Hypericum perforatum L. Nanoemulsion Mitigates Cisplatin-Induced Chemobrain via Reducing Neurobehavioral Alterations, Oxidative Stress, Neuroinflammation, and Apoptosis in Adult Rats

Cisplatin (Cis) is a potent chemotherapeutic agent; however, it is linked with oxidative stress, inflammation, and apoptosis, which may harmfully affect the brain. Hypericum perforatum L. (HP L.) is a strong medicinal plant, but its hydrophobic polyphenolic compounds limit its activity. Therefore, our study aimed to investigate the neuroprotective action of HP L. and its nanoemulsion (NE) against Cis-induced neurotoxicity. The prepared HP.NE was subjected to characterization. The droplet size distribution, surface charge, and morphology were evaluated. In addition, an in vitro dissolution study was conducted. Compared to Cis-intoxicated rats, HP L. and HP.NE-treated rats displayed improved motor activity and spatial working memory. They also showed an increase in their antioxidant defense system and a reduction in the levels of pro-inflammatory cytokines in the brain. Moreover, they showed an increase in the expression levels of the PON-3 and GPX genes, which are associated with a reduction in the brain levels of COX-2 and TP-53. These findings were confirmed by reducing the immunohistochemical expression of nuclear factor kappa (NF-ƘB) and enhanced Ki-67 levels. In conclusion, HP L. is a promising herb and could be used as an adjuvant candidate to ameliorate chemotherapeutic-induced neurotoxicity. Moreover, HP.NE has superior activity in lessening Cis-induced oxidative stress, inflammation, and apoptosis in brain tissue.


Introduction
Cisplatin (cis-diamminedichloroplatinum II) (Cis) is a universal anticancer agent with recognized neurotoxicity, nephrotoxicity, and hepatotoxicity [1,2]. It is a heavy metal containing a platinum atom surrounded by two chloride molecules and two ammonia molecules [3]. Plasma proteins, including albumin, gammaglobulin, and transferrin, are irreversibly linked to the platinum component of Cis [4]. Due to the prevalence of solid tumor treatment, Cis is one of the most extensively used and recommended anticancer drug [1]. Chemobrain or cognitive impairment is a typical adverse effect of chemotherapeutic medications regularly described by patients treated for cancers [5].

Development of Nanoemulsion (NE) 2.2.1. Construction of Pseudo-Ternary Phase Diagram
Pseudo-ternary phase diagrams were constructed to identify the NE region using the titration method. A mixture of surfactant (Tween 80) and co-surfactant (Transcutol HP) (Gattefosse, Lyon, France) was prepared at a 2:1 ratio (SCoSmix). Labrafac (Oil) (Gattefosse, France) and mixed with surfactant/co-surfactant in different volume ratios (from 1:9 to 9:1) in a transparent container. For each Oil/SCoSmix mixture, water was added slowly using the titration method under continuous stirring at room temperature until turbidity appeared [14,18]. The points up to the clarity transition occurred were defined as the phase boundaries. Phase diagrams were drawn using Sigma Plot ® software (version 14, Systat Software Inc., San Jose, CA, USA).

Selection of Formulations and Stability Tests
Different formulas were chosen from the NE region as shown in Table 1. Selected formulations were subjected to different stability tests, namely a centrifugation test and a dilution test. For the centrifugation test, the selected formulations were centrifuged at 3500 rpm for 30 min to test creaming or phase separation by visual observation. For the dilution test: the selected formulations were diluted with distilled water 100 times and then visually checked for phase separation and clarity. The symbol "-" and the word "clear" in the table denote zero.

Preparation of HP. Loaded NE (HP.NE)
The HP.NE was prepared by a spontaneous emulsification method, as shown in Table 1. HP L. extract (1.5% w/v) was mixed with oil, then surfactant and cosurfactant were added and mixed until a homogenous mixture was obtained. Water was added gradually under continuous stirring at room temperature. The prepared HP.NE was subjected to characterization. The droplet size distribution and surface charge were measured by Nano-ZS, Malvern Instruments, Malvern, UK. The morphology of the selected formula was visualized by transmission electron microscopy (TEM) (Joel, JEM-2100, Tokyo, Japan).

In Vitro Dissolution Study
The in vitro dissolution study was conducted using the dialysis method. About 5 mL of HP.NE was placed into a dialysis bag, placed in 900 mL water (USP II dissolution apparatus, Hanson, MA, USA), and maintained at 37 ± 0.5 • C and 75 rpm. A 5 mL aliquot was withdrawn at predetermined time intervals of 15, 30, 60, 120, and 240 min and replaced by the same volume of water. The HP L. released was measured by a UV/Visible spectrophotometer (Schimadzu, 1600, Kyoto, Japan) with 260 nm as the wave length for the total phenolic compounds, and was then compared with the extract solution.

