Geraniol Attenuates Oxidative Stress and Neuro-inammation Mediated Cognitive Impairment in D ‐ galactose ‐ induced Mouse Aging Model

D-galactose (D-gal) is a reducing sugar drug can induce arti�cial senescence and aging process that mimic natural aging along with the accompanying brain and liver injury in experimental animals. Therefore, chronic D-gal administration is widely used to induce cognitive impairment, Alzheimer disease and aging in rodents' models. Aging is a phenomenon in which oxidative stress and apoptosis play a vital role. Geraniol (GNL) belongs to the acyclic isoprenoid monoterpenes, presents in essential oils such as those from Cinnamomum tenuipilum and Valeriana o�cinalis. In the present study, we examined the effects of GNL on D-gal-induced oxidative stress and neuro-inammation mediated memory loss in mice. Analyzing the behavioral differences between control and treated groups, including the elderly mice, revealed that GNL signi�cantly improved memory in mice treated with D-gal-induced memory loss (supplementary videos are provided). The anti-inammatory and the anti-oxidative role of GNL were con�rmed by both histopathological investigations and biochemical analyses. Mechanistically, GNL appears to activate PI3K/Akt and thus upregulates the nuclear factor erythroid 2-related factor 2 (Nrf2) and the heme oxygenase 1 (HO-1) to reduce the oxidative stress and apoptosis induced after D-gal treatment leading to easing of neurological de�cits and cognitive dysfunction in D-gal-induced aging mouse models. Accordingly, our comprehensive behavioral analysis and bioassays suggest GNL as a promising agent preventing cognitive impairment and neurological de�cits associated with aging.


Introduction
Chronic oxidative stress is a major factor in neurodegenerative diseases and aging due to the accumulation of reactive oxygen species 1 .Aging is a natural phenomenon associated with an increase in reactive oxygen species, and then a decline in antioxidant defense mechanisms, leading to increased risk of diseases such as Alzheimer, cancer and heart diseases 2 .The decline of brain function and cognitive impairment by aging are attributed to oxidative stress, DNA damage and then mutations induction that cause abnormal protein aggregation, neuro-in ammation and neurodegeneration 3 4, 5 .
Several recent studies have clari ed that D-gal injection could induce brain aging through increasing oxidative damage, in ammation and apoptosis, as well as lowering brain function leading to cognitive impairment [6][7][8][9] .In fact, our body can synthesize D-gal, which is a reducing sugar constitutes a part of glycolipids and glycoproteins, obtained from a variety of foods.The in vivo accumulation of D-gal may result in the formation of advanced glycation end products (AGEs).D-gal can directly produce ROS, or indirectly triggers free radical production, which results in oxidative stress and then apoptosis 8,9 .On the other hand, cells have protective mechanisms and antioxidant system protects cells from oxidative and proteotoxic stress.One of these molecules is the nuclear factor (erythroid-derived-2)-like 2 (Nrf2).The antioxidant response element (ARE) is responsible for inducing NRF2 genes.The function of Nrf2 is to keep cells from being damaged by oxidative stress induced by reactive oxygen or nitrogen species [10][11][12] .
In the brain, Nrf2 and related pathways help to combat oxidative stress, making them appealing targets for attacking cognitive decline.In this line, several recent studies have documented that with aging, the decline in cognitive abilities is associated with a decrease in Nrf2 expression level.Therefore, the Nrf2 pathway is a promising target for treating neurodegenerative diseases [13][14][15][16] .In this line, to regulate oxidative stress and in ammatory response, Nrf2 was found to be a key factor of the phosphoinositide 3kinase/ protein kinase B, PKB (PI3K/Akt) downstream molecules 17 .In addition, D-gal-induced aging was reported to inhibit the PI3K/Akt pathway and inactivate Nrf2-mediated antioxidants expression in liver and brain 18,19 .
Geraniol (GNL) is one of the representatives of acyclic isoprenoid monoterpenes, which can be extracted from the aromatic plants' essential oils such as Cinnamomum tenuipilum and Valeriana o cinalis, and other plants 20 .Researchers found that GNL has a variety of pharmacological effects, including, antiin ammatory, antitumor, anti-oxidative and antimicrobial activities [21][22][23] .
In the present study, GNL was tested for its effects on oxidative stress and neuro-in ammation mediated cognitive impairment in dgalinduced aged mice model.Mechanistic action of GNL on Nrf2 and AKT signaling pathways have been explored.Biochemical and histopathological data reveled a neuroprotective effect of GNL on aged mice model.This has been clearly con rmed through evaluating behavioral analysis (videos are supplemented) based on the spatial learning and memory loss in aged model and GNL treated aged model relative to the control mice and old mice.

