The role of annexin A1 peptide in regulating PI3K/Akt signaling pathway to reduce lung injury after cardiopulmonary bypass in rats

Introduction Cardiopulmonary bypass (CPB) –induced lung ischemia-reperfusion (I/R) injury remains a large challenge in cardiac surgery; up to date, no effective treatment has been found. Annexin A1 (AnxA1) has an anti-inflammatory effect, and it has been proven to have a protective effect on CPB-induced lung injury. However, the specific mechanism of AnxA1 in CPB-induced lung injury is not well studied. Therefore, we established a CPB-induced lung injury model to explore the relevant mechanism of AnxA1 and try to find an effective treatment for lung protection. Methods Male rats were randomized into five groups (n = 6, each): sham (S group), I/R exposure (I/R group), I/R + dimethyl sulfoxide (D group), I/R + Ac2-26 (AnxA1 peptide) (A group), and I/R + LY294002 (a PI3K specific inhibitor) (AL group). Arterial blood gas analysis and calculation of the oxygenation index, and respiratory index were performed. The morphological changes in lung tissues were observed under light and electron microscopes. TNF-α and IL-6 and total protein in lung bronchoalveolar lavage fluid were detected via enzyme-linked immunosorbent assay. The expressions of PI3K, Akt, and NF-κB (p65) as well as p-PI3K, p-Akt, p-NF-κB (p65), and AnxA1 were detected via western blotting. Results Compared with the I/R group, the A group showed the following: lower lung pathological damage score; decreased expression of IL-6 and total protein in the bronchoalveolar lavage fluid, and TNF-α in the lung; increased lung oxygenation index; and improved lung function. These imply the protective role of Ac2-26, and show that LY294002 inhibited the ameliorative preconditioning effect of Ac2-26. Conclusion This finding suggested that the AnxA1 peptide Ac2-26 decreased the inflammation reaction and CPB-induced lung injury in rats, the lung protective effects of AnxA1may be correlated with the activation of PI3K/Akt signaling pathway.


Introduction
With the improvement of cardiac surgery and cardiopulmonary bypass (CPB)-induced technology, postoperative complications in patients with heart disease have been significantly reduced. However, almost all patients have varying degrees of lung injury after surgery, 1 and the fatality rate can be as high as 40-70%. 2 The systemic inflammatory response induced after CPB activates the complement system, which is the main cause of CPBinduced lung injury. 3,4 To reduce complications and improve the quality of life of patients after surgery, clinically or experimentally, researchers have been looking for the best lung-protection measures.
Annexin A1 (AnxA1) is a type of calcium-dependent phospholipid binding protein. 5,6 After proteolysis of the full-length AnxA1, the N-terminus is broken, and a peptide chain fragment Ac2-26 comprising 26 amino acids of the N-segment is formed. 7 The N-terminus of AnxA1 mimics the peptide Ac2-26 and has similar biological effects. 8 In the lipopolysaccharide (LPS)induced pulmonary endotoxemia model, pretreatment with the AnxA1 peptide Ac2-26 reduced the leakage of leukocytes and the release of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-1, which has a protective effect on the lungs. 9 AnxA1 and Ac2-26 play important anti-inflammatory and protective roles in the lungs. However, the effect of AnxA1 on the mechanism of inflammatory signaling pathway in CPB-induced lung injury was still unclear. The PI3K/Akt signaling pathway is widely present in various cells throughout the body and participates in regulating various pathophysiological processes of the body. Therefore, the aim of this study was to establish a CPB-induced left lung ischemia reperfusion (I/R) injury model to observe the effects of AnxA1 peptide Ac2-26 on rat lung tissue injury and PI3K/Akt signaling pathway, and try to provide a novel therapeutic strategy for CPBinduced lung injury.

Materials and methods Materials
The following materials were used: Ac2- 26  Preparation of the cardiopulmonary bypass pipeline 10 Cardiopulmonary bypass tubing comprised a blood reservoir (20 mL syringe), silicone tubing, a peristaltic pump, arteriovenous tubing, and rat lung membranes. Before the animal experiment, the CPB pipeline was connected, oxygen was connected, and the CPB pipeline was prefilled with 1 mL of sodium potassium magnesium calcium glucose injection, 9 mL of hydroxyethyl starch, 1 mL of 5% sodium bicarbonate, and 1 mL of mannitol.

Establishment of a model of cardiopulmonary bypass-induced ischemia reperfusion injury in the left lung of rats
This experimental model established a model of extracorporeal circulation I/R injury using the protocol described by He et al. 11 A detailed description of the experimental model is added in detail in the Supplemental Material.

