Synergistic effect of spermidine and ciprofloxacin against Alzheimer disease in male rat via ferroptosis modulation

doi:10.21203/rs.3.rs-3238520/v1

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Synergistic effect of spermidine and ciprofloxacin against Alzheimer disease in male rat via ferroptosis modulation

https://doi.org/10.21203/rs.3.rs-3238520/v1

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Alzheimer's disease (AD) is a prevalent form of neurodegenerative disease with a complex pathophysiology that remains not fully understood, and the exact mechanism of neurodegeneration is uncertain. Ferroptosis, a programmed cell death mechanism mediated by iron, has been linked to the progression of degenerative diseases observed in clinical AD models. In this study, we aimed to explore the synergistic effects of spermidine and/or ciprofloxacin in combating Alzheimer's disease (AD). We investigated AD-related biomarkers, including neurotoxic Aβ, arginaseI, and serotonin.Spermidine demonstrated an anti-ferroptotic effect in the AD model, evident from the modulation of ferroptosis parameters such as hippocampus iron levels, reduced protein expression of transferrin receptor 1 (TRF1), and arachidonate 15-lipoxygenase (ALOX15). Additionally, the administration of spermidine led to a significant increase in protein expression of phosphorylated nuclear factor erythroid 2-related factor 2 (p-Nrf2) and upregulation of Cystine/glutamate transporter (SLC7A11) gene expression. Moreover, spermidine notably decreased p53 protein levels, acrolein, and gene expression of spermidine/spermine N1-acetyltransferase 1 (SAT1). The histopathological examination of hippocampus tissue corroborated these results obtained from molecular biochemical inspection. Overall, our findings suggest that spermidine and/or ciprofloxacin may offer potential benefits against Alzheimer's disease (AD) by modulating ferroptosis. Furthermore, spermidine enhanced the antioxidant efficacy of ciprofloxacin and reduced its toxic effects by increasing antioxidant enzymes, thereby enhancing its potency against oxidative stress.

D-galactose

Spermidine

ciprofloxacin

ferroptosis

oxidative stress

antioxidant

Alzheimer’s disease

aluminum chloride.

Alzheimer's disease (AD) is characterized by progressive neuronal loss and gradual cognitive impairment, but its complex pathophysiology is not yet fully understood(Habieb et al., 2023). The disease is associated with the accumulation of β-amyloid (Aβ) plaques and tau-containing neurofibrillary tangles in the brain as it advances(Hippius & Neundörfer, 2022). The exact mechanism of neurodegeneration remains uncertain, leading to several postulated explanations for AD pathogenesis. In the early stages of the disease, deficits in memory and cognitive functions are primarily influenced by changes in the hippocampus, where up to 80% of the neurons may die during AD progression(Scheltens et al., 2021).

Iron accumulation in the brain of AD patients has been linked to brain oxidative stress, associated with disturbances in brain iron metabolism(Ryan et al., 2023). This iron metabolism disorder contributes to Aβ misfolding and tau hyperphosphorylation, resulting in oxidative stress and iron ion toxicity, both of which are implicated in AD(Mishima et al., 2022). Ferroptosis, a cell death mechanism characterized by iron-mediated phospholipid peroxidation, has been observed in AD and is affected by iron homeostasis disturbances(Peng et al., 2021). The intricate balance of iron regulation at both systemic and cellular levels affects ferroptosis sensitivity. Glutathione peroxidase 4 (GPX4) plays a crucial role in inhibiting ferroptosis and reducing lipid peroxidation. Inhibition of GPX4 activity or expression leads to ferroptosis, which contributes to neural cell death in neurodegenerative disorders like AD(Rodrigue et al., 2020). Iron chelators, including deferoxamine, deferrone, and ferrite, can suppress ferroptosis and are used to treat iron overload diseases(Gutbier et al., 2020).

Polyamines (PAs) are organic polycationic alkylamines with diverse functions, mainly related to cell growth, survival, and proliferation. Putrescine, spermidine (SPD), and spermine (Spm) are examples of polyamines strictly controlled in levels, synthesized from L-ornithine by amino acid decarboxylation(Zou et al., 2022). SPD, found in various living organisms and certain foods, has been associated with age-related autophagy deficiency leading to decreased intracellular concentrations(Madeo et al., 2018). Recent attention has been directed towards SPD's potential therapeutic benefits in neurodegenerative diseases like AD(Steichen et al., 2023). Studies have shown that SPD can extend lifespan in an autophagy-dependent manner, reduce soluble amyloid beta plaques in AD mouse models, and alleviate oxidative stress and inflammation while enhancing memory and cognitive function(Lumkwana et al., 2022). Ciprofloxacin (CPFX), a fluoroquinolone antibiotic, contains potential donor atoms that can coordinate with metal ions like iron(Telfer, 2020). This study investigates the beneficial effects of SPD and/or ciprofloxacin supplementation against an AD model induced by D-galactose or aluminum chloride via ferroptosis modulation in male rats.

