The protective effects of angiotensin-converting enzyme inhibitor against cecal ligation and puncture-induced sepsis via oxidative stress and inflammation

Journal Pre-proof The Protective Effects of Angiotensin-Converting Enzyme Inhibitor against Cecal Ligation and Puncture-Induced Sepsis via Oxidative Stress and Inflammation Ugur Kostakoglu, Atilla Topcu, Mehtap Atak, Levent Tumkaya, Tolga Mercantepe, Huseyin Avni Uydu PII:

S0024-3205(19)30978-6

DOI:

https://doi.org/10.1016/j.lfs.2019.117051

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LFS 117051

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Life Sciences

Received Date: 4 September 2019 Revised Date:

6 November 2019

Accepted Date: 7 November 2019

Please cite this article as: U. Kostakoglu, A. Topcu, M. Atak, L. Tumkaya, T. Mercantepe, H.A. Uydu, The Protective Effects of Angiotensin-Converting Enzyme Inhibitor against Cecal Ligation and Puncture-Induced Sepsis via Oxidative Stress and Inflammation, Life Sciences, https://doi.org/10.1016/ j.lfs.2019.117051. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

The Protective Effects of Angiotensin-Converting Enzyme Inhibitor against Cecal Ligation and Puncture-Induced Sepsis via Oxidative Stress and Inflammation Ugur Kostakoglu1*, Atilla Topcu2, Mehtap Atak3, Levent Tumkaya4, Tolga Mercantepe4, Huseyin Avni Uydu3 Affiliations: 1

Department of Department of Infectious Diseases and Clinical Microbiology, Faculty of

Medicine, Recep Tayyip Erdogan University, 53100 Rize, Turkey 2

Department of Pharmacology, Recep Tayyip Erdogan University, Faculty of Medicine,

53100 Rize, Turkey 3

Department of Medical Biochemistry, Faculty of Medicine, Recep Tayyip Erdogan

University, 53100 Rize, Turkey 4

Department of Histology and Embryology, Faculty of Medicine, Recep Tayyip Erdogan

University, 53100 Rize, Turkey

*Corresponding Author: Dr. Ugur Kostakoglu Recep Tayyip Erdogan University, Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, Rize, Turkey, 53100 Tel: +90 464 212 3009 Fax: +90 464 212 3015 e-mail:[email protected]

Running Title: Effects of Perindopril on Sepsis-Induced Lung Tissue Injury

Conflict of Interest: No authors have any conflict of interest to declare.

Abstract Aims: Sepsis is a severe public health problem affecting millions of individuals, with global mortality rates caused by lower respiratory tract infections are approximately 2.38 million people a year die from respiratory failure caused by infection. Although ACE is known to contribute to damage in septicemia, the pathophysiological mechanisms of sepsis remain unclear. While mortality can be significantly reduced through effective and sensitive antibiotic therapy, antibiotic resistance restricts the use of these drugs, and the investigation of novel agents and targets is therefore essential. Our aim was to determine whether Perindopril (PER) has anti-inflammatory and antioxidant capable of preventing these adverse conditions resulting in injury in previous studies. Main methods: Sprague Dawley rats were randomly assigned into the control group, received oral saline solution alone for four days. the cecal ligation and puncture (CLP) group, underwent only cecal ligation and puncture induced sepsis, while the CLP+PER (2 mg/kg) underwent cecal ligation and puncture-induced sepsis together with oral administration of 2 mg/kg PER for four days before induction of sepsis. Key findings: Malondialdehyde (MDA), tumor necrosis factor-alpha (TNF-α), Caspase-3 and nuclear factor kappa B (NF-kβ/p65) levels increased in the CLP group. On the other hand, PER (2 mg/kg) oral administration to septic rats decreased MDA, TNF-α and increase glutathione (GSH) in the lung tissue. In addition, PER administration also decreased the lung tissue NF-κB and Caspase-3 immunopositivity against sepsis. Significance: PER treatment may represent a promising means of preventing sepsis-induced lung injury via antioxidant and anti-inflammation effects. Keywords: Cecal Ligation and Puncture, inflammation, Lung, Oxidative stress, Perindopril, Rat, Sepsis

1. Introduction Sepsis is a severe public health problem affecting millions of individuals, with high mortality rates resulting in death in many cases[1,2]. Global mortality rates caused by lower respiratory tract infections are high[3]. One systemic analysis reported that approximately 2.38 million people a year die from respiratory failure caused by infection and that this is the sixth most important cause of all deaths[4]. While mortality can be significantly reduced through effective and sensitive antibiotic therapy, antibiotic resistance restricts the use of these drugs, and the investigation of novel agents and targets is therefore essential[5]. Proinflammatory cytokine levels rising in association with secondary organ failure, shock, and hypotension caused by advanced sepsis are one of the leading causes of all deaths[6]. Although the application of auxiliary therapeutic methods such as fluid support and vasopressor agents in addition to traditional antibiotherapy in septic patients is successful in some patients, it also causes various complications and treatment resistance, and mortality rates are rising in consequence[1,7]. One of the most important organs affected by sepsis is lung[8]. Acute lung injury (ALI) is seen in many patients with severe sepsis, and acute respiratory distress syndrome (ARDS) emerges as sepsis complications worsen[9]. ALI also plays a triggering role in the development of a series of complex events [10]. The most important of these are cell migration and increased proinflammatory cytokine synthesis and levels[10,11]. Neutrophil and macrophage cells exacerbate the scale of the injury by damaging the endothelia with the cytokines they express[12]. Increased neutrophil migration and activity in the lungs thus causes a significant increase in reactive oxygen species (ROS) together with pro-inflammatory cytokines and results in exacerbation of oxidative stress injury that worsens with inflammation[13]. Previous studies have shown an increase in important proinflammatory cytokines, particularly interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) in sepsis-induced ALI

and ARDS[14–16]. In addition, the effect of nuclear factor kappa B (NF-κB), an important transcript transcription factor that increases the synthesis of the proinflammatory cytokines discussed above, can be clearly seen when we consider the pathology of the disease[16]. Feng et al. determined an increase in NF-κB expression in pulmonary tissue in polyliposaccharideinduced sepsis, decreasing in association with downregulation following treatment, and also showed a decrease in TNF-α levels[17]. It therefore appears that NF-κB plays a regulatory role in sepsis. In addition to an increase in proinflammatory cytokine levels, inflammation also causes an increase in the production of free oxygen radicals by inducing oxidative stress[18]. Malondialdehyde (MDA), a component of lipid peroxidation, is an important product of oxidative stress arising as a result of inflammation[19]. Previous studies have shown that sepsis-induced through cecal ligation and puncture causes an increase in MDA levels in pulmonary tissue[20,21]. Glutathione (GSH) is an important component of the antioxidant defense mechanism that reduces the effect of oxidative stress[22]. Previous studies have shown that GSH also exhibits protective effects against proinflammatory injury developing in pulmonary tissue[18,23]. Increasing inflammatory and oxidative stress in the lungs also contributes to the development of ALI by inducing caspase-mediated apoptosis[24]. Caspase-3 is situated at the beginning of the apoptotic pathway in the cell and is one of the important components of intracellular signaling[20]. Previous studies have revealed that caspase-3 expression increases in sepsis-induced pulmonary injury, but that following treatment, downregulation occurs in caspase-3 levels in association with decreasing proinflammatory levels and increasing antioxidant effects[20,25]. The use of broad-spectrum antibiotics is generally preferred in the current treatment of sepsis[26]. Despite symptomatic treatments aimed at coping with emerging complications,

safe and effective novel agents need to be discovered and developed to eliminate the inflammation, oxidative stress, and apoptosis occurring in ARDS through different mechanisms. The cecal ligation and puncture (CLP)-induced sepsis model is one of the most commonly used methods in experimental studies in that context[15,18]. The renin-angiotensin system (RAS) is known to exhibit regulatory effects in several organs[27]. One of the most important effects of angiotensin II, one of the endogenous molecules in RAS, is that it increases inflammation[28]. Previous studies have shown that NF-Κβ-mediated activation of angiotensin II triggers the proinflammatory cascade[29]. In addition, suppression of angiotensin II production with angiotensin-converting enzyme (ACE) inhibitors has been reported to reduce oxidative stress[30]. Perindopril (PER), which has long been used in the treatment of chronic cardiovascular diseases, is an ACE inhibitor[31]. Previous research has revealed that PER exhibits anti-inflammatory and antioxidant effects[32,33]. Shalkami AS et al. showed that it exhibits significant inflammation-preventing effects in cisplatin-induced renal injury[34]. However, the effects and effect mechanisms of ACE inhibitors on sepsis induced by CLP are not yet fully understood. Sepsis-induced through CLP causes injury in pulmonary tissues through mechanisms involving oxidative stress, inflammation, and apoptosis. Our aim was to use biochemical, histochemical, and immunohistochemical methods to determine whether PER is capable of preventing these adverse conditions resulting in injury in previous studies. 2. Materials and Methods 2.1. Experimental Animals Twenty-four female Sprague Dawley rats weighing 290±10 g were used for biochemical, histopathological, and immunohistochemical analyses. Care was provided in line with the principles of the Guide for the Care and Use of Laboratory Animals published by the