Animals and Treatments
Thirty-six albino male mice weighing 25-30 gm were purchased from Charles River Laboratories (Écully, France) provided.All methods were carried out in accordance with relevant guidelines and according to regulations of King Faisal University.All experimental protocols were approved by King Faisal University Research Ethics Committee (KFU-REC/2021-01-15) before starting experiments and then the informed consent was obtained according to legal guardians.In a normal lab atmosphere, the mice were kept at a temperature of 22±2°C with a 12/12 h light/dark cycle.Six groups (6 mice each) were randomly selected.While mice of the control group received equal amounts of physiological saline once daily for 9 weeks, the 2nd group (D-gal control, II) was given orally D-gal (150 mg/wt) dissolved in normal saline solution (0.9%, w/v) for 9 weeks.Mice of the third group (III) were treated with GNL (40 mg/kg wt) alone in the rst week and starting from the second week up till the 10th week, mice were treated with oral gavage (without anesthesia) with D-gal (150 mg/wt) and with GNL (50 mg/kg wt) 4 h later.Mice of the fourth group (the drug control group, IV), were treated with GNL from the second week up till the end of the experiment.In addition, 6 mice of each sex were used as two untreated groups at 4 months (group 5) and 16 months (group 6) of age to analyze the cognitive impairment differences between mature and elderly mice.This was also done to determine whether treatment prevented or reversed the kinetics of cognitive decline.Mice were sedated with ether and sacri ced at the end of the experiment.To measure proin ammatory cytokines and biochemical markers, blood was collected via cardiac puncture and serum was separated and stored at -80°C.Using a sensitive balance (Nimbus, MK, UK), brains and spleen were excised and weighed before being xed or for histopathological analysis or homogenized for biochemical investigations.For biochemical evaluation, the brain hippocampus was homogenized in 0.1 M Tris-HCl buffer (pH 7.4).

Behavioral Analysis
Next, we used different testes including Morris' water maze test, Y-maze test and object-place recognition and open-eld test to study the behavior of control and treated mice (n = 6/group).2.4 Morris' Water Maze Test Morris' water maze test was used to estimate the spatial learning and memory after GNL and D-gal treatment.A circular tank has a diameter of 100 centimeters and a height of 20 centimeters was used.A white ink to the water (23±1°C) to make the tank opaque.A small platform was submerged 12 cm below the surface of water at the center of the third quadrant.MWM entailed a navigation training trial and a probe test.Each mouse was subjected to four tests per day for 4 days straight as part of the learning and memory training.Whenever the mice couldn't nd the platform in 45 seconds, they were arti cially guided to it and stayed there for 15 seconds to memorize it.The probe test without the platform was conducted for 45 seconds in the tank after the training trial.

Y-Maze Test
Studies have shown that excessive spontaneous alteration behavior is linked to enhanced cognitive performance.Then, a Y-maze made of brown pointed sheet with three arms that were 60 cm long, 15 cm high, and 15 cm wide at the bottom and top was used to analyze mice behavior.Three 5-minute sessions were given to the mice at the center.In the arms of the Y, theirs food.The mouse capability to remember if food is located in one arm or the other.By this way conditions could affect learning and memory and help to analyze the mice behavior.For the food identi cation, each mouse's arm position was recorded manually 3 times in different days for 5 minutes each.

