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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 63  |  Issue : 5  |  Page : 218-226

Combination of ellagic acid and trans-cinnamaldehyde alleviates aging-induced cognitive impairment via modulation of mitochondrial function and inflammatory and apoptotic mediators in the prefrontal cortex of aged rats


1 Department of Neurosurgery, Binzhou Central Hospital, Binzhou, Shandong, China
2 Department of Neurosurgery, Binzhou People's Hospital, Binzhou, Shandong, China
3 Department of Neurosurgery, The Second Affiliated Hospital of Xi'an Medical University, Xi'an, Shaanxi, China

Date of Submission11-Jul-2020
Date of Acceptance08-Oct-2020
Date of Web Publication27-Oct-2020

Correspondence Address:
Dr. Fenglu Wang
Department of Neurosurgery, Second Affiliated Hospital of Xi'an Medical University, No. 167 Fangdong Street, Baqiao District, Xi'an, Shaanxi, 710038
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/CJP.CJP_55_20

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  Abstract 

Cognitive impairments are associated with advancing age. Trans-cinnamaldehyde (CIN) and ellagic acid (ELA) have multiplex activities to reduce various age-related cognitive disorders. In this study, we investigated the effects of these compounds separately or in combination on the cognitive outcomes, mitochondrial function, and inflammatory and apoptotic mediators in aged male Wistar rats. Thirty-two old (22 months old) and eight young (5 months old) rats were randomly allocated to five groups of young control, aged control, ELA-aged, CIN-aged, and ELA + CIN-aged. ELA (15 mg/kg, orally) and CIN (50 mg/kg, intraperitoneally) separately or in combination were administered for 1 month in aged animals. Spatial memory and cognitive activity were evaluated by the Barnes maze and novel object recognition tests. Mitochondrial function (its reactive oxygen species [ROS], mitochondrial membrane potential and ATP level), pro-inflammatory cytokines such as interleukin (IL)-1β and IL-6 and pro-apoptotic caspase 3 and Bax, and anti-apoptotic Bcl2 levels and their ratio were assessed in the prefrontal cortex. Behavioral results revealed that CIN separately or in combination with ELA significantly alleviates aging-induced memory impairment. Moreover, co-administration of agents effectively decreased inflammatory cytokines, cleaved-caspase 3, Bax and Bax/Bcl2 levels, mitochondrial ROS production, and mitochondrial membrane depolarization and increased Bcl2 and ATP level as compared with untreated aged control rats. Combination therapy was greater than those of individual treatments in all parameters. Therefore, combination therapy with CIN and ELA improved aging-induced cognitive impairment through anti-inflammatory, anti-apoptotic, and mitochondrial-boosting effects in aged rats.

Keywords: Aging, apoptosis, cinnamaldehyde, ellagic acid, inflammation, learning and memory, mitochondrial function


How to cite this article:
Pan Z, He X, Zhou X, Li X, Rong B, Wang F. Combination of ellagic acid and trans-cinnamaldehyde alleviates aging-induced cognitive impairment via modulation of mitochondrial function and inflammatory and apoptotic mediators in the prefrontal cortex of aged rats. Chin J Physiol 2020;63:218-26

How to cite this URL:
Pan Z, He X, Zhou X, Li X, Rong B, Wang F. Combination of ellagic acid and trans-cinnamaldehyde alleviates aging-induced cognitive impairment via modulation of mitochondrial function and inflammatory and apoptotic mediators in the prefrontal cortex of aged rats. Chin J Physiol [serial online] 2020 [cited 2020 Nov 29];63:218-26. Available from: https://www.cjphysiology.org/text.asp?2020/63/5/218/299249


  Introduction Top


Aging is a normal process associated with the manifestation of several physiological phenomena and most importantly contributes to worse quality of life.[1],[2] The rapidly growing elderly population in most countries necessitates the assessment of the mechanisms associated with age-related cognitive disorders.[3] Alterations in structure, function, and signaling of synapses and several neuronal networks with aging, lead to cognitive changes in the elderly population. Prefrontal cortex which controls the executive function and working memory, is one of the most important parts of the brain, susceptible to aging-induced changes.[2],[3] Understanding the underlying mechanisms and finding appropriate therapeutics are necessary approaches to reduce these aging-induced burdens.

