Pifithrin-μ Attenuates Acute Sickness Response to Lipopolysaccharide in C57BL/6J Mice
Rongping Zhanga Jili Wangb, c Yanling Hub, c Xu Lub, c Bo Jiangb, c
Wei Zhangb, c Chao Huangb
a Department of Endocrinology, Affiliated Hospital of Nantong University, b Department of Pharmacology, School of Pharmacy and c Key Laboratory of Inflammation and Molecular Drug Targets of Jiangsu Province, Nantong University, Nantong, Jiangsu, China
Key Words : Sickness behavior · Pifithrin-μ · Lipopolysaccharide · Mice · Heat shock protein 70
Abstract
Sickness behavior is a coordinated set of behavioral changes that happen as a response to acute infectious pathogens. Its well-known benefit is to reorganize the organism’s priorities to cope with infection, but the uncontrolled development of sickness behavior may trigger negative feelings or chronic depressive events. This study aims at investigating the po- tential effect of pifithrin-μ, an inhibitor of heat shock protein 70 substrate binding activity, on lipopolysaccharide (LPS)- induced sickness response. C57BL/6J mice were submitted to the forced swimming test (FST), tail suspension test (TST), open field test (OFT) and light-dark box test. Food intake and body weight were also evaluated. The serum corticosterone level was measured using an ELISA kit. Treatment of mice with LPS (0.33 mg/kg, i.p.) markedly increased the floating and immobility time in the FST and TST, respectively, and depressed locomotor activity in the OFT. LPS administration prolonged the latency to first transition and reduced the to- tal number of transitions in the light-dark box test. In addi- tion, LPS induced anorexia and increased serum corticoste- rone levels. Pretreatment with pifithrin-μ (1 or 5 mg/kg) attenuated behavioral changes induced by LPS in the FST, TST, OFT and light-dark box test. Pifithrin-μ also prevented the formation of anorexia as well as the increase in serum corti- costerone levels in LPS-treated mice. Our previous studies showed that pifithrin-μ prevents the production of pro-in- flammatory factors in both microglia and macrophages. These findings presented here extend the role of pifithrin-μ beyond an anti-inflammatory molecule to a modulator of sickness behavior.
Introduction
Sickness behavior, a well-established physiological re- sponse triggered by the activation of the innate immune system [1], is characterized by depression-like behaviors such as anorexia, anhedonia, and exploratory behavior and locomotor activity decrease [2]. It can be triggered by the pro-inflammatory cytokines and/or prostaglan- dins released from activated cells in the peripheral or central immune system [3, 4]. Peripherally released in- flammatory cytokines such as interleukin (IL)-6 and tu- mor necrosis factor-α (TNF-α) mainly act on the neuro- nal receptors through the activation of primary afferent nerves or direct diffusion into the brain parenchyma [5,
6]. Cytokines released from the central nervous system mediate sickness behavior through a paracrine effect [1, 4].
As an important physiological adaptive response, sick- ness behavior protects the body against various infec- tions. But if it persists, numerous adverse consequences will occur [7]. For example, exaggerated and prolonged sickness behavior has been shown to induce memory def- icits [8]. Administration of IL-1β or lipopolysaccharide (LPS) significantly impairs spatial and learning memories [9, 10]. The chronic administration of cytokines can in- duce a group of sickness symptoms, including anxiety, cognitive dysfunction and depressed mood [11, 12]. Iden- tifying agents to mitigate these symptoms would be ben- eficial for improving the quality of life of patients with sickness behavior.
Pifithrin-μ (2-phenylethynesul-fonamide), a small compound with strong and effective anti-cancer effects, was first developed by Leu et al. [13]. Pifithrin-μ exerts its pharmacological effects mainly through the suppression of the substrate-binding activity of heat shock protein 70 (Hsp70) [13]. Given the inhibitory role of pifithrin-μ in pro-inflammatory factor production [14, 15], we specu- late that pifithrin-μ may attenuate sickness behavior. To test this possibility, the LPS-induced sickness model in C57BL/6J mice was employed in this study. Results showed that pifithrin-μ markedly attenuates LPS-in- duced sickness symptoms, suggesting that pifithrin-μ may be an appropriate agent that can be selected to at- tenuate sickness behavior.
