Olanzapine Ameliorates Ischemic Stroke-like Pathology in Gerbils and H2O2-Induced Neurotoxicity in SH-SY5Y Cells via Inhibiting the MAPK Signaling Pathway

Olanzapine (OLNZ) is used to treat psychotic disorders. To look into the neurological basis of this phenomenon, we investigated the neuroprotective effects of OLNZ in gerbils and SH-SY5Y cells. Gerbils were subjected to transient global cerebral ischemia (TGCI) by blocking both common carotid arteries, and OLNZ (10 mg/kg) was injected intraperitoneally. Hydrogen peroxide (H2O2) was used to induce oxidative-stress-mediated damage in the SH-SY5Y cells. The results indicated that OLNZ administration markedly reduced neuron damage and glial cell triggering within CA1 zone of the hippocampus. We used RNA sequencing to assess the numbers of up-and downregulated genes involved in TGCI. We found that OLNZ treatment downregulated the expression of complement-component-related genes and the expression of mitogen-activated protein kinases (MAPKs) in the hippocampus. In cells, OLNZ co-treatment significantly improved cell viability and reduced lactate dehydrogenase (LDH), and reactive oxygen species (ROS) generation. Expression of antioxidant superoxide dismutase-1,2 enzymes (SOD-1, SOD-2) was also intensely upregulated by OLNZ, while the expression of MAPKs and NF-κB were reduced. Co-incubation with OLNZ also regulated apoptosis-related proteins Bax/Bcl-2 expression. Finally, the results demonstrated that treatment with OLNZ showed neuroprotective effects and that the MAPK pathway could involve in the protective effects.


Introduction
Cerebral-ischemia-mediated ischemic stroke is a frequently occurring disorder due to sudden obstruction of the blood supply to the cerebrum. Cerebral ischemic injury leads to several cellular alterations that are influenced by free radical generation, excitotoxicity, and metabolic disorder [1][2][3]. Neuronal cell death and glial cell activation in the ischemic penumbra are considered the main cellular features of cerebral ischemic insult that could be targeted by administration of neuroprotectants within 6 h of induction of stroke [4].
The balance between intracellular oxidation and reduction (i.e., redox status) is an important physiological parameter of cells [5]. When this balance is altered by abnormal stimuli, cells may undergo oxidative stress, leading to the production of a huge amount of reactive oxygen species (ROS) by mitochondria [6]. Excessive production of ROS (including the superoxide radical, O 2 · − ) contributes to neuronal cell damage by disrupting cell membranes, damaging DNA, and triggering cellular apoptosis [7][8][9]. Antioxidant superoxide dismutase enzymes (SODs) are able to convert O 2 · − into hydrogen peroxide

Method of TI Induction in Gerbils
We implemented a technique to induce TI in gerbils that was described previously [35,36]. In brief, inhalation anesthesia was implemented with a combination of nitrous oxide, oxygen, and isoflurane (68%, 32%, and 2.5%, respectively). Then, the surgical site (midline of the neck) was cleaned, and an incision was made to expose the bilateral common carotid arteries (BCCA). The BCCA were isolated from surrounding muscle and nerve fibers and blocked for 5 min using aneurysm clips (Yasargil FE 723K, Aesculap, Tuttlingen, Germany). Immediately after the obstruction period, OLNZ was injected intraperitoneally (10 mg/kg body) in the TI + OLNZ treatment group.

Tissue Processing for Histological Study
As described in a previously established investigational procedure [35], 5 days after induction of TI, all gerbils in the experiment were sacrificed using 30% urethane. Immediately following euthanasia, intracardial perfusion by 0.1 M PBS, pH 7.4 followed, using 4% paraformaldehyde (PFA) for tissue fixation. All brains were carefully removed from the brain cavity in an intact state and transferred to a conical tube containing 4% PFA for 12 h. After the fixation period, the 4% PFA was changed, and 30% sucrose solution was added overnight. A cryostat (CM1900 UV, Leica, Wetzlar, Germany) device was used to section the brain into 30-µm-thick sections.

Cresyl Violet Staining
To assess neuronal cell destruction induced by TI in the brain, cresyl violet (CV) staining was implemented. Brain sections were placed on pre-coated gelatin slides. Cresyl violet acetate (Sigma, MO, USA) subjected to solved in distilled water (DW) and glacial acetic acid. Slides were dipped into the CV solution for staining and successively submerged into increasing concentrations of ethanol for dehydration. The stained slides were observed under a microscope.

Fluoro-Jade B Histofluorescent Stain
Degenerating neurons in the brain sections was assessed using Fluoro-Jade B (F-J B) staining, as described previously [37]. Briefly, slides of hippocampal sections were immersed in a 0.06% potassium permanganate solution (dissolved in DW) for 20 min. Subsequently, the slides were washed with DW and then dipped into a 0.0004% solution of

Method of TI Induction in Gerbils
We implemented a technique to induce TI in gerbils that was described previously [35,36]. In brief, inhalation anesthesia was implemented with a combination of nitrous oxide, oxygen, and isoflurane (68%, 32%, and 2.5%, respectively). Then, the surgical site (midline of the neck) was cleaned, and an incision was made to expose the bilateral common carotid arteries (BCCA). The BCCA were isolated from surrounding muscle and nerve fibers and blocked for 5 min using aneurysm clips (Yasargil FE 723K, Aesculap, Tuttlingen, Germany). Immediately after the obstruction period, OLNZ was injected intraperitoneally (10 mg/kg body) in the TI + OLNZ treatment group.