Object Place Recognition
Based on previous studies, we modi ed a standard object recognition task to test the recognition of shortterm object-place. 24.Through the experiment, each mouse was allowed to adapt for 10 min in an open box (120X 80 cm).Then they began training at 30-min intervals.Each mouse was given 5 minutes to practice object-place recognition in a quadratic box (150X100 cm W, 25 cm H).In the training session, two identical plastic objects were positioned diagonally 120 cm from the wall in the apparatus.Between the training and testing, there was a one-day rest period.Two identical objects were placed 120 cm from the wall and the mice during the test.Animals' behavior was evaluated by how much time they spent looking at each object.

Protein Extraction from brain hippocampus
Homogenized hippocampus tissues were centrifuged at 13,000 r.p.m. at 4°C for 25 minutes with a cocktail of phosphatase and protease inhibitors.Supernatants of all samples were stored at -80°C to be used for biochemical investigations.

Biochemical Analysis
Spectrophotometry was used to measure CAT (U/mg protein), SOD (U/mg protein), MDA (nmol/mg protein) and GPX (U/mg protein) in the serum and hippocampus tissue.MDA, GSH, SOD, CAT and Gpx assay kits (Cayman Chemical, Ann Arbor, MI, USA) were utilized for analysis 25 .

AChE Evaluation
Using Elabscience Biotechnology Inc.'s (Houston, TX, USA), we measured the levels of AChE in the hippocampal homogenates using a quantitative sandwich ELISA.

Histopathological Evaluations
We collected brain, liver and spleen from mice, xed them in 4% polyformaldehyde at 4°C for 4 h, soaked them overnight in 100 mM sodium phosphate buffer (containing 30% sucrose), and embedded them in para n.After that, sections were stained with haematoxylin and eosin (H&E) and Nissl staining to be investigated under microscope.

Western Blot Analysis
RIPA lysis buffer was used to extract proteins from brain hippocampus tissue homogenates.Using a Bio-Rad protein assay, and bovine serum albumin (BSA) as a reference standard, tissue homogenates were collected by centrifugation at 12,000 rpm for 30 min at 4°C.We isolated equal amounts of protein (40*g) on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred it to PVDF membranes.Block it with 5% nonfat dry milk in Tris-buffered saline with Tween 20 (TBST, 150 mM NaCl, 20 mM Tris-HCl, and 0.1% Tween 20) for 1 hour at room temperature.IgG labeled goat antirabbit IgG (1:2000) or anti-mouse IgG (1:4000) was used as a secondary antibody after membranes were washed three times with TBST.Incubation time was 1.5 hours.The samples were examined with a LI-COR chemiluminescence imaging system (3600-00-C-Digit Blot Scanner).Graphs of the densitometric band intensities were generated and analyzed using LI-COR Biosciences Image Studio Lite software (Lincoln, NE, USA) normalized to the untreated control band, which was set to 1.

Effect of GNL on body weight and organ index
As shown in Fig. 1B and after week 10 of treatment, the d-gal model group had a signi cant decrease on body weight relative to the control group (p < 0.05), however the other administrated groups reversed dgal-induced weight loss (p < 0.05).In the meantime, brain index, liver (mg/g) index of the d-gal group were decreased signi cantly compared with the control group (p < 0.05).However, the administration of GNL (40mg/kg) signi cantly attenuated the weight loss and increased spleen indexes in comparison to the D-gal group.In group V and group VI no signi cant variation was found.These results indicated that GNL modulated the reduction of body weight and the decrease in brain and liver indexes in D-gal model.