National Research Council and approved by the local ethical committee. Before and during the study, rats were housed in standard plastic cages with a sawdust floor under normal temperature conditions of 22±1 ºC and 55-65% humidity, under controlled lighting(12/12 h dark/light cycle), with ad libitum access to standard rat chow and tap water. Animal experiments and procedures were performed in accordance with the national guidelines for the use and care of laboratory animals. The study protocol was approved by the local animal care committee of Recep Tayyip Erdogan University (Approval number:2019/0131.01.2019). 2.2. Chemicals PER (Coversyl 10 mg 30 Film Tablets) was obtained from Servier Ilaç ve Araştırma A.S. (Istanbul, Turkey) under license from Les Laboratory Services (France). All animals were anesthetized using ketamine hydrochloride (Ketalar®, 50 mg/kg, Pfizer Đlaçları Ltd. Şti., Istanbul, Turkey) and sedative xylazine hydrochloride (Rompun®, 10 mg/kg, Bayer, Turkey). All chemicals used for laboratory experiments were obtained from Sigma Chemical Co. and Merck (Germany). 2.3. Experimental protocol Rats were randomly assigned into three groups of eight animals each. Group 1(n=8), the control group, received oral saline solution alone for four days. Group 2(n=8), the CLP group, underwent only cecal ligation and puncture induced sepsis, while Group 3(n=8), the CLP+PER(2 mg/kg) underwent cecal ligation and puncture-induced sepsis together with oral administration of 2 mg/kg PER for four days before induction of sepsis[35]. 2.4. Cecal Ligation and Puncture-Induced Sepsis Model Sepsis was induced in rats using the CLP-induced sepsis model of Rittirsch D et al. [36]. All surgical procedures were carried out under sterile conditions. Rats were anesthetized with 50 mg/kg ketamine HCL injection and 10 mg/kg xylazine HCl. Once anesthesia was

confirmed, a 2.5-cm incision was made to the abdominal midline. The abdominal organs and the cecum were isolated and ligated distal to the ileocecal valve using 3/0 silk sutures. The cecum content was brought into contact with the peritoneum by opening two holes in the distal from the mesentery to the opposite side with a 22-gauge needle. The abdominal incision was then closed with two layers of sterile 4/0 synthetic absorbable sutures. The wound was then washed with 1% lidocaine solution for analgesia. The experiment was terminated 16 hours after the related treatments and surgery[37]. At the end of the experiment, rats were sacrificed by euthanasia with the high-dose anesthetic. One lung was stored for use in biochemical analysis together with the serum specimens at -80ºC. The other lung lobe was divided into two halves and placed into 10% neutral formalin. 2.5. Biochemical Procedure 2.5.1. Tissue Sampling and Homogenization A mixture of 20 mM 1L sodium phosphate + 140 mM potassium chloride was prepared (pH 7.4)[38]. One hundred milligrams of lung tissue was homogenized with 1 ml homogenate buffer for 5 min at 30 Hz using a Tissue Lyser II device (Qiagen, Hilden, Germany), after which 800g was centrifuged for 10 min at 4oC. MDA, GSH and TNF-α assays were performed with the resulting supernatant. 2.5.2. Standard solutions Briefly, 82.5 µL 1,1,3,3-tetra methoxy propane was added to 0.01 M 50 mL HCl solution. The solution was then left to incubate for 1 h at 50°C. The concentration of this main stock solution was 10 µmol/mL. Next, 20, 10, 5, 2.5, 1.25, and 0.625 nmol/mL solutions were then prepared from this main stock solution.

2.5.3. Malondialdehyde (MDA) Concentration Assay MDA assay was performed as described by Ohkawa et al.[39]. A mixture of 200 µL tissue supernatant, 50 µL 8.1% SDS (sodium dodecyl sulfate), 375 µL 20% acetic acid (v/v) pH 3.5, and 375 µL 0.8% thiobarbituric acid (TBA) was vortexed and left to incubate for 1 h in a boiling water bath. Following incubation, the mixture was cooled in ice water for 5 min and centrifuged for 10 min at 750g. The resulting pink color was read on a spectrophotometer at 532 nm. The results were calculated as nmol/mg prt. 2.5.4. Reduced Glutathione (GSH) Assay –SH groups were assayed using Ellman’s reagent. Briefly, 250 µL supernatant was added 1000 µL 3M Na2HPO4 and 250 µL DTNB (4 mg DTNB was prepared in 1% 10 mL sodium citrate solution). This was then vortexed, and absorbance was measured at 412 nm. The results were calculated using a 1000 µM-62.5 µM reduced glutathione standard curve and were expressed as nmol/mg prt. 2.5.5. Total Protein Assay Since the quantitative analysis results were planned for expression for individual proteins, protein level assay was conducted using the Lowry method[40]. 2.6. Histopathological Analysis Lung tissue specimens were fixed in 10% phosphate-buffered formalin (Sigma Aldrich, Germany) solution for 36 h. After the fixation procedure, routine histological procedures were performed for dehydration (with increasing alcohol series, Merck GmbH, Darmstadt, Germany), mordanting (xylol, Merck GmbH, Darmstadt, Germany), embedding in soft paraffin (Merck GmbH, Darmstadt, Germany), and finally preparation of hard wax blocks (Merck GmbH, Darmstadt, Germany). Sections 4-5 µm in thickness were obtained from the

paraffin blocks using a rotary microtome (Leica RM2525, Lecia, Germany) and stained with Harris hematoxylin and Eosin G (H&E; Merck GmbH, Darmstadt, Germany). 2.6.1. Immunohistochemistry (IHC) Analysis Procedure NF-kβ/-p65 (rabbit polyclonal, ab16502, Abcam, UK) with secondary antibody (Goat AntiRabbit IgG H&L (HRP), ab205718, Abcam, UK) kits were used to determine proinflammatory cytokines. Caspase-3 primary antibody (rabbit polyclonal, ab44976, Abcam, UK) with secondary antibody (Goat Anti-Rabbit IgG H&L (HRP), ab205718, Abcam, UK) kits were employed to determine apoptotic pneumocytes. Lung tissue sections were subjected to deparaffinization in line with the manufacturing company guideline. Following the antigen retrieval procedure, sections were incubated with primary and secondary antibodies for 60 min. Finally, pulmonary tissue sections were stained with diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical, St. Louis, MO, USA) and Harris hematoxylin (Merck GmbH, Darmstadt, Germany). 2.6.2. Semi-Quantitative Analysis Semi-quantitative analysis was performed under the headings alveolar and interstitial neutrophil accumulation, alveolar debris accumulation, and alveolar septal wall thickness. Lung tissue damage score (LDS) was calculated using Matute-Bello et al.’s lung injury score (Table 2)[41]. Thirty-five different areas on each pulmonary tissue section were evaluated by two histopathologists blinded to the study groups. As shown in Table 3, NF-kβ/-p65 and Caspase-3 positive type I and type II pneumocytes were analyzed by two histopathologists blinded to the experimental groups. Thirty-five different areas were randomly selected for each section, and IHC positivity was thus measured in 210 different areas in each group.

2.7. Statistical Analysis Quantitative, semi-quantitative and biochemical data were analyzed using SPSS 18.0 statistical software (IBM Corp., Armonk, NY, USA). Non-parametric data obtained from semi-quantitative analysis were calculated as median with interquartile range values. Nonparametric data were analyzed using the Kruskal Wallis test followed by the Tamhane T2 test. Parametric data obtained from biochemical analysis were calculated as mean±standard deviation, and differences between groups were subjected to one-way analysis of variance (ANOVA), followed by the Duncan test. p values <0.05 were regarded as significant. 3. Results 3.1. TBARS Analysis Results Statistical analysis of the study groups’ TBARS levels revealed statistically significant differences between the sepsis group and both the control and treatment groups (p=0.009 and p=0.029, respectively; Figure 1; Table 1). The TBARS level in the control group, 13.24±2.34 nmol/mg tissue, increased to 16.04±1.89 nmol/mg tissue in the sepsis only group (p=0.009; Figure 1; Table 1). However, perindopril reduced this increased TBARS level to 13.77±0.74 nmol/mg tissue (p=0.029; Figure 1; Table 1). No statistically significant difference was observed between the control and treatment groups. 3.2. GSH Analysis Results Analysis of the groups’ total thiol levels revealed a statistically significant difference between the control group and the sepsis group (p= 0.007; Figure 1; Table 1). The total thiol level in the control group, 10.26±2.06 µmol/g tissue, increased to 14.07±2.46 µmol/g tissue in the group with induced sepsis (p=0.007; Figure 1: Table 1). Thiol levels in the sepsis group treated with PER were 10.84±2.42 µmol/g tissue. However, no difference was determined between the control and treatment groups. However, a statistically significant difference was

observed between the sepsis only group and the treated sepsis group (p=0.019; Figure 1; Table 1). 3.3. TNF-α Analysis Results Another parameter measured in this study was TNF-α. The TNF-α level in the control group was 0.81±0.16 ng/g tissue, rising to 0.98±0.21 ng/g tissue in the sepsis the only group. Levels decreased in the treated sepsis group, to 0.87±0.20 ng/g tissue. Statistical analysis of TNF-α levels revealed no significant difference between the groups (p>0.05; Figure 1; Table 1). 3.4. Histopathological Analysis Results We observed a normal structure in control group pulmonary tissue. Typical type I and type II pneumocytes were observed, particularly in the respiratory bronchioles and alveolar sac (Figure 2a-b, LDS median: 0.5 (0-1). In contrast, vascular congestion, pulmonary hyaline membrane accumulation, and diffuse neutrophil accumulations in interstitial spaces were present in pulmonary tissue sections from the CLP group (Figure 2d-f). We also observed alveolar debris accumulation and thickening of the alveolar septal wall (Figure 2d-f; LDS median: 6 (4.5-6). A decreased alveolar wall thickness and typical type I pneumocytes (arrow) and type II pneumocytes (tailed arrow) were present in pulmonary tissue sections from the CLP+PER treatment group (Figure 2g-i; LDS median: 1(0-2). 3.5.Quantitative Analysis Results The alveolar septal wall thickness of 8.26±1.73 µm in the control group increased to 19.24±5.97 µm in the CLP group (Figure 2a-f; Table 4; p=0.000). However, wall thickness of 19.24±5.97 µm in the CLP group decreased to 12.74±4.05 µm in the CLP+PER treatment group (Figure 2d-i; Table 4; p=0.017).

