Citrate‐coated silver nanoparticles loaded with agomelatine provide neuronal therapy in acute cerebral ischemia/reperfusion of rats by inhibiting the oxidative stress, endoplasmic reticulum stress, and P2X7 receptor‐mediated inflammasome

Cerebral ischemia and reperfusion are related to various situations like injuries after various traumas, oxidative stress, increased calcium ion, capillary hypoperfusion, microvascular hyperpermeability, leukocyte infiltration, and blood–brain barrier disruption. An antidepressant Agomelatine which is a melatonin receptor (MT1/MT2) agonist and serotonin receptor (5‐HT2C) antagonist has been reported by studies to have antioxidant and anti‐inflammatory effects. In our study, we aimed to detect the effects of citrate‐coated silver nanoparticle‐loaded agomelatine application on neurodegeneration, endoplasmic reticulum stress, autophagic and apoptotic cell death, inflammation, and P2X7R expression in the cerebral ischemia–reperfusion model to facilitate the passage of blood–brain barrier. Forty two Sprague–Dawley rats in total were divided into six equal groups (n:7) and applications were performed. Acute cerebral injury in the ischemia–reperfusion model was created 2 h after internal carotid artery ligation in rats and then at the 2nd hour of reperfusion citrate‐coated silver nanoparticles loaded with Agomelatine were applied. Twenty four hours later, neurologic analysis on animals in experimental groups was performed, animals were decapitated and GSH, GPx, SOD, CAT, MDA, IL‐1β, and TNF‐α parameters were examined after taking blood and the cerebral tissue samples. As a result, it was determined that ischemia–reperfusion caused endoplasmic reticulum stress in the cerebral tissues and thus caused cellular injury.

changes.In particular, Ca +2 infiltration takes place from intracellular stores to the cytoplasm.In cells, cytochrome c and apoptosispromoting factors cause inflammation and oxidative stress as a result of excessive Ca +2 . 7,8This situation causes activation of the endoplasmic reticulum which plays an important role in intracellular Ca +2 homeostasis.The endoplasmic reticulum also causes folded protein response and stress, which in turn leads to cell death. 9,10[13] Agomelatine is a melatonin receptor (MT1/MT2) agonist and a serotonin receptor (5-HT2C) antagonist. 14Agomelatine is a circadian antidepressant agent and is the first effective agent with a biological rhythm that regulates sleep efficiency and quality out of the monoamine system.In adults, it has been reported to be an antidepressant developed in particular for depression treatment and also has potential effects in treatments of mood and anxiety disorders. 15Because agomelatine has a high affinity to melatonin receptors as much as melatonin, it has been reported in various studies that it is analog with melatonin-like effects and has even more effective properties. 16,17e presence of melatonin's anti-inflammatory and antioxidant effects has provided the basis for the exploration of agomelatine's melatoninlike potent effects. 18Thus, it has been reported in various studies that agomelatine provides protection by using antioxidant and antiinflammatory effects in various tissues that underwent ischemia and reperfusion injury. 14,19,20Agents or metabolites that could be used to efficiently treat cerebral injury should pass the blood-brain barrier.In recent studies, it was shown that loading nanoparticles to various agents could facilitate the passage through the blood-brain barrier and thus increase their therapeutic efficacy. 21Silver nanoparticles (AgNPs) are the molecules that are being currently investigated as drug carriers and have very low toxicity to the body.In several studies, it was suggested that AgNPs could easily pass the blood-brain barrier. 22,23 addition, AgNPs were reported to suppress inflammation in microglia cells. 24In line with this information, agomelatine, a melatonin analog, was loaded with citrate-coated silver nanoparticles.It aimed to examine its effects on oxidative stress, inflammation, apoptosis, endoplasmic reticulum stress, and P2X7 receptor expression in rats' cerebral ischemia/reperfusion injury model.

