Antidepressant-like Effects of Combined Fluoxetine and Zinc Treatment in Mice Exposed to Chronic Restraint Stress Are Related to Modulation of Histone Deacetylase

Chronic stress is the key factor contributing to the development of depressive symptoms. Chronic restraint stress (CRS) is well validated and is one of the most commonly used models to induce depressive-like behavior in rodents. The present study aimed to evaluate whether fluoxetine (FLU 5 mg/kg) and zinc (Zn 10mg/kg) given simultaneously induce a more pronounced antidepressant-like effect in the CRS model than both those compounds given alone. Behavioral assessment was performed using the tail suspension and splash tests (TST and ST, respectively). Furthermore, the effects of CRS, FLU and Zn given alone and combined treatment with FLU + Zn on the expression of proteins involved in the apoptotic, inflammatory, and epigenetic processes were evaluated in selected brain structures (prefrontal cortex, PFC; and hippocampus, Hp) using Western blot analysis or enzyme-linked immunosorbent assays (ELISA). The results obtained indicated that three hours (per day) of immobilization for 4 weeks induced prominent depressive symptoms that manifested as increased immobility time in the TST, as well as decreased number and grooming time in the ST. Behavioral changes induced by CRS were reversed by both FLU (5 and 10 mg/kg) or Zn (10 mg/kg). Zinc supplementation (10 mg/kg) slightly increases the effectiveness of FLU (5 mg/kg) in the TST. However, it significantly increased the activity of FLU in the ST compared to the effect induced by FLU and Zn alone. Biochemical studies revealed that neither CRS nor FLU and Zn given alone or in combined treatment alter the expression of proteins involved in apoptotic or inflammatory processes. CRS induced major alterations in histone deacetylase (HDAC) levels by increasing the level of HADC1 and decreasing the level of HADC4 in the PFC and Hp, decreasing the level of HADC6 in the PFC but increasing it in Hp. Interestingly, FLU + Zn treatment reversed CRS-induced changes in HDAC levels in the Hp, indicating that HDAC modulation is linked to FLU + Zn treatment and this effect is structure-specific.


Introduction
According to the latest global statistics and the World Health Organization (WHO) report, major depressive disorder (MDD) is the most common mental disorder worldwide. It is characterized by mood and sleep disturbances, lack of interest and concentration, feeling of guilt, and suicidal ideation [1]. MDD pharmacotherapy is based on limited knowledge of its pathomechanism (primarily based on assumptions of monoaminergic theory), which is best has relatively low efficacy (only 30-45% of patients achieve remission) [2].
It is proven that chronic stress is the most critical factor contributing to the development of suicidal and depressive-like behaviors [3][4][5]. Stress, a state of impaired homeostasis Several clinical studies have shown a relationship between dietary zinc deficiency and depression. Clinical studies also suggest the potential benefits of zinc supplementation as an adjunct to antidepressant therapy or as a stand-alone therapy for the prevention of depressive symptoms [34]. The beneficial effects of zinc treatment were also reported in preclinical studies. Zinc induced an antidepressant-like effect (reduction in immobility time) in both the FST and TST [35]. Zinc was also active in different models of depression: olfactory bulbectomy (OB) [36]; CMS [37]; CUS [38]. Furthermore, zinc was found to improve the effects of standard antidepressants (imipramine, fluoxetine, paroxetine, bupropion, or citalopram) on TST in mice; FST and CUS in rats [see [35,39]].
Based on these data, we decided to further evaluate the role of CRS in the induction of depressive-like behavior, proapoptotic and inflammatory processes, and epigenetic changes. Furthermore, Zn supplementation and FLU in the reversion of CRS-induced depressive-like alterations were evaluated. Finally, we decided to check whether zinc supplementation will increase the antidepressant effect of FLU in the CRS model and what mechanisms this combination may trigger.

Establishing the Chronic Restraint Stress (CRS) Protocol Behavioral Studies
The first aim of this study was to establish a standardized protocol for CRS. Therefore, we investigated the induction of depressive effects of CRS in mice using 3-and 6-h daily immobility for 28 days. For this, the tail suspension test (TST) and the splash test (ST) were used to measure the depressive-like effects of CRS. The experimental schedule is shown in Figure 1A. The results obtained indicate that 3 but not 6 h of immobilization/day/28 days increased the immobility time in TST in mice (p < 0.05), exhibiting its depressive-like effects ( Figure 1B). In the ST ( Figure 1C-E), 3 h of immobilization did not reduce latency time (p > 0.05) however decrease in grooming number (p < 0.001), and grooming time (p < 0.0001) was observed. In comparison, 6-hr daily immobility for 28 days decreased the number of grooming (p < 0.001) but did not influence the latency and grooming time (p > 0.05), confirming 3-h as the ideal time for the induction of depressive-like effects in the CRS model in mice.