Inflammation and activation of inflammatory cytokines play crucial roles in the development of aging-related complications.[4],[5] Secretion of several inflammatory compounds consisting of chemokines and cytokines such as interleukin (IL)-6, IL-1β, and tumor necrosis factor-α (TNF-α) is the final trait of senescent cells and metabolic conditions.[4],[6] Chronic inflammation caused by cytokines is responsible for neurotoxicity and age-related learning and cognition disorders.[7] It is also well established that the apoptosis contributes to neuronal dysfunction and death in the brain of rats with neurological disorders.[8] The increased expression of pro-apoptotic Bax and decreased expression of anti-apoptotic Bcl2 play an important role in caspase 3-dependent cell apoptosis during aging. In addition, inhibition of the inflammatory pathway can protect neurons from apoptosis in cerebral and myocardial ischemic injuries via the modulation of Bax/Bcl-2 expression.[9],[10] These data suggest the important interaction of inflammatory cytokines and cell apoptosis in cerebral function and disorders.

Mitochondria, as a machine producing ATP for electrochemical neurotransmission, cell maintenance, and cell repair, are actively present in neuronal cells, and the accumulation of dysfunctional mitochondria is likely to be observed in most cell types during aging in the brain.[11] Animal studies on brain mitochondria changes during aging reveal numerous dysfunctions in this organelle such as enlargement or fragmentation of the mitochondria, dysfunction in the electron transport chain, remarkable depolarization of mitochondrial membrane, and oxidative damage to mitochondrial DNA.[12],[13] Increased oxidative stress and inflammatory reactions during aging can cause mitochondrial dysfunction, which in turn, lead to release of cytochrome-c into the cytosol and subsequent activation of apoptotic pathways and oxidative injury.[13],[14],[15] These findings justify the new therapeutic approaches focusing on mitochondria as one of the important cellular targets for protecting the aging-induced cognitive disorders.

Due to the dysfunction of multiple cell survival mechanisms in aging, modification and manipulation of these mechanisms by various ways can delay aging process and reduce its complications. Because of the multifactorial nature of aging, monotherapy or single-drug therapies may not have sufficient potency to produce efficient preventive or therapeutic impact; the use of combination therapies with less side effects can be considered a good alternative strategy. Today, herbal medicinal agents such as bioflavonoids and phytochemicals are gaining greater attention for their usage during aging because of their ability to potentially improve cellular and metabolic phenotypes. The anti-aging effect of these natural ingredients is as a result of their multiplex activities. Trans-cinnamaldehyde (CIN) is a natural major constituent isolated from cinnamon, and it has been generally regarded as a safe molecule, according to the Food and Drug Administration.[16],[17],[18] In addition, ellagic acid (ELA) is a natural polyphenolic compound excreted from many plant species, fruits, and nuts.[19],[20] These two compounds (ELA and CIN) have been shown to suppress oxidative stress, inflammation, apoptosis, and necrosis in a number of in vitro and in vivo models of pathological conditions.[16],[17],[18],[19],[20]

Because neural cells are abundantly rich in mitochondria, they are susceptible to cellular injury resulting from mitochondrial damage and dysfunction.[11] Previous studies have shown that both CIN and ELA have potent beneficial effects on mitochondrial function, dynamics, and biogenesis.[21],[22],[23] Notably, loss of neural cells activity and cognitive function of the brain during aging have been substantially associated with mitochondrial dysfunction. Both ELA and CIN have shown that they can improve the neurological outcomes,[24],[25],[26] mitochondrial functions,[19],[27] and inflammatory status[24],[26] in the brain and neural cells of adult young animals. All the effects of these two compounds indicate that their combination can significantly reduce cellular, molecular, and metabolic changes in cognitive impairment of the brain during aging process. Therefore, to evaluate this hypothesis in this study, we evaluated the individual and combined effects of ELA and CIN on aging-induced cognitive impairment and mechanistically implication of inflammatory and apoptotic pathways, as well as mitochondrial function in the prefrontal cortex of aged rats.


  Materials and Methods Top


Animals

Forty male Wistar rats (32 aged rats at the age of 22 months old and 8 young rats at the age of 5 months old) were used in this study. Three rats in each cage were housed in animal room under a room temperature of 24°C ± 2°C and a 12:12 h lightness and darkness cycle. Animals had free access to tap water and standard pellet food. This study was ethically approved by the local ethics committee under ethic number of BZCH2019120037. The experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH; Publication No. 85–23, revised 1985).