Tissue Processing for Histological Study
As described in a previously established investigational procedure [35], 5 days after induction of TI, all gerbils in the experiment were sacrificed using 30% urethane. Immediately following euthanasia, intracardial perfusion by 0.1 M PBS, pH 7.4 followed, using 4% paraformaldehyde (PFA) for tissue fixation. All brains were carefully removed from the brain cavity in an intact state and transferred to a conical tube containing 4% PFA for 12 h. After the fixation period, the 4% PFA was changed, and 30% sucrose solution was added overnight. A cryostat (CM1900 UV, Leica, Wetzlar, Germany) device was used to section the brain into 30-µm-thick sections.

Cresyl Violet Staining
To assess neuronal cell destruction induced by TI in the brain, cresyl violet (CV) staining was implemented. Brain sections were placed on pre-coated gelatin slides. Cresyl violet acetate (Sigma, St. Louis, MO, USA) subjected to solved in distilled water (DW) and glacial acetic acid. Slides were dipped into the CV solution for staining and successively submerged into increasing concentrations of ethanol for dehydration. The stained slides were observed under a microscope.

Fluoro-Jade B Histofluorescent Stain
Degenerating neurons in the brain sections was assessed using Fluoro-Jade B (F-J B) staining, as described previously [37]. Briefly, slides of hippocampal sections were immersed in a 0.06% potassium permanganate solution (dissolved in DW) for 20 min. Subsequently, the slides were washed with DW and then dipped into a 0.0004% solution of Fluro-Jade B (Histochem, Jefferson, AR, USA) (dissolved in DW and acetic acid) for 45 min. The tissue sections were dehydrated in an oven (60 • C) and mounted by dibutyl phthalate polystyrene xylene (DPX; Sigma, St. Louis, MO, USA).

RNA Isolation and Whole Transcriptome Sequencing (RNAseq)
Total RNA isolation from the hippocampal area of the brain (n = 3/group) and cells was executed using RiboEx™ total RNA isolation kits (GeneAll, Seoul, Korea), respectively, followed by manufacturer's indications. A Biospec-nano spectrophotometer was used to determine the quantity and quality of retrieved RNA (Shimadzu Biotech, Tokyo, Japan). Oligo (dT) magnetic beads were used to purify and make a fragment of mRNA molecules from total RNA. The fragmented mRNAs were used to synthesize cDNAs, and cDNA libraries were amplified using a polymerase chain reaction (PCR). The cDNA libraries were measured by an Agilent 2100 BioAnalyzer (Agilent, Santa Clara, CA, USA) and KAPA library quantification kit (Kapa Biosystems, Wilmington, MA, USA), respectively. Finally, RNA sequencing (n = 1/group) was completed in a paired-end configuration (2 × 150 bp) using an Illumina Novaseq 6000 (Illumina, San Diego, CA, USA).

Quantitative Real-Time PCR (qPCR) Analyses
To create cDNA (n = 3/group), total RNA was extracted and measured. Then, realtime PCR was performed using a Thermal Cycler Dice ® Real-Time System III (Takara, Shiga Japan) and TB Green Premix Ex Taq as the real-time PCR master mix. Subsequently, primers were used to perform RT-PCR (Table 1).

Cell Culture and Cell Viability Assay
The Korean Cell Line Bank provided the human neuroblastoma cell line SH-SY5Y. The cells were cultured in equal parts of EMEM (ATCC 30-2003, Manassas, VA, USA) and F12 medium (Gibco, Waltham, CA, USA). To measure the cell viability, the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test (Sigma-Aldrich, St. Louis, MO, USA) was utilized [39]. The SH-SY5Y cells were incubated with OLNZ (1, 5, 25, or 100 µg/mL) for 2 h before induction of cytotoxicity by adding 300 µM H 2 O 2 and co-culturing for another 24 h. After adding the MTT solution, the formation of blue color formazone crystals was detected in each well. A VersaMax™ microplate reader (Molecular Devices, San Jose, CA, USA) was employed to measurement the absorbance at 570 nm.

LDH and ROS Determination Test
Generation of LDH was estimated via an LDH cytotoxicity measurement kit, as directed by the manufacturer (Takara, Shiga, Japan). SH-SY5Y cells subjected to cultured overnight in 96-well plates, pretreated with OLNZ for 2 h with various doses, and then co-cultured with H 2 O 2 for another 24 h. The relative concentration of LDH from the supernatant of the cells was evaluated by determining the absorbance at 490 nm with an automated microplate reader.
Intracellular ROS generation was distinguished using a fluorometric intracellular ROS detection Kit (Sigma-aldrich, St. Louis, MO, USA). SH-SY5Y cells were incubated overnight in 96-well plates, pretreated with OLNZ for 2 h with various doses, and then co-cultured with H 2 O 2 for another 12 h. Hanks' Balanced Salt Solution (HBSS) was used for washing cells. After adding the master reaction mix (100 µL/well), the cells were incubated in the incubator (5% CO 2 , 37 • C) for 30 min. Then, we measured the fluorescence intensity (λ ex = 640/λ em = 675 nm).