Effect of FLU Treatment on the CRS Model in Mice
Chronic treatment with FLU at a dose of 10 mg/kg was effective in reversing the CRSinduced increase in immobility time in the TST in mice ( Figure 2B, p < 0.001). Moreover, FLU was found to normalize CRS-induced behavioral alterations in the ST ( Figure 2C-E) such as decreased latency to grooming (p < 0.05), and increased number (p < 0.0001) and time of grooming (p < 0.001). The experimental schedule is shown in Figure 2A  All data were analyzed by two-way ANOVA and Newman-Keuls multiple comparison test. Values are expressed as mean ± SEM; n = 8-9. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. CON, # p < 0.05, #### p < 0.0001 vs. CRS. CON-control group; CRS-chronic restraint stress; Accl.-an acclimation period.

Supplementation with Zn and FLU Treatment in the CRS Model in Mice-Antidepressant Effects
The main part of this work was to investigate whether Zn supplementation enhances the effect of FLU therapy in normalizing depressive-like behavior in the CRS model and describe the mechanisms underlying these effects. Therefore, the effect of chronic (14 days) zinc supplementation (10 mg/kg) to FLU (5 mg/kg) treatment in the CRS model (3 h immobilization/day /28 days) was studied. FLU (5 mg/kg) and zinc (10 mg/kg) given alone decreased the immobility time in the TST in mice subjected to CRS (p < 0.01), in-dicating antidepressant activity of each treatment ( Figure 3B). However, the combined treatment of FLU + Zn induced a stronger effect in TST (p < 0.0001). A significant positive effect of Zn supplementation was evident in the ST ( Figure 3C-E). Treatment with FLU + Zn reversed the CRS induced behavioral changes observed as decreased latency time to grooming (p < 0.01) and an increased number (p < 0.01), and grooming time of grooming (p < 0.001) ( Figure 3C). The experimental schedule is shown in Figure 3A. The two-way ANOVA showed: TST (B): effect of stress: F (1100) = 13.02, p = 0.0005; effect of treatment: F (3100) = 0.0002; but not significant interaction: F (3100) = 2.099, p = 1.051; Splash test: (C) latency: effect of stress F (1100) = 9.121, p = 0.0032, no effect of treatment F (3100) = 2.491, p = 0.0645; and significant interaction F (3100)   To eliminate false positive results in TST, the effects of the CRS procedure and the compounds studied, the general locomotor activity of the mice were measured (Table 1). There were no changes in locomotor activity of mice subjected to CRS (3/6 h/day/28 days) only (A, B, C) FLU (5, 10 mg/kg) alone (B,C); Zn (10 mg/kg) alone, and FLU (5 mg/kg) + Zn (10 mg/kg). One way ANOVA showed F = 1.736, p= 0.1493 Two-way ANOVA showed:  The values represent the mean ± SEM (n = 8-13 mice per group) and were evaluated by one (A) or two-way ANOVA (B, C) followed by the Newman-Keuls multiple comparison test.

Supplementation with Zn and Treatment with FLU in the CRS Model in Mice-Effect on the Level of BDNF Protein
CRS induced a significant decrease in BDNF protein levels in the PFC ( Figure 4A) and Hp ( Figure 4B) of mice (p < 0.001 and p < 0.01, respectively). The CRS induced decrease in BDNF protein level was reversed by FLU (5 mg/kg), Zn (10 mg/kg), and combined FLU + Zn chronic treatment (p < 0.05, p < 0.01, p < 0.05, respectively). Representative western blots are shown in Figure

Zinc Supplementation and FLU Treatment in the CRS Model in Mice-Effect on Pro-and Anti-Apoptotic Protein Levels.
No significant changes were observed in caspase-3, Bid, and Bax protein levels in the PFC ( Figure 5A-D) of mice subjected to CRS. However, the level of the Bcl-2 protein in the PFC decreased significantly decreased ( Figure 5D, p < 0.05). Furthermore, none of the drugs altered the levels of any of the proteins in the Hp ( Figure 6A-D). The representative Western blots are shown in Figure 5E-H (PFC) and Figure 6E-H (Hp).

