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The ameliorating effect of cannabinoid type 2 receptor activation on brain, lung, liver and heart damage in cecal ligation and puncture-induced sepsis model in rats
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The ameliorating effect of cannabinoid type 2 receptor activation on brain, lung, liver and heart damage in cecal ligation and puncture-induced sepsis model in rats ⁎
Murat Çakıra, , Suat Tekinb, Aslı Okanc, Pınar Çakand, Züleyha Doğanyiğitc a
Department of Physiology, Faculty of Medicine, Yozgat Bozok University, Yozgat, Turkey Department of Physiology, Faculty of Medicine, Inonu University, Malatya, Turkey c Department of Histology and Embryology, Faculty of Medicine, Yozgat Bozok University, Yozgat, Turkey d Department of Physiology, Hamidiye Faculty of Medicine, Health Sciences University, Istanbul, Turkey b
A R T I C LE I N FO
A B S T R A C T
Keywords: Sepsis Cecal ligation and puncture Cannabinoid type 2 receptor JWH-133
Uncontrolled infection and increased inflammatory mediators might cause systemic inflammatory response. It is already known that Cannabinoid Type 2 (CB2) receptors, which are commonly expressed in immune cells and in many other tissues, have an effect on the regulation of immune response. In the present study of ours, the effects of CB2 receptor agonist JWH-133 was investigated on cecal ligation and puncture (CLP)-induced polymicrobial sepsis model in rats. In the present study, Sprague-Dawley rats were divided into 5 groups (i.e. the Sham, CLP, JWH-133 0.2 mg/kg, JWH-133 1 mg/kg, and JWH-133 5 mg/kg Groups). Except for the Sham Group, the CLPinduced sepsis model was applied to all groups. The histopathological damage in brain, lung, liver and, heart was examined and the caspase-3, p-NF-κB, TNF-α, IL-1β and IL-6 levels were determined immunohistochemically. The serum TNF-α, IL-1β, IL-6, IL-10 levels were examined with the ELISA Method. The JWH-133 treatment decreased the histopathological damage in brain, heart, lung, and liver and reduced the caspase-3, p-NF-κB, TNFα, IL-1β, IL-6 levels in these tissues. In addition to this, JWH-133 treatment also decreased the serum TNF-α, IL1β, IL-6 levels, which were increased due to CLP, and increased the anti-inflammatory cytokine IL-10 levels. In the present study, it was determined that the CB2 receptor agonist JWH-133 decreases the CLP-induced inflammation, and reduces the damage in brain, lung, liver and heart. Our findings show the therapeutic potential of the activation of CB2 receptors with JWH-133 in sepsis.
1. Introduction Sepsis and septic shock are major health problems that affect millions of people every year. Although antimicrobial therapy, fluid therapy, vasoactive medications, resuscitation and mechanical ventilation are carried out in sepsis, more than one quarter of the patients that suffer from sepsis die [1]. Uncontrolled infection and increased inflammatory mediators might cause systemic inflammatory response [2]. In sepsis, exaggerated inflammatory response results in the production of proinflammatory cytokines, chemokines and other inflammatory mediators [3]. The extreme increase in the levels of inflammatory mediators causes sepsis, which is characterized with severe hypotension and multiple organ failure [4,5]. The production of the cytokines like sepsis-induced tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6) results in further increase of the inflammatory response [2,6]. Nuclear factor κB (NF-κB) activation, ⁎
which plays key roles in the transcription of inflammatory cytokine genes, triggers the inflammation cascade [7,8]. The endocannabinoid system consists of endogenous ligands, Cannabinoid Type 1 (CB1), Cannabinoid Type 2 (CB2) receptors, and the enzymes that perform the endocannabinoid synthesis and degradation [9]. While CB1 receptors are predominantly expressed in the Central Nervous System, CB2 receptors are mostly expressed in peripheral tissue [10]. It is already known that CB2 receptors have broad expression in immune cells, and have effects on the regulation of the immune response [11]. The psychoactive effect and abuse potential of CB1 receptor agonists limit their therapeutic potentials in clinical practice. The lack of psychoactive effect of CB2 agonists is an advantage for using them in treating inflammatory and autoimmune diseases [12]. It was shown in previous studies that the activation of CB2 receptors inhibit pro-inflammatory cytokine/chemokine production, and increase the production of anti-inflammatory cytokines [9]. We believe that
Corresponding author. E-mail address: [email protected] (M. Çakır).
https://doi.org/10.1016/j.intimp.2019.105978 Received 16 August 2019; Received in revised form 10 September 2019; Accepted 13 October 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Murat Çakır, et al., International Immunopharmacology, https://doi.org/10.1016/j.intimp.2019.105978
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2.3. Histologic analysis
JWH-133, which is an agonist of CB2 receptors whose anti-inflammatory effects have been reported, can alleviate the inflammation that is caused by sepsis, and cure tissue damage. For this reason, the purpose of the present study was to examine the potential protective effects of JWH-133 in cecal ligation and puncture (CLP)-induced polymicrobial sepsis model.
After male rats were sacrificed, the liver, lung, heart and brain tissues were removed. For routine paraffin embedding, samples were fixed in 10% formalin for 24 h. Then tissues were dehydrated through a graded series of ethanol, cleared with xylene, and finally, embedded in paraffin. Following paraffin embedding, paraffin sections at 5 μm thickness were stained with Harris hematoxylin and eosin (H&E) and were examined under light microscopy (Olympus BX53) for histopathologic evaluation. We evaluated hyperemia/congestion, intra-alveolar hemorrhage, cellular infiltration and cellular abnormal proliferation in lung tissue [14]; numerous damaged hepatocytes with vacuoles and pyknotic, sinusoidal dilatation and congestion and infiltration of inflammatory cells in the venules and sinusoids in liver tissue [15]; increase cell gap, hemorrhage, inflammatory cell infiltration and cell degeneration in heart tissue [16]; neuron degeneration, pericellular/perivascular edema at cerebral cortex and hippocampus in brain tissue [17]. Histopathologic results in each category were scored as follows: 0 = none, 1 = mild, 2 = moderate and 3 = severe.
2. Methods All the procedures in this study were carried out in line with the experimental protocol that was approved by the Ethical Committee on Animal Research of Inonu University (no: 2018/A-24). A total of 45 male Sprague-Dawley rats that were within the 250–320 g were obtained from Inonu University, Experimental Animals Reproduction and Research Center. The rats were kept under conditions that had 12-hr light and 12-hr dark cycle at a temperature of 21 ± 2 °C. The experimental animals were fed ad libitum with commercial standard pellet feeds.
2.4. Immunohistochemical analysis 2.1. Forming the CLP-induced experimental sepsis model The Avidin-Biotin-peroxidase method was used to detect the immunoreactivity of active caspase 3, TNF-α, phospho-NFκB-p65 (Ser536), IL-6 and IL-1β (Elabscience, Wuhan, China) in the lung, liver, heart and brain tissues. Briefly, after deparaffinization of 5 μm sections, a citrate buffer (pH:6.0) was used for antigen retrieval. Then, the slides were placed in 3% hydrogen peroxide in methanol to block endogenous peroxidase. Ultra V block was applied to block non-specific staining. After removing the blocking solution, the slides were incubated with primary antibodies caspase 3, TNF-α, phospho-NFκB-p65 (Ser536), IL-6 and IL-1β overnight at 4 °C. Biotinylated secondary streptavidin-HRP and DAB chromogens were applied respectively and then the slides were counterstained with Gill Hematoxylin, dehydrated, and mounted in entellan. The sections were examined by Olympus BX53 light microscope. The evaluation of the immunoreactivity levels was performed by the Image J program. For each slide, 10 different areas were evaluated from each group.
In the present study of ours, the CLP experimental model that was introduced by Rittirsch et al. was used [13]. The rats were anesthetized intraperitoneally with 70 mg/kg Ketamine (Ketalar, Eczacıbaşı, Istanbul, Turkey), and 8 mg/kg xylazine (Rompun, Bayer, Istanbul, Turkey). The abdominal region was shaved and cleaned with povidone iodine solution. The organs were reached after the abdominal region was entered by opening the peritoneum with a 1-cm incision from the midline of the abdomen, and then by opening the peritoneum. The cecum was isolated, and ligated with 4/0 silk ligature at the distal area of the ileo cecal valve. The cecum was punctured twice (in total, 4 punctures) via a 21-gauge needle in a distal way across the ligation area to pass to the opposite side of the mesentery. Then, the cecum was placed in the peritoneal cavity, and the areas that were incised were closed by using 4/0 sterile synthetic absorbable sutures. After the surgical procedures were completed, 1-ml saline injection was administered subcutaneously to all animals.
2.5. Statistical analyses The SPSS 20 (Inc. Chicago, IL, USA) Package Program was used for the analyses of the data. Whether the data showed normal distribution was tested according to the Kolmogorov-Smirnov Test. After it was determined that the data had normal distribution, the One-Way ANOVA test was used for statistical analyses. The Post-hoc Tukey Test was used for the comparisons among the groups. Arithmetic mean ± Standard Deviation (SD) was used for the biochemical data. Arithmetic mean ± Standard error of the mean (SEM) was used for the histopathological and immunohistochemical data. A P < 0.05 was considered to be statistically significant.
2.2. The design of the experimental groups The SHAM Group (n = 9): Without applying CLP, the cecum was reached with an incision through the abdomen area. After the cecum was manipulated, the incision area was closed. The rats were decapitated after 24 h. The CLP Group (n = 9): After induction of CLP, vehicle injection was made intraperitoneally. The JWH-133 0.2 mg/kg Group (n = 9): JWH-133 (Biorbyt, California, the USA) was dissolved in DMSO and diluted with 99% phosphate buffer. After induction of CLP immediately, 0.2 mg/kg JWH133 was administered intraperitoneally. The JWH-133 1 mg/kg Group (n = 9): After induction of CLP immediately, 1 mg/kg JWH-133 was administered intraperitoneally. The JWH-133 5 mg/kg Group (n = 9): After induction of CLP immediately, 5 mg/kg JWH-133 was administered intraperitoneally. After 24 h of the CLP induction, all the animals were decapitated under anesthesia. Brain, lung, liver, and heart tissues of the rats were taken to 10% formaldehyde solution for histopathological and immunohistochemical analyses. Care was taken to carry out these steps in a fast way. The serum was obtained from the bloods of the rats. By using commercial kits, the TNF-α, IL-1β, IL-6 and interleukin-10 (IL-10) (Thermo Fisher Scientific, Waltham, MA, USA) levels were measured from the sera with the Enzyme-Linked Immuno Sorbent Assay (ELISA) Method according to the kit protocols.
