table in the supine position. The abdominal cavity was cut open along the abdomen midline to separate tissues and expose abdominal aorta. Then the rats were killed by cardiac puncture and bloodletting, and blood was placed in GEM Premier 3000 blood gas analyzer to detect pH and partial pressures of oxygen (PaO 2 ) and carbon dioxide (PaCO 2 ). Thereafter, the chest skin was separated to fully expose the thoracic cavity and trachea. Next, right and left lung tissues were separated from 3 rats in each group to measure the wet weight/dry weight (W/D) ratio. For the remaining rats, the bronchus was ligated, and 22-G needle was inserted and connected with a syringe containing 1 mL of saline.
Subsequently, the cold saline was slowly injected into the lung, and bronchoalveolar lavage uid (BALF) was collected after repeated suction and centrifuged at 3000 rpm for 10 min. Supernatant was stored at -80°C until analysis. Following lavage, the whole lung tissues were placed on the surface of ice, followed by isolation of right and left lung tissues, with sections of lung tissue xed in 10% neutral formalin solution for hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and immuno uorescence (IF) detection. The remaining lung lobes were stored frozen under liquid nitrogen.

Determination of W/D ratio of lung tissues
The blood and impurities covering the surface of the whole lung tissues of rats were removed, and then the tissues were weighed using an electronic balance to obtain the wet weight. Next, the tissues were dried in a 65°C oven for 24 h, and dry weight determined. Finally, the ratio of W to D was calculated to quantify the edema of the lung tissues.

H&E staining
Formalin-xed lung tissue samples were placed sequentially into 70% alcohol for 12 h, 80% ethyl alcohol for 12 h, then 90% alcohol for 30 min, 95% alcohol for 30 min (repeated once), absolute alcohol for 30 min (repeated once), and nally xylene for 5 min. Subsequently, samples were soaked in para n.
Para n-embedded samples were sliced, subjected to H&E staining, and sealed with neutral gum, followed by observation under an optical microscope (CX33, Olympus Corporation, Japan).
Lung tissue slices were observed microscopically according to Smith's scoring standard [12]. Zero to four points were given, respectively, according to the extent of localized necrosis of the lung, thickening of the alveolar septum, enlargement of the alveolar cavity, and formation of pulmonary hyaline membranes according to the scale: 0 point for no damage, 1 point for lesion range <25%, 2 points for lesion range = 25-50%, 3 points for lesion range = 50-75 %, and 4 points for lesion range >75%. The average score was recorded from two pathologists who independently observed and analyzed 10 high power elds in each sample slice.

TUNEL staining
A TUNEL staining kit (C1086, Beyotime, China) was used to detect apoptosis in lung tissue cells. Lung tissue slices were depara nized by two cycles of xylene, for 5 min each time. After gradient alcohol soaking, these slices were dropwise added with 20 μg/mL DNase-free proteinase K (20 mg/mL) for 30 min of incubation at 37°C, and washed by phosphate-buffered saline (PBS) twice. Each slice was incubated in 50 μL of TUNEL test solution for 1 hr at 37°C in the dark, followed by PBS washing 3 times. At last, the slices were observed under a uorescence microscope after mounting using anti-uorescence quenching mounting solution, and pictures were taken.
Enzyme-linked immunosorbent assay (ELISA) ELISA kit (USCN, China) was utilized to detect the content of ICAM-1 TNF-α IL-1β IL-6 MDA MPO SOD ChAT and AchE in BALF according to instructions. Speci cally, 50 μL of appropriate standard was added to the rst and second wells of the rst row, respectively, on the ELISA plate, and diluted at multiple ratios in sequence. Then 40 μL of sample diluent was added to each sample well, and 10 μL of samples were added and incubated at 37°C for 30 min. Washing liquid was added to each well, incubated for30 s, then the liquid was discarded, which was repeated 5 times. Thereafter, 50 μL of enzyme-labeled reagents were added for 30 min of incubation at 37°C, and after washing, 50 μL of chromogenic reagent A and 50 μL of chromogenic reagent B were added, respectively, for 15 min of color development at 37°C in the dark.
Finally, 50 μL of stop buffer was added to each well, the absorbance at 450 nm was detected using a microplate reader, and the sample content was calculated according to the standard curve.