Discussion
Many different procedures have been described in the literature for the CRS animal model of depression. Generally, immobilization for 2-6 h/once daily /10-28 days was found to induce depressive-and anxiety-like symptoms in rodents [18,23,40,41]. Our laboratory adopted 3-6 h of daily immobilization for 28 days. To evaluate CRS-induced depressive-like symptoms, the TST and ST were used. TST is the most commonly used behavioral test for screening potential antidepressant drugs. However, it is also the standard tool for despair testing [42,43]. One of the symptoms accompanying depression is apathy, defined as a lack of interest in different aspects of life, including normal daily tasks and social activities [44]. In rodents, signs of apathy include impaired self-care measured by the ST. ST consists of sprinkling a 10% sucrose solution on a mouse's coat in its home cage. The viscosity of sucrose solution imitates dirt on the rodent fur and induces grooming behavior. The delay in time between sprinkling and the initiation of grooming and decreased grooming frequency are considered reduced motivational and self-care behaviors associated with the depression-like phenotype [42].
The most prominent depressive effects in our study were observed with the 3-h daily immobilization. Therefore, the 3 h/day/28 days protocol of CRS was chosen for further experiments. The next task was to verify that the depression-like changes induced by CRS will be normalized by treatment with an antidepressant drug. Fluoxetine (FLU), a selective serotonin reuptake inhibitor (SSRI), decreased immobility times in the TST and FST in mice following chronic and acute administration [45]. Chronic treatment with FLU also decreased immobility time in the TST and latency to eat in the novelty-induced hypophagia (NIH) test in adult female mice [46]. Moreover, chronic (21 days) FLU administration reduced depression-like behavior in the social interaction test and FST in the CRS model in mice [47]. Our results mirror those described above. Additionally, in our model, FLU at 10 mg/kg dose reversed all CRS-induced behavioral deficits observed in the TST and ST.
As mentioned above, zinc induced an antidepressant-like effect in the FST and TST in mice [34]. Zinc was also active in different models of depression, such as olfactory bulbectomy (OB), CMS, and CUS. Furthermore, Zn was also shown to improve the effects of standard antidepressants (imipramine, fluoxetine, paroxetine, bupropion, or citalopram) in the TST and CRS in mice and FST and CUS in rats [48,49]. To find out whether Zn supplementation may improve the effect of FLU, a lower dose of FLU (5 mg/kg) was administered jointly with Zn (10 mg/kg) chronically for 14 days. No synergistic effect of Zn + FLU was observed in the TST. All Zn, FLU, and Zn + FLU treatments decreased the CRS-induced increased immobility time in mice. However, the beneficial effect of Zn supplementation was observed in the splash test. Zn + FLU treatment normalized the CRS-induced increased latency to start grooming and extended the amount and time of grooming. This result may suggest that zinc supplementation can improve the antidepressant efficacy of FLU treatment in patients suffering from depression and apathy.
One of the mechanisms of antidepressant action is the modulation of brain-derived neurotrophic factor (BDNF) [50][51][52], a common feature of antidepressant drug action [53]. Our earlier studies showed that the CUS-induced reduction in the BDNF gene expression was antagonized by chronic treatment with zinc [54]. Furthermore, chronic treatment with zinc induced an increase in the BDNF mRNA and protein level in the hippocampus of rats [37]. Finally, our recent studies showed that acute co-administration of IMI and Zn induced antidepressant-like activity in the FST in mice, and this effect correlated with an increase in the level of BDNF protein in the PFC [55]. The modulation of BDNF expression is also associated with the antidepressant action of FLU [56,57]. In the present study, we found that CRS decreased BDNF protein levels in the PFC and Hp of mice; however, the reversal of these changes by FLU, Zn, and Zn + FLU was observed only in the PFC with a trend towards normalization in the Hp. These results further confirm that the mechanism of antidepressant action of Zn and FLU is associated with the modulation of BDNF signaling. However, no synergistic effect of Zn and FLU was seen in the increased BDNF levels.
Inflammation, oxidative stress, and apoptosis are important factors in the pathophysiology of depression and stress-related disorders [58][59][60][61]. The apoptotic process is programmed and controlled by the balance between pro-(Bax) and anti-(Bcl-2) apoptotic proteins within the cell [62]. Data on Bcl-2 and Bax protein levels under restraint stress are inconsistent. In mice, immobility (30 min/28 days) does not change the Bcl-2 protein level; however, the level of caspase-3 increased [63]. Mice stressed for 2 h daily for 21 days (CRS) exhibited an increased Bax/Bcl-2 ratio, caspase-3, IL-1β, and TNF-α in the PFC and Hp [64]. Finally, mice immobilized for 1.5 hr for 7 days showed decreased caspases-3 and no changes in Bcl-2 and Bax protein levels [65]. The only effect observed in our model is the decreased level of the Bcl-2 protein in the PFC, suggesting that CRS-induced apoptotic-like effects are dependent on the duration of stress. However, induction of the antiapoptotic processes is not associated with the antidepressant-like action of FLU + Zn seen in our experiments. The combined administration of Zn and FLU did not cause changes in the level of any of the proteins studied.
There is now ample evidence that the levels of pro-inflammatory cytokines (IL-1β, IL-6, or TNF-α) are elevated in depressed patients and animal models of depression [9,10,66,67]. Interleukins 6 and 1β are inflammatory cytokines, with the former touted as a biomarker for depression [68]. It was found that administration of FLU decreased IL-6 levels in the brain and serum, whereas FLU treatment in the CUMS model decreased IL-6 protein concentration and mRNA level in the Hp [69]. In vitro, Zn (10 and 30 µM) in cell culture decreased TNF-α and IL-6 after LPS administration [70]. We showed that IL-1beta concentration was unchanged in the PFC and Hp, but CRS induced up-regulation of IL-6 in the Hp. Conversely, IL-6 concentration in the PFC decreased following treatment with FLU and Zn alone in control and stressed mice. To summarize this section of our work, CRS for 3 h/28 days induced an inflammatory response in the Hp but not PFC. Zn + FLU treatment does not induce anti-inflammatory effects in the CRS model.