Experimental groups and treatments

After 1 week of adaptation in the animal room, the rats were randomly divided into the following groups (n = 8/each group): (1) control young rats, (2) control aged rats, (3) ELA-treated aged rats, (4) CIN-treated aged rats, and (5) ELA + CIN-treated aged rats. Control rats received normal saline (0.9% NaCl) (1 mL/kg; i.p.) in both control groups. ELA and CIN (Sigma-Aldrich, St. Louis, MO, USA) were administered at the dosages of 15 mg/kg (by oral gavage) and 50 mg/kg (i.p.) for 1 month, respectively. Interventions continued until the day of habituation of behavioral tests and were stopped on the days of testing. After behavioral experiments, the animals were anesthetized and sampling was performed immediately.

Behavioral tests

Novel object recognition test

To evaluate the episodic memory of aged rats, the novel object recognition test in three successive phases of habituation, training, and retention was employed. In this test, 24 h after habituation of rats with open field arena in a 70 cm × 70 cm × 30 cm test chamber, they were allowed to explore two similar objects for 10 min (training trial). For testing retention phase in the next day, one of the objects was replaced with a novel object and the exploration time of rats to find the new object was measured using a video camera placed above the chamber and connected to an automated Etho Vision video-tracking software (Noldus, Wageningen, the Netherlands). The displacement index (DI) was used to determine the novel object recognition as follows: DI = (NF)/(N + F) in which N is the novel and F is the familiar objects' exploration time.

Barnes test

The spatial learning and memory of rats was assayed using Barnes maze. For adaptation of the rats in the 1st day, a rat was placed at the center of a wooden circular-disc maze (120 cm in diameter) having twenty holes around the maze each of them with 10 cm in diameter. Then, the 80 dB white noise was triggered to guide the rat to the escape box placed under the goal hole. The white noise was switched off after entering the rat into the escape box. In the following days, the rats performed this task without the guidance. For evaluation of the reference memory, the escape box was removed at the probe test session. Then, the reversal training was started after probe trial so that the escape hole was rotated 180° from its location, and the similar procedure was done. A digital camera was placed above the maze and connected to an automated Etho Vision video-tracking software (Noldus, Netherlands) to record the rat behaviors. The time needed for finding the escape box (latency time) during the training trials was recorded as the working memory indicator. The time spent in the target quadrant, and the correct to wrong time (time spent in target hole/time spent in non-target holes) during the probe trials, were indicated as reference memory.

Tissue sampling and brain mitochondrial isolation

At the end of behavioral tests, the rats were anesthetized with ketamine (60 mg/kg) and xylazine (10 mg/kg). The rat brains were immediately isolated, and then the prefrontal cortex from the right hemispheres of the brains was dissected, and one section was transferred into −80°C deep freezer, another section was transferred to a homogenizer containing mitochondrial isolation buffer (containing 70 mM sucrose, 210 mM mannitol, and 1 mM EDTA in 50 mM Tris-HCl, pH 7.4). After homogenization and centrifugation at 1300 ×g for 10 min in 4°C, the resultant supernatant liquid was transferred to another tube and re-centrifuged at 12,000 ×g with similar conditions. Then, the mitochondrial pellet was suspended in storage buffer (containing 70 mM sucrose and 210 mM mannitol in 50 mM Tris-HCl, pH 7.4 at final volume of 100 μl). Nanodrop method was employed to determine the protein concentration of the samples.

Mitochondrial reactive oxygen species generation

The mitochondrial reactive oxygen species (ROS) generation of prefrontal samples was fluorometrically assessed by dichlorohydro-fluorescein diacetate (DCFDA) method. After incubation of mitochondrial pellets in storage buffer with 2 μM of DCFDA dye for 30 min, their fluorescence was detected at λexcitation= 480 nm, and λemission= 530 nm through a fluorescent microplate reader. The fluorescence intensities (FIs) were normalized to protein content of samples and the mitochondrial ROS levels were calculated and reported as FI/mg protein.

