Keywords

12.1 Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that is characterized by impairment of memory and disturbance in at least one of the thinking functions. It is considered the most common cause of dementia, affecting more than 25 million people worldwide [5] and considered the fifth leading cause of death for those aged more than 65 years. The most common hypothesis behind AD is the deposition of neurotic plaques extracellularly and neurofibrillary tangles intracellularly in the brain of AD patients. Several major theories are involved in the pathogenesis of AD, such as neurotic plaques hypothesis [45], tauopathy [30], inflammation [37], and oxidative stress [41].

Neurotic plaque hypothesis is considered the primary hallmark of AD in which APP (protein normally found in brain essential for the growth and survival of the neurons) is cleaved by mutant form of β-secretase then followed by γ- secretase which results in generation of the Aβ peptide which aggregates and deposit on the surface of the neurons [32].

Different mechanisms were implemented in order to decrease the level of beta amyloid plaques, one of them is altering catabolism of Aβ levels in the brains of AD, by using many proteases and peptidases that have the capability of cleaving Aβ either in vitro or in vivo. The following proteinases have the abilities of degrading Aβ peptide. One of the primary degrading enzyme which targets the degradation of beta amyloid peptides is Neprilysin enzyme (NEP) which is found to be abundant in the brain areas vulnerable to amyloid plaque deposition [23]. It is considered the major amyloid degrading enzyme and plays a major role in the clearance of amyloid beta peptides (Aβ) from the brain [22, 36]. NEP was found to degrade both monomeric and oligomeric forms of Aβ40 and Aβ42 in intracellular and extracellular compartments of the brain [16, 56].

Estrogen deprivation has been implicated in the pathogenesis of AD [54]. Furthermore, there is a strong evidence that AD pathology and AD-related cognitive decline are strongly related to decrease in estrogen level [43, 53]. Estrogen plays an important role in protection against neuron cell death [48, 52] and has the ability of inhibition of different aspects of AD neuropathology including Aβ accumulation [4, 42].

Interestingly NEP was found to be mainly upregulated by estrogen hormone [19]. Moreover it has been proven that estrogen deprivation caused a lowering of NEP level and that estrogen replacement therapy restored NEP level to control values in ovariectomized rat brains. It has been speculated that estrogen regulates the expression of NEP [13]. Furthermore NEP activity was found to be lower in the hippocampus, cerebellum, and caudate of ovariectomized rats than in non-ovariectomized rats, and this effect can be reversed by exogenous 17β-estradiol [59].

Lipopolysaccharide (LPS) is a gram negative bacterial endotoxin that was found to induce neuroinflammation which exhibits AD-like features when systemically injected to rodents [7]. Moreover it has been proven that intraperitoneal (i.p.) injection of LPS induces cognitive impairment in mice as it induces memory loss and amyloidogenesis in vivo and in vitro as a consequence of systemic inflammation [46, 51] by production of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α [38, 17].

Resveratrol (RSV) (3,4′,5-trihydroxystilbene), a secondary metabolite, belongs to a class of polyphenolic compounds called stilbenes [49]. It was first isolated from the roots of Veratrum grandiflorum [29]. Several plant species are known to produce RSV (classified as polyphenol) such as vine plant, peanuts, berries but was found to be in large amounts in skin of red grapes [11, 40].

RSV possesses many biological activities that are protective against atherosclerosis, including antioxidant, anti-inflammatory, antiplatelet, and vasorelaxant activities, and has been used in applying cardioprotective effect [35]. In addition, it is identified as a cancer chemotherapeutic agent [26] and has recently been reported to mimic effects of dietary restriction and extends the life span of lower organisms [18, 58].

It has been demonstrated that RSV can lessen amyloid-β peptide-induced toxicity, protect against cerebral ischemic injury, and shield neurons from induced excitotoxicity [28]. Moreover, RSV promotes the non-amyloidogenic pathway and enhances the cleavage of the amyloid precursor protein. Furthermore it enhances clearance of amyloid beta peptides, and reduces neuronal damage [31].

