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Glycyrrhizin attenuates isoflurane-induced cognitive deficits in neonatal rats via its anti-inflammatory activity
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GLYCYRRHIZIN ATTENUATES ISOFLURANE-INDUCED COGNITIVE DEFICITS IN NEONATAL RATS VIA ITS ANTI-INFLAMMATORY ACTIVITY
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W. WANG, X. CHEN, J. ZHANG, Y. ZHAO, S. LI, L. TAN, J. GAO, X. FANG AND A. LUO *
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Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
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Abstract—Children exposed to general anesthetics such as isoflurane are maybe at an increased risk of cognitive impairment. Recent studies have indicated that this kind of cognitive decline is associated with neuroinflammation in the hippocampus of neonatal rodents. Glycyrrhizin is a naturally available compound for the treatment of inflammatory and neurodegenerative diseases. We therefore aimed to investigate the effects of glycyrrhizin on the isoflurane-induced cognitive deficits and hippocampal neuroinflammation in the neonatal rats. Seven day-old rats were exposed to 1.8% isoflurane for 4 h. Saline and glycyrrhizin solution was injected intraperitoneally 30 min prior to isoflurane or control gas exposure. The effects of isoflurane and glycyrrhizin treatment on memory performance were examined using Morris Water Maze (MWM) task. The protein expression of high-mobility group box 1 (HMGB1), NFjB, Bcl-2, Bax and cleaved (active) caspase-3 were determined by Western blot assay. The protein levels of TNF-a and IL-1b were detected by enzyme-linked immunosorbent assay (ELISA). The combination of ELISA and Western blot results showed that glycyrrhizin attenuated isoflurane-induced increases of proinflammatory cytokines (TNF-a and IL-1b) and activation of HMGB1/NFjB signaling pathway in the hippocampus of neonatal rats. Furthermore, glycyrrhizin treatment prevented the deficits in spatial memory induced by neonatal exposure to isoflurane. Consistent with these observations, we found that glycyrrhizin alleviated isoflurane-induced neuroapoptosis and down-regulations of PSD-95 and SNAP-25 in the hippocampus of neonatal rats. These results suggest that glycyrrhizin may be a potential therapeutic agent for developmental neurotoxicity and subsequent cognitive decline induced by neonatal exposure to general anesthetics. Ó 2015 Published by Elsevier Ltd. on behalf of IBRO.
Key words: glycyrrhizin, hippocampus, HMGB1, isoflurane, NFjB.
*Corresponding author. Address: Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan 430030, China. Tel: +86-27-83665480. E-mail address: [email protected] (A. Luo). Abbreviations: AD, Alzheimer’s disease; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; HMGB1, high-mobility group box 1; LPS, lipopolysaccharide; MWM, Morris Water Maze. http://dx.doi.org/10.1016/j.neuroscience.2015.11.001 0306-4522/Ó 2015 Published by Elsevier Ltd. on behalf of IBRO. 1
INTRODUCTION
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General anesthetics such as isoflurane are widely used in pediatric surgery, and their safe uses in children have become a major issue of interest. However, several population studies have demonstrated that children exposed to general anesthetics display learning disabilities during childhood (Wilder et al., 2009). However, the underlying mechanisms are still not clearly understood. Recently, neuroinflammation has come to be recognized as an important underlying mechanism in general anesthetics-induced cognitive impairments in neonatal rodents (Lu et al., 2010; Shen et al., 2013). In vitro studies have demonstrated that isoflurane exposure may cause the activation of classic pro-inflammatory factor NFjB p65 in neuronal and microglia cultures (Zhang et al., 2013). This phenomenon was further supported by animal studies showing that anesthesia with isoflurane or sevoflurane in neonatal mice could result in overproductions of pro-inflammatory cytokines such as TNF-a and IL-1b in the hippocampus, which may contribute to cognitive impairment in the adulthood (Lu et al., 2010; Shen et al., 2013). Thus, the prevention of neuroinflammation might be a new and effective therapeutic approach for isoflurane-induced cognitive impairment in neonatal rodents. Glycyrrhizin, a triterpenoid saponin compound, is the main component of Glycyrrhiza glabra which possesses potent anti-oxidant, anti-inflammatory and immunemodulatory properties (Lee et al., 2007; Mollica et al., 2007). Glycyrrhizin is capable of inhibiting the chemoattractant activity and mitogenic activity of high-mobility group box 1 (HMGB1) (Mollica et al., 2007; Ohnishi et al., 2011). HMGB1 has been identified as an endogenous danger signal molecule in the brain and the inhibition of HMGB1 activities can provide the brain with protective effects by attenuating neuroinflammation in ischemic injury (Kim et al., 2006; Faraco et al., 2007) and neurodegenerative diseases (Gao et al., 2011; Li et al., 2013a,b). Furthermore, it has also been demonstrated that the treatment with glycyrrhizin can alleviate b-amyloid (Ahn et al., 2006; Zhao et al., 2013) or systemic lipopolysaccharide (LPS)-induced (Song et al., 2013) cognitive impairment via inhibition of neuroflammation. Therefore, we hypothesize that glycyrrhizin protects against isoflurane-induced cognitive impairment in neonatal rats via its anti-inflammatory property. In the present study, we report that, after neonatal exposure to isoflurane, glycyrrhizin reduces apoptosis and restores
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synaptic protein synthesis in the hippocampus of neonatal rats by inhibiting HMGB1/NFjB signaling pathwaydependent pro-inflammatory cytokine production, resulting in improvement of cognitive functions.
EXPERIMENTAL PROCEDURES
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Animals
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In the present study, mother Sprague–Dawley rats (n = 28) with litters containing male pups (n = 158, from the Center of Experimental Animal of Tongji Medical College) were cross-fostered before starting the experiment. When pups were 7 days old (weighting 11–16 g), they were divided into four experimental groups (defined in the section ‘‘Experimental groups”) and subjected to corresponding treatment. After experimental treatment, the four groups were distributed equally among litters. All rats were kept under standard lab housing with 12-h light/dark cycle. All experimental protocols and animal handling procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) revised 1996 and the experimental protocols were approved by the committee of experimental animals of Tongji Medical College.
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Isoflurane exposure
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Rats randomized to the anesthesia group received isoflurane (1.8%, approximate 1 MAC for P7 rats) (Stratmann et al., 2009) flushed with 60% oxygen (balanced with air) for 4 h. Rats in the control group received control gas (60% oxygen, balanced with air) in a similar chamber for 4 h. The size of the anesthesia chamber in the study was 20 20 10 cm. The chamber was kept in a homeothermic incubator to maintain the experimental temperature at 37 °C. An infrared probe (OhmedaS/5 Compact, Datex-Ohmeda, Louisville, CO, USA) was adopted to continuously monitor the concentrations of
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oxygen, carbon dioxide and isoflurane in the exhalant gas. After exposure to isoflurane or control gas, all rats were returned to mother female rats. To determine adequacy of ventilation, arterial blood was sampled at the end of isoflurane anesthesia by obtaining a single sample (100 ll) via cardiac puncture. PH, arterial oxygen, and carbon dioxide tensions, base excess, and blood glucose was analyzed by a blood gas analyzer (Kent Scientific Corp., Torrington, CT, USA).
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Experimental groups
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All neonatal rats were randomly divided into four groups: (1) control group (CON): rats were intraperitoneally injected with 100 ll of saline followed by control gas exposure for 4 h; (2) isoflurane group (ISO): rats were intraperitoneally injected with100 ll of saline followed by 1.8% isoflurane exposure for 4 h; (3) glycyrrhizin group (Gly): rats were intraperitoneally injected with 20 mg/kg glycyrrhizin (100 ll) followed by control gas exposure for 4 h; (4) isoflurane + glycyrrhizin group (ISO + Gly): rats were intraperitoneally injected with 20 mg/kg of glycyrrhizin (100 ll) followed by 1.8% isoflurane exposure for 4 h. The regimen of glycyrrhizin treatment was selected based on a previous study (Ohnishi et al., 2011). Glycyrrhizin was prepared with sterile saline. Saline and glycyrrhizin solution was injected intraperitoneally (i.p.) 30 min prior to isoflurane or control gas exposure. The number of animals used for each experimental group is provided in Table 1.
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MWM
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MWM test was performed as described in our previous work (Li et al., 2013a,b). The rats (n = 10 per group) prepared for MWM were weaned at P22 and started the training at P31. The training protocol for the task of MWM test consisted of three trials (60 s maximum; interval 20 min) each day for five consecutive days. The probe trial was performed 1 h after the end of the
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Table 1. Study population Animals
Groups
Sample size
Read-out (number of rats)
P7 rats (n = 158)
CON
n = 49
ISO
n = 49
Gly
n = 19
ISO + Gly
n = 41
WB (30) ELISA (5) MWM (10) Blood gas analysis WB (30) ELISA (5) MWM (10) Blood gas analysis WB (10) ELISA (5) Blood gas analysis WB (22) ELISA (5) MWM (10) Blood gas analysis
(4)
(4)
(4)
(4)
CON, control group; ELISA, enzyme-linked immunosorbent assay; Gly, glycyrrhizin group; ISO, isoflurane group; ISO + Gly, Isoflurane + glycyrrhizin group; MWM, Morris Water Maze; P7, postnatal day 7; WB, Western blot analysis.
