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Ketamine attenuates sepsis-induced acute lung injury via regulation of HMGB1-RAGE pathways
International Immunopharmacology 34 (2016) 114–128
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Ketamine attenuates sepsis-induced acute lung injury via regulation of HMGB1-RAGE pathways Kehan Li a,⁎, Jianxue Yang b, Xuechang Han a a b
Department of Anesthesiology, The First Affiliated Hospital of Henan Science and Technology University, Luoyang, Henan, China Department of Neurology, The First Affiliated Hospital of Henan Science and Technology University, Luoyang, Henan, China
a r t i c l e
i n f o
Article history: Received 10 September 2015 Received in revised form 5 January 2016 Accepted 21 January 2016 Available online xxxx Keywords: Ketamine CLP HMGB1 RAGE ALI
a b s t r a c t High mobility group box protein 1 (HMGB1) and receptor for the advanced glycation end product (RAGE) play important roles in the development of sepsis-induced acute lung injury (ALI). Ketamine is considered to confer protective effects on ALI during sepsis. In this study, we investigated the effects of ketamine on HMGB1-RAGE activation in a rat model of sepsis-induced ALI. ALI was induced in wild type (WT) and RAGE deficient (RAGE−/−) rats by cecal ligation and puncture (CLP) or HMGB1 to mimic sepsis-induced ALI. Rats were randomly divided to six groups: sham-operation + normal saline (NS, 10 mL/kg), sham-operation + ketamine (10 mg/kg), CLP/ HMGB1 + NS (10 mL/kg), CLP/HMGB1 + ketamine (5 mg/kg), CLP/HMGB1 + ketamine (7.5 mg/kg), and CLP/ HMGB1 + ketamine (10 mg/kg) groups. NS and ketamine were administered at 3 and 12 h after CLP/HMGB1 via intraperitoneal injection. Pathological changes of lung, inflammatory cell counts, expression of HMGB1and RAGE, and concentrations of various inflammatory mediators in bronchoalveolar lavage fluids (BALF) and lung tissue were then assessed. Nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathways in the lung were also evaluated. CLP/HMGB1 increased the wet to dry weight ratio and myeloperoxidase activity in lung, the number of total cells, neutrophils, and macrophages in the BALF, and inflammatory mediators in the BALF and lung tissues. Moreover, expression of HMGB1and RAGE in lung tissues was increased after CLP. Ketamine inhibited all the above effects. It also inhibited the activation of IκB-α, NFκB p65, and MAPK. Ketamine protects rats against HMGB1-RAGE activation in a rat model of sepsis-induced ALI. These effects may partially result from reductions in NF-κB and MAPK. Crown Copyright © 2016 Published by Elsevier B.V. All rights reserved.
1. Introduction Sepsis is a complex clinical syndrome resulting from a harmful host inflammatory response to infection [1]. Numerous advances have been made in diagnostic procedures, antimicrobial treatment, and supportive care; however, sepsis remains a major cause of morbidity and mortality in adult and pediatric intensive care units [2]. Lung is the most vulnerable, critical and sensitive organ during sepsis process [3]. During the development of sepsis, bacterial components, such as lipopolysaccharide (LPS), could lead to activate an inflammatory cascade resulting in the release of inflammatory mediators [4]. The overproduction of inflammatory mediators induces endothelial and epithelial injury, vascular leakage, edema and vasodilatation, subsequently causing the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) [5]. Previous studies have shown that inflammatory mediators play a key role in the pathogenesis of ALI/ARDS [6]. ALI is characterized ⁎ Corresponding author at: No. 24 Jinghua Road, Luoyang, Henan, China. E-mail address: [email protected] (K. Li).
http://dx.doi.org/10.1016/j.intimp.2016.01.021 1567-5769/Crown Copyright © 2016 Published by Elsevier B.V. All rights reserved.
by a local inflammatory response, and receptor for advanced glycation end-products (RAGE) has an important function in the HMGB1mediated activation of innate immunity [7]. Based on the key role of inflammatory mediators in the pathogenesis of ALI, pharmacological treatments have been aimed at modulating RAGE signaling and alleviating nonspecific inflammatory reactions that may as a potential therapeutic strategy for ALI. Extracellular HMGB1 is an inflammatory mediator; elevated HMGB1 levels have been associated with diseases such as sepsis, hemorrhagic shock, ALI, and ischemia–reperfusion injury [8–11]. RAGE, mainly located in the alveolar type I cells, is a multiligand-binding receptor with an extracellular, transmembrane, and cytosolic domain, which senses a variety of signaling molecules including advanced glycation end products (AGEs) and HMGB1 [12,13]. Binding of HMGB1 to its transmembrane receptor RAGE activates multiple signaling cascades, including NF-κB and mitogen-activated protein kinase (MAPK) [14]. Activation of these cascades is known to result in the production and release of inflammatory mediators such as cytokines, e.g. TNF-α, IL-6 and IL-1β, and HMGB1 [15]. This indicates that activation of HMGB1-
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RAGE pathway amplifies the inflammatory responses in vivo and plays a central role in the pathogenesis of inflammatory diseases. Recent studies have shown that HMGB1-RAGE signaling pathway is also involved in the development of ALI [16], suggesting that targeting HMGB1-RAGE pathway might be a novel strategy in the treatment of ALI. Ketamine has been shown to protect animals against injury by its anti-inflammatory and immunomodulatory effects [17–19]. Ketamine has been used in clinic to treat sepsis [20,21], and also has potential value in treatment of animal models of several other diseases, including ischemia/reperfusion injury [22], ALI [23]. However, whether ketamine inhibits HMGB1-RAGE pathway and confers to its suppression of ALI is still unclear. In the present study, we used rat model of CLP-induced
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ALI to evaluate the effects of ketamine on the inflammatory responses, particularly HMGB1-RAGE pathway, and to further explore its potential molecular mechanisms. 2. Materials and methods 2.1. Animals Male Sprague–Dawley (wild-type, WT) rats (Henan Science and Technology University Experimental Animal Center, Luoyang, China) and male Sprague–Dawley (RAGE-deficient, RAGE−/−) rats (Beijing Biocytogen Co., Ltd., Beijing, China) were used in this study. The rats
Fig. 1. Effects of ketamine on the injury indicators of CLP-induced ALI in WT rats. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. The lung W/D ratio (A) and MPO activity (B) were determined in lung tissue dissected at 24 h after CLP. BALF was collected at 24 h after CLP to measure the total protein concentration (C) and the number of total cells (D), neutrophils (E), and macrophage (F). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05, #P b 0.01vs. CLP.
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Fig. 2. Effects of ketamine on the injury indicators of HMGB1-induced ALI in WT rats. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after HMGB1. The lung W/D ratio (A) and MPO activity (B) were determined in lung tissue dissected at 24 h after HMGB1. BALF was collected at 24 h after HMGB1 to measure the total protein concentration (C) and the number of total cells (D), neutrophils (E), and macrophage (F). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05, #P b 0.01vs. HMGB1.
were exposed to 12 h of light and 12 h of darkness each day, and were given access to food and water ad libitum. The animal protocol was approved by the Institutional Animal Ethics Committee of the First Affiliated Hospital of Henan Science and Technology University. 2.2. Animal model and experimental groups 2.2.1. CLP/HMGB1-induced ALI model Rats were randomly assigned to six groups (n = 10): shamoperation + normal saline (NS, 10 mL/kg), shamoperation + ketamine (10 mg/kg), CLP/HMGB1 + NS (10 mL/kg), CLP/HMGB1 + ketamine (5 mg/kg), CLP/HMGB1 + ketamine
(7.5 mg/kg), and CLP/HMGB1 + ketamine (10 mg/kg). Sodium pentobarbital (50 mg/kg, Sigma-Aldrich, St. Louis., MO, USA) was injected intraperitoneally (i.p.) to anesthetize the animals before the surgical procedures. Rats were instilled intratracheally with 50 μg of HMGB1 (Sigma-Aldrich, St. Louis, MO, USA) diluted in 0.25 mL of sterile phosphatebuffered saline (PBS) [24]. Polymicrobial sepsis was induced by CLP as described previously [19]. In brief, a midline incision about 2 cm was made on the anterior abdomen. The cecum was carefully isolated and the distal 20% was ligated. Then the cecum was punctured twice with a sterile 21-gauge needle, and was squeezed to extrude the cecal contents from the wounds. The cecum was placed back and the abdominal
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Fig. 3. Effects of ketamine on the production of inflammatory cytokines in the BALF and lung tissue from WT rats with CLP-induced ALI. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. BALF and lung tissue were collected at 24 h after CLP to analyze the inflammatory cytokines TNF-α (A, B), IL-1β (C, D), and IL-6 (E, F). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05, #P b 0.01vs. CLP.
incision was closed. The sham control animals were treated in an identical manner, but no cecal ligation or puncture was performed. All rats were administered 1 mL sterile NS immediately after surgery for fluid resuscitation. At 3 h and 12 h after CLP/HMGB1, rats were treated with ketamine (ketamine hydrochloride, Hengrui, Inc., Nanjing, China) 5, 7.5 or 10 mg/kg (i.p.) or NS (10 mL/kg, i.p.). At 24 h after CLP/HMGB1, the rats were sacrificed. Then the lungs of rats were harvested and stored in liquid nitrogen for later use. To clarify the role of RAGE signaling in the release of inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6, and HMGB1), rats were treated with an intraperitoneal injection of anti-RAGE Ab (10 mg/kg) 1 h before CLP/HMGB1, and then the rats were treated with ketamine (10 mg/kg, i. p.) or an equal vehicle (NS) at 3 h and 12 h after CLP/HMGB1; Rats received intratracheal injection of 300 μL of NS or 300 μL (3 × 109 PFU) of RAGE siRNA. Seven days later, Ketamine (10 mg/kg, i.p.) was
administrated at 3 h and 12 h after CLP/HMGB1. BALF was collected at 24 h after CLP/HMGB1 to analyze the inflammatory cytokines. 2.2.2. RAGE-deficient model The WT and RAGE−/− rats were independently and randomly divided into 6 groups (n = 10): sham-operation + NS (10 mL/kg) in WT or RAGE−/− rats, CLP + NS (10 mL/kg) in WT or RAGE−/− rats, and CLP + ketamine (10 mg/kg) in WT or RAGE−/− rats. Polymicrobial sepsis was induced by CLP as described above. At 24 h after CLP, the rats were sacrificed. Then the BALF and lung tissues of rats were harvested and stored in liquid nitrogen for later use. 2.3. Lung wet to dry weight (W/D) ratio and MPO activity measurement After the rats were euthanized, the lungs were removed and the wet weight was determined. The lung tissue was placed in an oven at 60 °C
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Fig. 4. Effects of ketamine on the production of inflammatory cytokines in the BALF and lung tissue from WT rats with HMGB1-induced ALI. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after HMGB1. BALF and lung tissue were collected at 24 h after HMGB1 to analyze the inflammatory cytokines TNF-α (A, B), IL-1β (C, D), and IL-6 (E, F). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05, #P b 0.01vs. HMGB1.
for 24 h to obtain the dry weight. The ratio of the wet lung to the dry lung was calculated to assess tissue edema. MPO activity, reflecting the parenchymal infiltration of neutrophils and macrophages, was determined using test kits (BioLegend, Inc. Camino Santa Fe, Suite E San Diego, CA, USA) according to the instructions. The right lungs were excised. Lung tissues of 100 mg were homogenized and fluidized in extraction buffer to obtain 5% of homogenate. The sample including 0.9 mL homogenate and 0.1 mL of reaction buffer was heated to 37 °C in water for 15 min, on which occasion; The enzymatic activity was determined by measuring the change in absorbance at 460 nm using a 96well plate reader (Molecular devices, Sunnyvale, CA, USA).