Morris water maze
The effects of GNL on spatial memory and learning were studied using the Morris water maze 26 .Chronic D-gal administration resulted in a signi cant longer time for mice to reach the platform (P<0.05), while GNL therapy induced a signi cant reduction in escape latencies (Fig. 2A) (Video S1) (P<0.05).On the other hand, D-gal-treated aging mice spent signi cantly less time in the goal area than the control group in the spatial probe trial (Video S1) (P<0.05).In these poor conditions, mice in the D-gal plus GNL group showed remarkable reversion.Additionally, the aging mimic mice in the model group displayed similar behavior to the group VI.On the other hand, aged animals have a longer latency to reach the platform compared with young animals under 4 months (Group IV).These ndings demonstrated that GNL could restore the D-gal-induced de cit in memory and learning abilities.

Y-maze task
Using the Y-maze task, we analyzed spatial working memory using spontaneous alteration.D-gal treated mice were less likely to enter food rewards placed around its arms, which indicates no exploratory behavior.A signi cant increase in spontaneously altered behavior was observed in mice which received GNL either alone or in combination with D-gal (P<0.05),indicating that GNL improved memory in D-galtreated mice (Fig. 2B) (Video S2).Thus, GNL may improve working memory.It doesn't appear that there are any signi cant differences between groups III (Figure 2B) and control group in behavior to enter food rewards placed arms (Figure 2B).In addition, compared to 4-month-old young animals, animals in group VI are signi cantly less likely to enter the food rewards arm.Therefore, GNL appears to attenuate D-galinduced ageing in mice model.

GNL improves the D-gal-induced decline in object recognition
We explored the effect of treating with GNL on novel object recognition (NOR).When comparing the NOR test to the training session, a noticeable increase in object recognition index was observed in the control group.D-gal treatment also exacerbated the amnesic effect (Figure 2C) (Video S3), while pretreatment with GNL (40 mg/kg) (p <0.05) ameliorated it (Video S3).The long-term exposure to D-gal can lead to a recognition de cit in the NOR test, but you can improve it by pre-treatment with GNL.GNL could improve the memory impairment results in by D-gal treatment.The young animals in Group V moved quickly and used active recognition, while the old animals moved slowly, often stood still, and didn't explore.

Effects of GNL on Acetylcholinesterase (AChE) activity in aged mice model
Acetylcholinesterase (AChE) affects learning andmemory and controls cholinergic synapses and its activity, and therefore is a reliable indicator of cholinergic activity 27 .As indicated in Fig. 2D the AChE activity in the D-gal group is signi cantly higher than in the control group.Following supplementation with GNL, the levels of AChE in aging mice induced by D-gal were markedly reduced compared to the GNL group with D-gal.In group VI, the levels were signi cantly higher than those in group V.This proved that GNL can improve cognitive decline through modulating the level of AChE that has been dysregulated after D-gal treatment.

Effect of GNL on pro-in ammatory cytokines induced by D-gal
In here, the D-gal treated mice overexpress in ammatory cytokines, which is in line with previous reports 28, 29 .We found that pro-in ammatory cytokines such as TNF-α, IL-6, and IL-1β were about two times higher in the D-gal-treated group than in the control group.However, in D-gal treated group, serum levels of TNF-α, IL-6, and IL-1β were lower than D-gal model group after GNL administration (P < 0.05).This indicated an anti-in ammatory role for GNL on D-gal induced immune defect on mice.

Effect of GNL on antioxidants level and activities of MDA induced by D-gal
Increasing evidence indicated that oxidative stress is connected to the biology of aging 30,31 .Therefore, we investigated several antioxidants that could scavenge ROS in the serum and hippocampus of D-gal induced mice, including SOD, CAT, and GPX.According to Figs. 3A and B, there was a noticeable decrease in SOD, CAT, and GPx levels after D-gal exposure compared to the vehicle group (P < 0.05).However, in serum and hippocampal, GNL treatment reversed the decline due to its antioxidant activity.Group IV mice treated with GNL alone showed no signi cant difference in serum and hippocampus CAT, GSH-Px, and SOD.GNL treatment enhanced a better antioxidant activity in the hippocampus, according to our results.
On the other hand, MDA levels in serum and hippocampus homogenates signi cantly increased in the Dgal group, whereas GNL treatment at 40 mg/wt signi cantly abated the rise in serum and hippocampus MDA levels (Fig. 3A and B).In addition, as seen in Figure 3A and B, the MDA levels of aged mice (Group VI) are much higher than those of young control mice (V).Together these data revealed a protective effect of GNL on D-gal induced oxidative stress in Brain and serum of mice.