Please cite this article in press as: Wang W et al. Glycyrrhizin attenuates isoflurane-induced cognitive deficits in neonatal rats via its anti-inflammatory activity. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.11.001
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fifth-day training and the duration of time spent in each quadrant was determined.
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Western blot analysis
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At the time points of sacrifice, the rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and then killed by CO2 asphyxiation. The brains were rapidly removed and the hippocampus of neonatal rat from each group (n = 5) were dissociated and harvested at 4 °C. The tissues were then homogenized at 4 °C for 30 min in RIPA lysis buffer containing 1% Nonidet P-40, 20 mM Tris, pH 8.0, 137 mM NaCl, 0.5 mM EDTA, 10% glycerol, 10 mM Na2P2O7, 10 mM NaF, 1 g/ml aprotinin, 10 ng/ml leupeptin, 1 mM vanadate, and 1 mM PMSF. The protein levels of the supernatant were determined by a BCA assay kit (Boster, Wuhan, China). Equal amounts of protein sample (40 lg) was separated by SDS–PAGE and transferred to polyvinylidene difluoride (Millipore, Bedford, MA, USA) by electrophoresis. The membranes were blocked with 5% nonfat skim milk in TBST (0.1% Tween 20 in TBS) for 0.5 h at room temperature and then incubated overnight at 4 °C with anti-b-actin antibody (1:500; Boster, Wuhan, China), anti-HMGB1 antibody (1:500; Abcam, Cambridge, MA, USA), anti-NFjB p65 antibody (1:800; Abcam), anti-IjBa antibody (1:500; Santa Cruz Biotechnology, Inc., USA), anti-laminB antibody (1:500; Bioworld technology, Inc., Louis Park, MN, USA), anti-Bax (1:500; Abcam), anti-Bcl-2 (1:1000; Abclonal technology, Wuhan, China), anti-active (cleaved) caspase-3 (1:500; Cell Signaling Technology, Danvers, MA, USA), anti-total caspase-3 (1:1000; Cell Signaling Technology), anti-PSD-95 (1:1000; Abcam) or anti-synapsin I (1:1000; Cell Signaling Technology). After incubation overnight, the membranes were further incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG antibody (1:3000; Boster) for 2 h at room temperature. Labeled proteins were detected with the ChemiDocXRS chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA). Bands were quantified using lab imaging software. The experiments were repeated in triplicate.
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Enzyme-linked immunosorbent assay (ELISA)
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The dissociation and protein extraction procedure for hippocampi of neonatal rats were the same to that in Western blot. The protein levels of TNF-a and IL-1b in the hippocampus of neonatal rats were determined by commercial ELISA kits (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions. All samples were assayed in duplicate.
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Statistical analysis
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Values were represented as mean ± SEM, and analyzed by SPSS software version 16.0 (SPSS Inc., Chicago, IL, USA). Changes of escape latency in MWM test were analyzed by a two-way ANOVA (treatment and time). Other data were analyzed by a one-way ANOVA and followed by the Dunnett test. P value less than 0.05 was considered statistically significant.
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RESULTS
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Physiological parameters
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Systemic parameters including pH, PaCO2, PaO2, glucose and SaO2 were all within normal range and there were no differences among the four experimental groups in the present study (Table 2).
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Glycyrrhizin attenuates the isoflurane-induced increase in hippocampal protein expressions of HMGB1 in neonatal rats
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To determine the effects of isoflurane (1.8%, 4 h) on HMGB1expression in neonatal rats, we first detected the hippocampal protein expressions of HMGB1 by Western blot at 0, 6, 12 and 48 h after isoflurane exposure. As shown in Fig. 1A, hippocampal protein expressions of HMGB1 increased after isoflurane exposure, peaked at 12 h, and then began to decrease (P < 0.05 or P < 0.01, n = 4 per group). Glycyrrhizin is capable of inhibiting cytokine-like activities of HMGB1 via direct down-regulation of its protein expression (Mollica et al., 2007; Ohnishi et al., 2011). Therefore, we tested whether glycyrrhizin could attenuate isoflurane-induced increase in protein levels of hippocampal HMGB1 in neonatal rats. Our data showed that the treatment of 20 mg/kg or 30 mg/kg glycyrrhizin significantly reduced the isoflurane-induced increase in hippocampal HMGB1 protein expressions at 12 h after exposure to isoflurane (1.8%, 4 h) (Fig. 1B, P < 0.01, n = 4 per group). Based on the above observations, a dose of 20 mg/kg was selected in further work. Isoflurane-induced neurotoxicity in neonatal rats is associated with the release of pro-inflammatory cytokines such as IL-1b and TNF-a (Wu et al., 2012). Thus, we determined the effects of isoflurane (1.8%, 4 h) or glycyrrhizin 20 mg/kg on the production of hippocampal IL-1b and TNF-a by ELISA assays. Our data showed that the hippocampal protein levels of IL-1b and TNF-a in neonatal rats increased after isoflurane exposure, whereas the increase of IL-1b and TNF-a productions could be reversed in the presence of glycyrrhizin (20 mg/kg, i.p.) treatments (Fig. 1C, D, P < 0.05 or P < 0.01, n = 5 per group).
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Spatial memory deficits induced by neonatal exposure to isoflurane are alleviated by the treatment of glycyrrhizin
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Next, we tested the behavioral effects of isoflurane (1.8%, 4 h) and glycyrrhizin (20 mg/kg, i.p.) by performing the Morris water maze (MWM) task at 24 d after isoflurane exposure (P31) to evaluate spatial learning and memory (Liang et al., 2010). Isoflurane-exposed rats displayed cognitive impairment as indicated by prolonged escape latency (Fig. 2C, P < 0.001, F = 29.684, n = 10 per group, from the training trials), longer traveled distance (Fig. 2D, P = 0.009, F = 8.552, n = 10 per group, from the training trials), less time in the target quadrant (Fig. 2E, P < 0.05, n = 10 per group, from the probe trials) and decreased platform crossings (Fig. 2F, P < 0.01, n = 10 per group, from the probe trials). Importantly, the
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Please cite this article in press as: Wang W et al. Glycyrrhizin attenuates isoflurane-induced cognitive deficits in neonatal rats via its anti-inflammatory activity. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.11.001
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Table 2. Effects of isoflurane exposure on physiological parameters of arterial blood gas analysis in neonatal rats
pH PaCO2 (mmHg) PaO2 (mmHg) Glucose (mmol/L) SaO2 (%)
Control
ISO
Gly
ISO + Gly
7.33 ± 0.04 37.2 ± 3.7 110 ± 10 4.1 ± 0.3 99 ± 1.4
7.32 ± 0.05 39 ± 3.6 106 ± 11 4.2 ± 0.4 98 ± 0.7
7.35 ± 0.06 38.2 ± 3.9 108 ± 14 3.9 ± 0.6 99 ± 1.2
7.34 ± 0.05 38.3 ± 3.0 107 ± 15 3.8 ± 0.3 99 ± 0.8
The pH, PaCO2, PaO2, glucose and SaO2 levels did not differ significantly among the four groups. Data are presented as mean ± SEM (n = 4). PaO2, arterial oxygen tension; PaCO2, arterial carbon dioxide tension; SaO2, arterial oxygen saturation.
Fig. 1. Glycyrrhizin attenuates the isoflurane-induced increase in hippocampal protein expressions of HMGB1 in neonatal rats. (A) The effects of isoflurane (1.8%, 4 h) on hippocampal protein expressions of HMGB1 in neonatal rats were detected by Western blot analysis. Hippocampal protein expressions of HMGB1 increased after isoflurane exposure, peaked at 12 h, then began to decrease. (B) Administration of glycyrrhizin (20 and 30 mg/kg, i.p.) significantly attenuated the isoflurane-induced increase in hippocampal protein expressions of HMGB1 in neonatal rats. (C) The effects of isoflurane (1.8%, 4 h) and glycyrrhizin (20 mg/kg, i.p.) on hippocampal protein expressions of IL-1b in neonatal rats were detected by ELISA analysis. Application of glycyrrhizin significantly attenuated the isoflurane-induced increase in hippocampal protein expressions of IL-1b in neonatal rats. (D) The effects of isoflurane (1.8%, 4 h) and glycyrrhizin (20 mg/kg, i.p.) on hippocampal protein expressions of TNF-a in neonatal rats were detected by ELISA analysis. Administration of glycyrrhizin significantly attenuated the isoflurane-induced increase in hippocampal protein expressions of TNF-a in neonatal rats. Data are presented as mean ± SEM (n = 4/group). *P < 0.05 or **P < 0.01. CON, control group; Gly, glycyrrhizin group; HMGB1, high mobility group box-1 protein; IL-1b, Interleukine-1 b; ISO, isoflurane group; TNF-a, tumor necrosis factor a. 245 246 247 248 249 250 251 252 253 254 255 256
impairment in learning and memory displayed in isoflurane-exposed rats can be prevented by the treatment of glycyrrhizin as indicated by decreased escape latency (Fig. 2C, P < 0.001, F = 28.593, n = 10 per group, from the probe trials), shorter traveled distance (Fig. 2D, P = 0.014, F = 7.43, n = 10 per group, from the training trials), more time in the target quadrant (Fig. 2E, P < 0.05, n = 10 per group, from the probe trials) and increased platform crossings (Fig. 2F, P < 0.01, n = 10 per group, from the probe trials). These results indicated that glycyrrhizin could attenuate cognitive impairment induced by neonatal isoflurane anesthesia.