3. Results 3.1. Histopathological evaluation To elucidate the effects of different doses of JWH-133 on septic tissues (lung, liver, heart and brain), severity of the injury was scored as described in the material methods. According to the histological evaluation of lung samples; histological injury like severe congestion, interalveolar hemorrhage and neutrophil infiltrations were observed mostly in the CLP group than the JWH-133 treated groups (Fig. 1A). Among groups, in the JWH-133 0,2, 1 and 5 mg/kg groups, severity of lung injury was lower than CLP (P < 0.05). And JWH-133 5 mg/kg groups’ injury score was lower than JWH-133 0,2 and 1 mg/kg (P < 0.05). Although there was no statistically significant difference 2
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Fig. 1. (A) Histopathologic evaluation of the lung, liver, heart, cerebral cortex and hippocampal areas of the brain in rats by H&E staining method. Representative section images for sham, CLP, JWH-133 0,2 mg/kg, JWH-133 1 mg/kg and JWH-133 5 mg/kg treatment groups are shown at X40 magnification and bar = 20 μm. In the lung, blue arrow: neutrophils; astral: hyperemia/congestion, intra-alveolar hemorrhage. In the liver, yellow arrow: damaged hepatocytes; astral: sinusoidal dilatation and congestion; blue arrow: neutrophils. In the heart, black arrow: cell gap; yellow arrow: cell degeneration; astral: hemorrhage. In the brain, yellow arrow: neuron degeneration; astral: vascular congestion. (B) Histological damage scores of the lung, liver, heart, cerebral cortex and hippocampal areas of the brain in each experimental group were quantified as described in materials methods. Data are expressed as mean ± SEM and compared by one-way ANOVA and TUKEY’s multiple comparisons test (•P < 0.05 vs. sham group; ϕ P < 0.05 vs. CLP group; Ψ P < 0.05 vs. JWH-133 0,2 mg/kg group; δ P < 0.05 vs. JWH-133 1 mg/kg group). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 3
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groups (p < 0.05). The liver IL-6 expression level was decreased in JWH-133 1 and 5 mg/kg treated groups compared with the CLP and JWH-133 0,2 mg/kg treated groups (p < 0.05). Liver tissue TNF-α levels were significantly lower in all other groups compared to the CLP group (p < 0.05). The expression level of TNF-α, in the liver, significantly decreased in JWH-133 5 mg/kg treated groups compared with the CLP, JWH-133 0.2 and 1 mg/kg treated groups (p < 0.05). There was no difference between sham and JWH-133 5 mg/kg groups in terms of TNF-α expression level. Liver tissue IL-1β and p-NF-κB levels were significantly lower in all other groups compared to the CLP group (p < 0.05). Immunoreactivity of liver tissue IL-1β and p-NF-κB in JWH-133 5 mg/kg group was lower than JWH-133 0.2 and 1 mg/kg groups. As shown in Fig. 4, TNF-α, IL-1β, IL-6, caspase-3 and p-NF-κB levels in heart tissue were significantly increased in the CLP, JWH-133 0.2 mg/kg, JWH-133 1 mg/kg and JWH-133 5 mg/kg groups compared to the sham group (p < 0.05). IL-6, caspase-3 and p-NF-κB levels in heart tissue were significantly decreased in all JWH treated groups compared to the CLP group (p < 0.05). IL-1β and TNF-α levels were significantly decreased in heart tissue in JWH-133 1 mg/kg and JWH133 5 mg/kg groups compared to the CLP group. The levels of caspase3, IL-6, IL-1β and p-NF-κB decreased significantly in heart tissue in the JWH-133 5 mg/kg group compared to the JWH-133 0.2 mg/kg group (p < 0.05). There was a significant difference between JWH-133 0.2 mg/kg group and JWH-133 1 mg/kg group in terms of caspase-3 and IL-6 levels (p < 0.05). As shown in Fig. 5, TNF-α, IL-1β, IL-6, caspase-3 and p-NF-κB levels in the brain cortex were significantly higher in all other groups compared to the sham group (p < 0.05) (Fig. 5). TNF-α, IL-6, caspase-3 and p-NF-κB levels in the brain cortex decreased significantly in all groups treated with JWH-133 compared to the CLP group (p < 0.05). Brain cortex IL-1β level decreased in JWH-133 1 mg/kg and JWH-133 5 mg/kg groups compared to the CLP group (p < 0.05). Brain cortex tissue of the JWH-133 1 mg/kg and JWH-133 5 mg/kg groups TNF-α, IL-1β, IL-6, caspase-3 and p-NF-κB levels were lower than JWH-133 0.2 mg/kg group (p < 0.05). As shown in Fig. 6, TNF-α, IL-1β, IL-6, caspase-3 and p-NF-κB levels in hippocampus tissue increased in the CLP group compared to the sham group (p < 0.05) (Fig. 6). In all doses, JWH-133 administration significantly reduced the levels of TNF-α, IL-1β, IL-6, caspase-3 and pNF-κB in the hippocampus compared to the CLP group (p < 0.05). The levels of hippocampus Caspase-3 were significantly lower in both JWH133 1 mg/kg and JWH-133 5 mg/kg groups compared to the JWH-133 0.2 mg/kg group (p < 0.05). The level of IL-1β in the JWH-133 5 group was lower than the JWH-133 0.2 group (p < 0.05). IL-6 levels of hippocampus tissue were lower in the JWH-133 1 mg/kg group compared to the JWH-133 0,2 mg/kg group (p < 0.05). As a result, compared with the sham group, expressions of caspase 3, IL-6, TNF-α, IL-1β and p-NF-κB were significantly increased in the CLP group, whereas this increased expression level was significantly reduced by JWH-133 at a dose-dependent manner (P < 0.05) (Figs. 2–6).
between the sham and JWH-133 5 mg/kg groups in terms of histological score, a statistically significant difference was found in the groups of JWH-133 0,2 and JWH-133 1 mg/kg compared to the sham group (Fig. 1B). When we evaluated the liver samples, we observed that damaged hepatocytes, increased sinusoidal dilatation and infiltration of neutrophils were mostly in the CLP group (p < 0.05) (Fig. 1A). According to our observation, histological damage score of liver was higher in all treatment groups than sham (P < 0.05). The histopathological scores of the JWH-133 1 and 5 mg/kg groups were lower than those of the CLP and JWH-133 0.2 mg/kg groups (P < 0.05) (Fig. 1B). Increased cell gap, hemorrhage, inflammatory cell infiltration and cell degeneration in heart tissue were higher in the CLP, JWH-133 0,2 and 1 mg/kg treated groups than the sham and JWH-133 5 mg/kg groups (P < 0.05) (Fig. 1A). Histopathological damage was significantly decreased in the JWH-133 0.2 and JWH-133 1 mg/kg groups in the heart compared to the CLP group (p < 0.05). Reduced cell gap and hemorrhage and observed normal cells were seen markedly in JWH-133 5 mg/kg groups (Fig. 1B). In terms of groups histological damage score was the same in both brain regions (cerebral cortex and hippocampus). The severity of neuron degeneration and pericellular/perivascular edema score of both brain regions were higher in the CLP, JWH-133 0,2, JWH-133 1 and JWH-133 5 mg/kg groups compared with the sham (P < 0.05) (Fig. 1A). JWH-133 1 mg/kg group damage score was lower than the CLP; and JWH-133 5 mg/kg groups’ damage score was lower than the CLP and JWH-133 0,2 groups (P < 0.05) (Fig. 1B). As a result, as shown in Fig. 1A, serious pathological changes observed in the CLP group improved with the administration of increasing doses of JWH133. Compared with the sham group, all tissues examined in the CLP group were found to have significantly high histological damage score, whereas JWH treated groups showed improvement score with increasing dose of JWH-133 (Fig. 1B). 3.2. Immunohistochemical findings As shown in Fig. 2, in the lung samples, compared with the CLP group, the expression level of TNF-α, IL-1β and phospho-NFκB (p-NFκB) were statistically decreased in all treatment groups (P < 0.05). Among groups, in the lung, the expression level of caspase 3 was not different between the CLP and JWH-133 0,2 mg/kg groups, whereas it was different in JWH-133 1 mg/kg group compared to the CLP (P < 0.05). And also, in JWH-133 5 mg/kg, it was different compared to the CLP and JWH-133 0,2 mg/kg groups. Lung tissue IL-6 levels were significantly higher in all other groups compared to the sham group (p < 0.05). The expression level of IL-6 was higher in the lung of the CLP group than in the JWH-133 1 and 5 mg/kg groups (p < 0.05). However, there was no statistically significant difference between the CLP and JWH-133 0.2 mg/kg groups. TNF levels in lung tissue were significantly higher in all other groups compared to the sham group (p < 0.05). TNF-α expression levels were higher in the lung of CLP compared to all JWH-133 treatment groups. Among JWH-133 treatment groups, in JWH-133 1 and 5 mg/kg treated groups, TNF-α expression levels were lower compared to JWH-133 0,2 mg/kg group (p < 0.05). Finally, the lung tissue p-NF-κB level was significantly higher in all other groups compared to the sham group. The p-NF-κB expression in the lung of CLP group was higher compared with the JWH-133-treated groups (p < 0.05). At the same time, the p-NF-κB level was significantly lower in the JWH-133 5 mg/kg group compared to the JWH-133 0.2 and 1 mg/kg groups (p < 0.05). As shown in Fig. 3, liver tissue caspase-3 levels were significantly higher in the CLP, JWH-133 0.2 and JWH-133 1 mg/kg groups compared to the sham group (p < 0.05). The expression level of caspase 3 in liver tissues was lower in all groups treated with JWH-133 compared to the CLP group (p < 0.05). In addition, the level of caspase-3 in the JWH-133 5 mg group was lower than in the JWH-133 0.2 and 1 mg/kg
3.3. Serum cytokine levels The serum TNF-α, IL-1β, IL-6 and IL-10 levels are shown in Fig. 7. In our study, compared to the Sham Group, the serum IL-1β and IL-10 levels increased at a significant level (P < 0.05) in all groups to which the CLP and JWH-133 treatment was applied. The serum TNF-α and IL6 levels increased in the CLP and JWH-133 0.2 mg/kg Group at a significant level compared to the Sham Group (P < 0.05). The serum TNF-α, IL-1β and IL-6 levels decreased at a significant level in the JWH133 1 mg/kg and JWH-133 5 mg/kg Groups when compared to the CLP Group (P < 0.05). The level of IL-10, which is an anti-inflammatory cytokine, increased in the JWH-133 1 mg/kg and JWH-133 5 mg/kg Groups when compared to the CLP Group (P < 0.05). 4
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Fig. 2. (A) Caspase 3, IL-6, TNF-α, IL-1β and p-NF-κB images of immunostaining in rat lungs. Representative section images for sham, CLP, JWH-133 0,2 mg/kg, JWH-133 1 mg/kg and JWH-133 5 mg/kg treatment groups are shown at X40 magnification and bar = 20 μm. (B) Histograms represent the intensity values in percent of immunostaining obtained using Image J software. The bar graph data are expressed as mean ± SEM, and compared by one-way ANOVA and TUKEY’s multiple comparisons test (•P < 0.05 vs. sham group; ϕ P < 0.05 vs. CLP group; Ψ P < 0.05 vs. JWH-133 0,2 mg/kg group; δ P < 0.05 vs. JWH-133 1 mg/kg group).