2.4. The total protein concentration and inflammatory cell counts in the BALF To evaluate vascular permeability in the airways, the BALF was collected and protein content was determined by BCA protein assay kit (Pierce, Rockford, IL, USA). The principle of BCA method is as follow: The peptide bond of amino acids can bind with Cu2+ to form a complex in the basic solution and then the Cu2 + is deoxidized to Cu+. Bicinchoninic acid and its sodium salt are water-soluble. In the alkaline environment, it can bind with Cu2+ to form a dark purple compound, which has strong absorbance maximum at 562 nm wavelength. Because
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Fig. 5. Effect of ketamine on histopathological changes in lung tissues in CLP-induced ALI in WT rats. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. Lung tissues (n = 10) from each experimental group were processed for histological evaluation at 24 h after CLP. Representative histological changes of lung tissues obtained from rats of different groups. (A) Control group, (B) ketamine (10 mg/kg) group, (C) CLP group, (D) CLP + ketamine (5 mg/kg) group, (E) CLP + ketamine (7.5 mg/kg) group, (F) CLP + ketamine (10 mg/kg) group (H&E staining, magnification 200×).
the brightness is coordinated with the concentration of protein, we can use matching method to determine protein concentration. Proteins were expression in milligram protein per milliliter BALF. The fluid recovered from each sample was centrifuged (4 °C, 3000 rpm,10 min) to pellet the cells. The cell pellets were resuspended in PBS for total cell counts using a hemocytometer. Cytospins were prepared for differential cell counts by staining with the Wright–Giemsa staining method. 2.5. Inflammatory mediators assays The concentrations of TNF-α, IL-1β, IL-6, HMGB1, and soluble receptor for advanced glycation end product (sRAGE) in the BALF and lung
tissue were measured by ELISA kits according to the instruction recommended by the manufactures (BioLegend, Inc. Camino Santa Fe, Suite E San Diego, CA, USA).
2.6. Histopathological evaluation of the lung tissue Histopathological examination was performed on rats that were not subjected to BALF collection. Lungs were fixed with10% buffered formalin, imbedded in paraffin and sliced. After hematoxylin and eosin (H&E) staining, pathological changes of lung tissues were observed under a light microscope.
Fig. 6. Effect of ketamine on histopathological changes in lung tissues in HMGB1-induced ALI in WT rats. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after HMGB1. Lung tissues (n = 10) from each experimental group were processed for histological evaluation at 24 h after HMGB1. Representative histological changes of lung tissues obtained from rats of different groups.(A) Control group, (B) ketamine (10 mg/kg) group, (C) HMGB1 group, (D) HMGB1 + ketamine (5 mg/kg) group, (E) HMGB1 + ketamine (7.5 mg/kg) group group, (F) HMGB1 + ketamine (10 mg/kg) group (H&E staining, magnification 200×).
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Fig. 7. Effects of ketamine on the expression of HMGB1 and RAGE in the BALF and lung tissue from WT rats with CLP-induced ALI. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. HMGB1 (A) and sRAGE (B) protein expression in BALF were determined by ELISA; HMGB1(C) and RAGE (D) protein expression in lung tissues were determined by Western Blot; relative HMGB1(E) and RAGE (F) mRNA expression in lung tissues were determined by real-time PCR. The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05, #P b 0.01vs. CLP.
2.7. Western blot analysis Proteins were extracted from the lungs using T-PER Tissue Protein Extraction Reagent Kit according to the manufacturer's instructions. Protein concentrations were determined by BCA protein assay kit. The proteins were separated on12% SDS-Polyacrylamide gel and transferred on to the PVDF membrane. After blocking the nonspecific site with blocking solution (5% nonfat dry milk), the membrane was incubated overnight with specific primary antibody at 4 °C. Subsequently, the membrane
was washed with PBS-T followed by incubation with the secondary antibody conjugated with horseradish peroxidase at room temperature for 1 h. Blots were again washed with PBS-T and then developed with the ECL Plus Western Blotting Detection System (Amersham Life Science, UK). The following primary antibodies were used: phospho-p38, p38, phospho-ERK, ERK, phospho-JNK, JNK, phospho-IκB-α, IκB-α, NF-κB p65, Histone H3.1, RAGE, β-actin (all from Cell Signaling Technology, Beverly, MA, USA). Protein densities were standardized to β-actin and Histone H3.1 for total lysates and nuclear fractions, respectively.
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Fig. 8. Effect of anti-RAGE Ab on the production of inflammatory cytokines in the BALF from WT rats with CLP-induced ALI. Rats were treated with an intraperitoneal injection of anti-RAGE Ab (10 mg/kg) 1 h before CLP, and then the rats were treated with ketamine (10 mg/kg, i.p.) or an equal volume of vehicle (NS) at 3 h and 12 h after CLP. BALF was collected at 24 h after CLP to analyze the inflammatory cytokines TNF-α (A), IL-1β (B), IL-6 (C),and HMGB1 (D). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05 vs. CLP, #P b 0.05 vs. CLP + anti-RAGE Ab + ketamine.
2.8. Quantitative real-time polymerase chain reaction analysis (real-time PCR) Total RNA was extracted from lung tissues using Trizol (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer's instructions and reverse transcribed with Moloney Murine Leukemia Virus reverse transcriptase (Promega, Madison, WI). Quantitative real-time polymerase chain reaction (Real-Time PCR) was performed using a LightCycler 2.0 Real-Time PCR System (Roche Applied Science, Indianapolis, IN). Complementary DNA was amplified using specific primers for inflammatory mediator's gene expression, and the results were normalized to β-actin gene expression. The relative mean fold change of inflammatory mediator's gene expression in the experimental group was calculated using the 2ΔΔCt method and compared with the control group [25]. 2.9. Determination of NF-κB nuclear translocation Alveolar macrophages (2 × 106/mL) were isolated from BALF of each group. The cells were then fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.5% Triton X-100 for 15 min. After 1 h incubation with blocking buffer (5% BSA in PBS), cells were incubated with anti-NF-κB/p65 antibody diluted at 1:100 in blocking buffer overnight at 4 °C, washed, and then incubated with FITC-conjugated anti-rabbit IgG diluted at 1:1000 in blocking buffer for 1 h. In order to identify the nucleus, the FITC-conjugated-labeled samples were counterstained with a 1:500 dilution of Hoechst for 2 min. Fluorescent images were observed under the Leica TCS SP2 laser scanning spectral confocal microscope (Leica, Germany). 2.10. Statistical analysis Values are presented as mean ± standard deviation (S.D.) from three independent experiments. Data were analyzed using one-way
analysis of variance (ANOVA) followed by Tukey's post hoc test in SPSS11.0 (Chicago, IL, USA). P b 0.05 was considered to be significant between groups. 3. Result 3.1. Effects of ketamine on the injury indicators of CLP/HMGB1-induced ALI in WT rats The lung W/D ratio and MPO activity were determined in lung tissue dissected at 24 h after CLP/HMGB1. As shown in Fig. 1 and Fig. 2, the lung W/D ratio and MPO activity in lung tissue increased significantly in the CLP/HMGB1 group compared with that of the control group. However, this increase was apparently reduced by ketamine (5, 7.5, and 10 mg/kg) (P b 0.05, Fig. 1A, B; Fig. 2A, B). The total protein concentration and the number of total cells, neutrophils, and macrophages in BALF were analyzed at 24 h after CLP/ HMGB1. As shown in Fig. 1 and Fig. 2, CLP/HMGB1 significantly increased the number of total cells, neutrophils and macrophages compared with that of the control group. However, treatment with ketamine (5, 7.5, and10mg/kg) significantly decreased the number of total cells, neutrophils, and macrophages (P b 0.05, Fig. 1C–F; Fig.2 C–F). 3.2. Effects of ketamine on the production of inflammatory cytokines in the BALF and lung tissue from WT rats with CLP/HMGB1-induced ALI The effect of ketamine on inflammatory cytokines production was analyzed at 24 h after CLP/HMGB1 by ELISA. Compared with the control group, the levels of inflammatory cytokines TNF-α, IL-1β, and IL-6 in BALF and lung tissue were significantly increased after CLP/HMGB1. Ketamine (5, 7.5, and10mg/kg) reduced inflammatory cytokines production compared with that of the CLP/HMGB1 group (P b 0.05, Fig. 3; Fig. 4).