| Animals
The protocol of this study was approved by the Kafkas University Animal Experiments and Local Ethics Committee (KAÜ-HADYEK/2022-039).In the experimental study, a total of 42 Sprague-Dawley adult male rats (12-week-old) were obtained from Atatürk University Experimental Research Center (ATADEM) and were divided into equal six groups (n:6).The rats were fed in separate cages, each containing three rats and housed at an ambient temperature of 22 (±2) C, and the light pattern was adjusted to provide a 12-h light and 12-h dark cycle.Rats in all groups were allowed free access to water and food.

| Synthesis, characterization of citrate-coated silver nanoparticles (AgNPs) and interaction with agomelatine
For the synthesis of AgNPs, 0.0167 g of silver nitrate (AgNO 3 ) was dissolved in 100 mL of distilled water.Then, 0.020 g sodium citrate (Na 3 C 6 H 5 O 7 ) was dissolved in 20 mL distilled water in a flask.AgNO 3 was allowed to stir until boiling.Five milliliters Na 3 C 6 H 5 O 7 was added dropwise to the boiling AgNO 3 solution and boiled for about 1 h in a heated magnetic stirrer until a color change was observed.Finally, it was allowed to cool at room temperature and stored ready for use 25 FT-IR, UV, and TEM, spectroscopies were used for the characterization of AgNPs.As a result of the FT-IR analysis, spectra originating from O H groups at a wavelength of 3287 cm À1 were shown.There are strong bands originating from CH groups at a wavelength of 1634 cm À1 , 1275 cm À1 , 1260 cm À1 and 764 cm À1 , 749 cm À1 , 575 cm À1 , 567 cm À1 show stretching vibrations in C O groups and C C groups as well (Figure 1).The strong absorbance of the obtained AgNPs at approximately 400 nm (a typical band for AgNPs) is compatible with the literature (Figure 2A).Also, the shape of AgNPs was determined by TEM. Figure 2B shows the TEM image of AgNPs.In this study, the agomelatine molecule interacted with the negatively charged synthesized citrate-coated silver nanoparticles (AgNPs).For this purpose, Agomelatine was kept in the synthesized AgNPs solution for 3 h.As a result, stable structures with citrate-coated silver nanoparticles were attached to agomelatine molecules by electrostatic attraction (Figure 3). 26

| Experimental design
Forty-two male rats (6-month-old, 240-260 g) were randomly divided into six equal groups, containing seven rats in each group.The groups and the experimental applications to be made to the rats in each group are given in Table 1.

| Experimental transient middle cerebral artery occlusion (tMCAO) model
Transient middle cerebral artery occlusion (tMCAO) was performed using the intraluminal filament procedure according to the previous study. 27In line with this method; briefly, rats were anesthetized with F I G U R E 1 FT-IR spectrum of citratecoated silver nanoparticles (AgNPs).
F I G U R E 2 (A) UV spectrum of citrate-coated silver nanoparticles (AgNPs).(B) The transmission electron microscopy (TEM) spectrum of AgNPs.Rompun (Bayer AG., Germany) and Ketalar (Pfizer Inc., USA) anesthesia (0.01 and 0.04 mg/g per body weight, respectively).The rectal temperature was kept constant at 37 ± 1 C by placing it on the heating pad.A midline cervical incision was made, and the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed and each artery was ligated with cotton silk (2-0) for Cerebral ischemia model.Then, after 2 h of cerebral ischemia, the ligatures were removed with the help of two-knot separators to allow reperfusion of blood from the carotid arteries for 2 h.All surgical procedures were performed under sterile conditions.After the AgNPs and/or Agomelatine administrations, the neurological status of the animals in each group was evaluated, the animals were decapitated 24 h after the reperfusion, and blood and tissue samples were collected for laboratory analyses.

| Evaluation of neurological status in experimental groups
The behaviors of animals were examined neurologically 24 h after the applications.Motor and behavioral changes were evaluated by scoring between 0 and 4 points using previous studies. 28,29According to this scoring; 0: no neurological deficit.1: loss of flexion and strength in the foot opposite the lesion.2: circling in the direction of the loss of strength while walking.3: falling on the side of the power loss due to light pushing.
4: inability to walk spontaneously and decreased level of consciousness.
After evaluation of the neurological status, the rats were euthanized and the brain and blood samples of the sacrificed animals were removed.

