Beneficial effects of tannic acid on comorbid anxiety in cecal ligation and puncture-induced sepsis in rats and potential underlying mechanisms

Sepsis-associated encephalopathy (SAE), a neurological dysfunction caused by sepsis, is the most common complication among septic ICU patients. Given the major role of inflammation in the pathophysiology of sepsis-induced anxiety, an extreme and early manifestation of SAE, the present study examined whether tannic acid, as an anti-inflammatory agent, has anxiolytic effects in cecal ligation and puncture (CLP)-induced sepsis. Forty male Wistar rats were assigned to four groups: (1) sham; (2) sham + tannic acid; (3) sepsis and (4) sepsis + tannic acid. Sepsis was induced by cecal ligation and puncture model. Animals in the sham + tannic acid and sepsis + tannic acid groups received tannic acid (20 mg/kg, i.p.), 6, 12, and 18 h after the sepsis induction. Twenty-four hours after the sepsis induction, systolic blood pressure and sepsis score were assessed. Anxiety-related behaviors were evaluated using elevated plus-maze and dark–light transition tests. Moreover, inflammatory markers (TNF-α and IL-6) and oxidative stress parameters (MDA and SOD) were measured in the brain tissue while protein levels (GABAA receptors and IL-1β) were assessed in the hippocampus. Administration of tannic acid significantly improved sepsis score and hypotension induced by sepsis. Anxiety-related behaviors showed a significant decrease in the sepsis + tannic acid group compared to the sepsis group. Tannic acid caused a significant decrease in the brain inflammatory markers and a remarkable improvement in the brain oxidative status compared to the septic rats. Tannic acid prevented animals from decreasing GABAA receptors and increasing IL-1β protein levels in the hippocampus compared to the sepsis group. This study indicated that tannic acid mitigated anxiety-related behaviors through decreasing inflammation and oxidative stress and positively modifying IL-1β/GABAA receptor pathway. Therefore, tannic acid shows promise as an efficacious treatment for comorbid anxiety in septic patients.


Introduction
Sepsis as a systemic inflammatory response to infection may lead to multiple organ failure (Slikke et al. 2020). Since the intensive care unit (ICU) is a suitable place to manage organ failure, sepsis is a frequent cause of hospitalization in the ICU, and, here, sepsis-associated encephalopathy (SAE) is the most common complication among the patients (Czempik et al. 2020). SAE is defined as an acute brain dysfunction secondary to sepsis without overt infection of the central nervous system (CNS) and is characterized by neurological symptoms of varying severity, from sickness behavior and delirium to coma (Chung et al. 2020). Importantly, most SAE studies have focused on the most severe symptoms. Nevertheless, since many patients with less severe SAE symptoms survive and are discharged from the ICU, studies should also focus on them, as these symptoms severely affect the patients' functional status and quality of life (Heming et al. 2017).
Anxiety is a manifestation of sickness behavior caused by sepsis, which based on the available evidence occurs significantly in 47% of the patients (Lamar et al. 2011). Although the underlying mechanisms of anxiety comorbidity with sepsis are not fully understood, they appear to involve a combination of several factors, mainly within the limbic system. In the pathophysiology of sepsis, it is suggested that inflammation plays an important role in comorbid anxiety since increased entry of pro-inflammatory cytokines into the brain during sepsis leads to nerve damage (Zaghloul et al. 2017). Also, inflammation, by increasing the production of reactive oxygen species (ROS) and decreasing antioxidant activity, critically contributes to the development and progression of oxidative stress during sepsis and thus exacerbates neuronal damage (Prauchner 2017). Moreover, recent research on SAE has revealed a pathological change in the expression of some receptors such as γ-aminobutyric acid type A (GABAA) receptors which are the predominant inhibitory neurotransmitter receptors in the CNS and mediate the anxiolytic effects (Serantes et al. 2006;Mattei et al. 2019). Interestingly, pro-inflammatory cytokines, especially interleukin-1 beta , have been suggested to play a major role in this change in GABA receptor expression (Arulselvan et al. 2016). Thus, therapeutic modifications of these mechanisms may be promising approaches for alleviating anxiety-related behaviors in septic patients.
Plant-derived antioxidants have long been studied and proven to have anti-inflammatory properties in the context of different diseases (Sharma et al. 2019). Tannic acid is a polyphenol found in a wide range of plants including green tea, coffee, and grapes and has many biological activities (Soyocak et al. 2019). Tannic acid has been shown to have anti-inflammatory and neuroprotective effects (Mohd et al. 2020;Turkan et al. 2019). This natural product is also a potent antioxidant due to its effects on reducing ROS levels and increasing antioxidant enzyme activity (Luo et al. 2020). In addition to these benefits, another important issue with tannic acid is that the compound is recognized as safe by the US Food and Drug Administration (FDA) (Akhondzadeh et al. 2020).
Based on the above findings as well as the fact that anxiety-related behaviors occur early in patients with sepsis, in the current study, we examined these behaviors shortly after induction of an experimental sepsis and then assessed the anxiolytic effects of tannic acid along with its underlying mechanisms.