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Fig. 3. (A) Caspase 3, IL-6, TNF-α, IL-1β and p-NF-κB images of immunostaining in rat livers. Representative section images for sham, CLP, JWH-133 0,2 mg/kg, JWH-133 1 mg/kg and JWH-133 5 mg/kg treatment groups are shown at X40 magnification and bar = 20 μm. (B) Histograms represent the intensity values in percent of immunostaining obtained using Image J software. The bar graph data are expressed as mean ± SEM, and compared by one-way ANOVA and TUKEY’s multiple comparisons test (•P < 0.05 vs. sham group; ϕ P < 0.05 vs. CLP group; Ψ P < 0.05 vs. JWH-133 0,2 mg/kg group; δ P < 0.05 vs. JWH-133 1 mg/kg group).
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Fig. 4. (A) Caspase 3, IL-6, TNF-α, IL-1β and p-NF-κB images of immunostaining in rat hearts. Representative section images for sham, CLP, JWH-133 0,2 mg/kg, JWH-133 1 mg/kg and JWH-133 5 mg/kg treatment groups are shown at X40 magnification and bar = 20 μm. (B) Histograms represent the intensity values in percent of immunostaining obtained using Image J software. The bar graph data are expressed as mean ± SEM, and compared by one-way ANOVA and TUKEY’s multiple comparisons test (•P < 0.05 vs. sham group; ϕ P < 0.05 vs. CLP group; Ψ P < 0.05 vs. JWH-133 0,2 mg/kg group; δ P < 0.05 vs. JWH-133 1 mg/kg group).
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Fig. 5. Caspase 3, IL-6, TNF-α, IL-1β and p-NF-κB images of immunostaining in the cerebral cortex of rat brains. Representative section images for sham, CLP, JWH133 0,2 mg/kg, JWH-133 1 mg/kg and JWH-133 5 mg/kg treatment groups are shown at X40 magnification and bar = 20 μm. (B) Histograms represent the intensity values in percent of immunostaining obtained using Image J software. The bar graph data are expressed as mean ± SEM, and compared by one-way ANOVA and TUKEY’s multiple comparisons test (•P < 0.05 vs. sham group; ϕ P < 0.05 vs. CLP group; Ψ P < 0.05 vs. JWH-133 0,2 mg/kg group; δ P < 0.05 vs. JWH-133 1 mg/kg group).
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Fig. 6. Caspase 3, IL-6, TNF-α, IL-1β and p-NF-κB images of immunostaining in the hippocampus of rat brains. Representative section images for sham, CLP, JWH133 0,2 mg/kg, JWH-133 1 mg / kg and JWH-133 5 mg / kg treatment groups are shown at X40 magnification and bar = 20 μm. (B) Histograms represent the intensity values in percent of immunostaining obtained using Image J software. The bar graph data are expressed as mean ± SEM, and compared by one-way ANOVA and TUKEY’s multiple comparisons test (•P < 0.05 vs. sham group; ϕ P < 0.05 vs. CLP group; Ψ P < 0.05 vs. JWH-133 0,2 mg/kg group; δ P < 0.05 vs. JWH-133 1 mg/kg group).
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Fig. 7. Serum IL-6, TNF-α, IL-1β and IL10 levels (A–D). JWH-133 treatment at doses of 1 mg/kg and 5 mg/kg decreased serum IL-6, TNF-α, IL-1β levels compared to CLP group and increased IL-10 levels. Data are expressed as mean ± SD and compared by one-way ANOVA and TUKEY’s multiple comparisons test (•P < 0.05 vs. sham group; ϕ P < 0.05 vs. CLP group).
applied. Parallel to this finding, JWH-133 application also decreased the TNF-α, IL-1β and IL-6 levels in the same tissues. It was reported in previous studies that the activation of caspase-3, which plays a central role in the programmed cell death in sepsis, is associated with mortality. In experimental sepsis models, the caspase-3 activity increased in many tissues [26,27]. In our study, in the CLP group, increased caspase3 levels decreased with JWH-133 administration. We found similar findings in the histopathological examination. The levels of TNF-α, IL1β, IL-6, which are among proinflammatory cytokines, decreased in the serum of the groups that were treated with JWH-133. The IL-10 is an anti-inflammatory cytokine which plays important roles in negative regulation of immune response [28]. In our study, the JWH-133 administration increased the serum IL-10 levels. According to our findings, JWH-133 showed its effects in a dose-dependent way. We observed that the most effective dose was 5 mg/kg. CB2 receptor agonists have therapeutic potentials for many diseases which include sepsis [29]. Lehmann et al. conducted a study and reported that although CB2 agonist HU308 reduced the systemic inflammation and Leukocyte adhesion, the CB2 antagonist AM630 did not show the same effect [30]. In another study, it was reported that the administration of CB2 agonist Gp1a in CLP-induced sepsis model increased survival and decreased bacteremia, IL-6 levels and lung injury. In the same study, bacteremia, IL-6 levels, and lung injury increased in CB2-knock-out (KO) mice that were exposed to CLP, and the survival decreased [31]. In their study, Gui et al. reported that the administration of CB2 agonist GW405833 increased survival and decreased serum proinflammatory cytokine levels in LPS-treated mice [32]. JWH-133 is a selective CB2 receptor agonist [33]. The number of studies that used JWH-133 in experimental sepsis models is limited. Gamal et al. showed in their study that the administration of JWH-133 in LPS-induced endotoxemia reduced IL-6, vascular cell adhesion molecule (VCAM), E-selectin levels in the brain and serum, and
4. Discussion In our study, JWH-133, which is a CB2 receptor agonist, decreased the proinflammatory cytokine level by inhibiting the NF-κB activity in the CLP-induced sepsis model. It also reduced the damage in brain, heart, liver and lung. Although there were extensive investigations in the past, sepsis is still an important health problem whose pathophysiology has not yet been fully elucidated. The incidence of sepsis, which can lead to multiple organ failure, is increasing with each passing day [18,19]. In the past, several animal models were developed that mimicked the pathophysiological changes in septic patients to understand the underlying mechanisms of sepsis and systemic inflammatory response [20]. The CLP is a realistic model, which is used to establish polymicrobial sepsis in an empirical way to examine the underlying mechanisms of sepsis [13]. The activation of the NF-κB in cells causes the production of various inflammatory mediators such as TNF-α, IL-1β, IL-6 by triggering the inflammatory cascade [21,22]. The phosphorylation of NF-κB controls its activation. NF-κB has different subunits. The p65 is the most prominent NF-κB subunit in NF-κB phosphorylation studies conducted so far. Many studies focused on S536, which is one of the best understood phosphorylation targets in p65 [23]. The p65 (ser536) plays key roles in inflammatory events in CLP-induced sepsis [24]. In our study, a parallelism was detected between the NF-κB p65 (ser536) and pro-inflammatory cytokine immunoreactivity in the brain, lung, liver and heart tissues. In sepsis, high NF-κB activation level is associated with higher mortality and worse clinical outcomes. In sepsis, the inhibition of NF-κB signal prevents the expression of multiple proinflammatory genes and multiple organ injury. It also prolongs the survival [25]. In our study, p-NF-κB immunoreactivity decreased in lung, heart, liver, brain cortex and hippocampus of the groups to which JWH-133 was
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endotoxemia-associated brain dysfunction [34]. In the literature review, no studies were detected on the effects of JWH-133 in CLP-induced sepsis models. Sun et al. showed that JWH-133 reduced the surgery-induced increased IL-1β, TNF-α, Monocyte chemotactic protein (MCP-1) levels, and alleviated the neuroinflammation in the brain cortex and in the hippocampus [35]. In a study in the stroke model, JWH-133 reduced IL-6, IL-1β, TNF-α levels and infarct volume in the brain, while the administration of CB2 antagonist prevented these effects [36]. In another study, JWH-133 reduced the immune cell infiltration in the brain in a cerebral ischemia model [37]. Tomar et al. reported that JWH-133 had a reducing effect on liver damage in galactosamine/lipopolysaccharide-induced acute liver failure [38]. In another study, it was shown that JWH-133 suppressed the NF-kB activation in paraquat-induced acute lung injury, decreased TNF-α and IL1β, and prevented lung injury [39]. Montecucco et al. showed that JWH-133 administration reduced leukocyte infiltration, inflammatory mediator level, and infarct volume in cardiac ischemia [40].