Chemicals and Drugs:

Spermidine, D-galactose, aluminum chloride, and ciprofloxacin were purchased from Sigma-Aldrich, USA.

Animals:

Seventy adult Wistar Albino male rats (190–220 g) were obtained from the Faculty of Veterinary Medicine, Mansoura University, Egypt. The rats were acclimated for a week under controlled conditions before the study.

Experimental design:

Rats were classified into seven groups (n=10) as follows: Gp1(Normal control): Rats were injected with 0.5 mL salin i.p.; Gp2(ciprofloxacin control): Rats were injected i.p. with 14 mg/kg ciprofloxacin (dissolved in saline) day after day for 4 weeks(Elmi et al., 2009); Gp3(spermidine control): Rats were injected i.p. with 20mg/kg spermidine (dissolved in saline) day after day for 4 weeks(Yadav et al., 2018); Gp4(Alzheimer disease):Rats were injected i.p. with 120mg/kg D-gal (dissolved in saline) and ALCL3 50 mg/ kg bw(dissolved in dis.wt) orally daily for one month(Beltagy et al., 2021); Gp5(ciprofloxacin co treatment): rats received D-gal and ALCL3as in Gp4, along with injected i.p. with ciprofloxacin as in Gp2 day after day for 4 weeks, Gp6(spermidine co treatment): rats received D-gal and ALCL3as in Gp4, along with injected i.p. with spermidine as in Gp3 day after day for 4 weeks, and Gp7(Combination co treatment): rats received D-gal and ALCL3as in Gp4 then injected with CPFX and SPD day after day for 4 weeks. All rats were sacrificed 24 hours after the last dose of the treatment.After rats being anaesthetized by intraperitoneal injection of 1.2 mg/kg bw urethane(Flecknell, 1993), Blood samples were taken of each animal by cardiac puncture and centrifuged to obtain serum for biochemical assays. The brain specimens were quickly removed, perfused with isotonic saline to exclude the blood cells and then blotted on filter paper to dry, divided mid-sagitally in to two halves separated hippocampus from whole brain. A portion was added to 10% neutral formalin for histopathological and immunochemical studies. The other part of the hippocampus tissue was homogenized and frozen at −20 °C for further analyses.

Sample collection and analysis:

After anesthesia, blood samples were obtained by cardiac puncture for biochemical assays, and brain specimens were removed for further analysis. Serum analysis included blood glucose levels, while hippocampus serotonin, ArginaseI, malondialdehyde (MDA), reduced glutathione, catalase activity, glutathione peroxidase activity, superoxide dismutase, total iron, and acrolein were analyzed in the hippocampus. Western blot analysis was conducted to determine protein expression of TfR1, ALOX15, p-Nrf2, and β-amyloid. Quantitative real-time RT-PCR was used to measure hippocampus SAT1 and SLC7A11 gene expression. The study was conducted following the guidelines approved by the Research Ethical Committee (Faculty of Science, Tanta University, Egypt) (IACUC-SCI-TU-0233).

Serum analysis:

Blood glucose level was determined using kits from (Vitro scient- Diagnostics, Egypt) according to Colorimetric Method described by (Trinder, 1969).

Biochemical analysis

Hippocampus serotonin was measured by utilizing Rat serotonin (SERT) Elisa kit purchased from (Elabscience Cat. No: E-EL-R1140), Hippocampus ArginaseI was detected using Rat ArginaseI (ARG1) ELISA Kit (MyBioSource, USA Cat.No MBS917512). Hippocampus malondialdehyde (MDA) was estimated using Rat Malondialdehyde (MDA) ELISA Kit (CUSABIO, USA, Cat. NO. CSB-E08558r) following the manufacturer’s instructions, hippocampus Reduced glutathione was estimated using GSH Assay Kit (Colorimetric) (Abcam, USA Cat. NO. ab239727). Hippocampus Catalase activity was estimated using Catalase colorimetric kit (Abcam, USA Cat. NO. ab83464), hippocampus Glutathione Peroxidase activity was estimated using Glutathione Peroxidase activity Assay Kit Colorimetric (Abcam, USA Cat. NO. ab102530), hippocampus Superoxide Dismutase was estimated using Superoxide Dismutase Activity Assay kit (Colorimetric) (Abcam, USA Cat. NO. ab65354). Hippocampus total iron was determined according to colorimetric method using kits from (Bio-diagnostic, Egypt Cat. No IR 15 10) described by (Dreux, 1977). Hippocampus acroline was detected using Rat Acrolein (ACR) Elisa kit following the protocol (MyBioSource, USA Cat.No MBS7238523).