Detection of reactive oxygen species (ROS) via IF
Frozen lung tissues were sliced on a freezing microtome. Slices were incubated in antigen retrieval buffer for 10 min. Next, the normal goat serum was added for 15 min incubation at room temperature, and then decanted. After that, Mito-Tracker Red CMXRos working solution (C1049, Beyotime, China) was added, and then slices were washed by PBS thoroughly. DAPI solution was added after the slices were slightly dried for incubation at room temperature for 10 min away from light, followed by PBS washing 3 times.
Finally, anti-uorescence quenching mounting medium was applied for mounting, and photos were taken using the uorescence microscope.

Western blotting
Lung tissues were taken out from the liquid nitrogen, added with radioimmunoprecipitation assay (RIPA) lysate containing protease inhibitor for 30 min of lysis and centrifuged. Then the supernatant was taken to measure protein concentration, and SDS-PAGE was performed. Following proteintransfer to PVDF membranes and blocking, nuclear factor kappa B (NF-κB), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), PI3K, Akt, phosphorylated (p)-Akt, HO-1, Nrf2, thioredoxin reductase 1 (Trx-1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies diluted at 1:1000 were added for incubation at 4°C overnight. Then membranes were washed by Tris-buffered saline with Tween 20 (TBST), and horseradish peroxidase-labeled secondary antibodies were added for incubation at room temperature for 2 h. Subsequently, enhanced chemiluminescence (ECL) kit and gel imaging system were employed for color development of proteins, and Image Tools was used for analysis.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
A total of 1 mL of TRIzol (15596026, Invitrogen, USA) was added to the lung tissues in liquid nitrogen, and ribonucleic acids (RNAs) were extracted by chloroform method and reverse transcribed (K1622, Invitrogen, USA). Then HO-1, Nrf2, PKC-α and GAPDH primers were added separately, and qRT-PCR kit (204057, Qiagen, USA) was used to prepare the reaction system. Reaction conditions: pre-denaturation at 95°C for 30 s, and PCR at 95°C for 5 s and 60°C for 20 s for 40 cycles. The results were expressed via the 2 -△△Ct method. Primer sequences are listed in Table 1. Results α7nAChR agonist reduced endotoxic shock-induced ALI Due to the stimulation of toxins during septic shock, catecholamines in the body are increased, and blood ow perfusion in microcirculation, cardiac output, and blood pressure are decreased. As time goes on, there is constriction of capillary sphincters, tissue perfusion is reduced, tissue ischemia and hypoxia appear, and acidic products of anaerobic metabolism build up, resulting in a drop in blood pH, pulmonary capillary exudation, pulmonary edema formation, and hypoxemia. In this experiment, it was found that an α7nAChR agonist could prevent the drop in PaO 2 and pH, reduce PaCO 2 (vs. ALI group, p<0.05) (Figure 1-A) and W/D ratio, and relieve pulmonary edema in ALI rats (Figure 1-B). Using H&E staining, such histopathological changes as in ammatory cell in ltration, interstitial edema, and obvious thickening of the alveolar septum in the lung of ALI rats were observed. After the α7nAChR agonist was given, the degree of in ammatory cell in ltration in the lung tissues was lowered, interstitial edema was alleviated, alveolar septum thickening was decreased (Figure 1-C), and the score of lung injury was reduced (vs. ALI group, p<0.05) (Figure 1-D). The above results indicate that α7nAChR agonist can effectively treat endotoxic shock-induced ALI.