Grouping and specimen collection
The animals were divided into five groups (n = 6 per group): Sham operation group (S group), Left lung I/R injury group (I/R group), Dimethyl sulfoxide group (D group), Ac2-26 group (A group), Ac2-26 + LY294002 (LY294002; phosphoinositide 3-kinase inhibitor (PI3K) specific inhibitor) group (AL group). In the S group, after vascular catheterization, only mechanical ventilation was performed, and the rats did not receive any other treatment. The other four groups had their chests opened 10 min after the establishment of CPB, their left hilum was blocked, and lung I/R in rats was simulated. In the I/R group, an equal volume of normal saline was injected through the tail vein 10 min before the left hilum was blocked. In the D group, an equal volume of DMSO was injected through the tail vein 10 min before the left hilum was blocked. In the A group, 1 mg/kg Ac2-26 12 was administered through the tail vein 10 min before the left hilum was blocked. In the AL group, to establish a model of left lung CPB-induced I/R, 0.3 mg/kg LY294002 13 and 1 mg/kg of Ac2-26 were simultaneously administered through the tail vein 10 min before left hilum occlusion. PH2O is the saturated water vapor pressure, which is 47 mmHg under standard conditions. FiO2 (%) is the concentration of inhaled oxygen, and the experimental model of this animal is 99%. P is the actual atmospheric pressure, which is 680 mmHg in the Zunyi area.
PaCO2 is the partial pressure of carbon dioxide. Pathological observation. At the end of the experiment, the left lung tissue was taken and divided into three parts, one of which was stored at À80°C for inspection. A part of the left lung tissue was fixed with formaldehyde solution, embedded in paraffin, dehydrated, and deparaffinized. This tissue was further stained with hematoxylin and eosin and mounted and the morphology of the lung tissue was observed using a light microscope. Three high-powered visual fields were randomly selected to observe the lung tissue morphology, and images were captured. The average value was calculated using the protocol described by from Cheng et al. 14 Scoring standard, which reflects the degree of lung injury, is shown in Table 1. A portion of the left lung tissue was placed in 2% glutaraldehyde for 24 h, washed with phosphate buffer, fixed with 2% osmium acid for 1 h, dehydrated, embedded, and stained. Cell morphology and ultrastructure were observed under an electron microscope.
Bronchoalveolar lavage fluid (BALF). At the end of the experiment, the trachea and lungs were stripped, the right main bronchus was blocked, and 5 mL of 0.9% sodium chloride was injected through the trachea to the left main bronchus and left lung. The same was repeated, with the left lung three times and at least 3 mL of the lavage fluid was recovered, and centrifuged at 3000 rmp/ min for 10 min, supernatant was removed and stored at À80°C for further testing.
Enzyme-linked immunosorbent assay (ELISA). The frozen lung tissue was thawed at 4°C, and the specimen was maintained at a temperature of 2-8°C after thawing. Phosphate buffered saline (pH 7.4) was added to the sample, and the specimen was thoroughly ground until homogenized. The concentration of TNF-α, IL-6 and total protein were detected by the ELISA with the specific ELISA kit according to the instructions.
Western blotting. Immediately after the experiment, the lung tissue was excised and frozen at À80°C. The samples were then centrifuged in ice-cold lysis buffer at 12,000 g for 5 min at 4°C. The samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes. After blocking with Tris-buffered saline with 0.1% Tween ® 20 Detergent containing 5% (w/v) nonfat milk, the membranes were incubated with specific primary antibodies against PI3K (Abcam, Cambridge, UK. No. ab191606), Akt (Abcam, Cambridge, UK. No. ab8805), NF-κB (p65) (Abcam, Cambridge, UK. No. ab16502), p-PI3K (Abcam, Cambridge, UK. No. ab182651), p-Akt (Abcam, Cambridge, UK. No. ab8805), p-NF-κB (p65) (Abcam, Cambridge, UK. No. ab16502), and AnxA1 (Abcam, Cambridge, UK. No. ab214486) at 4°C overnight in blocking solution. All antibodies were diluted 1:1000. After washing the membranes three times with TBST, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Chemiluminescence was detected using the ECL-chemiluminescent kit with Protein Simple. The protein content of the supernatant was determined using a BCA protein assay kit.

