DNA-Aptamer Raised against Receptor for Advanced Glycation End Products Improves Survival Rate in Septic Mice

Despite remarkable scientific advances in the understanding of molecular mechanisms for sepsis, therapeutic options are far from satisfactory. High mobility group box 1 (HMGB1), one of the ligands of receptor for advanced glycation end products (RAGE), is a late mediator of lethality in septic mice. We have recently found that the DNA-aptamer raised against RAGE (RAGE-aptamer) significantly blocks experimental diabetic nephropathy and melanoma growth and metastasis. We examined the effects of RAGE-aptamer on sepsis score, survival rate, and inflammatory and oxidative stress responses in serum, peripheral monocytes, kidneys and livers of lipopolysaccharide- (LPS-) injected mice, and on LPS-exposed THP-1 cells. RAGE-aptamer inhibited the binding of HMGB1 to RAGE in vitro. RAGE-aptamer significantly (P = 0.002) improved sepsis score at 8 hours after LPS injection and survival rate at 24 hours (P < 0.01, 70%) in septic mice compared with LPS+vehicle- or LPS+control-aptamer-treated mice. RAGE-aptamer treatment significantly decreased expression of p-NF-κB p65, an active form of redox-sensitive transcriptional factor, NF-κB and gene or protein expression of TNF-α, IL-1β, IL-6, and HMGB1 in serum, peripheral monocytes, and kidneys of septic mice in association with the reduction of oxidative stress and improvement of metabolic acidosis, renal and liver damage. LPS-induced oxidative stress, inflammatory reactions, and growth suppression in THP-1 cells were significantly blocked by RAGE-aptamer. Our present study suggests that RAGE-aptamer could attenuate multiple organ damage in LPS-injected septic mice partly by inhibiting the inflammatory reactions via suppression of HMGB1-RAGE interaction.


Introduction
Sepsis is a public health burden to human health and its care system affects an estimated 30-50 million people all over the world with approximately 5-10 million deaths annually [1][2][3]. The prevalence of severe sepsis is especially high in critically ill patients with an incidence rate of 270 per 100,000 person-years, and its hospital mortality rate is about 25% [1][2][3].
Sepsis is defined as maladaptive systemic inflammatory responses to infection, which could lead to potentially lifethreatening multiple organ failures [4,5]. The initial inflammatory response is caused by invading pathogen-derived molecules, such as bacterial lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria [4][5][6], which could be involved in a myriad of inflammatory responses in sepsis through the interaction with pattern recognition receptors (PRRs) on immune cells [6]. Despite remarkable scientific advances in the understanding of molecular mechanisms for sepsis, therapeutic options are extremely limited and far from satisfactory, and clinical trials targeting invading bacteria, LPS, or Toll-like receptor-4, one of the main PRRs that could mediate the signaling pathway of LPS, have been disappointing [4][5][6][7][8]. Since most of the failed trials target molecules involved in early inflammatory responses in sepsis, development of a novel therapeutic strategy that specially targets the maladaptive pathway lately activated by sepsis may be urgently needed [9].

Preparation of LPS Solution.
The lyophilized powder of LPS was dissolved in saline (0.9% NaCl) under sterile conditions followed by heating at 70°C for 1 minute on the day of use.
2.5. Animals. Male 8-week-old BALB/c mice (Charles River Laboratories Japan, Inc., Yokohama, Japan) were used. Animal procedures in this study were approved by the Animal Research Committee of Kurume University School of Medicine (Approval number: 2019-230, 2020-167, and 2021-175).
2.6. Animal Experiment 1. In order to determine the optimal dose of LPS to cause sepsis in mice, various doses of LPS (15, 20, or 30 μg/g-body weight (BW)) were injected intraperitoneally to the mice. After the injection of LPS, mice were monitored every 2 hours for 24 hours and assessed by the sepsis scoring system according to the method of Shrum et al. [21]. Survival was determined using the Kaplan-Meier method and a log-rank test.

