Protective effects of gingerol on streptozotocin-induced sporadic Alzheimer’s disease: emphasis on inhibition of β-amyloid, COX-2, alpha-, beta - secretases and APH1a

Protective effects of gingerol on streptozotocin-induced sporadic Alzheimer’s disease: emphasis... www.nature.com/scientificreports OPEN Protective effects of gingerol on streptozotocin-induced sporadic Alzheimer’s disease: emphasis on Received: 22 November 2016 inhibition of β-amyloid, COX-2, Accepted: 26 April 2017 Published: xx xx xxxx alpha-, beta - secretases and APH1a 1 2,3 1,4 1 Ali M. El Halawany , Nesrine S. EL Sayed , Hossam M. Abdallah & Riham Salah El Dine Gingerol is a major dietary compound that occurs in several plants belonging to the Zingiberaceae family. In the current study, the protective effect of gingerol on STZ-induced sporadic Alzheimer’s disease (SAD) was determined. Gingerol was isolated from the seeds of Aframomum melegueta K. Schum and tested at doses of 10 and 20 mg/kgbwt for its possible effect on the SAD model in mice, using celecoxib (30 mg/kg bwt) as a reference standard. The curative effects of gingerol were assessed through measurement of β-amyloid (Aβ-42), α-, β- secretases, APH1a and COX-2 levels. In addition, improvement in the cognitive deficit in mice after treatment was confirmed using the water maze and Y-maze with intra-maze cues. Gingerol improved the cognitive and behavioral impairment and AD-like pathology in streptozotocin model mice. These beneficial effects occurred with an increase in α-secretase activity and a decrease in cerebral Aβ-42, β- secretase, APH1a activity and COX-2-linked neuro-inflammation. Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by loss of cognitive function as well as pathological deposition of β-amyloid (Aβ) senile plaques and neurob fi rillary tangles . AD is classified into late-onset sporadic AD (SAD) and early onset familial AD (FAD). The majority of AD cases are SAD, which involves many etiopathogenic mechanisms including environmental, genetic and metabolic factors . e Th intracerebroventricular (ICV) injection of streptozotocin results in a well-established mouse model show - ing many aspects of SAD including neuroinflammation, brain insulin resistance, cholinergic deficits, accumula- tion of β-amyloid and tau proteins, and oxidative stress as well as memory and learning impairment . In AD, the amyloidogenic pathway results in the production of Aβ through the cleavage of amyloid precursor protein (APP) by β-secretase followed by the APH1a subunit of the γ-secretase complex. However, α-secretase produces the non-amyloidogenic soluble amyloid precursor protein α (sAPPα) . Accumulating evidence has demonstrated that COX inhibitors are involved in the management of AD through their action on the down- stream ee ff cts of the insulin signaling pathway inhibiting neuroinflammation and oxidative stress . Moreover, it was previously suggested that expression of cyclooxygenases (COXs) may ae ff ct β -amyloid peptide generation through mechanisms that involve the PGE2-mediated potentiation of γ-secretase activity, further supporting a role for COX-2 in the neuropathology of AD . Natural products are a well-known source of most of the discovered medicines. Most of the active compounds that have shown protective effects against SAD are from plants, with little contribution from marine and micro- bial origins . Most of the discovered plant-derived compounds have shown activity as acetyl cholinesterase inhib- itors (AChEIs), but they only address the symptoms of AD. However, certain plant extracts and compounds with antioxidant and anti-inflammatory effects have also been reported to alleviate AD . 1 2 Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt. Department of Pharmacology &Toxicology, Faculty of pharmacy, Cairo University, 11562, Cairo, Egypt. Department of Pharmacology &Toxicology, Faculty of pharmacy & Biotechnology, German University in Cairo, 11835, Cairo, Egypt. Department of Natural Products, Faculty of Pharmacy, King Abdulaziz University, Jeddah, 21589, Saudi Arabia. Correspondence and requests for materials should be addressed to H.M.A. (email: hmafifi2013@gmail.com ) Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 1 www.nature.com/scientificreports/ Figure 1. Effect of STZ, celecoxib and gingerol on the mean escape latency in the Morris water maze. Animals were divided into 5 groups. Group 1 received a single ICV dose of saline; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. Group 2 received a single injection of STZ (3 mg/kg, i.c.v.), and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. Group A was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. Group B received a single injection of STZ (3 mg/kg, i.c.v.), and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals from groups A, B, and C were subjected rd to the behavioral tests aer t ft he 3 week of injections. Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way ANOVA followed by the Tukey-Kramer multiple *** comparison test with the aid of GraphPad Prism Software version 5. Significantly different from the normal ++ +++ group P < 0.001. Significantly different from the STZ group P < 0.01. Significantly different from the STZ group P < 0.001. 6-Gingerol is a major phenolic constituent and hydroxyphenyl alkane of several plants belonging to the Zingiberaceae family such as ginger, cardamom and grain of paradise. The formerly mentioned plants are widely used in Middle Eastern and Asian cuisine as spices and every-day beverages. According to Arabian folklore, ginger has been claimed to improve memory and has been traditionally used as an ingredient for cognitive enhancement . 6-Gingerol is reported to display several biochemical and pharmacologic activity such as anticarcinogenic, 9 10 11, 12 antimutagenic, anti-apoptotic , antioxidant, anti-inflammatory , and cardio- and hepatoprotective effects . It is also known to inhibit nitric oxide synthase and cyclo-oxygenase and the expression of tumor necrosis factor alpha (TNF-α) . The antioxidant, anti-apoptotic and anti-inflammatory potentials of 6-gingerol suggest its pos- sible effect for protection against some SAD manifestations. e o Th bjective of this study is to investigate the effect of gingerol on some molecular biomarkers of SAD includ- ing anestimation of the level of β-amyloid, α- and β-secretases and the APH1a subunit of γ-secretase as well as the cognitive skills, suggesting that it could be a promising component of a clinical nutrition regime or used as 4–6 an adjuvant therapy . Results 1 13 Purified gingerol was identified using H and C NMR analysis and comparison of the data with that of the 15, 16 reported literature (Supplementary data, Figs 1–3) . All experiments were designed to test the possible effect of gingerol at two dosages (10 and 20 mg/kg bwt) on the improvement of cognitive function as well as Aβ forma- tion in the brain in STZ-induced cognitive impairment and amyloid genesis in a mouse model. As no significant difference was found during the pilot study between the two gingerol (10 mg/kg and 20 mg/kg) groups and the saline-treated group (Supplementary data, Tables 1–7), only the saline control group was represented in all of the following graphs. The possible anti-inflammatory effect of gingerol was tested in comparison with the effect of the anti-inflammatory celecoxib as a reference standard. Morris water maze. Cognitive impairment was induced by ICV injection of STZ as manifested in the water maze test and modified Y maze test. The Morris water maze is used to investigate the spatial reference learning and memory of mice. During the training phase, all mice learned the platform location, as revealed by a decrease in the latency and the distance travelled to locate the submerged platform. However, the training was delayed for the ICV-STZ mice as they needed more time and travelled a longer distance to find the platform than the control mice, which clearly indicated a short-term memory impairment in the STZ-treated mice. The treated groups (groups A, B and C) showed a