Effect of GNL on D-gal induced changes in the expression of PI3K/Akt
Next, we measured AKT and PI3K expression levels in control and treated groups.Although total PI3K and AKT protein levels were similar among all groups, D-gal exposure prevented PI3K signaling, as re ected by the reduced expression of pPI3K and pAKT expression.This inhibition of pPI3K and pAKT by D-gal was signi cantly alleviated by GNL treatment (Fig. 4A).In 4-month-old animals (Fig. 4B), the expression of PI3K and AKT showed insigni cant variation relative to group I, but in group VI (old mice), the level of phosphorylated of PI3K/AKT showed signi cant decrease.

Effects of GNL on Nrf2, HO-1 and NQO-1 expressions in mouse hippocampus
In agreement with the inhibition of PI3K, the expression of Nrf2, HO-1 and NQO-1 was reduced in GNL with D-gal treated mice (Fig. 5a) compared to that of D-gal model mice.Further, Nrf2, HO-1, and NQO-1 expression in natural aging mice (Fig. 5a) decreased in similar way to D-gal induced aging in mice.In group III (D-gal with GNL) treated mice activation of this protein was signi cantly elevated (p<0.05).However, GNL alone, vehicle control and young (4 months old) weren't signi cantly different (Fig. 5b).

GNL on expression of RAGE and BACE-1 Proteins
The D-gal was reported to increase RAGE protein levels, which stimulates hippocampus cells in neuroin ammatory neurodegeneration 32,33 .To analyze GNL's effect on RAGE and BACE-1, we conducted western blots analysis using protein extract from all experimental groups.The immunoblots showed that D-gal alone signi cantly increased RAGE and BACE-1 protein expression levels in hippocampus regions in comparison with the control group.However, co-administration of GNL + D-gal markedly reduced RAGE, and BACE-1 expression (Figure 6A).Actually, the immunoblot results showed that GNL is not toxic to normal mice brains hippocampus because no signi cant difference was found between normal control mice and mice treated with GNL by itself (Figure 6A).RAGE and BACE-1 expression were also up in natural aging mice (16 month old) (Fig. 6b), similar to D-gal induced aging mice.In contrast, GNL alone, vehicle control and young (4 months old) had no effect (Fig. 6b).

GNL supplementation inhibited D-gal-induced hippocampal apoptosis
D-gal induced aging was tied to hippocampal apoptosis, according to accumulating evidence 34,35 .Here, we performed western blot to check the ability of GNL supplementation to inhibit apoptosis.As shown in Fig. 7A and B, the expression of caspase-3 and Bax/Bcl-2 are signi cantly higher after D-gal treatment compared to the control group.By supplementing GNL, the upregulation of these apoptosis-related proteins was dramatically attenuated.Natural aging mice also showed an increase in the Bax/Bcl-2 ratio (Fig. 7b).However, GNL alone, vehicle control and young (4 months old) had no signi cant effect (Fig. 7a  and b).This revealed a protective role for GNL on D-gal inducing hippocampal apoptosis.