Materials and Methods
Animals
All experiments were approved by the University Animal Eth- ics Committee of Nantong University (permit number 2110836). Male mice (8–10 weeks) were housed in temperature (23 ± 1 °C) controlled room, and allowed to habituate to the housing facilities for at least 1 week before experiments. Mice had free access to food and water. Behavioral studies were conducted between 9:00 and 11:00 a.m. to avoid circadian variation.
Experimental Procedures
In the testing room, the mice were pretreated with pifithrin-μ or vehicle (i.p.) for 30 min, and then LPS (Saint Louis, Mo., USA,
0.33 mg/kg, i.p.) or saline (0.9% NaCl, i.p.) were injected into the mice. This dosage of LPS has been used in previous studies to in- duce sickness behavior [16]. The behavioral tests were chosen on the basis of previous behavioral, neurochemical and endocrine studies, and performed 2 h after the LPS administration. The dos- es of pifithrin-μ (San Diego, Calif., USA) used in this study have been tested in our previous studies [16].
Forced Swimming Test
For forced swimming test (FST), mice (n = 10) were individu- ally placed into a clear glass cylinder (height 25 cm, diameter 10 cm) filled with 25 °C water to a depth of 16 cm for 6 min. The du- ration of floating was recorded during the last 4-min by an inves- tigator blind to the study. The water was replaced after each trial. Immobility time was defined as the time spent by the mouse float- ing in the water without struggling, and making only those move- ments necessary to keep its head above the water.
Tail Suspension Test
For FST, mice (n = 10) were suspended 50 cm above the floor for 6 min by adhesive tape placed approximately 1 cm from the tip of the tail after 30 min of single drug injection. The duration of immobility was recorded during the last 4-min by an investigator blind to the study. Mice were considered immobile only when they hung passively and were completely motionless, and any mice that did climb their tails were removed from the experimental analysis.
Open Field Test
For open field test (OFT), mice (n = 10) were placed individu- ally in the middle of an open-field apparatus (40 cm height, 100 cm width, 100 cm length) with 25 (5 × 5 cm) squares delineated on the floor in the beginning of this test. The apparatus was illuminated with a red bulb (50 W) on the ceiling. Thirty minutes after single drug injection, mice were placed in the central sector. The squares that each mouse crossed were counted over a 5-min period under dim light conditions by an investigator blind to the study. The open-field apparatus was thoroughly cleaned after each trial.
Light-Dark Box Test
The apparatus consisted of a Plexiglas rectangular box (48 cm length, 24 cm width, 24 cm height) divided into a light region (24 cm length) and a dark region (24 cm length). The light and dark regions were separated by an opening (8.0 × 8.0 cm) allowed the animals to move freely between the 2 compartments. The light portion of this apparatus was made of white Plexiglas with a 60 W light over it. The dark portion was made of black Plexiglas and covered a black lid. During the experiment, each mouse (n = 10) was placed in the light compartment. The behavior was video-recorded for a total of 5 min, and the videotapes were scored for the latency to first transition and the number of transitions between the light and dark compartments.
Measurement of Food Intake and Body Weight
The C57BL/6J mice (n = 10) fasted for 12 h before receiving injections. Immediately after injections, a fresh supply of pre- weighted food was given. Food intake was calculated at 3, 6, 12, and 24 h after the injection by measuring the difference between the pre-weighted chow available and the weight of chow and spilled crumbs at each time point. Changes in body weight were measured by weighing the animals at the beginning of the experiment as well as before and after an experimental day.
Measurement of Serum Corticosterone
To measure serum corticosterone levels, mice were killed by decapitation, and trunk blood was collected 2 h after LPS or saline injections into chilled heparinized tubes. Samples were centri- fuged at 3,000 rpm for 15 min at 4°C, and the serum was removed. Levels of serum corticosterone were determined using an ELISA kit (Assaypro, Kfar Saba, Israel).