significant improvement starting from the first day as they exhibited a significantly lower escape latency than those in the STZ group (Fig. 1). In the probe test, The STZ-treated group showed a significant decrease in the mean time spent in the target quadrant by approximately 70% compared to that of the normal group. Moreover, the treated groups A, B and C showed a significant increase in the mean time spent in the target quadrant by approximately 60% compared to that of the STZ-treated group (Fig. 2). es Th e results suggest an impairment to encode and remember the spatial coordinates of the platform within the environment. Retrieval of spatial memory was more specifically analyzed with the probe trials performed 24 hours after the last training trial. The ICV-STZ mice crossed the former platform site much less often than Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 2 www.nature.com/scientificreports/ Figure 2. Effect of STZ, celecoxib, and gingerol (10 and 20 mg/kg) on the Morris water maze probe test. Animals were divided into 5 groups. Group 1 received a single dose of saline ICV; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. Group 2 received a single injection of STZ (3 mg/kg, i.c.v.), and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. Group A was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. Group B animals were injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals from groups A, B, and C were subjected rd to the behavioral tests aer t ft he 3 week of injections. Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way ANOVA followed by a Tukey-Kramer multiple comparison test with the aid of GraphPad Prism Software version 5. ***Significantly different from the normal +++ +++ group P < 0.001. Significantly different from the STZ group P < 0.001. Significantly different from the STZ group P < 0.001. Figure 3. Effect of STZ, celecoxib, and gingerol (10 and 20 mg/kg) on the novel arm count in the intra-maze cue version of the Y-maze. Animals were divided into 5 groups. Group 1 received a single ICV dose of saline; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. Group 2 received a single injection of STZ (3 mg/kg, i.c.v.), and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. Group A was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. Group B animals were injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals from groups rd A, B, and C were subjected to the behavioral tests aer t ft he 3 week of injections. Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way ANOVA followed by a Tukey-Kramer multiple comparison test with the aid of GraphPad Prism Software version 5. ***Significantly +++ different from the normal group P < 0.001. Significantly different from the STZ group P < 0.001. Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 3 www.nature.com/scientificreports/ Figure 4. Effect of STZ, celecoxib, and gingerol (10 and 20 mg/kg) on the mean mouse amyloid beta 42 concentration using an ELISA assay. Animals were divided into 5 groups. Group 1 received a single ICV dose of saline; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. Group 2 received a single injection of STZ (3 mg/kg, i.c.v.), and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. Group A was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. Group B animals were injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals rd from groups A, B, and C were subjected to tests aer t ft he 3 week of injections. Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way ANOVA followed by Tukey- Kramer multiple comparison test with the aid of GraphPad Prism Software version 5. ***Significantly different +++ from the normal group P < 0.001. Significantly different from the STZ group P < 0.001. the control mice and did not show marked preference toward the target quadrant. es Th e results indicate that the ICV-STZ mice have a decreased ability to encode the environment as a spatial map and to localize the platform, suggesting an impairment in spatial reference memory. Intra-maze cue version of the Y-maze. The effect of STZ (3 mg/kg, ICV) was investigated on the novel arm count in the intra-maze cue version of the Y-maze and demonstrated significant impairment in novelty-seeking behavior and spatial memory compared with that of the control mice. Treatment of mice with the positive control (celecoxib, 30 mg/kg, i.p.) and gingerol (10 mg/kg and 20 mg/kg, i.p.) for 7 days resulted in significant improvement in both the novelty-seeking behavior as well as spatial memory (Fig.  3). STZ-induced Aβ-42 generation in mouse brains. To further investigate the anti-amyloid genesis ee ff cts of gingerol, the inhibitory effect on the levels of Aβ-42 as well as α-, β- and γ-secretases was examined. e m Th ean levels of Aβ-42 in mouse brains were evaluated by a mouse Aβ-42 ELISA kit. The STZ-treated mice showed a significantly higher concentration of amyloid beta than the normal group. However, treatment with celecoxib or gingerol (10 mg/kg and 20 mg/kg) resulted in a significant reduction in the concentration of Aβ -42 when compared with that in the STZ-treated mice (Fig. 4). STZ decreased α-secretase activity but increased β-secretase activity and the APH1a con- centration. Tumor necrosis factor alpha (TNF-α)-converting enzyme (TACE), also called ADAM17 or α-secretase, belongs to the ADAM (A Disintegrin and Metalloprotease) family of proteins involved in neurogen- esis through the ectodomain shedding of cell surface proteins . TACE, the protease responsible for the generation of mature TNF-α, plays an important role in acute and chronic inflammation. The inhibition of TACE by a phar - macological agent may represent an alternative approach to modulate the effect of TNF-α . TACE is also respon- sible for the proteolytic cleavage of amyloid precursor proteins, L-selectin and transforming growth factor-α . Moreover, APH1a encodes a component of the γ-secretase complex that cleaves integral membrane proteins such as β-amyloid precursor protein (APP). Accordingly, the APH1a concentration was used in the present study as a way to represent the level of γ-secretase in the brains of mice. Following a single ICV injection of STZ, the β-secretase activity and APH1a concentration increased, whereas the activity of α-secretase was reduced in mouse brains. Intraperitoneal injection of celecoxib orgingerol (10 mg/kg and 20 mg/kg) resulted in a significant reduction in the level of β and APH1a and a significant increase in the activity of α-secretase (Figs  5, 6 and 7). Immunohistochemistry: Effect of various drug treatments on the expression of COX-2 in the brain. ICV injection of STZ showed a marked increase in the expression of COX-2 in the mouse brains Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 4 www.nature.com/scientificreports/ Figure 5. Effect of STZ, celecoxib, andgingerol (10 and 20 mg/kg) on the mean mouse APH1a concentration using an ELISA assay. Animals were divided into 5 groups. Group 1 received a single ICV dose of saline; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. Group 2 received a single injection of STZ (3 mg/kg, i.c.v.). and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. Group A was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. Group B animals were injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals from groups rd A, B, and C were subjected to tests aer t ft he 3 week of injections. Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way ANOVA followed by Tukey-Kramer multiple comparison test with the aid of GraphPad Prism Software version 5. ***Significantly different from the +++ normal group P < 0.001. Significantly different from the STZ group P < 0.001. compared to that in normal animals. However, COX-2 expression was markedly decreased in the celecoxib and gingerol (10 mg/kg and 20 mg/kg) -treated animal groups compared to that in the STZ-treated animals (Fig. 8). Discussion AD is an age-linked neurodegenerative disease characterized by severe dementia. Symptoms of AD occur over a long period of time, and therefore, treatments should target this silent period of the disease to retard the disease progression towards dementia . Centrally administered STZ is known to cause SAD-like memory deterioration, 21, 22 and STZ-treated mice are therefore one of the non-transgenic mice models used to represent AD . ICV-STZ-treated rats showed an impaired learning and memory performance. Cognitive impairment after ICV-STZ administration is induced by the degradation of phospholipids and the resulting increases in the free fatty acid, arachidonic acid. COX is a rate-limiting enzyme in the metabolism of arachidonic acid to prostanoids, particularly prostaglandins (PGs), which significantly contribute to neuro-inflammation with oxidative stress. PGs such as PGE1 and PGE2 have also been reported to cause neurotoxicity and stimulate the release of gluta- mate from astrocytes. In a recent study, the use of COX inhibitors, such as indomethacin, has been shown to cause some improvement in the symptoms of AD, and the mechanism has been hypothesized to involve a reduction in inflammation and oxidative stress. In addition, celecoxib (a selective COX-2 inhibitor) aids in slowing the progression of the disease through its anti-inflammatory property resulting in reduced neuronal cell death . Accordingly, it was used as a positive control in this study. In this study, the neuroprotective effects of gingerol on the molecular biomarkers of SAD and cognitive skills were investigated. Its protective effect was assessed through measuring the levels of Aβ , α-and β-secretases, APH1a (a component of γ-secretase) and COX-2. In addition, the improvement in the cognitive skills ofmice aer t ft reatment was confirmed using the Y and water maze. In the present study, fourteen days following a single ICV injection of STZ, the level of Aβ-42 as well as the activity of β-secretase and the APH1a concentration in the whole brain increased, whereas that of α-secretase decreased. Treatment with celecoxib and gingerol resulted in a significant reduction in the levels of Aβ -42, APH1a and β-secretase activity as well as a significant increase in the activity of α-secretase. Aβ precursor protein (APP) is generally known to be a limiting reagent in the cell, and it has been pos- tulated that α- and β-secretases compete for APP proteolysis . Reduced β-secretase activity could therefore be responsible for the alleviation of the cerebral Aβ pathology in gingerol-treated mice. In this regard, it was previously demonstrated that another polyphenol, epigallocatechin gallate (EGCG), was also able to promote non-amyloidogenic APP metabolism in the Tg2576 cerebral amyloidosis mouse model. Interestingly, the mech- anism for the beneficial effects of EGCG on non-amyloidogenic APP processing relied on promoting the activity of the candidate α-secretase, a disintegrin and metalloprotease 10 . Recently, nutraceuticals, including the green 26 27 28 29 tea polyphenol and citrus bioflavonoid EGCG , luteolin , grape-derived polyphenols , and caein ff e have been found to exhibit anti-amyloidogenic properties. Moreover, a plant-derived polyphenol, tannic acid (TA), has been Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 5 www.nature.com/scientificreports/ Figure 6. Effect of STZ, celecoxib, and gingerol (10 and 20 mg/kg) on the mean mouse α–secretase activity using an ELISA assay. Animals were divided into 5 groups. Group 1 received a single ICV dose of saline; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. Group 2 received a single injection of STZ (3 mg/kg, i.c.v.), and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. Group A was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. Group B animals were injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals from groups rd A, B, and C were subjected to tests aer t ft he 3 week of injections. Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way ANOVA followed by Tukey-Kramer multiple comparison test by the aid of Graph Pad Prism Software version 5. ***Significantly different from +++ normal group P < 0.001. Significantly different from STZ group P < 0.001. shown to oppose the behavioral impairment and AD-like pathology in transgenic mice. These beneficial effects occur with a reduction in cerebral Aβ pathology, cleaved β-CTF, sAPP-β, BACE1 (beta-site amyloid precursor protein cleaving enzyme 1) protein expression and activity, and neuro-inflammation . Neuro-inflammation plays a crucial role in the pathogenesis of SAD. In the current study, the expression levels of COX-2 were observed to be significantly up-regulated in STZ-induced AD mice and down-regulated strongly by gingerol compared with that in control mice. Similarly, it was previously demonstrated that the STZ-induced learning and memory impairments in mice are due to stimulation of the neuro-inflammatory response . e p Th rotective effect of gingerol was further confirmed through immunohistochemistry, in which we studied the effects of these compounds on expression of COX-2 in the brain. The protective effect of gingerol on Aβ -42, β-secretase and APH1a levels was accompanied by a marked decrease in the expression of COX-2 in the brain tissue. Recently, the effects of shogaol, a structurally related compound to gingerol and isolated from Zingiber officinale , on microglia activation induced by lipopolysaccharide (LPS) were studied both in primary cortical neuron-glia cultures and in an in vivo neuro-inflammatory model. Shogaol exerted an anti-inflammatory effect by inhibiting the production of PGE2 and by down-regulating COX-2 expression better than gingerol . In addition, a recent study done by Ilic and his colleagues on the ethanolic extract of grains of paradise (A. melegueta), the source of gingerol in the current study, showed that the major phenolics from the extract,6-paradol, 6-gingerol, and 6-shogaol, reduced inflammation, which is in part due to the inhibition of COX-2 enzyme activity and the expression of pro-inflammatory genes . In addition, the improvement in the cognitive skills of mice aer t ft reatment was established using the Y and water maze. In the present study, the MWM was used to observe the effects of STZ and gingerol on cognitive dysfunction in a mouse model. Our results indicated that STZ can significantly increase the escape latency and th decrease the percentage of time spent in the 4 quadrant, which is in agreement with previous studies that con- cluded that ICV-STZ-treated mice had deficits in spatial learning and memory indicated by impaired acquisition and retention in the MWM and passive avoidance tasks . Moreover, during the probe trial done on last day with the platform removed, mice failed to remember the precise location of the platform, spending significantly less time in the target quadrant than the normal groups; this result was proven earlier . Similarly, STZ resulted in severe significant impairment in novelty-seeking behavior and spatial memory in the modified Y-maze test. This finding coincides with a recent study that proved that the deficits in Y-maze performance following chronic stress reflect neurochemical and/or neurobiological changes underlying spatial memory ability . In addition, the relationship between higher expression of inflammatory cytokines and STZ-caused cognitive impairment has been widely acknowledged. Previous studies have shown that cognitive function will be improved if the excessive Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 6 www.nature.com/scientificreports/ Figure 7. Effects of STZ, celecoxib, and gingerol (10 and 20 mg/kg) on the mean mouse β-secretase cleavage activity using an ELISA assay. Animals were divided into 5 groups. Group 1 received a single ICV dose of saline; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. Group 2 received a single injection of STZ (3 mg/kg, i.c.v.), and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. Group A was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. Group B animals were injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals from groups rd A, B, and C were subjected to tests aer t ft he 3 week of injections. Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way ANOVA followed by a Tukey-Kramer multiple comparison test with the aid of GraphPad Prism Software version 5. ***Significantly different from the +++ normal group P < 0.001. Significantly different from the STZ group P < 0.001. activation of the inflammatory response has been corrected . This indicates that the STZ model is beneficial as a mouse model of cognitive impairment. In this light, recent focus has been given to a group of phenolics such as flavonoids, which have been found to be potentially anti-amyloidogenic . The beneficial behavioral neuroprotec- tive effects of the studied phenolics and gingerol coincide with a recent finding that pretreatment with hesperidin improved the cognitive impairment induced by ICV injection of STZ in the MWM test. This was concluded to be mediated by inhibiting the overexpression of inflammatory markers such as COX-2 , and their efficacies were found to be at the level of celecoxib, used as a positive control in this study. Finally, it could be concluded from the present work that gingerol prevented memory impairments in a SAD mouse model, which could be attributable to the amelioration of STZ- induced neuroinflammation and amyloi- dogenesis. This study gives a perspective on the wide variety of interactions between the inflammatory COX-2 and APP secretases and the beneficial effect of gingerol. On the one hand, overexpression of COX-2by STZ is able to increase the activity of β-secretase and γ-secretase and to decrease that of α-secretase. On the other hand, gingerol and celecoxib are able to modulate the activity of the secretases by regulating their levels in the case of BACE1 and APH1a or by directly shifting the cleavage site of γ-secretase. This study is the first, to the best of our knowledge, to report the protective effects of gingerol against STZ-induced biochemical changes and behavioral impairments, rendering gingerol a promising compound to treat or prevent AD. Methods General experimental procedures. Nuclear magnetic resonance (NMR) spectra were recorded on a 1 13 Bruker Unity INOVA 850 instrument at 850 MHz for H- and 213 MHz for C-NMR (Bruker BioSpin, Billerica, MA, USA) using CDCl as the solvent. ESIMS was recorded on an Agilent 1200 system connected to an Agilent 6320 Ion Trap HPLC-ESI-MS (Agilent Technology, Waldbronn, Germany). TLC was carried out on a pre-coated silica gel 60 F (0.25 mm, Merck) and RP-18 F S (0.25 mm, Merck Co., Darmstadt, Germany). Column chro- 254 254 matography (CC) was carried out on a silica gel 60 for column chromatography (Fluka, 70-230 mesh, Germany). The plant material and the isolation and purification methods of gingerol can be seen in the Supplementary Information. Biological Study. Experimental Design. Adult male Swiss albino mice weighing between 20–25 g were uti- lized. Mice were obtained from the animal state of the National Institute of Research (NRC) (Giza, Egypt). Mice were housed in a temperature-controlled room (23–24 °C) and presented with 12-hour dim/light cycles. Free access to food and water was permitted. All procedures were done following the guide for the treatment and care Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 7 www.nature.com/scientificreports/ Figure 8. Effects of STZ, celecoxib, and gingerol (10 and 20 mg/kg) on the immunohistochemical analysis of COX-2 expression in the brain. (A) The normal group received a single ICV dose of saline; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. (B) The control group received a single injection of STZ (3 mg/kg, i.c.v.), and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. (C) The celecoxib group received STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. (D) The gingerol (10 mg/kg) group received STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. (E) The gingerol (20 mg/kg) group received STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Scale bar, 60 µm (A–E ). of laboratory animals published by US National Institute of Health and approved by the Ethics Committee at the German University in Cairo. All actions were made to minimize mouse suffering. A pilot study was performed whereby two doses of gingerol (10 and 20 mg/kg) were employed in addition to the saline control group. e b Th ehavioral and biochemical tests were performed on the three control groups. e Th findings of this study revealed that there were no statistically significant differences noted in the results among the three control groups. Therefore, the normal saline control group was the only group used in the experimental design and the description of the results. Mice were then randomly isolated into five groups, and every group comprised ten mice. These groups were defined as follows: Group 1: Mice received a solitary ICV infusion of saline; then, following 2 weeks, they received intraperitoneal injections with the vehicle (saline) for seven days. Group 2: Mice were infused with STZ (3 mg/kg, i.c.v.) once, and then received intraperitoneal injections of saline aer 2 w ft eeks for seven days. Group A: Mice were infused with STZ (3 mg/kg, i.c.v.) once, and following 2 weeks, they were treated with celecoxib (30 mg/kg, i.p.) for 7 days. Group B: Mice were injected with STZ (3 mg/kg, i.c.v.) once and following 2 weeks, they were treated with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C: Animals were injected with STZ (3 mg/kg, i.c.v.) once, and following 2 weeks, they were treated with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals from groups A, B, and C were subjected to the behavioral and biochemical tests aer t ft he third week of infusions. All mice were subjected to the Morris water maze and Ymi behavioral tests toward the end of the already spec- ified medications. Aer t ft hat, they were euthanized, and their brains were harvested. Two whole brains from each Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 8 www.nature.com/scientificreports/ group were placed in 4% formaldehyde to be utilized for immunohistochemical examination of COX-2 expres- sion in the brain, and the remaining brains were separated into 2 hemispheres and solidified at −80 °C to be later homogenized and utilized for ELISA investigation for Aβ content and α-, β- and γ-secretase activity. STZ-induced SAD. SAD was induced in mice by intracerebroventricular (ICV) injection of STZ (3 mg/kg) dis- solved in saline . The detailed procedures can be reviewed in the Supplementary Information. Morris water maze. e M Th orris water maze (MWM) test was performed to assess spatial reference learning and memory as previously described . The detailed procedures can be reviewed in the Supplementary Information. Ymitest. e Y Th -maze included remarkable intra-maze cues (Ymi). In this prompted version, one vast wooded, painted object (unique in relation to those utilized as a part of the object recognition test and sufficiently heavy to keep the mice from moving it) was set toward the end of every arm. The detailed procedures can be reviewed in the Supplementary Information. ELISA measurement of amyloid β-42. e m Th ean of the Aβ-42 concentration present in mouse brains was calcu- lated by using a mouse Aβ-42 ELISA kit (Anaspec, Germany) . The detailed procedures can be reviewed in the Supplementary Information. Estimation of gamma-secretase in the brain homogenate. Quantitative determination of the mouse gamma secretase subunit APH-1a concentration in the brain homogenate was done using a mouse gamma-secretase subunit APH-1a ELISA kit (Catalog No: E0406m, EIAab, China).The detailed procedures can be reviewed in the Supplementary Information. Estimation of alpha-secretase in the brain homogenate. e t Th otal activity of α-secretase or tumor necrosis factor alpha-converting enzyme (TACE) present in mouse brain homogenates was determined using a commercially available Sensolyte520 TACE (α-secretase) activity kit (Anaspec, CA, USA), according to the provided instruc- tions. The detailed procedures can be reviewed in the Supplementary Information. Estimation of beta-secretase in the brain homogenate. In this assay, a beta-secretase-specific peptide was used and conjugated to two reporter molecules, EDANS and DABCYL. Then, the fluorescent emissions from EDANS were quenched by the physical proximity of the DABCYL moiety in the un-cleaved form. The detailed procedures can be reviewed in the Supplementary Information. Immunohistochemistry for COX-2 deposition in the brain. The immunohistochemistry procedures for COX-2 deposition in the brain were performed according to Ramos-Vara et al. . 