Statistical methods
Data analysis and statistical evaluation were performed using IBM SPSS 18.0. All results are means and standard deviations of at least three experiments conducted in triplicate. Data are shown as mean ± standard error of the mean (SEM). Repeated measurements were performed within this group using analysis of variance. One-way analysis of variance (ANOVA) and post-hoc (LSD) ANOVA were used among the groups. p value of <0.05 was considered statistically significant. Interstitial and alveolar hemorrhage edema range<25% 2 Interstitial widening, alveolar hemorrhage edema range 25-50% 3 Interstitial significantly widened, alveolar hemorrhage edema range 50-75% 4 Interstitial significantly widened, alveolar hemorrhage edema range >75% Inflammation 0 None 1 Interstitial small amount of neutrophils 2 Interstitial and some alveolar spaces have more neutrophils 3 Neutrophils and agglomerates in most of the alveolar spaces

Changes in oxygenation index and respiratory index in rats
Changes in oxygenation index. At T1, there were no significant differences in oxygenation index (OI) between the groups. At T2 and T3, the OI of rats in the I/R, D, and AL groups were significantly reduced compared to S group (p < 0.05) with the lowest value observed in AL group (Figure 1(a)).
Changes in respiratory index (RI). At T1, there was no significant difference in RI of rats in each group.
At T2 and T3, the RI of rats in the D and AL groups were significantly increased compared to S group (p < 0.05) with the highest value observed in AL group (Figure 1(b)).

Light microscopy results and pathological damage score of lung tissue
The lung tissue structure of the S group was clear, and the alveolar wall was relatively complete (Figure 2(a)). In the I/R group and D groups, the lung tissue structure was disordered, some alveolar wall was broken, and the alveolar cavity was filled with edema fluid, showing moderate inflammation; infiltration of cells and red blood cells and the degree of lung injury was more severe than that in the S group (Figure 2(b), Figure 2(c)). In the A group, the lung tissue structure was complete, and some alveolar walls were broken, and there was a small amount of inflammatory cell infiltration. The degree of lung injury was significantly less than that in the I/R group (Figure 2(d)). The lung tissue structure of the AL group was obviously damaged, the alveolar wall was severely broken, and a large number of inflammatory cells, red blood cells, and edema fluid were seen in the alveolar cavity (Figure 2(e)).
Compared with the S group, the lung injury scores of the other four groups were higher than those of the S group (p < 0.05). The lung injury scores of A group were significantly lower than that of the I/R group (p < 0.05). Compared with the A group, the lung injury score of AL group increased (p < 0.05) (Figure 2(f)).

Lung tissue electron microscopy results
Under the microscope, the structure of the lamellar bodies in the S group was basically intact, and the mitochondria were not swollen (Figure 3(a)). In the I/R and D groups, the structure of lamellar corpuscles was damaged, some lamellar corpuscles and mitochondria were swollen, and there was moderate inflammatory exudation in the alveolar cavity, which was significantly worse than that in the S group (Figure 3(b), Figure 3(c)). Under the electron microscope in the A group, it was seen that the lamellar structure was slightly damaged, the mitochondria were not significantly swollen, and there was a small amount of inflammatory exudation in the alveolar cavity, which was significantly less damaged than the I/R group (Figure 3(d)). In the AL group, the lamellar structure was damaged, some mitochondria were swollen, and there was a large amount of inflammatory exudation in the alveolar cavity, which was more severe than that in the A group (Figure 3(e)).

Levels of tumor necrosis factor-alpha in lung tissues and those of IL-6 and total protein in bronchoalveolar lavage fluid at T3
At the end of the experiment, the level of TNF-α in the lung tissue from the I/R, D, A, and AL groups significantly increased compared with the S group (p < 0.05). The level of TNF-α in the A group was significantly lower than that in the I/R and D groups (p < 0.05). The level of TNF-α in the AL group was significantly higher than that in A group (p < 0.05) (Figure 4(a)).
At the end of the experiment, the levels of IL-6 and total protein in the left BALF from the lung tissue in the I/R, D, A, and AL groups were significantly increased compared with those in the S group (p < 0.05). The level of IL-6 and total protein in the A group was significantly lower than that in the I/R, D, and AL groups (p < 0.05). (Figure 4(b), Figure 4