tilting toward immunosuppression, Nat. Med. 15 (5) (2009) 496–497. [6] J.A. Smith, P.R. Mayeux, R.G. Schnellmann, Delayed mitogen-activated protein kinase/extracellular signal-regulated kinase inhibition by Trametinib attenuates systemic inflammatory responses and multiple organ injury in murine sepsis, Crit. Care Med. 44 (8) (2016) e711–e720. [7] K. Zhang, X.F. Jiao, J.X. Li, X.W. Wang, Rhein inhibits lipopolysaccharide-induced intestinal injury during sepsis by blocking the toll-like receptor 4 nuclear factorkappaB pathway, Mol. Med. Rep. 12 (3) (2015) 4415–4421. [8] A. Tanyeli, E. Eraslan, M.C. Guler, S.O. Sebin, D. Celebi, F.B. Ozgeris, E. Toktay, Investigation of biochemical and histopathological effects of tarantula cubensis D6 on lung tissue in cecal ligation and puncture-induced polymicrobial sepsis model in rats, Medicine (2019). [9] Q. He, F. Xiao, Q. Yuan, J. Zhang, J. Zhan, Z. Zhang, Cannabinoid receptor 2: a potential novel therapeutic target for sepsis? Acta Clin. Belg. 74 (2) (2019) 70–74. [10] R.P. Picone, D.A. Kendall, Minireview: from the bench, toward the clinic: therapeutic opportunities for cannabinoid receptor modulation, Mol. Endocrinol. 29 (6) (2015) 801–813. [11] K.R. Kasten, J. Tschop, M.H. Tschop, C.C. Caldwell, The cannabinoid 2 receptor as a potential therapeutic target for sepsis, Endocr. Metab. Immune. Disord. Drug. Targets. 10 (3) (2010) 224–234. [12] R. Mechoulam, L.A. Parker, The endocannabinoid system and the brain, Annu. Rev. Psychol. 64 (2013) 21–47. [13] D. Rittirsch, M.S. Huber-Lang, M.A. Flierl, P.A. Ward, Immunodesign of experimental sepsis by cecal ligation and puncture, Nat. Protocols 4 (1) (2009) 31–36. [14] Y. Hirano, M. Aziz, W.L. Yang, Z. Wang, M. Zhou, M. Ochani, A. Khader, P. Wang, Neutralization of osteopontin attenuates neutrophil migration in sepsis-induced acute lung injury, Crit. Care 19 (2015) 53. [15] A. Dadkhah, F. Fatemi, A. Rasooli, M.R. Mohammadi Malayeri, F. Torabi, Assessing the effect of Mentha longifolia essential oils on COX-2 expression in animal model of sepsis induced by caecal ligation and puncture, Pharm. Biol. 56 (1) (2018) 495–504. [16] J. Liu, J. Li, P. Tian, B. Guli, G. Weng, L. Li, Q. Cheng, H2S attenuates sepsis-induced cardiac dysfunction via a PI3K/Akt-dependent mechanism, Exp. Ther. Med. 17 (5) (2019) 4064–4072. [17] D. Arslan, A. Ekinci, A. Arici, E. Bozdemir, E. Akil, H.H. Ozdemir, Effects of Ecballium elaterium on brain in a rat model of sepsis-associated encephalopathy, Libyan J. Med. 12 (1) (2017) 1369834. [18] G.S. Martin, D.M. Mannino, S. Eaton, M. Moss, The epidemiology of sepsis in the United States from 1979 through 2000, N. Engl. J. Med. 348 (16) (2003) 1546–1554. [19] D.F. Gaieski, J.M. Edwards, M.J. Kallan, B.G. Carr, Benchmarking the incidence and mortality of severe sepsis in the United States, Crit. Care Med. 41 (5) (2013) 1167–1174. [20] D. Rittirsch, L.M. Hoesel, P.A. Ward, The disconnect between animal models of sepsis and human sepsis, J. Leukoc Biol. 81 (1) (2007) 137–143. [21] P.N. Moynagh, The NF-kappaB pathway, J. Cell Sci. 118 (Pt 20) (2005) 4589–4592. [22] T. Lawrence, The Nuclear Factor NF-kappa B Pathway in Inflammation, Cold Spring Harbor Perspect. Biol. 1 (6) (2009). [23] F. Christian, E.L. Smith, R.J. Carmody, The regulation of NF-kappaB subunits by phosphorylation, Cells 5 (1) (2016). [24] J. Walker, E. Dichter, G. Lacorte, D. Kerner, B. Spur, A. Rodriguez, K. Yin, Lipoxin a4 increases survival by decreasing systemic inflammation and bacterial load in sepsis, Shock 36 (4) (2011) 410–416. [25] S.F. Liu, A.B. Malik, NF-kappa B activation as a pathological mechanism of septic shock and inflammation, Am. J. Physiol. Lung Cell Mol. Physiol. 290 (4) (2006) L622–L645. [26] L. Lorente, M.M. Martin, J. Ferreres, J. Sole-Violan, L. Labarta, C. Diaz, A. Jimenez, J.M. Borreguero-Leon, Serum caspase 3 levels are associated with early mortality in severe septic patients, J. Crit. Care 34 (2016) 103–106. [27] C.M. Comim, T. Barichello, D. Grandgirard, F. Dal-Pizzol, J. Quevedo, S.L. Leib, Caspase-3 mediates in part hippocampal apoptosis in sepsis, Mol. Neurobiol. 47 (1) (2013) 394–398. [28] S. Rutz, W. Ouyang, Regulation of interleukin-10 expression, Adv. Exp. Med. Biol. 941 (2016) 89–116. [29] R.G. Pertwee, Targeting the endocannabinoid system with cannabinoid receptor agonists: pharmacological strategies and therapeutic possibilities, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 367 (1607) (2012) 3353–3363. [30] C. Lehmann, M. Kianian, J. Zhou, I. Kuster, R. Kuschnereit, S. Whynot, O. Hung, R. Shukla, B. Johnston, V. Cerny, D. Pavlovic, A. Spassov, M.E. Kelly, Cannabinoid receptor 2 activation reduces intestinal leukocyte recruitment and systemic inflammatory mediator release in acute experimental sepsis, Crit. Care 16 (2) (2012) R47. [31] J. Tschop, K.R. Kasten, R. Nogueiras, H.S. Goetzman, C.M. Cave, L.G. England, J. Dattilo, A.B. Lentsch, M.H. Tschop, C.C. Caldwell, The cannabinoid receptor 2 is critical for the host response to sepsis, J. Immunol. 183 (1) (2009) 499–505. [32] H. Gui, Y. Sun, Z.M. Luo, D.F. Su, S.M. Dai, X. Liu, Cannabinoid receptor 2 protects against acute experimental sepsis in mice, Mediators Inflamm. 2013 (2013) 741303. [33] J.W. Huffman, CB2 receptor ligands, Mini. Rev. Med. Chem. 5 (7) (2005) 641–649. [34] M. Gamal, J. Moawad, L. Rashed, W. El-Eraky, D. Saleh, C. Lehmann, N. Sharawy, Evaluation of the effects of Eserine and JWH-133 on brain dysfunction associated with experimental endotoxemia, J. Neuroimmunol. 281 (2015) 9–16. [35] L. Sun, R. Dong, X. Xu, X. Yang, M. Peng, Activation of cannabinoid receptor type 2 attenuates surgery-induced cognitive impairment in mice through anti-inflammatory activity, J. Neuroinflamm. 14 (1) (2017) 138. [36] J.G. Zarruk, D. Fernandez-Lopez, I. Garcia-Yebenes, M.S. Garcia-Gutierrez,
5. Conclusion To sum up, CB2 receptors modulate the immune response. CB2 receptor activation causes reductions in leukocyte recruitment, chemotaxis and in proinflammatory cytokine levels [11]. It was also shown in different experimental models that CB2 activation reduced organ damage. In this study, we found that JWH-133, which is a CB2 agonist, prevents brain, heart, lung and liver damage in CLP-induced sepsis in a dose-dependent manner. JWH-133 also reduced the proinflammatory cytokine levels through the inhibition of apoptosis and NF-κB. Our results show that activation of CB2 receptor with JWH-133 might be a potential therapeutic target in the treatment of inflammatory diseases like sepsis. Disclosure statement Authors declare no conflict of interest. Authors contributions M.Ç. projected and conducted the study, analyzed the data, and wrote the study. M.Ç. and S. T. dealt with the rats. Z. D. and A. O. O. performed histopathological and immunohistochemical analyzes. P. Ç. made ELISA analysis. Funding We were supported by Yozgat Bozok University, Department of Scientific Research Projects, Turkey (Project no: 6602c-TF/18-229). References [1] A. Rhodes, L.E. Evans, W. Alhazzani, M.M. Levy, M. Antonelli, R. Ferrer, A. Kumar, J.E. Sevransky, C.L. Sprung, M.E. Nunnally, B. Rochwerg, G.D. Rubenfeld, D.C. Angus, D. Annane, R.J. Beale, G.J. Bellinghan, G.R. Bernard, J.D. Chiche, C. Coopersmith, D.P. De Backer, C.J. French, S. Fujishima, H. Gerlach, J.L. Hidalgo, S.M. Hollenberg, A.E. Jones, D.R. Karnad, R.M. Kleinpell, Y. Koh, T.C. Lisboa, F.R. Machado, J.J. Marini, J.C. Marshall, J.E. Mazuski, L.A. McIntyre, A.S. McLean, S. Mehta, R.P. Moreno, J. Myburgh, P. Navalesi, O. Nishida, T.M. Osborn, A. Perner, C.M. Plunkett, M. Ranieri, C.A. Schorr, M.A. Seckel, C.W. Seymour, L. Shieh, K.A. Shukri, S.Q. Simpson, M. Singer, B.T. Thompson, S.R. Townsend, T. Van der Poll, J.L. Vincent, W.J. Wiersinga, J.L. Zimmerman, R.P. Dellinger, Surviving sepsis campaign: international guidelines for management of sepsis and septic shock: 2016, Intensive Care Med. 43 (3) (2017) 304–377. [2] B.G. Chousterman, F.K. Swirski, G.F. Weber, Cytokine storm and sepsis disease pathogenesis, Semin. Immunopathol. 39 (5) (2017) 517–528. [3] M. Aziz, A. Jacob, W.L. Yang, A. Matsuda, P. Wang, Current trends in inflammatory and immunomodulatory mediators in sepsis, J. Leukoc. Biol. 93 (3) (2013) 329–342. [4] R.C. Bone, C.J. Grodzin, R.A. Balk, Sepsis: a new hypothesis for pathogenesis of the disease process, Chest 112 (1) (1997) 235–243. [5] R.S. Hotchkiss, C.M. Coopersmith, J.E. McDunn, T.A. Ferguson, The sepsis seesaw:
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cannabinoid receptor 2 activation in galactosamine/lipopolysaccharide-induced acute liver failure through regulation of macrophage polarization and microRNAs, J. Pharmacol. Exp. Ther. 353 (2) (2015) 369–379. [39] Z. Liu, Y. Wang, H. Zhao, Q. Zheng, L. Xiao, M. Zhao, CB2 receptor activation ameliorates the proinflammatory activity in acute lung injury induced by paraquat, Biomed. Res. Int. 2014 (2014) 971750. [40] F. Montecucco, S. Lenglet, V. Braunersreuther, F. Burger, G. Pelli, M. Bertolotto, F. Mach, S. Steffens, CB(2) cannabinoid receptor activation is cardioprotective in a mouse model of ischemia/reperfusion, J. Mol. Cell Cardiol. 46 (5) (2009) 612–620.