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3.3. Effect of ketamine on histopathological changes in lung tissues in CLP/ HMGB1-induced ALI in WT rats Histologic assessment of the effects of ketamine on sepsis-induced lung inflammation and injury was performed at 24 h. Lungs (n = 10) from each experimental group were processed for histologic evaluation after H&E staining (magnification 200×). As shown in Fig. 5 and Fig. 6 the lung tissues from the control group showed normal histological features. Isolated lung tissues from the CLP/HMGB1 group showed characteristics of acute alveolar damage and acute inflammation. These features included congestion, accumulation of neutrophils, and markedly thickened alveolar walls. These changes were observed in all lung tissues from this group. Furthermore, isolated lung tissues in the ketamine-treated group showed that ketamine treatment prevented CLP/HMGB1-induced damage (Fig. 5; Fig. 6). 3.4. Effects of ketamine on the expression of HMGB1 and RAGE in the BALF and lung tissue from WT rats with CLP-induced ALI ELISA showed that HMGB1 protein levels in the BALF were significantly higher in the CLP group than in the control group. Ketamine treatment was found to significantly down-regulate the levels of
HMGB1 relative to the CLP group at 24 h (P b 0.05, Fig. 7A). However, ELISA showed that sRAGE protein levels in the BALF were significantly higher in the CLP group than in the control group. Ketamine treatment was not found to significantly regulate the levels of sRAGE relative to the CLP group at 24 h (P N 0.05, Fig. 7B). Western blot analysis and Real-Time PCR showed that the expression of HMGB1 and RAGE in lung tissues were significantly higher in the CLP group than in the control group. Ketamine treatment was found to significantly downregulate the expression of HMGB1 and RAGE relative to the CLP group at 24 h (P b 0.05,Fig. 7C–F). 3.5. Effect of anti-RAGE Ab on the production of inflammatory cytokines in the BALF from WT rats with CLP/HMGB1-induced ALI To clarify the role of RAGE signaling in the release of inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6, and HMGB1), rats were treated with an intraperitoneal injection of anti-RAGE Ab (10 mg/kg) 1 h before CLP/HMGB1, and then the rats were treated with ketamine (10 mg/kg, i. p.) or an equal vehicle (NS) at 3 h and 12 h after CLP/HMGB1. BALF was collected at 24 h after CLP/HMGB1 to analyze the inflammatory cytokines. We found that CLP/HMGB1-induced releases of inflammatory cytokines were significantly inhibited by both anti-RAGE Ab treatment
Fig. 9. Effect of anti-RAGE Ab on the production of inflammatory cytokines in the BALF from RAGE siRNA-transfected WT rats with HMGB1-induced ALI. Rats were treated with an intraperitoneal injection of anti-RAGE Ab (10 mg/kg) 1 h before HMGB1, and then the rats were treated with ketamine (10 mg/kg, i.p.) or an equal volume of vehicle (NS) at 3 h and 12 h after HMGB1; rats received intratracheal injection of 300 μL of NS or 300 μL (3 × 109 PFU) of RAGE siRNA. Seven days later, ketamine (10 mg/kg, i.p.) was administrated at 3 h and 12 h after HMGB1. BALF was collected at 24 h after HMGB1 to analyze the inflammatory cytokines TNF-α (A, B), IL-1β (C, D), and IL-6 (E, F). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05 vs. HMGB1, #P b 0.05 vs. HMGB1 + anti-RAGE Ab/RAGE siRNA + ketamine.
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Fig. 10. Effect of ketamine on the production of inflammatory cytokines in the BALF from RAGE siRNA-transfected WT rats with CLP-induced ALI. Rats received intratracheal injection of 300 μL of NS or 300 μL (3 × 109 PFU) of RAGE siRNA. Seven days later, ketamine (10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. BALF was collected at 24 h after CLP to analyze the inflammatory cytokines TNF-α (A), IL-1β (B), IL-6 (C),and HMGB1 (D). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05 vs. CLP, #P b 0.05 vs. CLP + RAGE siRNA + ketamine.
and ketamine treatment alone (*P b 0.05, Fig. 8; Fig. 9); furthermore, when both ketamine treatment and anti-RAGE Ab treatment were performed to the CLP/HMGB1-induced ALI in rats, the inflammatory mediators releasing was most strongly inhibited (#P b 0.05, Fig. 8; Fig. 9). These results implicate RAGE as a likely effector of ketamine-mediated inhibition of CLP/HMGB1-induced inflammatory signaling and the effects of ketamine treatment may be not completely removed by blocking RAGE signaling pathway.
siRNA. Seven days later, Ketamine (10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP/HMGB1. BALF was collected at 24 h after CLP/HMGB1 to analyze the inflammatory cytokines. We found that silencing RAGE with siRNA inhibited production of inflammatory cytokines in the BALF from rats with CLP/HMGB1-induced ALI (*P b 0.05, Fig. 9; Fig. 10), and the ketamine treatment strongly increased the inhibition effect of RAGE siRNA (#P b 0.05, Fig. 9; Fig. 10).
3.6. Effect of ketamine on the production of inflammatory cytokines in the BALF from RAGE siRNA-transfected WT rats with CLP/HMGB1 -induced ALI
3.7. Effect of ketamine on the activation of NF-κB in the lung tissue and alveolar macrophages from WT rats with CLP-induced ALI
To clarify the role of RAGE signaling in the release of inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6, and HMGB1), rats received intratracheal injection of 300 μL of NS or 300 μL (3 × 109 PFU) of RAGE
Our results showed that CLP-induced ALI in rats significantly increased IκB-α phosphorylation and nuclear NF-κB (p65) activation in lung tissues (P b 0.05, Fig. 11), and induced nuclear translocation of
Fig. 11. Effect of ketamine on the activation of NF-κB in the lung tissue from WT rats with CLP-induced ALI. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. Protein expression of NF-κB (p65) in the nucleus (A) and the cytosol (B) and p-IκB-α (C) were respectively measured by Western blot. The quantification of relative band intensities was determined by densitometry. The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05, #P b 0.01vs. CLP.
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Fig. 12. Effect of ketamine on the nuclear translocation of NF-κB (p65) in alveolar macrophages from WT rats with CLP-induced ALI. Ketamine (10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. Alveolar macrophages (2 × 106 cells/mL) were isolated from BALF of each group. Samples were prepared for confocal microscopy analysis. The values presented are the mean ± S.D. (n = 10 in each group).
NF-κB (p65) in alveolar macrophages from BALF (Fig. 12). Ketamine inhibited all the above effects (P b 0.05, Fig. 11; Fig. 12). 3.8. Effect of ketamine on the phosphorylation of the MAPK signaling pathway in the lung tissue from WT rats with CLP-induced ALI Because the MAPK signaling molecules investigated here are activated by phosphorylation, we detected the phosphorylated forms of ERK, p38 MAPK, and JNK by Western-blotting. After rats were treated with CLP for 24 h, the phosphorylation of ERK, p38 MAPK and JNK in the lung tissue from rats with CLP-induced ALI was significantly increased compared with their respective control groups. However,
Ketamine remarkably attenuated these effects in a concentrationdependent manner. These results suggest that the MAPK signaling pathway is involved in ketamine-mediated inhibition of CLP-induced ALI in rats (P b 0.05, Fig. 13). 3.9. Effects of ketamine on the injury indicators of CLP-Induced ALI in WT and RAGE−/− rats The lung W/D ratio and MPO activity were determined in lung tissue dissected at 24 h after CLP. As shown in Fig. 14, the lung W/D ratio and MPO activity in lung tissue increased significantly in the CLP group compared with that of the control group in WT and RAGE−/− rats. However,
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Fig. 13. Effect of ketamine on the phosphorylation of the MAPK signaling pathway in the lung tissue from WT rats with CLP-induced ALI. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. Western blot analysis was subsequently used to detect the expression of ERK, p38 MAPK, and JNK. Phosphorylated proteins were also detected. The quantification of relative band intensities was determined by densitometry. The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05, #P b 0.01vs. CLP.
Fig. 14. Effects of ketamine on the injury indicators of CLP-induced ALI in WT and RAGE−/− rats. Ketamine (10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. The lung W/D ratio (A) and MPO activity (B) were determined in lung tissue dissected at 24 h after CLP. BALF was collected at 24 h after CLP to measure the total protein concentration (C) and the number of total cells (D), neutrophils (E), and macrophage (F). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05 vs. CLP in WT or RAGE−/− rats, #P b 0.05 vs. CLP + ketamine in WT rats.
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Fig. 15. Effects of ketamine on the production of inflammatory cytokines in the BALF and lung tissue from WT and RAGE−/− rats with CLP-induced ALI. Ketamine (10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. BALF and lung tissue were collected at 24 h after CLP to analyze the inflammatory cytokines TNF-α (A, B), IL-1β (C, D), and IL-6 (E, F). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05 vs. CLP in WT or RAGE−/− rats, #P b 0.05 vs. CLP + ketamine in WT rats.
Fig. 16. Effect of ketamine on histopathological changes in lung tissues in CLP-induced ALI in WT and RAGE−/− rats. Ketamine (10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. Lung tissues (n = 10) from each experimental group were processed for histological evaluation at 24 h after CLP. Representative histological changes of lung tissues obtained from WT and RAGE−/− rats of different groups. (A) Control group in WT rats, (D) control group in RAGE−/− rats, (B) CLP group in WT rats, (E) CLP group in RAGE−/− rats, (C) CLP + ketamine (10 mg/kg) group in WT rats, (F) CLP + ketamine (10 mg/kg) group in RAGE−/− rats (H&E staining, magnification 200×).