Our data suggest a CRS-induced modulation of deacetylation. CRS increased the levels of HDAC1 protein in the PFC and HDAC1, HDAC6, and Sirt2 proteins in the Hp. However, HDAC4, HDAC6, and Sirt2 protein levels decreased in the PFC and HDAC4 in the Hp. Several works document the level of HDACs in patients with depressive disorders. Peripheral white blood cells of MDD patients exhibited significantly elevated HDAC2 and 5 [71,72] and decreased Sirt1, 2, and 6 protein levels [73]. On the other hand, in the depressive phase in BD patients, increased HDAC4 and decreased HDAC6, 8, and Sirt 1, 2, and 6 protein levels were noted [71,73]. Interestingly, Convington et al. (2009) found decreased levels of HDAC2 in the nucleus accumbens of depressed humans suggesting that peripheral changes in HDAC levels may contradict changes in the brain [74]. Similarly, results from different brain regions in animal models of depression seem contradictory. Most of the available data are related to the social or mild stress procedure. Generally, in these animal models of depression, HDAC2 and HDAC5 were elevated in the Hp but decreased in the nucleus accumbens [27,75]. In the young adult offspring of mice subjected to the gestational stress model, elevated HDAC1 and HDAC2 were correlated with decreased BDNF levels [30]. It is thus not surprising that CRS also decreased the BDNF protein level in our study. Han et al. (2014) confirmed a decrease in HDAC2 and HDAC5 protein levels, but not HDAC 1, 3, and 4 in the Hp after CRS (2 h/14 days) [76]. We showed that HADAC1 increased, but HDAC4 decreased in the Hp and PFC after CRS. HDAC6 is regulated by stress and is elevated in neurodegenerative disorders [77]. Fukada et al. (2012) found that Hdac6 deficient mice exhibit less anxiety in the EPM test and antidepressant-like behavior in the TST, while administration of the Hdac6-specific inhibitor replicated antidepressant-like behavior in mice [77]. This study was confirmed by [78], in which CF (cystic fibrosis) mice with a deletion of HDAC6 restored wild-type growth and inflammatory phenotype. Moreover, decreased levels of HDAC6 impacted mice behavior such that they exhibited anti-depressive properties in the tail suspension test [78]. Our study showed elevated HDAC6 in the Hp of mice subjected to CRS with a decrease in the PFC. The decrease in HDAC6 (and also HDAC2) gene expression in the Hp was also observed by Martin et al. (2017) using CSDS [79].
Sirt2 plays a role in mitochondrial stress and the inflammatory response. In the VGLUT1+/-model of depression, Sirt2 was elevated, and its inhibitor 33i acted as an antidepressant in the sucrose preference test (Munoz-Cobo et al., 2017). Another study with 33i confirmed that inhibition of Sirt2 reversed anhedonia and social avoidance in chronic mild stress [80]. Zhang et al., 2018 found that a lack of Sirt2 blocked the development of stress-induced depressive behavior in social defeat [81]. However, in the CUS model of depression, Sirt2 expression in the Hp decreased and was reversed by fluoxetine treatment [82]. Other authors also confirmed a decrease in hippocampal Sirt2 expression in mice subjected to the CUS and CRS procedures [83]. In contrast, our study showed an increase in Sirt2 protein level in the Hp and a decrease in the PFC. The observed discrepancies may be due to the different stress schedules (2 h/day/14 days vs. 3 h/day/28 days) in both studies. Sirt2 is a member of the HDACIII family and plays a vital role in controlling cell cycle progression, genome stability, and autophagy in neuronal cells. Sirt2 inhibits the transcriptional activation of p53/TP53 and regulates oxidative stress [84]. The increased level of Sirt-2 in the Hp of mice subjected to CRS and the elevated level of inflammatory IL-6 indicated that CRS indeed induced some inflammatory and oxidative stress processes and that this effect is brain region-specific. Chronic treatment with Zn, FLU and Zn + FLU decreased Sirt2 levels in the PFC of naïve mice, indicating that epigenetic modulations might also underlie the antidepressant effect of compounds studied.
The only mechanism that seems to be specific for the effects of Zn + FLU treatment in the CRS model is the modulation of HDACs. Co-administration of FLU + Zn normalized CRS-induced increases in HDAC1 in the PFC and HDAC1, 4, and 6 protein levels in the Hp. In addition, FLU and Zn alone normalized HADC4 levels in the Hp. Furthermore, in the PFC, zinc (not FLU) restored the protein level of HDAC1.
Studies with Balb/c mice indicated that histone deacetylase (HDAC) inhibition enhances the efficacy of FLU. More precisely, treatment with FLU and a class I HDACs (1 and 3) inhibitor elicited antidepressant effects; additional inhibition of class II HDACs was necessary for the anxiolytic effects of FLU. Moreover, the same studies showed that treatment with fluoxetine and various HDAC inhibitors led to significantly enhanced enrichment of acH4K12 at the Bdnf gene promoter 3 and increased expression of Bdnf transcript variant 3 [85]. We can only speculate that the increase in BDNF protein level observed after Zn + FLU treatment might be due to decreased HDAC1 in the PFC. Further studies, e.g., assays measuring the catalytic activity of HADCs are needed to fully unravel the involvement of the modulation of HADCs in the mechanism of the antidepressant-like activity of Zn + FLU treatment.
Despite the different results obtained with the different stress models, CRS in our paradigm appears to be independent of apoptosis but induces inflammation. Despite the limitation mentioned above, our studies suggested that epigenetic processes might underlie the CRS-induced depressive-like effects and antidepressant-like activity of combined Zn + FLU treatment. Our data also indicated that the effects induced both by CRS and Zn + FLU treatment differ between brain structures.