3.6. Semi-Quantitative Analysis Results We determined statistically significant increases in neutrophil accumulation in interstitial spaces, pulmonary hyaline membrane accumulation, and alveolar debris accumulation in the CLP group compared to the control group (Figures 2a-f; Table 5; p=0.024, p=0.000, and p=0.005; respectively). There was no difference between the CLP and control groups in terms of neutrophil accumulation in alveolar regions (Figures 2a-f; Table 5). In contrast, we observed significant decreases in neutrophil accumulation in interstitial spaces, pulmonary hyaline membrane accumulation, and alveolar debris accumulation in the CLP+PER treatment group compared to the CLP group (Figures 2d-i; Table 5; p=0.024; p=0.001; p=0.017; respectively). 3.7. Immunohistochemical (IHC) Analysis Results Analysis of pro-inflammatory cytokines using the IHC method revealed a significant increase in NF-kβ/p65 positivity in type I and II pneumocytes in the CLP group compared to the control group (Figure 3a-d; Table 6; p=0.000; p=0.000; respectively). In contrast, NF-kβ/p65 positivity in type I and type II pneumocytes decreased significantly in the CLP+PER treatment group compared to the CLP group (Figure 3c-f; Table 6; p=0.000; p=0.002; respectively). Analysis of apoptotic cells using the IHC method revealed an increase in Caspase-3 positivity in apoptotic type I and II pneumocytes in the CLP group compared to the control group (Figure 4a-d; Table 6; decreased in apoptotic type I and II pneumocytes in the CLP+PER treatment group (Figure 3c-f; Table 6; p=0.007, and p=0.000, respectively).

3.8. Mortality One (12.5%) of the eight rats in the CLP-induced sepsis group died between 12 and 16 h after sepsis induction. No mortality was observed throughout the experiments in the CLP+PER and control groups. 4. Discussion This study employed biochemical and histopathological methods to investigate the effects of PER, an ACE inhibitor, on preventing injury occurring in sepsis-associated ALI, which is frequently seen in clinical practice and difficult to treat. The findings showed that PER prevents sepsis-induced injury in the rat lung by exhibiting antioxidant and anti-inflammatory effects. In addition, levels of caspase-3 that increased with sepsis in lung tissue were also lowered with PER, thus revealing antiapoptotic effects. This is one of the first studies to investigate the effects of PER in a CLP-induced sepsis model. The poor prognosis caused by sepsis, involving increased inflammation together with secondary organ failure, shock, and hypotension, followed by mortality in the majority of patients, is an important evidence of the dramatic outcomes of sepsis. The treatment of sepsis is also costly, and there is a growing need for new therapeutic discoveries [42]. Several experimental models have been established in order to test different agents in sepsis. We used a CLP-induced ALI model and a polymicrobial sepsis model, which was previously used in other studies and with proven efficacy[43–45]. ACE, which plays an important role in the regulation of vascular function, increases the production of free radicals and pro-inflammatory cytokines in the endothelium via AT1 receptors as a result of the conversion of angiotensin I to angiotensin II [46–49]. Thus, oxidative stress and inflammation cause endothelial dysfunction [49,50]. Control of ACE with an inhibitor such as perindopril, may prevent oxidative and inflammatory damage.

Accordingly, in our study, increased oxidative stress and inflammation due to the effect of sepsis on the lung tissue decreased in the treatment groups and this finding is in parallel with previous studies. The lung is one of the organs most affected in sepsis-related inflammation[51]. The severe injury occurs in the lungs as a result of oxidative stress and proinflammatory cytokine release developing in association with polymicrobial sepsis[52]. Increasing synthesis and secretion of proinflammatory cytokines activate the oxidative mechanism. The oxidantantioxidant balance is impaired in association with redox activity, leading to tissue damage[18]. MDA is one of the most important markers of injury caused by oxidative stress[7]. MDA is a reactive metabolite that appears with the degradation of lipids, the basic component of the cell membrane, and is capable of inflicting severe tissue injury if not halted[53]. When we compared the group with CLP-induced sepsis with the control group, MDA levels were higher in the CLP group. Consistent with our findings, previous studies have shown that MDA levels increase in pulmonary tissue in CLP-induced sepsis[18,52,54]. We also observed a decrease in MDA levels in the group with induced sepsis treated with PER[34,55,56]. Reactive oxygen species that increases for environmental reasons or following various reactions inside the cell trigger the synthesis of the natural antioxidant GSH. Previous research has shown that failure to eliminate the causes leading to such injury causes a continuous decrease in GSH, followed by total depletion, and that tissue damage therefore continues to rise[7,52]. In contrast, we observed an increase in GSH levels in lung tissue in the CLP-induced sepsis group compared to the healthy control group. In contrast to previous studies, our finding shows that GSH levels are activated as a natural defense mechanism in as little as 16 h and that there is no rapid decrease in the early period. These data are consistent with previous research by Topcu A. et al. and Aktoz T. et al.[57,58]. Previous studies

involving PER have also shown that it raises GSH levels and has significant antioxidant capacity, while in our study it reduced GSH levels to a value close to that of the healthy control group[35,55]. ALI in sepsis-induced with CLP is one of the main problems needing to be overcome[59,60]. Endothelial injury and tissue damage occurring with sepsis trigger ALI development by causing intensive inflammatory cell migration to the lungs and severe proinflammatory cytokine release[61]. One of the principal proinflammatory cytokines emerging with the induction of sepsis is TNF-α[62]. TNF-α also causes the secretion of several proinflammatory cytokines by stimulating inflammatory cells[63]. Previous similar research has also shown that sepsis increases TNF-α levels compared to healthy control groups[37,64]. Consistent with other studies, we also observed an increased TNF-α level in our sepsis group. However, that elevation was not statistically significant. This may be because the various stages of the inflammatory process are not completed within this acute period. In contrast, PER treatment exhibits anti-inflammatory effects by reducing TNF-α levels. In agreement with previous studies, this finding shows that the drug has antiinflammatory effects[33,65]. Previous research has shown that PER possesses both anti-inflammatory and antioxidant properties[33,65]. The increasing inflammatory process following sepsis caused by CLP leads to the emergence of severe ALI [15,52]. Histopathological examination of the ALI process in our research was compatible with Cinar et al’s histopathological examinations and revealed marked neutrophil accumulation in interstitial spaces, vascular congestion, and hyaline membrane accumulation[37]. Following treatment with PER, we observed a decrease in alveolar wall thickness, and typical type I and II pneumocytes in the alveolar sac, showing that PER exhibits important effects in reversing injury in ALI induced with sepsis.

Previous studies have revealed that NF-κβ upregulation plays an important role in the development of the inflammatory process following sepsis-induced lung damage[37,66]. Consistent with previous studies, NF-κβ expression also increased in the sepsis groups in the present study. One previous piece of research involving PER reduced the levels of the transcription factor NF-κβ through an inhibitory effect on the NF-κβ pathway in mice with diethyl nitrosamine-induced hepatocellular carcinoma[67]. Similarly in the present study, NFκβ that increased with sepsis in the lung decreased following PER treatment. In addition, at biochemical examination, tissue TNF-α levels changed similarly to those of NF-κβ in both the sepsis and treatment groups. Based on that finding we may conclude that NF-κβ contributes to the regulation of the inflammatory process by TNF-α, one of the principal proinflammatory cytokines. Another parameter investigated in this research was tissue caspase-3 expression. Caspase-3 is a protein that plays the most important role in apoptosis[68]. Impairment of the balance between the damage and repair mechanisms following the inflammatory process leads to a manifestation of severe ARDS[69]. Through apoptosis, cells can be eliminated with minimal or no damage to other cells, thus halting the increasing inflammatory process[70,71]. Increasing NF-κβ and TNF-α protect the tissue against the effects of inflammation by triggering apoptosis[37]. Similarly to previous studies, caspase-3 levels increased similarly to tissue NF-κβ and TNF-α following sepsis and approached normal levels with PER treatment. Induction of apoptosis with ARDS may be urgently required in order to alleviate and eliminate increasingly severe inflammation[72]. Angiotensin II (AngII), one of the principal regulators of the cardiovascular system, is also responsible for the regulation of several other physiological events[73]. In addition, recent research has shown that AngII plays important role in the inflammatory response[74]. AngII can also activate transcription factors such as NF-κβ by inducing redox activation[75].

PER, an ACE inhibitor, inhibits one step of AngII synthesis. However, in contrast to other ACE inhibitors, PER is also known to exhibit antioxidant, anti-inflammatory and antiapoptotic effects by increasing the synthesis of NO[76]. In agreement with previous studies, PER exhibited antioxidant, anti-inflammatory, and antiapoptotic effects through the above mechanisms in the present research. 5. Conclusion In conclusion, increased oxidative stress, inflammation, and apoptosis caused severe tissue damage following CLP-induced sepsis in lung tissue. Additionally, we observed that the resulting damage was suppressed the application of PER as a protective agent, through antioxidant, anti-inflammatory, and antiapoptotic effects. In light of this information, we may predict that mortality can probably be reduced and important contributions to treatment can be made in sepsis developing in patients receiving chronic PER therapy due to cardiovascular causes. Limitations of our study include the fact that only single PER dose effectiveness was investigated, and that no comparison was made with an effective antibiotic. In addition, ours is a pilot study aimed at clarifying pathways to be studied in future research at the cellular level and needs to be supported by studies also involving intracellular pathways and mechanisms and mitochondrial calcium levels. Authors’ Contributions UK and AT conceived and designed the research. UK, AT and LT conducted the experiments. MA, HAU and TM contributed new reagents or analytical tools. UK, AT, MA, HAU and TM analyzed the data. AT, UK, MA and TM wrote the manuscript. All authors read and approved the manuscript.