| Oxidative stress parameters
Brain tissue samples washed with PBS were lysed with Qiagen Tissue Lyser II at 30 Hz for 3 min by adding liquid nitrogen.Then, a 0.

| Glutatyon peroksidaz (GPx) enzyme activity measurement
The method developed by Beutler was used to determine GPx activity in brain tissues obtained from experimental groups.Glutathione reduced by hydrogen peroxide (GSH) is oxidized to oxidase glutathione (GSSG) by GPx.Then, in the reverse direction, NADPH (nicotinamide adenine dinucleotide phosphate) is used during the reduction of GSSG to the enzyme glutathione reductase.GPx activity is calculated by measuring the decrease in absorbance during the oxidation of NADPH to NADP+ at 340 nm. 33A B L E 1 The groups and experimental protocols.

| Histopathologic analysis
Obtained brain tissues were fixed in 10% natural buffered formaldehyde solution for 24 h, then the tissues were passed graded alcohols series and c, cleaned with xylene, and embedded in paraffin blocks.The blocks were cut at 5 μm thickness and stained with Crossman Modified Triple staining for histopathologic assessment.The slides were evaluated and photographed with Trinocular Microscope (Zeiss, Axio1, German).

| Immunohistochemical analysis
Sections 5-μm thick of paraffin-embedded brain tissue passed graded alcohol and xylene series and were heated for 20 min in a microwave at 600 kWa with 10 mM citrate buffer pH 6.0.Nonspecific binding was blocked with 30% normal goat serum diluted in PBS for 1 h at RT.The slides were incubated with an anti-CHOP primary antibody (AF6277, Affinity Biotech) overnight at 4 C.The sections were then incubated polymer according to the manufacturer's instructions (Labeled Polymer-Dako REAL Envision-HRP K011, Dako, Denmark).
After Staining, the slides were incubated with 3,3 0 -diaminobenzidine for 2 min and counterstained in Mayers hematoxylin for 20 s.For the negative control, an isotype-specific immunoglobulin was used as a substitute for the primary antibody in all experiments; no immunostaining was detected in these sections.

| Stereological immune reactivity analysis
To determine whether immunohistochemical staining was specific, all sections had negative control performed with all processes performed under the same conditions using PBS instead of primary antibody.
Immunohistochemical investigations evaluated the tissue slides under a light microscope (Olympus Bx53) and then photographs were taken.
Semiquantitative analyses for all groups investigated the immunoreactivity degree at the cellular level.The numerical density values of immune reactive cells of lung tissue were determined and calculated using a stereology workstation, consisting of a modified light microscope (Leica DM4000B; Leica Instruments) and stereology software (Microbrightfield Stereo-Investigator software v. 9.0; Microbrightfield, Williston, VT, USA), as described previous study. 35Each section was sampled using the fractionator principle of the stereology software.
Cells were counted using a 40Â Leica Plan Apo objective (NA = 1.40), which allowed accurate recognition.

| Western blot analysis
Before western blot analysis, the acquired brain tissue samples were kept at À80 C in a deep freezer.The brain tissue samples were weighted and crushed in nitrogen gas, treated with radioimmunoprecipitation (RIPA buffer, Ecotech Bio, Turkey) supplemented with protease and phosphatase inhibitors, and homogenized using a tissue lyser device (Qiagen, USA) at 30 Hz for 20 s to determine the relative protein expressions of IL-1β, TNF-α, P2X7R, Caspase-3, Bcl-2, CREB, Nf-kB-p65, LC3, Beclin-1, ATF6 and IRE1.A protein assay kit was used to quantify brain tissue's total protein (Pierce BCA, Thermo Sci., USA).
Thirty micrograms of protein were then put into the PVDF membrane after being separated by 10% SDS-PAGE.First, at room temperature, 5% bovine serum albumin was used to block the membranes for 90 min.Then, the membranes were incubated at 4 C overnight with the appropriate primary antibodies.After primary antibody incubation, the PVDF membranes were washed with TBST and then incubated for an additional 90 min at room temperature with the second antibody (Santa Cruz, sc-2004/sc-2005) coupled to horseradish peroxidase.
Then, the protein bands were captured using the enhanced chemiluminescence reagent Western ECL substrate (Thermo, 3405), visualized and analyzed by Image Lab™ Software (Bio-Rad, Hercules, CA, USA). 3 | RESULTS