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Invest. 20, 393–413 (2008). Acknowledgements This project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant no. (250/166/1434). The authors, therefore, acknowledge with thanks DSR technical and financial support. Author Contributions A.M.E., H.M.A. and R.S.E. all performed the isolation and identification of gingerol and shared in designing the experiments and editing the manuscript. N.S.E. also helped to design the experiments, performed the pharmacological study, carried out the data analysis and graphical presentation, and shared in editing the manuscript. Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-02961-0 Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Protective effects of gingerol on streptozotocin-induced sporadic Alzheimer’s disease: emphasis on inhibition of β-amyloid, COX-2, alpha-, beta - secretases and APH1a

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www.nature.com/scientificreports OPEN Protective effects of gingerol on streptozotocin-induced sporadic Alzheimer’s disease: emphasis on Received: 22 November 2016 inhibition of β-amyloid, COX-2, Accepted: 26 April 2017 Published: xx xx xxxx alpha-, beta - secretases and APH1a 1 2,3 1,4 1 Ali M. El Halawany , Nesrine S. EL Sayed , Hossam M. Abdallah & Riham Salah El Dine Gingerol is a major dietary compound that occurs in several plants belonging to the Zingiberaceae family. In the current study, the protective effect of gingerol on STZ-induced sporadic Alzheimer’s disease (SAD) was determined. Gingerol was isolated from the seeds of Aframomum melegueta K. Schum and tested at doses of 10 and 20 mg/kgbwt for its possible effect on the SAD model in mice, using celecoxib (30 mg/kg bwt) as a reference standard. The curative effects of gingerol were assessed through measurement of β-amyloid (Aβ-42), α-, β- secretases, APH1a and COX-2 levels. In addition, improvement in the cognitive deficit in mice after treatment was confirmed using the water maze and Y-maze with intra-maze cues. Gingerol improved the cognitive and behavioral impairment and AD-like pathology in streptozotocin model mice. These beneficial effects occurred with an increase in α-secretase activity and a decrease in cerebral Aβ-42, β- secretase, APH1a activity and COX-2-linked neuro-inflammation. Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by loss of cognitive function as well as pathological deposition of β-amyloid (Aβ) senile plaques and neurob fi rillary tangles . AD is classified into late-onset sporadic AD (SAD) and early onset familial AD (FAD). The majority of AD cases are SAD, which involves many etiopathogenic mechanisms including environmental, genetic and metabolic factors . e Th intracerebroventricular (ICV) injection of streptozotocin results in a well-established mouse model show - ing many aspects of SAD including neuroinflammation, brain insulin resistance, cholinergic deficits, accumula- tion of β-amyloid and tau proteins, and oxidative stress as well as memory and learning impairment . In AD, the amyloidogenic pathway results in the production of Aβ through the cleavage of amyloid precursor protein (APP) by β-secretase followed by the APH1a subunit of the γ-secretase complex. However, α-secretase produces the non-amyloidogenic soluble amyloid precursor protein α (sAPPα) . Accumulating evidence has demonstrated that COX inhibitors are involved in the management of AD through their action on the down- stream ee ff cts of the insulin signaling pathway inhibiting neuroinflammation and oxidative stress . Moreover, it was previously suggested that expression of cyclooxygenases (COXs) may ae ff ct β -amyloid peptide generation through mechanisms that involve the PGE2-mediated potentiation of γ-secretase activity, further supporting a role for COX-2 in the neuropathology of AD . Natural products are a well-known source of most of the discovered medicines. Most of the active compounds that have shown protective effects against SAD are from plants, with little contribution from marine and micro- bial origins . Most of the discovered plant-derived compounds have shown activity as acetyl cholinesterase inhib- itors (AChEIs), but they only address the symptoms of AD. However, certain plant extracts and compounds with antioxidant and anti-inflammatory effects have also been reported to alleviate AD . 1 2 Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt. Department of Pharmacology &Toxicology, Faculty of pharmacy, Cairo University, 11562, Cairo, Egypt. Department of Pharmacology &Toxicology, Faculty of pharmacy & Biotechnology, German University in Cairo, 11835, Cairo, Egypt. Department of Natural Products, Faculty of Pharmacy, King Abdulaziz University, Jeddah, 21589, Saudi Arabia. Correspondence and requests for materials should be addressed to H.M.A. (email: hmafifi2013@gmail.com ) Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 1 www.nature.com/scientificreports/ Figure 1. Effect of STZ, celecoxib and gingerol on the mean escape latency in the Morris water maze. Animals were divided into 5 groups. Group 1 received a single ICV dose of saline; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. Group 2 received a single injection of STZ (3 mg/kg, i.c.v.), and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. Group A was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. Group B received a single injection of STZ (3 mg/kg, i.c.v.), and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals from groups A, B, and C were subjected rd to the behavioral tests aer t ft he 3 week of injections. Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way ANOVA followed by the Tukey-Kramer multiple *** comparison test with the aid of GraphPad Prism Software version 5. Significantly different from the normal ++ +++ group P < 0.001. Significantly different from the STZ group P < 0.01. Significantly different from the STZ group P < 0.001. 6-Gingerol is a major phenolic constituent and hydroxyphenyl alkane of several plants belonging to the Zingiberaceae family such as ginger, cardamom and grain of paradise. The formerly mentioned plants are widely used in Middle Eastern and Asian cuisine as spices and every-day beverages. According to Arabian folklore, ginger has been claimed to improve memory and has been traditionally used as an ingredient for cognitive enhancement . 