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

References [1]

R.P. Dellinger, M.M. Levy, A. Rhodes, D. Annane, H. Gerlach, S.M. Opal, J.E. Sevransky, C.L. Sprung, I.S. Douglas, R. Jaeschke, T.M. Osborn, M.E. Nunnally, S.R. Townsend, K. Reinhart, R.M. Kleinpell, D.C. Angus, C.S. Deutschman, F.R. Machado, G.D. Rubenfeld, S. Webb, R.J. Beale, J.L. Vincent, R. Moreno, L. Aitken, H. Al Rahma, G.R. Bernard, P. Biban, J.F. Bion, T. Calandra, J.A. Carcillo, T.P. Clemmer, J. V. Divatia, B. Du, S. Fujishima, S. Gando, C.G. Bruch, G. Guyatt, J.A. Hazelzet, H. Hirasawa, S.M. Hollenberg, J. Jacobi, I. Jenkins, E. Jimenez, A.E. Jones, R.M. Kacmarek, W. Kern, S.O. Koh, J. Kotani, M. Levy, F. Machado, J. Marini, J.C. Marshall, H. Masur, S. Mehta, J. Muscedere, L.M. Napolitano, M.M. Parker, J.E. Parrrillo, H. Qiu, A.G. Randolph, J. Rello, E. Resende, E.P. Rivers, C.A. Schorr, K. Shukri, E. Silva, M.D. Soth, A.E. Thompson, J.S. Vender, S.A. Webb, T. Welte, J.L. Zimmerman, Surviving sepsis campaign: International guidelines for management of severe sepsis and septic shock, 2012, Intensive Care Med. 39 (2013) 165–228. doi:10.1007/s00134-012-2769-8.

[2]

J. Fu, G. Li, X. Wu, B. Zang, Sodium Butyrate Ameliorates Intestinal Injury and Improves Survival in a Rat Model of Cecal Ligation and Puncture-Induced Sepsis, Inflammation. 42 (2019) 1276–1286. doi:10.1007/s10753-019-00987-2.

[3]

C. Troeger, B. Blacker, I.A. Khalil, P.C. Rao, J. Cao, S.R.M. Zimsen, S.B. Albertson, A. Deshpande, T. Farag, Z. Abebe, I.M.O. Adetifa, T.B. Adhikari, M. Akibu, F.H. Al Lami, A. Al-Eyadhy, N. Alvis-Guzman, A.T. Amare, Y.A. Amoako, C.A.T. Antonio, O. Aremu, E.T. Asfaw, S.W. Asgedom, T.M. Atey, E.F. Attia, E.F.G.A. Avokpaho, H.T. Ayele, T.B. Ayuk, K. Balakrishnan, A. Barac, Q. Bassat, M. Behzadifar, M. Behzadifar, S. Bhaumik, Z.A. Bhutta, A. Bijani, M. Brauer, A. Brown, P.A.M. Camargos, C.A. Castañeda-Orjuela, D. Colombara, S. Conti, A.F. Dadi, L. Dandona,

R. Dandona, H.P. Do, E. Dubljanin, D. Edessa, H. Elkout, A.Y. Endries, D.O. Fijabi, K.J. Foreman, M.H. Forouzanfar, N. Fullman, A.L. Garcia-Basteiro, B.D. Gessner, P.W. Gething, R. Gupta, T. Gupta, G.B. Hailu, H.Y. Hassen, M.T. Hedayati, M. Heidari, D.T. Hibstu, N. Horita, O.S. Ilesanmi, M.B. Jakovljevic, A.A. Jamal, A. Kahsay, A. Kasaeian, D.H. Kassa, Y.S. Khader, E.A. Khan, M.N. Khan, Y.H. Khang, Y.J. Kim, N. Kissoon, L.D. Knibbs, S. Kochhar, P.A. Koul, G.A. Kumar, R. Lodha, H. Magdy Abd El Razek, D.C. Malta, J.L. Mathew, D.T. Mengistu, H.B. Mezgebe, K.A. Mohammad, M.A. Mohammed, F. Momeniha, S. Murthy, C.T. Nguyen, K.R. Nielsen, D.N.A. Ningrum, Y.L. Nirayo, E. Oren, J.R. Ortiz, M. PA, M.J. Postma, M. Qorbani, R. Quansah, R.K. Rai, S.M. Rana, C.L. Ranabhat, S.E. Ray, M.S. Rezai, G.M. Ruhago, S. Safiri, J.A. Salomon, B. Sartorius, M. Savic, M. Sawhney, J. She, A. Sheikh, M.S. Shiferaw, M. Shigematsu, J.A. Singh, R. Somayaji, J.D. Stanaway, M.B. Sufiyan, G.R. Taffere, M.H. Temsah, M.J. Thompson, R. Tobe-Gai, R. Topor-Madry, B.X. Tran, T.T. Tran, K.B. Tuem, K.N. Ukwaja, S.E. Vollset, J.L. Walson, F. Weldegebreal, A. Werdecker, T.E. West, N. Yonemoto, M.E.S. Zaki, L. Zhou, S. Zodpey, T. Vos, M. Naghavi, S.S. Lim, A.H. Mokdad, C.J.L. Murray, S.I. Hay, R.C. Reiner, Estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory infections in 195 countries, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016, Lancet Infect. Dis. 18 (2018) 1191–1210. doi:10.1016/S1473-3099(18)30310-4. [4]

M. Naghavi, A.A. Abajobir, C. Abbafati, K.M. Abbas, F. Abd-Allah, S.F. Abera, V. Aboyans, O. Adetokunboh, J. Ärnlöv, A. Afshin, A. Agrawal, A.A. Kiadaliri, A. Ahmadi, M.B. Ahmed, A.N. Aichour, I. Aichour, M.T.E. Aichour, S. Aiyar, A. AlEyadhy, F. Alahdab, Z. Al-Aly, K. Alam, N. Alam, T. Alam, K.A. Alene, S.D. Ali, R. Alizadeh-Navaei, J.M. Alkaabi, A. Alkerwi, F. Alla, P. Allebeck, C. Allen, R. Al-

Raddadi, U. Alsharif, K.A. Altirkawi, N. Alvis-Guzman, A.T. Amare, E. Amini, W. Ammar, Y.A. Amoako, N. Anber, H.H. Andersen, C.L. Andrei, S. Androudi, H. Ansari, C.A.T. Antonio, P. Anwari, M. Arora, A. Artaman, K.K. Aryal, H. Asayesh, S.W. Asgedom, T.M. Atey, L. Avila-Burgos, E.F.G.A. Avokpaho, A. Awasthi, B.P.A. Quintanilla, Y. Béjot, T.K. Babalola, U. Bacha, K. Balakrishnan, A. Barac, M.A. Barboza, S.L. Barker-Collo, S. Barquera, L. Barregard, L.H. Barrero, B.T. Baune, N. Bedi, E. Beghi, B.B. Bekele, M.L. Bell, J.R. Bennett, I.M. Bensenor, A. Berhane, E. Bernabé, B.D. Betsu, M. Beuran, S. Bhatt, S. Biadgilign, K. Bienhof, B. Bikbov, D. Bisanzio, R.R.A. Bourne, N.J.K. Breitborde, L.N.B. Bulto, B.R. Bumgarner, Z.A. Butt, R. Cárdenas, L. Cahuana-Hurtado, E. Cameron, J.C. Campuzano, J. Car, J.J. Carrero, A. Carter, D.C. Casey, C.A. Castañeda-Orjuela, F. Catalá-López, F.J. Charlson, C.E. Chibueze, O. Chimed-Ochir, V.H. Chisumpa, A.A. Chitheer, D.J. Christopher, L.G. Ciobanu, M. Cirillo, A.J. Cohen, D. Colombara, C. Cooper, B.C. Cowie, M.H. Criqui, L. Dandona, R. Dandona, P.I. Dargan, J. Das Neves, D. V. Davitoiu, K. Davletov, B. De Courten, L. Degenhardt, S. Deiparine, K. Deribe, A. Deribew, S. Dey, D. Dicker, E.L. Ding, S. Djalalinia, H.P. Do, D.T. Doku, D. Douwes-Schultz, T.R. Driscoll, M. Dubey, B.B. Duncan, M. Echko, Z.Z. El-Khatib, C.L. Ellingsen, A. Enayati, H.E. Erskine, S. Eskandarieh, A. Esteghamati, S.P. Ermakov, K. Estep, C.S. Sa Farinha, A. Faro, F. Farzadfar, V.L. Feigin, S.M. Fereshtehnejad, J.C. Fernandes, A.J. Ferrari, T.R. Feyissa, I. Filip, S. Finegold, F. Fischer, C. Fitzmaurice, A.D. Flaxman, N. Foigt, T. Frank, M. Fraser, N. Fullman, T. Fürst, J.M. Furtado, E. Gakidou, A.L. GarciaBasteiro, T. Gebre, G.B. Gebregergs, T.T. Gebrehiwot, D.Y. Gebremichael, J.M. Geleijnse, R. Genova-Maleras, H.A. Gesesew, P.W. Gething, R.F. Gillum, I.A.M. Ginawi, A.Z. Giref, M. Giroud, G. Giussani, W.W. Godwin, A.L. Gold, E.M. Goldberg, P.N. Gona, S.V. Gopalani, H.N. Gouda, A.C. Goulart, M. Griswold, P.C.