Animals and Drugs Administration
Experiments were carried out on male C57BL/6J mice (~25 g; Charles River, Germany). The animals were kept in a natural day/night cycle at room temperature (21 to 23 • C), with free access to food and water (excluding the restraint period). The mice were housed in groups. After a 1-week acclimatization period to laboratory conditions, mice from stressed groups were subjected to the CRS procedure (3 or 6 h of immobilization/4 weeks) and then to one-day treatment with fluoxetine hydrochloride (5 or 10 mg/kg; i.p.; Tocris) and/or zinc hydroaspartate (10 mg/kg; i.p.; Farmapol) for 14 days. Control groups (CON) received 0.9% NaCl. The injections were carried out between 8:00 a.m. and 2:00 p.m. To verify CRS-induced depressive-like behavior and influence of FLU/FLU + Zn treatment, behavioral tests were performed. At the end of the experiment, the mice were sacrificed to isolate biological material for further research.

Chronic Restraint Stress Procedure
Mice were divided into two groups: Control (not stressed) and stress (subjected to CRS procedure). Control mice were kept in home cages in separate rooms (they did not have contact with stressed animals). Stressed mice were restrained for 3 (8 a.m. to 11 a.m.) or 6 (8 a.m. to 2 p.m.) hours daily for four consecutive weeks using 50 mL tubes enabling easy breathing during the entire period of stress. At the end of each stress session, mice were returned to home cages with unlimited access to food and water. The same steps were repeated every day for 4 weeks.