GNL on Ameliorated Histopathological Alterations of the Brain, Liver and Spleen
Hippocampus of brain (regions CA1 and CA3) of treated mice was stained with H&E (Fig. 8) to investigate the pathological changes relative to the control.As indicated, D-gal causes neurodegeneration characterized by focal disappearance and a decrease in density, as well as a necrosis represented by shrunken neurons displaying dark nuclear staining (arrows) relative to the control group.In the meantime, in group III (6 weeks), GNL showed a protective effect where mild neurodegeneration was seen (thin arrows) with highly pronounced normal neurons (thick arrow).Both of control group and group IV (the drug control) showed normal appearance of neurons shown in G4 (6 weeks).However, in group V (4 months), mild decrease in neuronal density was seen relative to the control.On the other hand, severe neural loss, degeneration, and necrosis (arrows) were seen in old mice (16 months).In the same line, Dgal induced histopathological alteration in liver and spleen (supplementary data), these changes have been modulated by GNL treatment.Together these data support protective effects on GNL at the histological level that in synchronization with what we have seen using behavioral and biochemical analysis.

Discussion
Life expectancy in the 21st century is rising, resulting in more age-related illnesses, such as memory impairment and Alzheimer's disease.Herein, GNL was studied for its protective effect on D-galactoseinduced aging in mice.We found that GNL was able to improve spatial location learning and to reduce the extent of memory impairment (please see suppl videos) as well as it protected neurons in the hippocampus from oxidative damage and apoptosis induced by D-galactose.Mechanistically, to alleviate D-gal effect on brain, GNL appears to activate Nrf2, the key factor among PI3K/Akt downstream through modulating the phosphorylation of PI3K/Akt.Therefore, GNL activates Nrf2 via the PI3K/AKT pathway to enhance the activity of antioxidant enzymes, like HO-1, NQO-1, and SOD.
The effect of GNL on memory impairment and learning in the D-galactose-induced aging model is demonstrated here for the 1st time.Interestingly, consistent with our results, GNL was reported to enhance a neuro-protective effect against ischemic injury in the brain and to pass e ciently through the blood-brain barrier 36,37 .In the present study, we provide robust evidence (suppl videos) to show that GNL is a potential drug in can modulate behavior changes induced in mice after D-gal treatment.By applying the Y-maze and open eld tests, we found signi cant variations between the vehicle and D-galtreated mice, suggesting that D-gal injection causes motor abnormalities that led to impairments of novelty-induced exploratory behavior.Furthermore, our data showed that treating mice with GNL for 8 weeks can reverse the D-gal-induced behavioral changes and then GNL can protect against D-gal-induced memory loss and spatial learning impairment.In consistent with our results, recently, GNL was suggested as a promising therapeutic agent in improving cognitive function and neurotoxicity induced in rats by ZnO-NPson, behavioral and biochemical evidence were provided 38 .
Several age-associated characteristics were found to be related to the increase in reactive oxygen species, and thus leading to the induction of oxidative stress and in ammation 39,40 .Interestingly, in the present study, both aging model and old mice displayed these characteristics including oxidative damage, apoptosis, and induction in ammatory response.The increase in ROS can cause NF-кB to move rapidly from the cytosol to the nucleus, resulting in an increase in in ammatory cytokines such as TNF-α, IL-6, and IL-1β. 3.In this line, when we gave mice D-gal for 9 weeks orally, the serum concentrations of TNF-α, IL-1β, and IL-6 were markedly increased in model animal.However, GNL substantially inhibited in ammatory cytokine production, and hence GNL was able to attenuate the induced in ammatory response in D-gal-treated mice.In addition, D-gal attenuated brain hippocampus antioxidant enzymes; SOD, CAT, and GPX in mice serum and hippocampi.to the exhaustion of antioxidant enzymes is due to the increase of ROS production with aging explaining the induction of age-related degenerative disease by D-gal 41 .However, the increase of brain hippocampus and serum SOD, CAT, and GPx activities in D-gal treated mice