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this increase was apparently reduced by ketamine (10 mg/kg) (P b 0.05, Fig. 14A, B). Inhibition effects of ketamine in RAGE−/− rats had higher lung W/D ratio and MPO activity at 24 h of CLP when compared with those in WT rats (P b 0.05, Fig. 14A, B). The total protein concentration and the number of total cells, neutrophils, and macrophages in BALF were analyzed at 24 h after CLP. As shown in Fig. 14, CLP significantly increased the number of total cells, neutrophils and macrophages compared with that of the control group in WT and RAGE−/− rats. However, treatment with ketamine (10 mg/kg) significantly decreased the number of total cells, neutrophils, and macrophages (P b 0.05, Fig. 14C–F). Inhibition effects of ketamine in RAGE−/− rats had higher total protein concentrations and the number of total cells, neutrophils, and macrophages in BALF at 24 h of CLP when compared with those in WT rats (P b 0.05, Fig. 14C–F). 3.10. Effects of ketamine on the production of inflammatory cytokines in the BALF and lung tissue from WT and RAGE−/− rats with CLP-induced ALI The effect of ketamine on inflammatory cytokines production was analyzed at 24 h after CLP by ELISA. Compared with the control group, the levels of inflammatory cytokines TNF-α, IL-1β, and IL-6 in BALF and lung tissues were significantly increased after CLP in WT and RAGE−/− rats. Ketamine (10 mg/kg) reduced inflammatory cytokines production compared with that of the CLP group (P b 0.05, Fig. 15). Inhibition effects of ketamine in RAGE−/− rats had higher inflammatory cytokines levels in BALF and lung tissues at 24 h of CLP when compared with those in WT rats (P b 0.05, Fig. 15). 3.11. Effect of ketamine on histopathological changes in lung tissues in CLPInduced ALI in WT and RAGE−/− rats Histologic assessment of the effects of ketamine on sepsis-induced lung inflammation and injury was performed at 24 h. Lungs (n = 10) from each experimental group were processed for histologic evaluation after H&E staining (magnification 200×). As shown in Fig. 16, the lung tissues from the control group showed normal histological features. Isolated lung tissues from the CLP group showed characteristics of acute alveolar damage and acute inflammation. These changes were observed in all lung tissues from this group. Furthermore, isolated lung tissues in the ketamine-treated group showed that ketamine treatment prevented CLP-induced damage (Fig. 16). Inhibition effects of ketamine in RAGE−/− rats had severer histopathological changes in lung tissues at 24 h of CLP when compared with those in WT rats (Fig. 16). 4. Discussion In the present study, we investigate the underlying mechanisms of anti-inflammatory effect of ketamine and observe the effect of ketamine on HMGB1 and RAGE expression during CLP-induced ALI in rats. The results of this study have demonstrated that ketamine reduces pathological changes of rat with CLP-induced ALI and mitigates inflammatory responses in the alveolar space, such as neutrophil infiltration and edema formation. Meanwhile, ketamine treatments on CLP-induced ALI in rats downregulate HMGB1 and RAGE expression in the BALF and lung tissues, as well as production of IL-1β, IL-6, and TNF-α in the BALF and lung tissues. These findings suggest that ketamine could be protective against the development of ALI by inhibiting HMGB1 and its receptor-RAGE expression and subsequently reducing inflammatory mediators production. Our study also shows that the CLP-induced release of inflammatory mediators in BALF was suppressed by antiRAGE Ab and RAGE siRNA. Furthermore, treatment with ketamine significantly inhibited the phosphorylation of MAPK and the activation of NF-κB p65 in lung tissues. HMGB1, a nuclear non-histone DNA-binding protein, stabilizes nucleosome formation and acts as a transcription-factor-like protein that regulates the expression of several genes [26]. However, when released
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into the extracellular milieu, HMGB1 has recently been shown to be an important late mediator of endotoxin shock, intra-abdominal sepsis, and ALI, and a promising therapeutic target of severe sepsis [8,27,28]. Meanwhile, previous studies showed that lung HMGB1 levels increased in the development of CLP-induced ALI in rats [29], and neutralizing antibodies as well as siRNA targeting HMGB1 significantly improve sepsisinduced ALI [27,30]. In this study, to further investigate whether HMGB1 acts as a stimulating factor in the development of ALI, HMGB1 was used to induce ALI. We found that ketamine inhibited the production of inflammatory cytokines in the BALF and lung tissue from WT rats with HMGB1-induced ALI, and attenuated the injury indicators and histopathological changes in lung tissues in HMGB1-induced ALI in WT rats. Therefore, this study provides evidence that HMGB1 is a potential important mediator in the development of ALI, and ketamine act through inhibiting HMGB1 to attenuate ALI. RAGE, a transmembrane receptor of the immunoglobulin superfamily, play critical roles in the innate immune system by interacting with microbial pathogens as well as endogenous molecules released during sepsis [31]. RAGE has been proposed to be involved in the pathogenesis of sepsis due to its role in transmitting signals from pathogen substrates to activate cells during the onset and perpetuation of inflammation [31]. Ligation of RAGE on the cellular surface triggers a series of cellular signaling events, including the activation and translocation to the nucleus of transcription factor NF-κB, leading to the production of proinflammatory cytokines, chemokines, adhesion molecules and oxidative stress and causing inflammation [32]. Inhibition of the RAGE products increases survival in experimental models of severe sepsis [12]. In this study, to further investigate whether RAGE acts as its major mediator in the development of ALI, RAGE-deficient rat was used. We noted that the inhibition effects of ketamine in RAGE−/− rats had higher injury indicators and inflammatory cytokines levels in BALF and lung tissues at 24 h of CLP when compared with those in WT rats. Inhibition effects of ketamine in RAGE−/− rats had severer histopathological changes in lung tissues at 24 h of CLP when compared with those in WT rats. Therefore, this study provides evidence that RAGE is a potential important mediator in the development of ALI, and ketamine act through inhibiting RAGE to attenuate ALI. RAGE possesses a secretory isoform known as sRAGE, which maintains the extracellular ligand-binding domain but lacks the cytosolic and transmembrane domains [33]. sRAGE has the same ligand binding specificity and competes with cell-bound RAGE, serving as a decoy that abolishes cell activation [33]. sRAGE is up-regulated during LPSinduced lung injury, which was ameliorated by recombinant sRAGE [34]. sRAGE may be secreted as a decoy receptor and contribute to the suppression of excessive inflammatory response during ALI [34]. In this study, we found that sRAGE is up-regulated during CLP-induced ALI, which was not influenced by ketamine. Activation of the RAGE signaling pathways results in the activation of NF-κB to induce expression of pro-inflammatory cytokines [35,36]. Previous studies demonstrated a significant decrease in lung NF-κB activation as well as blockade of RAGE by soluble RAGE in mice following induction of lung injury by LPS [34]. Simultaneously, the activated RAGE also stimulates MAPK pathway, one of the most widespread mechanisms of cell regulation, leading to upregulation of pro-inflammatory mediators and cell death [37]. ERKs, p38 MAPK, and JNKs are three major groups of the MAPK family [38]. In CLP-induced ALI, activated MAPKs contribute to the inflammatory response following CLP [39]. In our current study, CLP significantly increased the activation of NF-κB as well as the phosphorylation of ERK, p38 MAPK, and JNK. These changes were attenuated by post-treatment with ketamine. Our results suggest that the anti-inflammatory response following ketamine post-treatment is associated with suppression of NF-κB and MAPK activation. In summary, the present study demonstrates that ketamine ameliorates CLP-induced ALI through suppression of HMGB1 and RAGEmediated inflammatory signaling pathways. Thus, ketamine might be
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useful as a potential therapeutic medication for preventing ALI and ARDS. Author contributions Kehan Li: conception, design, analysis and interpretation of data; writing the manuscript. Jianxue Yang: conception, design, analysis and interpretation of data; writing the manuscript. Xuechang Han: conception, design, analysis and interpretation of data; writing the manuscript. Conflicts of interest The authors report no proprietary or commercial interest in any product mentioned, or concept discussed, in this article. References [1] R.D. Ramnath, S.W. Ng, A. Guglielmotti, M. Bhatia, Role of MCP-1 in endotoxemia and sepsis, Int. Immunopharmacol. 8 (2008) 810–818. [2] J. Garnacho-Montero, T. Aldabo-Pallas, C. Garnacho-Montero, A. Cayuela, R. Jimenez, S. Barroso, et al., Timing of adequate antibiotic therapy is a greater determinant of outcome than are TNF and IL-10 polymorphisms in patients with sepsis, Crit. Care 10 (2006) R111. [3] Z. Hasan, K. Palani, M. Rahman, H. Thorlacius, Targeting CD44 expressed on neutrophils inhibits lung damage in abdominal sepsis, Shock 35 (2011) 567–572. [4] A.M. Van Nuffel, V. Sukhatme, P. Pantziarka, L. Meheus, V.P. Sukhatme, G. Bouche, Repurposing Drugs in Oncology (ReDO)-clarithromycin as an anti-cancer agent, ecancermedicalscience 9 (2015) 513. [5] E. Letsiou, S. Sammani, W. Zhang, T. Zhou, H. Quijada, L. Moreno-Vinasco, et al., Pathologic mechanical stress and endotoxin exposure increases lung endothelial microparticle shedding, Am. J. Respir. Cell Mol. Biol. 52 (2015) 193–204. [6] H. Ueno, T. Matsuda, S. Hashimoto, F. Amaya, Y. Kitamura, M. Tanaka, et al., Contributions of high mobility group box protein in experimental and clinical acute lung injury, Am. J. Respir. Crit. Care Med. 170 (2004) 1310–1316. [7] Q. Wang, X. Wu, X. Tong, Z. Zhang, B. Xu, W. Zhou, Xuebijing ameliorates sepsis-induced lung injury by downregulating HMGB1 and RAGE expressions in mice, Evid. Based Complement. Alternat. Med. 2015 (2015) 860259. [8] Y.M. Yao, Y.Y. Luan, Q.H. Zhang, Z.Y. Sheng, Pathophysiological aspects of sepsis: an overview, Methods Mol. Biol. 1237 (2015) 5–15. [9] Y. Zhou, Y. Li, T. Mu, HMGB1 neutralizing antibody attenuates cardiac injury and apoptosis induced by hemorrhagic shock/resuscitation in rats, Biol. Pharm. Bull. 38 (2015) 1150–1160. [10] S.Y. Wang, Z.J. Li, X. Wang, W.F. Li, Z.F. Lin, Effect of ulinastatin on HMGB1 expression in rats with acute lung injury induced by sepsis, Genet. Mol. Res. 14 (2015) 4344–4353. [11] H. Hu, C. Zhai, G. Qian, A. Gu, J. Liu, F. Ying, et al., Protective effects of tanshinone IIA on myocardial ischemia reperfusion injury by reducing oxidative stress, HMGB1 expression, and inflammatory reaction, Pharm. Biol. 53 (2015) 1752–1758. [12] E.C. Lutterloh, S.M. Opal, D.D. Pittman, J.C. Keith Jr., X.Y. Tan, B.M. Clancy, et al., Inhibition of the RAGE products increases survival in experimental models of severe sepsis and systemic infection, Crit. Care 11 (2007), R122. [13] R. Clynes, K. Herold, A.M. Schmidt, RAGE: exacting a toll on the host in response to polymicrobial sepsis and Listeria monocytogenes, Crit. Care 11 (2007) 183. [14] J. Xie, J.D. Mendez, V. Mendez-Valenzuela, M.M. Aguilar-Hernandez, Cellular signalling of the receptor for advanced glycation end products (RAGE), Cell. Signal. 25 (2013) 2185–2197. [15] D.J. Weber, Y.M. Allette, D.S. Wilkes, F.A. White, The HMGB1-RAGE inflammatory pathway: implications for brain injury-induced pulmonary dysfunction, Antioxid. Redox Signal. (2015).