Mitochondrial membrane potential

The JC-1 method was used to measure the changes of mitochondrial membrane potential (ΔΨm), according to the manufacturer's protocol (Sigma-Aldrich, St. Louis, MO, USA). Briefly, isolated mitochondrial pellet (3 μg) was diluted in 100 μl JC-1 assay buffer and incubated for 15 min at 25° in dark situation. In healthy cells, JC-1 penetrates the mitochondrial membrane and emits red fluorescence. Following ΔΨm dissipation, red fluorescence changes to the green. Red fluorescence (JC-1 aggregates) were exited at 525 nm and emitted at 590 nm, and green fluorescence (JC-1 monomers) were exited at 485 nm and emitted at 530 nm using a fluorometer. The ΔΨm was determined by the ratio of red/green intensity ratio and normalized to the proteins of samples. Decreased ratio of red/green intensity represents mitochondrial depolarization.

ATP production levels

A related bioluminescent assay kit (MAK190, Sigma, USA) was used for the determination of cellular ATP content, according to the manufacturer's instructions. In brief, approximately 10-mg prefrontal cortex samples were lysed in 100 μl of ATP assay buffer. Thereafter, the ATP probe was added to the solution in the presence of developer, and the absorbance of the solution was read at 570 nm. The ATP contents were calculated from the absorbance intensities and reported in nmol/mg protein.

Measurement of cytokines

The changes of pro-inflammatory cytokines such as IL-6 and IL-1β in brain samples were assessed via a specific ELISA kit, according to the instructions (MyBioscience, Inc., USA). The relative absorbance of cytokines was read at 450 nm wavelength using a microplate ELISA reader, and the final concentration was normalized to the protein content of each sample. The cytokines levels were reported as pg/mg of protein.

Western blotting

The expression of caspase 3, Bax, and Bcl2 proteins was detected by Western blotting technique, as described previously.[8] In brief, SDS-gel electrophoresed proteins of the samples were transferred to a PVDF membrane and the membrane was blocked in 5% skim milk containing 0.1% Tween-20 for 1 h. Then, the blocked membrane was incubated overnight with primary antibodies for cleaved-caspase 3, Bax, Bcl2 (1:1000, Cell Signaling), and β-actin (1:500, Cell Signaling) at 4°C. After washing steps with Tris buffer saline, the HRP-conjugated secondary antibody (1:3000, Cell Signaling) was added for an hour. Then, the membrane was washed and incubated with the enhanced chemiluminescence (Amersham) reagents in dark room and exposed to an X-ray film. The protein bands were visualized and the related intensities were quantified using Image J software (IJ 1.46r version, NIH, USA) and normalized to the intensity of β-actin in each sample.

Statistical analysis

The data were reported as mean ± SE. Two-way and/or one-way analysis of variance (ANOVA) tests and Tukey post hoc test were used to detect the difference between groups. Statistically significant differences were determined when P < 0.05.


  Results Top


Behavioral tests

[Figure 1] shows the exploratory preference of the animals in the retention phase of novel object recognition test, having a significant difference between groups. The exploratory preference of aged animals for a novel object was significantly lower than those of young group (P < 0.001). Individual treatments with ELA or CIN significantly increased the exploratory preference of the animals in comparison to untreated aged group (P < 0.05). In addition, the combined treatment with ELA and CIN significantly improved cognitive impairment induced by aging (P < 0.001) [Figure 1]. There was no significant difference between the combination treatment and each of the individual treatments. Furthermore, no significant differences were seen in the locomotor activity and exploratory preference between different groups during the habituation and training phases.
Figure 1: Discrimination index for exploratory preference during the retention phase of novel object recognition test. Data are presented as mean ± standard error mean, (n = 8). ***P < 0.001 versus Young group; ##P < 0.01, and ###P < 0.001 versus Control-aged group. The differences between combination therapy and each of individual treatment were not significant. ELA: ellagic acid, CIN: cinnamaldehyde.

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[Figure 2] shows the findings of Barnes maze test among experimental groups. Between-groups post hoc analysis of the time of escape latency revealed that aging reduced the ability of rats to find the escape box in the 4th (acquisition) and 7th (new learning) days of the training session in comparison to the young control group (P < 0.01). Although ELA administration could not significantly affect the time of escape latency, CIN alone (P < 0.05) or in combination with ELA (P < 0.01) significantly reduced the escape latency time on the 4th day of the training session as compared to that of the control aged group. The differences between the effects of combination therapy and each of individual treatments on escape latency were not statistically significant [Figure 2]a. Furthermore, during probe session, the time spent in the target quadrant [P < 0.01; [Figure 2]b] as well as correct/wrong relative time [P < 0.001; [Figure 2]c] in control-aged group was significantly lesser than those of young animals. Administration of ELA and CIN individually to aged rats improved the times spent in target quadrant and correct to wrong relative times in comparison to the untreated aged rats (P < 0.05 and P < 0.01, respectively). In addition, the combination of ELA and CIN significantly increased both these parameters as compared to those of ELA-aged group (P < 0.05). There was no significant difference between the combination therapy and CIN-aged group [[Figure 2]b and c].
Figure 2: (a) Escape latency in the training sessions, (b) time spent in the target quadrant, and (c) correct/wrong relative time in probe sessions of Barnes test. Data are presented as mean ± standard error mean, (n = 8). **P < 0.01, and ***P < 0.001 versus Young group; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. Control-aged group; +P < 0.05 vs. ELA-aged group. The differences between combination therapy and CIN-aged group were not significant. ELA: ellagic acid, CIN: cinnamaldehyde.