Animal Experiment 2.
To examine the effects of RAGEaptamer on the survival rate of septic mice, controlaptamer (40 pmol/g-BW) or RAGE-aptamer (2, 10, 20, or 40 pmol/g-BW) was administered intraperitoneally in mice 5 minutes after the injection of LPS (20 μg/g-BW). Control mice were given saline twice at 5-minute intervals. Sepsis 2 Oxidative Medicine and Cellular Longevity scores and survival rates were assessed in the same methods as in Animal Experiment 1. Eight hours after the injection of LPS or first saline (vehicle), blood was collected from the left ventricular of mice. Blood gas measurement was performed immediately after the collection using the GASTAT-710 (Techno Medica Co., Ltd., Kanagawa, Japan). Blood lactate was measured using a Lactate Pro2 LT-1730 (ARKRAY, Inc., Kyoto, Japan). Complete blood counts were measured using a VetScan HM5 (Abaxis, Inc., Union City, CA, USA). Serum electrolytes, alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), creatinine, and lactate dehydrogenase (LDH) were analyzed using a FUJI DRI-CHEM 4000 V chemistry analyzer (Fujifilm Ltd., Tokyo, Japan). Kidneys and livers were obtained from mice at 0.5, 2, 4, 6, or 8 hours after the injection of LPS or first saline for morphological analyses, and urine was collected for measurements of 8-OHdG and KIM-1.

Immunostaining and Morphological
Analysis. The specimen of kidneys and livers were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned at 4 μm intervals and mounted on glass slides. The sections were left in 0.3% hydrogen peroxide for 30 minutes to block endogenous peroxidase activity and incubated overnight at 4°C with primary antibodies, and the reactions were visualized with a Histofine Simple Stain Mouse MAX PO (MULTI) kit (Cat# 424152, Nichirei Bioscience Inc., Tokyo, Japan) as described before [19]. Ten images were captured per section from individual mice in each group. Immunohistoreactivity fields in each sample were measured by cellSens version 1.14 software (Olympus Corp., Tokyo, Japan). Three-micrometer paraffin sections were stained with hematoxylin and eosin (HE) for light microscopic analysis. Severity grade for tubular degeneration/necrosis score in the HE-stained kidney specimen was assessed according to the method of Bellinger et al. [22]. Hepatic necrosis area accompanied with inflammatory cell aggregation in the HE-stained liver specimen was calculated with cellSens version 1.14 software.
2.13. Measurement of NADPH Oxidase Activity. Renal NADPH oxidase activity and cultured THP-1 cells were measured by a luminescence assay as described previously [15].
2.14. Statistical Analysis. All values were expressed as mean ± standard deviation. Survival curve by the Kaplan-Meier method was evaluated by log-rank test (Figures 1(c) and 1(e)). Comparisons of LPS-induced sepsis scores between control-aptamer-treated and RAGE-aptamer-treated mice were performed in the proportional hazard model by including sepsis score and group interaction terms (Table 1). Statistical comparisons were performed using ANOVA followed by the Steel-Dwass test (  Table 2 (PaO 2 , pH, lactate, and potassium)). The rest of the data were analyzed by the Tukey-Kramer test. P < 0:05 was considered significant. All statistical analyses

Effects of RAGE-Aptamers on HMGB1-vRAGE
Interaction. We have already found that RAGE-aptamer clones #1, #2, and #3 inhibit the binding of AGEs to vRAGE [15]. Therefore, we examined the effects of these aptamers on HMGB1-vRAGE interaction. As shown in Figure 1(a), all three clones significantly inhibited the binding of HMGB1 to vRAGE. HMGB1 was bound to RAGE with a dissociation constant (K d ) of 48.3 nM. In the presence of RAGE-aptamer #1, the K d value exhibited two orders of magnitude higher level [16]. Since we have reported that RAGE-aptamer clone #1 did not exert nonspecific toxic effects in both cell culture and animal model systems [15,19], we used RAGEaptamer clone #1 for the following experiments.  (Table 1). Since P value of HR was the smallest in 20 μg-BW LPStreated mice, we chose the condition in the following experiments.