| Neurological deficit score
As a result of the neurological examination in the experimental groups, there was a statistically significant difference between the sham and IR groups in terms of mean neurological deficit ( p < .05).
There was no change in the Sham and AgNPs groups.The neurological deficit score in IR and AgNPs+IR groups was higher than all other groups ( p < .05).It was observed that the application of agomelatine loaded with agomelatine and citrate-coated silver nanoparticles reduced the neurological deficit (Figure 4).

| Oxidative stress parameters
In the oxidative stress parameters, it was observed that the MDA level was similar in the Sham and AgNPs groups, while the MDA levels were significantly the highest in the IR group (p < .05).In addition, a significant decrease was determined in the Ago + IR and AgNPs + IR and Ago + AgNPs + IR groups compared to the IR group, but no significant difference was determined among these groups.SOD, GSH, GPX, and CAT levels were similar in the Sham and AgNPs groups, but there was a significant decrease in the IR group.It was determined that it was significantly higher in the Ago + AgNPs + IR group than in the IR group ( p < .05),(Table 2).

| Serum Inflammation parameters
In the serum obtained from the experimental groups after the study, the TNF-α and IL-1β levels were low in the Sham and AgNPs groups TNF-α and IL-1β levels were similar in Sham and AgNPs groups.While these levels were highest in the IR group compared to other groups (p < .05).In addition, a significant decrease was observed in the Ago + IR and AgNPs + IR and Ago + AgNPs + IR groups compared to the IR group, but no significant difference was found among them (Figure 5).

| Histologic analysis results
In the histopathological evaluation, no histopathological abnormalities were observed in the Sham and Ago + AgNPs groups, and the sections did not show clear cell contours, integrated tissue structures, prominent nucleoli, and intracellular edema in the cerebral cortex.
However, many cells in the penumbra of the ischemic cerebral cortex area in the Ago + IR, IR, and AgNPs + IR groups showed shrinkage with deeply stained and condensed nuclei (karyopycnosis) and additionally, edematous cells, loose cytoplasm, and severe cell deformation were also observed.On the other hand, in the Ago + AgNPs + IR group, edematous cell density, loose cytoplasm, and cellular deformations were decreased compared to the IR group.The histopathologists of all groups are shown in Figures 6 and 7.

| Immunohistochemical analysis
In the stereological evaluation of immune reactive cell densities,  Note: All data were expressed as mean ± SD.The letterings show the statistical difference between the groups in the same column.
lower in the Sham and Ago + AgNPs groups but significantly upregulated in the IR group, with its expression levels being the highest ( p < .05).In the Ago + IR, AgNPs + IR, and Ago + AgNPs + IR groups, the CHOP expression was significantly upregulated compared with the Sham and Ago + AgNPs groups (Figure 8 and Table 3) but downregulated considerably compared with the IR group ( p < .05).
The immune reactivity of CHOP was observed to be widely expressed around the hippocampus.

| Western blot analysis results
In evaluating brain tissue's relative protein expression levels, the ATF6 expression level was low in the Sham and AgNPs groups, and there was no significant difference between them.While LC3, IRE1, TNF-α, Caspase-3, IL-1β, Beclin-1, and NfkB-p65 expressions were similar in Sham and AgNPs groups, these levels were found to be significantly highest in the IR group (p < .05).In addition, a significant decrease