Western blot analysis

Hippocampus determination of TfR1, ALOX15, p-Nrf2 and β-amyloid protein expression. Proteins were separated by SDS-Polyacrylamide gel electrophoresis as described byLillehoj and Malik (1994). About 30 to 40 μg of protein was separated. After protein separation the gel is transferred to PVDF membrane. The PVDF were blocked with skimmed milk powder (5%) for 12 h at room temperature. Following blocking, the blocking solution was decanted and the membrane was briefly rinsed in Tris Buffer Solution Tween20 (TBS-T) and incubated for overnight in Tris-buffered saline (TBS) containing suitably diluted primary antibody for Anti-Transferrin Receptor antibody (Abcam, USA Cat No. ab84036), Anti-15 Lipoxygenase 1 antibody [EP1808Y] (Abcam, USA Cat No. ab244205), Nrf2 (Phospho-S40) antibody (biorbyt, Cambridge Cat No. orb336606,), or Anti-beta Amyloid antibody [DE2B4] (Abcam, USA Cat No. ab11132). Subsequently after primary incubation the membrane was washed with TBS-T and further it was incubated with HRP-conjugated secondary antibody. The protein expression was detected by chemiluminescence and corresponding chemiluminescence and corresponding densitometry analysis was performed. β-actin was loaded as the internal reference gene an Anti-beta Actin antibody (Abcam, USA Cat No. ab8224), using the same protocol as mentioned(Mahmood & Yang, 2012)..

Molecular investigation

Determination of hippocampus SAT1 and SLC7A11 gene expression using quantitative real-time RT-PCR: Quantitative real-time PCR (qRT-PCR) was used to measure the mRNA gene expression of SAT1 and SLC7A11 in addition to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Total RNA was isolated from hippocampus tissues using the RNeasy Mini Kit (Cat No. 74104) obtained from Qiagen Inc. (Valencia, CA, USA) following the manufacturer protocol (Monis & Giglio, 2006).Single-stranded cDNA was synthesized from the reverse transcription of 0.5–2 μg total RNA using random hexamer primers in a total volume of 50 μl with High Capacity cDNA Reverse Transcription Kit (Cat No. K1621) purchased from Applied Biosystems (Foster City, CA, USA). Synthesis of cDNA was performed at 37 °C for 6 min and 1 h for elongation, followed by reverse transcriptase enzyme inactivation for 10 min at 95 °C(Monis & Giglio, 2006).The synthesized cDNA was stored at − 20 °C pending quantitative real-time polymerase chain reaction (qRT-PCR) analyses of gene expression. PCR primers were designed with Gene Runner Software (Hasting Software Inc., Hasting, NY, USA) from RNA sequences from GenBank (Table:1). qRT-PCR amplifications were performed using Applied Biosystems with software version 3.1 (StepOne™, USA). The qRT-PCR mixture consisted of: 3 µL cDNA template (10-20 ng/ μL), 2X Maxima SYBR Green/ROX qPCR Master Mix (12.5 µL), 1 µl Primer forward (10 μM), 1 µl Primer reverse (0.1- 0.5 μM), 7.5 µL Water, nuclease-free in a total volume of 25 μl. The final reaction mixture was placed in a Step One Plus real-time thermal cycler and the PCR program was performed in 45 Cycles with the PCR conditions as follow (Initial denaturation 95˚C/ 10 min, Denaturation 95˚C/ 15 sec, Annealing 60˚C/ 30 sec and Extension72˚C/ 30 sec). The quantities critical thresholds (Ct) of the target gene were normalized with quantities (Ct) of the house-keeping gene by used the 2-∆∆Ct method (Livak & Schmittgen, 2001).

Histopathological investigation

The brains were excised and fixed at 10 % neutral formalin for twenty four hours; and then washed by running water over night. Via a graded ethanol sequence, fixed brains were dehydrated and embedded in paraffin according to standard procedures. On gelatin chromium-coated glass slides, paraffin sections (5μm thick) were mounted and used as a routine method for haematoxylin and eosin stains (Bancroft & Stevens, 1996).