Animals
The current study was carried out with 28 male albino Wistar rats weighing about 170-200 g. The rats were housed in a plastic cage under standard temperature (25 ± 2 • C), humidity (50 ± 5%), and 12:12 h light-dark (LD) cycles. They were provided with a commercial standard diet and water ad libitum.

Experimental Protocol
A total of 28 male rats were randomly distributed into 4 groups, with 7 animals in each, and all groups of rats were treated in the following manner: Group I-Saline/Normal control rats were administered with normal saline (2 mL/kg, p.o. per day) for 14 days and then injected with saline i.p on day 14, followed by oral gavage of saline for another 7 days. Group II-Cis rats were administered with normal saline (2 mL/kg, p.o. per day) for 14 days and injected with Cis (10 mg/kg; Sigma-Aldrich, Inc., St. Louis, MO, USA) i.p on day 14, followed by oral gavage of saline for another 7 days. Group III-Hypericum (HP 100 mg/kg + Cis) rats were treated with Hypericum perforatum L. (100 mg/kg, p.o. per day) for 14 days and injected with Cis (10 mg/kg) i.p on day 14, followed by oral gavage of HP L. (100 mg/kg) for another 7 days. Group IV-Hypericum perforatum L. loaded NE (HP.NE 100 mg/kg + Cis) rats were treated with HP.NE (100 mg/kg, p.o. per day) for 14 days and injected with Cis (10 mg/kg) i.p on day 14, followed by oral gavage of HP.NE (100 mg/kg) for another 7 days. Twenty-four h after the last dose, all groups were subjected to behavioral assessment at 10 a.m., and this continued for 3 days. The dose of Cis was selected according to previous studies [19,20], while the HP L. dose was selected based on our previous research [16].

Neurobehavioral Assessments
Rats were submitted for neurobehavioral assessments, including psychomotor activity using the open field test and spatial working memory using a Y-maze test. In the open field test, rats were placed in the corner of a square wooden arena (70 cm × 70 cm) and allowed to move freely for 3 min. The numbers of crossing squares and rearing frequencies were the measuring parameters [21,22]. In the Y-maze test, each rat was allowed to explore a Y-shaped wooden maze for 5 min. The measuring parameters were the number of arm entries and spontaneous alternation percentage (SAP), as previously reported [22]. All the test devices were cleaned and touched with 70% alcohol to remove any olfactory cues. The assessments were performed by an experienced observer blinded to the experimental groups.

Euthanasia and Brain Sampling
After the last behavioral test, rats were euthanized by cervical dislocation. Brains were excised gently, and half of the brains were rinsed with physiological buffer saline (100 mM Na2HPO4/NaH2PO4, 0.16 mg/mL heparin, pH 7.4) to remove RBCs and clot residues. Then, 1 g of brain sample was homogenized in 5 mL of cold phosphate-buffered saline (50 mM potassium phosphate, 1 mM of ethylenediaminetetraacetic acid [EDTA], pH 7.5) using a sonic homogenizer. All set homogenates were centrifuged at 14,000× g for 15 min at 4 • C. The supernatant was used to measure the oxidative biomarkers and proinflammatory cytokines. The other halves of the brains were preserved in neutral buffer formalin 10% for subsequent histopathological investigation.