J. Vivancos, F. Nombela, M. Torres, M.C. Burguete, J. Manzanares, I. Lizasoain, M.A. Moro, Cannabinoid type 2 receptor activation downregulates stroke-induced classic and alternative brain macrophage/microglial activation concomitant to neuroprotection, Stroke; J. Cerebral Circul. 43 (1) (2012) 211–219. [37] S. Murikinati, E. Juttler, T. Keinert, D.A. Ridder, S. Muhammad, Z. Waibler, C. Ledent, A. Zimmer, U. Kalinke, M. Schwaninger, Activation of cannabinoid 2 receptors protects against cerebral ischemia by inhibiting neutrophil recruitment, FASEB J. 24 (3) (2010) 788–798. [38] S. Tomar, E.E. Zumbrun, M. Nagarkatti, P.S. Nagarkatti, Protective role of
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
The ameliorating effect of cannabinoid type 2 receptor activation on brain, lung, liver and heart damage in cecal ligation and puncture-induced sepsis model in rats ⁎
Murat Çakıra, , Suat Tekinb, Aslı Okanc, Pınar Çakand, Züleyha Doğanyiğitc a
Department of Physiology, Faculty of Medicine, Yozgat Bozok University, Yozgat, Turkey Department of Physiology, Faculty of Medicine, Inonu University, Malatya, Turkey c Department of Histology and Embryology, Faculty of Medicine, Yozgat Bozok University, Yozgat, Turkey d Department of Physiology, Hamidiye Faculty of Medicine, Health Sciences University, Istanbul, Turkey b
A R T I C LE I N FO
A B S T R A C T
Keywords: Sepsis Cecal ligation and puncture Cannabinoid type 2 receptor JWH-133
Uncontrolled infection and increased inflammatory mediators might cause systemic inflammatory response. It is already known that Cannabinoid Type 2 (CB2) receptors, which are commonly expressed in immune cells and in many other tissues, have an effect on the regulation of immune response. In the present study of ours, the effects of CB2 receptor agonist JWH-133 was investigated on cecal ligation and puncture (CLP)-induced polymicrobial sepsis model in rats. In the present study, Sprague-Dawley rats were divided into 5 groups (i.e. the Sham, CLP, JWH-133 0.2 mg/kg, JWH-133 1 mg/kg, and JWH-133 5 mg/kg Groups). Except for the Sham Group, the CLPinduced sepsis model was applied to all groups. The histopathological damage in brain, lung, liver and, heart was examined and the caspase-3, p-NF-κB, TNF-α, IL-1β and IL-6 levels were determined immunohistochemically. The serum TNF-α, IL-1β, IL-6, IL-10 levels were examined with the ELISA Method. The JWH-133 treatment decreased the histopathological damage in brain, heart, lung, and liver and reduced the caspase-3, p-NF-κB, TNFα, IL-1β, IL-6 levels in these tissues. In addition to this, JWH-133 treatment also decreased the serum TNF-α, IL1β, IL-6 levels, which were increased due to CLP, and increased the anti-inflammatory cytokine IL-10 levels. In the present study, it was determined that the CB2 receptor agonist JWH-133 decreases the CLP-induced inflammation, and reduces the damage in brain, lung, liver and heart. Our findings show the therapeutic potential of the activation of CB2 receptors with JWH-133 in sepsis.
1. Introduction Sepsis and septic shock are major health problems that affect millions of people every year. Although antimicrobial therapy, fluid therapy, vasoactive medications, resuscitation and mechanical ventilation are carried out in sepsis, more than one quarter of the patients that suffer from sepsis die [1]. Uncontrolled infection and increased inflammatory mediators might cause systemic inflammatory response [2]. In sepsis, exaggerated inflammatory response results in the production of proinflammatory cytokines, chemokines and other inflammatory mediators [3]. The extreme increase in the levels of inflammatory mediators causes sepsis, which is characterized with severe hypotension and multiple organ failure [4,5]. The production of the cytokines like sepsis-induced tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6) results in further increase of the inflammatory response [2,6]. Nuclear factor κB (NF-κB) activation, ⁎
which plays key roles in the transcription of inflammatory cytokine genes, triggers the inflammation cascade [7,8]. The endocannabinoid system consists of endogenous ligands, Cannabinoid Type 1 (CB1), Cannabinoid Type 2 (CB2) receptors, and the enzymes that perform the endocannabinoid synthesis and degradation [9]. While CB1 receptors are predominantly expressed in the Central Nervous System, CB2 receptors are mostly expressed in peripheral tissue [10]. It is already known that CB2 receptors have broad expression in immune cells, and have effects on the regulation of the immune response [11]. The psychoactive effect and abuse potential of CB1 receptor agonists limit their therapeutic potentials in clinical practice. The lack of psychoactive effect of CB2 agonists is an advantage for using them in treating inflammatory and autoimmune diseases [12]. It was shown in previous studies that the activation of CB2 receptors inhibit pro-inflammatory cytokine/chemokine production, and increase the production of anti-inflammatory cytokines [9]. We believe that
Corresponding author. E-mail address: [email protected] (M. Çakır).
https://doi.org/10.1016/j.intimp.2019.105978 Received 16 August 2019; Received in revised form 10 September 2019; Accepted 13 October 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Murat Çakır, et al., International Immunopharmacology, https://doi.org/10.1016/j.intimp.2019.105978
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2.3. Histologic analysis
JWH-133, which is an agonist of CB2 receptors whose anti-inflammatory effects have been reported, can alleviate the inflammation that is caused by sepsis, and cure tissue damage. For this reason, the purpose of the present study was to examine the potential protective effects of JWH-133 in cecal ligation and puncture (CLP)-induced polymicrobial sepsis model.
After male rats were sacrificed, the liver, lung, heart and brain tissues were removed. For routine paraffin embedding, samples were fixed in 10% formalin for 24 h. Then tissues were dehydrated through a graded series of ethanol, cleared with xylene, and finally, embedded in paraffin. Following paraffin embedding, paraffin sections at 5 μm thickness were stained with Harris hematoxylin and eosin (H&E) and were examined under light microscopy (Olympus BX53) for histopathologic evaluation. We evaluated hyperemia/congestion, intra-alveolar hemorrhage, cellular infiltration and cellular abnormal proliferation in lung tissue [14]; numerous damaged hepatocytes with vacuoles and pyknotic, sinusoidal dilatation and congestion and infiltration of inflammatory cells in the venules and sinusoids in liver tissue [15]; increase cell gap, hemorrhage, inflammatory cell infiltration and cell degeneration in heart tissue [16]; neuron degeneration, pericellular/perivascular edema at cerebral cortex and hippocampus in brain tissue [17]. Histopathologic results in each category were scored as follows: 0 = none, 1 = mild, 2 = moderate and 3 = severe.
2. Methods All the procedures in this study were carried out in line with the experimental protocol that was approved by the Ethical Committee on Animal Research of Inonu University (no: 2018/A-24). A total of 45 male Sprague-Dawley rats that were within the 250–320 g were obtained from Inonu University, Experimental Animals Reproduction and Research Center. The rats were kept under conditions that had 12-hr light and 12-hr dark cycle at a temperature of 21 ± 2 °C. The experimental animals were fed ad libitum with commercial standard pellet feeds.
2.4. Immunohistochemical analysis 2.1. Forming the CLP-induced experimental sepsis model The Avidin-Biotin-peroxidase method was used to detect the immunoreactivity of active caspase 3, TNF-α, phospho-NFκB-p65 (Ser536), IL-6 and IL-1β (Elabscience, Wuhan, China) in the lung, liver, heart and brain tissues. Briefly, after deparaffinization of 5 μm sections, a citrate buffer (pH:6.0) was used for antigen retrieval. Then, the slides were placed in 3% hydrogen peroxide in methanol to block endogenous peroxidase. Ultra V block was applied to block non-specific staining. After removing the blocking solution, the slides were incubated with primary antibodies caspase 3, TNF-α, phospho-NFκB-p65 (Ser536), IL-6 and IL-1β overnight at 4 °C. Biotinylated secondary streptavidin-HRP and DAB chromogens were applied respectively and then the slides were counterstained with Gill Hematoxylin, dehydrated, and mounted in entellan. The sections were examined by Olympus BX53 light microscope. The evaluation of the immunoreactivity levels was performed by the Image J program. For each slide, 10 different areas were evaluated from each group.
In the present study of ours, the CLP experimental model that was introduced by Rittirsch et al. was used [13]. The rats were anesthetized intraperitoneally with 70 mg/kg Ketamine (Ketalar, Eczacıbaşı, Istanbul, Turkey), and 8 mg/kg xylazine (Rompun, Bayer, Istanbul, Turkey). The abdominal region was shaved and cleaned with povidone iodine solution. The organs were reached after the abdominal region was entered by opening the peritoneum with a 1-cm incision from the midline of the abdomen, and then by opening the peritoneum. The cecum was isolated, and ligated with 4/0 silk ligature at the distal area of the ileo cecal valve. The cecum was punctured twice (in total, 4 punctures) via a 21-gauge needle in a distal way across the ligation area to pass to the opposite side of the mesentery. Then, the cecum was placed in the peritoneal cavity, and the areas that were incised were closed by using 4/0 sterile synthetic absorbable sutures. After the surgical procedures were completed, 1-ml saline injection was administered subcutaneously to all animals.
2.5. Statistical analyses The SPSS 20 (Inc. Chicago, IL, USA) Package Program was used for the analyses of the data. Whether the data showed normal distribution was tested according to the Kolmogorov-Smirnov Test. After it was determined that the data had normal distribution, the One-Way ANOVA test was used for statistical analyses. The Post-hoc Tukey Test was used for the comparisons among the groups. Arithmetic mean ± Standard Deviation (SD) was used for the biochemical data. Arithmetic mean ± Standard error of the mean (SEM) was used for the histopathological and immunohistochemical data. A P < 0.05 was considered to be statistically significant.
2.2. The design of the experimental groups The SHAM Group (n = 9): Without applying CLP, the cecum was reached with an incision through the abdomen area. After the cecum was manipulated, the incision area was closed. The rats were decapitated after 24 h. The CLP Group (n = 9): After induction of CLP, vehicle injection was made intraperitoneally. The JWH-133 0.2 mg/kg Group (n = 9): JWH-133 (Biorbyt, California, the USA) was dissolved in DMSO and diluted with 99% phosphate buffer. After induction of CLP immediately, 0.2 mg/kg JWH133 was administered intraperitoneally. The JWH-133 1 mg/kg Group (n = 9): After induction of CLP immediately, 1 mg/kg JWH-133 was administered intraperitoneally. The JWH-133 5 mg/kg Group (n = 9): After induction of CLP immediately, 5 mg/kg JWH-133 was administered intraperitoneally. After 24 h of the CLP induction, all the animals were decapitated under anesthesia. Brain, lung, liver, and heart tissues of the rats were taken to 10% formaldehyde solution for histopathological and immunohistochemical analyses. Care was taken to carry out these steps in a fast way. The serum was obtained from the bloods of the rats. By using commercial kits, the TNF-α, IL-1β, IL-6 and interleukin-10 (IL-10) (Thermo Fisher Scientific, Waltham, MA, USA) levels were measured from the sera with the Enzyme-Linked Immuno Sorbent Assay (ELISA) Method according to the kit protocols.