Survival Rate in Animal Experiment 2.
We examined the effects of the indicated doses of RAGE-aptamer on the survival rate of septic mice (Figure 1(d)). As shown in Figure 1(e), compared with vehicle or 40 pmol/g-BW control-aptamer, treatment with the same concentration of RAGE-aptamer significantly improved the survival rate of 20 μg/g-BW LPS-injected septic mice, while median survival time and survival rate at 24 hours after LPS injection in the control-aptamer-treated group were 16 hours and 0%, respectively; the latter was drastically extended to 70% in RAGE-aptamer-treated mice. Those in vehicle-treated septic mice were 14 hours and 0%, respectively.
Then, we compared the effects of sepsis score measured at 8 hours after LPS injection on the survival time between the 40 pmol/g-BW RAGE-aptamer-and 40 pmol/g-BW control-aptamer-treated groups by the proportional hazard model with sepsis score and group interaction terms. As shown in Table 3, the likelihood ratio test indicated a significant interaction (likelihood ratio = 5:8, degrees of freedom = 1, P = 0:016); there was a significant difference in sepsis score effects between the two groups. Based on the estimate of hazard ratio, high sepsis score was associated with the increased risk of death (HR = 2:6, P = 0:005), and average sepsis score in the 40 pmol/g-BW RAGE-aptamer-treated group was significantly lower (P = 0:002) than that in the 40 pmol/g-BW control-aptamer-treated group (6:6 ± 1:6 vs. 10:3 ± 2:7).

3.4.
Effects of RAGE-Aptamer on Blood Gas, Electrolytes, and Biochemical Parameters. As shown in Table 2, compared with control nonseptic mice, PaO 2 , lactate, anion gap, red blood cells, hemoglobin, BUN, creatinine, AST, and ALT were significantly increased in vehicle-treated-or controlaptamer-treated septic mice, while HCO 3 -, pH, base excess, white blood cells, and platelets were decreased. RAGEaptamer treatment significantly restored HCO 3 -, pH, base excess, and platelets and simultaneously decreased lactate, anion gap, BUN, creatinine, AST, and ALT levels. There was a significant difference of anion gap between vehicletreated-and control-aptamer-treated septic mice.

Effects of RAGE-Aptamer on Cytokine Expression in Septic
Mice. Serum and PBMCs were obtained from septic mice at the indicated time periods after LPS or first saline injection. As shown in Figure 2, p-NF-κB p65, TNF-α, IL-1β, IL-6, and HMGB1 mRNA levels in PBMCs isolated from vehicle-treated-or control-aptamer-treated septic mice were significantly increased compared with control nonseptic mice, which were associated with the increases in serum levels of TNF-α, IL-1β, IL-6, and HMGB1 ( Figure 2). RAGE-aptamer treatment significantly reduced these inflammatory parameters in both PBMCs and serum obtained from septic mice.
3.6. Effects of RAGE-Aptamer on Renal and Liver Injuries in Septic Mice. Gene and protein expression levels of TNF-α, IL-1β, IL-6, HMGB1, and KIM-1, a marker of renal damage, and urinary KIM-1 levels were significantly increased in vehicle-treated-or control-aptamer-treated septic mice (Figures 3(a)-3(k)). Treatment of RAGE-aptamer significantly decreased all of these parameters compared with LPS plus control-aptamer-treated mice (Figures 3(a)-3(k)). Furthermore, renal 8-OHdG, a marker of oxidative stress, NADPH oxidase-derived superoxide generation, and gene expression of components of NADPH oxidase except for p67phox were significantly increased in vehicle-treated-or control-aptamer-treated septic mice in association with the elevation of urinary 8-OHdG levels (Figures 3(l)-3(r)).    Oxidative Medicine and Cellular Longevity RAGE-aptamer treatment significantly reduced the expression levels of these oxidative stress markers in the kidneys of septic mice compared with control-aptamer-treated septic mice (Figures 3(l)-3(r)). HE staining revealed that renal tubular degeneration and hepatic necrosis area accompanied with inflammatory cell aggregation were increased in vehicletreated-or control-aptamer-treated septic mice, both of which were ameliorated by RAGE-aptamer (Figures 3(s)-3(t)). There was a significant difference of Nox2 mRNA levels in the kidneys between vehicle-treated-and controlaptamer-treated septic mice.