Biochemical Assay Oxidative Biomarkers Assay
The antioxidant enzymatic activities of superoxide dismutase (SOD), glutathione -stransferase (GST), and catalase (CAT), with the level of reduced glutathione (GSH), and the lipid peroxidation marker, malondialdehyde (MDA), nitric oxide concentration (NO) level were evaluated in the supernatant as previously mentioned according to the manufacturer's guide notes (Laboratory Biodiagnostic Co., Giza, Egypt).
Quantitative of COX-2, TP-53, PON-3, and GPX Gene Expression by qRT-PCR RNA was purified from 100 mg of rat brain tissue using the RNA purification kit (Thermo Scientific, Waltham, MA, USA, GeneJET #K0734) according to the manufacturer's protocol. The concentration of the total RNA was assessed spectrophotometrically (Thermo Scientific, USA). The isolated RNA was reverse transcribed into cDNA and used for PCR with specific primers. The sequences of these primers were designed by https://www. ncbi.nlm.nih.gov/tools/primer-blast/ access date 9/2022, and are as follows: COX-2 forward 5 -AAA GCC TCGTCCAGATGC TA-3 , reverse 5 -ATGGTGGCTGTCTTGGTAGG-3 ; TP-53 forward 5 -GTG GTA CCG TAT GAG CCA CC-3 , reverse 5 -CAA CCT GGC ACA CAG CTT CC-3 ; PON-3 forward5 -CATCCAGGATCCTTTGTCAGATAA3 , reverse 5 CACGGTGCTGCCCTGAAG-3 ; GPX forward 5 CGGTTTCCCGTGCAATCAGT3 , reverse 5 -ACACCGGGGACCAAATGATG-3 ; and β-Actin (housekeeping gene) forward 5 CAC-CCGCGAGTACAACCTTC 3 , reverse 5 CCCATACCCACCATCACACC 3 . SYBR Green PCR Master Mix was used to evaluate the studied genes. cDNA was added to an SYBR Green qPCR Master Mix (Thermo Scientific, USA) containing 30 pg/mL of each primer. The product was amplified by 40 cycles of denaturation at 95 • C for 15 s, annealing at 60 • C for 15 s, and extension at 72 • C for 45 s. During the first cycle, the 95 • C steps were extended to 1 min. The β-Actin gene was amplified in the same reaction to serve as the reference gene. Every sample was analyzed in duplicate. The gene expression level was calculated using the comparative cycle threshold method [23].

Histopathological Analysis
Sagittal sections from brain tissue were fixed in paraformaldehyde (4%) and embedded in paraffin blocks. After that, blocks were cut into thin slices (5-6 µm) and stained with hematoxylin and eosin staining [24]. Different brain regions were examined in each animal by using an Olympus light microscope (BX43, USA) connected to a DP27 digital camera and CellSens dimensions software. The histological damage score was assessed in each animal according to previous studies [25,26]. Briefly, variant lesions, including neuronal degeneration, neurophagia, gliosis, and necrosis, were scored in different brain regions according to the severity as follows: 0 = absent, 1 = sporadic neuron (<25%), 2 = distributed neurons (25-50%), 3 = distributed neurons (>75), 4 = multifocal (>75). The final score for each animal was obtained by summation of the total score for multiple lesions (4-5 fields/200×).

Immunohistochemistry (IHC) Analysis for Inflammation and Cell Proliferation Biomarkers
Deparaffinized brain tissues were rehydrated and submitted to peroxidase-blocking procedures. Subsequently, slides were incubated with the primary antibody of NF-KB or Ki-67 (1:100). Later, the slides were rinsed with PBS three times and incubated with the secondary antibody. Color enrichment was carried out by diaminobenzidine (2 mL, 15 min), and hematoxylin was used as counterstain. Positive results were obtained as area % using Image J software version 1.47 (http://imagej.en.softonic.com/, accessed on 3 February 2023).

Statistical Analysis
Neurobehavioral parameters and biochemical data were expressed as mean ± standard error (SE), while histopathological scoring was expressed as median ± standard deviation. The statistical analyses were performed using the one-way analysis of variance (ANOVA) followed by Tukey's post hoc test or the Kruskal-Wallis test, followed by Dunn's multiple comparison tests for histopathological brain scoring. Histograms were plotted using Graph Pad Prism 9.4.1 (Graphpad Software, San Diego, CA, USA). Differences were accepted when p was <0.05, <0.01, or <0.001.

Development of HP. NE
The pseudo-ternary phase diagram was constructed to identify the NE region, where 9 trials were investigated using oil and SCoSmix in different volume ratios (from 1:9 to 9:1). According to Figure 1a, only three trials showed a clear transparent NE with low oil percentage and high SCoSmix percentage. Oil percentage (7.5% to 26.1%), and SCoSmix (60.9% to 67.5%) were used to draw the borderline for the NE region ( Figure 1a). Hence, three formulations in the middle of NE were selected for further investigations. The first step was testing formulation stability by centrifugation test using visual observation of creaming or phase separation along with a dilution test to visually check for phase separation and clarity. As shown in Table 1 Figure 1e). The dissolution profile of F4 with 10% oil and 80% SCoSmix showed the fastest dissolution, with around 50% and 100% released after 1 h and 4 h, respectively. Decreasing SCoSmix to 70% slightly slowed the release, as shown for F5. The slowest formula was F6 with 20% oil and 70% SCoSmix, where around 25% and 80% were released after 1 h and 4 h, respectively. Therefore, F4 with 10% oil and 80% SCoSmix was selected for further investigation. A TEM image of F4, represented in Figure 1f, shows spherical NE globules with sizes near to the size measured by the dynamic light scattering technique.