Immunohistochemical investigation

Immunohistochemical staining of hippocampus tumor protein p53 (p53):

Tissue sections were cut at thickness of 4 μm. After that, deparaffanize with xylene, rehydrate in graded ethanol concentration (100-95-75%) and epitope retrieve; the preferred method was the use of heat Induced Epitope Retrieval (HIER) technique using Cell Marque triology in conjunction with a pressure cooker. Upon completion, rinse with 5 changes of distilled water and phosphate-buffered saline (PBS). Antigen retrieval eas enhanced by heating in citrate buffer PH (6.0) then, 3% hydrogen peroxide was used to block endogenous peroxidase activity. After that, we incubate the tissue sections with antibodies for 60 minutes against p53 rabbit polyclona (1:1600; Cat.No. GB11626, Servicebio, china). Then, Incubate with UltraVision One HRP Polymer for 15 minute. Then, Substrate/Chromogen Prepare Ready-To-Use DAB substrate solution. Add DAB Chromogen Solution (Reagent B1) to DAB Buffer Solution (Reagent B2) and mix the two solutions in a 1:1 ratio with the volume determined by the number of slides to stain. In general, 200 µL of mixed substrate solution is sufficient to cover one tissue slide. After that, we washed tissue sections with distilled water then counterstain with hematoxylin. Then, the step of dehydration using ascending grades of alcohol and xylene takes place. The final step in processing was mounting the slides with mounting medium and cover with cover slide(Aprikian et al., 1994).

Statistical analysis

All the data were expressed as means ±S.E. The statistical significance was evaluated by one-way analysis of variance (ANOVA) using SPSS, 18.0 software, 2011 and therefore the individual comparisons were obtained by Duncan's multiple range test (DMRT). Values were considered statistically significant when p <0.05.

At a high dose of spermidine (50mg/kg bw) alone, it led to the death of more than 50% of the rat groups. There was no significant change in the initial and final body weight of the normal control animals. However, the animals treated with D-gal and ALCL3 showed a significant (p < 0.001) decrease in body weight on the last day (30th day) compared to the normal control group. Treatment with spermidine and/or ciprofloxacin significantly (p < 0.05) restored the body weight compared to the D-gal and ALCL3-treated groups (Table 2).

The serum glucose levels significantly increased (p < 0.05) in the untreated AD group compared with the normal control group. These elevated levels were markedly reduced (p < 0.05) in all co-treated groups, with significant differences observed between the normal control group and the CPFX control group. The SPD-CPFX combination group exhibited the most significant decrease (Table 2).

Regarding the effect of spermidine on AD biomarkers, the hippocampus serotonin levels were significantly (p < 0.05) decreased in the untreated AD group compared with the normal control group. These levels were significantly (p < 0.05) increased in all co-treated groups, with the best improvement observed in the combination group. Additionally, significant differences were observed between the normal control group (GP1) and the CPFX control group (GP2) (Table 3).

The hippocampus levels of arginaseI were significantly (p < 0.05) increased in the untreated AD group compared with the normal control group. These levels were significantly (p < 0.05) decreased in all co-treated groups, with the smallest decrease in the combined group. Moreover, significant differences (p < 0.05) were observed between the normal control group (GP1) and the CPFX group (GP2), while the SPD co-treated group (G6) showed a remarkable reduction (p ≤ 0.05) compared to the CPFX co-treated group (G5) (Table 3).

The protein level of β-amyloid in the untreated AD group showed a marked upregulation (p < 0.05) compared with the normal control group. Co-treatment with CPFX, SPD, or the combination significantly decreased (p < 0.05) these levels, with the most substantial decrease observed in the combination group. Significant differences (p < 0.05) were also observed between the normal control group (GP1) and the CPFX or SPD control groups (GP2, GP3), respectively (Fig. 1).

Regarding the effect of spermidine on ferroptosis biomarkers, the protein levels of TfR1 and ALOX15 were upregulated (p < 0.05), while the protein level of p-Nrf2 was downregulated (p < 0.05) in the AD group compared with the normal control group. Treatment with CPFX, SPD, or the combination resulted in a significant decrease in TfR1 and ALOX15 expression and a significant increase (p < 0.05) in p-Nrf2 expression, with the maximum recovery observed in the combination group)Fig. 2).

The hippocampus levels of iron significantly increased (p ≤ 0.05) in rats treated with D-gal and AlCl3 (AD group, G4) compared with the control group (G1). These increased levels were significantly reduced (p ≤ 0.05) following co-treatment with CPFX (G5), SPD (G6), and the SPD/CPFX combination group (G7), with the combination group showing the most remarkable decrease. Significant differences were observed between SPD (G3), CPFX (G2), and the normal control (Table 2).