Glycyrrhizin mitigates the isoflurane-induced activation of NFjB p65 signaling pathway in the hippocampus of neonatal rats
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In vitro studies have demonstrated that isofluraneinduced release of pro-inflammatory cytokines may be related to the activation of NFjB p65 signaling pathway and the degradation of IjBa (an inhibitory factor of NFjB p65) (Zhang et al., 2013). Our data showed that isoflurane exposure also induced an increase in the nuclear NFjB p65 expressions in the hippocampus of neonatal rats, which was attenuated by the treatment of glycyrrhizin (20 mg/kg, i.p.) (Fig. 3A, P < 0.01, n = 5
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Please cite this article in press as: Wang W et al. Glycyrrhizin attenuates isoflurane-induced cognitive deficits in neonatal rats via its anti-inflammatory activity. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.11.001
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Fig. 2. Spatial memory deficits induced by neonatal exposure to isoflurane are alleviated by the treatments of glycyrrhizin (20 mg/kg, i.p.). (A) The experimental design of behavioral study. (B) Representative swim paths obtained during trial 3 (session 4) from rats in the three experimental group. (C) Escape latency in the Morris water maze plotted against the training days. n = 10 per group. Repeated measures ANOVA followed by a post hoc Bonferroni multiple comparison test: CON vs. ISO, P < 0.001, F = 29.684; ISO vs. ISO + Gly, P < 0.001, F = 28.593. (D) Escape path length in the Morris water maze plotted against the training days. n = 10 per group. Repeated measures ANOVA followed by a post hoc Bonferroni multiple comparison test: CON vs. ISO, P = 0.009, F = 8.552; ISO vs. ISO + Gly, P = 0.014, F = 7.43. (E) The analysis of the time spent in each quadrant during the probe test of MWM. (F) The platform crossing times during a 60-s probe trial of MWM test. Data are presented as mean ± SEM (n = 10/group). *P < 0.05 or **P < 0.01.
Fig. 3. Glycyrrhizin mitigates the isoflurane-induced activation of NFjB p65 signaling pathway in the hippocampi of neonatal rats. (A) A graph representative of Western blot analysis of nuclear NFjB p65 expressions in the hippocampus. The statistical analysis showed that application of glycyrrhizin (20 mg/kg, i.p.) mitigated the isoflurane-induced increased nuclear NFjB p65 expressions in the hippocampi of neonatal rats. (B) A graph representative of Western blot analysis of cytosolic NFjB p65 expressions in the hippocampus. The statistical analysis showed that application of glycyrrhizin (20 mg/kg, i.p.) attenuated the isoflurane-induced down-regulations of cytosolic NFjB p65 expressions in the hippocampi of neonatal rats. (C) A graph representative of Western blot analysis of cytosolic IjBa expressions in the hippocampus. The statistical analysis showed that application of glycyrrhizin (20 mg/kg, i.p.) attenuated the isoflurane-induced down-regulations of cytosolic IjBa expressions in the hippocampi of neonatal rats. Data are presented as mean ± SEM (n = 5/group). *P < 0.05 or **P < 0.01. CON: control group; Gly: glycyrrhizin; HMGB1: high mobility group box-1 protein; NF-jB: nuclear factor kappa-light-chain-enhancer of activated B cells; IjBa: nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha; ISO: isoflurane group. 269 270 271 272 273
per group). Additionally, isoflurane exposure also induced decreases in cytoplasmic NFjB p65 and IjBa expressions, whereas these changes could be attenuated by the treatment of glycyrrhizin (Fig. 3B, C, P < 0.05 or P < 0.01, n = 5 per group).
Glycyrrhizin mitigates isoflurane-induced apoptosis in the hippocampus of neonatal rats
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Previous studies have demonstrated that neonatal exposure to isoflurane result in increased
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neuroapoptosis in the hippocampus, which may contribute to isoflurane-induced long-term cognitive deficits (Loepke et al., 2009). Therefore, we test whether glycyrrhizin could reduce isoflurane-induced hippocampal neuroapoptosis in neonatal rats. As shown in Fig. 4A, we found that hippocampal active (cleaved) caspase-3 expressions in isoflurane-exposed rats were higher than those of saline-treated rats (Fig. 4A, P < 0.01, n = 5 per group). However, the treatment of glycyrrhizin (20 mg/kg, i.p.) significantly attenuated isofluraneinduced increase in hippocampal active (cleaved) caspase-3 expressions of neonatal rats (Fig. 4A, P < 0.01, n = 5 per group). In addition, we also detect the effects of isoflurane and glycyrrhizin on the protein levels of pro-apoptotic factor Bax and anti-apoptotic factor Bcl-2 in the hippocampus of neonatal rats (Wei et al., 2005; Wang et al., 2014). As expected, we found that hippocampal Bax protein expressions increased while Bcl-2 protein expressions decreased in neonatal rats exposed to isoflurane (Fig. 4B, C, P < 0.05 or P < 0.01, n = 5 per group). In contrast, the isoflurane-induced effects on hippocampal Bax and Bcl-2 protein expression could be attenuated by the treatment of glycyrrhizin (Fig. 4 B and C, P < 0.05 or P < 0.01, n = 5 per group).
Glycyrrhizin reverses the isoflurane-induced decrease in hippocampal PSD-95 expressions in neonatal rats It has been shown that general anesthetics-induced cognitive deficits are associated with dysfunction in synthesis of vital synaptic proteins (Yan et al., 2012; Wang et al., 2013). To test whether glycyrrhizin can restore the synthesis of vital synaptic proteins in neonatal rats exposed to isoflurane, we detected the effects of isoflurane (1.8%, 4 h) and glycyrrhizin (20 mg/kg, i.p.) on hippocampal protein expressions of PSD-95, SNAP25 and synapsin I in neonatal rats by Western blot analysis. We found that the hippocampal protein levels
of PSD-95 and SNAP-25 in the isoflurane-treated rats were lower than those in the saline-treated rats (Fig. 5A, B, P < 0.05 or P < 0.01, n = 4 per group), whereas the effect of isoflurane on hippocampal protein levels of synapsin I was not observed (Fig. 5C, P > 0.05). In addition, our results showed that glycyrrhizin could restore hippocampal PSD-95 and SNAP-25 protein expressions (Fig. 5A, B, P < 0.05 or P < 0.01, n = 4 per group), but didn’t alter hippocampal synapsin I protein expressions in neonatal rats exposed to isoflurane (Fig. 5C, P > 0.05).