Western Blot Analyses
RIPA buffer (Biosesang, Gyeonggi-do, South Korea), and T-Per tissue protein extraction reagent (Thermo Scientific, Rockford, LA, USA) was applied to extract proteins from SH-SY5Y cells and the hippocampal area of brain tissue, correspondingly. Lysed cells were centrifuged at 13,000× g for 15 min after being sonicated, and the supernatant was collected. Using a PierceTM BCA protein assay reagent (Thermo Scientific, Rockford, IL, USA), the total protein content of lysed cells was calculated. Then, protein samples were separated using 10-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and added to transfer in a nitrocellulose membrane. To prevent nonspecific binding of primary and secondary antibodies, the membranes were blocked with 5% bovine serum albumin (BSA; Sigma, St. Louis, MO, USA) for 2 h at room temperature. Primary antibodies were mixed with 5% BSA and Tris-buffered saline with Tween ® -20 (TBST). Membranes were incubated with primary antibodies overnight, and then secondary antibodies were added for 2 h (goat anti-rabbit IgG-HRP; Santa Cruz Biotechnology, Inc. Dallas, TX, USA). Finally, membranes were placed in an ImageQuant™ LAS-500 image system (GE Healthcare, Little Chalfont, UK) to capture band images.

Statistical Analyses
In this experiment, all the measurements and comparative analyses among the groups were analyzed using Graph Pad Prism version 5.0 (Graph Pad Software, Inc., San Diego, CA, USA) and are presented as mean ± standard error (SEM). Analysis of variance (ANOVA) and subsequent Tukey's post hoc tests were used for the statistical analyses. A minimum p-value of 0.05 was deemed significant.

Neuroprotective Effects of OLNZ against TI-Mediated Neuronal Cell Death
Following obstruction of the BCCA, neuronal damage in the CA1 domain of the hippocampus was observed by histological analysis of selected CV-stained sections in all three groups. Examination of the CV-stained cells revealed that induction of TI significantly reduced the number of live neuronal cells in the pyramidal layer in the CA1 domain ( Figure 2B,b), compared with the sham group. Intraperitoneal (I/P) injection of OLNZ (n = 5) immediately after induction of TI resulted in a greater number of large pyramidalshaped CV-positive cells in the CA1 domain, compared to the TI-induced group ( Figure 2C,c). Cells that were positive for the neuronal degenerative marker F-J B+ could not be distinguished in the group's hippocampus either low or high magnification ( Figure 2); however, in the TI-induced stroke group, the CA1 area contained large numbers of F-J B+ degenerative cells ( Figure 2E,e). In contrast, the expression pattern and the number of F-J B+ cells were notably diminished in the TI-induced stroke + OLNZ treatment group ( Figure 2F,f) 6 days after induction of TI.
Immunohistochemistry (IHC) using NeuN immunoreactivity was performed to identify the nuclear protein expressed by neuronal cells in the CA1 area of the hippocampus ( Figure 3). Few NeuN+ neuronal cells were found under either low or high magnification in the TI-induced stroke group ( Figure 3B,b), in contrast to the sham group. A single dose of OLNZ (10 mg/kg) significantly increased the NeuN immunoreactive cells in the hippocampus, as confirmed by the well-stained pyramidal-shaped nucleus of the neurons with lighter staining of the cytoplasm ( Figure 3C,c).

Neuroprotective Effects of OLNZ by Inactivation of TI-Induced Neuroglia Cells
The activation status of both astrocytes and microglial cells was assessed either by immunoreactivity of GFAP+ or Iba-1+ cells in the hippocampus of all three groups after 5 days of TI induction ( Figure 4). All TI-induced animals exhibited GFAP+ and Iba-1+ cells in the entire focus of the hippocampus. Neither the sham group nor the OLNZ treatment group showed expression of GFAP+ or Iba-1+ cells, even in the most vulnerable area of the hippocampus ( Figure 4).

Neuroprotective Effects of OLNZ by Inhibiting Oxidative Stress
Oxidative stress-induced free radicles attack lipids that provoked lipid peroxidation [40]. Lipid peroxidation marker 4-HNE immunoreactivity was significantly increased in CA1 pyramidal neurons of the TI-induced group ( Figure 5B,b) and decreased by OLNZ treatment ( Figure 5C,c) after TI. We measured the expression of free radical scavenging enzyme (SOD-2) proteins in the gerbil's brain by immunoblotting analysis ( Figure 5E). SOD-2 expression was decreased in hippocampal neurons of the TI-induced group and increased by OLNZ treatment after TI (not significantly).
2C,c). Cells that were positive for the neuronal degenerative marker F-J B+ could not be distinguished in the group's hippocampus either low or high magnification ( Figure 2); however, in the TI-induced stroke group, the CA1 area contained large numbers of F-J B+ degenerative cells ( Figure 2E,e). In contrast, the expression pattern and the number of F-J B+ cells were notably diminished in the TI-induced stroke + OLNZ treatment group (  In the TI-mediated stroke gerbils, the number of CV positive cells in the CA1 domain was reduced compared with the sham group, although a greater number of F-J B+ degenerative cells were distinguished in the hippocampus of the ischemic group compared to both the sham and TI-induced stroke + OLNZ treatment groups. (G) Graphs represent the relative numbers of CV+ and (H) F-J B+ cells. Data were expressed as the mean ± SEM, n = 5 in each group. After performing one-way ANOVA with Tukey's post hoc tests, p < 0.05 for comparisons of the treatment groups with the sham group (#) and p < 0.05 for comparisons with the TI-induced stroke group (*) were considered significant.