(c))
Expression of PI3K, p-PI3K, Akt, p-Akt (Ser473), NF-κB(p65), p-NF-κB(p65), and annexin A1 in rat lung tissue PI3K was set as an internal reference, and the ratio of p-PI3K/PI3K was calculated to indicate the expression level of p-PI3K. 15 Compared with the I/R group, the ratio of p-PI3K/PI3K in the A group significantly increased (p < 0.05). The ratio of p-PI3K/PI3K in the AL group was significantly lower than that in the A group (p < 0.05) ( Figure 5(a), Figure 5(b)). Compared with the I/R group, the ratio of p-Akt/Akt in the A group was significantly increased (p < 0.05). The p-Akt/Akt ratio in the AL group was significantly lower than that in the A group (p < 0.05) (Figure5(a), Figure 5(c)).
The expression of NF-κB (p65) in the lung tissues of rats in the I/R, D, A, and AL groups was upregulated (p < 0.05). Compared with the I/R group, the ratio of p-NF-κB/NF-κB (p65) in the A group was significantly lower (p < 0.05). Compared with the A group, the ratio of p-NF-κB/NF-κB (p65) in the AL group was significantly higher (p < 0.05) ( Figure 5(d), Figure 5(e)).
Compared with the S group, the expression of AnxA1 in the lung tissues of the I/R, D, A, and AL groups significantly increased (p < 0.05). Compared with the I/R group, the expression of AnxA1 in the A group increased (p < 0.05). Compared with the A group, the expression of AnxA1 decreased in the AL group (p < 0.05). Compared with S group, the expression of AnxA1 (37 kD) in the lung tissues of the I/R, D, A, and AL groups decreased (p < 0.05), whereas the expression of AnxA1 (33 kD) in the lung tissues increased (p < 0.05) ( Figure 5(f), Figure 5(g)).
Discussion CPB-induced lung injury, one of the main complications after cardiovascular surgery, a dominant factor of postoperative death. 16 Studies have revealed that tumor necrosis and interleukin produced after CPB directly damage endothelial cells, increase capillary permeability, cause pulmonary edema, and promote infiltration of neutrophils and macrophage. The resulting release of cytotoxic enzymes aggravates lung injury. 15 Clinically, lung I/R injury is difficult to be controlled with current therapies. According to this experiment, the levels of total protein and IL-6 in BAFL increased significantly after CPB-induced lung I/R injury, suggesting that lung inflammation and vascular permeability increased. In addition, pathology and electron microscopy showed severe lung injury. Meanwhile, in this experiment, a CPB-induced left lung I/R injury model was successfully established.
As a member of the structurally related calciumdependent phospholipid binding protein superfamily, AnxA1 comprises a highly conserved central domain and an N-terminal sequence that bears a unique function 17 and its function involves all aspects of cell life activities, including cell secretion, signal transduction, inflammatory response, and apoptosis. 18 Under the action of inflammatory factors, neutrophils and capillary endothelial cells are combined to stimulate the translocation of a large amount of AnxA1 to the cell surface and anchor in the cell in a calcium-dependent manner. The plasma membrane interacts with adhesion molecules to inhibit the migration of leukocytes to the inflammation site and regulate the body's anti-inflammatory effects as well. 19 Ju et al. 20 found that Ac2-26 reduced LPS-induced lung injury, promoted alveolar-capillary permeability, ameliorated the local and systemic inflammation, and inhibited the oxidative stress response and apoptosis. In our previous study, it was found that 11 by binding to formyl peptide receptors inhibit, Ac2-26 inhibited inflammatory cytokines and reduced CPB-induced lung I/R injury. Similarly, it was also observed in this study that under Ac2-26 treatment, the pathological injury score of lung tissue decreased, the expression of inflammatory factor IL-6 and TNF-α decreased, and the lung function was improved. Furthermore, it follows that Ac2-26 plays a protective role in lung through anti-inflammatory effects. However, in terms of lung protection, the effects of AC2-26 on the inflammatory signaling pathway are not clear when it comes to lung protection.
The PI3K/Akt signaling pathway, a classic signaling pathway in cells, participates in the body's I/R injury, inflammation, and other processes. 21 PI3K is a heterodimer composed of a catalytic subunit (p110) and a regulatory subunit (P85). Extracellular signals, such as growth factors, growth hormones, endotoxins, and I/R injury, can activate PI3K, which makes cell membrane to produce the second messenger PIP3 that could bind to Akt and PDK in the cell, thus ensuring the phosphorylation and activation of Akt at Ser473 and Thr308. Phosphorylation at Ser473 can maximize Akt biological activity, 22 and p-Akt can cause the phosphorylation cascade of downstream effector molecules and the interaction between the target protein. 23 Being the downstream target protein of the PI3K/Akt signaling pathway, NF-κB regulates inflammatory mediators, cell adhesion molecules, and cytokines. 