Gupta, R. Gupta, T. Gupta, V. Gupta, J.A. Haagsma, N. Hafezi-Nejad, A.D. Hailu, G.B. Hailu, R.R. Hamadeh, M.T. Hambisa, S. Hamidi, M. Hammami, J. Hancock, A.J. Handal, G.J. Hankey, Y. Hao, H.L. Harb, H.A. Hareri, M.S. Hassanvand, R. Havmoeller, S.I. Hay, F. He, M.T. Hedayati, N.J. Henry, I.B. Heredia-Pi, C. Herteliu, H.W. Hoek, M. Horino, N. Horita, H.D. Hosgood, S. Hostiuc, P.J. Hotez, D.G. Hoy, C. Huynh, K.M. Iburg, C. Ikeda, B.V. Ileanu, A.A. Irenso, C.M.S. Irvine, M. Jürisson, K.H. Jacobsen, N. Jahanmehr, M.B. Jakovljevic, M. Javanbakht, S.P. Jayaraman, P. Jeemon, V. Jha, D. John, C.O. Johnson, S.C. Johnson, J.B. Jonas, Z. Kabir, R. Kadel, A. Kahsay, R. Kamal, A. Karch, S.M. Karimi, C. Karimkhani, A. Kasaeian, N.A. Kassaw, N.J. Kassebaum, S.V. Katikireddi, N. Kawakami, P.N. Keiyoro, L. Kemmer, C.N. Kesavachandran, Y.S. Khader, E.A. Khan, Y.H. Khang, A.T.A. Khoja, A. Khosravi, M.H. Khosravi, J. Khubchandani, C. Kieling, D. Kievlan, D. Kim, Y.J. Kim, R.W. Kimokoti, Y. Kinfu, N. Kissoon, M. Kivimaki, A.K. Knudsen, J.A. Kopec, S. Kosen, P.A. Koul, A. Koyanagi, B.K. Defo, X.R. Kulikof, G.A. Kumar, P. Kumar, M. Kutz, H.H. Kyu, D.K. Lal, R. Lalloo, T.L.N. Lambert, Q. Lan, V.C. Lansingh, A. Larsson, P.H. Lee, J. Leigh, J. Leung, M. Levi, Y. Li, D.L. Kappe, X. Liang, M.L. Liben, S.S. Lim, A. Liu, P.Y. Liu, Y. Liu, R. Lodha, G. Logroscino, S. Lorkowski, P.A. Lotufo, R. Lozano, T.C.D. Lucas, S. Ma, E.R.K. Macarayan, E.R. Maddison, M.M. Abd El Razek, M. Majdan, R. Majdzadeh, A. Majeed, R. Malekzadeh, R. Malhotra, D.C. Malta, H. Manguerra, T. Manyazewal, C.C. Mapoma, L.B. Marczak, D. Markos, J. Martinez-Raga, F.R. Martins-Melo, I. Martopullo, C. McAlinden, M. McGaughey, J.J. McGrath, S. Mehata, T. Meier, K.G. Meles, P. Memiah, Z.A. Memish, M.M. Mengesha, D.T. Mengistu, B.G. Menota, G.A. Mensah, A. Meretoja, T.J. Meretoja, A. Millear, T.R. Miller, S. Minnig, M. Mirarefn, E.M. Mirrakhimov, A. Misganaw, S.R. Mishra, K.A. Mohammad, A. Mohammadi, S. Mohammed, A.H.

Mokdad, G.L.D. Mola, S.K. Mollenkopf, M. Molokhia, L. Monasta, J.C.M. Hernandez, M. Montico, M.D. Mooney, M. Moradi-Lakeh, P. Moraga, L. Morawska, S.D. Morrison, C. Morozof, C. Mountjoy-Venning, K.B. Mruts, K. Muller, G.V.S. Murthy, K.I. Musa, J.B. Nachega, A. Naheed, L. Naldi, V. Nangia, B.R. Nascimento, J.T. Nasher, G. Natarajan, I. Negoi, J.W. Ngunjiri, C.T. Nguyen, G. Nguyen, M. Nguyen, Q. Le Nguyen, T.H. Nguyen, E. Nichols, D.N.A. Ningrum, V.M. Nong, J.J.N. Noubiap, F.A. Ogbo, I.H. Oh, A. Okoro, A.T. Olagunju, H.E. Olsen, B.O. Olusanya, J.O. Olusanya, K. Ong, J.N. Opio, E. Oren, A. Ortiz, M. Osman, E. Ota, P.A. Mahesh, R.E. Pacella, S. Pakhale, A. Pana, B.K. Panda, S. Jonas, C. Papachristou, E.K. Park, S.B. Patten, G.C. Patton, D. Paudel, K. Paulson, D.M. Pereira, F. Perez-Ruiz, N. Perico, A. Pervaiz, M. Petzold, M.R. Phillips, D.M. Pigott, C. Pinho, D. Plass, M.A. Pletcher, S. Polinder, M.J. Postma, F. Pourmalek, C. Purcell, M. Qorbani, A. Radfar, A. Rafay, V. Rahimi-Movaghar, M. Rahman, M.H. Ur Rahman, R.K. Rai, C.L. Ranabhat, Z. Rankin, P.C. Rao, G.K. Rath, S. Rawaf, S.E. Ray, J. Rehm, R.C. Reiner, M.B. Reitsma, G. Remuzzi, S. Rezaei, M.S. Rezai, M.B. Rokni, L. Ronfani, G. Roshandel, G.A. Roth, D. Rothenbacher, G.M. Ruhago, S.A. Rizwan, S. Saadat, P.S. Sachdev, N. Sadat, M. Safdarian, S. Saf, S. Safiri, R. Sagar, R. Sahathevan, J. Salama, P. Salamati, J.A. Salomon, A.M. Samy, J.R. Sanabria, M.D. Sanchez-Niño, D. Santomauro, I.S. Santos, M.M.S. Milicevic, B. Sartorius, M. Satpathy, S. Shahraz, M.I. Schmidt, I.J.C. Schneider, S. Schulhofer-Wohl, A.E. Schutte, D.C. Schwebel, F. Schwendicke, S.G. Sepanlou, E.E. Servan-Mori, K.A. Shackelford, M.A. Shaikh, M. Shamsipour, M. Shamsizadeh, S.M.S. Islam, J. Sharma, R. Sharma, J. She, S. Sheikhbahaei, M. Shey, P. Shi, C. Shields, M. Shigematsu, R. Shiri, S. Shirude, I. Shiue, H. Shoman, M.G. Shrime, I.D. Sigfusdottir, N. Silpakit, J.P. Silva, A. Singh, J.A. Singh, E. Skiadaresi, A. Sligar, A. Smith, D.L. Smith, M. Smith, B.H.A. Sobaih,

S. Soneji, R.J.D. Sorensen, J.B. Soriano, C.T. Sreeramareddy, V. Srinivasan, J.D. Stanaway, V. Stathopoulou, N. Steel, D.J. Stein, C. Steiner, S. Steinke, M.A. Stokes, M. Strong, B. Strub, M. Subart, M.B. Sufyan, B.F. Sunguya, P.J. Sur, S. Swaminathan, B.L. Sykes, R. Tabarés-Seisdedos, S.K. Tadakamadla, K. Takahashi, J.S. Takala, R.T. Talongwa, M.R. Tarawneh, M. Tavakkoli, N. Taveira, T.K. Tegegne, A. TehraniBanihashemi, M.H. Temsah, A.S. Terkawi, J.S. Thakur, O. Thamsuwan, K.R. Thankappan, K.E. Thomas, A.H. Thompson, A.J. Thomson, A.G. Thrift, R. Tobe-Gai, R. Topor-Madry, A. Torre, M. Tortajada, J.A. Towbin, B.X. Tran, C. Troeger, T. Truelsen, D. Tsoi, E.M. Tuzcu, S. Tyrovolas, K.N. Ukwaja, E.A. Undurraga, R. Updike, O.A. Uthman, B.S.C. Uzochukwu, J.F.M. Van Boven, T. Vasankari, N. Venketasubramanian, F.S. Violante, V.V. Vlassov, S.E. Vollset, T. Vos, T. Wakayo, M.T. Wallin, Y.P. Wang, E. Weiderpass, R.G. Weintraub, D.J. Weiss, A. Werdecker, R. Westerman, B. Whetter, H.A. Whiteford, T. Wijeratne, C.S. Wiysonge, B.G. Woldeyes, C.D.A. Wolfe, R. Woodbrook, A. Workicho, D. Xavier, Q. Xiao, G. Xu, M. Yaghoubi, B. Yakob, Y. Yano, M. Yaseri, H.H. Yimam, N. Yonemoto, S.J. Yoon, M. Yotebieng, M.Z. Younis, Z. Zaidi, M. El Sayed Zaki, E.A. Zegeye, Z.M. Zenebe, T.A. Zerfu, A.L. Zhang, X. Zhang, B. Zipkin, S. Zodpey, A.D. Lopez, C.J.L. Murray, Global, regional, and national age-sex specifc mortality for 264 causes of death, 19802016: A systematic analysis for the Global Burden of Disease Study 2016, Lancet. 390 (2017) 1151–1210. doi:10.1016/S0140-6736(17)32152-9. [5]

M.S. Rangel-Frausto, The epidemiology of bacterial sepsis, Infect. Dis. Clin. North Am. 13 (1999) 299–312. doi:10.1016/S0891-5520(05)70076-3.

[6]

M.E. Mikkelsen, C. V. Shah, N.J. Meyer, D.F. Gaieski, S. Lyon, A.N. Miltiades, M. Goyal, B.D. Fuchs, S.L. Bellamy, J.D. Christie, The epidemiology of acute respiratory distress syndrome in patients presenting to the emergency department with severe

sepsis, Shock. 40 (2013) 375–381. doi:10.1097/SHK.0b013e3182a64682. [7]

C.H. Liu, W.D. Zhang, J.J. Wang, S.D. Feng, Senegenin Ameliorate Acute Lung Injury Through Reduction of Oxidative Stress and Inhibition of Inflammation in Cecal Ligation and Puncture-Induced Sepsis Rats, Inflammation. 39 (2016) 900–906. doi:10.1007/s10753-016-0322-6.