Discussion
LPS, also known as endotoxin, has been shown to play a central role in the pathogenesis of Gram-negative bacteriainduced sepsis [6,23]. In order to investigate the efficacy of RAGE-aptamer on the survival rate of septic mice, we chose the condition of intraperitoneal injection of 20 μg/g-BW LPS because the effect of sepsis score measured at 8 hours on survival time was the largest after this dose of LPS injection. In the present study, we found for the first time that RAGEaptamer significantly inhibited the binding of HMGB1 to vRAGE in vitro and that intraperitoneal administration of

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Oxidative Medicine and Cellular Longevity 40 pmol/g-BW RAGE-aptamer improved the sepsis score measured at 8 hours after LPS injection and survival rate in LPS-injected septic mice. Furthermore, RAGE-aptamer treatment significantly attenuated oxidative stress and inflammatory reactions, including HMGB1 expression in serum, PBMCs, and kidneys of LPS-injected septic mice in association with the improvement of renal and liver injuries and metabolic acidosis. We also found here that RAGEaptamer inhibited the LPS-induced oxidative stress generation in, and secretion of cytokines and HMGB1 by, THP-1 cells. Therefore, the present study suggests that RAGEaptamer could attenuate multiple organ damage in LPSinjected septic mice partly by inhibiting the inflammatory reactions via suppression of HMGB1-RAGE interaction.
Although the pathological role of RAGE in sepsis has been evaluated in several kinds of animal models, there is some controversy about the effects of RAGE gene deficiency on sepsis in experimental models [24][25][26][27][28][29][30]. A couple of papers have reported that RAGE-deficient mice exhibited a significant protection against inflammation and lethality due to polymicrobial sepsis caused by cecal ligation and puncture or intranasal inoculation with Streptococcus pneumoniae [25][26][27]. However, others have shown that survival rate is not improved, or rather deteriorated, in septic mice with intratracheal instillation of LPS or Escherichia coli, intravenous injection of Streptococcus pneumoniae, or intranasal inoculation of LPS or Klebsiella pneumoniae [28][29][30]. In addition, as far as we know, there are only two papers to show that administration of anti-RAGE antibody significantly improves the survival rate in septic mice of cecal ligation and puncture model or intratracheal infection with Streptococcus pneumoniae [25,31]. In this study, we showed first that RAGE-aptamer administration just after LPS injection not only ameliorated the sepsis score and survival rate, but also attenuated critical organ damage in septic mice, thus suggesting that RAGE is a therapeutic target for sepsis.
Nucleic acid aptamers are short single-stranded oligonucleotides that are able to bind to various kinds of target proteins like antibodies, which are selected by systemic evolution of ligands by exponential enrichment [32,33]. Compared with neutralizing antibodies, aptamers are more easily prepared and could more efficiently penetrate into various tissues with less immunogenicity and more thermal stability [32,33]. Its advantages over antibodies for blocking target proteins make nucleic acid aptamers a very attractive tool for in vivo-therapeutic application. Indeed, pegaptanib (Macugen), an RNA-aptamer raised against vascular endothelial growth factor 165 , has already been approved by the U.S. Food and Drug Administration for the treatment of the wet type of age-related macular degeneration, while several types of aptamers directed against coagulation systems have undergone clinical trials [33,34]. In addition to these things, we found here that RAGE-aptamer treatment after the LPS injection exhibited protective effects on organ damage and death in septic mice without adverse side effects. Given that HMGB1 is a late lethality mediator in sepsis [10,11], our present findings may support the clinical feasibility of RAGE-aptamer for treatment of severe sepsis.