24 Inhibiting the activity of NF-κB can inhibit the transcription of cytokines such as TNF-α and IL-6 and reduce tissue I/R injury. 25,26 Studies have proved that the activation of the PI3K/Akt pathway plays a key role in CPB-induced lung injury. 27 In rats with acute lung injury, oxidative stress can be inhibited by activating the PI3K/Akt signaling pathway, which can reduce inflammatory mediators. 28 In patients with chronic obstructive pulmonary disease, elevated AnxA1 can  downregulate NF-κB and reduce IL-6 levels as well as collagen deposition induced by inflammatory cytokines in lung tissue and protect the lung. 29 One study gives the information that an acute lung inflammatory response to LPS, thereby leading to a distinct increase in p-PI3K, P-Akt and P-mTOR, 30 which was consistent with the results obtained in our study. The expressions of p-PI3K and p-AKT were upregulated after lung I/R injury. Moreover, the use of Ac2-26 can increase the expression of p-PI3K and p-AKT, reduce the expression of p-NF-κB (p65), inhibit the production of inflammatory factors, improve pulmonary vascular permeability, and reduce the pathological damage of CPB in lung tissue. However, the protective effects of Ac2-26 on rat lung tissue were reversed after the use of PI3K specific inhibitors, further proving that this protective mechanism involved the activation of PI3K/Achter signaling pathway. In summary, Ac2-26 can upregulate p-PI3K and p-Akt expression, decrease p-NF-κB(p65) expression, inhibit inflammatory factors' production, improve pulmonary vascular permeability, and reduce the pathological damage caused by CPB in lung tissue.
The experiment also obtained an interesting finding. Compared with the expression of AnxA1 in lung tissues of S group, it gets increased in I/R group, D group, A group and AL group which indicates that CPB should be blamed for this result because it could activate a systemic inflammatory response and induce I/R injury. The expression of AnxA1 of A group gets increased than that of I/R group. The lung function tests exhibited that the lungs of group A work better. Meanwhile, the biomarkers of inflammation involved in the lung injury were evaluated. These evidences suggested that Ac2-26 can regulate the expression of AnxA1 during CPB and play similar antiinflammatory effect of AnxA1. In inflammatory lung disease, AnxA1 was completely degraded to a protein that had a molecular weight of 33 kD compared with the 37 kD AnxA1. 31 Bruna et al. 32 proposed that in the lung injury model induced by I/R, Ac2-26 treatment can reduce lung inflammation. Besides, within treatment groups, the expression of AnxA1 with a molecular weight of 37 kDa decreased and that of 33 kDa increased. Our study is consistent with previous findings drawn by Damico et al. 33 : After the treatment of Ac2-26, the expression of AnxA1 (37 kDa) in the lung tissue of A group was lower than that of group S, while the expression of AnxA1 (33 kDa) was just the opposite, which gives us a hint that Ac2-26 can regulate the conversion of endogenous AnxA1 between 37 kDa and 33 kDa.
The protective effects of Ac2-26, a mimetic peptide of ANXA1, in the lung I/R injury model after CPB were investigated, and the regulatory mechanism of Ac2-26 on PI3K/Akt signaling pathway was explored. However, there are a few limitations to the experiments. Therefore, further investigations are being pursued, including cytological experiments and the exploration of the relevant mechanisms of lung protection.

Conclusion
The AnxA1 peptide Ac2-26 can improve lung ventilation function in rats after CPB, reduce the degree of lung pathological damage and lung inflammation, and exert certain lung-protection effects after CPB in rats. The mechanism may be related to the activation of the PI3K/ Akt signaling pathway, upregulation of the phosphorylation levels of PI3K and Akt, inhibition of p-NF-κB (p65) expression in lung tissue, and reduction of the release of inflammatory factors.

Acknowledgments
I would like to express my gratitude to all those who helped me during the writing of this thesis. I gratefully acknowledge the help of my supervisor, HZ, who has offered me valuable suggestions in the academic studies. In the preparation of the thesis, she has spent much time reading through each draft and provided me with inspiring advice. Without her patient instruction, insightful criticism and expert guidance, the completion of this thesis would not have been possible.

Author Contributions
YZ designed the experimental project, conducted most of the experiments, and prepared the article. YH participated in the establishment of an in vitro lung IR injury model and participated in the writing and revision of the article. HW and JL completed the analysis of part of the experimental data, and CC as the instructor completed most of the experimental operation techniques. HZ revised the article and had the primary responsibility for the final content. All authors read and approved the final article.