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Mitochondrial tests

The levels of ROS production from mitochondria isolated from the samples of prefrontal cortex were quantified by the FI of DCF. [Figure 3]a shows that ROS production level in control aged rats was significantly higher than that of the young group (P < 0.001). ROS production levels had significantly reduced in ELA-aged and CIN-aged groups in comparison with those of untreated aged animals (P < 0.05). Moreover, the combined treatment reduced the mitochondrial ROS level more remarkably as compared with that of the control aged group (P < 0.001). The mitochondrial ROS level in combination therapy was significantly lower than those of ELA-aged and CIN-aged groups (P < 0.05) [Figure 3]a.
Figure 3: Mitochondrial tests: (a) mitochondrial reactive oxygen species production level, (b) red/green intensity of JC-1 staining indicating the mitochondrial membrane potential (ΔΨMito), and (c) mitochondrial production of ATP level in the prefrontal cortex of animals in different groups. Data are presented as mean ± standard error mean, (n = 5). **P < 0.01, and ***P < 0.001 vs. Young group; #P < 0.05, and ###P < 0.001 vs. Control-aged group; +P < 0.05 vs. ELA-aged group; $P < 0.05 versus CIN-aged group. ELA: ellagic acid, CIN: cinnamaldehyde.

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Mitochondrial membrane potential changes (ΔΨ) were expressed as the ratio of red/green JC-1 fluorescent intensity in isolated mitochondria from prefrontal cortex samples [Figure 3]b. The lower red/green ratio correlates with membrane depolarization. The ratio in control-aged group was significantly lower than that of young group (P < 0.01), indicating the collapsed membrane potential in aged animals. Although ELA alone or CIN alone tended to increase the red/green intensity ratio, these effects were not statistically significant when compared with those of control-aged group. However, the ratio of fluorescent intensity in combination treatment was significantly higher than those of control-aged group (P < 0.05), demonstrating the increased efficiency of this treatment on mitochondrial functional preservation [Figure 3]b.

Furthermore, ATP levels had significantly decreased in the prefrontal cortex of aged rats in comparison to that of the young ones [P < 0.01; [Figure 3]c]. CIN alone or in combination with ELA significantly restored the ATP levels as compared with control-aged group (P < 0.05). The difference between the effect of combination therapy and each of the individual treatments on mitochondrial ΔΨ and ATP level was not statistically significant [Figure 3]b and [Figure 3]c.

Inflammatory cytokines

The changes in the levels of pro-inflammatory cytokines IL-6 and IL-1β were evaluated to estimate the inflammatory response in prefrontal cortex of aged rats to the treatments [Figure 4]. The levels of IL-1β were significantly higher in control-aged rats than that of young rats [P < 0.05; [Figure 4]b], but there was no significant difference in IL-6 [Figure 4]a. Each of the individual treatments could significantly reduce the levels of both cytokines (IL-6 and IL-1β) in comparison to the control-aged group (P < 0.05 and P < 0.01, respectively). In addition, concomitant administration of both ELA and CIN to aged rats significantly reduced the myocardial levels of both IL-6 (P < 0.01) and IL-1β (P < 0.01) as compared with untreated control aged rats. Combination therapy significantly reduced the level of IL-1β as compared to that of CIN-aged group (P < 0.05), but this effect was not statistically significant in comparison to the effect of ELA alone [Figure 4]. Furthermore, the differences of IL-6 levels between treatment groups were not significant.
Figure 4: Inflammatory cytokines: (a) the levels of IL-6, and (b) the levels of IL-1β in the prefrontal cortex of animals in different groups. Data are presented as mean ± standard error mean, (n = 6). *P < 0.05 vs. Young group; #P < 0.05, and ##P < 0.01 vs. Control-aged group. $p < 0.05 vs. CIN-aged group. The differences between combination therapy and ELA-aged group were not significant. ELA: ellagic acid, CIN: cinnamaldehyde.