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DISCUSSION
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Our study elucidated that glycyrrhizin protected against isoflurane-induced cognitive impairment in neonatal rats via its anti-inflammatory properties. This effect was identified by the improvement in the performance in MWM trials. Glycyrrhizin prevented HMGB1/NFjB signaling-mediated neuroinflammation that may lead to neurodegeneration and decrease of synaptic protein synthesis in the hippocampus of isoflurane-exposed neonatal rats. The findings support the concept that the treatment of glycyrrhizin inhibition might be effective therapeutic measurement for long-term cognitive impairment induced by neoantal exposure to volatile anesthetics. This work builds on previous findings demonstrating that neonatal exposure to volatile anesthetics induces hippocampal neuroinflammation in neonatal rodents, leading to subsequent cognitive decline (Shen et al., 2013). Therefore, these findings led us to predict that pharmacological targeting on neuroinflammation would be neuroprotective against isoflurane-induced neurotoxicity in the immature brain. It is well established that glycyrrhizin could provide neuroprotective effects in in vitro or in vivo models via its anti-inflammation actions (Cherng et al., 2006; Kim et al., 2008; Zhang et al., 2014). Indeed, our data showed that the treatment of glycyrrhizin reversed isoflurane-induced up-regulation of
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Fig. 4. Glycyrrhizin mitigates isoflurane-induced apoptosis in the hippocampus of neonatal rats. (A) The treatment of glycyrrhizin (20 mg/kg, i.p.) significantly reduced isoflurane (1.8%, 4 h)-induced up-regulation of active (cleaved) caspase-3 in the hippocampus of neonatal rats. (B) Application of glycyrrhizin (20 mg/kg, i.p.) significantly reduced isoflurane (1.8%, 4 h)-induced enhancement of Bax in the hippocampus of neonatal rats. (C) Administration of glycyrrhizin (20 mg/kg, i.p.) significantly attenuated isoflurane (1.8%, 4 h)-induced down-regulation of Bcl-2 in the hippocampus of neonatal rats. Data are presented as mean ± SEM (n = 5/group). *P < 0.05 or **P < 0.01. Bax: Bcl-2-associated X protein; CON, control group; Gly, glycyrrhizin; ISO, isoflurane group. Please cite this article in press as: Wang W et al. Glycyrrhizin attenuates isoflurane-induced cognitive deficits in neonatal rats via its anti-inflammatory activity. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.11.001
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Fig. 5. Glycyrrhizin reverses the isoflurane-induced decrease in hippocampal PSD-95 expressions in neonatal rats. The effects of isoflurane (1.8%, 4 h) and glycyrrhizin (20 mg/kg, i.p.) on hippocampal protein expressions of PSD-95(A), SNAP-25 (B) or synapsin I (C) in neonatal rats were detected by Western blot analysis. (A) Isoflurane exposure induced a significant decrease in hippocampal protein expressions of PSD-95, whereas the decrease was reversed by the application of glycyrrhizin. (B) Isoflurane exposure reduced hippocampal protein expressions of SNAP-25, whereas the reduction was attenuated by the treatment of glycyrrhizin. (C) Isoflurane exposure or the combined treatment of glycyrrhizin didn’t alter the hippocampal protein levels of synapsin I in neonatal rats. Data are presented as mean ± SEM (n = 4/group). *P < 0.05 or **P < 0.01. CON, control group; Gly, glycyrrhizin; ISO: isoflurane group; PSD-95, postsynaptic density protein 95; SNAP-25, synaptosomal-associated protein 25. 353 354 355 356 357 358 359 360 361 362 363 364 365 366
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hippocampal TNF-a and IL-1b in neonatal rats. In consistent with previous studies employing the Morris water maze to assess cognition following isoflurane anesthesia (Callaway et al., 2012; Wang et al., 2014), the memory deficit in isoflurane exposed rats was also observed in the present study, indicated by a longer escape latency/path length, less time spent in the target quadrant and fewer original platform crossings. However, this memory deficit induced by early exposure to isoflurane was significantly improved in the presence of glycyrrhizin treatment. This result is consistent with previous reports showing that glycyrrhizin treatment could attenuate memory deficit in LPS-induced brain injury (Song et al., 2013) and Ab1– 42-induced Alzheimer’s disease (AD) (Zhu et al., 2012) models. Thus, our results indicated that glycyrrhizin treatment could protect against cognitive deficits in neonatal isoflurane-exposed rats via its anti-inflammatory actions. The cognitive deficits induced by neonatal exposure to anesthetics have been attributed to the increased apoptosis and synaptic dysfunctions in the hippocampus (Simon et al., 2001; Xie et al., 2006; Zhong et al., 2015). Accumulated evidence also suggests that neuroinflammation may cause synaptic dysfunction and neuronal cell death (Agostinho et al., 2010; Xuan et al., 2012). Therefore, we hypothesized that the anti-inflammatory activities of glycyrrhizin may also benefit to improve hippocampal cell survival and synaptic function in isoflurane-exposed neonatal rats. Indeed, we found that glycyrrhizin significantly attenuated isoflurane-induced hippocampal apoptosis, as indicated by the decreased expressions of active caspase-3 and pro-apoptotic factor Bax as well as the increased expression of anti-apoptotic factor Bcl-2. These results were in accordance with a previous study showing that glycyrrhizin attenuates kainic acid-induced neuronal apoptosis in the hippocampus (Luo et al., 2013). Furthermore, glycyrrhizin treatment also restored isoflurane-induced down-regulation of hippocampal PSD-95 and SNAP-25 expressions, suggesting that glycyrrhizin could restore hippocampal synaptic pro-
tein synthesis in isoflurane-exposed neonatal rats. These results were in consistent with previous reports that intraperitoneal injection of glycyrrhizin could alleviate the decrease of hippocampal PSD-95 expressions in an AD animal model (Jang et al., 2013). Taken together, our data suggested that glycyrrhizin could attenuate isoflurane-induced apoptosis and synaptic protein loss in the hippocampus of neonatal rats. The mechanisms by which neonatal exposure to anesthesia induces neuroinflammation remain largely unknown. HMGB1 is a highly conserved nuclear protein with a pivotal role in inducing neuroinflammation in various CNS diseases (Kim et al., 2006; Fang et al., 2012). Under pathological conditions, HMGB1 translocates into cytoplasm where it interacts with various cellsurface receptors such as RAGE (receptor for advanced glycation end products), resulting in activation of transcription factor NFjB (Palumbo et al., 2007; Yang and Tracey, 2010). Interestingly, a previous study has indicated that hippocampal HMGB1, TNF-a and IL-1b can be up-regulated by isoflurane anesthesia or surgical stimuli in aged rats, which may contribute to neuronal damage and subsequent cognitive dysfunction (He et al., 2012). Here, our results showed that isoflurane exposure induced the increase of cytosolic HMGB1, IjB degradation and NFjB p65 translocation into nucleus, which are in paralleled with the increase in TNF-a and IL-1b. Remarkably, previous studies have demonstrated that by direct binding to HMGB1, glycyrrhizin can attenuate HMGB1-dependent downstream activation of NFjB signaling pathway and neuroinflammation (Mollica et al., 2007; Ohnishi et al., 2011). Consistently, our results showed that the administration of glycyrrhizin not only reduced the hippocampal production of TNF-a and IL-1b, but also reversed isoflurane-induced HMGB1 cytosolic expression, IjB degradation and NFjB p65 nuclear translocation. Therefore, our results indicated that glycyrrhizin is dependent on the HMGB-1/NFjB signal pathway to exert anti-inflammatory actions.
Please cite this article in press as: Wang W et al. Glycyrrhizin attenuates isoflurane-induced cognitive deficits in neonatal rats via its anti-inflammatory activity. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.11.001
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Although the improvements of memory deficits and the attenuation of neuroinflammation have been found, it is still not conclusive that the behavioral benefits by glycyrrhizin treatments are completely due to its antiinflammatory activity. Besides neuroinflammation, hippocampal b-amyloid deposition (Xie et al., 2007; Zhen et al., 2009) and neurogenesis impairments (Stratmann et al., 2009; Zhu et al., 2010) have also been recognized as important contributors to anesthesiarelated learning and memory abnormalities. Interestingly, previous studies have reported the beneficial cognitive effects of glycyrrhizin by protecting against b-amyloid deposition (Zhu et al., 2012) and promoting neurogenesis (Lei et al., 2013). Therefore, further studies are required to investigate the potential relationship between bamyloid deposition/neurogenesis and the protective effects of glycyrrhizin against learning and memory deficits induced by early anesthesia exposure. The finding in the present study that there was no difference in blood gas parameters between anesthetized and non-anesthetized rat pups may contradict with many other studies. The study by (Murphy and Baxter, 2013) indicated that P7 rats displayed acidosis and hypercapnia after 2 h of 3% isoflurane anesthesia (pure oxygen as carrier gas). In a more specific study by (Stratmann et al., 2009), it was shown that hypercapnia and acidosis developed immediately after induction of isoflurane (1 MAC as determined by tail clamping) anesthesia and a steady decrease over the 4-h anesthesia duration. The discrepancy between their and our results could be attributed to the lower concentration of isoflurane, and the sampling time point for blood gas analysis (the end of the 4-h period of anesthesia) when respiratory depression effects caused by isoflurane were not as evident as immediately after anesthesia induction. Furthermore, our data were consistent with a series of studies from other laboratories (Loepke et al., 2009; Brambrink et al., 2010; Liang et al., 2010), where neither hypercapnia nor acidosis was observed in isofluraneanesthetized animals.
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CONCLUSION
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Our results showed that glycyrrhizin protects against cognitive impairment induced by neonatal exposure to isoflurane via inhibition of HMGB-1/NFjB signal pathway and downstream production of proinflammatory cytokines. These protective effects of glycyrrhizin relate to the attenuation of apoptosis and restore of synaptic protein synthesis in the hippocampus of neonatal rats. Therefore, we believe that treatment of glycyrrhizin could be beneficial in clinical treatment for developmental neurotoxicity and subsequent cognitive decline induced by early exposure to volatile anesthetics.
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CONFLICT OF INTEREST
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None.
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Acknowledgments—The present work was supported by a grant from the National Natural Science Foundation of China (Nos. 81571047, 81271233, 81400882 and 81200880), and also sup-
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ported by 2010 Clinical Key Disciplines Construction Grant from the Ministry of Health of China.