3. Results 3.1. Histopathological evaluation To elucidate the effects of different doses of JWH-133 on septic tissues (lung, liver, heart and brain), severity of the injury was scored as described in the material methods. According to the histological evaluation of lung samples; histological injury like severe congestion, interalveolar hemorrhage and neutrophil infiltrations were observed mostly in the CLP group than the JWH-133 treated groups (Fig. 1A). Among groups, in the JWH-133 0,2, 1 and 5 mg/kg groups, severity of lung injury was lower than CLP (P < 0.05). And JWH-133 5 mg/kg groups’ injury score was lower than JWH-133 0,2 and 1 mg/kg (P < 0.05). Although there was no statistically significant difference 2
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Fig. 1. (A) Histopathologic evaluation of the lung, liver, heart, cerebral cortex and hippocampal areas of the brain in rats by H&E staining method. Representative section images for sham, CLP, JWH-133 0,2 mg/kg, JWH-133 1 mg/kg and JWH-133 5 mg/kg treatment groups are shown at X40 magnification and bar = 20 μm. In the lung, blue arrow: neutrophils; astral: hyperemia/congestion, intra-alveolar hemorrhage. In the liver, yellow arrow: damaged hepatocytes; astral: sinusoidal dilatation and congestion; blue arrow: neutrophils. In the heart, black arrow: cell gap; yellow arrow: cell degeneration; astral: hemorrhage. In the brain, yellow arrow: neuron degeneration; astral: vascular congestion. (B) Histological damage scores of the lung, liver, heart, cerebral cortex and hippocampal areas of the brain in each experimental group were quantified as described in materials methods. Data are expressed as mean ± SEM and compared by one-way ANOVA and TUKEY’s multiple comparisons test (•P < 0.05 vs. sham group; ϕ P < 0.05 vs. CLP group; Ψ P < 0.05 vs. JWH-133 0,2 mg/kg group; δ P < 0.05 vs. JWH-133 1 mg/kg group). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 3
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groups (p < 0.05). The liver IL-6 expression level was decreased in JWH-133 1 and 5 mg/kg treated groups compared with the CLP and JWH-133 0,2 mg/kg treated groups (p < 0.05). Liver tissue TNF-α levels were significantly lower in all other groups compared to the CLP group (p < 0.05). The expression level of TNF-α, in the liver, significantly decreased in JWH-133 5 mg/kg treated groups compared with the CLP, JWH-133 0.2 and 1 mg/kg treated groups (p < 0.05). There was no difference between sham and JWH-133 5 mg/kg groups in terms of TNF-α expression level. Liver tissue IL-1β and p-NF-κB levels were significantly lower in all other groups compared to the CLP group (p < 0.05). Immunoreactivity of liver tissue IL-1β and p-NF-κB in JWH-133 5 mg/kg group was lower than JWH-133 0.2 and 1 mg/kg groups. As shown in Fig. 4, TNF-α, IL-1β, IL-6, caspase-3 and p-NF-κB levels in heart tissue were significantly increased in the CLP, JWH-133 0.2 mg/kg, JWH-133 1 mg/kg and JWH-133 5 mg/kg groups compared to the sham group (p < 0.05). IL-6, caspase-3 and p-NF-κB levels in heart tissue were significantly decreased in all JWH treated groups compared to the CLP group (p < 0.05). IL-1β and TNF-α levels were significantly decreased in heart tissue in JWH-133 1 mg/kg and JWH133 5 mg/kg groups compared to the CLP group. The levels of caspase3, IL-6, IL-1β and p-NF-κB decreased significantly in heart tissue in the JWH-133 5 mg/kg group compared to the JWH-133 0.2 mg/kg group (p < 0.05). There was a significant difference between JWH-133 0.2 mg/kg group and JWH-133 1 mg/kg group in terms of caspase-3 and IL-6 levels (p < 0.05). As shown in Fig. 5, TNF-α, IL-1β, IL-6, caspase-3 and p-NF-κB levels in the brain cortex were significantly higher in all other groups compared to the sham group (p < 0.05) (Fig. 5). TNF-α, IL-6, caspase-3 and p-NF-κB levels in the brain cortex decreased significantly in all groups treated with JWH-133 compared to the CLP group (p < 0.05). Brain cortex IL-1β level decreased in JWH-133 1 mg/kg and JWH-133 5 mg/kg groups compared to the CLP group (p < 0.05). Brain cortex tissue of the JWH-133 1 mg/kg and JWH-133 5 mg/kg groups TNF-α, IL-1β, IL-6, caspase-3 and p-NF-κB levels were lower than JWH-133 0.2 mg/kg group (p < 0.05). As shown in Fig. 6, TNF-α, IL-1β, IL-6, caspase-3 and p-NF-κB levels in hippocampus tissue increased in the CLP group compared to the sham group (p < 0.05) (Fig. 6). In all doses, JWH-133 administration significantly reduced the levels of TNF-α, IL-1β, IL-6, caspase-3 and pNF-κB in the hippocampus compared to the CLP group (p < 0.05). The levels of hippocampus Caspase-3 were significantly lower in both JWH133 1 mg/kg and JWH-133 5 mg/kg groups compared to the JWH-133 0.2 mg/kg group (p < 0.05). The level of IL-1β in the JWH-133 5 group was lower than the JWH-133 0.2 group (p < 0.05). IL-6 levels of hippocampus tissue were lower in the JWH-133 1 mg/kg group compared to the JWH-133 0,2 mg/kg group (p < 0.05). As a result, compared with the sham group, expressions of caspase 3, IL-6, TNF-α, IL-1β and p-NF-κB were significantly increased in the CLP group, whereas this increased expression level was significantly reduced by JWH-133 at a dose-dependent manner (P < 0.05) (Figs. 2–6).
between the sham and JWH-133 5 mg/kg groups in terms of histological score, a statistically significant difference was found in the groups of JWH-133 0,2 and JWH-133 1 mg/kg compared to the sham group (Fig. 1B). When we evaluated the liver samples, we observed that damaged hepatocytes, increased sinusoidal dilatation and infiltration of neutrophils were mostly in the CLP group (p < 0.05) (Fig. 1A). According to our observation, histological damage score of liver was higher in all treatment groups than sham (P < 0.05). The histopathological scores of the JWH-133 1 and 5 mg/kg groups were lower than those of the CLP and JWH-133 0.2 mg/kg groups (P < 0.05) (Fig. 1B). Increased cell gap, hemorrhage, inflammatory cell infiltration and cell degeneration in heart tissue were higher in the CLP, JWH-133 0,2 and 1 mg/kg treated groups than the sham and JWH-133 5 mg/kg groups (P < 0.05) (Fig. 1A). Histopathological damage was significantly decreased in the JWH-133 0.2 and JWH-133 1 mg/kg groups in the heart compared to the CLP group (p < 0.05). Reduced cell gap and hemorrhage and observed normal cells were seen markedly in JWH-133 5 mg/kg groups (Fig. 1B). In terms of groups histological damage score was the same in both brain regions (cerebral cortex and hippocampus). The severity of neuron degeneration and pericellular/perivascular edema score of both brain regions were higher in the CLP, JWH-133 0,2, JWH-133 1 and JWH-133 5 mg/kg groups compared with the sham (P < 0.05) (Fig. 1A). JWH-133 1 mg/kg group damage score was lower than the CLP; and JWH-133 5 mg/kg groups’ damage score was lower than the CLP and JWH-133 0,2 groups (P < 0.05) (Fig. 1B). As a result, as shown in Fig. 1A, serious pathological changes observed in the CLP group improved with the administration of increasing doses of JWH133. Compared with the sham group, all tissues examined in the CLP group were found to have significantly high histological damage score, whereas JWH treated groups showed improvement score with increasing dose of JWH-133 (Fig. 1B). 3.2. Immunohistochemical findings As shown in Fig. 2, in the lung samples, compared with the CLP group, the expression level of TNF-α, IL-1β and phospho-NFκB (p-NFκB) were statistically decreased in all treatment groups (P < 0.05). Among groups, in the lung, the expression level of caspase 3 was not different between the CLP and JWH-133 0,2 mg/kg groups, whereas it was different in JWH-133 1 mg/kg group compared to the CLP (P < 0.05). And also, in JWH-133 5 mg/kg, it was different compared to the CLP and JWH-133 0,2 mg/kg groups. Lung tissue IL-6 levels were significantly higher in all other groups compared to the sham group (p < 0.05). The expression level of IL-6 was higher in the lung of the CLP group than in the JWH-133 1 and 5 mg/kg groups (p < 0.05). However, there was no statistically significant difference between the CLP and JWH-133 0.2 mg/kg groups. TNF levels in lung tissue were significantly higher in all other groups compared to the sham group (p < 0.05). TNF-α expression levels were higher in the lung of CLP compared to all JWH-133 treatment groups. Among JWH-133 treatment groups, in JWH-133 1 and 5 mg/kg treated groups, TNF-α expression levels were lower compared to JWH-133 0,2 mg/kg group (p < 0.05). Finally, the lung tissue p-NF-κB level was significantly higher in all other groups compared to the sham group. The p-NF-κB expression in the lung of CLP group was higher compared with the JWH-133-treated groups (p < 0.05). At the same time, the p-NF-κB level was significantly lower in the JWH-133 5 mg/kg group compared to the JWH-133 0.2 and 1 mg/kg groups (p < 0.05). As shown in Fig. 3, liver tissue caspase-3 levels were significantly higher in the CLP, JWH-133 0.2 and JWH-133 1 mg/kg groups compared to the sham group (p < 0.05). The expression level of caspase 3 in liver tissues was lower in all groups treated with JWH-133 compared to the CLP group (p < 0.05). In addition, the level of caspase-3 in the JWH-133 5 mg group was lower than in the JWH-133 0.2 and 1 mg/kg
3.3. Serum cytokine levels The serum TNF-α, IL-1β, IL-6 and IL-10 levels are shown in Fig. 7. In our study, compared to the Sham Group, the serum IL-1β and IL-10 levels increased at a significant level (P < 0.05) in all groups to which the CLP and JWH-133 treatment was applied. The serum TNF-α and IL6 levels increased in the CLP and JWH-133 0.2 mg/kg Group at a significant level compared to the Sham Group (P < 0.05). The serum TNF-α, IL-1β and IL-6 levels decreased at a significant level in the JWH133 1 mg/kg and JWH-133 5 mg/kg Groups when compared to the CLP Group (P < 0.05). The level of IL-10, which is an anti-inflammatory cytokine, increased in the JWH-133 1 mg/kg and JWH-133 5 mg/kg Groups when compared to the CLP Group (P < 0.05). 4
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Fig. 2. (A) Caspase 3, IL-6, TNF-α, IL-1β and p-NF-κB images of immunostaining in rat lungs. Representative section images for sham, CLP, JWH-133 0,2 mg/kg, JWH-133 1 mg/kg and JWH-133 5 mg/kg treatment groups are shown at X40 magnification and bar = 20 μm. (B) Histograms represent the intensity values in percent of immunostaining obtained using Image J software. The bar graph data are expressed as mean ± SEM, and compared by one-way ANOVA and TUKEY’s multiple comparisons test (•P < 0.05 vs. sham group; ϕ P < 0.05 vs. CLP group; Ψ P < 0.05 vs. JWH-133 0,2 mg/kg group; δ P < 0.05 vs. JWH-133 1 mg/kg group).
