Protective Effects of Antioxidant Peptide SS-31 Against Multiple Organ Dysfunctions During Endotoxemia

Abstract—Oxidative stress causes mitochondrial impairment, the failure of energy production, and c- onsequent organ dysfunctions. The aim of the present study was to investigate the potential therapeutic effects of mitochondrial antioxidant SS-31 on sepsis-induced organ dysfunctions and to explore the possible mechanism. Sepsis was induced by cecal ligation and puncture. Immediately and at 5 h after the operation, SS-31 (5 mg/kg) or vehicle was administered intraperitoneally. The levels of organ dysfunc- tions, malondialdehyde, superoxide dismutase, proinflammatory cytokines, pulmonary wet-to-dry weight ratio, myeloperoxidase activity, histological scores, nuclear factor kappa B p65, inducible nitric oxide synthase, reactive oxygen species, adenosine triphosphate, and terminal deoxynucleotidyl trans- ferase dUTP nick end labeling (TUNEL)-positive cells were assessed at the indicated time points. The 7- day survival rate was estimated by the Kaplan-Meier method. In the present study, SS-31 treatment significantly improved sepsis-induced organ dysfunctions as evidenced by decreased histological scores, increased arterial partial oxygen tension, and deceased serum alanine aminotransferase, urea nitrogen, and creatinine levels, which was accompanied by decreased levels of malondialdehyde, tumor necrosis factor-alpha, pulmonary myeloperoxidase activity, nuclear factor kappa B p65, inducible nitric oxide synthase, reactive oxygen species, and TUNEL-positive cells. In conclusion, our data suggested that the protective effects of SS-31 on sepsis-induced organ dysfunctions were associated with the inhibition of proinflammatory cytokines, oxidative stress, and apoptosis.

KEY WORDS: sepsis; mitochondria; inflammation; oxidative stress; apoptosis.

INTRODUCTION

Despite increased understanding of the complex path- ophysiology of sepsis, it remains one of the major causes of morbidity and mortality in the intensive care units [1–4]. Sepsis represents an exaggerated inflammatory response and oxidative stress to infection that can progress to multiorgan dysfunction syndromes (MODS), which is characterized by a massive deregulated inflammatory re- sponse, overproduction of reactive oxygen species (ROS),
and reactive nitrogen species (RNS) in the circulation and the affected organs [4–6]. However, current therapeutic options for sepsis-associated organ dysfunctions are limit- ed, thus the development of new drug is urgently needed. Accumulating evidence has demonstrated that mito- chondrial dysfunction that occurs as a result of oxidative stress can result in the failure of energy production and consequent organ dysfunctions [7, 8]. This notion is sup- ported by the evidence that antioxidants that act preferen- tially in mitochondria can reduce mitochondrial damage and organ dysfunctions in a rat model of acute sepsis [5, 9]. SS-31 (D-Arg-Dmt-Lys-Phe-NH2) is a new and innovative mitochondria-targeted antioxidant that has an alternating aromatic-cationic structure, which allows it to freely cross the cell membrane and concentrate >1000 fold in the mitochondrial inner membrane independently of mito- chondrial membrane potential [9, 10]. It has been sug- gested that SS-31 can scavenge mitochondrial ROS, pro- mote mitochondrial function, reduce mitochondrial ROS generation, inhibit mitochondrial permeability transition, and prevent apoptosis [9–12]. In addition to targeting mi- tochondria, SS-31 is reported to scavenge hydrogen per- oxide (H2O2), hydroxyl radical, and peroxynitrite in a dose-dependent manner [13, 14]. Our previous study sug- gested that treatment with SS-31 ameliorates the cognitive deficits in a mouse model of sepsis [9]. Based on these findings, it is hypothesized that mitochondrial targeting with antioxidant peptide SS-31 represents a potential ther- apeutic option for the treatment of sepsis-induced organ dysfunctions.In the present study, we investigated the potential therapeutic effects of SS-31 on biomarkers of organ dys- functions and to explore its underlying mechanisms in a mouse model induced by cecal ligation and puncture (CLP).

MATERIALS AND METHODS

Animals and Ethics

The present study was approved by the Ethics Committee of Jinling Hospital, Nanjing University, and experiments were performed in adherence to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. Adult male C57BL/6 mice weighing 25–32 g were purchased from the Animal Center of Jinling Hospital, Nanjing, China. Animals were housed in standard conditions and maintained in a 12 h light/12 h dark cycle with food and water ad libitum.