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(Accepted 1 November 2015) (Available online xxxx)
Please cite this article in press as: Wang W et al. Glycyrrhizin attenuates isoflurane-induced cognitive deficits in neonatal rats via its anti-inflammatory activity. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.11.001
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GLYCYRRHIZIN ATTENUATES ISOFLURANE-INDUCED COGNITIVE DEFICITS IN NEONATAL RATS VIA ITS ANTI-INFLAMMATORY ACTIVITY
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W. WANG, X. CHEN, J. ZHANG, Y. ZHAO, S. LI, L. TAN, J. GAO, X. FANG AND A. LUO *
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Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
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Abstract—Children exposed to general anesthetics such as isoflurane are maybe at an increased risk of cognitive impairment. Recent studies have indicated that this kind of cognitive decline is associated with neuroinflammation in the hippocampus of neonatal rodents. Glycyrrhizin is a naturally available compound for the treatment of inflammatory and neurodegenerative diseases. We therefore aimed to investigate the effects of glycyrrhizin on the isoflurane-induced cognitive deficits and hippocampal neuroinflammation in the neonatal rats. Seven day-old rats were exposed to 1.8% isoflurane for 4 h. Saline and glycyrrhizin solution was injected intraperitoneally 30 min prior to isoflurane or control gas exposure. The effects of isoflurane and glycyrrhizin treatment on memory performance were examined using Morris Water Maze (MWM) task. The protein expression of high-mobility group box 1 (HMGB1), NFjB, Bcl-2, Bax and cleaved (active) caspase-3 were determined by Western blot assay. The protein levels of TNF-a and IL-1b were detected by enzyme-linked immunosorbent assay (ELISA). The combination of ELISA and Western blot results showed that glycyrrhizin attenuated isoflurane-induced increases of proinflammatory cytokines (TNF-a and IL-1b) and activation of HMGB1/NFjB signaling pathway in the hippocampus of neonatal rats. Furthermore, glycyrrhizin treatment prevented the deficits in spatial memory induced by neonatal exposure to isoflurane. Consistent with these observations, we found that glycyrrhizin alleviated isoflurane-induced neuroapoptosis and down-regulations of PSD-95 and SNAP-25 in the hippocampus of neonatal rats. These results suggest that glycyrrhizin may be a potential therapeutic agent for developmental neurotoxicity and subsequent cognitive decline induced by neonatal exposure to general anesthetics. Ó 2015 Published by Elsevier Ltd. on behalf of IBRO.
Key words: glycyrrhizin, hippocampus, HMGB1, isoflurane, NFjB.
*Corresponding author. Address: Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan 430030, China. Tel: +86-27-83665480. E-mail address: [email protected] (A. Luo). Abbreviations: AD, Alzheimer’s disease; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; HMGB1, high-mobility group box 1; LPS, lipopolysaccharide; MWM, Morris Water Maze. http://dx.doi.org/10.1016/j.neuroscience.2015.11.001 0306-4522/Ó 2015 Published by Elsevier Ltd. on behalf of IBRO. 1
INTRODUCTION
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General anesthetics such as isoflurane are widely used in pediatric surgery, and their safe uses in children have become a major issue of interest. However, several population studies have demonstrated that children exposed to general anesthetics display learning disabilities during childhood (Wilder et al., 2009). However, the underlying mechanisms are still not clearly understood. Recently, neuroinflammation has come to be recognized as an important underlying mechanism in general anesthetics-induced cognitive impairments in neonatal rodents (Lu et al., 2010; Shen et al., 2013). In vitro studies have demonstrated that isoflurane exposure may cause the activation of classic pro-inflammatory factor NFjB p65 in neuronal and microglia cultures (Zhang et al., 2013). This phenomenon was further supported by animal studies showing that anesthesia with isoflurane or sevoflurane in neonatal mice could result in overproductions of pro-inflammatory cytokines such as TNF-a and IL-1b in the hippocampus, which may contribute to cognitive impairment in the adulthood (Lu et al., 2010; Shen et al., 2013). Thus, the prevention of neuroinflammation might be a new and effective therapeutic approach for isoflurane-induced cognitive impairment in neonatal rodents. Glycyrrhizin, a triterpenoid saponin compound, is the main component of Glycyrrhiza glabra which possesses potent anti-oxidant, anti-inflammatory and immunemodulatory properties (Lee et al., 2007; Mollica et al., 2007). Glycyrrhizin is capable of inhibiting the chemoattractant activity and mitogenic activity of high-mobility group box 1 (HMGB1) (Mollica et al., 2007; Ohnishi et al., 2011). HMGB1 has been identified as an endogenous danger signal molecule in the brain and the inhibition of HMGB1 activities can provide the brain with protective effects by attenuating neuroinflammation in ischemic injury (Kim et al., 2006; Faraco et al., 2007) and neurodegenerative diseases (Gao et al., 2011; Li et al., 2013a,b). Furthermore, it has also been demonstrated that the treatment with glycyrrhizin can alleviate b-amyloid (Ahn et al., 2006; Zhao et al., 2013) or systemic lipopolysaccharide (LPS)-induced (Song et al., 2013) cognitive impairment via inhibition of neuroflammation. Therefore, we hypothesize that glycyrrhizin protects against isoflurane-induced cognitive impairment in neonatal rats via its anti-inflammatory property. In the present study, we report that, after neonatal exposure to isoflurane, glycyrrhizin reduces apoptosis and restores
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synaptic protein synthesis in the hippocampus of neonatal rats by inhibiting HMGB1/NFjB signaling pathwaydependent pro-inflammatory cytokine production, resulting in improvement of cognitive functions.
EXPERIMENTAL PROCEDURES
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Animals
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In the present study, mother Sprague–Dawley rats (n = 28) with litters containing male pups (n = 158, from the Center of Experimental Animal of Tongji Medical College) were cross-fostered before starting the experiment. When pups were 7 days old (weighting 11–16 g), they were divided into four experimental groups (defined in the section ‘‘Experimental groups”) and subjected to corresponding treatment. After experimental treatment, the four groups were distributed equally among litters. All rats were kept under standard lab housing with 12-h light/dark cycle. All experimental protocols and animal handling procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) revised 1996 and the experimental protocols were approved by the committee of experimental animals of Tongji Medical College.
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Isoflurane exposure
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Rats randomized to the anesthesia group received isoflurane (1.8%, approximate 1 MAC for P7 rats) (Stratmann et al., 2009) flushed with 60% oxygen (balanced with air) for 4 h. Rats in the control group received control gas (60% oxygen, balanced with air) in a similar chamber for 4 h. The size of the anesthesia chamber in the study was 20 20 10 cm. The chamber was kept in a homeothermic incubator to maintain the experimental temperature at 37 °C. An infrared probe (OhmedaS/5 Compact, Datex-Ohmeda, Louisville, CO, USA) was adopted to continuously monitor the concentrations of
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oxygen, carbon dioxide and isoflurane in the exhalant gas. After exposure to isoflurane or control gas, all rats were returned to mother female rats. To determine adequacy of ventilation, arterial blood was sampled at the end of isoflurane anesthesia by obtaining a single sample (100 ll) via cardiac puncture. PH, arterial oxygen, and carbon dioxide tensions, base excess, and blood glucose was analyzed by a blood gas analyzer (Kent Scientific Corp., Torrington, CT, USA).
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Experimental groups
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All neonatal rats were randomly divided into four groups: (1) control group (CON): rats were intraperitoneally injected with 100 ll of saline followed by control gas exposure for 4 h; (2) isoflurane group (ISO): rats were intraperitoneally injected with100 ll of saline followed by 1.8% isoflurane exposure for 4 h; (3) glycyrrhizin group (Gly): rats were intraperitoneally injected with 20 mg/kg glycyrrhizin (100 ll) followed by control gas exposure for 4 h; (4) isoflurane + glycyrrhizin group (ISO + Gly): rats were intraperitoneally injected with 20 mg/kg of glycyrrhizin (100 ll) followed by 1.8% isoflurane exposure for 4 h. The regimen of glycyrrhizin treatment was selected based on a previous study (Ohnishi et al., 2011). Glycyrrhizin was prepared with sterile saline. Saline and glycyrrhizin solution was injected intraperitoneally (i.p.) 30 min prior to isoflurane or control gas exposure. The number of animals used for each experimental group is provided in Table 1.