α7nAChR agonist inhibited in ammatory responses in ALI rats
The activation and aggregation of in ammatory cells in the lung tissues are the main causes of ALI/ARDS. When nAChR on immune cells are activated, local or systemic immune responses can be downregulated, thus inhibiting in ammation. In this study, after ALI-induced rats were treated with an α7nAChR agonist, ChAT and AChE were released (Figure 2-A), and the expression of NF-κB was inhibited (Figure 2-B). However, the inhibition of NF-κB could down-regulate ICAM-1, and block the release of proin ammatory cytokines, TNF-and IL-1β. Rats in PHA group exhibited reduced content of ICAM-1 IL-6, TNF-and IL-1β in BALF (Figure 2-C), suggesting that α7nAChR is able reduce the in ammatory lung responses of ALI rats.

α7nAChR agonist suppressed oxidative stress injury in ALI rats
Under the action of endotoxins, the body produces a large amount of reactive oxygen species (ROS), which cause cellular and organ damage. Based on this, ELISA was rstly carried out to detect the content of MDA, MPO and SOD in BALF. The α7nAChR agonist promoted SOD release and inhibited MDA and MPO (vs. ALI group, p<0.05) (Figure 3-A). IF revealed that the ROS level, compared to control group, was increased in ALI group and decreased in PHA group (Figure 3-B). It can be concluded from the above results that α7nAChR agonist inhibits oxidative stress injury in ALI rats. α7nAChR agonist activated the PI3K/Akt signaling pathway to alleviate lung cell apoptosis in ALI rats When ALI occurred, in ammatory cytokines were released, which elevated the expression of proapoptosis factor Bax in the lung, reduced the expression of anti-apoptosis factor Bcl-2, and thus increased the rate of apoptosis in lung tissue cells (Figure 4-A). Studies have shown that the activation of the PI3K/Akt signaling pathway can lower the apoptosis rate of alveolar type II cells and inhibit the expression of Bax, thus protecting the lung. TUNEL staining results in this study demonstrated that α7nAChR agonist reduced the LPS-triggered apoptosis of lung cells during ALI (Figure 4-B). Western blotting con rmed that α7nAChR agonist could stimulate PI3K to phosphorylate Akt, and the activated Akt acted on the downstream target proteins of the signaling pathway (Figure 4-C), blocked cell apoptosis, and protected lung tissues.
α7nAChR agonist activated the Nrf2/HO-1 signaling pathway When ALI caused lung oxidative stress, Nrf2 was activated with a decreased degradation rate, and the content of Nrf2 in the nucleus was increased signi cantly ( Figure 5-A). In addition, α7nAChR agonist was found to facilitate the entry of Nrf-2 to the nucleus and initiate the expression of downstream antioxidant proteins, HO-1 and Trx-1 ( Figure 5-B).
α7nAChR agonist relieved ALI through the Nrf2/HO-1 signaling pathway To further explore involvement the Nrf2/HO-1 signaling pathway, the rats were co-administered Nrf2 inhibitor with α7nAChR agonist, and the protective effect of α7nAChR agonist against ALI was observed. The pulmonary alveolar wall was thickened, in ammatory cell in ltration was observed, and injury scores were increased in ML group (vs. PHA group, p<0.05) (Figure 6-A). Moreover, the content of IL-6, TNF-α and IL-1β in BALF was increased ( Figure 6-B), the antioxidant factor SOD was decreased, and MDA and MPO were increased ( Figure 6-C) (vs. PHA group, p<0.05). Since the PI3K/Akt pathway is involved in the activation of Nrf2-ARE and the regulation of related dependent gene expressions, apoptosis and Akt activation were examined. It was discovered that Akt phosphorylation was inhibited ( Figure 6-D), and the apoptosis rate of lung tissue cells was raised in ML group (Figure 6-E). The above results suggest that α7nAChR agonist decreases the lung injury in ALI rats through the Nrf2/HO-1 signaling pathway.