Effect of HP L. and HP.NE Administration on the Neurobehavioral Parameters of the Cis-Intoxicated Rats
As displayed in Figure 2, Cis-intoxicated rats displayed a significant reduction in

Effect of HP L. and HP.NE Administration on the Neurobehavioral Parameters of the Cis-Intoxicated Rats
As displayed in Figure 2, Cis-intoxicated rats displayed a significant reduction in their motor activities, evidenced by a decrease in the number of crossing squares and the rearing frequency in the open field test and a marked reduction in the number of arm entries in the Y-maze test compared to control rats. However, administration of HP L. and its formulation, HP.NE, succeeded in improving the general locomotion of rats compared to Cis-intoxicated rats (Figure 2a-c). Concerning SAP in the Y-maze test, Cis-intoxicated rats exhibited a marked reduction in their SAP compared to control rats. On the other hand, HP L. displayed a marked increase in the SAP compared to Cis-intoxicated rats ( Figure 2d). Remarkably, HP.NE-treated rats displayed a noticeable improvement in all the aforementioned parameters over HP-treated rats. . Data are expressed as mean ± standard error (SE)using one-way analysis of variance (one-way ANOVA) followed by Tukey's post hoc test for seven rats in each group. * means significant from the Control group; @, significant from the Cis group; #, significant from the HP +Cis group.

Effect of HP L. and HP.NE Administration on the Levels of Oxidative Biomarkers of the Cis-Intoxicated Rats
As shown in Figure 3, SOD, CAT, GST, and GSH brain levels were significantly decreased in the brains of Cis-intoxicated rats compared to control rats. In contrast, HP L. . Data are expressed as mean ± standard error (SE)using one-way analysis of variance (one-way ANOVA) followed by Tukey's post hoc test for seven rats in each group. * means significant from the Control group; @, significant from the Cis group; #, significant from the HP + Cis group.

Effect of HP L. and HP.NE Administration on the Levels of Oxidative Biomarkers of the Cis-Intoxicated Rats
As shown in Figure 3, SOD, CAT, GST, and GSH brain levels were significantly decreased in the brains of Cis-intoxicated rats compared to control rats. In contrast, HP L. and HP.NE-treated rats exhibited a marked increase in the brain levels of SOD, CAT, GST, and GSH compared to Cis-intoxicated rats.
Toxics 2023, 11, x FOR PEER REVIEW 9 of 20 and HP.NE-treated rats exhibited a marked increase in the brain levels of SOD, CAT, GST, and GSH compared to Cis-intoxicated rats. Conversely, Cis-intoxicated rats showed a substantial increase in the brain levels of MDA, 8-OHdG, and NO compared to the control group. However, HP L. and HP.NEtreated rats exhibited a significant decrease in these levels compared to Cis-intoxicated rats ( Figure 4). Furthermore, HP.NE was found to be more effective in improving antioxidant mechanism enzymes as compared to the HP-treated rats. Data are expressed as mean ± SE (one-way ANOVA) followed by Tukey's post hoc test for seven rats in each group. * means significant from the Control group; @, significant from the Cis group, #, significant from the HP +Cis group. Data are expressed as mean ± SE (one-way ANOVA) followed by Tukey's post hoc test for seven rats in each group. * means significant from the Control group; @, significant from the Cis group, #, significant from the HP + Cis group.
Conversely, Cis-intoxicated rats showed a substantial increase in the brain levels of MDA, 8-OHdG, and NO compared to the control group. However, HP L. and HP.NEtreated rats exhibited a significant decrease in these levels compared to Cis-intoxicated rats ( Figure 4). Furthermore, HP.NE was found to be more effective in improving antioxidant mechanism enzymes as compared to the HP-treated rats.

Effect of HP L. and HP.NE Administration on the Levels of Pro-Inflammatory Cytokines Cis-Intoxicated Rats
As depicted in Figure 5, Cis-intoxicated rats' brains displayed a marked neuroinfla mation, evidenced by a substantial increase in the brain levels of IL-6, IL-1β, TNF-α, a caspase-3 compared to control rats. Treatment with HP L. and HP.NE exerted an an inflammatory action evidenced by decreasing the aforementioned pro-inflammatory tokines compared to Cis-intoxicated rats. Interestingly, HP.NE administration was fou to be more effective in modulating neuro-inflammation, as shown by a significant red tion in the brain levels of these pro-inflammatory cytokines.