Animals
In the present study, forty male Wistar rats weighing 250-300 g were used. Animals were placed in the animal laboratory in a controlled environment, a cycle of 12 h of darkness-light and temperature 22 ± 2 °C, with free access to food and water. All experiments were approved by the Ethics Committee of Tehran University of Medical Sciences (Project number: 98-01-139-41,863, Approval ID: IR.TUMS. NI.REC.1398.002).

Establishment of Sepsis by the CLP Model
First, during isoflurane inhalation, the abdominal cavity of animals was exposed by a 2-cm midline incision. Then, separation of the cecum was carefully performed without any injury to the blood vessels. Next, cecum ligation was stoutly made at the base of it with a 4-silk suture. After that, an 18-gauge needle was used to puncture the underside of the ileocecal valve twice. To extrude a small amount of feces into the peritoneal cavity, the perforations were gently pressed. Finally, the incision was closed in two layers by a 4-0 silk suture, and for fluid resuscitation, saline (3 mL/100 g body wt) was injected subcutaneously. Animals were placed into their cages, and 0.86 mg/kg ketorolac was administrated via intra-muscular injection to relieve pain (Pourmirzaei et al. 2021).

Study Design
Animals were randomly assigned to four groups of ten in each: (1) Sham: in this group, the lower part of the animals abdomen was incised in order to expose the cecum without induction of sepsis. (2) Sham + tannic acid: 6, 12, and 18 h after sham operation, animals were given 20 mg/kg tannic acid intraperitoneally (Shrum et al. 2014). (3) Sepsis: animals underwent the CLP surgery. (4) Sepsis + tannic acid: in this group, 6, 12, and 18 h after the sepsis induction, animals received tannic acid (20 mg/kg) via intraperitoneal injection (Shrum et al. 2014).

Sample Collection and Preparation
Twenty-four hours after the CLP surgery in rats, systolic blood pressure was measured by PowerLab Tail cuff system. Then, sepsis score was evaluated 12 and 24 h after the sepsis induction. Next, animal's anxiety-related behaviors were assessed using the elevated plus-maze and light-dark transition tests. Finally, animals were anesthetized via intraperitoneal injection of ketamine hydrochloride (100 mg/kg) and xylazine (10 mg/kg), and the brain tissue and hippocampus were collected for assessment of inflammatory markers (tumor necrosis factor-alpha (TNFα) and interleukin-6 (IL-6)), oxidative stress parameters (malondialdehyde (MDA) and superoxide dismutase (SOD) activity), and protein levels (GABAA receptors and IL-1β).

Measurement of Systolic Blood Pressure
Systolic blood pressure was measured non-invasively by PowerLab Tail cuff system at the end of the study prior to tissue sampling. For this purpose, a rat restrainer was used to place a conscious rat. The animal's tail was cleaned with moistened gauze; then, the tail cuff was located on. Next, the non-invasive blood pressure sensor was placed below the tail-cuff. After deflating the tail cuff, the blood pressure was monitored and recorded for three times consecutively.