Neuroprotective Effects of OLNZ by Blocking MAPK Pathway in the Hippocampus
To investigate the possible pathways involved in OLNZ-mediated neuroprotection, we measured the activation of proteins associated with neuronal cell death, specifically the regulation of MAPK cascades. Experimental results revealed that activation of MAPK (ERK1/2, JNK, and p38) family proteins were notably augmented in the hippocampus after induction of TI, but ERK1/2 and JNK were significantly reversed by treatment with OLNZ ( Figure 6). identify the nuclear protein expressed by neuronal cells in the CA1 area of the hippocampus ( Figure 3). Few NeuN+ neuronal cells were found under either low or high magnification in the TI-induced stroke group ( Figure 3B,b), in contrast to the sham group. A single dose of OLNZ (10 mg/kg) significantly increased the NeuN immunoreactive cells in the hippocampus, as confirmed by the well-stained pyramidal-shaped nucleus of the neurons with lighter staining of the cytoplasm ( Figure 3C,c).

Neuroprotective Effects of OLNZ by Inactivation of TI-Induced Neuroglia Cells
The activation status of both astrocytes and microglial cells was assessed either by immunoreactivity of GFAP+ or Iba-1+ cells in the hippocampus of all three groups after 5 days of TI induction ( Figure 4). All TI-induced animals exhibited GFAP+ and Iba-1+ cells in the entire focus of the hippocampus. Neither the sham group nor the OLNZ treatment group showed expression of GFAP+ or Iba-1+ cells, even in the most vulnerable area of the hippocampus (Figure 4).

Effects of OLNZ on DEGs Involved in the Ischemic Stroke Response in the Hippocampus
The RNA sequencing was accomplished using hippocampal RNA from the sham, TI-induced, and TI-induced + OLNZ-treated groups after 5 days of cerebral ischemia. Initially, we assessed up and downregulated genes to identify DEGs among the three groups of gerbils. Bioinformatics analyses identified a total of 932 DEGs in the sham vs. TI-induced stroke hippocampus, among which 714 genes were upregulated and 218 were downregulated by TI-induced stroke ( Figure 7A). In addition, 766 DEGs were identified in the TI-induced stroke vs. TI-induced stroke + OLNZ hippocampus, among which 521 genes were downregulated and 245 upregulated in the TI-induced stroke + OLNZ treatment group ( Figure 7B).
We recorded a certain number of differentially upregulated RNA genes and the downregulated RNA genes of sham vs. stroke and stroke vs. OLNZ treatment group respectively (Table 2), and a certain number of differentially downregulated RNA genes and the upregulated RNA genes of sham vs. stroke and stroke vs. OLNZ treatment group respectively (Table 3)   In the TI-mediated stroke group, Iba-1+ cells were notably augmented and GFAP+ cells exhibited bigger and denser processes in the CA1 area compared with OLNZ treatment groups. (G) Graphs signify the relative numbers of Iba1+ and (H) GFAP immunoreactive cells. Data were expressed as the mean ± SEM, n = 5 in each group. After performing a one-way ANOVA with Tukey's post hoc test, p < 0.05 for comparisons of treatment groups with the sham group (#) and p < 0.05 for comparisons with the TI-induced stroke group (*) were considered significant.

Neuroprotective Effects of OLNZ by Inhibiting Oxidative Stress
Oxidative stress-induced free radicles attack lipids that provoked lipid peroxidation [40]. Lipid peroxidation marker 4-HNE immunoreactivity was significantly increased in CA1 pyramidal neurons of the TI-induced group ( Figure 5B,b) and decreased by OLNZ treatment (Figure 5C,c) after TI. We measured the expression of free radical scavenging enzyme (SOD-2) proteins in the gerbil's brain by immunoblotting analysis ( Figure 5E). SOD-2 expression was decreased in hippocampal neurons of the TI-induced group and increased by OLNZ treatment after TI (not significantly). In the TI-mediated stroke group, Iba-1+ cells were notably augmented and GFAP+ cells exhibited bigger and denser processes in the CA1 area compared with OLNZ treatment groups. (G) Graphs signify the relative numbers of Iba1+ and (H) GFAP immunoreactive cells. Data were expressed as the mean ± SEM, n = 5 in each group. After performing a one-way ANOVA with Tukey's post hoc test, p < 0.05 for comparisons of treatment groups with the sham group (#) and p < 0.05 for comparisons with the TI-induced stroke group (*) were considered significant.

Effects of OLNZ on the Expression of Complement Component mRNA
Based on the DEG results, we selected five genes for verification by RT qPCR. To detect the similarity of complement system activation in the hippocampus after induction of TI (Figure 9), we investigated the expression of C1q, C2, C3, C4a, and C9 mRNAs. All complement components were upregulated by ischemic insult and downregulated by TI-induced stroke + OLNZ treatment.