Fig. 1. Effects of vehicle or pifithrin-μ (1 or 5 mg/kg) pretreatment on time spent floating in the FST (n = 10, a) and the immobile time in the TST (n = 10, b). The floating (a) and immobile (b) time were measured 2 h after administration of either LPS or saline (** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. vehicle/LPS group). Statistical Analysis All data were presented as the means ± SE. One-way analysis of variance followed by a post hoc LSD test was used for statistical evaluation. p < 0.05 was considered statistically significant. Results Effect of Pifithrin-μ on the Time Spend Floating in the FST and the Immobility Time in the TST In order to investigate whether pifithrin-μ alters the behavior in FST, mice were treated with different doses of pifithrin-μ at 1 and 5 mg/kg. As shown in figure 1a and b, pifithrin-μ at both concentrations did not alter the time spent floating in the FST as well as the immobile time in the tail suspension test (TST) in saline-treated mice. The administration of LPS induced a significant increase in the floating time in the FST (p < 0.01; fig. 1a) and the im- mobile time in the TST (p < 0.01; fig. 1b) in mice pre- treated with vehicle. Pretreatment of mice with pifithrin-μ (1 or 5 mg/kg) markedly reduced the floating and immo- bile time in comparison with the vehicle/LPS group (p < 0.05 or p < 0.01; fig. 1a, b). Effect of Pifithrin-μ on the Locomotor Activity in the OFT We next tested the number of crossings in the OFT after the administration of pifithrin-μ. As shown in figure 2a–c, treatment of mice with pifithrin-μ (1 or 5 mg/kg) did not alter the locomotor activity in the OFT 2 h after saline injection. On the other hand, LPS markedly de- creased the number of line crossings in the periphery (p < 0.01; fig. 2a) and in the central (p < 0.01; fig. 2b) as well as the total number of line crossings (p < 0.01; fig. 2c). Pifithrin-μ pretreatment (1 or 5 mg/kg) significantly re- versed LPS-induced decreases in the number of periph- eral (p < 0.01; fig. 2a) and central line crossings (p < 0.01; fig. 2b) as well as the total number of line crossings (p < 0.01; fig. 2c). Effect of Pifithrin-μ on the Behavior in the Light-Dark Box Test Figure 3 showed that LPS administration markedly in- creased the latency to first transition (p < 0.01), and re- duced the total number of transitions between the light and dark compartments (p < 0.01). Pretreatment of mice with pifithrin-μ (1 or 5 mg/kg) prior to LPS administra- tion caused a significant decrease in the latency to first transition (p < 0.01; fig. 3a) and an increase in the total number of transitions between the 2 compartments, com- pared to the vehicle/LPS group (p < 0.01; fig. 3b). Effect of Pifithrin-μ on Food Intake, Body Weight, and Corticosterone Levels As shown in figure 4a and b, LPS significantly de- creased food intake 3 h (p < 0.01), 6 h (p < 0.01), 12 h (p < 0.01), and 24 h (p < 0.01) after injections and body weight 24 h after injections (p < 0.05). LPS injections (2 h) also induced a significant increase in the serum corticosterone levels (p < 0.01; fig. 4c). Pifithrin-μ pretreatment at the concentration of 1 or 5 mg/kg markedly reversed the hypophagic effect (p < 0.05 or p < 0.01; fig. 4a), the loss of body weight (p < 0.01; fig. 4b), and the increase in the se- rum corticosterone levels (p < 0.01; fig. 4c), compared with the vehicle/LPS group. Treatment of mice with pifithrin-μ (1 or 5 mg/kg) did not alter the food intake (fig. 4a), the body weight (fig. 4b), or the serum corticos- terone levels (fig. 4c) after saline injection. Fig. 2. Effects of vehicle or pifithrin-μ (1 or 5 mg/kg) pretreatment on peripheral (a), central (b), and total (c) line crossings in the OFT (n = 10), which were measured 2 h after administration of either LPS or saline (* p < 0.05,** p < 0.01, vs. control group; ## p < 0.01 vs. vehicle/LPS group). Fig. 3. Effects of vehicle or pifithrin-μ (1 or 5 mg/kg) pretreatment on latency to first transition (a) and number of transitions (b) in the light-dark box test (n = 10 per group), which were measured 2 h after administration of either LPS or saline (** p < 0.01 vs. control group; ## p < 0.01 vs. vehicle/LPS group). Discussion The findings presented here provide evidence that an Hsp70 inhibitor pifithrin-μ efficiently improves explor- atory behavior and locomotor activity, and suppresses anorexia and serum corticosterone increase caused by LPS, suggesting a possible involvement of pifithrin-μ in attenuation of the sickness behavior in numerous pro- cesses