| DISCUSSION
Ischemic stroke is a significant public health problem with high morbidity and mortality. 36It is suggested that neurological problems may occur because of insufficient blood flow to the brain during the stroke. 37The cerebral ischemia-reperfusion model is a good example of the mechanism that operated during the stroke.In the cerebral ischemia model, the amount of energy metabolites like ATP and phosphocreatine decreases secondary to disruption of glucose metabolism, and lactate concentration increases depending on reduced blood flow to the cerebral tissues.As a result of depleted ATP, the cell membrane is depolarized, and thus permeability is impaired: Intracellular Na, Ca, Cl, and extracellular K concentrations increase. 38In addition, reperfusion after ischemia is a complex process that starts with tissue hypoxia and continues as an inflammatory response in which free radicals and the immune system play a role, resulting in organelle injury and apoptosis. 39,40Various studies suggested that agents that could pass the blood-brain barrier and have antioxidant and anti-inflammatory effects could prevent oxidative injury, inflammation, and apoptosis. 41,42With this study, we aimed to reveal the effects of citratecoated silver nanoparticle-loaded agomelatine on oxidative stress, inflammation, apoptosis, endoplasmic reticulum stress, and P2X7 receptor levels during the cerebral ischemia-reperfusion injury.
Oxidative stress is a process where a tissue injury develops due to an imbalance between the oxidant and antioxidant defense system due to excessive production or decreased clearance of reactive oxygen metabolites of metabolic processes. 43In an ischemia-reperfusion state, severe oxidative stress develops in tissue as a result of both undernourishment of tissues and insufficient blood flow.Here, one of the basic mechanisms of oxidative stress is increased MDA levels as a result of lipid peroxidation, decreased superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione (GSH), and catalase (CAT) enzyme activities that indicate antioxidant affectivity in tissues. 44,45 et al., in their study, reported that MDA increases, and CAT, SOD, GSH, and GPx decrease in the cerebral ischemia-reperfusion state. 46r study findings are similar to this study.We found that MDA increased, and CAT, SOD, GSH, and GPx decreased in the cerebral tissues of rats of the IR group.On the other hand, only agomelatine and citrate-coated silver nanoparticle-loaded agomelatine applications decreased MDA and increased CAT, SOD, GSH, and GPx in the cerebral tissues.In line with these findings, we concluded that agomelatine is protective against oxidative stress caused by acute cerebral ischemia-reperfusion, and citrate-coated silver nanoparticle-loaded agomelatine is more effective than agomelatine.
ER stress has an important role in the pathogenesis of ischemia and reperfusion injury.Studies have proven it. 47,48Decreasing ER stress is an effective method to prevent or treat IR-induced cerebral injury.ER stress is a process where unfolded or misfolded proteins protect cellular homeostasis.ATF6 and IRE1 are sensor proteins that show UPR.At the end of ER stress, PERK, IRE1, and ATF6 activations increase. 49,50This increased activation in turn increases the level of a proapoptotic protein, CHOP. 51In a study with rats, it was shown that experimental IR causes an increase in GRP78, ATF-6, PERK, and IRE1 expressions, thus stimulating CHOP.On the other hand, acupuncture treatment was reported to decrease previously mentioned stress markers. 52Our study findings are parallel to this study and indicate that ATF-6, IRE1, and CHOP expressions in the IR group are significantly higher than in other groups, and decrease in agomelatine groups.
Autophagy is a biological process that is under the influence of various genes and includes the lysis of impaired organelles and macromolecules through lysosomes.LC3 and Beclin-1 genes are markers of autophagy.As a result of various studies, it was suggested that cerebral ischemia-reperfusion induces autophagy and thus causes injury in the cerebral tissues. 53Various studies have proved the relationship between ER stress and autophagy. 54,55In our study, we determined that LC3 and Beclin-1 gene expression levels increase in the cerebral tissues of rats belonging to the IR group and decrease in agomelatine and citrate-coated silver nanoparticle-loaded agomelatine-applied groups.This finding is compatible with other studies.ER stress induced by IR injury is also related to apoptosis 47 ER stress-related caspase-12 and CHOP are the proteins that regulate apoptosis. 56In addition, it has been shown that activation of the PERK/eIF2α/ATF4 pathway decreases Bcl-2 protein expression, and then results in increased CHOP expression that leads to caspase-3 activation. 57 our study, we determined that Caspase-3 expression increases and BCL-2 expression decreases in the cerebral tissues of rats belonging to the IR group, and Caspase-3 expression decreases and BCL-2 expression increases in agomelatine-applied groups.The findings of various studies are in line with the fact that IR-induced ER stress triggers cellular inflammation.In a study, it was shown that ER stress activates the NF-κβ signal pathway that supports myocardial I/R injury and in turn, activates nucleotide-binding oligomerization domain-like receptor protein 1 (NLRP1), 58 and thus, causes proinflammatory and inflammatory cytokine (TNF-α and IL-1β) formation and secretion. 59 our study, we determined that the NFκβ-p65, TNF-α, and IL-1β expressions increase in the cerebral tissues of rats belonging to the IR Increased intracellular Ca 2+ in the cerebral ischemia activates some pathways and causes CREB-related transcription of genes like Bcl-2 and BDNF that protect from CREB phosphorylation and ischemia-induced neuronal death.Tanaka et al found increased CREB phosphorylation in the penumbra region around the ischemic nucleus using the cerebral ischemia-reperfusion model.60 In a global ischemia model that was formed with carotid artery ligation, similar results were obtained.61 In this case, CREB phosphorylation is increased in hippocampal dental gyrus (DG) but decreased in Cornus Amonis 1 (CA1) neurons that are sensitive to ischemic cell death.These findings indicate a correlation between the ability to maintain CREB phosphorylation and ischemic resistance.However, activation of the AKT/CREB signal pathway has provided improvement in many ischemia models by increasing anti-apoptotic and neurotropic factors.62 Accordingly, many protective drugs in ischemic models also induce the activation of this pathway.63,64 In our study, we determined that CREB expression decreases in the cerebral tissues of rats belonging to the IR group and increases in agomelatine and citrate-coated silver agomelatine-applied groups.This finding is compatible with previous studies and is proof that agomelatine application has cerebral protective effects against ischemic cerebral injury.
Purinergic receptors are the receptors that are first expressed in microglia and macrophages, have major effects on the central nervous system, and are activated by ATP. 65After the activation, these receptors regulate the secretion of the pro-inflammatory cytokine, interleukin-1 (IL-1) 66 Interleukin-1 is present as IL-lα and IL-1β is the main mediator of experimentally induced neurodegeneration. 67So, inhibition of IL-1 considerably limits neuronal injury secondary to cerebral ischemia. 68The P2X7 receptor plays an important role in IRinduced cerebral injury and serves as an important therapeutic target.
In various studies, it has been shown that IR injury in the cerebral and other tissues increases tissue P2X7R expression and various therapeutic agents suppress inflammation by decreasing P2X7R expression 69,70 Our findings are compatible with other studies.In our study, we determined that P2X7R expression increases in the cerebral