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Apoptotic markers

In control aged rats, Bax protein level was not significantly different from that of young rats, but Bcl2 protein level was statistically significantly lower (P < 0.01) and cleaved-caspase 3 (P < 0.05) and the ratio of Bax/Bcl2 (P < 0.001) were significantly higher than those in the young group [[Figure 5]a-d]. Administration of ELA had no effect on the expression of Bax, Bcl2, and cleaved-caspase 3 protein, but significantly reduced the Bax/Bcl2 ratio as compared to that of the control-aged group (P < 0.05). On the other hand, CIN alone had significant effects on decreasing Bax level (P < 0.05) and Bax/Bcl2 ratio (P < 0.01), and increasing Bcl2 levels (P < 0.05), without significant effect on cleaved-caspase 3 expression. Finally, combination treatment with ELA and CIN in aged rats significantly and more potently restored the levels of these apoptotic markers toward the values of young animals, as compared with those of untreated aged rats [P < 0.01 and P < 0.001; [Figure 5]a, [Figure 5]b, [Figure 5]c, [Figure 5]d]. The effect of combination therapy on the reduction of Bax/Bcl2 ratio and cleaved-caspase 3 level was significantly greater than those of ELA-aged group (P < 0.05) but not CIN-aged group. In the case of Bax and Bcl2 levels, there were no significant differences between the combination therapy and each of the individual treatments [Figure 5].
Figure 5: Apoptotic markers: (a) Bax protein expression levels, (b) Bcl2 protein expression levels, (c) the ratio of Bax/Bcl2 levels, and (d) cleaved-caspase 3 levels in the prefrontal cortex of animals in different groups. Data are presented as mean ± standard error mean, (n = 6). **P < 0.01, and ***P < 0.001 vs. Young group; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. Control-aged group; +P < 0.05 vs. ELA-aged group. The differences between combination therapy and CIN-aged group were not significant. ELA: ellagic acid, CIN: cinnamaldehyde.

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  Discussion Top


In this study, we investigated the effects of CIN and ELA, alone or in combination, on cognitive impairment and related mechanisms by investigating inflammatory, mitochondrial, and apoptotic mediators in aged rats. The results showed that the CIN alone improved the cognitive function of the old rats compared to the ELA and control groups. In addition, the combination of these two drugs could have stronger and consistent effects on the parameters related to the cognitive function of rats. In this context, combination therapy could significantly improve mitochondrial function by reducing the mitochondrial ROS production and mitochondrial membrane depolarization, increasing cellular ATP production, declining inflammatory cytokines (IL-1β and IL-6), and lessening cell apoptosis by reducing Bax/Bcl2 ratio and cleaved-caspase 3 expression in the prefrontal cortex of aged rats.

Inflammation is one of the proposed theories of senescence initiation in different cell types of the brain.[4],[5] Besides, inflammatory responses are closely and reciprocally linked to the apoptotic pathways, as a second theory of aging.[9] Increased expression of transcription factor nuclear factor-kappa B (NF-κB) through inflammatory reactions activates apoptotic mediators, which, in turn, can produce inflammatory cytokines such as IL-1β, IL-6, and TNF-α, thereby compromising cellular function.[7],[10] In the present study, the levels of both inflammatory cytokines and Bax apoptotic protein and Bax/Bcl2 ratio in prefrontal cortex of untreated aged rats were significantly higher than young rats, and these findings were associated with decreased cognitive function in these rats. Administration of ELA was able to reduce ILs and apoptosis indices, besides increasing the novel object recognition and spatial learning activity, but it did not have a significant effect on some other parameters. However, CIN alone did not only significantly restore all the parameters but also had stronger effects than ELA. More importantly, their combined effects were more potent than their individual effects. This suggests that concomitant administration of CIN can recover the loss of effectiveness of ELA on aging and thus provide a full neuroprotective effect. In support of this combination hypothesis, the combined therapy with nicotinamide and melatonin has been shown to increase the potency of each other in cardiac ischemia–reperfusion injury in aged rats and considerably reduce the size of myocardial infarction.[28] Here, the two compounds have had synergistic and additive effects.