such as cytokine administration and chronic neu- rodegenerative disorder therapy.Sickness behavior has been documented in all animal species that have been studied [2]. FST and TST, 2 behav- ioral despair tests, are generally used to examine the exploratory activity of sickness behavior. Our data showed that pifithrin-μ reversed the loss of mice exploration ac- tivity in the FST and TST. The locomotor activity is an- other important index that can be used to evaluate the performance of mice in the FST and TST. Here, LPS ad- ministration depressed locomotor activity in the OFT, and pifithrin-μ treatment reversed this effect. The light- dark box test is a characteristic tool used in the assessment of behaviors associated with anxiety [17, 18]. Our data showed that LPS elicited an anxiogenic-like response in the light-dark box test, exemplified by a significant in- crease in the latency to first transition and a significant decrease in the total number of transitions. Pifithrin-μ remarkably improved these behaviors in the LPS-stimulated mice. Fig. 4. Effects of vehicle or pifithrin-μ (1 or 5 mg/kg) pretreatment on change of food intake (a), body weight (b), and corticosterone serum levels (c), at different time points after administration of either LPS or saline (* p < 0.05, ** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. vehicle/LPS group). Sickness behavior is a well-accepted motivational sta- tus that reorganizes an organism’s priorities to cope with infectious pathogens; however, the mechanisms underly- ing its development remain unclear. Studies in humans and rodents have identified pro-inflammatory cytokines, such as TNF-α and IL-6, as the central mediators of sick- ness behavior [19, 20]. Cytokines diffuse into the brain through visceral sensory pathways or blood-brain-barrier deficient areas [5]. They also act on neurons with the as- sistance of endothelial cell transporters [6]. Our previous studies showed that pifithrin-μ attenuates the production of pro-inflammatory factors in both microglia and mac- rophages [14, 15], demonstrating that the anti-sickness effect of pifithrin-μ may be associated with its prevention on cytokine production. Reduction of food intake is one of the symptoms observed after the overproduction of pro-inflammatory cytokines in the innate immune system. The increase of food intake after pifithrin-μ admin- istration supports the potential role of pro-inflammatory cytokines in pifithrin-μ-mediated attenuation of sickness behavior in the FST, TST, OFT, and light-dark box test. Glucocorticoids have been suggested to play an impor- tant role in organisms coping with unpredictable events; during these events, glucocorticoids are released from the hypothalamic-pituitary-adrenal axis and they are used to maintain the homeostasis of organisms [21]. In this study, animals treated with pifithrin-μ prior to LPS administra- tion displayed a decrease in serum corticosterone levels. Previous studies showed that pifithrin-μ exerts its phar- macological effects mainly through targeting the Hsp70 C terminal substrate binding activity [13], and this targeting dissociates a battery of molecules such as apoptotic prote- ase activating facter-1 (APAF1) and Na+/H+ exchanger-1 from Hsp70 [13–15]. Hsp70 interacts physically with the intracellular glucocorticoid receptor (GR) and facilitates its activation by glucocorticoids [22, 23]. Glucocorticoids and Hsp70 are produced simultaneously in response to repeated stresses [24]. We thus postulate that Hsp70-GR interaction may be associated positively with the release of glucocorticoids under stresses. Pifithrin-μ reverses the in- crease in serum corticosterone under stresses possibly through the disassociation of GR from Hsp70. More stud- ies should be done to test this hypothesis. In summary, our findings show that pifithrin-μ atten- uates behavioral changes as well as anorexia in a mouse model for acute sickness response, and uncover the potential advantage of pifithrin-μ in cytokine-based disease therapy.
Acknowledgments
This work was supported by the Natural Science Foundation of China (No. 81571323) and the Natural Science Foundation of Jiangsu Province to Dr. C. Huang (No. BK20141240) and Profes- sor W. Zhang (No. BK20151276), and the Priority Academic Pro- gram Development of Jiangsu Higher Education Institutions.
Disclosure Statement
The authors declare that the research was conducted in the ab- sence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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