In the present study, RAGE-aptamer treatment significantly decreased expression of p-NF-κB p65, an active form of redox-sensitive transcriptional factor, NF-κB [35,36], and gene expression of TNF-α, IL-1β, and IL-6, and subsequently reduced HMGB1 mRNA levels in PBMCs isolated from septic mice. Furthermore, serum levels of these cytokines and HMGB1 were increased in LPS-injected septic mice, all of which were significantly blocked by RAGE-    16 Oxidative Medicine and Cellular Longevity   [37][38][39][40]. Moreover, oxidative stress and inflammatory reactions have been reported to stimulate HMGB1 release and activity [37][38][39][40]. These observations suggest a positive feedback loop between HMGB1-RAGE-induced ROS generation as well as inflammatory reactions and HMGB1 expression in septic mice. RAGE-aptamer could break the crosstalk between HMGB1-RAGE axis and ROS, thereby improving the sepsis score and survival rate in LPS-injected septic mice.
In this study, we also found that treatment with RAGEaptamer significantly attenuated gene and protein expression of TNF-α, IL-1β, and IL-6, and HMGB1 in the kidneys of septic mice at 2-8 hours after LPS injection. Subsequently, 8-OHdG, a marker of oxidative stress, NADPH oxidase activity, and gene expression of components of NADPH oxidase except for p67phox were increased in the kidneys of septic mice in association of elevation of urinary 8-OHdG and KIM-1, one of the markers of proximal tubular damage and acute kidney injury [41], renal dysfunction as evaluated by BUN and creatinine levels, metabolic acidosis, and renal tubular degeneration, all of which were inhibited by RAGEaptamer. Therefore, RAGE-aptamer could protect against renal damage in septic mice partly by suppressing the vicious cycle among HMGB1-RAGE axis, ROS generation, and inflammatory reactions in the kidneys. In addition, we found here that RAGE-aptamer significantly attenuated liver injury in septic mice, which were indicated by a decrease in serum levels of ALT and AST and amelioration of hepatic necrosis in HE staining.
We also found in the present study that LPS-induced increases in TNF-α, IL-1β, and IL-6, and HMGB1 gene and protein expression, ROS generation, activity and gene expression of components of NADPH oxidase, and p-NF-κB p65 in THP-1 cells were significantly blocked by RAGEaptamer. Moreover, RAGE-aptamer inhibited the LPSinduced decrease in viable cell number of THP-1 cells. These in vitro-findings further support the concept that a maladaptive feedforward loop between the HMGB1-RAGE axis and inflammatory reactions in immune cells could partly contribute to organ damage and death in LPS-induced sepsis and that blockade of the system by RAGE-aptamer may be a therapeutic target for the devastating disorder. Pyroptosis, an inflammatory form of programed cell death, has been shown to be involved in LPS-HMGB1-induced sepsis [13]. Since inflammatory reactions and reduced viable cell number of LPS-exposed THP-1 cells were simultaneously prevented by RAGE-aptamer, our present study suggests that RAGEaptamer may inhibit the pyroptosis of immune cells in LPSinduced septic mice.
There are a couple of limitations in the present study. It remains unclear whether other RAGE ligands other than HMGB1 could play a role in our model. It would be helpful to investigate the pathological role of other RAGE ligands other than HMGB1, such as S100 proteins in LPS-injected septic mice [42]. Furthermore, since the protein and mRNA levels of IL-6 treated with RAGE-aptamer were still rising from 6 to 12 hours, it would be also interesting to examine the effects of RAGE-aptamer on IL-6 expression on time points post 12 hours.

Conclusion
Our present study suggests that RAGE-aptamer could attenuate multiple organ damage in LPS-injected septic mice partly by inhibiting the inflammatory reactions via suppression of HMGB1-RAGE interaction.

Data Availability
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
There is no conflict of interests to declare in this paper.

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