One of the important features that RSV has been characterized with is being a phytoestrogen on the basis of its ability to bind to estrogen receptors α and β (ERs α and ER β) and elicits similar responses to endogenous estrogens. Moreover, the chemical structure of RSV is very similar to that of the synthetic estrogen agonist, diethylstilbestrol (DES). RSV is considered an estrogen agonist [6]. It was found that RSV in particular acts on estrogen receptor beta which has a major function in the cognitive processes, so by binding of RSV to estrogen receptor beta, this results in improvement of cognitive impairment which results from AD [10].

So due to the structural similarity between RSV and estradiol this study was conducted to investigate the strong correlation between RSV and NEP in the degradation of amyloid beta.

12.2 Methodology

After 1 week of acclimatization, mice were randomly allocated to four groups each consisting of 10–12 mice. One group served as normal and received (saline 0.9 % i.p.) once.

The second group mice were injected with LPS (0.8 mg/kg i.p in saline) once to induce systemic and neuroinflammation and then were subjected to the tests after 7 days [1, 47, 14]. The third group mice were injected with LPS (0.8 mg/kg i.p in saline) once, and after 30 min mice were injected with RSV (4 mg/kg in saline) for 7 days [8, 9]. RSV was dissolved in the least amount of DMSO and completed to the final volume with saline [12, 24]. Animals were subjected to the tests after the seventh day of all injections. The fourth group mice were injected with only RSV (4 mg/kg i.p in saline) and were subjected to the tests after the seventh day of injections. All Animals were subjected to different behavioral tests such as open field, object recognition, and Y-maze behavioral tests at the end of the previously mentioned treatments, then they were sacrificed, and their brains were harvested. The brains were divided into two hemispheres and were frozen at 80 °C to be later homogenized and used for ELISA Kit.

12.3 Relation Between Resveratrol as a Phytoestrogen and Neprilysin in Lipopolysaccharide Induced Alzheimer’s Disease

Behavioral tests were performed as a form of noninvasive method to detect changes in cognitive function after treating mice with LPS. Three behavioral tests were conducted: Y-maze test to detect working memory, open field test to measure locomotor functions, and object recognition test to measure nonspatial memory.

LPS-induced cognitive impairment was associated with a decrease in ambulation, rearing, and grooming frequencies compared to the normal animals in open field testing. It also resulted in significant decrease in working memory evaluated by significant decrease in MAP measured by Y-maze test. These results are in agreement with those of [14], in which administration of LPS once then testing mice after 7 days from injection significantly reduced locomotor activity as well as reduced MAP (Figs. 12.1 and 12.2).

Fig. 12.1
figure 1

Effects of LPS and resveratrol on the ambulation, grooming, and rearing frequencies

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Fig. 12.2
figure 2

Effect of LPS and resveratrol on the mean alternation percentage

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LPS-induced AD was associated with a decrease in nonspatial memory evaluated using new object recognition test. The results showed a significant decrease in the discrimination ratio compared to normal mice. This result is in agreement with those of [25] in which LPS injection showed significant nonspatial memory deficit evaluated by the impairment of object memory in new object recognition test. This object recognition memory deficit was evaluated to be due to systemic response induced by the i.p. injection of LPS (Fig. 12.3).

Fig. 12.3
figure 3

Effect of LPS and resveratrol on the discrimination ratio

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RSV, a polyphenolic compound found mainly in skin of red grapes, was found to induce in vivo protective properties against multiple illnesses, including cancer, cardiovascular disease, and ischemia, and was also found to confer resistance to stress and to extend life span [3].

Administration of RSV resulted in significant increase in locomotor activity evaluated by open field test. Our results are in agreement with a study in which RSV has showed that it can contribute to the preservation of cognitive function during aging as it was shown in the results that RSV was able to improve the locomotor function using open field test [44] (Fig. 12.1).