Sepsis Score
In all animals, 12 and 24 h after the sepsis induction, sepsis scores were assessed by one of the investigators who was not aware of the experimental groups. Each variable evaluated in this assessment (piloerection, consciousness level, amount of activity, response to auditory stimulus or touch, eyes, respiration rate, and respiration quality) was given a score ranging from 0 to 4 (Pellow and File 1986). There was a positive correlation between the severity of sepsis and the sepsis score amount.

Elevated Plus-Maze Test
In the present study, anxiety-related behaviors were evaluated using the elevated plus-maze (Zuluaga et al. 2005). The instrument consisted of four arms which were 35 cm long and 5 cm wide. The edges of two open arms were 0.5 cm high, and the dark walls of two closed arms were 15 cm high. The maze height from the floor was 50 cm.

Light-Dark Transition Test
The light-dark transition test was used to assess anxietyrelated behaviors (Esterbauer et al. 1991). The box contained two Plexiglas compartments, one of them with white walls illuminated by a 60-W bulb, the other one with the black walls which had no illumination. The size of the compartments was the same (30 × 40 × 40 cm). The time spent in the light side was recorded for 5 min.

Assessment of the Brain Tissue Oxidative Stress Markers (MDA Level and SOD Activity)
MDA level was evaluated according to the Esterbauer and Cheeseman technique (Jones et al. 2005). This measurement was based on the reaction of MDA with thiobarbituric acid which produced a pink substance with the maximum absorption at 532 nm.
To assess SOD activity, an ELISA kit (Navand Salamat Co., Iran) was provided. Briefly, prior to the assay, all materials, reagents, and the brain tissue samples were placed in the room temperature. Then, 100-mg brain tissue was homogenized with 500 μl lysing buffer. Then, this solution was centrifuged at 12,000 rpm for 4 min at 4 °C, and then, collected supernatants were used to evaluate SOD enzyme activity. First, 50-µl supernatants were added to the wells of the micro-titer plate. Then, 50 µl deionized water was added to the control wells. Next, 200 μl R1 reagent and then 50 μl R2 reagent were added to all wells respectively. After that, the ELISA kit was incubated at room temperature away from light for 5 min. The samples' light absorption was read by an ELISA reader at 405 nm. Finally, the level of enzyme activity was calculated using the following formula:

Assessment of the Brain Tissue Inflammatory Markers (TNF-α and IL-6)
The measurement of TNF-α and IL-6 levels in the brain tissues was performed using specific rat ELISA kits (Zellbio Co, Germany). All samples were evaluated in duplicate and according to the instructions provided by the manufacturer. The absorbance of samples was measured by a micro plate reader (Biotek, USA) and then the obtained data were compared to the standard curve, and the concentrations were calculated.

Assessment of the Hippocampal GABAA Receptors and IL-1β Protein Levels
To isolate lysate, the hippocampus was homogenized in lysis buffer (50 mM Tris-HCl pH 7.5, 137 mM NaCl, 0.5% Triton X-100, EDTA-free 1 × complete protease inhibitor mixture, Roche, Massachusetts, USA) and kept on ice for 15 min prior to centrifugation at the maximum speed in an Eppendorf centrifuge at 4 °C for 10 min. The total protein concentration was evaluated by the Bradford's method. The standard plot was provided by using bovine serum albumin. Then, the 12% SDS-PAGE gels (Bio-Rad Laboratories, USA) were used to electrophorese whole proteins and conveyed to polyvinylidene fluoride (PVDF) (Millipore, USA) membranes and probed with GABAA and IL-1β antibodies (ABNOVA, Taiwan) and then placed in secondary antibody (Cell Signaling Technology, USA). Bands were detected by chemiluminescence applying the electro-chemiluminescence reagent kit (Amersham Bioscience, USA). It was provided to recognize immunoreactive polypeptides and consequent autoradiography. The PVDF membranes were stripped and reused by applying an anti-β-actin antibody (Sigma-Aldrich, USA) to normalize protein loading and transfer. The density of bands on the radiography film was quantified by ImageJ software.