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Fig. 3. (A) Caspase 3, IL-6, TNF-α, IL-1β and p-NF-κB images of immunostaining in rat livers. Representative section images for sham, CLP, JWH-133 0,2 mg/kg, JWH-133 1 mg/kg and JWH-133 5 mg/kg treatment groups are shown at X40 magnification and bar = 20 μm. (B) Histograms represent the intensity values in percent of immunostaining obtained using Image J software. The bar graph data are expressed as mean ± SEM, and compared by one-way ANOVA and TUKEY’s multiple comparisons test (•P < 0.05 vs. sham group; ϕ P < 0.05 vs. CLP group; Ψ P < 0.05 vs. JWH-133 0,2 mg/kg group; δ P < 0.05 vs. JWH-133 1 mg/kg group).
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Fig. 4. (A) Caspase 3, IL-6, TNF-α, IL-1β and p-NF-κB images of immunostaining in rat hearts. Representative section images for sham, CLP, JWH-133 0,2 mg/kg, JWH-133 1 mg/kg and JWH-133 5 mg/kg treatment groups are shown at X40 magnification and bar = 20 μm. (B) Histograms represent the intensity values in percent of immunostaining obtained using Image J software. The bar graph data are expressed as mean ± SEM, and compared by one-way ANOVA and TUKEY’s multiple comparisons test (•P < 0.05 vs. sham group; ϕ P < 0.05 vs. CLP group; Ψ P < 0.05 vs. JWH-133 0,2 mg/kg group; δ P < 0.05 vs. JWH-133 1 mg/kg group).
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Fig. 5. Caspase 3, IL-6, TNF-α, IL-1β and p-NF-κB images of immunostaining in the cerebral cortex of rat brains. Representative section images for sham, CLP, JWH133 0,2 mg/kg, JWH-133 1 mg/kg and JWH-133 5 mg/kg treatment groups are shown at X40 magnification and bar = 20 μm. (B) Histograms represent the intensity values in percent of immunostaining obtained using Image J software. The bar graph data are expressed as mean ± SEM, and compared by one-way ANOVA and TUKEY’s multiple comparisons test (•P < 0.05 vs. sham group; ϕ P < 0.05 vs. CLP group; Ψ P < 0.05 vs. JWH-133 0,2 mg/kg group; δ P < 0.05 vs. JWH-133 1 mg/kg group).
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Fig. 6. Caspase 3, IL-6, TNF-α, IL-1β and p-NF-κB images of immunostaining in the hippocampus of rat brains. Representative section images for sham, CLP, JWH133 0,2 mg/kg, JWH-133 1 mg / kg and JWH-133 5 mg / kg treatment groups are shown at X40 magnification and bar = 20 μm. (B) Histograms represent the intensity values in percent of immunostaining obtained using Image J software. The bar graph data are expressed as mean ± SEM, and compared by one-way ANOVA and TUKEY’s multiple comparisons test (•P < 0.05 vs. sham group; ϕ P < 0.05 vs. CLP group; Ψ P < 0.05 vs. JWH-133 0,2 mg/kg group; δ P < 0.05 vs. JWH-133 1 mg/kg group).
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Fig. 7. Serum IL-6, TNF-α, IL-1β and IL10 levels (A–D). JWH-133 treatment at doses of 1 mg/kg and 5 mg/kg decreased serum IL-6, TNF-α, IL-1β levels compared to CLP group and increased IL-10 levels. Data are expressed as mean ± SD and compared by one-way ANOVA and TUKEY’s multiple comparisons test (•P < 0.05 vs. sham group; ϕ P < 0.05 vs. CLP group).
applied. Parallel to this finding, JWH-133 application also decreased the TNF-α, IL-1β and IL-6 levels in the same tissues. It was reported in previous studies that the activation of caspase-3, which plays a central role in the programmed cell death in sepsis, is associated with mortality. In experimental sepsis models, the caspase-3 activity increased in many tissues [26,27]. In our study, in the CLP group, increased caspase3 levels decreased with JWH-133 administration. We found similar findings in the histopathological examination. The levels of TNF-α, IL1β, IL-6, which are among proinflammatory cytokines, decreased in the serum of the groups that were treated with JWH-133. The IL-10 is an anti-inflammatory cytokine which plays important roles in negative regulation of immune response [28]. In our study, the JWH-133 administration increased the serum IL-10 levels. According to our findings, JWH-133 showed its effects in a dose-dependent way. We observed that the most effective dose was 5 mg/kg. CB2 receptor agonists have therapeutic potentials for many diseases which include sepsis [29]. Lehmann et al. conducted a study and reported that although CB2 agonist HU308 reduced the systemic inflammation and Leukocyte adhesion, the CB2 antagonist AM630 did not show the same effect [30]. In another study, it was reported that the administration of CB2 agonist Gp1a in CLP-induced sepsis model increased survival and decreased bacteremia, IL-6 levels and lung injury. In the same study, bacteremia, IL-6 levels, and lung injury increased in CB2-knock-out (KO) mice that were exposed to CLP, and the survival decreased [31]. In their study, Gui et al. reported that the administration of CB2 agonist GW405833 increased survival and decreased serum proinflammatory cytokine levels in LPS-treated mice [32]. JWH-133 is a selective CB2 receptor agonist [33]. The number of studies that used JWH-133 in experimental sepsis models is limited. Gamal et al. showed in their study that the administration of JWH-133 in LPS-induced endotoxemia reduced IL-6, vascular cell adhesion molecule (VCAM), E-selectin levels in the brain and serum, and
4. Discussion In our study, JWH-133, which is a CB2 receptor agonist, decreased the proinflammatory cytokine level by inhibiting the NF-κB activity in the CLP-induced sepsis model. It also reduced the damage in brain, heart, liver and lung. Although there were extensive investigations in the past, sepsis is still an important health problem whose pathophysiology has not yet been fully elucidated. The incidence of sepsis, which can lead to multiple organ failure, is increasing with each passing day [18,19]. In the past, several animal models were developed that mimicked the pathophysiological changes in septic patients to understand the underlying mechanisms of sepsis and systemic inflammatory response [20]. The CLP is a realistic model, which is used to establish polymicrobial sepsis in an empirical way to examine the underlying mechanisms of sepsis [13]. The activation of the NF-κB in cells causes the production of various inflammatory mediators such as TNF-α, IL-1β, IL-6 by triggering the inflammatory cascade [21,22]. The phosphorylation of NF-κB controls its activation. NF-κB has different subunits. The p65 is the most prominent NF-κB subunit in NF-κB phosphorylation studies conducted so far. Many studies focused on S536, which is one of the best understood phosphorylation targets in p65 [23]. The p65 (ser536) plays key roles in inflammatory events in CLP-induced sepsis [24]. In our study, a parallelism was detected between the NF-κB p65 (ser536) and pro-inflammatory cytokine immunoreactivity in the brain, lung, liver and heart tissues. In sepsis, high NF-κB activation level is associated with higher mortality and worse clinical outcomes. In sepsis, the inhibition of NF-κB signal prevents the expression of multiple proinflammatory genes and multiple organ injury. It also prolongs the survival [25]. In our study, p-NF-κB immunoreactivity decreased in lung, heart, liver, brain cortex and hippocampus of the groups to which JWH-133 was
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endotoxemia-associated brain dysfunction [34]. In the literature review, no studies were detected on the effects of JWH-133 in CLP-induced sepsis models. Sun et al. showed that JWH-133 reduced the surgery-induced increased IL-1β, TNF-α, Monocyte chemotactic protein (MCP-1) levels, and alleviated the neuroinflammation in the brain cortex and in the hippocampus [35]. In a study in the stroke model, JWH-133 reduced IL-6, IL-1β, TNF-α levels and infarct volume in the brain, while the administration of CB2 antagonist prevented these effects [36]. In another study, JWH-133 reduced the immune cell infiltration in the brain in a cerebral ischemia model [37]. Tomar et al. reported that JWH-133 had a reducing effect on liver damage in galactosamine/lipopolysaccharide-induced acute liver failure [38]. In another study, it was shown that JWH-133 suppressed the NF-kB activation in paraquat-induced acute lung injury, decreased TNF-α and IL1β, and prevented lung injury [39]. Montecucco et al. showed that JWH-133 administration reduced leukocyte infiltration, inflammatory mediator level, and infarct volume in cardiac ischemia [40].