2. 7 . 1 | 30 2. 7 . 2 | 31 2. 7 . 3 |
Superoxide dismutase (SOD) enzyme activity measurementThe method is based on the principle of inhibition of free radicals by the enzyme superoxide dismutase during the reduction of free oxygen radicals released by enzymatic reaction in the presence of Nitrobluetetrazolium (NBT) in the sample.The color change observed as a result of the reaction was measured at 560 nm with a spectrophotometer.Measurement of MDA levels Lipid peroxidation (LPO) measurement was performed by Ohkawa et al.31 Preparation of Homogenate: By adding 2.5 mL of 10% KCl to the ground tissue with 25 mg nitrogen, the mixtures were homogenized with Tissue Lyzer LT homogenizer at 30 Hz for 3 min.The homogenates were centrifuged at 4000 rpm, 4 C for 30 min, and These supernatants were read Absorbance values at 532 nm against a blank, and tissue MDA concentration was calculated using the formula: Tissue MDA (μmol/g protein): MDA (μmol/L)/Tissue protein (g/L).The determination of malondialdehyde (MDA) is based on the principle of measuring the absorbance of the pink-colored compound formed by the reaction of thiobarbituric acid (TBA) and MDA at a wavelength of 532 nm.Measurements of tissue GSH levels Glutathione (GSH) levels in brain tissues of rats were determined by spectrophotometric method according to Sedlak et al.32 The principle of the method is that the color intensity of dark yellow 5-thio2-nitrobenzoic acid (TNB) is 412 nm, which is revealed by the reduction of Ellman's reagent (5,5 0 -dithiobis (2-nitrobenzoic acid); DTNB] by free thiol groups.wavelength (2 GSH + DTNB !G-S-S-G + 2 TNB (dark yellow color)) Homogenate buffer: 50 mM pH 7.4, Tris-HCl (1.514 g Tris dissolved in 200 mL distilled water pH 7.4 The final volume was completed to 250 mL with distilled water.)The obtained values were evaluated by comparing between the groups.
Statistical analysis was performed using SPSS (version 25.0;IBM SPSS Inc, Chicago, IL, USA) package program.The normality of data was determined with the Kolmogorov-Smirnov test.Descriptive statistical analyses (mean ± SD) were used.One-way ANOVA test and post hoc Tukey test were performed to compare groups.P values less than 0.05 at the 95% confidence interval were considered statistically significant.