Sepsis Model

Polymicrobial sepsis was induced by CLP as we described previously [15, 16]. Briefly, male C57BL/6 mice were anesthetized with intraperitoneal injection of 2 % sodium pentobarbital (60 mg/kg; Sigma Chemical Co, St. Louis, MO). The cecum was isolated and ligated with 4.0 silk below the ileocecal junction, approximately 1 cm from the distal end. The cecum was then punctured twice on the anti-mesenteric side with a sterile 22-gauge needle and was gently squeezed to extrude the fecal contents into the peritoneal cavity. Finally, the cecum was placed back into the abdomen and the incision was closed with sutures in layers. Control animals underwent laparotomy and bowel manipulation without ligation or perforation. All mice were resuscitated with subcutaneous lactated Ringers 30 ml/kg and antibiotic therapy (ertapenem, 20 mg/kg; Merck Research Laboratory, USA) immediately after surgery then returned to their cages.

Experimental Protocols

Vehicle (normal saline) or SS-31 (5 mg/kg, China Peptides Co., Ltd., China) was intraperitoneally adminis- tered immediately and at 5 h after the operation, which yielded the following four groups: control + vehicle group (n = 25), control + SS-31 group (n =25), CLP + vehicle group (n = 50), or CLP + SS-31 group (n = 50). The dosage of SS-31 was selected based on our previous study dem- onstrating SS-31 at 5 mg/kg exerting maximum protective effects without any side-effects [9].

Multiorgan Injury-Associated Enzymatic Indicators

Twenty-four hours after the operation, the arte- rial blood was obtained from the carotid artery for the determination of arterial oxygen (PO2) and car- bon dioxide tension (PCO2) by a GEM Premier 3000 gas analyzer (Instrumentation Laboratory, Guangzhou, China). The serum levels of blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were evaluated by the Olympus AU5400 automatic bio- chemical analyzer (Olympus, Tokyo, Japan) using commercially available clinical assay kits.

Malondialdehyde Content and Superoxide Dismutase Activity

Blood, lung, liver, and kidney were collected at 24 h after operation and then were separated by centrifugation at 5000 g for 20 min at 4 °C. Subsequently, malondialdehyde (MDA) content in the supernatants was measured using a commercially available MDA assay kit (Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instruction.

The superoxide dismutase (SOD) activity was estimated using a commercially available assay kit (Jiancheng Bioengineering Institute) following the manufacturer’s instruction. The amount of enzyme required to produce 50 % inhibition was defined as one unit of enzyme activity.

Pulmonary Myeloperoxidase Activity and Wet-to-Dry Weight Ratio

Pulmonary myeloperoxidase (MPO) activity, a mark- er for polymorphonuclear neutrophil infiltration into the lung, was determined by a MPO assay kit (Nanjing Jiancheng Bioengineering Institute) according to the man- ufacturer’s protocol.

The lung wet-to-dry weight (W/D) ratio was calculat- ed as a parameter of lung edema. To do this, the lungs were removed, weighed, and then dried in an oven at 80 °C for 48 h to obtain lung W/D ratio.

Enzyme-Linked Immunosorbent Assay

Blood samples were obtained from cardiac puncture at 0 (baseline), 6, 12, and 24 h after CLP. Serum levels of TNF-α (Diaclone Research, Besanson Cedex, France), IL- 1β, IL-6, and IL-10 (R&D Systems, Minneapolis, MN, USA) were quantified using specific enzyme-linked im- munosorbent assay (ELISA) kits for mice according to the manufacturers’ instructions.

Western Blotting Analysis

Western blotting was performed as previously de- scribed [15–17]. The primary antibodies used to detect nuclear factor (NF)-κB p65 and nitric oxide synthase (iNOS) were from Santa Cruz Biotechnology. The anti- body for β-actin was from Cell Signaling Technology. We used the NIH Image J software (National Institutes of Health, Bethesda, MD, USA) to quantitate protein band concentrations.

Histological Analysis

The severity of microscopic injury in the lung was graded from 0 (normal) to 4 (severe) based on the following categories: neutrophil infiltration, interstitial edema, hemorrhage, hyaline membrane. The sum of all scores was combined to calculate a composite score as described previously [15–17]. Liver injury was graded from 0 (normal) to 4 (severe) in four categories as follows: hepatocellular necrosis, hepatic parenchymal inflammato- ry infiltrate, hemorrhage, and sinusoidal inflammatory in- filtrate. The score of renal injury was assessed by the loss of brush border, grading of tubular necrosis, cast formation, and tubular dilatation, which was judged as follows: 0 (none), 1 (≤10 %), 2 (11–25 %), 3 (26–45 %), 4 (46–75 %), and 5 (≥76 %).