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MWM
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MWM test was performed as described in our previous work (Li et al., 2013a,b). The rats (n = 10 per group) prepared for MWM were weaned at P22 and started the training at P31. The training protocol for the task of MWM test consisted of three trials (60 s maximum; interval 20 min) each day for five consecutive days. The probe trial was performed 1 h after the end of the
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Table 1. Study population Animals
Groups
Sample size
Read-out (number of rats)
P7 rats (n = 158)
CON
n = 49
ISO
n = 49
Gly
n = 19
ISO + Gly
n = 41
WB (30) ELISA (5) MWM (10) Blood gas analysis WB (30) ELISA (5) MWM (10) Blood gas analysis WB (10) ELISA (5) Blood gas analysis WB (22) ELISA (5) MWM (10) Blood gas analysis
(4)
(4)
(4)
(4)
CON, control group; ELISA, enzyme-linked immunosorbent assay; Gly, glycyrrhizin group; ISO, isoflurane group; ISO + Gly, Isoflurane + glycyrrhizin group; MWM, Morris Water Maze; P7, postnatal day 7; WB, Western blot analysis.
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fifth-day training and the duration of time spent in each quadrant was determined.
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Western blot analysis
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At the time points of sacrifice, the rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and then killed by CO2 asphyxiation. The brains were rapidly removed and the hippocampus of neonatal rat from each group (n = 5) were dissociated and harvested at 4 °C. The tissues were then homogenized at 4 °C for 30 min in RIPA lysis buffer containing 1% Nonidet P-40, 20 mM Tris, pH 8.0, 137 mM NaCl, 0.5 mM EDTA, 10% glycerol, 10 mM Na2P2O7, 10 mM NaF, 1 g/ml aprotinin, 10 ng/ml leupeptin, 1 mM vanadate, and 1 mM PMSF. The protein levels of the supernatant were determined by a BCA assay kit (Boster, Wuhan, China). Equal amounts of protein sample (40 lg) was separated by SDS–PAGE and transferred to polyvinylidene difluoride (Millipore, Bedford, MA, USA) by electrophoresis. The membranes were blocked with 5% nonfat skim milk in TBST (0.1% Tween 20 in TBS) for 0.5 h at room temperature and then incubated overnight at 4 °C with anti-b-actin antibody (1:500; Boster, Wuhan, China), anti-HMGB1 antibody (1:500; Abcam, Cambridge, MA, USA), anti-NFjB p65 antibody (1:800; Abcam), anti-IjBa antibody (1:500; Santa Cruz Biotechnology, Inc., USA), anti-laminB antibody (1:500; Bioworld technology, Inc., Louis Park, MN, USA), anti-Bax (1:500; Abcam), anti-Bcl-2 (1:1000; Abclonal technology, Wuhan, China), anti-active (cleaved) caspase-3 (1:500; Cell Signaling Technology, Danvers, MA, USA), anti-total caspase-3 (1:1000; Cell Signaling Technology), anti-PSD-95 (1:1000; Abcam) or anti-synapsin I (1:1000; Cell Signaling Technology). After incubation overnight, the membranes were further incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG antibody (1:3000; Boster) for 2 h at room temperature. Labeled proteins were detected with the ChemiDocXRS chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA). Bands were quantified using lab imaging software. The experiments were repeated in triplicate.
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Enzyme-linked immunosorbent assay (ELISA)
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The dissociation and protein extraction procedure for hippocampi of neonatal rats were the same to that in Western blot. The protein levels of TNF-a and IL-1b in the hippocampus of neonatal rats were determined by commercial ELISA kits (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions. All samples were assayed in duplicate.
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Statistical analysis
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Values were represented as mean ± SEM, and analyzed by SPSS software version 16.0 (SPSS Inc., Chicago, IL, USA). Changes of escape latency in MWM test were analyzed by a two-way ANOVA (treatment and time). Other data were analyzed by a one-way ANOVA and followed by the Dunnett test. P value less than 0.05 was considered statistically significant.
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RESULTS
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Physiological parameters
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Systemic parameters including pH, PaCO2, PaO2, glucose and SaO2 were all within normal range and there were no differences among the four experimental groups in the present study (Table 2).
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Glycyrrhizin attenuates the isoflurane-induced increase in hippocampal protein expressions of HMGB1 in neonatal rats
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To determine the effects of isoflurane (1.8%, 4 h) on HMGB1expression in neonatal rats, we first detected the hippocampal protein expressions of HMGB1 by Western blot at 0, 6, 12 and 48 h after isoflurane exposure. As shown in Fig. 1A, hippocampal protein expressions of HMGB1 increased after isoflurane exposure, peaked at 12 h, and then began to decrease (P < 0.05 or P < 0.01, n = 4 per group). Glycyrrhizin is capable of inhibiting cytokine-like activities of HMGB1 via direct down-regulation of its protein expression (Mollica et al., 2007; Ohnishi et al., 2011). Therefore, we tested whether glycyrrhizin could attenuate isoflurane-induced increase in protein levels of hippocampal HMGB1 in neonatal rats. Our data showed that the treatment of 20 mg/kg or 30 mg/kg glycyrrhizin significantly reduced the isoflurane-induced increase in hippocampal HMGB1 protein expressions at 12 h after exposure to isoflurane (1.8%, 4 h) (Fig. 1B, P < 0.01, n = 4 per group). Based on the above observations, a dose of 20 mg/kg was selected in further work. Isoflurane-induced neurotoxicity in neonatal rats is associated with the release of pro-inflammatory cytokines such as IL-1b and TNF-a (Wu et al., 2012). Thus, we determined the effects of isoflurane (1.8%, 4 h) or glycyrrhizin 20 mg/kg on the production of hippocampal IL-1b and TNF-a by ELISA assays. Our data showed that the hippocampal protein levels of IL-1b and TNF-a in neonatal rats increased after isoflurane exposure, whereas the increase of IL-1b and TNF-a productions could be reversed in the presence of glycyrrhizin (20 mg/kg, i.p.) treatments (Fig. 1C, D, P < 0.05 or P < 0.01, n = 5 per group).
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Spatial memory deficits induced by neonatal exposure to isoflurane are alleviated by the treatment of glycyrrhizin
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Next, we tested the behavioral effects of isoflurane (1.8%, 4 h) and glycyrrhizin (20 mg/kg, i.p.) by performing the Morris water maze (MWM) task at 24 d after isoflurane exposure (P31) to evaluate spatial learning and memory (Liang et al., 2010). Isoflurane-exposed rats displayed cognitive impairment as indicated by prolonged escape latency (Fig. 2C, P < 0.001, F = 29.684, n = 10 per group, from the training trials), longer traveled distance (Fig. 2D, P = 0.009, F = 8.552, n = 10 per group, from the training trials), less time in the target quadrant (Fig. 2E, P < 0.05, n = 10 per group, from the probe trials) and decreased platform crossings (Fig. 2F, P < 0.01, n = 10 per group, from the probe trials). Importantly, the
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Table 2. Effects of isoflurane exposure on physiological parameters of arterial blood gas analysis in neonatal rats
pH PaCO2 (mmHg) PaO2 (mmHg) Glucose (mmol/L) SaO2 (%)
Control
ISO
Gly
ISO + Gly
7.33 ± 0.04 37.2 ± 3.7 110 ± 10 4.1 ± 0.3 99 ± 1.4
7.32 ± 0.05 39 ± 3.6 106 ± 11 4.2 ± 0.4 98 ± 0.7
7.35 ± 0.06 38.2 ± 3.9 108 ± 14 3.9 ± 0.6 99 ± 1.2
7.34 ± 0.05 38.3 ± 3.0 107 ± 15 3.8 ± 0.3 99 ± 0.8
The pH, PaCO2, PaO2, glucose and SaO2 levels did not differ significantly among the four groups. Data are presented as mean ± SEM (n = 4). PaO2, arterial oxygen tension; PaCO2, arterial carbon dioxide tension; SaO2, arterial oxygen saturation.
Fig. 1. Glycyrrhizin attenuates the isoflurane-induced increase in hippocampal protein expressions of HMGB1 in neonatal rats. (A) The effects of isoflurane (1.8%, 4 h) on hippocampal protein expressions of HMGB1 in neonatal rats were detected by Western blot analysis. Hippocampal protein expressions of HMGB1 increased after isoflurane exposure, peaked at 12 h, then began to decrease. (B) Administration of glycyrrhizin (20 and 30 mg/kg, i.p.) significantly attenuated the isoflurane-induced increase in hippocampal protein expressions of HMGB1 in neonatal rats. (C) The effects of isoflurane (1.8%, 4 h) and glycyrrhizin (20 mg/kg, i.p.) on hippocampal protein expressions of IL-1b in neonatal rats were detected by ELISA analysis. Application of glycyrrhizin significantly attenuated the isoflurane-induced increase in hippocampal protein expressions of IL-1b in neonatal rats. (D) The effects of isoflurane (1.8%, 4 h) and glycyrrhizin (20 mg/kg, i.p.) on hippocampal protein expressions of TNF-a in neonatal rats were detected by ELISA analysis. Administration of glycyrrhizin significantly attenuated the isoflurane-induced increase in hippocampal protein expressions of TNF-a in neonatal rats. Data are presented as mean ± SEM (n = 4/group). *P < 0.05 or **P < 0.01. CON, control group; Gly, glycyrrhizin group; HMGB1, high mobility group box-1 protein; IL-1b, Interleukine-1 b; ISO, isoflurane group; TNF-a, tumor necrosis factor a. 245 246 247 248 249 250 251 252 253 254 255 256
impairment in learning and memory displayed in isoflurane-exposed rats can be prevented by the treatment of glycyrrhizin as indicated by decreased escape latency (Fig. 2C, P < 0.001, F = 28.593, n = 10 per group, from the probe trials), shorter traveled distance (Fig. 2D, P = 0.014, F = 7.43, n = 10 per group, from the training trials), more time in the target quadrant (Fig. 2E, P < 0.05, n = 10 per group, from the probe trials) and increased platform crossings (Fig. 2F, P < 0.01, n = 10 per group, from the probe trials). These results indicated that glycyrrhizin could attenuate cognitive impairment induced by neonatal isoflurane anesthesia.