The relative expression of the SCL7A11 gene significantly decreased (p < 0.05) in the untreated AD group, but gene expression was significantly increased (p < 0.05) in all co-treated groups, with the highest expression observed in the SPD-CPFX combination group (Fig. 2).

Assessment of MDA levels indicated that hippocampus MDA levels were significantly elevated (p < 0.05) in the untreated AD group compared with the normal control group. Co-treatment resulted in a marked decrease (p < 0.05) in MDA levels, with minimal diminishment observed in both the SPD co-treated group and the SPD-CPFX combination group, with significant differences (p < 0.05) between the normal control and CPFX control (Table 4).

Spermidine and/or ciprofloxacin significantly improved the hippocampus antioxidant capacity. In the untreated AD group, the activities of SOD, CAT, GPX4, and GSH levels were significantly decreased (p < 0.05) compared with the normal control group. These decreased activities were recovered in all co-treated groups, with significant differences (p < 0.05) between the normal control group and the CPFX or SPD control groups, with the combination group showing the maximum improvement (Table 4).

The results elucidated that there was a marked increase (p < 0.05) in the relative expression of SAT1gene in the AD group compared with normal control. This increased level was significantly (p < 0.05) brought down in the other co treated groups with a minimal diminish in the combination group. Moreover Assessment of acrolein concentration indictate that acrolein concentrations were significantly (p < 0.05) elevated in the AD group as compared of normal control group. The acrolein concentrations were markedly brought down (p < 0.05) in the all co treated groups with minimal diminish in both SPD-CPFX combination group with significant difference (p < 0.05) between the normal control and CPFX, SPD control groups (Fig. 3).

Histological assessment of the hippocampus revealed normal neurons in the control groups (G1, G3) and slightly mild neuronal degeneration in the CPFX control group (G2). Marked neuronal shrinkage and degeneration were observed in the hippocampal sections from the D-gal and ALCL3-induced AD model group (G4). Co-treatment with CPFX (G5) showed slightly decreased neuronal shrinkage and degeneration, while SPD co-treatment (G6) showed moderate neuronal shrinkage and degeneration. The SPD-CPFX combination group (G7) displayed mild neuronal shrinkage and degeneration (Magnification X: 400) (Fig. 4).

Immunohistochemical staining of the hippocampus against p53 showed negatively stained neurons in the control groups: normal control (G1), CPFX (G2), and SPD (G3). Prominent positive brown neuronal staining against p53 was observed in the hippocampal sections from the D-gal and ALCL3-induced AD model group (G4). Co-treatment with CPFX (G5) showed slightly decreased positive brown neuronal staining against p53. SPD co-treatment (G6) exhibited moderate positive brown neuronal staining against p53. The SPD-CPFX combination group (G7) displayed mild positive brown neuronal staining against p53 (Magnification X: 400) (Fig. 5)

The current study aimed to investigate the synergistic effect of spermidine and/or ciprofloxacin in a male rat model of Alzheimer's disease induced by D-galactose and aluminum chloride. The results showed that the hippocampus was atrophied in the AD rat model, successfully induced by D-galactose and aluminum chloride administration. However, spermidine alone or in combination with ciprofloxacin was able to mitigate the hippocampal damage induced by D-galactose and aluminum chloride in male rats. The AD model group exhibited distinct differences in histopathological, immunohistochemical, and biochemical indexes compared to other groups.

The study revealed a significant reduction in body weight in the AD model group compared to the normal healthy control rats, which aligns with previous findings(Mohamed et al., 2021) who indicated that there was a significant decline in body weight in AD group compared with normal healthy control rats. However, the co-treatment with spermidine and/or ciprofloxacin improved body weight; consistent with studies showed that spermidine treatment can prevent age-related weight loss(Filfan et al., 2020).

Iron accumulation in certain brain areas responsible for movement and cognitive function execution has been linked to neurodegenerative disorders like Alzheimer's disease(Pal et al., 2022). The results demonstrated a significant increase in hippocampus total iron in the AD model group, which may be attributed to the deposition of beta-amyloid(Viktorinova & Durfinova, 2021). Moreover, excessive iron accumulation due to long-term exposure to D-galactose increases oxidative stress, and reduces antioxidant activities that subsequently accelerate degenerative changes in the brain, as with normal aging (Lou et al., 2023). However, co-treatment with spermidine and/or ciprofloxacin normalized the iron levels in the AD group, indicating their iron-chelating effects and protection against oxidative stress-induced brain degeneration. Our result agree withLane et al. (2018) who illustrated that administration of putrescine, spermidine, and under some conditions by spermine could serve as iron chelators to inhibit proliferation

The present study indicated an elevation in hippocampus total iron in SPD control group compared with normal control group which is similar to the results was obtained by (Kumar et al., 2022) who demonstrated that spermidine mitigates oxidative stress in in the ΔspeG strain by lowering overall ROS levels. However, Giving too much spermidine can cause the body to produce excessive amounts of superoxide, a type of harmful free radical via enhancing catabolism of polyamine; thereby induced hydrogen peroxide and superoxide anions accumulation, resulting in redox balance and iron homeostasis disruption. These reflect that RNA-bound spermidine inhibits iron oxidation. Meanwhile, free spermidine interacts and oxidizes the iron so that regulation of PAs should be actively handled within the cells.