Fig. 1. Effects of SS-31 on organ dysfunction-associated indicators. CLP significantly increased decreased PO2 and increased serum levels of ALT, BUN, and Cr than those of the control + vehicle group. SS-31 treatment significantly attenuated the decreased PO2 and elevation of ALT, BUN, and Cr levels caused by CLP. Data are mean ± SEM (n = 6); *P <0.05 vs. the control + vehicle group; #P < 0.05 vs. the CLP + vehicle group.

ROS and ATP Content

Lung, kidney, and liver tissues were harvested 24 h after the operation and homogenized to quantify the amount of ROS and ATP as we previously described [9]. Intracellular ROS was detected using ROS assay kit (Genmed Scientifics Inc., Shanghai, China) by an oxidation-sensitive fluorescent probe (DCFH-DA) in a spectrofluorometer (excitation 490 nm, emission 520 nm). Measurement of relative ATP content based on the reaction of ATP with recombinant firefly luciferase and its substrate luciferin was performed using the ATP biolu- minescence assay kit (Beyotime Institute of Biotechnology, Shanghai, China) following the manufactory’s instructions.

Apoptosis Assessment

The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to measure the extent of DNA fragmentation as a measurement of apopto- sis in paraffin-embedded sections. The assay was per- formed according to the manufacturer’s instructions (Boehringer, Mannheim, Germany). Fluorescein- conjugated dUTP incorporated in nucleotide polymers were detected and quantified using fluorescence microsco- py (Zeiss LSM 410, Wetzlar, Germany).

Fig. 2. Effects of SS-31 on oxidative product and antioxidant enzymatic activities. CLP resulted in significantly increased MDA in serum and tissues when compared with those of the control + vehicle group. SS-31 treatment significantly attenuated the increased MDA levels caused by CLP. No difference in SOD was observed among groups. Data are mean±SEM (n = 6); *P <0.05 vs. the control + vehicle group; #P < 0.05 vs. the CLP + vehicle group.

Survival Rate

In the additional three groups of animals: control + vehicle group (n = 12), CLP + vehicle group (n =20), and CLP + SS-31 group (n = 20). Mice were then allowed to food and water ad libitum. Animals were frequently ob- served by a researcher blinded to the group assignment to determine the 7-day survival rates.

Statistical Analysis

Data are expressed as the mean±standard error of the mean (SEM). Statistical significance was determined by one-way, two-way, or repeated-measures of analysis of variance (ANOVA) followed by a Bonferroni test as appro- priate. The survival rate was estimated by Kaplan-Meier method and compared by the log-rank test. A P value <0.05 was regarded as statistically significant difference.

RESULTS

Effects of SS-31 on the Indicators of Organ Dysfunctions

To investigate the protective effects of SS-31 on sepsis-induced organ dysfunctions, we measured the arterial blood analysis data (PO2 and PCO2), serum ALT, AST, BUN, and Cr levels 24 h after the operation. CLP significantly decreased PO2 while increased serum levels of ALT, BUN, and Cr than those of the control groups (Fig. 1, P < 0.05). However, SS-31 treatment at 5 mg/kg reversed the decrease in PO2 and increase in ALT, BUN, and Cr levels caused by CLP (Fig. 1, P <0.05).

Effects of SS-31 on Oxidative Product and Antioxidant Enzymatic Activities

The levels of oxidative product (MDA) and the activ- ities of antioxidant enzymes (SOD) in the serum and tis- sues (lung, kidney, and liver) of all animals were measured at 24 h after the operation. As shown in Fig. 2, CLP resulted in significantly increased MDA levels in the serum and tissues when compared with those of the control groups (P < 0.05), whereas SS-31 treatment significantly attenuated the increased MDA levels induced by CLP (P < 0.05). However, no difference in SOD was observed among groups (P >0.05).

Effects of SS-31 on Inflammatory Cytokines

The serum level of TNF-α, IL-1β, and IL-6 were significantly increased at 6 h after CLP (Fig. 3a–c,P < 0.05). SS-31 treatment markedly suppressed only the increased TNF-α at 6 h after CLP (Fig. 3a, P < 0.05). However, there was no difference in IL-10 level among the four groups over time (Fig. 3d, P >0.05).