Effect of HP L. and HP.NE Administration on the Levels of Pro-Inflammatory Cytokines in Cis-Intoxicated Rats
As depicted in Figure 5, Cis-intoxicated rats' brains displayed a marked neuroinflammation, evidenced by a substantial increase in the brain levels of IL-6, IL-1β, TNF-α, and caspase-3 compared to control rats. Treatment with HP L. and HP.NE exerted an anti-inflammatory action evidenced by decreasing the aforementioned pro-inflammatory cytokines compared to Cis-intoxicated rats. Interestingly, HP.NE administration was found to be more effective in modulating neuro-inflammation, as shown by a significant reduction in the brain levels of these pro-inflammatory cytokines. Data are expressed as mean ± SE (one-way ANOVA) followed Tukey's post hoc test for seven rats in each group. *means significant from the Control group; significant from the Cis group; #, significant from the HP +Cis group.

Effect of HP L. and HP.NE Administration on the Expression Levels of PON-3 and GPX Genes in Cis-Intoxicated Rats
Cis-intoxicated rats displayed a substantial downgrade of the expression levels of t PON-3 and GPX genes compared to the control rats. Meanwhile, HP L. and HP.NE-treat rats displayed a marked upgrade in the expression levels of these genes compared to t Cis-intoxicated rats. Likewise, the administration of HP.NE was found to be more effe tive in increasing the expression levels of PON-3 and GPX genes in the brain than in t HP-treated rats (Figure 6a,b). Data are expressed as mean ± SE (one-way ANOVA) followed by Tukey's post hoc test for seven rats in each group. * means significant from the Control group; @, significant from the Cis group; #, significant from the HP + Cis group.

Effect of HP L. and HP.NE Administration on the Expression Levels of PON-3 and GPX Genes in Cis-Intoxicated Rats
Cis-intoxicated rats displayed a substantial downgrade of the expression levels of the PON-3 and GPX genes compared to the control rats. Meanwhile, HP L. and HP.NE-treated rats displayed a marked upgrade in the expression levels of these genes compared to the Cis-intoxicated rats. Likewise, the administration of HP.NE was found to be more effective in increasing the expression levels of PON-3 and GPX genes in the brain than in the HP-treated rats (Figure 6a,b). Data are expr as mean ± SE (one-way ANOVA) followed by Tukey's post hoc test for seven rats in each gro means significant from the Control group; @, significant from the Cis group; #, significant from HP +Cis group.

Effect of HP L. and HP.NE Administration on the Expression Levels of COX-2 and TP5 Genes in Cis-Intoxicated Rats
Cis-intoxicated rats displayed a significant increase in the expression levels of C 2 and TP53 in the brain compared to the control rats. Meanwhile, HP L. and HP treated rats displayed a marked reduction in the expression levels of these apoptotic g compared to the Cis-intoxicated rats. Moreover, the administration of HP.NE was fo to be more effective in decreasing the expression levels of TP53 and COX-2 genes in brain compared to the HP-treated rats (Figure 6c,d).

Histopathological Investigation
In our histological analysis, we focused on three different brain regions: cerebra tex, hippocampus, and cerebellum. As depicted in Figure 7, the control group reve normal histological structures of different brain sections. Briefly, the cerebral cortex sists of intact neurons and neuroglia cells. The hippocampus region showed regular struction of the subiculum, dentate gyrus, and cornus ammonis (CA) that split into Data are expressed as mean ± SE (one-way ANOVA) followed by Tukey's post hoc test for seven rats in each group. * means significant from the Control group; @, significant from the Cis group; #, significant from the HP + Cis group.

Effect of HP L. and HP.NE Administration on the Expression Levels of COX-2 and TP53 Genes in Cis-Intoxicated Rats
Cis-intoxicated rats displayed a significant increase in the expression levels of COX-2 and TP53 in the brain compared to the control rats. Meanwhile, HP L. and HP.NEtreated rats displayed a marked reduction in the expression levels of these apoptotic genes compared to the Cis-intoxicated rats. Moreover, the administration of HP.NE was found to be more effective in decreasing the expression levels of TP53 and COX-2 genes in the brain compared to the HP-treated rats (Figure 6c,d).