Glycyrrhizin mitigates the isoflurane-induced activation of NFjB p65 signaling pathway in the hippocampus of neonatal rats
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In vitro studies have demonstrated that isofluraneinduced release of pro-inflammatory cytokines may be related to the activation of NFjB p65 signaling pathway and the degradation of IjBa (an inhibitory factor of NFjB p65) (Zhang et al., 2013). Our data showed that isoflurane exposure also induced an increase in the nuclear NFjB p65 expressions in the hippocampus of neonatal rats, which was attenuated by the treatment of glycyrrhizin (20 mg/kg, i.p.) (Fig. 3A, P < 0.01, n = 5
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Fig. 2. Spatial memory deficits induced by neonatal exposure to isoflurane are alleviated by the treatments of glycyrrhizin (20 mg/kg, i.p.). (A) The experimental design of behavioral study. (B) Representative swim paths obtained during trial 3 (session 4) from rats in the three experimental group. (C) Escape latency in the Morris water maze plotted against the training days. n = 10 per group. Repeated measures ANOVA followed by a post hoc Bonferroni multiple comparison test: CON vs. ISO, P < 0.001, F = 29.684; ISO vs. ISO + Gly, P < 0.001, F = 28.593. (D) Escape path length in the Morris water maze plotted against the training days. n = 10 per group. Repeated measures ANOVA followed by a post hoc Bonferroni multiple comparison test: CON vs. ISO, P = 0.009, F = 8.552; ISO vs. ISO + Gly, P = 0.014, F = 7.43. (E) The analysis of the time spent in each quadrant during the probe test of MWM. (F) The platform crossing times during a 60-s probe trial of MWM test. Data are presented as mean ± SEM (n = 10/group). *P < 0.05 or **P < 0.01.
Fig. 3. Glycyrrhizin mitigates the isoflurane-induced activation of NFjB p65 signaling pathway in the hippocampi of neonatal rats. (A) A graph representative of Western blot analysis of nuclear NFjB p65 expressions in the hippocampus. The statistical analysis showed that application of glycyrrhizin (20 mg/kg, i.p.) mitigated the isoflurane-induced increased nuclear NFjB p65 expressions in the hippocampi of neonatal rats. (B) A graph representative of Western blot analysis of cytosolic NFjB p65 expressions in the hippocampus. The statistical analysis showed that application of glycyrrhizin (20 mg/kg, i.p.) attenuated the isoflurane-induced down-regulations of cytosolic NFjB p65 expressions in the hippocampi of neonatal rats. (C) A graph representative of Western blot analysis of cytosolic IjBa expressions in the hippocampus. The statistical analysis showed that application of glycyrrhizin (20 mg/kg, i.p.) attenuated the isoflurane-induced down-regulations of cytosolic IjBa expressions in the hippocampi of neonatal rats. Data are presented as mean ± SEM (n = 5/group). *P < 0.05 or **P < 0.01. CON: control group; Gly: glycyrrhizin; HMGB1: high mobility group box-1 protein; NF-jB: nuclear factor kappa-light-chain-enhancer of activated B cells; IjBa: nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha; ISO: isoflurane group. 269 270 271 272 273
per group). Additionally, isoflurane exposure also induced decreases in cytoplasmic NFjB p65 and IjBa expressions, whereas these changes could be attenuated by the treatment of glycyrrhizin (Fig. 3B, C, P < 0.05 or P < 0.01, n = 5 per group).
Glycyrrhizin mitigates isoflurane-induced apoptosis in the hippocampus of neonatal rats
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Previous studies have demonstrated that neonatal exposure to isoflurane result in increased
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neuroapoptosis in the hippocampus, which may contribute to isoflurane-induced long-term cognitive deficits (Loepke et al., 2009). Therefore, we test whether glycyrrhizin could reduce isoflurane-induced hippocampal neuroapoptosis in neonatal rats. As shown in Fig. 4A, we found that hippocampal active (cleaved) caspase-3 expressions in isoflurane-exposed rats were higher than those of saline-treated rats (Fig. 4A, P < 0.01, n = 5 per group). However, the treatment of glycyrrhizin (20 mg/kg, i.p.) significantly attenuated isofluraneinduced increase in hippocampal active (cleaved) caspase-3 expressions of neonatal rats (Fig. 4A, P < 0.01, n = 5 per group). In addition, we also detect the effects of isoflurane and glycyrrhizin on the protein levels of pro-apoptotic factor Bax and anti-apoptotic factor Bcl-2 in the hippocampus of neonatal rats (Wei et al., 2005; Wang et al., 2014). As expected, we found that hippocampal Bax protein expressions increased while Bcl-2 protein expressions decreased in neonatal rats exposed to isoflurane (Fig. 4B, C, P < 0.05 or P < 0.01, n = 5 per group). In contrast, the isoflurane-induced effects on hippocampal Bax and Bcl-2 protein expression could be attenuated by the treatment of glycyrrhizin (Fig. 4 B and C, P < 0.05 or P < 0.01, n = 5 per group).
Glycyrrhizin reverses the isoflurane-induced decrease in hippocampal PSD-95 expressions in neonatal rats It has been shown that general anesthetics-induced cognitive deficits are associated with dysfunction in synthesis of vital synaptic proteins (Yan et al., 2012; Wang et al., 2013). To test whether glycyrrhizin can restore the synthesis of vital synaptic proteins in neonatal rats exposed to isoflurane, we detected the effects of isoflurane (1.8%, 4 h) and glycyrrhizin (20 mg/kg, i.p.) on hippocampal protein expressions of PSD-95, SNAP25 and synapsin I in neonatal rats by Western blot analysis. We found that the hippocampal protein levels
of PSD-95 and SNAP-25 in the isoflurane-treated rats were lower than those in the saline-treated rats (Fig. 5A, B, P < 0.05 or P < 0.01, n = 4 per group), whereas the effect of isoflurane on hippocampal protein levels of synapsin I was not observed (Fig. 5C, P > 0.05). In addition, our results showed that glycyrrhizin could restore hippocampal PSD-95 and SNAP-25 protein expressions (Fig. 5A, B, P < 0.05 or P < 0.01, n = 4 per group), but didn’t alter hippocampal synapsin I protein expressions in neonatal rats exposed to isoflurane (Fig. 5C, P > 0.05).