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Ketamine attenuates sepsis-induced acute lung injury via regulation of HMGB1-RAGE pathways Kehan Li a,⁎, Jianxue Yang b, Xuechang Han a a b
Department of Anesthesiology, The First Affiliated Hospital of Henan Science and Technology University, Luoyang, Henan, China Department of Neurology, The First Affiliated Hospital of Henan Science and Technology University, Luoyang, Henan, China
a r t i c l e
i n f o
Article history: Received 10 September 2015 Received in revised form 5 January 2016 Accepted 21 January 2016 Available online xxxx Keywords: Ketamine CLP HMGB1 RAGE ALI
a b s t r a c t High mobility group box protein 1 (HMGB1) and receptor for the advanced glycation end product (RAGE) play important roles in the development of sepsis-induced acute lung injury (ALI). Ketamine is considered to confer protective effects on ALI during sepsis. In this study, we investigated the effects of ketamine on HMGB1-RAGE activation in a rat model of sepsis-induced ALI. ALI was induced in wild type (WT) and RAGE deficient (RAGE−/−) rats by cecal ligation and puncture (CLP) or HMGB1 to mimic sepsis-induced ALI. Rats were randomly divided to six groups: sham-operation + normal saline (NS, 10 mL/kg), sham-operation + ketamine (10 mg/kg), CLP/ HMGB1 + NS (10 mL/kg), CLP/HMGB1 + ketamine (5 mg/kg), CLP/HMGB1 + ketamine (7.5 mg/kg), and CLP/ HMGB1 + ketamine (10 mg/kg) groups. NS and ketamine were administered at 3 and 12 h after CLP/HMGB1 via intraperitoneal injection. Pathological changes of lung, inflammatory cell counts, expression of HMGB1and RAGE, and concentrations of various inflammatory mediators in bronchoalveolar lavage fluids (BALF) and lung tissue were then assessed. Nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathways in the lung were also evaluated. CLP/HMGB1 increased the wet to dry weight ratio and myeloperoxidase activity in lung, the number of total cells, neutrophils, and macrophages in the BALF, and inflammatory mediators in the BALF and lung tissues. Moreover, expression of HMGB1and RAGE in lung tissues was increased after CLP. Ketamine inhibited all the above effects. It also inhibited the activation of IκB-α, NFκB p65, and MAPK. Ketamine protects rats against HMGB1-RAGE activation in a rat model of sepsis-induced ALI. These effects may partially result from reductions in NF-κB and MAPK. Crown Copyright © 2016 Published by Elsevier B.V. All rights reserved.
1. Introduction Sepsis is a complex clinical syndrome resulting from a harmful host inflammatory response to infection [1]. Numerous advances have been made in diagnostic procedures, antimicrobial treatment, and supportive care; however, sepsis remains a major cause of morbidity and mortality in adult and pediatric intensive care units [2]. Lung is the most vulnerable, critical and sensitive organ during sepsis process [3]. During the development of sepsis, bacterial components, such as lipopolysaccharide (LPS), could lead to activate an inflammatory cascade resulting in the release of inflammatory mediators [4]. The overproduction of inflammatory mediators induces endothelial and epithelial injury, vascular leakage, edema and vasodilatation, subsequently causing the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) [5]. Previous studies have shown that inflammatory mediators play a key role in the pathogenesis of ALI/ARDS [6]. ALI is characterized ⁎ Corresponding author at: No. 24 Jinghua Road, Luoyang, Henan, China. E-mail address: [email protected] (K. Li).
http://dx.doi.org/10.1016/j.intimp.2016.01.021 1567-5769/Crown Copyright © 2016 Published by Elsevier B.V. All rights reserved.
by a local inflammatory response, and receptor for advanced glycation end-products (RAGE) has an important function in the HMGB1mediated activation of innate immunity [7]. Based on the key role of inflammatory mediators in the pathogenesis of ALI, pharmacological treatments have been aimed at modulating RAGE signaling and alleviating nonspecific inflammatory reactions that may as a potential therapeutic strategy for ALI. Extracellular HMGB1 is an inflammatory mediator; elevated HMGB1 levels have been associated with diseases such as sepsis, hemorrhagic shock, ALI, and ischemia–reperfusion injury [8–11]. RAGE, mainly located in the alveolar type I cells, is a multiligand-binding receptor with an extracellular, transmembrane, and cytosolic domain, which senses a variety of signaling molecules including advanced glycation end products (AGEs) and HMGB1 [12,13]. Binding of HMGB1 to its transmembrane receptor RAGE activates multiple signaling cascades, including NF-κB and mitogen-activated protein kinase (MAPK) [14]. Activation of these cascades is known to result in the production and release of inflammatory mediators such as cytokines, e.g. TNF-α, IL-6 and IL-1β, and HMGB1 [15]. This indicates that activation of HMGB1-
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RAGE pathway amplifies the inflammatory responses in vivo and plays a central role in the pathogenesis of inflammatory diseases. Recent studies have shown that HMGB1-RAGE signaling pathway is also involved in the development of ALI [16], suggesting that targeting HMGB1-RAGE pathway might be a novel strategy in the treatment of ALI. Ketamine has been shown to protect animals against injury by its anti-inflammatory and immunomodulatory effects [17–19]. Ketamine has been used in clinic to treat sepsis [20,21], and also has potential value in treatment of animal models of several other diseases, including ischemia/reperfusion injury [22], ALI [23]. However, whether ketamine inhibits HMGB1-RAGE pathway and confers to its suppression of ALI is still unclear. In the present study, we used rat model of CLP-induced
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ALI to evaluate the effects of ketamine on the inflammatory responses, particularly HMGB1-RAGE pathway, and to further explore its potential molecular mechanisms. 2. Materials and methods 2.1. Animals Male Sprague–Dawley (wild-type, WT) rats (Henan Science and Technology University Experimental Animal Center, Luoyang, China) and male Sprague–Dawley (RAGE-deficient, RAGE−/−) rats (Beijing Biocytogen Co., Ltd., Beijing, China) were used in this study. The rats
Fig. 1. Effects of ketamine on the injury indicators of CLP-induced ALI in WT rats. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. The lung W/D ratio (A) and MPO activity (B) were determined in lung tissue dissected at 24 h after CLP. BALF was collected at 24 h after CLP to measure the total protein concentration (C) and the number of total cells (D), neutrophils (E), and macrophage (F). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05, #P b 0.01vs. CLP.
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Fig. 2. Effects of ketamine on the injury indicators of HMGB1-induced ALI in WT rats. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after HMGB1. The lung W/D ratio (A) and MPO activity (B) were determined in lung tissue dissected at 24 h after HMGB1. BALF was collected at 24 h after HMGB1 to measure the total protein concentration (C) and the number of total cells (D), neutrophils (E), and macrophage (F). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05, #P b 0.01vs. HMGB1.
were exposed to 12 h of light and 12 h of darkness each day, and were given access to food and water ad libitum. The animal protocol was approved by the Institutional Animal Ethics Committee of the First Affiliated Hospital of Henan Science and Technology University. 2.2. Animal model and experimental groups 2.2.1. CLP/HMGB1-induced ALI model Rats were randomly assigned to six groups (n = 10): shamoperation + normal saline (NS, 10 mL/kg), shamoperation + ketamine (10 mg/kg), CLP/HMGB1 + NS (10 mL/kg), CLP/HMGB1 + ketamine (5 mg/kg), CLP/HMGB1 + ketamine
(7.5 mg/kg), and CLP/HMGB1 + ketamine (10 mg/kg). Sodium pentobarbital (50 mg/kg, Sigma-Aldrich, St. Louis., MO, USA) was injected intraperitoneally (i.p.) to anesthetize the animals before the surgical procedures. Rats were instilled intratracheally with 50 μg of HMGB1 (Sigma-Aldrich, St. Louis, MO, USA) diluted in 0.25 mL of sterile phosphatebuffered saline (PBS) [24]. Polymicrobial sepsis was induced by CLP as described previously [19]. In brief, a midline incision about 2 cm was made on the anterior abdomen. The cecum was carefully isolated and the distal 20% was ligated. Then the cecum was punctured twice with a sterile 21-gauge needle, and was squeezed to extrude the cecal contents from the wounds. The cecum was placed back and the abdominal
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Fig. 3. Effects of ketamine on the production of inflammatory cytokines in the BALF and lung tissue from WT rats with CLP-induced ALI. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. BALF and lung tissue were collected at 24 h after CLP to analyze the inflammatory cytokines TNF-α (A, B), IL-1β (C, D), and IL-6 (E, F). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05, #P b 0.01vs. CLP.
incision was closed. The sham control animals were treated in an identical manner, but no cecal ligation or puncture was performed. All rats were administered 1 mL sterile NS immediately after surgery for fluid resuscitation. At 3 h and 12 h after CLP/HMGB1, rats were treated with ketamine (ketamine hydrochloride, Hengrui, Inc., Nanjing, China) 5, 7.5 or 10 mg/kg (i.p.) or NS (10 mL/kg, i.p.). At 24 h after CLP/HMGB1, the rats were sacrificed. Then the lungs of rats were harvested and stored in liquid nitrogen for later use. To clarify the role of RAGE signaling in the release of inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6, and HMGB1), rats were treated with an intraperitoneal injection of anti-RAGE Ab (10 mg/kg) 1 h before CLP/HMGB1, and then the rats were treated with ketamine (10 mg/kg, i. p.) or an equal vehicle (NS) at 3 h and 12 h after CLP/HMGB1; Rats received intratracheal injection of 300 μL of NS or 300 μL (3 × 109 PFU) of RAGE siRNA. Seven days later, Ketamine (10 mg/kg, i.p.) was
administrated at 3 h and 12 h after CLP/HMGB1. BALF was collected at 24 h after CLP/HMGB1 to analyze the inflammatory cytokines. 2.2.2. RAGE-deficient model The WT and RAGE−/− rats were independently and randomly divided into 6 groups (n = 10): sham-operation + NS (10 mL/kg) in WT or RAGE−/− rats, CLP + NS (10 mL/kg) in WT or RAGE−/− rats, and CLP + ketamine (10 mg/kg) in WT or RAGE−/− rats. Polymicrobial sepsis was induced by CLP as described above. At 24 h after CLP, the rats were sacrificed. Then the BALF and lung tissues of rats were harvested and stored in liquid nitrogen for later use. 2.3. Lung wet to dry weight (W/D) ratio and MPO activity measurement After the rats were euthanized, the lungs were removed and the wet weight was determined. The lung tissue was placed in an oven at 60 °C
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Fig. 4. Effects of ketamine on the production of inflammatory cytokines in the BALF and lung tissue from WT rats with HMGB1-induced ALI. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after HMGB1. BALF and lung tissue were collected at 24 h after HMGB1 to analyze the inflammatory cytokines TNF-α (A, B), IL-1β (C, D), and IL-6 (E, F). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05, #P b 0.01vs. HMGB1.
for 24 h to obtain the dry weight. The ratio of the wet lung to the dry lung was calculated to assess tissue edema. MPO activity, reflecting the parenchymal infiltration of neutrophils and macrophages, was determined using test kits (BioLegend, Inc. Camino Santa Fe, Suite E San Diego, CA, USA) according to the instructions. The right lungs were excised. Lung tissues of 100 mg were homogenized and fluidized in extraction buffer to obtain 5% of homogenate. The sample including 0.9 mL homogenate and 0.1 mL of reaction buffer was heated to 37 °C in water for 15 min, on which occasion; The enzymatic activity was determined by measuring the change in absorbance at 460 nm using a 96well plate reader (Molecular devices, Sunnyvale, CA, USA).