Statistical Analysis
All data were presented as mean ± SEM. Sepsis score was analyzed by repeated-measures ANOVA, and the other SOD activity(U∕mg protein) = OD test∕OD control × 200 variables were analyzed by one-way ANOVA followed by Tukey's post hoc test. p < 0.05 was considered statistically significant.

Effect of Tannic Acid Administration on Systolic Blood Pressure in the Septic Rats
Hypotension induced by sepsis is an important and vital parameter in medical examinations. Measurement of this parameter may prevent the patients from the high mortality of sepsis. In this study, systolic blood pressure was assessed by PowerLab Tail cuff system. There was no significant difference in systolic blood pressure in the sham + tannic acid group compared to the sham group (Fig. 1). After the sepsis induction, systolic blood pressure significantly decreased compared to the sham group (p < 0.01; Fig. 1). Treatment with tannic acid in the sepsis + tannic acid group significantly prevented animals from hypotension compared to the sepsis group (p < 0.01; Fig. 1).

Effect of Tannic Acid Administration on Sepsis Score in the Septic Rats
No significant difference was seen in sepsis scores between the sham and sham + tannic acid groups (Fig. 2). Sepsis scores significantly increased in the sepsis group compared to the sham rats, 12 h (p < 0.01; Fig. 2) and 24 h (p < 0.001; Fig. 2) after the sepsis induction. Treatment with tannic acid, 24 h after the sepsis induction, significantly reduced sepsis score compared to the sepsis group (p < 0.01; Fig. 2).

Effect of Tannic Acid Administration on Behavioral Tests in the Septic Rats
There was no significant difference in behavioral tests (elevated plus-maze and light-dark transition tests) in the sham + tannic acid group in comparison with the sham group (Fig. 3).
Analysis of elevated plus-maze data showed that the percentage of time spent in the open arms remarkably decreased in the sepsis group compared to the sham group (p < 0.001; Fig. 3A). Administration of tannic acid in the sepsis + tannic acid group significantly increased the percentage of time spent in the open arms in comparison with the sepsis group (p < 0.001; Fig. 3A).
There was a significant decrease in the percentage of open arm entries in the sepsis group compared to the sham group (p < 0.01; Fig. 3B). Tannic acid significantly increased the percentage of open arm entries in comparison with the sepsis group (p < 0.01; Fig. 3B).  Fig. 1 Effect of tannic acid administration on systolic blood pressure in the septic rats. Twenty-four hours after the sepsis induction in rats, systolic blood pressure was measured by PowerLab Tail cuff system. Data are expressed as mean ± SEM. (n = 6 in each group) **p < 0.01 in comparison with the sham group. ##p < 0.01 in comparison with the sepsis group. In the sham + tannic acid group; 6, 12, and 18 h after sham operation, animals were given 20 mg/kg tannic acid intraperitoneally. In the sepsis group, sepsis induction was performed by the cecal ligation and puncture model. In the sepsis + tannic acid group, 6, 12, and 18 h after the sepsis induction, animals received tannic acid (20 mg/kg) via intraperitoneal injection Fig. 2 Effect of tannic acid administration on sepsis score in the septic rats. In all animals, 12 and 24 h after the sepsis induction, sepsis score was assessed by scoring some items such as piloerection, consciousness level, amount of activity, response to auditory stimulus or touch, eyes, respiration rate, and respiration quality on a scale ranging from 0 to 4. Data are expressed as mean ± SEM. (n = 10 in each group) *p < 0.05 in comparison with the sham group. **p < 0.01 in comparison with the sham group. ***p < 0.001 in comparison with the sham group. ##p < 0.01 in comparison with the sepsis group. In the sham + tannic acid group; 6, 12, and 18 h after sham operation, animals were given 20 mg/kg tannic acid intraperitoneally. In the sepsis group, sepsis induction was performed by the cecal ligation and puncture model. In the sepsis + tannic acid group, 6, 12, and 18 h after the sepsis induction, animals received tannic acid (20 mg/kg) via intraperitoneal injection The differences in total entries were not significant among animals in the sham, sepsis, and sepsis + tannic acid groups (Fig. 3C).
Spending time in the light side significantly decreased in the sepsis group compared to the sham group (p < 0.001; Fig. 3D). Spending time in the light side significantly increased in the sepsis + tannic acid group compared to the sepsis group (p < 0.001; Fig. 3D).