6-Gingerol is reported to display several biochemical and pharmacologic activity such as anticarcinogenic, 9 10 11, 12 antimutagenic, anti-apoptotic , antioxidant, anti-inflammatory , and cardio- and hepatoprotective effects . It is also known to inhibit nitric oxide synthase and cyclo-oxygenase and the expression of tumor necrosis factor alpha (TNF-α) . The antioxidant, anti-apoptotic and anti-inflammatory potentials of 6-gingerol suggest its pos- sible effect for protection against some SAD manifestations. e o Th bjective of this study is to investigate the effect of gingerol on some molecular biomarkers of SAD includ- ing anestimation of the level of β-amyloid, α- and β-secretases and the APH1a subunit of γ-secretase as well as the cognitive skills, suggesting that it could be a promising component of a clinical nutrition regime or used as 4–6 an adjuvant therapy . Results 1 13 Purified gingerol was identified using H and C NMR analysis and comparison of the data with that of the 15, 16 reported literature (Supplementary data, Figs 1–3) . All experiments were designed to test the possible effect of gingerol at two dosages (10 and 20 mg/kg bwt) on the improvement of cognitive function as well as Aβ forma- tion in the brain in STZ-induced cognitive impairment and amyloid genesis in a mouse model. As no significant difference was found during the pilot study between the two gingerol (10 mg/kg and 20 mg/kg) groups and the saline-treated group (Supplementary data, Tables 1–7), only the saline control group was represented in all of the following graphs. The possible anti-inflammatory effect of gingerol was tested in comparison with the effect of the anti-inflammatory celecoxib as a reference standard. Morris water maze. Cognitive impairment was induced by ICV injection of STZ as manifested in the water maze test and modified Y maze test. The Morris water maze is used to investigate the spatial reference learning and memory of mice. During the training phase, all mice learned the platform location, as revealed by a decrease in the latency and the distance travelled to locate the submerged platform. However, the training was delayed for the ICV-STZ mice as they needed more time and travelled a longer distance to find the platform than the control mice, which clearly indicated a short-term memory impairment in the STZ-treated mice. The treated groups (groups A, B and C) showed a significant improvement starting from the first day as they exhibited a significantly lower escape latency than those in the STZ group (Fig. 1). In the probe test, The STZ-treated group showed a significant decrease in the mean time spent in the target quadrant by approximately 70% compared to that of the normal group. Moreover, the treated groups A, B and C showed a significant increase in the mean time spent in the target quadrant by approximately 60% compared to that of the STZ-treated group (Fig. 2). es Th e results suggest an impairment to encode and remember the spatial coordinates of the platform within the environment. Retrieval of spatial memory was more specifically analyzed with the probe trials performed 24 hours after the last training trial. The ICV-STZ mice crossed the former platform site much less often than Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 2 www.nature.com/scientificreports/ Figure 2. Effect of STZ, celecoxib, and gingerol (10 and 20 mg/kg) on the Morris water maze probe test. Animals were divided into 5 groups. Group 1 received a single dose of saline ICV; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. Group 2 received a single injection of STZ (3 mg/kg, i.c.v.), and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. Group A was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. Group B animals were injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals from groups A, B, and C were subjected rd to the behavioral tests aer t ft he 3 week of injections. Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way ANOVA followed by a Tukey-Kramer multiple comparison test with the aid of GraphPad Prism Software version 5. ***Significantly different from the normal +++ +++ group P < 0.001. Significantly different from the STZ group P < 0.001. Significantly different from the STZ group P < 0.001. Figure 3. Effect of STZ, celecoxib, and gingerol (10 and 20 mg/kg) on the novel arm count in the intra-maze cue version of the Y-maze. Animals were divided into 5 groups. Group 1 received a single ICV dose of saline; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. Group 2 received a single injection of STZ (3 mg/kg, i.c.v.), and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. Group A was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. Group B animals were injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals from groups rd A, B, and C were subjected to the behavioral tests aer t ft he 3 week of injections. Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way ANOVA followed by a Tukey-Kramer multiple comparison test with the aid of GraphPad Prism Software version 5. ***Significantly +++ different from the normal group P < 0.001. Significantly different from the STZ group P < 0.001. Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 3 www.nature.com/scientificreports/ Figure 4. Effect of STZ, celecoxib, and gingerol (10 and 20 mg/kg) on the mean mouse amyloid beta 42 concentration using an ELISA assay. Animals were divided into 5 groups. Group 1 received a single ICV dose of saline; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. Group 2 received a single injection of STZ (3 mg/kg, i.c.v.), and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. Group A was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. Group B animals were injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals rd from groups A, B, and C were subjected to tests aer t ft he 3 week of injections. Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way ANOVA followed by Tukey- Kramer multiple comparison test with the aid of GraphPad Prism Software version 5. ***Significantly different +++ from the normal group P < 0.001. Significantly different from the STZ group P < 0.001. the control mice and did not show marked preference toward the target quadrant. es Th e results indicate that the ICV-STZ mice have a decreased ability to encode the environment as a spatial map and to localize the platform, suggesting an impairment in spatial reference memory. Intra-maze cue version of the Y-maze. The effect of STZ (3 mg/kg, ICV) was investigated on the novel arm count in the intra-maze cue version of the Y-maze and demonstrated significant impairment in novelty-seeking behavior and spatial memory compared with that of the control mice. Treatment of mice with the positive control (celecoxib, 30 mg/kg, i.p.) and gingerol (10 mg/kg and 20 mg/kg, i.p.) for 7 days resulted in significant improvement in both the novelty-seeking behavior as well as spatial memory (Fig.  3). STZ-induced Aβ-42 generation in mouse brains. To further investigate the anti-amyloid genesis ee ff cts of gingerol, the inhibitory effect on the levels of Aβ-42 as well as α-, β- and γ-secretases was examined. e m Th ean levels of Aβ-42 in mouse brains were evaluated by a mouse Aβ-42 ELISA kit. The STZ-treated mice showed a significantly higher concentration of amyloid beta than the normal group. However, treatment with celecoxib or gingerol (10 mg/kg and 20 mg/kg) resulted in a significant reduction in the concentration of Aβ -42 when compared with that in the STZ-treated mice (Fig. 4). STZ decreased α-secretase activity but increased β-secretase activity and the APH1a con- centration. Tumor necrosis factor alpha (TNF-α)-converting enzyme (TACE), also called ADAM17 or α-secretase, belongs to the ADAM (A Disintegrin and Metalloprotease) family of proteins involved in neurogen- esis through the ectodomain shedding of cell surface proteins . TACE, the protease responsible for the generation of mature TNF-α, plays an important role in acute and chronic inflammation. The inhibition of TACE by a phar - macological agent may represent an alternative approach to modulate the effect of TNF-α . TACE is also respon- sible for the proteolytic cleavage of amyloid precursor proteins, L-selectin and transforming growth factor-α . Moreover, APH1a encodes a component of the γ-secretase complex that cleaves integral membrane proteins such as β-amyloid precursor protein (APP). Accordingly, the APH1a concentration was used in the present study as a way to represent the level of γ-secretase in the brains of mice. Following a single ICV injection of STZ, the β-secretase activity and APH1a concentration increased, whereas the activity of α-secretase was reduced in mouse brains. Intraperitoneal injection of celecoxib orgingerol (10 mg/kg and 20 mg/kg) resulted in a significant reduction in the level of β and APH1a and a significant increase in the activity of α-secretase (Figs  5, 6 and 7). Immunohistochemistry: Effect of various drug treatments on the expression of COX-2 in the brain. ICV injection of STZ showed a marked increase in the expression of COX-2 in the mouse brains Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 4 www.nature.com/scientificreports/ Figure 5. Effect of STZ, celecoxib, andgingerol (10 and 20 mg/kg) on the mean mouse APH1a concentration using an ELISA assay. Animals were divided into 5 groups. Group 1 received a single ICV dose of saline; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. Group 2 received a single injection of STZ (3 mg/kg, i.c.v.). and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. Group A was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. Group B animals were injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals from groups rd A, B, and C were subjected to tests aer t ft he 3 week of injections. Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way ANOVA followed by Tukey-Kramer multiple comparison test with the aid of GraphPad Prism Software version 5. ***Significantly different from the +++ normal group P < 0.001. Significantly different from the STZ group P < 0.001. compared to that in normal animals. However, COX-2 expression was markedly decreased in the celecoxib and gingerol (10 mg/kg and 20 mg/kg) -treated animal groups compared to that in the STZ-treated animals (Fig. 8). Discussion AD is an age-linked neurodegenerative disease characterized by severe dementia. Symptoms of AD occur over a long period of time, and therefore, treatments should target this silent period of the disease to retard the disease progression towards dementia . Centrally administered STZ is known to cause SAD-like memory deterioration, 21, 22 and STZ-treated mice are therefore one of the non-transgenic mice models used to represent AD . ICV-STZ-treated rats showed an impaired learning and memory performance. Cognitive impairment after ICV-STZ administration is induced by the degradation of phospholipids and the resulting increases in the free fatty acid, arachidonic acid. COX is a rate-limiting enzyme in the metabolism of arachidonic acid to prostanoids, particularly prostaglandins (PGs), which significantly contribute to neuro-inflammation with oxidative stress. PGs such as PGE1 and PGE2 have also been reported to cause neurotoxicity and stimulate the release of gluta- mate from astrocytes. In a recent study, the use of COX inhibitors, such as indomethacin, has been shown to cause some improvement in the symptoms of AD, and the mechanism has been hypothesized to involve a reduction in inflammation and oxidative stress. In addition, celecoxib (a selective COX-2 inhibitor) aids in slowing the progression of the disease through its anti-inflammatory property resulting in reduced neuronal cell death . Accordingly, it was used as a positive control in this study. In this study, the neuroprotective effects of gingerol on the molecular biomarkers of SAD and cognitive skills were investigated. Its protective effect was assessed through measuring the levels of Aβ , α-and β-secretases, APH1a (a component of γ-secretase) and COX-2. In addition, the improvement in the cognitive skills ofmice aer t ft reatment was confirmed using the Y and water maze. In the present study, fourteen days following a single ICV injection of STZ, the level of Aβ-42 as well as the activity of β-secretase and the APH1a concentration in the whole brain increased, whereas that of α-secretase decreased. Treatment with celecoxib and gingerol resulted in a significant reduction in the levels of Aβ -42, APH1a and β-secretase activity as well as a significant increase in the activity of α-secretase. Aβ precursor protein (APP) is generally known to be a limiting reagent in the cell, and it has been pos- tulated that α- and β-secretases compete for APP proteolysis . Reduced β-secretase activity could therefore be responsible for the alleviation of the cerebral Aβ pathology in gingerol-treated mice. In this regard, it was previously demonstrated that another polyphenol, epigallocatechin gallate (EGCG), was also able to promote non-amyloidogenic APP metabolism in the Tg2576 cerebral amyloidosis mouse model. Interestingly, the mech- anism for the beneficial effects of EGCG on non-amyloidogenic APP processing relied on promoting the activity of the candidate α-secretase, a disintegrin and metalloprotease 10 . Recently, nutraceuticals, including the green 26 27 28 29 tea polyphenol and citrus bioflavonoid EGCG , luteolin , grape-derived polyphenols , and caein ff e have been found to exhibit anti-amyloidogenic properties. Moreover, a plant-derived polyphenol, tannic acid (TA), has been Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 5 www.nature.com/scientificreports/ Figure 6. Effect of STZ, celecoxib, and gingerol (10 and 20 mg/kg) on the mean mouse α–secretase activity using an ELISA assay. Animals were divided into 5 groups. Group 1 received a single ICV dose of saline; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. Group 2 received a single injection of STZ (3 mg/kg, i.c.v.), and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. Group A was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. Group B animals were injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals from groups rd A, B, and C were subjected to tests aer t ft he 3 week of injections. Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way ANOVA followed by Tukey-Kramer multiple comparison test by the aid of Graph Pad Prism Software version 5. ***Significantly different from +++ normal group P < 0.001. Significantly different from STZ group P < 0.001. shown to oppose the behavioral impairment and AD-like pathology in transgenic mice. These beneficial effects occur with a reduction in cerebral Aβ pathology, cleaved β-CTF, sAPP-β, BACE1 (beta-site amyloid precursor protein cleaving enzyme 1) protein expression and activity, and neuro-inflammation . Neuro-inflammation plays a crucial role in the pathogenesis of SAD. In the current study, the expression levels of COX-2 were observed to be significantly up-regulated in STZ-induced AD mice and down-regulated strongly by gingerol compared with that in control mice. Similarly, it was previously demonstrated that the STZ-induced learning and memory impairments in mice are due to stimulation of the neuro-inflammatory response . e p Th rotective effect of gingerol was further confirmed through immunohistochemistry, in which we studied the effects of these compounds on expression of COX-2 in the brain. The protective effect of gingerol on Aβ -42, β-secretase and APH1a levels was accompanied by a marked decrease in the expression of COX-2 in the brain tissue. Recently, the effects of shogaol, a structurally related compound to gingerol and isolated from Zingiber officinale , on microglia activation induced by lipopolysaccharide (LPS) were studied both in primary cortical neuron-glia cultures and in an in vivo neuro-inflammatory model. Shogaol exerted an anti-inflammatory effect by inhibiting the production of PGE2 and by down-regulating COX-2 expression better than gingerol . In addition, a recent study done by Ilic and his colleagues on the ethanolic extract of grains of paradise (A. melegueta), the source of gingerol in the current study, showed that the major phenolics from the extract,6-paradol, 6-gingerol, and 6-shogaol, reduced inflammation, which is in part due to the inhibition of COX-2 enzyme activity and the expression of pro-inflammatory genes . In addition, the improvement in the cognitive skills of mice aer t ft reatment was established using the Y and water maze. In the present study, the MWM was used to observe the effects of STZ and gingerol on cognitive dysfunction in a mouse model. Our results indicated that STZ can significantly increase the escape latency and th decrease the percentage of time spent in the 4 quadrant, which is in agreement with previous studies that con- cluded that ICV-STZ-treated mice had deficits in spatial learning and memory indicated by impaired acquisition and retention in the MWM and passive avoidance tasks . Moreover, during the probe trial done on last day with the platform removed, mice failed to remember the precise location of the platform, spending significantly less time in the target quadrant than the normal groups; this result was proven earlier . Similarly, STZ resulted in severe