2.4. The total protein concentration and inflammatory cell counts in the BALF To evaluate vascular permeability in the airways, the BALF was collected and protein content was determined by BCA protein assay kit (Pierce, Rockford, IL, USA). The principle of BCA method is as follow: The peptide bond of amino acids can bind with Cu2+ to form a complex in the basic solution and then the Cu2 + is deoxidized to Cu+. Bicinchoninic acid and its sodium salt are water-soluble. In the alkaline environment, it can bind with Cu2+ to form a dark purple compound, which has strong absorbance maximum at 562 nm wavelength. Because
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Fig. 5. Effect of ketamine on histopathological changes in lung tissues in CLP-induced ALI in WT rats. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. Lung tissues (n = 10) from each experimental group were processed for histological evaluation at 24 h after CLP. Representative histological changes of lung tissues obtained from rats of different groups. (A) Control group, (B) ketamine (10 mg/kg) group, (C) CLP group, (D) CLP + ketamine (5 mg/kg) group, (E) CLP + ketamine (7.5 mg/kg) group, (F) CLP + ketamine (10 mg/kg) group (H&E staining, magnification 200×).
the brightness is coordinated with the concentration of protein, we can use matching method to determine protein concentration. Proteins were expression in milligram protein per milliliter BALF. The fluid recovered from each sample was centrifuged (4 °C, 3000 rpm,10 min) to pellet the cells. The cell pellets were resuspended in PBS for total cell counts using a hemocytometer. Cytospins were prepared for differential cell counts by staining with the Wright–Giemsa staining method. 2.5. Inflammatory mediators assays The concentrations of TNF-α, IL-1β, IL-6, HMGB1, and soluble receptor for advanced glycation end product (sRAGE) in the BALF and lung
tissue were measured by ELISA kits according to the instruction recommended by the manufactures (BioLegend, Inc. Camino Santa Fe, Suite E San Diego, CA, USA).
2.6. Histopathological evaluation of the lung tissue Histopathological examination was performed on rats that were not subjected to BALF collection. Lungs were fixed with10% buffered formalin, imbedded in paraffin and sliced. After hematoxylin and eosin (H&E) staining, pathological changes of lung tissues were observed under a light microscope.
Fig. 6. Effect of ketamine on histopathological changes in lung tissues in HMGB1-induced ALI in WT rats. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after HMGB1. Lung tissues (n = 10) from each experimental group were processed for histological evaluation at 24 h after HMGB1. Representative histological changes of lung tissues obtained from rats of different groups.(A) Control group, (B) ketamine (10 mg/kg) group, (C) HMGB1 group, (D) HMGB1 + ketamine (5 mg/kg) group, (E) HMGB1 + ketamine (7.5 mg/kg) group group, (F) HMGB1 + ketamine (10 mg/kg) group (H&E staining, magnification 200×).
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Fig. 7. Effects of ketamine on the expression of HMGB1 and RAGE in the BALF and lung tissue from WT rats with CLP-induced ALI. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. HMGB1 (A) and sRAGE (B) protein expression in BALF were determined by ELISA; HMGB1(C) and RAGE (D) protein expression in lung tissues were determined by Western Blot; relative HMGB1(E) and RAGE (F) mRNA expression in lung tissues were determined by real-time PCR. The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05, #P b 0.01vs. CLP.
2.7. Western blot analysis Proteins were extracted from the lungs using T-PER Tissue Protein Extraction Reagent Kit according to the manufacturer's instructions. Protein concentrations were determined by BCA protein assay kit. The proteins were separated on12% SDS-Polyacrylamide gel and transferred on to the PVDF membrane. After blocking the nonspecific site with blocking solution (5% nonfat dry milk), the membrane was incubated overnight with specific primary antibody at 4 °C. Subsequently, the membrane
was washed with PBS-T followed by incubation with the secondary antibody conjugated with horseradish peroxidase at room temperature for 1 h. Blots were again washed with PBS-T and then developed with the ECL Plus Western Blotting Detection System (Amersham Life Science, UK). The following primary antibodies were used: phospho-p38, p38, phospho-ERK, ERK, phospho-JNK, JNK, phospho-IκB-α, IκB-α, NF-κB p65, Histone H3.1, RAGE, β-actin (all from Cell Signaling Technology, Beverly, MA, USA). Protein densities were standardized to β-actin and Histone H3.1 for total lysates and nuclear fractions, respectively.
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Fig. 8. Effect of anti-RAGE Ab on the production of inflammatory cytokines in the BALF from WT rats with CLP-induced ALI. Rats were treated with an intraperitoneal injection of anti-RAGE Ab (10 mg/kg) 1 h before CLP, and then the rats were treated with ketamine (10 mg/kg, i.p.) or an equal volume of vehicle (NS) at 3 h and 12 h after CLP. BALF was collected at 24 h after CLP to analyze the inflammatory cytokines TNF-α (A), IL-1β (B), IL-6 (C),and HMGB1 (D). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05 vs. CLP, #P b 0.05 vs. CLP + anti-RAGE Ab + ketamine.
2.8. Quantitative real-time polymerase chain reaction analysis (real-time PCR) Total RNA was extracted from lung tissues using Trizol (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer's instructions and reverse transcribed with Moloney Murine Leukemia Virus reverse transcriptase (Promega, Madison, WI). Quantitative real-time polymerase chain reaction (Real-Time PCR) was performed using a LightCycler 2.0 Real-Time PCR System (Roche Applied Science, Indianapolis, IN). Complementary DNA was amplified using specific primers for inflammatory mediator's gene expression, and the results were normalized to β-actin gene expression. The relative mean fold change of inflammatory mediator's gene expression in the experimental group was calculated using the 2ΔΔCt method and compared with the control group [25]. 2.9. Determination of NF-κB nuclear translocation Alveolar macrophages (2 × 106/mL) were isolated from BALF of each group. The cells were then fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.5% Triton X-100 for 15 min. After 1 h incubation with blocking buffer (5% BSA in PBS), cells were incubated with anti-NF-κB/p65 antibody diluted at 1:100 in blocking buffer overnight at 4 °C, washed, and then incubated with FITC-conjugated anti-rabbit IgG diluted at 1:1000 in blocking buffer for 1 h. In order to identify the nucleus, the FITC-conjugated-labeled samples were counterstained with a 1:500 dilution of Hoechst for 2 min. Fluorescent images were observed under the Leica TCS SP2 laser scanning spectral confocal microscope (Leica, Germany). 2.10. Statistical analysis Values are presented as mean ± standard deviation (S.D.) from three independent experiments. Data were analyzed using one-way
analysis of variance (ANOVA) followed by Tukey's post hoc test in SPSS11.0 (Chicago, IL, USA). P b 0.05 was considered to be significant between groups. 3. Result 3.1. Effects of ketamine on the injury indicators of CLP/HMGB1-induced ALI in WT rats The lung W/D ratio and MPO activity were determined in lung tissue dissected at 24 h after CLP/HMGB1. As shown in Fig. 1 and Fig. 2, the lung W/D ratio and MPO activity in lung tissue increased significantly in the CLP/HMGB1 group compared with that of the control group. However, this increase was apparently reduced by ketamine (5, 7.5, and 10 mg/kg) (P b 0.05, Fig. 1A, B; Fig. 2A, B). The total protein concentration and the number of total cells, neutrophils, and macrophages in BALF were analyzed at 24 h after CLP/ HMGB1. As shown in Fig. 1 and Fig. 2, CLP/HMGB1 significantly increased the number of total cells, neutrophils and macrophages compared with that of the control group. However, treatment with ketamine (5, 7.5, and10mg/kg) significantly decreased the number of total cells, neutrophils, and macrophages (P b 0.05, Fig. 1C–F; Fig.2 C–F). 3.2. Effects of ketamine on the production of inflammatory cytokines in the BALF and lung tissue from WT rats with CLP/HMGB1-induced ALI The effect of ketamine on inflammatory cytokines production was analyzed at 24 h after CLP/HMGB1 by ELISA. Compared with the control group, the levels of inflammatory cytokines TNF-α, IL-1β, and IL-6 in BALF and lung tissue were significantly increased after CLP/HMGB1. Ketamine (5, 7.5, and10mg/kg) reduced inflammatory cytokines production compared with that of the CLP/HMGB1 group (P b 0.05, Fig. 3; Fig. 4).