Splash Test
ST was performed as previously reported [86][87][88]. Each mouse was placed in a clean cage during the test session, and a 10% sucrose solution was spread on the dorsal coat of the animal. Three different parameters were monitored: latency, grooming times; and grooming frequency during a 5-min test period. ST was carried out in a dark room and the cages were lit with a 100 lm bulb. After the test, the mice were returned to their home cages.

Tail Suspension Test
The current study protocol was adapted from [89]. The mice were left hanging from the tip of the tail attached to the flat surface with medical adhesive tape, 30 cm above the floor. The experiment was carried out in dark rooms; otherwise, the rooms were lit centrally by a 100-lumen bulb. The total measurement time was 6 min for each mouse. During this period immobility (defined as freezing and hanging passively without movement) time was measured.

Locomotor Activity
Locomotor activity was measured using an Opto-Varimex-4 Auto-Track (Columbus Instruments, Ohio, USA). The movements of the mice were analyzed in transparent cages (3) covered with lids, a set of four infrared emitters with 16 laser beams each, and four detector tracking and monitoring [90]. Movement detection was analyzed for each mouse separately for 6 min, which is correlated with the TST test. The results were presented as the arithmetic average distance traveled by animals ± SEM for each group.

Western Blot Analysis
Western blot analyzes were performed according to [91]. For Western blot, brain structures such as Hp and the PFC were used. The brains were dissected 24 h after the last drug injection and stored at −80 • C. The total protein level was determined by the BCA method (Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Thirty micrograms of protein samples were loaded onto polyacrylamide gels with 2% SDS and sample buffer (Thermo Scientific Pierce Lane Marker Reducing Sample Buffer). Proteins present in the polyacrylamide gels were transferred to a nitrocellulose membrane, blocked in 1% blocking solution (BM Chemiluminescence Western Blotting Kit Mouse/Rabbit Roche) for 1 h, and incubated with specific primary antibodies overnight in 4 • C [rabbit anti-Bcl-2 (D17C4), rabbit anti-Bax (D3R2M), rabbit anti-Caspase-3, rabbit anti-Bid (Cell Signaling); rabbit anti-BDNF (N-20), mouse anti-HDAC1 (H-11), mouse anti-HDAC4 (A-4), mouse anti-HDAC6 (D-11), mouse anti-HDAC11 (C-5), mouse anti-Sirt2 (A-5) Santa Cruz Biotechnology]. After incubation, the membrane was washed 3 times in TBST for 10 min and incubated with secondary antibodies: anti-mouse IgG-peroxidase-conjugated antirabbit IgG-peroxidase-conjugated antibodies [diluted 1: 7,000; Roche kit (Roche, Mannheim, Germany) for 1 h at room temperature. Before detection, the membrane was washed 3 times for 10 min in TBST, then read on Fuji-Las 1000 system and measured using a Fuji Image Gauge v.4.0. software (FujiFilm, Tokyo, Japan). The proteins were compared to the optical density of β-actin (mouse anti-β-actin antibody Sigma Aldrich (USA)), 1:10,000) run on the same gel.

ELISA Assays
Levels of interleukin IL-1β and IL-6 were measured by commercial ELISA kits (mouse RayBioTech IL-1β and IL-6). Hp and PFC were sonicated in 2% SDS and used in appropriate protocols. The homogenate was diluted 5× and added to the wells, and incubated for 2.5 h. After incubation and washing, the biotinylated antibody was added. The streptavidin solution was prepared according to the manufacturer's protocol (ref). The plates were read immediately read at 450 nm. The IL-1β and IL-6 concentrations were calculated based on the total protein level in each probe.

Statistical Analysis
All values are expressed as mean ± standard error of the mean (SEM). The data were analyzed by repeated or factorial analysis of variance one-way ANOVA, followed by post hoc Bonferroni s or Dunnett s multiple comparisons tests and two-way ANOVA followed by post hoc Tukey multiple comparisons tests (main CRS experiment and Western blot, ELISA analysis). Statistical analysis was performed using the GraphPad Prism software ver. 8. (GraphPad, San Diego, CA, USA) p < 0.05 was considered statistically significant. Data Availability Statement: Data that support the findings of this study are available from the corresponding author upon reasonable request.