Fig. 3. Effect of SS-31 on inflammatory cytokines. The serum level of TNF-α, IL-1β, and IL-6 were significantly increased at 6 h after CLP. SS-31 treatment markedly suppressed only the increase of TNF-α at 6 h after CLP. There was no difference in IL-10 level among groups over time. Data are mean ±SEM (n = 6); *P <0.05 vs. the control + vehicle group; #P <0.05 vs. the CLP + vehicle group.

Effects of SS-31 on ROS and ATP Contents, MPO Activity, and W/D Ratio

CLP-induced increase in ROS and decrease in ATP levels were reversed by SS-31 treatment, suggesting the recovery of the mitochondrial function (Fig. 4a, c, P < 0.05). Pulmonary MPO activity and W/D ratio were increased significantly after CLP when compared with the control + vehicle group, whereas SS-31 treatment signifi- cantly decreased the MPO activity and W/D ratio (Fig. 4b, d, P <0.05).

Effects of SS-31 on Lung, Kidney, and Liver Histology Scores

Compared with the control + vehicle group, CLP caused increased lung, kidney, and liver histological scores, whereas SS-31 treatment significantly attenuated the lung, kidney, and liver histological scores (Fig. 5, P <0.05).

Effects of SS-31 on Lung, Kidney, and Liver NF-κB p65 and iNOS Expressions

Lung, kidney, and liver levels of NF-κB p65 and iNOS were significantly increased after CLP when com- pared with the control + vehicle group, whereas SS-31 treatment significantly inhibited the increased NF-κB p65 and iNOS expressions (Fig. 6, P < 0.05). In addition, our data showed that SS-31 also decreased some mea- surements of NF-κB p65 and iNOS in the control group. The reason may be that the laparotomy itself could up-regulate organ NF-κB p65 and iNOS ex- pressions while SS-31 decreased these two inflamma- tory regulators.

Effects of SS-31 on Lung, Kidney, and Liver TUNEL-Positive Cells

TUNEL-positive cells in the lung, kidney, and liver were significantly increased after CLP when compared with the control + vehicle group, while SS-31 treatment significantly decreased the TUNEL- positive cells (Fig. 7).

Fig. 4. Effects of SS-31 on organ ROS generation, ATP levels, MPO activity, and W/D ratio. CLP-induced increased ROS and decreased ATP levels were reversed by treatment of SS-31. Pulmonary MPO activity and W/D ratio were significantly increased after CLP, whereas SS-31 treatment significantly decreased the MPO activity and W/D ratio. Data are mean±SEM (n = 6); *P < 0.05 vs. the control + vehicle group; #P < 0.05 vs. the CLP + vehicle group.

Fig. 5. Effects of SS-31 on organ histological scores. CLP-induced increased lung, kidney, and liver histological scores, whereas SS-31 treatment significantly attenuated these increased histological scores (×200). Data are mean ± SEM (n =6); *P <0.05 vs. the control + vehicle group; #P < 0.05 vs. the CLP + vehicle group.

Effects of SS-31 Treatment on Survival Rate

No animal died in the control group. The 7-day sur- vival rate in the CLP + vehicle group and CLP + SS-31 groups was 60 and 70 %, respectively, and SS-31 treatment did not result in increased survival rate (Fig. 8, P >0.05).

Fig. 6. Effects of SS-31 on organ NF-κBp65 and iNOS expressions. Lung, kidney, and liver levels of NF-κB p65 and iNOS were significantly increased after CLP, whereas SS-31 treatment significantly inhibited the increased NF-κB p65 and iNOS expressions. Data are mean ±SEM (n = 4); *P < 0.05 vs. the control + vehicle group; #P <0.05 vs. the CLP + vehicle group.

DISCUSSION

CLP has been validated as an animal model to inves- tigate the settings of sepsis and related organ dysfunctions because it closely mimics the pathophysiology of clinical sepsis, which has been well established in our previous studies [15, 16]. By using this model, we demonstrated that treatment with SS-31 ameliorated oxidative stress, inflam- matory response, and apoptosis, which consequently improved pulmonary, hepatic and renal function during endotoxemia.