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3.3. Effect of ketamine on histopathological changes in lung tissues in CLP/ HMGB1-induced ALI in WT rats Histologic assessment of the effects of ketamine on sepsis-induced lung inflammation and injury was performed at 24 h. Lungs (n = 10) from each experimental group were processed for histologic evaluation after H&E staining (magnification 200×). As shown in Fig. 5 and Fig. 6 the lung tissues from the control group showed normal histological features. Isolated lung tissues from the CLP/HMGB1 group showed characteristics of acute alveolar damage and acute inflammation. These features included congestion, accumulation of neutrophils, and markedly thickened alveolar walls. These changes were observed in all lung tissues from this group. Furthermore, isolated lung tissues in the ketamine-treated group showed that ketamine treatment prevented CLP/HMGB1-induced damage (Fig. 5; Fig. 6). 3.4. Effects of ketamine on the expression of HMGB1 and RAGE in the BALF and lung tissue from WT rats with CLP-induced ALI ELISA showed that HMGB1 protein levels in the BALF were significantly higher in the CLP group than in the control group. Ketamine treatment was found to significantly down-regulate the levels of
HMGB1 relative to the CLP group at 24 h (P b 0.05, Fig. 7A). However, ELISA showed that sRAGE protein levels in the BALF were significantly higher in the CLP group than in the control group. Ketamine treatment was not found to significantly regulate the levels of sRAGE relative to the CLP group at 24 h (P N 0.05, Fig. 7B). Western blot analysis and Real-Time PCR showed that the expression of HMGB1 and RAGE in lung tissues were significantly higher in the CLP group than in the control group. Ketamine treatment was found to significantly downregulate the expression of HMGB1 and RAGE relative to the CLP group at 24 h (P b 0.05,Fig. 7C–F). 3.5. Effect of anti-RAGE Ab on the production of inflammatory cytokines in the BALF from WT rats with CLP/HMGB1-induced ALI To clarify the role of RAGE signaling in the release of inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6, and HMGB1), rats were treated with an intraperitoneal injection of anti-RAGE Ab (10 mg/kg) 1 h before CLP/HMGB1, and then the rats were treated with ketamine (10 mg/kg, i. p.) or an equal vehicle (NS) at 3 h and 12 h after CLP/HMGB1. BALF was collected at 24 h after CLP/HMGB1 to analyze the inflammatory cytokines. We found that CLP/HMGB1-induced releases of inflammatory cytokines were significantly inhibited by both anti-RAGE Ab treatment
Fig. 9. Effect of anti-RAGE Ab on the production of inflammatory cytokines in the BALF from RAGE siRNA-transfected WT rats with HMGB1-induced ALI. Rats were treated with an intraperitoneal injection of anti-RAGE Ab (10 mg/kg) 1 h before HMGB1, and then the rats were treated with ketamine (10 mg/kg, i.p.) or an equal volume of vehicle (NS) at 3 h and 12 h after HMGB1; rats received intratracheal injection of 300 μL of NS or 300 μL (3 × 109 PFU) of RAGE siRNA. Seven days later, ketamine (10 mg/kg, i.p.) was administrated at 3 h and 12 h after HMGB1. BALF was collected at 24 h after HMGB1 to analyze the inflammatory cytokines TNF-α (A, B), IL-1β (C, D), and IL-6 (E, F). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05 vs. HMGB1, #P b 0.05 vs. HMGB1 + anti-RAGE Ab/RAGE siRNA + ketamine.
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Fig. 10. Effect of ketamine on the production of inflammatory cytokines in the BALF from RAGE siRNA-transfected WT rats with CLP-induced ALI. Rats received intratracheal injection of 300 μL of NS or 300 μL (3 × 109 PFU) of RAGE siRNA. Seven days later, ketamine (10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. BALF was collected at 24 h after CLP to analyze the inflammatory cytokines TNF-α (A), IL-1β (B), IL-6 (C),and HMGB1 (D). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05 vs. CLP, #P b 0.05 vs. CLP + RAGE siRNA + ketamine.
and ketamine treatment alone (*P b 0.05, Fig. 8; Fig. 9); furthermore, when both ketamine treatment and anti-RAGE Ab treatment were performed to the CLP/HMGB1-induced ALI in rats, the inflammatory mediators releasing was most strongly inhibited (#P b 0.05, Fig. 8; Fig. 9). These results implicate RAGE as a likely effector of ketamine-mediated inhibition of CLP/HMGB1-induced inflammatory signaling and the effects of ketamine treatment may be not completely removed by blocking RAGE signaling pathway.
siRNA. Seven days later, Ketamine (10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP/HMGB1. BALF was collected at 24 h after CLP/HMGB1 to analyze the inflammatory cytokines. We found that silencing RAGE with siRNA inhibited production of inflammatory cytokines in the BALF from rats with CLP/HMGB1-induced ALI (*P b 0.05, Fig. 9; Fig. 10), and the ketamine treatment strongly increased the inhibition effect of RAGE siRNA (#P b 0.05, Fig. 9; Fig. 10).
3.6. Effect of ketamine on the production of inflammatory cytokines in the BALF from RAGE siRNA-transfected WT rats with CLP/HMGB1 -induced ALI
3.7. Effect of ketamine on the activation of NF-κB in the lung tissue and alveolar macrophages from WT rats with CLP-induced ALI
To clarify the role of RAGE signaling in the release of inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6, and HMGB1), rats received intratracheal injection of 300 μL of NS or 300 μL (3 × 109 PFU) of RAGE
Our results showed that CLP-induced ALI in rats significantly increased IκB-α phosphorylation and nuclear NF-κB (p65) activation in lung tissues (P b 0.05, Fig. 11), and induced nuclear translocation of
Fig. 11. Effect of ketamine on the activation of NF-κB in the lung tissue from WT rats with CLP-induced ALI. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. Protein expression of NF-κB (p65) in the nucleus (A) and the cytosol (B) and p-IκB-α (C) were respectively measured by Western blot. The quantification of relative band intensities was determined by densitometry. The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05, #P b 0.01vs. CLP.
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Fig. 12. Effect of ketamine on the nuclear translocation of NF-κB (p65) in alveolar macrophages from WT rats with CLP-induced ALI. Ketamine (10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. Alveolar macrophages (2 × 106 cells/mL) were isolated from BALF of each group. Samples were prepared for confocal microscopy analysis. The values presented are the mean ± S.D. (n = 10 in each group).
NF-κB (p65) in alveolar macrophages from BALF (Fig. 12). Ketamine inhibited all the above effects (P b 0.05, Fig. 11; Fig. 12). 3.8. Effect of ketamine on the phosphorylation of the MAPK signaling pathway in the lung tissue from WT rats with CLP-induced ALI Because the MAPK signaling molecules investigated here are activated by phosphorylation, we detected the phosphorylated forms of ERK, p38 MAPK, and JNK by Western-blotting. After rats were treated with CLP for 24 h, the phosphorylation of ERK, p38 MAPK and JNK in the lung tissue from rats with CLP-induced ALI was significantly increased compared with their respective control groups. However,
Ketamine remarkably attenuated these effects in a concentrationdependent manner. These results suggest that the MAPK signaling pathway is involved in ketamine-mediated inhibition of CLP-induced ALI in rats (P b 0.05, Fig. 13). 3.9. Effects of ketamine on the injury indicators of CLP-Induced ALI in WT and RAGE−/− rats The lung W/D ratio and MPO activity were determined in lung tissue dissected at 24 h after CLP. As shown in Fig. 14, the lung W/D ratio and MPO activity in lung tissue increased significantly in the CLP group compared with that of the control group in WT and RAGE−/− rats. However,
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Fig. 13. Effect of ketamine on the phosphorylation of the MAPK signaling pathway in the lung tissue from WT rats with CLP-induced ALI. Ketamine (5, 7.5, and10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. Western blot analysis was subsequently used to detect the expression of ERK, p38 MAPK, and JNK. Phosphorylated proteins were also detected. The quantification of relative band intensities was determined by densitometry. The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05, #P b 0.01vs. CLP.
Fig. 14. Effects of ketamine on the injury indicators of CLP-induced ALI in WT and RAGE−/− rats. Ketamine (10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. The lung W/D ratio (A) and MPO activity (B) were determined in lung tissue dissected at 24 h after CLP. BALF was collected at 24 h after CLP to measure the total protein concentration (C) and the number of total cells (D), neutrophils (E), and macrophage (F). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05 vs. CLP in WT or RAGE−/− rats, #P b 0.05 vs. CLP + ketamine in WT rats.
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Fig. 15. Effects of ketamine on the production of inflammatory cytokines in the BALF and lung tissue from WT and RAGE−/− rats with CLP-induced ALI. Ketamine (10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. BALF and lung tissue were collected at 24 h after CLP to analyze the inflammatory cytokines TNF-α (A, B), IL-1β (C, D), and IL-6 (E, F). The values presented are the mean ± S.D. (n = 10 in each group). *P b 0.05 vs. CLP in WT or RAGE−/− rats, #P b 0.05 vs. CLP + ketamine in WT rats.
Fig. 16. Effect of ketamine on histopathological changes in lung tissues in CLP-induced ALI in WT and RAGE−/− rats. Ketamine (10 mg/kg, i.p.) was administrated at 3 h and 12 h after CLP. Lung tissues (n = 10) from each experimental group were processed for histological evaluation at 24 h after CLP. Representative histological changes of lung tissues obtained from WT and RAGE−/− rats of different groups. (A) Control group in WT rats, (D) control group in RAGE−/− rats, (B) CLP group in WT rats, (E) CLP group in RAGE−/− rats, (C) CLP + ketamine (10 mg/kg) group in WT rats, (F) CLP + ketamine (10 mg/kg) group in RAGE−/− rats (H&E staining, magnification 200×).