Effect of Tannic Acid Administration on the Brain Oxidative Stress Markers in the Septic Rats
No significant difference was observed in brain oxidative stress markers (MDA and SOD) between the sham and sham + tannic acid groups (Fig. 4).
Sepsis caused a significant increase in the brain MDA level compared to the sham group (p < 0.05; Fig. 4A).
Administration of tannic acid significantly decreased MDA level compared to the sepsis group (p < 0.05; Fig. 4A).
SOD activity decreased in the septic rats compared to the sham group (p < 0.01; Fig. 4B). Tannic acid administration in the sepsis + tannic acid group significantly increased SOD enzyme activity compared to the sepsis group (p < 0.05; Fig. 4B).

Effect of Tannic Acid Administration on the Brain Inflammatory Markers in the Septic Rats
There was no significant difference in brain inflammatory markers (TNF-α and IL-6) in the sham + tannic acid group compared to the sham group (Fig. 5). The inflammatory markers significantly increased in the sepsis group compared to the sham group (both p < 0.001; Fig. 5A and B). Tannic acid administration in the sepsis + tannic acid group In the sham + tannic acid group; 6, 12, and 18 h after sham operation, animals were given 20 mg/kg tannic acid intraperitoneally. In the sepsis group, sepsis induction was performed by the cecal ligation and puncture model. In the sepsis + tannic acid group, 6, 12, and 18 h after the sepsis induction, animals received tannic acid (20 mg/kg) via intraperitoneal injection significantly decreased inflammatory markers compared to the sepsis group (both p < 0.05; Fig. 5A and B).

Effect of Tannic Acid Administration on the Hippocampal GABAA and IL-1β Protein Levels in the Septic Rats
The levels of GABAA receptors and IL-1β proteins were measured in the hippocampus by Western blotting. No significant difference was seen in the levels of GABAA receptors and IL-1β proteins between the sham and sham + tannic acid groups (Fig. 6). Induction of sepsis resulted in a significant decrease in GABAA receptor protein level and a significant increase in IL-1β protein level compared to the sham group (both p < 0.01; Fig. 6A and B). Administration of tannic acid in the sepsis + tannic acid group significantly prevented animals from a decrease in GABAA receptor protein level and an increase in IL-1β protein level compared to the sepsis group (both p < 0.01; Fig. 6A and B).