Histopathological Investigation
In our histological analysis, we focused on three different brain regions: cerebral cortex, hippocampus, and cerebellum. As depicted in Figure 7, the control group revealed normal histological structures of different brain sections. Briefly, the cerebral cortex consists of intact neurons and neuroglia cells. The hippocampus region showed regular construction of the subiculum, dentate gyrus, and cornus ammonis (CA) that split into four areas (CA1-CA4) according to the volume and distribution of pyramidal cells. Further, the cerebellum exhibited a normal molecular layer, a granule cell layer, and a large, active basophilic purkinje cell with a vesicular nucleus.  Results are expressed as median ± standard deviation (SD), determined using the Kruskal-Wallis test followed by Dunn's multiple comparison test. * means significant from the control group; @, significant from the Cis group; and #, significant from HP + Cis group.
Cis can induce undesirable changes in the histological structure of brain that present as massive neuronal degeneration with neuronophagia in the cerebral cortex. Regarding the hippocampal area, apoptosis of pyramidal cells, as well as distinct vacuolation, were detected in dentate gyrus. Furthermore, the Cis group revealed significant degeneration and nonappearance of neurons in different arenas of CA1 and CA3. Moreover, degenerated purkinje cells were observed in the cerebellum. Results are expressed as median ± standard deviation (SD), determined using the Kruskal-Wallis test followed by Dunn's multiple comparison test. * means significant from the control group; @, significant from the Cis group; and #, significant from HP + Cis group.
Cis can induce undesirable changes in the histological structure of brain that present as massive neuronal degeneration with neuronophagia in the cerebral cortex. Regarding the hippocampal area, apoptosis of pyramidal cells, as well as distinct vacuolation, were detected in dentate gyrus. Furthermore, the Cis group revealed significant degeneration and nonappearance of neurons in different arenas of CA1 and CA3. Moreover, degenerated purkinje cells were observed in the cerebellum.
Administration of HP L. and HP.NE to Cis-intoxicated rats improved and maintained the histological structure of the brain tissues against Cis toxicity. However, the greater protective effect against Cis was attributed to HP.NE.

Immunohistochemical Investigation of NF-KB and Ki-67 Protein Expression
As depicted in Figure 8, Cis and HP-treated rats revealed strong upregulation of NF-KB expression compared to the control group, while HP.NE-treated rats exhibited weak expression compared to the control and HP-treated rats. Remarkably, strong positive brown Ki-67 protein expression was recorded in both the control and HP.NE-treated rats, whereas marked expression reduction was detected in the Cis and HP-treated rats. It is worth mentioning that the expression of KI-67 in the group that received HP.NE was greater than those who received HP L. extract. Administration of HP L. and HP.NE to Cis-intoxicated rats improved and maintained the histological structure of the brain tissues against Cis toxicity. However, the greater protective effect against Cis was attributed to HP.NE..

Immunohistochemical Investigation of NF-ƘB and Ki-67 Protein Expression
As depicted in Figure 8, Cis and HP-treated rats revealed strong upregulation of NF-ƘB expression compared to the control group, while HP.NE-treated rats exhibited weak expression compared to the control and HP-treated rats. Remarkably, strong positive brown Ki-67 protein expression was recorded in both the control and HP.NE-treated rats, whereas marked expression reduction was detected in the Cis and HP-treated rats. It is worth mentioning that the expression of KI-67 in the group that received HP.NE was greater than those who received HP L. extract.

Discussion
In our study, we investigated the neuroprotective effect of HP L. and its nano-form (NE) against Cis-induced chemobrain while addressing the causes underlying this disorder, including locomotion disturbance, spatial working memory alterations, oxidative stress, and apoptosis. In addition, an emphasis on some regulatory genes, including PON-3, GPX, TP53, and COX2, has been reported. The selected HP.NE was composed of 10%