[8]

P. Andrews, E. Azoulay, M. Antonelli, L. Brochard, C. Brun-Buisson, D. De Backer, G. Dobb, J.Y. Fagon, H. Gerlach, J. Groeneveld, J. Mancebo, P. Metnitz, S. Nava, J. Pugin, M. Pinsky, P. Radermacher, C. Richard, R. Tasker, Year in review in intensive care medicine. 2005. I. Acute respiratory failure and acute lung injury, ventilation, hemodynamics, education, renal failure, Intensive Care Med. 32 (2006) 207–216. doi:10.1007/s00134-005-0027-z.

[9]

J.J. Zimmerman, S.R. Akhtar, E. Caldwell, G.D. Rubenfeld, Incidence and outcomes of pediatric acute lung injury, Pediatrics. 124 (2009) 87–95. doi:10.1542/peds.2007-2462.

[10] A. Bedirli, M. Kerem, H. Pasaoglu, N. Akyurek, T. Tezcaner, S. Elbeg, L. Memis, O. Sakrak, Beta-glucan attenuates inflammatory cytokine release and prevents acute lung injury in an experimental model of sepsis, Shock. 27 (2007) 397–401. doi:10.1097/01.shk.0000245030.24235.f1. [11] I.J. Laudes, R.F. Guo, N.C. Riedemann, C. Speyer, R. Craig, J.V. Sarma, P.A. Ward, Disturbed Homeostasis of Lung Intercellular Adhesion Molecule-1 and Vascular Cell Adhesion Molecule-1 during Sepsis, Am. J. Pathol. 164 (2004) 1435–1445. doi:10.1016/S0002-9440(10)63230-0. [12] J. Grommes, O. Soehnlein, Contribution of neutrophils to acute lung injury, Mol. Med. 17 (2011) 293–307. doi:10.2119/molmed.2010.00138.

[13] J. Bi, R. Cui, Z. Li, C. Liu, J. Zhang, Astaxanthin alleviated acute lung injury by inhibiting oxidative/nitrative stress and the inflammatory response in mice, Biomed. Pharmacother. 95 (2017) 974–982. doi:10.1016/j.biopha.2017.09.012. [14] G. Sun, W. Yang, Y. Zhang, M. Zhao, Esculentoside A ameliorates cecal ligation and puncture-induced acute kidney injury in rats, Exp. Anim. 66 (2017) 303–312. doi:10.1538/expanim.16-0102. [15] B. Polat, E. Cadirci, Z. Halici, Y. Bayir, D. Unal, B.C. Bilgin, T.N. Yuksel, S. Vancelik, The protective effect of amiodarone in lung tissue of cecal ligation and puncture-induced septic rats: A perspective from inflammatory cytokine release and oxidative stress, Naunyn. Schmiedebergs. Arch. Pharmacol. 386 (2013) 635–643. doi:10.1007/s00210-013-0862-3. [16] U. Siebenlist, G. Franzoso, K. Brown, Structure, regulation and function of NF-κB, Annu. Rev. Cell Biol. 10 (1994) 405–455. doi:10.1146/annurev.cb.10.110194.002201. [17] G. Feng, S. Liu, G.L. Wang, G.J. Liu, Lidocaine attenuates lipopolysaccharide-induced acute lung injury through inhibiting NF-κB activation, Pharmacology. 81 (2007) 32– 40. doi:10.1159/000107792. [18] A. Albayrak, Z. Halici, B. Polat, E. Karakus, E. Cadirci, Y. Bayir, S. Kunak, S.S. Karcioglu, S. Yigit, D. Unal, S.S. Atamanalp, Protective effects of lithium: A new look at an old drug with potential antioxidative and anti-inflammatory effects in an animal model of sepsis, Int. Immunopharmacol. 16 (2013) 35–40. doi:10.1016/j.intimp.2013.03.018. [19] G. Till, J.R. Hatherill, Lipid Peroxidation Acute Lung Injury to Skin Role for Hydroxyl Radical, (1985) 376–384.

[20] Y. Wang, X. Wang, L. Zhang, R. Zhang, Alleviation of acute lung injury in rats with sepsis by resveratrol via the phosphatidylinositol 3-kinase/nuclear factor-erythroid 2 related factor 2/heme oxygenase-1 (PI3K/NRF2/HO-1) pathway, Med. Sci. Monit. 24 (2018) 3604–3611. doi:10.12659/MSM.910245. [21] Q.Y. Peng, Y. Zou, L.N. Zhang, M.L. Ai, W. Liu, Y.H. Ai, Blocking cyclic adenosine diphosphate ribose-mediated calcium overload attenuates sepsis-induced acute lung injury in rats, Chin. Med. J. (Engl). 129 (2016) 1725–1730. doi:10.4103/03666999.185854. [22] I. Rahman, S. Biswas, L. Jimenez, M. Torres, H. Forman, Glutathione, stress responses, and redox signaling in lung inflammation, Antioxid. Redox Signal. 7 (2005) 42–59. [23] Y. Zong, H. Zhang, Amentoflavone prevents sepsis-associated acute lung injury through Nrf2-GCLc-mediated upregulation of glutathione, Acta Biochim. Pol. 64 (2017) 93–98. doi:10.18388/abp.2016_1296. [24] Y. Zhai, X. Zhou, Q. Dai, Y. Fan, X. Huang, Hydrogen-rich saline ameliorates lung injury associated with cecal ligation and puncture-induced sepsis in rats, Exp. Mol. Pathol. 98 (2015) 268–276. doi:10.1016/j.yexmp.2015.03.005. [25] W. Zhao, L. Jia, H.J. Yang, X. Xue, W.X. Xu, J.Q. Cai, R.J. Guo, C.C. Cao, Taurine enhances the protective effect of Dexmedetomidine on sepsis-induced acute lung injury via balancing the immunological system, Biomed. Pharmacother. 103 (2018) 1362– 1368. doi:10.1016/j.biopha.2018.04.150. [26] Q.L. Wang, L. Yang, Y. Peng, M. Gao, M.S. Yang, W. Xing, X.Z. Xiao, Ginsenoside Rg1 regulates SIRT1 to ameliorate sepsis-induced lung inflammation and injury via

inhibiting endoplasmic reticulum stress and inflammation, Mediators Inflamm. 2019 (2019) 1–10. doi:10.1155/2019/6453296. [27] W.C. De Mello, Local Renin Angiotensin Aldosterone Systems and Cardiovascular Diseases, Med. Clin. North Am. 101 (2017) 117–127. doi:10.1016/j.mcna.2016.08.017. [28] D.N. Muller, R. Dechend, E.M.A. Mervaala, J.K. Park, F. Schmidt, A. Fiebeler, J. Theuer, V. Breu, D. Ganten, H. Haller, F.C. Luft, NF-κB inhibition ameliorates angiotensin II-induced inflammatory damage in rats, Hypertension. 35 (2000) 193–201. [29] G. Wolf, U. Wenzel, K.D. Burns, R.C. Harris, R.A.K. Stahl, F. Thaiss, Angiotensin II activates nuclear transcription factor-κB through AT1 and AT2 receptors, Kidney Int. 61 (2002) 1986–1995. doi:10.1046/j.1523-1755.2002.00365.x. [30] E. Taskin, K.A. Tuncer, C. Guven, S.T. Kaya, N. Dursun, Inhibition of angiotensin-II production increases susceptibility to acute ischemia/reperfusion arrhythmia, Med. Sci. Monit. 22 (2016) 4587–4595. doi:10.12659/MSM.896350. [31] P.A. Todd, A. Fitton, Perindopril: A Review of its Pharmacological Properties and Therapeutic Use in Cardiovascular Disorders, Drugs. 42 (1991) 90–114. doi:10.2165/00003495-199142010-00006. [32] Z. Zheng, H. Chen, G. Ke, Y. Fan, H. Zou, X. Sun, Q. Gu, Protective Effect of Perindopril on Diabetic Retinopathy Is Associated With Decreased Vascular Endothelial Growth Factor–to–Pigment Epithelium–Derived, Diabetes. 58 (2009). doi:10.2337/db07-1524.Z.Z. [33] P. Raj, B.M. Aloud, X.L. Louis, L. Yu, S. Zieroth, T. Netticadan, Resveratrol is equipotent to perindopril in attenuating post-infarct cardiac remodeling and contractile dysfunction in rats, J. Nutr. Biochem. 28 (2016) 155–163.

doi:10.1016/j.jnutbio.2015.09.025. [34] A.G.S. Shalkami, M.I.A. Hassan, A.A. Abd El-Ghany, Perindopril regulates the inflammatory mediators, NF-κB/TNF-α/IL-6, and apoptosis in cisplatin-induced renal dysfunction, Naunyn. Schmiedebergs. Arch. Pharmacol. 391 (2018) 1247–1255. doi:10.1007/s00210-018-1550-0. [35] T. Mashhoody, K. Rastegar, F. Zal, Perindopril may improve the hippocampal reduced glutathione content in rats, Adv. Pharm. Bull. 4 (2014) 155–159. doi:10.5681/apb.2014.023. [36] D. Rittirsch, M.S. Huber-lang, M.A. Flierl, P.A. Ward, Immunodesing of experimental sepsis by cecal ligation and puncture, Nat Protoc. 4 (2009) 31–36. doi:10.1038/nprot.2008.214.Immunodesign. [37] I. Cinar, B. Sirin, P. Aydin, E. Toktay, E. Cadirci, I. Halici, Z. Halici, Ameliorative effect of gossypin against acute lung injury in experimental sepsis model of rats, Life Sci. 221 (2019) 327–334. doi:10.1016/j.lfs.2019.02.039. [38] D.B. Rojas, T. Gemelli, R.B. De Andrade, A.G. Campos, C.S. Dutra-Filho, C.M.D. Wannmacher, Administration of histidine to female rats induces changes in oxidative status in cortex and hippocampus of the offspring, Neurochem. Res. 37 (2012) 1031– 1036. doi:10.1007/s11064-012-0703-7. [39] H. Ohkawa, N. Ohishi, K. Yagi, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Anal. Biochem. 95 (1979) 351–358. doi:10.1016/00032697(79)90738-3. [40] R.J. Randall, A. Lewis, The folin by oliver, (n.d.).