Neuroprotective Effects of OLNZ by Blocking MAPK Pathway in the Hippocampus
To investigate the possible pathways involved in OLNZ-mediated neuroprotection, we measured the activation of proteins associated with neuronal cell death, specifically the regulation of MAPK cascades. Experimental results revealed that activation of MAPK (ERK1/2, JNK, and p38) family proteins were notably augmented in the hippocampus after induction of TI, but ERK1/2 and JNK were significantly reversed by treatment with OLNZ ( Figure 6).  . Protective role of OLNZ against the TI-mediated phosphorylation of ERK, JNK, p38 protein in the brain. The expression (% of sham) of (A) pERK, (B) pJNK proteins decreased significantly, and (C) pP38 proteins also decreased but not significantly after treatment with OLNZ in TI-induced gerbil hippocampus. Data were expressed as the mean ± SEM, n = 3/group. After performing a oneway ANOVA with Tukey's post hoc test, p < 0.05 for comparisons of treatment groups with the sham group (#) and p < 0.05 for comparisons with the stroke group (*) were considered significant.

Effects of OLNZ on DEGs Involved in the Ischemic Stroke Response in the Hippocampus
The RNA sequencing was accomplished using hippocampal RNA from the sham, TIinduced, and TI-induced + OLNZ-treated groups after 5 days of cerebral ischemia. Initially, we assessed up and downregulated genes to identify DEGs among the three groups of gerbils. Bioinformatics analyses identified a total of 932 DEGs in the sham vs. TI-induced stroke hippocampus, among which 714 genes were upregulated and 218 were downregulated by TI-induced stroke ( Figure 7A). In addition, 766 DEGs were identified in the TI-induced stroke vs. TI-induced stroke + OLNZ hippocampus, among which 521 Figure 6. Protective role of OLNZ against the TI-mediated phosphorylation of ERK, JNK, p38 protein in the brain. The expression (% of sham) of (A) pERK, (B) pJNK proteins decreased significantly, and (C) pP38 proteins also decreased but not significantly after treatment with OLNZ in TI-induced gerbil hippocampus. Data were expressed as the mean ± SEM, n = 3/group. After performing a one-way ANOVA with Tukey's post hoc test, p < 0.05 for comparisons of treatment groups with the sham group (#) and p < 0.05 for comparisons with the stroke group (*) were considered significant.  We recorded a certain number of differentially upregulated RNA genes and the downregulated RNA genes of sham vs. stroke and stroke vs. OLNZ treatment group respectively (Table 2), and a certain number of differentially downregulated RNA genes and the upregulated RNA genes of sham vs. stroke and stroke vs. OLNZ treatment group respectively (Table 3) in Gene ID forms. To comprehend how DEGs are distributed across

Effects of OLNZ on Functional Pathway Involved in the Ischemic Stroke Response in the Hippocampus
The KEGG pathways in the stroke vs. OLNZ treatment group were revealed ( Figure 8A,B). The analytical data showed that downregulated pathways by OLNZ treatment after induction of TI were mainly involved in chemokine and cytokine signaling.

Effects of OLNZ on the Expression of Complement Component mRNA
Based on the DEG results, we selected five genes for verification by RT qPCR. To detect the similarity of complement system activation in the hippocampus after induction of TI (Figure 9), we investigated the expression of C1q, C2, C3, C4a, and C9 mRNAs. All complement components were upregulated by ischemic insult and downregulated by TIinduced stroke + OLNZ treatment.  In TI-induced stroke, gene expression of C1q, C2, C3, C4a, and C9, was significantly upregulated, whereas treatment with OLNZ markedly downregulated the expression of these genes. Data were expressed as the mean ± SEM, n = 3 in each group. After performing a one-way ANOVA with Tukey's post hoc test, p < 0.05 for comparisons of treatment groups with the sham group (#) and p < 0.05 for comparisons with the stroke group (*) were considered significant.

Neuroprotective Effects of OLNZ against SH-SY5Y Cell Toxicity, LDH, and ROS Release
We used the MTT assay to evaluate the neuroprotective efficacy of the OLNZ using SH-SY5Y cells incubated with cytotoxic concentrations of H2O2. To identify the optimum non-toxic dose of OLNZ, SH-SY5Y cells were incubated with several concentrations of OLNZ drug (1, 5, 25, or 100 µg/mL) for 24 h. A noticeable reduction in cell viability was recorded at a high concentration of OLNZ (100 µg/mL) as related to control cultures (Figure 10A). We found that 300 µM/mL of H2O2 increased the SH-SY5Y cytotoxicity compared to control cultures. The viability of cells incubated with H2O2 increased significantly in a concentration-dependent manner in SH-SY5Y cultures co-incubated with OLNZ (1, 5, 25 µg/mL; (Figure 10B.). We examined intracellular LDH and ROS release caused by H2O2 in the human neuroblastoma cell line SH-SY5Y. Our data demonstrated that H2O2 incubation greatly increased intracellular LDH and ROS formation in SH-SY5Y cells (p >0.05) compared with control cultures; however, OLNZ co-incubation notably (p >0.05) reduced LDH ( Figure 10C) and ROS ( Figure 10D) accumulation inside the cells. In TI-induced stroke, gene expression of C1q, C2, C3, C4a, and C9, was significantly upregulated, whereas treatment with OLNZ markedly downregulated the expression of these genes. Data were expressed as the mean ± SEM, n = 3 in each group. After performing a one-way ANOVA with Tukey's post hoc test, p < 0.05 for comparisons of treatment groups with the sham group (#) and p < 0.05 for comparisons with the stroke group (*) were considered significant.