are in-line with several previous studies that indicated the antioxidant properties of GNL.For instance, Lin and colleagues studied the effect of GNL on methicillin-resistant Staphylococcus aureus (MRSA) infections in mice where GNL increased the antioxidant activity of SOD, and reduced ROS and MDA in the kidneys 42 .In addition, Farokhcheh et al.Showed that GNL reduced ROS in neurotoxicity cells in brain tissue 38 .As a result, this implies that the effect of GNL on aging may be mediated by modulating the imbalance between free radicals and antioxidants.PI3K/Akt signaling is considered as an important upstream pathway regulating Nrf2 43,44 .Activation of PI3K/Akt signaling pathway promotes the dissociation downstream molecules including Nrf2 and Keap1, thereby regulating the nuclear expression of Nrf2 45,46 .Numerous studies hinted that the PI3K/Akt signaling pathway protects against D-gal-induced brain injury by modulating Nrf2 46, 47 .Additionally, several natural compounds were found to improve the behavioral dysfunction and neurological de cits in D-galactose-induced aging in mice via activation of PI3K/Akt/Nrf2 pathway [48][49][50] .Results of the current work demonstrated that phosphorylated levels of PI3K and Akt are signi cantly upregulated after GNL treatment, which revealed that GNL can activate PI3K/Akt signaling pathway to promote Nrf2 nuclear translocation via reduction D-galactose-induced oxidative stress.Neurodegenerative diseases are treated by targeting Nrf2 pathway 51 .Keap1, a cysteine-rich protein present in the cytoplasm, binds to Nrf2 under normal conditions.However, when Keap1 denatures, Nrf2 translocate into the nucleus, binds to antioxidant elements (ARE), and turns on antioxidant enzyme genes, like SOD and CAT 52 .Studies have shown that aged animals have less nuclear translocation of Nrf2 45 .This model showed similar results to previous studies where D-galactose treatment inhibited Nrf2 translocation to the nucleus 48 , however, treatment the model with GNL have shown good results.
The neuroprotective effect can be attributed to NQO-1 and HO-1, antioxidant enzymes 53 .In aged model treated with GNL, the accumulation nuclear factor Nrf2, increased expression of NQO-1 and HO-1, the downstream proteins of Nrf2, which con rms that Nrf2 is involved, but also demonstrates how critical they are for protecting GNL from free radical damage.Rodent D-gal brain neurotoxicity is also attributed to oxidative stress-induced apoptosis 46 .Herein, brain hippocampus of mice treated with D-gal showed elevated level of Bax and caspase-3, and lowere expression of Bcl-2 54 .D-gal induced aging mice showed that normal neuronal morphology was damaged, and expression was increased in the hippocampus.GNL, however, restored cell architecture and morphology and reduced neuronal apoptosis.This indicates that GNL protects hippocampal neurons from D-galactose-induced apoptosis.That has been con rmed by histopathological analysis where GNL reduced the neurodegeneration, necrotic injury and decreased numbers of shrunken neurons that were seen in the aging model.

Conclusion
Results of the current study demonstrate, for the rst time, that GNL exhibits a strong antioxidant activity managed to signi cantly attenuate brain hippocampus injury induced in vivo by D-gal.Through activation of PI3K/Akt/Nrf2 it reduces apoptosis and oxidative stress and then helps in easing cognitive dysfunction and in reducing neurological de cits in D-galactose-induced aging mouse models.According to our study, GNL might be a promising therapeutic agent for age-related diseases.solution (0.9%, w/v) for 9 weeks (II).Animals in the third group of experiments were treated with GNL (40 mg/kg wt) alone in the rst week, and then from the second to the 10th week by oral gavage (without anesthesia) with D-gal (150 mg/wt) followed by GNL (50 mg/kg wt) 4 h later (III).Animals in the fourth group were given GNL from the second week until the end of the experiment (IV).We included 6 mice from two untreated groups at 4 months (V) and 6 months (IV) of age for analysis of cognitive impairment.This was done to determine whether treatment prevented or reversed the kinetics of cognitive decline.(B), Body weight (gm), (C), Brain weight (gm) (D), Spleen weight (mg).Data represent mean ± SD. *P<0.05 vs control group, #P<0.05vs D-gal alone treated mice.@ P<0.05 young vs old mice.Figure 8

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