Discussion
Imbalance in the regulatory function on immune in ammatory responses is a main cause of damage from sepsis. Under the action of endotoxins, the body produces a large amount of ROS, which leads to cellular damage and release of pro-in ammatory cytokines, thereby further causing the dysfunction of multiple organs, including the brain, lung and liver [13]. Therefore, in theory, the occurrence and mortality rate of ALI could be reduced by boosting intracellular anti-in ammatory and anti-oxidative stress pathways. In this study, a rat model of endotoxic shock-induced ALI was established by tail vein injection of LPS, and the effects of treatment with an α7nAChR agonist observed on lung injury, in ammatory responses, oxidative stress injury, and apoptosis. The α7nAChR agonist reduced endotoxic shocktriggered ALI and reduced in ammatory responses and oxidative stress injury, and this effect was prevented by inhibition of the Nrf2/HO-1 signaling pathway.
Nerves can exerting local or systemic anti-in ammatory effects by releasing acetylcholine, which combines with α7nAChRs on the surface of various immune cells to block the synthesis and release of pro-in ammatory cytokines by inhibiting or up-regulating intracellular downstream signaling pathways [14], thus ultimately alleviating tissue damage from excessive in ammation. After interfering with the production of a7nAChRs or knocking out a7nAChRs in rats, neither electric stimulation nor a7nAchR can inhibit the release of in ammatory cytokines such as TNF-α [15]. In the present study, α7nAChR agonist elevated PaO 2 and pH in ALI rats, alleviated pulmonary edema, and effectively inhibited in ammatory cytokine release and oxidative stress injury, suggesting that α7nAChR stimulation can effectively reduce lung injury in ALI rats.
The cholinergic anti-in ammatory pathway (CAP) can protectively downregulate immune responses. It has been con rmed that α7nAChRs are core receptors mediating CAP. When α7nAChRs are activated, cholinergic anti-in ammatory signals can play anti-in ammatory roles in cells through NF-κB, Janus activated kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and PI3K/Akt pathways [10,14]. As a speci c α7nChRA agonist, PHA568487 stimulates CAP activity through speci c binding to α7nAChRs [16]. In this study PHA568487 was given to ALI-induced rats, which effectively stimulated the release of AChE and ChAT, and inhibited NF-κB. PHA568487 activated PI3K to promote Akt phosphorylation, thus inhibiting the expression of Bax and reducing the apoptosis rate of lung cells.
Under normal physiological conditions, Nrf2 binds to cytoplasmic linker protein Keap1, contributing to Nrf2 degradation [9]. Under oxidative stress, proteasomes degradation is blocked Nrf2, resulting in Nrf2 accumulation. In this study, Nrf2 levels in the lung of ALI rats were increased, consistent with the involvement of Nrf2 in oxidative stress injury. HO-1 is a Nrf2-regulated gene that prevents vascular in ammation, and plays a crucial role in anti-apoptosis, anti-tumor, anti-in ammation, and antioxidant effects [9,17]. In the current research, it was found that both nuclear Nrf2 expression and HO-1 expression in the lung of ALI rats was up-regulated after PHA568487 intervention [18]. It has been illustrated in studies that the up-regulation of HO-1 protein expression and subsequent increase in CO concentration in the liver, kidney and lung tissues of rats with endotoxic shock can reduce the mortality rate of rats, thereby protecting the above organs. Correspondingly, Nrf2 inhibitor caused a rise in in ammatory cytokines, aggravation of oxidative stress injury, and increase in apoptosis by blocking the effect of α7nAChR agonist, implying that α7nAChR agonist may relieve the lung injury in ALI rats through the Nrf2/HO-1 signaling pathway.
To sum up, α7nAChR agonist activates the CAP and the Nrf2/HO-1 signaling pathway to inhibit in ammatory responses, relieve oxidative stress injury, lower the apoptosis rate and alleviate pulmonary edema, and thus thus should be further investigated as a potential method for treatment of endotoxic shock-induced ALI.