Neuroprotective Effects of OLNZ against SH-SY5Y Cell Toxicity, LDH, and ROS Release
We used the MTT assay to evaluate the neuroprotective efficacy of the OLNZ using SH-SY5Y cells incubated with cytotoxic concentrations of H 2 O 2 . To identify the optimum non-toxic dose of OLNZ, SH-SY5Y cells were incubated with several concentrations of OLNZ drug (1, 5, 25, or 100 µg/mL) for 24 h. A noticeable reduction in cell viability was recorded at a high concentration of OLNZ (100 µg/mL) as related to control cultures ( Figure 10A). We found that 300 µM/mL of H 2 O 2 increased the SH-SY5Y cytotoxicity compared to control cultures. The viability of cells incubated with H 2 O 2 increased significantly in a concentration-dependent manner in SH-SY5Y cultures co-incubated with OLNZ (1, 5, 25 µg/mL; ( Figure 10B). We examined intracellular LDH and ROS release caused by H 2 O 2 in the human neuroblastoma cell line SH-SY5Y. Our data demonstrated that H 2 O 2 incubation greatly increased intracellular LDH and ROS formation in SH-SY5Y cells (p > 0.05) compared with control cultures; however, OLNZ co-incubation notably (p > 0.05) reduced LDH ( Figure 10C) and ROS ( Figure 10D) accumulation inside the cells.

Anti-Oxidant Activity of OLNZ in SH-SY5Y Cells
We measured the expression of free radical scavenging enzyme (SOD-1 and SOD-2) genes and proteins in SH-SY5Y cells by RT-qPCR and immunoblotting analysis, respectively ( Figure 11). Incubation with H 2 O 2 stimulated oxidative stress that led to decreased expression of SOD-1 and SOD-2 mRNA (Figure 11A,B) and proteins ( Figure 11C,D) compared to control cultures. In contrast, co-incubation with OLNZ and H 2 O 2 significantly (p < 0.05) amplified the expression of both antioxidant enzymes. Figure 10. Effects of OLNZ in H2O2-induced neuronal cell death in vitro: (A) The non-toxic concentration of OLNZ in SH-SY5Y cells was estimated using the MTT assay; (B) Cell viability in H2O2mediated toxicity was also evaluated using the MTT assay; (C) The release of cytotoxic marker LDH (%); and (D) the oxidative stress marker ROS was examined. Data were expressed as the mean ± SEM, and the experiment was repeated three times. # and * indicate a statistically significant difference compared with the control and H2O2 treatment alone, respectively, p < 0.05. the oxidative stress marker.

Anti-Oxidant Activity of OLNZ in SH-SY5Y Cells
We measured the expression of free radical scavenging enzyme (SOD-1 and SOD-2) genes and proteins in SH-SY5Y cells by RT-qPCR and immunoblotting analysis, respectively ( Figure 11). Incubation with H2O2 stimulated oxidative stress that led to decreased expression of SOD-1 and SOD-2 mRNA (Figure 11A,B) and proteins ( Figure 11C,D) compared to control cultures. In contrast, co-incubation with OLNZ and H2O2 significantly (p < 0.05) amplified the expression of both antioxidant enzymes. The non-toxic concentration of OLNZ in SH-SY5Y cells was estimated using the MTT assay; (B) Cell viability in H 2 O 2 -mediated toxicity was also evaluated using the MTT assay; (C) The release of cytotoxic marker LDH (%); and (D) the oxidative stress marker ROS was examined. Data were expressed as the mean ± SEM, and the experiment was repeated three times. # and * indicate a statistically significant difference compared with the control and H 2 O 2 treatment alone, respectively, p < 0.05. the oxidative stress marker.

Neuroprotective Effects of OLNZ to Prevent MAPK Cascade and NF-kB Activation
We found that H 2 O 2 -exposed SH-SY5Y cells elevated phosphorylation levels of MAPK (ERK1/2, JNK, and p38) ( Figure 12A-C) cascade and NF-kB ( Figure 12D) in comparison to the control. Meanwhile, activation of ERK, JNK, P38, and NF-kB proteins was considerably reduced (p < 0.05) after OLNZ pretreatment in a concentration-dependent pattern.

Neuroprotective Effects of OLNZ to Prevent MAPK Cascade and NF-kB Activation
We found that H2O2-exposed SH-SY5Y cells elevated phosphorylation levels of MAPK (ERK1/2, JNK, and p38) ( Figure 12A-C) cascade and NF-kB ( Figure 12D) in comparison to the control. Meanwhile, activation of ERK, JNK, P38, and NF-kB proteins was considerably reduced (p < 0.05) after OLNZ pretreatment in a concentration-dependent pattern.