The study also revealed that the hippocampus total iron level was elevated in the ciprofloxacin group compared to the normal control group. It was indicated that repeated pharmacological doses of CPFX were associated with iron elevation related oxidative stress which adversely affect central nervous system via depletion of endogenous antioxidant(Al-Naely et al., 2022). Our result was supported by histopathological changes which were observed in brain tissue of rats received only ciprofloxacin at dose (14 mg/kg per day) compared to the control group.

The expression of beta-amyloid was significantly increased in the AD model group compared to the normal control group, attributed to free iron accumulation in hippocampus neuronal cells and oxidative stress-induced damage(Zhang et al., 2021). Co-treatment with spermidine and/or ciprofloxacin reduced the expression of beta-amyloid, suggesting their potential to degrade neurotoxic soluble beta-amyloid and inhibit neuroinflammation, which is associated with Alzheimer's disease(Freitag et al., 2022).

Serotonin, a neurotransmitter regulating memory and learning, was significantly reduced in the hippocampus of the AD model group. Spermidine and/or ciprofloxacin co-treatment increased serotonin levels, possibly due to their antioxidant effects, which counteracted oxidative stress-induced alterations in neurotransmitters(Ghosh et al., 2020).

The current study showed a significant increase in the concentration of arginaseI after administration of D-gal and ALCL₃ (AD group) compared by normal control group may be due to an increase in free iron inside cells can lead to excessive oxidative stress, which can cause Aβ to deposit and change the expression levels of genes, such as arginaseI. (Polis et al., 2018). Our result is in accordance with the results of(Banerjee et al., 2022) who reported that arginaseI dysregulated in aged brain, suggesting their role in the pathogenesis of age-related diseases. These elevated levels were significantly decrease upon co treatment with SPD or CPFX with minimal diminish in the SPD-CPFX combination group and this result was similar to the results obtained by(Avtandilyan et al., 2013) who demonstrate that SPD influence exactly on the special stereospecific regulatory site of arginase .Thus, the change of arginase activity could be a consequence of quantitative changes of polyamines.

Our results illustrated a significant elevation in blood glucose level in D-gal and ALCL₃ group compared with normal control which may be attributed abnormality of insulin and disruption of insulin signaling such as insulin growth factor type I and II (IGF-I and IGF-II), resulting in enhancing a brain-specific form of diabetes involved in the Alzheimer disease model(Kakoty et al., 2023). Administration of spermidine and / or ciprofloxacin along with AD group modulated serum glucose levels which suggests that spermidine and/or ciprofloxacin co–administration restore insulin signaling which subsequently modulated glucose levels. Our results are in line withMéndez and Balderas (2006) who indicated that spermidine exerted an inhibitory effect on hemoglobin glycation and lipid peroxidation in vivo, which may be implicated in prevention of diabetic complications.

Ciprofloxacin administration may cause hypoglycemia by increasing insulin release, even in non-diabetic patients(Gupta et al., 2020), which can explain the reduction in blood glucose levels seen in the ciprofloxacin group.

In this study, oxidative stress damage was increased in the AD model group, as evidenced by elevated hippocampus MDA levels and decreased antioxidant activities. Co-treatment with spermidine and/or ciprofloxacin restored antioxidant enzyme activities and reduced MDA levels, indicating their antioxidant effects against hippocampus injury induced by D-galactose and aluminum chloride(Nahar et al., 2017). Moreover, the result obtained by (Salomon-Zimri et al., 2022) indicated that ciprofloxacin exhibited antioxidant effect against ferric reducing antioxidant power.

The expression of ALOX15, an enzyme involved in ferroptosis, was significantly increased in the AD model group, indicating its association with iron accumulation and lipid peroxidation (Zhao et al., 2023). However, co-treatment with spermidine and/or ciprofloxacin decreased ALOX15 expression, suggesting their anti-ferroptotic effects.