Administration of RSV resulted in significant increase in mean alternation percentage evaluated by Y-maze test. This result is in accordance with a study in which RSV was examined to see its effect on learning and working memory in Y-maze in diabetic rat; results showed that RSV elevated MAP significantly and improved learning and working memory [39] (Fig. 12.2).

Administration of RSV resulted in significant increase in nonspatial memory evaluated by object recognition test. This result is in harmony with a study in which RSV has been administrated to assess its effect on subarachnoid and intracranial hemorrhage, ischemia, and stroke. Results showed that administration of RSV was able to improve nonspatial memory dramatically after trauma and that posttraumatic memory was restored to 66 % compared to uninjured control [50] (Fig. 12.3).

Previous studies proved that estrogen treatment inhibits microglial activation following exposure to the inflammatory stimuli (LPS) in LPS-treated mice, and that resulted in increasing cell viability and attenuating the inflammatory effect of LPS [55].

Administration of LPS (0.8 mg/kg i.p.) once significantly decreased estrogen level when measured by estradiol ELISA kit. These results are in agreement with a study done by [57] in which LPS and TNF-α suppress ovarian cell function and decreased estrogen level significantly (Fig. 12.4).

Fig. 12.4
figure 4

Effect of LPS and resveratrol on estradiol level

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Administration of LPS (0.8 mg/kg i.p.) once significantly decreased NEP level when measured by NEP ELISA kit. These results are in agreement with a study done by [15] in which administration of LPS resulted in significant decrease in the level of NEP (Fig. 12.5).

Fig. 12.5
figure 5

Effect of LPS and resveratrol on neprilysin level

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RSV, a phytoestrogen, has been proven to be able to confer a significant improvement in spatial memory, and protect animals from Aβ-induced neurotoxicity. These neurological protection effects of resveratrol were associated with elevated estrogen level together with reduced iNOS level and oxidative stress [20].

Administration of RSV a phytoestrogen (4 mg/kg i.p) for 7 days either in the group of mice that were injected with LPS to induce AD or in the other group that was injected with RSV, both groups showed a very high significant increase in estrogen level when measured by estradiol ELISA kit based on its structural similarity to diethylstilbestrol. Similar to our results, different studies were conducted to prove that administration of RSV led to elevation in the level of estradiol [2, 27] (Fig. 12.4).

NEP is a zinc metalloendopeptidase that regulates the activity of a number of physiological peptides. Evidence has accumulated that NEP is involved in the clearance of amyloid beta peptides in the brain. Several studies provide evidence that show a strong relation between estrogen and NEP enzyme which is considered the major degrading enzyme for beta amyloid plaques in AD brain. Previous studies have shown that NEP gene responds to progesterone, androgen, and glucocorticoids, but primarily its activity is regulated by estrogen [21].

It has been proven that experimental depletion of endogenous NEP activity by pharmacological inhibitors or reduction of NEP expression by molecular approaches results in neural accumulation of Aβ [33].

Administration of RSV, a phytoestrogen, resulted in significant increase in NEP level when measured by NEP ELISA kit. These results are in agreement with a study by Marambaud et al. where RSV promoted the clearance of Alzheimer’s disease amyloid beta peptides through upregulation of NEP level [34] (Fig. 12.5).

12.4 Results

Normal animals group were injected once with saline 7 days before subjecting them to open field testing (Group 1). Another group (4) was injected only with RSV (4 mg/kg) for 7 days and was subjected to open field after 7 days. Alzheimer’s disease was induced in groups 2 and 3 by intraperitoneal injection of LPS (0.8 mg/kg) 7 days before subjecting them to open field testing. Group 3 was injected with RSV (4 mg/kg i.p.) for 7 days after 30 min from injection the LPS, and was subjected to open field test on the seventh day. Analysis was carried out using unpaired t-test to compare each two groups.