Anti-Apoptotic Effects of OLNZ in SH-SY5Y Cells
We used Western blotting to see if H 2 O 2 and/or OLNZ had any effect on the expression of Bcl-2 and Bax proteins. As compared to the control, H 2 O 2 considerably decreased the expression of Bcl-2 protein in SH-SY5Y cells, while significantly increasing the expression of Bax protein. Furthermore, pretreatment with OLNZ markedly (p < 0.05) reduced Bax and increased Bcl-2 proteins in a dose-dependent manner ( Figure 13).

Anti-Apoptotic Effects of OLNZ in SH-SY5Y Cells
We used Western blotting to see if H2O2 and/or OLNZ had any effect on the expression of Bcl-2 and Bax proteins. As compared to the control, H2O2 considerably decreased the expression of Bcl-2 protein in SH-SY5Y cells, while significantly increasing the expression of Bax protein. Furthermore, pretreatment with OLNZ markedly (p < 0.05) reduced Bax and increased Bcl-2 proteins in a dose-dependent manner ( Figure 13).

Discussion
During cerebral ischemia, excessive release of dopamine and serotonin induces excitotoxicity and energy deprivation and reduces blood flow in the ischemic brain, leading to irreversible brain damage [41][42][43][44]. Olanzapine is a well-known dopamine and serotonin receptor antagonist [45]. Therefore, olanzapine may be a potential neuroprotective compound; although the exact mechanism of neuroprotection by OLNZ in vivo is unclear. Further experiments are needed to investigate the actual mechanism involved. In this study, gerbils were used as an in vivo model for transient global cerebral ischemia-mediated neuronal death, and SH-SY5Y cells were used as a model for in vitro neuroprotection and potential mechanisms of action.
Transient cerebral ischemia in gerbils is brought on by blocking both common carotid arteries for 5 min [46][47][48]. In our previous study, severe neuronal cell damage was identified 5 days after induction of ischemic insult [36,49]. We also found neuronal cell death and activation of microglial cells and astrocytes in the most vulnerable part of the brain (hippocampus) after 5 days of TI. The number of CV-positive cells in the hippocampus of the OLNZ treatment group was higher as compared to the TI-induced group, but lower than in the sham group. The same pattern of NeuN immunoreactive neuronal cell expression was evident in all three groups, although the numbers of neuronal degenerative response indicator F-JB-positive cells were markedly increased in the CA1 region after induction of TI and decreased by OLNZ treatment. A previous study reported that olanzapine treatment after permanent focal cerebral ischemia in mice reduced brain damage as assessed by triphenyl tetrazolium chloride staining [34]. Other studies have revealed that oral administration of olanzapine increased the proliferation of subventricular zone neurons and decreased dendritic spine loss in rats [33,50]. Kainic acid-induced hippocampal