tilting toward immunosuppression, Nat. Med. 15 (5) (2009) 496–497. [6] J.A. Smith, P.R. Mayeux, R.G. Schnellmann, Delayed mitogen-activated protein kinase/extracellular signal-regulated kinase inhibition by Trametinib attenuates systemic inflammatory responses and multiple organ injury in murine sepsis, Crit. Care Med. 44 (8) (2016) e711–e720. [7] K. Zhang, X.F. Jiao, J.X. Li, X.W. Wang, Rhein inhibits lipopolysaccharide-induced intestinal injury during sepsis by blocking the toll-like receptor 4 nuclear factorkappaB pathway, Mol. Med. Rep. 12 (3) (2015) 4415–4421. [8] A. Tanyeli, E. Eraslan, M.C. Guler, S.O. Sebin, D. Celebi, F.B. Ozgeris, E. Toktay, Investigation of biochemical and histopathological effects of tarantula cubensis D6 on lung tissue in cecal ligation and puncture-induced polymicrobial sepsis model in rats, Medicine (2019). [9] Q. He, F. Xiao, Q. Yuan, J. Zhang, J. Zhan, Z. Zhang, Cannabinoid receptor 2: a potential novel therapeutic target for sepsis? Acta Clin. Belg. 74 (2) (2019) 70–74. [10] R.P. Picone, D.A. Kendall, Minireview: from the bench, toward the clinic: therapeutic opportunities for cannabinoid receptor modulation, Mol. Endocrinol. 29 (6) (2015) 801–813. [11] K.R. Kasten, J. Tschop, M.H. Tschop, C.C. Caldwell, The cannabinoid 2 receptor as a potential therapeutic target for sepsis, Endocr. Metab. Immune. Disord. Drug. Targets. 10 (3) (2010) 224–234. [12] R. Mechoulam, L.A. Parker, The endocannabinoid system and the brain, Annu. Rev. Psychol. 64 (2013) 21–47. [13] D. Rittirsch, M.S. Huber-Lang, M.A. Flierl, P.A. Ward, Immunodesign of experimental sepsis by cecal ligation and puncture, Nat. Protocols 4 (1) (2009) 31–36. [14] Y. Hirano, M. Aziz, W.L. Yang, Z. Wang, M. Zhou, M. Ochani, A. Khader, P. Wang, Neutralization of osteopontin attenuates neutrophil migration in sepsis-induced acute lung injury, Crit. Care 19 (2015) 53. [15] A. Dadkhah, F. Fatemi, A. Rasooli, M.R. Mohammadi Malayeri, F. Torabi, Assessing the effect of Mentha longifolia essential oils on COX-2 expression in animal model of sepsis induced by caecal ligation and puncture, Pharm. Biol. 56 (1) (2018) 495–504. [16] J. Liu, J. Li, P. Tian, B. Guli, G. Weng, L. Li, Q. Cheng, H2S attenuates sepsis-induced cardiac dysfunction via a PI3K/Akt-dependent mechanism, Exp. Ther. Med. 17 (5) (2019) 4064–4072. [17] D. Arslan, A. Ekinci, A. Arici, E. Bozdemir, E. Akil, H.H. Ozdemir, Effects of Ecballium elaterium on brain in a rat model of sepsis-associated encephalopathy, Libyan J. Med. 12 (1) (2017) 1369834. [18] G.S. Martin, D.M. Mannino, S. Eaton, M. Moss, The epidemiology of sepsis in the United States from 1979 through 2000, N. Engl. J. Med. 348 (16) (2003) 1546–1554. [19] D.F. Gaieski, J.M. Edwards, M.J. Kallan, B.G. Carr, Benchmarking the incidence and mortality of severe sepsis in the United States, Crit. Care Med. 41 (5) (2013) 1167–1174. [20] D. Rittirsch, L.M. Hoesel, P.A. Ward, The disconnect between animal models of sepsis and human sepsis, J. Leukoc Biol. 81 (1) (2007) 137–143. [21] P.N. Moynagh, The NF-kappaB pathway, J. Cell Sci. 118 (Pt 20) (2005) 4589–4592. [22] T. Lawrence, The Nuclear Factor NF-kappa B Pathway in Inflammation, Cold Spring Harbor Perspect. Biol. 1 (6) (2009). [23] F. Christian, E.L. Smith, R.J. Carmody, The regulation of NF-kappaB subunits by phosphorylation, Cells 5 (1) (2016). [24] J. Walker, E. Dichter, G. Lacorte, D. Kerner, B. Spur, A. Rodriguez, K. Yin, Lipoxin a4 increases survival by decreasing systemic inflammation and bacterial load in sepsis, Shock 36 (4) (2011) 410–416. [25] S.F. Liu, A.B. Malik, NF-kappa B activation as a pathological mechanism of septic shock and inflammation, Am. J. Physiol. Lung Cell Mol. Physiol. 290 (4) (2006) L622–L645. [26] L. Lorente, M.M. Martin, J. Ferreres, J. Sole-Violan, L. Labarta, C. Diaz, A. Jimenez, J.M. Borreguero-Leon, Serum caspase 3 levels are associated with early mortality in severe septic patients, J. Crit. Care 34 (2016) 103–106. [27] C.M. Comim, T. Barichello, D. Grandgirard, F. Dal-Pizzol, J. Quevedo, S.L. Leib, Caspase-3 mediates in part hippocampal apoptosis in sepsis, Mol. Neurobiol. 47 (1) (2013) 394–398. [28] S. Rutz, W. Ouyang, Regulation of interleukin-10 expression, Adv. Exp. Med. Biol. 941 (2016) 89–116. [29] R.G. Pertwee, Targeting the endocannabinoid system with cannabinoid receptor agonists: pharmacological strategies and therapeutic possibilities, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 367 (1607) (2012) 3353–3363. [30] C. Lehmann, M. Kianian, J. Zhou, I. Kuster, R. Kuschnereit, S. Whynot, O. Hung, R. Shukla, B. Johnston, V. Cerny, D. Pavlovic, A. Spassov, M.E. Kelly, Cannabinoid receptor 2 activation reduces intestinal leukocyte recruitment and systemic inflammatory mediator release in acute experimental sepsis, Crit. Care 16 (2) (2012) R47. [31] J. Tschop, K.R. Kasten, R. Nogueiras, H.S. Goetzman, C.M. Cave, L.G. England, J. Dattilo, A.B. Lentsch, M.H. Tschop, C.C. Caldwell, The cannabinoid receptor 2 is critical for the host response to sepsis, J. Immunol. 183 (1) (2009) 499–505. [32] H. Gui, Y. Sun, Z.M. Luo, D.F. Su, S.M. Dai, X. Liu, Cannabinoid receptor 2 protects against acute experimental sepsis in mice, Mediators Inflamm. 2013 (2013) 741303. [33] J.W. Huffman, CB2 receptor ligands, Mini. Rev. Med. Chem. 5 (7) (2005) 641–649. [34] M. Gamal, J. Moawad, L. Rashed, W. El-Eraky, D. Saleh, C. Lehmann, N. Sharawy, Evaluation of the effects of Eserine and JWH-133 on brain dysfunction associated with experimental endotoxemia, J. Neuroimmunol. 281 (2015) 9–16. [35] L. Sun, R. Dong, X. Xu, X. Yang, M. Peng, Activation of cannabinoid receptor type 2 attenuates surgery-induced cognitive impairment in mice through anti-inflammatory activity, J. Neuroinflamm. 14 (1) (2017) 138. [36] J.G. Zarruk, D. Fernandez-Lopez, I. Garcia-Yebenes, M.S. Garcia-Gutierrez,
5. Conclusion To sum up, CB2 receptors modulate the immune response. CB2 receptor activation causes reductions in leukocyte recruitment, chemotaxis and in proinflammatory cytokine levels [11]. It was also shown in different experimental models that CB2 activation reduced organ damage. In this study, we found that JWH-133, which is a CB2 agonist, prevents brain, heart, lung and liver damage in CLP-induced sepsis in a dose-dependent manner. JWH-133 also reduced the proinflammatory cytokine levels through the inhibition of apoptosis and NF-κB. Our results show that activation of CB2 receptor with JWH-133 might be a potential therapeutic target in the treatment of inflammatory diseases like sepsis. Disclosure statement Authors declare no conflict of interest. Authors contributions M.Ç. projected and conducted the study, analyzed the data, and wrote the study. M.Ç. and S. T. dealt with the rats. Z. D. and A. O. O. performed histopathological and immunohistochemical analyzes. P. Ç. made ELISA analysis. Funding We were supported by Yozgat Bozok University, Department of Scientific Research Projects, Turkey (Project no: 6602c-TF/18-229). References [1] A. Rhodes, L.E. Evans, W. Alhazzani, M.M. Levy, M. Antonelli, R. Ferrer, A. Kumar, J.E. Sevransky, C.L. Sprung, M.E. Nunnally, B. Rochwerg, G.D. Rubenfeld, D.C. Angus, D. Annane, R.J. Beale, G.J. Bellinghan, G.R. Bernard, J.D. Chiche, C. Coopersmith, D.P. De Backer, C.J. French, S. Fujishima, H. Gerlach, J.L. Hidalgo, S.M. Hollenberg, A.E. Jones, D.R. Karnad, R.M. Kleinpell, Y. Koh, T.C. Lisboa, F.R. Machado, J.J. Marini, J.C. Marshall, J.E. Mazuski, L.A. McIntyre, A.S. McLean, S. Mehta, R.P. Moreno, J. Myburgh, P. Navalesi, O. Nishida, T.M. Osborn, A. Perner, C.M. Plunkett, M. Ranieri, C.A. Schorr, M.A. Seckel, C.W. Seymour, L. Shieh, K.A. Shukri, S.Q. Simpson, M. Singer, B.T. Thompson, S.R. Townsend, T. Van der Poll, J.L. Vincent, W.J. Wiersinga, J.L. Zimmerman, R.P. Dellinger, Surviving sepsis campaign: international guidelines for management of sepsis and septic shock: 2016, Intensive Care Med. 43 (3) (2017) 304–377. [2] B.G. Chousterman, F.K. Swirski, G.F. Weber, Cytokine storm and sepsis disease pathogenesis, Semin. Immunopathol. 39 (5) (2017) 517–528. [3] M. Aziz, A. Jacob, W.L. Yang, A. Matsuda, P. Wang, Current trends in inflammatory and immunomodulatory mediators in sepsis, J. Leukoc. Biol. 93 (3) (2013) 329–342. [4] R.C. Bone, C.J. Grodzin, R.A. Balk, Sepsis: a new hypothesis for pathogenesis of the disease process, Chest 112 (1) (1997) 235–243. [5] R.S. Hotchkiss, C.M. Coopersmith, J.E. McDunn, T.A. Ferguson, The sepsis seesaw:
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M. Çakır, et al.
cannabinoid receptor 2 activation in galactosamine/lipopolysaccharide-induced acute liver failure through regulation of macrophage polarization and microRNAs, J. Pharmacol. Exp. Ther. 353 (2) (2015) 369–379. [39] Z. Liu, Y. Wang, H. Zhao, Q. Zheng, L. Xiao, M. Zhao, CB2 receptor activation ameliorates the proinflammatory activity in acute lung injury induced by paraquat, Biomed. Res. Int. 2014 (2014) 971750. [40] F. Montecucco, S. Lenglet, V. Braunersreuther, F. Burger, G. Pelli, M. Bertolotto, F. Mach, S. Steffens, CB(2) cannabinoid receptor activation is cardioprotective in a mouse model of ischemia/reperfusion, J. Mol. Cell Cardiol. 46 (5) (2009) 612–620.
J. Vivancos, F. Nombela, M. Torres, M.C. Burguete, J. Manzanares, I. Lizasoain, M.A. Moro, Cannabinoid type 2 receptor activation downregulates stroke-induced classic and alternative brain macrophage/microglial activation concomitant to neuroprotection, Stroke; J. Cerebral Circul. 43 (1) (2012) 211–219. [37] S. Murikinati, E. Juttler, T. Keinert, D.A. Ridder, S. Muhammad, Z. Waibler, C. Ledent, A. Zimmer, U. Kalinke, M. Schwaninger, Activation of cannabinoid 2 receptors protects against cerebral ischemia by inhibiting neutrophil recruitment, FASEB J. 24 (3) (2010) 788–798. [38] S. Tomar, E.E. Zumbrun, M. Nagarkatti, P.S. Nagarkatti, Protective role of
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