Discussion
In our study, we investigated the neuroprotective effect of HP L. and its nano-form (NE) against Cis-induced chemobrain while addressing the causes underlying this disorder, including locomotion disturbance, spatial working memory alterations, oxidative stress, and apoptosis. In addition, an emphasis on some regulatory genes, including PON-3, GPX, TP53, and COX2, has been reported. The selected HP.NE was composed of 10% Labrafac, 53.3% Tween 80, 26.6% Transcutol, and 10% water, whereas HP L. was 1.5% w/v. The globule size was 125.2 ± 24 nm, and the surface charge was −16.94 ± 3 mV due to the presence of polyphenolic compounds. A mixture of surfactant (Tween 80) and co-surfactant (Transcutol HP) was used at a 2:1 ratio (SCoSmix). Transcutol HP is usually used at a low ratio when mixed with oil due to its ability to spontaneously develop NE, as previously reported with Tadalafil NE (Oil: Capmul and SCoSmix: Labrasol/Transcutol HP) [18]. For HP encapsulation, a study encapsulated HP (concentration 1.5 to 6 mg/mL) in NE using 8% Lipoid, 2% egg lecithin, 1% tween 80 as SCoSmix, and ethanol +acetone as a solvent mixture. Although this method successfully prepared NE with a size of less than 200 nm, it requires the evaporation step of organic solvent and testing the residual solvent [27]. Another study reported self-NE loaded with HP L. for post-myocardial infarction depression. This study used self-nano-emulsification and in situ nanosuspension as a way to solubilize HP L. using 10% triacetin, 57% Tween 20, and 33% PEG 400 as surfactants and cosurfactants. The size was 258.65 nm, and the charge was −8.03 mV. The HP self-NE released 72.3% of HP L. in 5 min, which indicates a significant improvement in HP L. solubility [16]. These findings are in contrast with our study, where the development of HP.NE controlled the dissolution profile over 4 h. This could be attributed to the differences in the nature of the oil and surfactant system used in the current study.
The exposure of rats to Cis (10 mg/kg) caused a marked behavioral alteration, including locomotion disturbance, as portrayed by the decrease the number of crossing squares and the rearing frequency in the open field test, as well as the decrease the number of arm entries in the Y-maze test, compared to the control group. An open field test is an experimental test used to measure the locomotor activity level, anxiety, and exploratory behavior [22]. This test depends on the assumption of the natural fear of rodents to open illuminated areas. The reduction in the number of crossing squares reflects motor deficits and a high state of emotionality, and decreasing the rearing frequency reflects a reduction in exploratory behavior. These results are in agreement with a previous study conducted by Kandeil et al. [28], who investigated the locomotor and memory impairment associated with Cis administration. In addition, Ongnok et al. discussed the pathogenesis of chemotherapeutic medications on the brain in their study [29]. Besides locomotor disturbance, Cis treatment caused impairments in cognitive function and learning abilities [30]. Herein, we used the Y-maze test, a hippocampal-dependent test, to measure the spatial working memory in response to Cis toxicity. Memory impairment was evidenced by a decrease in the percentage of SAP. This parameter depends on the spontaneous ability of rats to alternate between three different arms [31]. Working memory processing depends on the intactness of several brain regions, such as the prefrontal cortex, hippocampus, and striatum [32]. This finding agrees with a previous study by Ongnok et al. [29].
These neurobehavioral alterations were confirmed by examining different brain regions, including the cerebral cortex, hippocampus, and cerebellum. Cis induced several histopathological alterations in the brain tissue [7]. Similarly, in our study, the exposure of rats to Cis induced degeneration and neurophagia in the cerebral cortex, as well as vacuolation and loss of pyramidal neuron in the hippocampal area. Furthermore, deeply stained Purkinje cells were detected in the cerebellum compared to control group. This could be due to the ability of Cis to cross the blood-brain barrier (BBB) and suppress neural stem cell proliferation, causing neurotoxicity [4]. Several pathways for Cis neurotoxicity have been proposed, with oxidative damage being one of the most significant in Cis and other chemotherapeutic medications [33]. Oxidative stress affects cell structure and function while decreasing antioxidant mechanisms and damaging DNA [2]. In this study, our data demonstrated that Cis considerably induces severe cerebral oxidative stress, as indicated by the severe depletion of SOD, GST, CAT, and GSH levels, along with the concurrent augmentation of MDA, 8-OHdG, and NO. These results align with previously reported studies [34,35].
PON-3 and GPX are essential antioxidant genes; their capacity to prevent lipid oxidation makes them necessary for the myelination of nerves in the brain. Their protein products interact with high-density lipoproteins in the blood. Xie et al. demonstrated that oxidative stress induced by Cis is associated with decreased PON-3 and GPX expression in brain tissue [36]. Similarly, the Cis-intoxicated group displayed a marked reduction in PON-3 and GPX expression compared to the control group.