Mitochondria are normally protected from oxidative damage by a multilayer network of mitochondrial antiox- idant systems [18], but they can undergo oxidative damage when ROS production exceeds the antioxidant capacity of mitochondria [5]. The burst of ROS can trigger the opening of the mitochondrial permeability transition pore, resulting in mitochondrial depolarization, decreased ATP synthesis, and increased ROS production [9]. Consistently, antioxi- dants have been demonstrated to attenuate mortality or prevent organ dysfunctions in endotoxemic animal models and are helpful in protecting against MODS in patients with septic shock [4–6]. During endotoxemia, the inflammatory cells have the potential to produce large amounts of ROS and RNS by various mechanisms [5, 6], which in turn can lead to amplified inflammatory re- sponses. Oxidative stress induces peroxidation of mem- brane lipids, affecting the biological properties of the cel- lular membrane and impairing normal cellular function [5, 6]. In support with this hypothesis, it has been suggested that non-surviving septic patients showed higher serum MDA levels, which serve as a marker of lipid oxidative damage than surviving ones [19], while N-acetylcysteine, vitamins, and statins exert antioxidant capacity to show beneficial effects in sepsis [20–22]. In the present study, the presence of high ROS, MDA levels, and inflammatory mediators support the notion that increased oxidative stress and inflammatory response contributes to sepsis-induced organ dysfunctions. Our results were consistent with the notion that increased oxidative stress and inflammatory biomarkers are associated with poor outcomes in critically ill patients [23].

Fig. 7. Effects of SS-31 on TUNEL-positive cells. TUNEL-positive cells in the lung, kidney, and liver were significantly increased after CLP when compared with the control + vehicle group, while SS-31 treatment significantly decreased the TUNEL-positive cells (×400).

Fig. 8. Effects of SS-31 treatment on survival rate. SS-31 treatment did not result in increased survival rate after CLP. The survival rates were estimated by the Kaplan-Meier method and compared by log-rank test (n = 12–20).

ROS had been shown to be involved in activating the transcription factor NF-κB, which is a central regulator of immunity, inflammation, and cell survival [15, 16]. It also served as a second messenger in different signaling events, including induction of iNOS [24, 25]. Overproduction of nitric oxide by iNOS protein reacts with superoxide anion to produce the more potent reactive oxygen metabolite, the peroxynitrite anion, which oxidizes sulfhydryl groups and generates the hydroxyl radical [24]. Both nitric oxide and peroxynitrite anion are capable of causing cellular lipid peroxidation, protein oxidation, and mitochondria devasta- tion, which resulted in further impairments to tissues and induce cell death, leading to the progression of circulatory failure and MODS [6]. In the present study, we demon- strated that treatment with SS-31 can suppress sepsis- induced iNOS expression and TNF-α release, which may be mediated via suppression of NF-κB signaling pathway. In addition, the activation of apoptotic pathway is an important early pathophysiological event in the develop- ment of ALI after sepsis [17]. In this study, activation of apoptotic pathways in the lung, kidney, and liver was evaluated by measuring the TUNEL-positive cells. Notably, treatment with SS-31 effectively reduced LPS- induced increase in TUNEL-positive cells in all the organs tested. Our results were supported by previous studies demonstrating that SS-31 has the anti-apoptotic prop- erty in various pathological states [13, 26]. Furthermore, it has been suggested that blockade of caspase-3 can reduce subsequent proinflammatory cy- tokine levels [27], implying that the anti-apoptotic effect of SS-31 may contribute to the reduced inflam- matory response.

Unfortunately, SS-31 did not confer beneficial effects in survival rate, which can be explained by many reasons. Firstly, SS-31 treatment only attenuated the inflammatory mediator TNF-α, and did not prevent the increase in IL-1β and IL-6; this may help to explain why SS-31 treatment only showed partial beneficial effects on the survival ben- efit. Secondly, our previous study suggested that daily treatment with mitochondria-targeted peptide SS-31 for seven consecutive days reduces mortality rate in a sepsis animal model using the same paradigm, suggesting that the beneficial effects of SS-31 on survival rate may be dose and time dependant. Finally, since we did not perform these biochemical data 24 h after the operation, we did not exclude the possibility that the effects of SS-31 are short-lived. Therefore, future studies are needed to better understand the pharmacological effects of SS-31 on organ dysfunctions during sepsis.

In conclusion, SS-31 effectively attenuated the proin- flammatory response, oxidative stress, and apoptosis, which ultimately attenuated organ dysfunctions during endotoxemia. Thus,Elamipretide SS-31 might be considered as a novel therapeutic strategy in the prevention of MODS during endotoxemia.