It is well known that Nrf 2 is a critical modulator of oxidative homeostasis and is produced in response to elevated oxidative stress, which plays a crucial role in modulating ferroptotic response, as it induces the expression of SLC7A11 to protect hippocampus tissue against ferroptosis(Liu et al., 2020). In our study p-Nrf2 protein expression levels and mRNA gene expression levels of SLC7A11 significantly deceased in D-gal and ALCL₃ compared with normal control group which suggested that initially when the levels of ROS exceed cellular antioxidant capacity could results in down-regulation of antioxidant regulator Nrf2, which consequently leads to neuronal antioxidant depletion(Kasai et al., 2020). In this study the improvement of p-Nrf2 protein expression levels and mRNA gene expression levels of SLC7A11 have shown in all co treaded group with maximum increase in SPD along with CPFX combination group. This was confirmed by Liu et al. (2019) who indicated that SPD acts as a noncanonical Nrf2 inducer via activation of microtubule associated protein 1S (MAP1S)–mediated autophagy, which acts as an activator noncanonical pathway of Nrf2.

The expression of TfR1, involved in iron uptake, was significantly elevated in the AD model group. Co-treatment with spermidine and/or ciprofloxacin modulated TfR1 expression, possibly through mTOR signaling regulation to inhibit TfR1 overexpression, leading to ferroptosis modulation(Ding et al., 2021).

Furthermore, Immunohistochemical analysis showed positive staining for p53 in the hippocampus of the AD model group, which may be related to neuronal cell death and accumulation of p53 oligomers(Farmer et al., 2020). Co-treatment with spermidine and/or ciprofloxacin decreased p53 levels.

It is known that mutation of p53 is mediated activation Spermidine/ Spermine N1 acetyltransferase-1 (SAT1), an enzyme of polyamine catabolism induces lipid peroxidation, contributes to the death of neuronal cells (Hasan et al., 2023). In the present study, there was a significant increase in SAT1 mRNA gene expression levels in AD rat group induced by D-gal and ALCL₃ compared with normal group. Administration of spermidine and/or ciprofloxacin significantly decreased SAT1 mRNA gene expression compared with AD group. Other results reported that depletion of iron decrease polyamine levels by a conserved mechanism involving the downregulation of polyamine anabolic pathway as well as the upegulation of the rate-limiting catabolic enzyme, SAT1, resulting in neurotoxicity induced by elevation in hydrogen peroxide and acrolein in the hippocampus of the brain(Lane et al., 2018).

Acrolein is a highly active unsaturated aldehyde involved in neurodegenerative diseases such as AD as a result of elevating oxidative stress, polyamine metabolism, neuronal damage (Chang et al., 2022)

Our results indicated a significant increase in acrolein levels in D-gal and ALCL₃ (AD model) compared with control group which may be due to elevated intracellular iron concentration and a depletion of antioxidant enhance ROS levels which consequently induce formation of many secondary products, such as acrolein (Wang et al., 2022).Furthermore, the induction of acrolein in neuronal cells could be related to increase the expression of spermidine/spermine N1-acetyltransferase (SAT1) (Đorđievski et al., 2023).

In conclusion, spermidine and/or ciprofloxacin co-treatment exhibited a synergistic effect in mitigating Alzheimer's disease progression induced by D-galactose and aluminum chloride via modulation of ferroptosis. Spermidine showed greater efficiency in protecting against Alzheimer's disease progression compared to ciprofloxacin alone, possibly due to its unique antioxidative and neuroprotective properties.

Acknowledgements: The authors appreciate the support of Tanta University and Agricultural Research Centre that was provided to complete this work.

Authors' contributions:

All authors contributed to the study conception and design. Material preparation and data collection were performed by Menna Allah M. Youssef andMai M. El-Keiy. Data analysis and visualization were performed by Menna Allah M. Youssef,Mai M. El-Keiy,Tarek M. Mohamed,Azza A. Bakry. Supervision was underTarek M. Mohamed andAzza A. Bakry. The first draft of the manuscript was written by Menna Allah M. Youssef andMai M. El-Keiy, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Availability of data and material: The data will be available upon reasonable request.

Ethics approval and consent to participate: The animals were handled and the experiments were done according to the suggested National ethical guidelines for the care of laboratory animals and the Animal Ethics Committee of the Faculty of Science, Tanta University (IACUC-SCI-TU-0233).

Consent for publication: The authors approve publication

Competing interests: The authors have no conflicts of interest.