[41] G. Matute-Bello, G. Downey, B.B. Moore, S.D. Groshong, M.A. Matthay, A.S. Slutsky, W.M. Kuebler, An official american thoracic society workshop report: Features and measurements of experimental acute lung injury in animals, Am. J. Respir. Cell Mol. Biol. 44 (2011) 725–738. doi:10.1165/rcmb.2009-0210ST. [42] Z. Zador, A. Landry, M.D. Cusimano, N. Geifman, Multimorbidity states associated with higher mortality rates in organ dysfunction and sepsis: A data-driven analysis in critical care, Crit. Care. 23 (2019) 1–11. doi:10.1186/s13054-019-2486-6. [43] X. Chen, T. Wang, L. Song, X. Liu, Activation of multiple Toll‑like receptors serves different roles in sepsis‑induced acute lung injury, Exp. Ther. Med. (2019) 443–450. doi:10.3892/etm.2019.7599. [44] J. Zhou, Y. Fu, K. Liu, L. Hou, W. Zhang, miR‑206 regulates alveolar type�II epithelial cell Cx43 expression in sepsis‑induced acute lung injury, Exp. Ther. Med. (2019) 296–304. doi:10.3892/etm.2019.7551. [45] P. Störmann, N. Becker, J.T. Vollrath, K. Köhler, A. Janicova, S. Wutzler, F. Hildebrand, I. Marzi, B. Relja, Early Local Inhibition of Club Cell Protein 16 Following Chest Trauma Reduces Late Sepsis-Induced Acute Lung Injury, J. Clin. Med. 8 (2019) 896. doi:10.3390/jcm8060896. [46] C. Rosendorff, The renin-angiotensin system and vascular hypertrophy, J. Am. Coll. Cardiol. 28 (1996) 803–812. doi:10.1016/S0735-1097(96)00251-3. [47] A. Sirker, M. Zhang, C. Murdoch, A.M. Shah, Involvement of NADPH oxidases in cardiac remodelling and heart failure, Am. J. Nephrol. 27 (2007) 649–660. doi:10.1159/000109148.

[48] K.K. Griendling, C.A. Minieri, J.D. Ollerenshaw, R.W. Alexander, Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells, Circ. Res. 74 (1994) 1141–1148. doi:10.1161/01.RES.74.6.1141. [49] D. Agarwal, M. Haque, S. Sriramula, N. Mariappan, R. Pariaut, J. Francis, Role of Proinflammatory Cytokines and Redox Homeostasis in Exercise-Induced Delayed Progression of Hypertension in Spontaneously Hypertensive Rats, Hypertension. 54 (2009) 1393–1400. doi:10.1161/HYPERTENSIONAHA.109.135459. [50] A.C. Montezano, M. Dulak-Lis, S. Tsiropoulou, A. Harvey, A.M. Briones, R.M. Touyz, Oxidative stress and human hypertension: Vascular mechanisms, biomarkers, and novel therapies, Can. J. Cardiol. 31 (2015) 631–641. doi:10.1016/j.cjca.2015.02.008. [51] Y. Butt, A. Kurdowska, T.C. Allen, Acute lung injury: A clinical and molecular review, Arch. Pathol. Lab. Med. 140 (2016) 345–350. doi:10.5858/arpa.2015-0519RA. [52] E. Akpinar, Z. Halici, E. Cadirci, Y. Bayir, E. Karakus, M. Calik, A. Topcu, B. Polat, What is the role of renin inhibition during rat septic conditions: Preventive effect of aliskiren on sepsis-induced lung injury, Naunyn. Schmiedebergs. Arch. Pharmacol. 387 (2014) 969–978. doi:10.1007/s00210-014-1014-0. [53] E. Niki, Y. Yoshida, Y. Saito, N. Noguchi, Lipid peroxidation: Mechanisms, inhibition, and biological effects, Biochem. Biophys. Res. Commun. 338 (2005) 668–676. doi:10.1016/j.bbrc.2005.08.072. [54] E. Cadirci, B.Z. Altunkaynak, Z. Halici, F. Odabasoglu, M.H. Uyanik, C. Gundogdu, H. Suleyman, M. Halici, M. Albayrak, B. Unal, α-LIPOIC acid as a potential target for

the treatment of lung injury caused by cecal ligation and puncture-induced sepsis model in rats, Shock. 33 (2010) 479–484. doi:10.1097/SHK.0b013e3181c3cf0e. [55] E.A.M. El-Shoura, B.A.S. Messiha, S.M.Z. Sharkawi, R.A.M. Hemeida, Perindopril ameliorates lipopolysaccharide-induced brain injury through modulation of angiotensin-II/angiotensin-1-7 and related signaling pathways, Eur. J. Pharmacol. 834 (2018) 305–317. doi:10.1016/j.ejphar.2018.07.046. [56] T. Jawaid, S. Jahan, M. Kamal, A comparative study of neuroprotective effect of angiotensin converting enzyme inhibitors against scopolamine-induced memory impairments in rats, J. Adv. Pharm. Technol. Res. 6 (2015) 130–135. doi:10.4103/2231-4040.161514. [57] A. Topcu, F. Mercantepe, S. Rakici, L. Tumkaya, H.A. Uydu, T. Mercantepe, An investigation of the effects of N-acetylcysteine on radiotherapy-induced testicular injury in rats, Naunyn. Schmiedebergs. Arch. Pharmacol. 392 (2019) 147–157. doi:10.1007/s00210-018-1581-6. [58] T. Aktoz, M. Caloglu, V. Yurut-Caloglu, O. Yalcin, N. Aydogdu, D. Nurlu, E. Arda, O. Inci, Histopathological and biochemical comparisons of the protective effects of amifostine and l-carnitine against radiation-induced acute testicular toxicity in rats, Andrologia. 49 (2017) 1–7. doi:10.1111/and.12754. [59] W.Y. Kim, S.B. Hong, Sepsis and acute respiratory distress syndrome: Recent update, Tuberc. Respir. Dis. (Seoul). 79 (2016) 53–57. doi:10.4046/trd.2016.79.2.53. [60] E.J. Seeley, M.A. Matthay, P.J. Wolters, Inflection points in sepsis biology: From local defense to systemic organ injury, Am. J. Physiol. - Lung Cell. Mol. Physiol. 303 (2012). doi:10.1152/ajplung.00069.2012.

[61] D.H. Seitz, U. Niesler, A. Palmer, M. Sulger, S.T. Braumüller, M. Perl, F. Gebhard, M.W. Knöferl, Blunt chest trauma induces mediator-dependent monocyte migration to the lung, Crit. Care Med. 38 (2010) 1852–1859. doi:10.1097/CCM.0b013e3181e8ad10. [62] J.Y. Li, H.X. Wu, G. Yang, Pachymic acid improves survival and attenuates acute lung injury in septic rats induced by cecal ligation and puncture, Eur. Rev. Med. Pharmacol. Sci. 21 (2017) 1904–1910. [63] W. Schulte, J. Bernhagen, R. Bucala, Cytokines in sepsis: Potent immunoregulators and potential therapeutic targets - An updated view, Mediators Inflamm. 2013 (2013). doi:10.1155/2013/165974. [64] J. Hu, Z. Tang, J. Xu, W. Ge, Q. Hu, F. He, G. Zheng, L. Jiang, Z. Yang, W. Tang, The inhibitor of interleukin-3 receptor protects against sepsis in a rat model of cecal ligation and puncture, Mol. Immunol. 109 (2019) 71–80. doi:10.1016/j.molimm.2019.03.002. [65] W. Gilowski, R. Krysiak, B. Marek, B. Okopien, The effect of short-term perindopril and telmisartan treatment on circulating levels of anti-inflammatory cytokines in hypertensive patients, Endokrynol. Pol. 69 (2018) 667–974. [66] A. Aderem, J. Ulevitch, Toll-like receptors in the induction of the innate immune response, Nature. 406 (2000) 782–787. [67] S. Saber, A.A.A. Mahmoud, R. Goda, N.S. Helal, E. El-ahwany, R.H. Abdelghany, Perindopril, fosinopril and losartan inhibited the progression of diethylnitrosamineinduced hepatocellular carcinoma in mice via the inactivation of nuclear transcription factor kappa-B, Toxicol. Lett. 295 (2018) 32–40. doi:10.1016/j.toxlet.2018.05.036. [68] H. Mou, Y. Zheng, P. Zhao, H. Bao, W. Fang, N. Xu, Celastrol induces apoptosis in non-small-cell lung cancer A549 cells through activation of mitochondria- and