Furthermore, mitochondria are a very important and effective organelle in the aging process, and their dysfunction is directly related to both inflammatory and apoptotic outcomes so that the number and function of mitochondria in neuronal cells decreases progressively during aging.[11] Moreover, overactivation of inflammatory events and cellular oxidative stress impair normal mitochondrial function, leading to the loss of mitochondrial membrane integrity and opening of permeability transition pores. These events are followed by the subsequent activation of pro-apoptotic and pro-inflammatory mediators. In addition, mitochondria starts producing oxygen-free radicals and thus, the oxidative stress in the cell again intensifies.[13],[14],[15] The mitochondrial function and thereby, whole cell function, will no longer be corrected or less corrected if these processes enter a vicious circle.

The two compounds, each having positive effects on the function, dynamics, and biogenesis of mitochondria in various cells such as the cardiac and neuronal cells, are able to prevent the development of the above-mentioned vicious circle. Individual administration of CIN or its combination with ELA significantly reduced the mitochondrial ROS production and membrane depolarization and increased the available ATP levels. This suggests that in the presence of these two agents at the same time, mitochondria are in a more normal state, reducing the exacerbation of cellular apoptosis and inflammatory events, leading to enhanced learning and cognitive function in aged rats. In support to this hypothesis, previous studies reported that EA protects neuronal cells from arsenic neurotoxicity and D-galactose-induced aging of human neuroblastoma cell line through restoring the neuro-inflammation and mitochondrial Dysfunction-associated apoptosis.[19],[20] This agent was also able to antagonize BNIP3-mediated mitochondrial injury and necrotic cell death in myocardial cells.[22] Similarly, CIN has been shown to stimulate mitochondrial biogenesis and decrease mitochondrial respiratory enzyme activities and apoptosis in different tissues and cell types.[21],[29] Therefore, the combined treatment with ELA + CIN in aging conditions can be considered a reliable protective approach.

Based on the previous documents, both ELA and CIN are the multi-target bioactive compounds acting on different mediators and receptors in CNS and other systems. Studies have reported that both CIN and ELA can inhibit the activity of Toll-like receptor-4 (TLR-4) and NF-κB.[30],[31] Therefore, combination therapy in the present study reduces the release of inflammatory cytokines more likely by affecting these upstream targets of the pro-inflammatory pathway, even though other targets may also mediate the effects of these compounds, as seen in different cell types.[32],[33] Reduction in cellular inflammation can prevent mitochondrial depolarization and thus have a positive effect on mitochondrial oxidative function. Improving the oxidative status of the mitochondria, in turn, prevents the mitochondrial membrane potential collapse.[7],[34] Following these changes, the release of pro-apoptotic factors such as cytochrome C into the cytosol is reduced to inhibit cell apoptosis (as indicated by reduced expression of cleaved-caspase 3 and Bax/Bcl2 ratio). Therefore, combined administration of CIN with ELA may considerably improve the aging-induced cognitive impairment through inhibition of inflammatory/mitochondrial dysfunction/apoptosis signaling axis. However, all of the above-mentioned mediators are interrelated and can moderate or enhance each other's effects. Therefore, to understand their causal relationship, it is necessary to design a supplementary study using specific inhibitors or transgenic animals. Finding the exact target or targets of this combination therapy in mitochondria and mitochondrial end-effectors would have a significant impact in determining the dose–response relationship and efficacy of this therapeutic approach, which needs to be clarified in future studies. On the other hand, due to the parallel changes and cross-links of cellular oxidative stress and inflammatory pathways in aging, it is desirable to investigate the interactive effects of these pathways on the efficacy of the combination therapy in aged rats.

In conclusion, the results of the present study showed that individual administration of CIN and ELA, and more specifically their combination therapy, restored the cognitive impairment of aged rats through reducing the activity of inflammatory cytokines and cellular apoptosis as well as protecting the mitochondrial function in the prefrontal cortex of rats. The potency and ability of combination therapy was greater than that of single treatments. Therefore, their combined application is suggested to delay aging and its complications under similar conditions.

Acknowledgment

The authors would like to thank the Department of Neurosurgery, Binzhou Central Hospital, Binzhou, and The Second Affiliated Hospital of Xi'an Medical University, Shaanxi, China, for their assistance in experiments and providing research equipment.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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