Discussion
During cerebral ischemia, excessive release of dopamine and serotonin induces excitotoxicity and energy deprivation and reduces blood flow in the ischemic brain, leading to irreversible brain damage [41][42][43][44]. Olanzapine is a well-known dopamine and serotonin receptor antagonist [45]. Therefore, olanzapine may be a potential neuroprotective compound; although the exact mechanism of neuroprotection by OLNZ in vivo is unclear. Further experiments are needed to investigate the actual mechanism involved. In this study, gerbils were used as an in vivo model for transient global cerebral ischemia-mediated neuronal death, and SH-SY5Y cells were used as a model for in vitro neuroprotection and potential mechanisms of action.
Transient cerebral ischemia in gerbils is brought on by blocking both common carotid arteries for 5 min [46][47][48]. In our previous study, severe neuronal cell damage was identified 5 days after induction of ischemic insult [36,49]. We also found neuronal cell death and activation of microglial cells and astrocytes in the most vulnerable part of the brain (hippocampus) after 5 days of TI. The number of CV-positive cells in the hippocampus of the OLNZ treatment group was higher as compared to the TI-induced group, but lower than in the sham group. The same pattern of NeuN immunoreactive neuronal cell expression was evident in all three groups, although the numbers of neuronal degenerative response indicator F-JB-positive cells were markedly increased in the CA1 region after induction of TI and decreased by OLNZ treatment. A previous study reported that olanzapine treatment after permanent focal cerebral ischemia in mice reduced brain damage as assessed by triphenyl tetrazolium chloride staining [34]. Other studies have revealed that oral administration of olanzapine increased the proliferation of subventricular zone neurons and decreased dendritic spine loss in rats [33,50]. Kainic acid-induced hippocampal neuronal loss in neonatal rats was also blocked by high doses of olanzapine [51]. Therefore, we can say that OLNZ treatment can prevent neuronal loss in the hippocampus induced by TI.
Many research studies have confirmed that brain ischemia, trauma, tumor growth, or neurodegenerative disease induce excessive activation of glial cells [52,53]. In ischemic insult, neuroinflammatory responses induce the secretion of various inflammatory mediators [54,55]. Our data revealed that Iba1+ microglia and GFAP+ astrocytes were significantly active across the hippocampus's focal area in the TI-induced stroke group, in comparison with those of the OLNZ treatment and sham groups. Handi Zhang et al. showed that OLNZ alleviated astrocyte gliosis in a cuprizone (CPZ)-induced model of demyelination in C57BL/6 mice [56]. These experimental data strongly indicate that the neuroprotective effects of OLNZ are due to the modulation of TI-induced glial cell activation.
To confirm the TI-induced neuronal modification and neuroprotective effects of OLNZ, DEGs of RNA seq were evaluated. Some chemokine ligands and interferon-induced proteins (Ccl8, Cd69, Sell and Ccl7) were aggravated by induction of TI and regulated by OLNZ treatment. Ccl8 and Ccl7 are kinds of chemokines that attract monocytes to the site of trauma, infection, toxin exposure, and ischemia [57]. Cd69 triggers monocyte and NK cell activation [58] and sell recruits leukocytes in inflammatory lesions [59]. ALOX15 gene is a strong suppressor of inflammation that was downregulated by TI and upregulated by OLNZ treatment [60]. In addition, the ischemic state appeared to increase the expression of complement components that stimulate apoptosis [19][20][21]. Our qPCR data also supported a role for upregulated complement component expression in global cerebral ischemia, and that OLNZ treatment downregulated that expression. Hence, OLNZ can block the complement components mediated damage after induction of TI.
The H 2 O 2 -induced model of neurotoxicity in SH-SY5Y human neuroblastoma cells has been widely used to assess the neuroprotective effects of various compounds against oxidative stress [61,62]. Here, the same model of neurotoxicity was applied to investigate the protective effects of OLNZ, and OLNZ pretreatment was found to significantly mitigate H 2 O 2mediated SH-SY5Y cell death. Similarly, OLNZ pretreatment reduced LDH (a cell membrane disintegrity marker) and ROS release. Earlier experiments showed that co-incubation with OLNZ increased cell viability in β-amyloid peptide 25-35-, N-methyl-4-phenyl pyridinium ion-, and hydrogen peroxide-mediated SH-SY5Y and PC12 cell death [63,64]. So, OLNZ can regulate H 2 O 2 -induced neurotoxicity in SH-SY5Y.
Oxidative-stress-induced cytotoxicity by H 2 O 2 is associated with increased production of ROS that damage intracellular molecules including lipids, proteins, and DNA [65,66]. Besides, TI-induced oxidative stress-mediated free radicles increase lipid peroxidation in the CA1 region of the hippocampus [40,67]. Opposed to SODs are enzymes, which stabilize most of the superoxide radicals (O 2 − ) by oxidative stress, and thus protect cells from ROS-mediated cytotoxicity [68]. induced We investigated whether SOD-1 or SOD-2 expression was reduced by H 2 O 2 or TI-induced oxidative stress, and whether OLNZ treatment blocked this reduction of SODs protein and gene expression. Early intervention explored pre-incubation with 10 and 100 uM/L olanzapine upregulated SOD-1 mRNA expression in PC12 cell cultures after 48 h of incubation [69]. Another experiment confirmed that the antioxidant activity of olanzapine is due to the scavenging of superoxide anions formed during the respiratory burst and augmentation of the antioxidant enzymes SODs, catalase, and glutathione peroxidase [64,70]. Therefore, OLNZ could be an important factor against oxidative stress-induced cell death.
Oxidative stress initiated by ROS production and inflammation-induced cell and tissue injuries are important factors in the activation of the MAPK cascade [71]. Activation of the MAPK cascade can be predicted based on ERK, JNK, and p38 kinase activation [72]. When the MAPK cascade is activated, it triggers apoptosis by DNA damage or caspase-8 activation [14,73]. In our study, OLNZ treatment significantly downregulated transient ischemia and H 2 O 2-mediated phosphorylation of MAP kinases in the hippocampus and SH-SY5Y cells. Previous studies have demonstrated that olanzapine can trigger the activation of MAPKs in a dose-and time-dependent manner [30,74,75]. The phosphorylation of NF-κB is an important event in the stress response and can be triggered by a wide range of stimuli, including oxidative stress. As such, H 2 O 2 can rapidly activate NF-κB, which plays an important part in ROS-induced apoptosis [76]. Our results indicated that OLNZ efficiently suppressed H 2 O 2 -induced phosphorylation of NF-κB. Taken together, our findings suggest that OLNZ has neuroprotective effects against oxidative stress-induced neuronal damage in both in vivo and in vitro models by regulating MAPK signaling pathway.
Bcl-2 family proteins (Bax and Bcl-2) are directly related to the mitochondrial apoptosis process [77]. These proteins control the absorbency of the mitochondrial outer membrane as well as the delivery of apoptotic mediators from the intermembranous mitochondrial region [77]. In this study, OLNZ pretreatment boosted Bcl-2 protein expression and decreased Bax protein expression in H 2 O 2 -mediated apoptosis of SH-SY5Y cells. These findings suggest that OLNZ can control apoptotic pathways that are dependent on Bax and Bcl-2.

Conclusions
In summary, OLNZ mitigates neuronal cell death and maintains almost normal hippocampal integrity. Our result authenticated that olanzapine attributed its neuroprotective impacts against TI-induced neuronal damage and H 2 O 2 -mediated neurotoxicity in SH-SY5Y cells by decreasing neuronal apoptosis, as well as oxidative stress by regulating MAPK signaling pathway. These findings provide opportunities for further research on olanzapine as a promising medicine against ischemic stroke. Data Availability Statement: All experimental data produced during this investigation has been inserted in this main manuscript.

Conflicts of Interest:
The authors declare that they have no competing interest.