Fas/FasL-mediated pathways, Toxicol. Vitr. 25 (2011) 1027–1032. doi:10.1016/j.tiv.2011.03.023. [69] R. Zhai, M.N. Gong, W. Zhou, T.B. Thompson, P. Kraft, L. Su, D.C. Christiani, Genotypes and haplotypes of the VEGF gene are associated with higher mortality and lower VEGF plasma levels in patients with ARDS, Thorax. 62 (2007) 718–722. doi:10.1136/thx.2006.069393. [70] L. Fialkow, L.F. Filho, M.C. Bozzetti, A.R. Milani, E.M. Rodrigues Filho, R.M. Ladniuk, P. Pierozan, R.M. de Moura, J.C. Prolla, E. Vachon, G.P. Downey, Neutrophil apoptosis: A marker of disease severity in sepsis and sepsis-induced acute respiratory distress syndrome, Crit. Care. 10 (2006) 1–14. doi:10.1186/cc5090. [71] P.C.A. Kam, N.I. Ferch, Apoptosis: Mechanisms and clinical implications, Anaesthesia. 55 (2000) 1081–1093. doi:10.1046/j.1365-2044.2000.01554.x. [72] R.Ş. Fodor, A.M. Georgescu, B.L. Grigorescu, A.D. Cioc, M. Veres, O.S. Cotoi, P. Fodor, S.M. Copotoiu, L. Azamfirei, Caspase 3 expression and plasma level of fas ligand as apoptosis biomarkers in inflammatory endotoxemic lung injury, Rom. J. Morphol. Embryol. 57 (2016) 951–957. [73] G. Nguyen, F. Delarue, C. Burcklé, L. Bouzhir, T. Giller, J.D. Sraer, Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin, J. Clin. Invest. 109 (2002) 1417–1427. doi:10.1172/JCI0214276. [74] Y. Suzuki, M. Ruiz-Ortega, O. Lorenzo, M. Ruperez, V. Esteban, J. Egido, Inflammation and angiotensin II, Int. J. Biochem. Cell Biol. 35 (2003) 881–900. doi:10.1016/S1357-2725(02)00271-6. [75] J. Barnes, M. Karin, Nuclear factor-B. A pivotal transcription factor in choronic

inflammatory diseases, N. Engl. J. Med. 336 (1997) 1066–1071. [76] J.J. Dinicolantonio, C.J. Lavie, J.H. O’Keefe, Not all angiotensin-converting enzyme inhibitors are equal: focus on ramipril and perindopril., Postgrad. Med. 125 (2013) 154–168. doi:10.3810/pgm.2013.07.2687.

FIGURE LEGENDS Figure 1. Biochemical Analysis Results. Figure 2. Light microscopic images representing pulmonary tissue sections stained with H&E. A(x10; bar length: 100 µm)-B(x20; bar length: 50 µm)-C(x40; bar length: 20 µm): Control group lung tissue sections showing typical type I (arrow) and type II pneumocytes (tailed arrow) in a normal alveolar sac. Respiratory bronchiole (rb). (LDS median: 0.5 (0-1). D(x10; bar length: 100 µm)-E(x20; bar length: 50 µm)-F(x40; bar length: 20 µm): CLP group lung tissue sections showing vascular congestion (c), hyaline membrane formation (curved arrow), and neutrophil accumulations in interstitial spaces (I). In addition, debris accumulation can be seen in the alveoli (asterisk) and thickening in the alveolar septal wall (arrowhead). (LDS median: 6 (4.5-6). G(x10; bar length: 100 µm)-H(x20; bar length: 50 µm)I(x40; bar length: 20 µm): PER treatment lung tissue sections show a decreased alveolar wall thickness (spiral arrow), and typical type I (arrow) and type II pneumocytes (tailed arrow) in the alveolar sac. Respiratory bronchiole (rb). (LDS median: 1(0-2).

Figure 3. Representative light microscopic images of lung tissue treated with NF-kβ/p65 primary antibody. A(x20; bar length: 50 µm)-B(x40; bar length: 20 µm): Control group lung tissue sections showing normal type I (arrow) and type II pneumocytes (tailed arrow). Respiratory bronchiole (rb). (NF-kβ/p65 positivity score median: 0.00(0-0). C(x20; bar length: 50 µm)-D(x40; bar length: 20 µm): CLP group lung tissue sections showing type I (blue arrow) (NF-kβ/p65 positivity score median: 3.00 (2-3)) and type II pneumocyte (blue tailed arrow) NF-kβ/p65 positivity (NF-kβ/p65 positivity score median: 2.00 (2-2.5)). E(x20; bar length: 50 µm)-F(x40; bar length: 20 µm): PER treatment group sections showing decreased type I (blue arrow) (NF-kβ/p65 positivity score median: 1.00 (1-1.50) and type II pneumocyte tip (blue tailed arrow) (NF-kβ/p65 positivity score median: 1.00(1-1.50)) NFkβ/p65 positivity, together with diffuse typical type I (arrow) and type II pneumocytes (tailed arrow) in the aveolar sacs. Respiratory bronchiole (rb).

Figure 4. Representative light microscopic images of lung tissue sections treated with caspase-3 primary antibody. A(x20; bar length: 50 µm)-B(x40; bar length: 20 µm): Control group lung tissue sections showing normal type I (arrow) and type II pneumocytes (tailed arrow). Respiratory bronchiole (rb). (Caspase-3 positivity score median: 0.00 (0-0.5). C(x20; bar length: 50 µm)-D(x40; bar length: 20 µm): CLP group lung tissue sections exhibiting apoptotic type I (blue arrow) (Caspase-3 positivity score median: 2.00 (2-2)) and type II pneumocytes (blue tailed arrow) (Caspase-3 positivity score median: 2.00 (2-2.5)). E(x20; bar length: 50 µm)-F(x40; bar length: 20 µm): PER treatment group lung tissue sections showing typical type I (arrow) (Caspase-3 positivity score median: 1.00 (1-1)) and type II pneumocytes (tailed arrow) (Caspase-3 positivity score median: 1.00 (1-1.5)). Respiratory bronchiole (rb).

Figure 1. Biochemical Analysis Results.

Figure 2. Representative light microscopy images of lung tissue sections stained with H&E

Figure 3. Representative light microscopy image of lung tissue sections treated with NFkβ/p65 primary antibody

Figure 4. Representative light microscopy image of lung tissue sections treated with caspase3 primary antibody

TABLES Table 1: Biochemical Analysis Results Measured Parameters

Study Groups

Control CLP CLP+PER

TBARS

Total SH

TNF-α

(nmol/g tissue)

(µmol/g tissue)

(ng/g tissue)

13.24 ± 2.34 a*

10.26 ± 2.06 a*

0.81 ± 0.16

16.04 ± 1.89

14.07 ± 2.46

0.98 ± 0.21

13.77 ± 0.74 a**

10.84 ± 2.42 a**

0.87 ± 0.20

*: p<0.01 **: p<0.05 a: Sepsis development group differed statistically significantly from the other groups

Table 2. Matute-Bello et al. Lung Damage Score (LDS)

Findings

Score 0

1

2

A. Neutrophils in alveolar areas B. Neutrophils in interstitial areas

None

1-5

>5

None

1-5

>5

C. Hyaline membrane

None

1

>1

D. Alveolar debris accumulation

None

1

>1

E. Alveolar septum thickness (Treatment Group/Control Group)

˂X2

2X-4X

>X4

Table 3. IHC Staining Positivity Scores None Mild Moderate Severe

0 + ++ +++

˂5% ˂5-25% ˂26-50% ˃51%

Table 4. Quantitative results. Group

a

Alveolar wall thickness (µm) (median+standard deviation)

Matute-Bello et al. modified alveolar septum thickness score

Control

8.26±1.73

1.00

0 (˂X2)

CLP

19.24±5.97a

2.33

2 (≥X2)

CLP+PER

12.74±4.05b

1.54

0 (˃X2)

p=0.000 Compared with the control group, p=0.017 Compared with the CLP group, One-Way ANOVA-Tukey test. b

Alveolar septum thickness (Treatment Group/Control Group)

Table 5. Semi-quantitative resutls. Group

Neutrophils Neutrophils Hyaline Alveolar Alveolar LDS debris septum in alveolar in membrane areas interstitial accumulation thickness areas (MatuteBello et al.) 0(0-0) 0(0-0.5) 0(0-0.0) 0(0-0.0) 0.5(0Control 0 1) 0(0-0) 1(1-1.5)a 1.5(1-2)c 1(1-2)e 6(4.5CLP 2c 6)c 0(0-0) 0(0-0.5)b 0(0-0.5)d 0(0-0.5)f 1(0-2)g CLP+PER 0f Values are given as median (interquartile range) a p=0.024 Compared with the control group, b p=0.024 Compared with the CLP group, c p=0.000 Compared with the control group, d p=0.001 Compared with the CLP group, e p=0.005 Compared with the control group, f p=0.017 Compared with the CLP group, g p=0.000 Compared with the CLP group, Kruskal Wallis -Tamhane’s T2 test

Table 6. IHC Positivity Grade Score Results (median with interquartile range). Group

Type I pneumocytes NF-kβ/p65 Caspase-3 Positivity Score Positivity Score 0.00 (0-0) 0.00 (0-0.5) Control a 3.00 (2-3) 2.00 (2-2)a CLP 1.00 (1-1.50)b 1.00 (1-1)c CLP+PER a p=0.000 Compared with the control group, b p=0.000 Compared with the CLP group, c p=0.007 Compared with the CLP group, d p=0.002 Compared with the CLP group, Kruskal Wallis-Tamhane T2

Type II pneumocytes NF-kβ/p65 Caspase-3 Positivity Score Positivity Score 0.00 (0-0) 0.00 (0-0.5) a 2.00 (2-2.5) 2.00 (2-2)a 1.00(1-1.50)d 0(0-1)b

Figure 1. Biochemical Analysis Results.

Figure 2. Representative light microscopy images of lung tissue sections stained with H&E

Figure 3. Representative light microscopy image of lung tissue sections treated with NFkβ/p65 primary antibody

Figure 4. Representative light microscopy image of lung tissue sections treated with caspase3 primary antibody