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DISCUSSION
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Our study elucidated that glycyrrhizin protected against isoflurane-induced cognitive impairment in neonatal rats via its anti-inflammatory properties. This effect was identified by the improvement in the performance in MWM trials. Glycyrrhizin prevented HMGB1/NFjB signaling-mediated neuroinflammation that may lead to neurodegeneration and decrease of synaptic protein synthesis in the hippocampus of isoflurane-exposed neonatal rats. The findings support the concept that the treatment of glycyrrhizin inhibition might be effective therapeutic measurement for long-term cognitive impairment induced by neoantal exposure to volatile anesthetics. This work builds on previous findings demonstrating that neonatal exposure to volatile anesthetics induces hippocampal neuroinflammation in neonatal rodents, leading to subsequent cognitive decline (Shen et al., 2013). Therefore, these findings led us to predict that pharmacological targeting on neuroinflammation would be neuroprotective against isoflurane-induced neurotoxicity in the immature brain. It is well established that glycyrrhizin could provide neuroprotective effects in in vitro or in vivo models via its anti-inflammation actions (Cherng et al., 2006; Kim et al., 2008; Zhang et al., 2014). Indeed, our data showed that the treatment of glycyrrhizin reversed isoflurane-induced up-regulation of
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Fig. 4. Glycyrrhizin mitigates isoflurane-induced apoptosis in the hippocampus of neonatal rats. (A) The treatment of glycyrrhizin (20 mg/kg, i.p.) significantly reduced isoflurane (1.8%, 4 h)-induced up-regulation of active (cleaved) caspase-3 in the hippocampus of neonatal rats. (B) Application of glycyrrhizin (20 mg/kg, i.p.) significantly reduced isoflurane (1.8%, 4 h)-induced enhancement of Bax in the hippocampus of neonatal rats. (C) Administration of glycyrrhizin (20 mg/kg, i.p.) significantly attenuated isoflurane (1.8%, 4 h)-induced down-regulation of Bcl-2 in the hippocampus of neonatal rats. Data are presented as mean ± SEM (n = 5/group). *P < 0.05 or **P < 0.01. Bax: Bcl-2-associated X protein; CON, control group; Gly, glycyrrhizin; ISO, isoflurane group. Please cite this article in press as: Wang W et al. Glycyrrhizin attenuates isoflurane-induced cognitive deficits in neonatal rats via its anti-inflammatory activity. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.11.001
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Fig. 5. Glycyrrhizin reverses the isoflurane-induced decrease in hippocampal PSD-95 expressions in neonatal rats. The effects of isoflurane (1.8%, 4 h) and glycyrrhizin (20 mg/kg, i.p.) on hippocampal protein expressions of PSD-95(A), SNAP-25 (B) or synapsin I (C) in neonatal rats were detected by Western blot analysis. (A) Isoflurane exposure induced a significant decrease in hippocampal protein expressions of PSD-95, whereas the decrease was reversed by the application of glycyrrhizin. (B) Isoflurane exposure reduced hippocampal protein expressions of SNAP-25, whereas the reduction was attenuated by the treatment of glycyrrhizin. (C) Isoflurane exposure or the combined treatment of glycyrrhizin didn’t alter the hippocampal protein levels of synapsin I in neonatal rats. Data are presented as mean ± SEM (n = 4/group). *P < 0.05 or **P < 0.01. CON, control group; Gly, glycyrrhizin; ISO: isoflurane group; PSD-95, postsynaptic density protein 95; SNAP-25, synaptosomal-associated protein 25. 353 354 355 356 357 358 359 360 361 362 363 364 365 366
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hippocampal TNF-a and IL-1b in neonatal rats. In consistent with previous studies employing the Morris water maze to assess cognition following isoflurane anesthesia (Callaway et al., 2012; Wang et al., 2014), the memory deficit in isoflurane exposed rats was also observed in the present study, indicated by a longer escape latency/path length, less time spent in the target quadrant and fewer original platform crossings. However, this memory deficit induced by early exposure to isoflurane was significantly improved in the presence of glycyrrhizin treatment. This result is consistent with previous reports showing that glycyrrhizin treatment could attenuate memory deficit in LPS-induced brain injury (Song et al., 2013) and Ab1– 42-induced Alzheimer’s disease (AD) (Zhu et al., 2012) models. Thus, our results indicated that glycyrrhizin treatment could protect against cognitive deficits in neonatal isoflurane-exposed rats via its anti-inflammatory actions. The cognitive deficits induced by neonatal exposure to anesthetics have been attributed to the increased apoptosis and synaptic dysfunctions in the hippocampus (Simon et al., 2001; Xie et al., 2006; Zhong et al., 2015). Accumulated evidence also suggests that neuroinflammation may cause synaptic dysfunction and neuronal cell death (Agostinho et al., 2010; Xuan et al., 2012). Therefore, we hypothesized that the anti-inflammatory activities of glycyrrhizin may also benefit to improve hippocampal cell survival and synaptic function in isoflurane-exposed neonatal rats. Indeed, we found that glycyrrhizin significantly attenuated isoflurane-induced hippocampal apoptosis, as indicated by the decreased expressions of active caspase-3 and pro-apoptotic factor Bax as well as the increased expression of anti-apoptotic factor Bcl-2. These results were in accordance with a previous study showing that glycyrrhizin attenuates kainic acid-induced neuronal apoptosis in the hippocampus (Luo et al., 2013). Furthermore, glycyrrhizin treatment also restored isoflurane-induced down-regulation of hippocampal PSD-95 and SNAP-25 expressions, suggesting that glycyrrhizin could restore hippocampal synaptic pro-
tein synthesis in isoflurane-exposed neonatal rats. These results were in consistent with previous reports that intraperitoneal injection of glycyrrhizin could alleviate the decrease of hippocampal PSD-95 expressions in an AD animal model (Jang et al., 2013). Taken together, our data suggested that glycyrrhizin could attenuate isoflurane-induced apoptosis and synaptic protein loss in the hippocampus of neonatal rats. The mechanisms by which neonatal exposure to anesthesia induces neuroinflammation remain largely unknown. HMGB1 is a highly conserved nuclear protein with a pivotal role in inducing neuroinflammation in various CNS diseases (Kim et al., 2006; Fang et al., 2012). Under pathological conditions, HMGB1 translocates into cytoplasm where it interacts with various cellsurface receptors such as RAGE (receptor for advanced glycation end products), resulting in activation of transcription factor NFjB (Palumbo et al., 2007; Yang and Tracey, 2010). Interestingly, a previous study has indicated that hippocampal HMGB1, TNF-a and IL-1b can be up-regulated by isoflurane anesthesia or surgical stimuli in aged rats, which may contribute to neuronal damage and subsequent cognitive dysfunction (He et al., 2012). Here, our results showed that isoflurane exposure induced the increase of cytosolic HMGB1, IjB degradation and NFjB p65 translocation into nucleus, which are in paralleled with the increase in TNF-a and IL-1b. Remarkably, previous studies have demonstrated that by direct binding to HMGB1, glycyrrhizin can attenuate HMGB1-dependent downstream activation of NFjB signaling pathway and neuroinflammation (Mollica et al., 2007; Ohnishi et al., 2011). Consistently, our results showed that the administration of glycyrrhizin not only reduced the hippocampal production of TNF-a and IL-1b, but also reversed isoflurane-induced HMGB1 cytosolic expression, IjB degradation and NFjB p65 nuclear translocation. Therefore, our results indicated that glycyrrhizin is dependent on the HMGB-1/NFjB signal pathway to exert anti-inflammatory actions.
Please cite this article in press as: Wang W et al. Glycyrrhizin attenuates isoflurane-induced cognitive deficits in neonatal rats via its anti-inflammatory activity. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.11.001
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Although the improvements of memory deficits and the attenuation of neuroinflammation have been found, it is still not conclusive that the behavioral benefits by glycyrrhizin treatments are completely due to its antiinflammatory activity. Besides neuroinflammation, hippocampal b-amyloid deposition (Xie et al., 2007; Zhen et al., 2009) and neurogenesis impairments (Stratmann et al., 2009; Zhu et al., 2010) have also been recognized as important contributors to anesthesiarelated learning and memory abnormalities. Interestingly, previous studies have reported the beneficial cognitive effects of glycyrrhizin by protecting against b-amyloid deposition (Zhu et al., 2012) and promoting neurogenesis (Lei et al., 2013). Therefore, further studies are required to investigate the potential relationship between bamyloid deposition/neurogenesis and the protective effects of glycyrrhizin against learning and memory deficits induced by early anesthesia exposure. The finding in the present study that there was no difference in blood gas parameters between anesthetized and non-anesthetized rat pups may contradict with many other studies. The study by (Murphy and Baxter, 2013) indicated that P7 rats displayed acidosis and hypercapnia after 2 h of 3% isoflurane anesthesia (pure oxygen as carrier gas). In a more specific study by (Stratmann et al., 2009), it was shown that hypercapnia and acidosis developed immediately after induction of isoflurane (1 MAC as determined by tail clamping) anesthesia and a steady decrease over the 4-h anesthesia duration. The discrepancy between their and our results could be attributed to the lower concentration of isoflurane, and the sampling time point for blood gas analysis (the end of the 4-h period of anesthesia) when respiratory depression effects caused by isoflurane were not as evident as immediately after anesthesia induction. Furthermore, our data were consistent with a series of studies from other laboratories (Loepke et al., 2009; Brambrink et al., 2010; Liang et al., 2010), where neither hypercapnia nor acidosis was observed in isofluraneanesthetized animals.
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CONCLUSION
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Our results showed that glycyrrhizin protects against cognitive impairment induced by neonatal exposure to isoflurane via inhibition of HMGB-1/NFjB signal pathway and downstream production of proinflammatory cytokines. These protective effects of glycyrrhizin relate to the attenuation of apoptosis and restore of synaptic protein synthesis in the hippocampus of neonatal rats. Therefore, we believe that treatment of glycyrrhizin could be beneficial in clinical treatment for developmental neurotoxicity and subsequent cognitive decline induced by early exposure to volatile anesthetics.
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CONFLICT OF INTEREST
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None.
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Acknowledgments—The present work was supported by a grant from the National Natural Science Foundation of China (Nos. 81571047, 81271233, 81400882 and 81200880), and also sup-
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ported by 2010 Clinical Key Disciplines Construction Grant from the Ministry of Health of China.
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(Accepted 1 November 2015) (Available online xxxx)
Please cite this article in press as: Wang W et al. Glycyrrhizin attenuates isoflurane-induced cognitive deficits in neonatal rats via its anti-inflammatory activity. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.11.001
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