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Table (1): The sequences of the forward and reverse oligonucleotide primers were as follows:

Reverse primer	Forward primer	accession no.	Gene
5′-CCCACGAGAGAAAAAGTCG-3′	5′-CATCTCTCCTAAGGGCGTGC-3′	NC_051337.1	SLC7A11
5′-TCCATCCCTCTTCACTGGAC-3′	5′-GCTGCAGCAGTATGCACTTC-3′	NM_0011007667.3	SAT1
5′-AGGGGCCAT CCACAGTCTTC-3′	5′-AGAAGG CTGGGGCTCATTTG-3′	NC_051339.1	GAPDH

Table (2): Body weight, glucose levels and hippocampus total iron levels in different experimental rat groups

Animal groups	Intial b.wt Mean± SEM	Finil b.wt Mean± SEM	Body weight gain Mean± SEM	glucose level (mg/dl) Mean± SEM	Total iron level (µg/dl) Mean± SEM
Normal control	203±4.55	259±5.57^a	54±5.09^a	116.92±1.78^e	75.45±3.88^e
CPFX control	204±6.67	232±3.39^b	24±4.30^cd	97.80±1.25^f	109.41±7.48^d
SPD control	206±10.09	241±2.45^b	32±8.46^bc	125.500±4.33^de	95.61±8.35^d
D-gal and ALCL₃ (AD model)	211±2.00	148.33±4.59^e	-63.33±4.77^a	228.65±4.25^a	183.33±7.50^a
CPFX co treatment	210±6.00	165±7.07^d	-47±7.35^ab	190.75±5.74^b	161.78±4.87^b
SPD co treatment	212±4.00	200.8±2.85^c	-11±3.59^d	150.15±5.36^c	140.45±7.77^c
Combination treatment	211±2.00	194±4.85^c	-17±5.61^cd	132.20 ±7.51^d	131.21±4.02^c

Data are presented as mean ± SEM (standard error of mean), n = 8 per group. Mean values with different superscript letters in the same column are significantly different at (p≤0.05).

Table (3): Serotonin &ArginaseI levels in hippocampus of different experimental rat groups

Animal groups	Serotonin (ng/g tissue) Mean± SEM	arginaseI (ng/g tissue) Mean± SEM
Normal control	562±24.32^a	3.20±0.02^f
CPFX control	467.66±17.38^cd	4.55±0.11^ed
SPD control	532±9.17^ab	3.50±0.25^ef
D-gal and ALCL₃ (AD model)	337.5±8.34^e	9.31±0.75^a
CPFX co treatment	417.67±9.69^d	7.77±0.26^b
SPD co treatment	480±23.02^bc	5.86±0.43^c
Combination treatment	496.60±28.31^bc	5.24±0.17^cd

Data are presented as mean ± SEM (standard error of mean), n = 8 per group. Mean values with different superscript letters in the same column are significantly different at (p≤0.05).

Table (4): Oxidative stress and antioxidant parameters in hippocampus tissue in different experimental rat groups

Animal groups	MDA (nmol/g tissue) Mean ± SEM	SOD (U/g tissue) Mean ± SEM	CAT (U/g tissue) Mean ± SEM	GPX4 (U/g tissue) Mean ± SEM	GSH (mg/ gtissue) Mean ± SEM
Normal control	6.31 ±0.34^e	66.75±1.88^a	5.46 ±0.17^a	147.4 ±0.63^a	40.20 ±0.92^a
CPFX control	10.01 ±1.02^d	38.095 ±1.79^d	4.24 ±0.11^b	125.32 ±0.82^bc	22.39 ±1.07^e
SPD control	8.71 ±0.31^ed	48.07 ±4.00^c	3.596 ±0.3^bc	127.71 ±0.47^b	32.77 ±0.33^cd
D-gal and ALCL₃ (AD model)	32 ±1.21^a	26.00 ±3.38^e	1.55 ±0.24^e	66.15 ±0.61^f	11.06 ±0.08^g
CPFX co treatment	19.35 ±0.89^b	34.69 ±2.03^d	2.57 ±0.21^d	91.25 ±0.66^e	18.53 ±0.64^f
SPD co treatment	13.91 ±0.61^c	49.46±0.61^c	3.23 ±0.23^c	106.11 ±1.33^d	30.72 ±0.90^d
Combination treatment	16.27 ±0.83^c	53.88±1.84^bc	3.76 ±0.08^bc	122.89 ±1.5^c	37.27 ±0.54^b

Data are presented as mean ± SEM (standard error of mean), n = 8 per group. Mean values with different superscript letters in the same column are significantly different at (p≤0.05).

Table (5): Histopathological score of hippocampal sections of different experimental groups

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Editorial decision: Reject
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Synergistic effect of spermidine and ciprofloxacin against Alzheimer disease in male rat via ferroptosis modulation

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