Inflammation is another pathway of Cis-induced neurotoxicity [37]. NF-KB is considered the master regulator of inflammation and it can mediate the upregulation of pro-inflammatory cytokines and chemokines [38]. In our study, Cis-intoxicated rats showed an increase in the expression levels of brain NF-KB, which stimulates the production of pro-inflammatory cytokines, including IL-6, IL-1β, TNF-α, and caspase-3, compared to control rats. Furthermore, Cis produced a marked increase in the brain levels of COX2 and TP53 compared to the control group. Under normal conditions, the apoptotic gene (COX2) is expressed in the CNS [39]. Additionally, the TP53 gene mediates the regulation of the COX2 gene [40]. The overexpression of COX2 and TP53 are both signs and causes of brain injury [39]. The excessive production of NO as a result of neurotoxicity may be the primary cause of COX2 and TP53 overexpression. Furthermore, Cis induces downregulation in the expression levels of the Ki-67 marker, which is responsible for the ability of brain tissue to survive restoration following injury [41]. This finding is attributed to the oxidative stress and inflammation elicited by Cis that could be consistent with the inhibition of neurogenesis [41].
Natural products are sources of antioxidant and anti-inflammatory components, including phenolic compounds such as chlorogenic acid [42][43][44], caffeoylquinic acid derivatives [45], procyanidins [46], and catechins [47]. Flavonoids are phytochemicals which have attracted considerable attention because of their many pharmacological interests and health-promoting uses. Myricetin and its glycosides, which were previously identified in our extract ( Figure S1 and Table S1) [16], have great antioxidant, anti-inflammatory, and antiapoptotic potential [48,49]. Rutin is also the major compound in our extract; it exhibited several pharmacological effects and was reported as a good alternative to conventional drugs for oxidative stress and inflammation [50]. HP L. is a phenolics-enriched extract; thus, it could inhibit the production of ROS and, in consequence, prevent apoptosis. Rutin, myricetin, hyperoside, quercetin, kamepherol, hyperforin, and hypericin are among the identified metabolites in our extract ( Figure S1 and Table S1) [16]. The role of polyphenolics as antiapoptotic agents has been reported in many studies [51][52][53]. Incorporation of HP L. into an NE system was found to effectively ameliorate the neurobehavioral alterations caused by Cis, represented by increasing the number of crossing squares and the rearing frequency in the open field test and increasing the number of arm entries and SAP in the Y-maze test. Several studies have investigated the protective benefits of HP L. against cognitive impairment in neurodegenerative disorders [16,54].
Moreover, the antioxidant property of HP L. and HP.NE was evidenced by lowering the brain levels of MDA, 8-OHdG, and NO and increasing in the cerebral antioxidant defense of SOD, GST, CAT, and GSH. The antioxidant activities of HP L. and HP.NE are correlated with their ability to act as free radical scavengers by inhibiting lipid peroxidation. These findings were associated with promoting the expression of PON-3 and GPX antioxidant genes in the brain tissue. As we mentioned before, this could be attributed to the bio-flavonoid content of HP L. and HP.NE, which has a direct scavenging effect on ROS [55].
Furthermore, the anti-inflammatory property of the HP L. and HP.NE was portrayed by decreasing the brain levels of IL-6, IL-1β, TNF-α, and caspase-3. This finding may be explained by the ability of HP L. and HP.NE to scavenge free radicals and inhibit enzyme leakage across plasma membranes [49]. Notably, the pro-inflammatory cytokines in HP.NE treated rats were comparable to those in control rats. In addition, the expression levels of COX-2 and TP-53 were decreased in the HP L. and HP.NE-treated rats. Wu et al. revealed that antioxidants inhibit COX2 expression in brain tissue [56] by inhibiting activator protein1 (AP1), which regulates the promoters of different inflammatory genes (such as COX2 and TP53). Furthermore, Cui et al. showed that antioxidants down-regulate COX2 and TP53 production via inhibiting NF-κ activity in the same sequence [57]. The antiinflammatory activity of HP L. and HP.NE may be partially attributable to their ability to modulate COX2 and TP53 expression [58]. Alternatively, it has been demonstrated that hyperforin acts as a dual inhibitor of TP53 and COX2 in intact cells and on the catalytic activity of the enzymes, suggesting therapeutic promise for inflammatory diseases [59]. These findings were further confirmed by assessing the expression levels of the Ki-67 marker that was considered a marker for newly formed cells in brain tissue after exposure to damage [60]. Treatment with HP L. and HP.NE promoted proliferation in the brain tissue after Cis administration via significant improvement in the Ki-67 marker. These results were consistent with previous studies that demonstrated the anti-inflammatory and preservative action of HP L. [16,61]. Based on the previously mentioned results, utilizing antioxidant, anti-inflammatory, and anti-apoptotic agents such as HP L. and HP.NE to guard against Cis-induced cerebral apoptosis is a logical strategy.

Conclusions
In conclusion, the administration of HP L. and HP.NE appeared to be an effective therapy for counteracting the neurobehavioral alterations, brain oxidative stress, inflammation, and apoptosis induced by Cis, as evidenced by the marked improvement in the behavioral profile of treated rats, significant reduction in lipid peroxidation markers, pro-inflammatory cytokines, elevated PON-3 and GPX, and suppression of TP53 and COX2 brain levels. In addition, a reduction in the expression of brain levels of NF-KB associated with an increase in the brain levels of Ki-67 was confirmed. Similarly, the brain architecture was restored. These results could be attributed to the phytochemical profile of HP L., which possesses antioxidant, anti-inflammatory, and antiapoptotic compounds. The medicinal activity of HP L. was improved by incorporating HP L. into the NE system, where the nanosized and lipophilic nature of the carrier improved the absorption of the phytochemical components of HP L.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/toxics11020159/s1, Figure S1: Total ion chromatogram of HP L. ethanolic extract in positive ionization mode and Table S1: Metabolites identified from the ethanolic extract of HP L.