5
Serum TNF-α and IL-1β levels of all groups.The values are given as mean ± SEM (n = 7) and analyzed by one-way ANOVA followed by the Tukey test.Different letters indicate statistically significant differences, p < .05.

F I G U R E 7
Illustration of histopathological changes in rat cerebral tissues of rats, the arrows indicate the circle indicates the edematous cell density, loose cytoplasm, and cellular deformations (Crossman modified Triple staining).groupand decrease in agomelatine-applied groups.The fact that these changes are in line with ER stress markers proves that IR-induced ER stress is related to autophagy, apoptosis, and inflammation, while agomelatine and citrate-coated silver nanoparticle-loaded agomelatine applications treat IR-induced ER stress and thus prevent autophagy, apoptosis and inflammation in cells.

F I G U R E 8
Representative Immunohistochemical staining images of anti-CHOP in the cerebrum of rats after IR model, (Streptavidinperoxidase staining).T A B L E 3 Stereological evaluation of immune reactive cell densities of CHOP in the brain tissues of rats for all groups.

5 | 9
CONCLUSIONBased on our findings, we thought that citrate-coated silver nanoparticle and/or agomelatine reduces oxidative stress, and by the way, alleviates the cerebral I/R injury by inhibiting ER stress through IRE1 and ATF6 signal pathways.In addition, citrate-coated silver nanoparticle and/or agomelatine treatments suppress autophagy, inflammation, and apoptosis depending on ER stress during cerebral I/R injury.Furthermore, citrate-coated silver nanoparticles and/or agomelatine reduced the P2X7 receptor expression in the cerebral I/R injury.Thus, our findings shed light on the protective mechanisms of citrate-coated silver nanoparticles and/or agomelatine in ischemic stroke treatment.Relative expression of proteins for ATF6, LC3, BECL _ IN-1, P2X7R, IRE1, Bcl-2, TNF-α, IL-1B, CREB, Caspase-3, Nfkb-p65.The values are given as mean ± SEM (n = 7) and analyzed by one-way ANOVA followed by the Tukey test.Different letters indicate statistically significant differences, p < .05.
The method developed by Aebi was used to determine the Catalase activity in the brain tissues obtained from the experimental groups.
levels were measured using commercial ELISA kits to determine inflammation in serum samples obtained from the experimental groups.
Biochemical parameters of oxidant and antioxidant parameters for all groups.
Macroscopic view of brains of rats after IR model.wasdetermined in the Ago + IR and AgNPs + IR and Ago + AgNPs + IR groups compared to the IR group.Still, no significant difference was determined among these groups regarding LC3, IRE1, TNF-α, Caspase-3, IL-1β, Beclin-1, and NfkB-p65 expression levels.The P2X7R expression level was similar in the Sham and AgNPs groups, but a significant increase was determined in Ago + IR, IR, and AgNPs + IR groups.In the Ago + AgNPs + IR group, it was determined that Ago + IR was significantly lower than the IR and AgNPs + IR groups ( p < .05).Bcl-2 expression was the lowest in Ago + IR, IR,