Discussion
The present study aimed to examine for the first time, to the best of our knowledge, whether tannic acid is able to alleviate sepsis-associated encephalopathy (SAE), in particular anxiety during sepsis. As a starting point, we evaluated the beneficial effects of tannic acid on the typical symptoms of Fig. 4 Effect of tannic acid administration on the brain oxidative stress markers in the septic rats. A Malondialdehyde (MDA) level in the brain tissue samples was assessed by spectrophotometry method. B Superoxide dismutase (SOD) activity in the brain tissue samples was measured by an ELISA kit. Data are expressed as mean ± SEM. (n = 6 in each group) *p < 0.05 in comparison with the sham group. **p < 0.01 in comparison with the sham group. #p < 0.05 in comparison with the sepsis group. In the sham + tannic acid group, 6, 12, and 18 h after sham operation, animals were given 20 mg/kg tannic acid intraperitoneally. In the sepsis group, sepsis induction was performed by the cecal ligation and puncture model. In the sepsis + tannic acid group, 6, 12, and 18 h after the sepsis induction, animals received tannic acid (20 mg/kg) via intraperitoneal injection sepsis. Initially, the systolic blood pressure was recorded non-invasively using a PowerLab Tail cuff system because hypotension is highly suggestive of sepsis as the etiology of shock (Huang et al. 2021). The severity of sepsis was then measured according to a reliable scoring system for rodents since this parameter was a key determinant of SAE (Grondman et al. 2020). Our findings showed that 24 h after the CLP surgery, there was a significant decrease in the systolic blood pressure and a significant increase in the sepsis score, which was considerably reversed by the administration of tannic acid. In the next step, we specifically assessed the protective effects of tannic acid on the anxiety-related behavioral symptoms of sepsis using two well-validated tests of anxiety, the elevated plus-maze and light-dark transition, since anxiety was considered as an extreme and early manifestation of SAE (Heming et al. 2017;Pellow and File 1986;Zuluaga et al. 2005). In our study, a significant increase in anxiety-related behaviors was observed in rats shortly after the CLP surgery, which was interestingly improved by tannic acid administration.
Achieving such promising results from tannic acid exceedingly encouraged us to elucidate some of the underlying molecular mechanisms. In this regard, we began by evaluating the beneficial effects of tannic acid on brain inflammatory status because inflammation has been known to play a major role in the pathophysiology of sepsis. Following infection, a variety of pro-inflammatory cytokines are released into the periphery, which, upon Effect of tannic acid administration on the brain inflammatory markers in the septic rats. A Tumor necrosis factor-alpha (TNF-α) level in the brain tissue samples was measured by an ELISA kit. B Interleukin-6 (IL-6) level in the brain tissue samples was assessed by an ELISA kit. Data are expressed as mean ± SEM. (n = 6 in each group) *p < 0.05 in comparison with the sham group. **p < 0.01 in comparison with the sham group. ***p < 0.001 in comparison with the sham group. #p < 0.05 in comparison with the sepsis group. In the sham + tannic acid group, 6, 12, and 18 h after sham operation, animals were given 20 mg/kg tannic acid intraperitoneally. In the sepsis group, sepsis induction was performed by the cecal ligation and puncture model. In the sepsis + tannic acid group, 6, 12, and 18 h after the sepsis induction, animals received tannic acid (20 mg/kg) via intraperitoneal injection  (Seemann et al. 2017). In the brain, these cytokines cause vascular endothelial damage and activation of microglial cells, resulting in neuron damage (Zaghloul et al. 2017). In addition, pro-inflammatory cytokines secreted by infiltrating immune cells recruit leukocytes to sites of inflammation, leading to complex immune responses and the accumulation of neurotoxic agents, thereby exacerbating neuronal damage (Gonzalez et al. 2014). Our results showed that 24 h after the CLP surgery, there was a significant increase in TNF-α and IL-6 levels in the brain, which was markedly reduced by the administration of tannic acid.
Then, we went to assess the protective effects of tannic acid on brain oxidative stress status since inflammation and oxidative stress have been found to be closely related pathophysiological processes (Ahmad and Ahsan 2020). Oxidative stress is an imbalance between the production of ROS and their removal by antioxidant defense systems (Pisoschi et al. 2021). When compared to other organs, the brain is particularly vulnerable to oxidative damage due to its high oxygen consumption, high lipid content, and small amount of antioxidant enzymes (Bhatt et al. 2020). Under septic conditions, leukocytes recruited to inflammatory sites in the brain generate excessive ROS Fig. 6 Effect of tannic acid administration on the hippocampal GABAA receptors and IL-1β protein levels in the septic rats. A Assessment of the hippocampal GABAA receptor protein level by Western blot technique. B Assessment of the hippocampal IL-1β protein level by Western blot technique. Data are