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this increase was apparently reduced by ketamine (10 mg/kg) (P b 0.05, Fig. 14A, B). Inhibition effects of ketamine in RAGE−/− rats had higher lung W/D ratio and MPO activity at 24 h of CLP when compared with those in WT rats (P b 0.05, Fig. 14A, B). The total protein concentration and the number of total cells, neutrophils, and macrophages in BALF were analyzed at 24 h after CLP. As shown in Fig. 14, CLP significantly increased the number of total cells, neutrophils and macrophages compared with that of the control group in WT and RAGE−/− rats. However, treatment with ketamine (10 mg/kg) significantly decreased the number of total cells, neutrophils, and macrophages (P b 0.05, Fig. 14C–F). Inhibition effects of ketamine in RAGE−/− rats had higher total protein concentrations and the number of total cells, neutrophils, and macrophages in BALF at 24 h of CLP when compared with those in WT rats (P b 0.05, Fig. 14C–F). 3.10. Effects of ketamine on the production of inflammatory cytokines in the BALF and lung tissue from WT and RAGE−/− rats with CLP-induced ALI The effect of ketamine on inflammatory cytokines production was analyzed at 24 h after CLP by ELISA. Compared with the control group, the levels of inflammatory cytokines TNF-α, IL-1β, and IL-6 in BALF and lung tissues were significantly increased after CLP in WT and RAGE−/− rats. Ketamine (10 mg/kg) reduced inflammatory cytokines production compared with that of the CLP group (P b 0.05, Fig. 15). Inhibition effects of ketamine in RAGE−/− rats had higher inflammatory cytokines levels in BALF and lung tissues at 24 h of CLP when compared with those in WT rats (P b 0.05, Fig. 15). 3.11. Effect of ketamine on histopathological changes in lung tissues in CLPInduced ALI in WT and RAGE−/− rats Histologic assessment of the effects of ketamine on sepsis-induced lung inflammation and injury was performed at 24 h. Lungs (n = 10) from each experimental group were processed for histologic evaluation after H&E staining (magnification 200×). As shown in Fig. 16, the lung tissues from the control group showed normal histological features. Isolated lung tissues from the CLP group showed characteristics of acute alveolar damage and acute inflammation. These changes were observed in all lung tissues from this group. Furthermore, isolated lung tissues in the ketamine-treated group showed that ketamine treatment prevented CLP-induced damage (Fig. 16). Inhibition effects of ketamine in RAGE−/− rats had severer histopathological changes in lung tissues at 24 h of CLP when compared with those in WT rats (Fig. 16). 4. Discussion In the present study, we investigate the underlying mechanisms of anti-inflammatory effect of ketamine and observe the effect of ketamine on HMGB1 and RAGE expression during CLP-induced ALI in rats. The results of this study have demonstrated that ketamine reduces pathological changes of rat with CLP-induced ALI and mitigates inflammatory responses in the alveolar space, such as neutrophil infiltration and edema formation. Meanwhile, ketamine treatments on CLP-induced ALI in rats downregulate HMGB1 and RAGE expression in the BALF and lung tissues, as well as production of IL-1β, IL-6, and TNF-α in the BALF and lung tissues. These findings suggest that ketamine could be protective against the development of ALI by inhibiting HMGB1 and its receptor-RAGE expression and subsequently reducing inflammatory mediators production. Our study also shows that the CLP-induced release of inflammatory mediators in BALF was suppressed by antiRAGE Ab and RAGE siRNA. Furthermore, treatment with ketamine significantly inhibited the phosphorylation of MAPK and the activation of NF-κB p65 in lung tissues. HMGB1, a nuclear non-histone DNA-binding protein, stabilizes nucleosome formation and acts as a transcription-factor-like protein that regulates the expression of several genes [26]. However, when released
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into the extracellular milieu, HMGB1 has recently been shown to be an important late mediator of endotoxin shock, intra-abdominal sepsis, and ALI, and a promising therapeutic target of severe sepsis [8,27,28]. Meanwhile, previous studies showed that lung HMGB1 levels increased in the development of CLP-induced ALI in rats [29], and neutralizing antibodies as well as siRNA targeting HMGB1 significantly improve sepsisinduced ALI [27,30]. In this study, to further investigate whether HMGB1 acts as a stimulating factor in the development of ALI, HMGB1 was used to induce ALI. We found that ketamine inhibited the production of inflammatory cytokines in the BALF and lung tissue from WT rats with HMGB1-induced ALI, and attenuated the injury indicators and histopathological changes in lung tissues in HMGB1-induced ALI in WT rats. Therefore, this study provides evidence that HMGB1 is a potential important mediator in the development of ALI, and ketamine act through inhibiting HMGB1 to attenuate ALI. RAGE, a transmembrane receptor of the immunoglobulin superfamily, play critical roles in the innate immune system by interacting with microbial pathogens as well as endogenous molecules released during sepsis [31]. RAGE has been proposed to be involved in the pathogenesis of sepsis due to its role in transmitting signals from pathogen substrates to activate cells during the onset and perpetuation of inflammation [31]. Ligation of RAGE on the cellular surface triggers a series of cellular signaling events, including the activation and translocation to the nucleus of transcription factor NF-κB, leading to the production of proinflammatory cytokines, chemokines, adhesion molecules and oxidative stress and causing inflammation [32]. Inhibition of the RAGE products increases survival in experimental models of severe sepsis [12]. In this study, to further investigate whether RAGE acts as its major mediator in the development of ALI, RAGE-deficient rat was used. We noted that the inhibition effects of ketamine in RAGE−/− rats had higher injury indicators and inflammatory cytokines levels in BALF and lung tissues at 24 h of CLP when compared with those in WT rats. Inhibition effects of ketamine in RAGE−/− rats had severer histopathological changes in lung tissues at 24 h of CLP when compared with those in WT rats. Therefore, this study provides evidence that RAGE is a potential important mediator in the development of ALI, and ketamine act through inhibiting RAGE to attenuate ALI. RAGE possesses a secretory isoform known as sRAGE, which maintains the extracellular ligand-binding domain but lacks the cytosolic and transmembrane domains [33]. sRAGE has the same ligand binding specificity and competes with cell-bound RAGE, serving as a decoy that abolishes cell activation [33]. sRAGE is up-regulated during LPSinduced lung injury, which was ameliorated by recombinant sRAGE [34]. sRAGE may be secreted as a decoy receptor and contribute to the suppression of excessive inflammatory response during ALI [34]. In this study, we found that sRAGE is up-regulated during CLP-induced ALI, which was not influenced by ketamine. Activation of the RAGE signaling pathways results in the activation of NF-κB to induce expression of pro-inflammatory cytokines [35,36]. Previous studies demonstrated a significant decrease in lung NF-κB activation as well as blockade of RAGE by soluble RAGE in mice following induction of lung injury by LPS [34]. Simultaneously, the activated RAGE also stimulates MAPK pathway, one of the most widespread mechanisms of cell regulation, leading to upregulation of pro-inflammatory mediators and cell death [37]. ERKs, p38 MAPK, and JNKs are three major groups of the MAPK family [38]. In CLP-induced ALI, activated MAPKs contribute to the inflammatory response following CLP [39]. In our current study, CLP significantly increased the activation of NF-κB as well as the phosphorylation of ERK, p38 MAPK, and JNK. These changes were attenuated by post-treatment with ketamine. Our results suggest that the anti-inflammatory response following ketamine post-treatment is associated with suppression of NF-κB and MAPK activation. In summary, the present study demonstrates that ketamine ameliorates CLP-induced ALI through suppression of HMGB1 and RAGEmediated inflammatory signaling pathways. Thus, ketamine might be
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useful as a potential therapeutic medication for preventing ALI and ARDS. Author contributions Kehan Li: conception, design, analysis and interpretation of data; writing the manuscript. Jianxue Yang: conception, design, analysis and interpretation of data; writing the manuscript. Xuechang Han: conception, design, analysis and interpretation of data; writing the manuscript. Conflicts of interest The authors report no proprietary or commercial interest in any product mentioned, or concept discussed, in this article. References [1] R.D. Ramnath, S.W. Ng, A. Guglielmotti, M. Bhatia, Role of MCP-1 in endotoxemia and sepsis, Int. Immunopharmacol. 8 (2008) 810–818. [2] J. Garnacho-Montero, T. Aldabo-Pallas, C. Garnacho-Montero, A. Cayuela, R. Jimenez, S. Barroso, et al., Timing of adequate antibiotic therapy is a greater determinant of outcome than are TNF and IL-10 polymorphisms in patients with sepsis, Crit. Care 10 (2006) R111. [3] Z. Hasan, K. Palani, M. Rahman, H. Thorlacius, Targeting CD44 expressed on neutrophils inhibits lung damage in abdominal sepsis, Shock 35 (2011) 567–572. [4] A.M. Van Nuffel, V. Sukhatme, P. Pantziarka, L. Meheus, V.P. Sukhatme, G. Bouche, Repurposing Drugs in Oncology (ReDO)-clarithromycin as an anti-cancer agent, ecancermedicalscience 9 (2015) 513. [5] E. Letsiou, S. Sammani, W. Zhang, T. Zhou, H. Quijada, L. Moreno-Vinasco, et al., Pathologic mechanical stress and endotoxin exposure increases lung endothelial microparticle shedding, Am. J. Respir. Cell Mol. Biol. 52 (2015) 193–204. [6] H. Ueno, T. Matsuda, S. Hashimoto, F. Amaya, Y. Kitamura, M. Tanaka, et al., Contributions of high mobility group box protein in experimental and clinical acute lung injury, Am. J. Respir. Crit. Care Med. 170 (2004) 1310–1316. [7] Q. Wang, X. Wu, X. Tong, Z. Zhang, B. Xu, W. Zhou, Xuebijing ameliorates sepsis-induced lung injury by downregulating HMGB1 and RAGE expressions in mice, Evid. Based Complement. Alternat. Med. 2015 (2015) 860259. [8] Y.M. Yao, Y.Y. Luan, Q.H. Zhang, Z.Y. Sheng, Pathophysiological aspects of sepsis: an overview, Methods Mol. Biol. 1237 (2015) 5–15. [9] Y. Zhou, Y. Li, T. Mu, HMGB1 neutralizing antibody attenuates cardiac injury and apoptosis induced by hemorrhagic shock/resuscitation in rats, Biol. Pharm. Bull. 38 (2015) 1150–1160. [10] S.Y. Wang, Z.J. Li, X. Wang, W.F. Li, Z.F. Lin, Effect of ulinastatin on HMGB1 expression in rats with acute lung injury induced by sepsis, Genet. Mol. Res. 14 (2015) 4344–4353. [11] H. Hu, C. Zhai, G. Qian, A. Gu, J. Liu, F. Ying, et al., Protective effects of tanshinone IIA on myocardial ischemia reperfusion injury by reducing oxidative stress, HMGB1 expression, and inflammatory reaction, Pharm. Biol. 53 (2015) 1752–1758. [12] E.C. Lutterloh, S.M. Opal, D.D. Pittman, J.C. Keith Jr., X.Y. Tan, B.M. Clancy, et al., Inhibition of the RAGE products increases survival in experimental models of severe sepsis and systemic infection, Crit. Care 11 (2007), R122. [13] R. Clynes, K. Herold, A.M. Schmidt, RAGE: exacting a toll on the host in response to polymicrobial sepsis and Listeria monocytogenes, Crit. Care 11 (2007) 183. [14] J. Xie, J.D. Mendez, V. Mendez-Valenzuela, M.M. Aguilar-Hernandez, Cellular signalling of the receptor for advanced glycation end products (RAGE), Cell. Signal. 25 (2013) 2185–2197. [15] D.J. Weber, Y.M. Allette, D.S. Wilkes, F.A. White, The HMGB1-RAGE inflammatory pathway: implications for brain injury-induced pulmonary dysfunction, Antioxid. Redox Signal. (2015).
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