Each value represents Mean ± Standard Error of Mean. *Significantly different from Group 1 at P < 0.05. +Significantly different from Group 2 at P < 0.05.

Normal animals group were injected once with saline 7 days before subjecting them to Y-maze testing (Group 1). Another group (4) was injected only with RSV (4 mg/kg) for 7 days and was subjected to Y-maze test after 7 days. Alzheimer’s disease was induced in groups 2 and 3 by intraperitoneal injection of LPS (0.8 mg/kg) 7 days before subjecting them to Y-maze test. Group 3 was injected with RSV (4 mg/kg) for 7 days after 30 min from injection the LPS, and was subjected to Y-maze test on the seventh day. Analysis was carried out using unpaired t-test to compare each two groups.

Each value represents Mean ± Standard Error of Mean. *Significantly different from Group 1 at P < 0.05. +Significantly different from Group 2 at P < 0.05.

MAP = Mean Alternation Percentage.

Normal animals group were injected once with saline 7 days before subjecting them to object recognition testing (Group 1). Another group (4) was injected only with RSV (4 mg/kg) for 7 days and was subjected to object recognition test after 7 days. Alzheimer’s disease was induced in groups 2 and 3 by intraperitoneal injection of LPS (0.8 mg/kg) 7 days before subjecting them to object recognition test. Group 3 was injected with RSV (4 mg/kg) for 7 days after 30 min from injection the LPS, and was subjected to object recognition test on the seventh day. Analysis was carried out using unpaired t-test to compare each two groups.

Each value represents Mean ± Standard Error of Mean. *Significantly different from Group 1 at P < 0.05. +Significantly different from Group 2 at P < 0.05.

Discrimination Ratio = DR

Animals were divided into four groups; group1 is the normal animal group injected only with 0.9 % saline i.p once. Alzheimer’s disease was induced in groups 2 and 3 by intraperitoneal injection of LPS (0.8 mg/kg i.p) once. Group 2 is the untreated group while the treated group is (group 3) that was injected with RSV (4 mg/kg i.p) for 7 days after 30 min from injection with LPS. Another group of mice injected with RSV only (4 mg/kg i.p) for 7 days (group 4). After performing the behavioral tests, brains were obtained and homogenized as 10 % homogenate in PBS, centrifuged, and supernatants were obtained. Fifty microliters of the supernatants from six samples of each group was diluted to 50 μl with assay buffer, added on it reaction mix and assayed. Then substituted by the absorbance in the straight line equation to get the concentrations and calculated as Sa/SV. Statistical analysis of estradiol concentrations was carried out using unpaired t-test to compare each two groups.

Each value represents Mean ± Standard Error of Mean. *Significantly different from Group 1 at P < 0.05. +Significantly different from Group 2 at P < 0.05.

Animals were divided into four groups; group1 is the normal animal group injected only with 0.9 % saline i.p. once. Alzheimer’s disease was induced in groups 2 and 3 by intraperitoneal injection of LPS (0.8 mg/kg i.p) once. Group 2 is the untreated group while the treated group is (group 3) that was injected with RSV (4 mg/kg i.p) for 7 days after 30 min from injection with LPS. Another group of mice injected with RSV only (4 mg/kg i.p) for 7 days (group 4). After performing the behavioral tests, brains were obtained and homogenized as 10 % homogenate in PBS, centrifuged, and supernatants were obtained. Fifty microliters of the supernatants from six samples of each group was diluted to 50 μl with assay buffer, added on it reaction mix and assayed. Then substituted by the absorbance in the straight line equation to get the concentrations and calculated as Sa/SV. Statistical analysis of estradiol concentrations was carried out using unpaired t-test to compare each two groups.

Each value represents Mean ± Standard Error of Mean. *Significantly different from Group 1 at P < 0.05. +Significantly different from Group 2 at P < 0.05.

Conclusion

  • LPS can be a perfect model for induction of AD in rodents.

  • RSV can be a promising drug in management of AD.