expressed as mean ± SEM. (n = 4 in each group) **p < 0.01 in comparison with the sham group. ##p < 0.01 in comparison with the sepsis group. In the sham + tannic acid group, 6, 12, and 18 h after sham operation, animals were given 20 mg/kg tannic acid intraperitoneally. In the sepsis group, sepsis induction was performed by the cecal ligation and puncture model. In the sepsis + tannic acid group, 6, 12, and 18 h after the sepsis induction, animals received tannic acid (20 mg/kg) via intraperitoneal injection which, in addition to impaired mitochondrial function and thereby limited oxygen delivery, cause lipid peroxidation (McGarry et al. 2018). In our study, a significant increase in the level of MDA, a byproduct of lipid peroxidation, and a significant decrease in the activity of SOD, a potent antioxidant enzyme, were observed in the brain shortly after the sepsis induction, which was considerably reversed by tannic acid administration.
Finally, we wished to evaluate the beneficial effects of tannic acid on hippocampal GABAA receptor protein level because a pathological change in the expression of these receptors has been proposed in the pathophysiology of sepsis, and more importantly, it is well known to be associated with anxiety disorders (Serantes et al. 2006;Mattei et al. 2019). In the GABAergic system, GABAA receptors are a family of ligand-gated ion channels that transmit the effects of the major inhibitory neurotransmitter GABA on fast inhibitory synaptic transmission throughout the brain (Ghit et al. 2021). From the perspective of pathophysiological mechanisms, although unknown, it has been suggested that pro-inflammatory cytokines produced during sepsis play a critical role in altering GABAA receptor expression (Chen et al. 2019). In this regard, the hippocampus, one of the key brain regions involved in modulation of anxiety, seems to be particularly vulnerable to altered GABAA receptor expression following sepsis since it is known to have the highest density of proinflammatory cytokine receptors compared to other parts of the brain (Chen et al. 2019;Gao et al. 2017). Our findings showed that 24 h after the CLP surgery, there was a significant decrease in the expression level of GABAA receptors in the hippocampus, which was markedly increased by the administration of tannic acid. In line with this result, an important question arose as to which of the pro-inflammatory cytokines production was reduced by tannic acid and thus the level of GABAA receptors was increased. Due to both the major involvement of IL-1β in the CNS in response to sepsis and an IL-1β-induced decrease in the function of GABAA receptors (Wang et al. 2000), we assessed the protein level of IL-1β and showed a significant increase in the expression level of this cytokine in the hippocampus, which was considerably decreased by tannic acid administration.
Considering all the beneficial effects of tannic acid mentioned above, an important question that arises is how this compound exerts its biological effects. Tannic acid is a high molecular weight compound that does not readily penetrate into cells or freely cross the blood-brain barrier (BBB). Nevertheless, tannic acid is able to be metabolized to absorbable tannins of much lower molecular weight, which are biologically active in different organs such as the brain. Flavonoids, a family of polyphenols, have been shown to enter the brain in rodents (Mori et al. 2012). Therefore, the flavonoid fraction is a possible candidate for mediating the bioactivity of tannic acid in our system. However, future study is necessary to determine which chemical structure in tannic acid plays an important role in improving comorbid anxiety in sepsis.
Regarding the sham + tannic acid group, it is not surprising that the administration of tannic acid did not make significant differences compared to the sham group, because since tannic acid is a polyphenol found in a wide range of plants, it is unlikely to have harmful effects on the body. On the other hand, under physiological conditions, since there is no oxidative stress in the body, tannic acid as a natural antioxidant is not expected to have beneficial effects.
Considering the fact that there is a correlation between Akt activation and protein level of GABAA receptors, two limitations of the present study were lack of measurement of Akt protein level and GABA concentration in the hippocampus. In addition, since the aim of this study was not to investigate neural plasticity, which is an index of brain function evaluation, the morphological data was not assessed.

Conclusion
The present study demonstrated that anxiety comorbidity with sepsis may occur due to increased inflammation and oxidative stress in the brain, and negative modifying IL-1β/ GABAA receptor pathway in the hippocampus. Nonetheless, treatment with tannic acid was able to ameliorate all the mentioned parameters almost close to the levels measured before the induction of sepsis. Therefore, tannic acid administration may be considered as a potential therapeutic strategy in the context of comorbid anxiety during sepsis. However, further investigations are needed to elucidate the exact mechanisms underlying the beneficial effects of tannic acid.