significant impairment in novelty-seeking behavior and spatial memory in the modified Y-maze test. This finding coincides with a recent study that proved that the deficits in Y-maze performance following chronic stress reflect neurochemical and/or neurobiological changes underlying spatial memory ability . In addition, the relationship between higher expression of inflammatory cytokines and STZ-caused cognitive impairment has been widely acknowledged. Previous studies have shown that cognitive function will be improved if the excessive Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 6 www.nature.com/scientificreports/ Figure 7. Effects of STZ, celecoxib, and gingerol (10 and 20 mg/kg) on the mean mouse β-secretase cleavage activity using an ELISA assay. Animals were divided into 5 groups. Group 1 received a single ICV dose of saline; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. Group 2 received a single injection of STZ (3 mg/kg, i.c.v.), and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. Group A was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. Group B animals were injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C was injected with STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals from groups rd A, B, and C were subjected to tests aer t ft he 3 week of injections. Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way ANOVA followed by a Tukey-Kramer multiple comparison test with the aid of GraphPad Prism Software version 5. ***Significantly different from the +++ normal group P < 0.001. Significantly different from the STZ group P < 0.001. activation of the inflammatory response has been corrected . This indicates that the STZ model is beneficial as a mouse model of cognitive impairment. In this light, recent focus has been given to a group of phenolics such as flavonoids, which have been found to be potentially anti-amyloidogenic . The beneficial behavioral neuroprotec- tive effects of the studied phenolics and gingerol coincide with a recent finding that pretreatment with hesperidin improved the cognitive impairment induced by ICV injection of STZ in the MWM test. This was concluded to be mediated by inhibiting the overexpression of inflammatory markers such as COX-2 , and their efficacies were found to be at the level of celecoxib, used as a positive control in this study. Finally, it could be concluded from the present work that gingerol prevented memory impairments in a SAD mouse model, which could be attributable to the amelioration of STZ- induced neuroinflammation and amyloi- dogenesis. This study gives a perspective on the wide variety of interactions between the inflammatory COX-2 and APP secretases and the beneficial effect of gingerol. On the one hand, overexpression of COX-2by STZ is able to increase the activity of β-secretase and γ-secretase and to decrease that of α-secretase. On the other hand, gingerol and celecoxib are able to modulate the activity of the secretases by regulating their levels in the case of BACE1 and APH1a or by directly shifting the cleavage site of γ-secretase. This study is the first, to the best of our knowledge, to report the protective effects of gingerol against STZ-induced biochemical changes and behavioral impairments, rendering gingerol a promising compound to treat or prevent AD. Methods General experimental procedures. Nuclear magnetic resonance (NMR) spectra were recorded on a 1 13 Bruker Unity INOVA 850 instrument at 850 MHz for H- and 213 MHz for C-NMR (Bruker BioSpin, Billerica, MA, USA) using CDCl as the solvent. ESIMS was recorded on an Agilent 1200 system connected to an Agilent 6320 Ion Trap HPLC-ESI-MS (Agilent Technology, Waldbronn, Germany). TLC was carried out on a pre-coated silica gel 60 F (0.25 mm, Merck) and RP-18 F S (0.25 mm, Merck Co., Darmstadt, Germany). Column chro- 254 254 matography (CC) was carried out on a silica gel 60 for column chromatography (Fluka, 70-230 mesh, Germany). The plant material and the isolation and purification methods of gingerol can be seen in the Supplementary Information. Biological Study. Experimental Design. Adult male Swiss albino mice weighing between 20–25 g were uti- lized. Mice were obtained from the animal state of the National Institute of Research (NRC) (Giza, Egypt). Mice were housed in a temperature-controlled room (23–24 °C) and presented with 12-hour dim/light cycles. Free access to food and water was permitted. All procedures were done following the guide for the treatment and care Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 7 www.nature.com/scientificreports/ Figure 8. Effects of STZ, celecoxib, and gingerol (10 and 20 mg/kg) on the immunohistochemical analysis of COX-2 expression in the brain. (A) The normal group received a single ICV dose of saline; then, aer 2 w ft eeks, animals were intraperitoneally injected with saline for seven days. (B) The control group received a single injection of STZ (3 mg/kg, i.c.v.), and then, aer 2 w ft eeks, they were intraperitoneally injected with saline for 7 days. (C) The celecoxib group received STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with celecoxib (30 mg/kg, i.p.) for 7 days. (D) The gingerol (10 mg/kg) group received STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (10 mg/kg/day, i.p.) for 7 days. (E) The gingerol (20 mg/kg) group received STZ (3 mg/kg, i.c.v.) once, and aer 2 w ft eeks, they were injected with gingerol (20 mg/kg/day, i.p.) for 7 days. Scale bar, 60 µm (A–E ). of laboratory animals published by US National Institute of Health and approved by the Ethics Committee at the German University in Cairo. All actions were made to minimize mouse suffering. A pilot study was performed whereby two doses of gingerol (10 and 20 mg/kg) were employed in addition to the saline control group. e b Th ehavioral and biochemical tests were performed on the three control groups. e Th findings of this study revealed that there were no statistically significant differences noted in the results among the three control groups. Therefore, the normal saline control group was the only group used in the experimental design and the description of the results. Mice were then randomly isolated into five groups, and every group comprised ten mice. These groups were defined as follows: Group 1: Mice received a solitary ICV infusion of saline; then, following 2 weeks, they received intraperitoneal injections with the vehicle (saline) for seven days. Group 2: Mice were infused with STZ (3 mg/kg, i.c.v.) once, and then received intraperitoneal injections of saline aer 2 w ft eeks for seven days. Group A: Mice were infused with STZ (3 mg/kg, i.c.v.) once, and following 2 weeks, they were treated with celecoxib (30 mg/kg, i.p.) for 7 days. Group B: Mice were injected with STZ (3 mg/kg, i.c.v.) once and following 2 weeks, they were treated with gingerol (10 mg/kg/day, i.p.) for 7 days. Group C: Animals were injected with STZ (3 mg/kg, i.c.v.) once, and following 2 weeks, they were treated with gingerol (20 mg/kg/day, i.p.) for 7 days. Animals from groups A, B, and C were subjected to the behavioral and biochemical tests aer t ft he third week of infusions. All mice were subjected to the Morris water maze and Ymi behavioral tests toward the end of the already spec- ified medications. Aer t ft hat, they were euthanized, and their brains were harvested. Two whole brains from each Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 8 www.nature.com/scientificreports/ group were placed in 4% formaldehyde to be utilized for immunohistochemical examination of COX-2 expres- sion in the brain, and the remaining brains were separated into 2 hemispheres and solidified at −80 °C to be later homogenized and utilized for ELISA investigation for Aβ content and α-, β- and γ-secretase activity. STZ-induced SAD. SAD was induced in mice by intracerebroventricular (ICV) injection of STZ (3 mg/kg) dis- solved in saline . The detailed procedures can be reviewed in the Supplementary Information. Morris water maze. e M Th orris water maze (MWM) test was performed to assess spatial reference learning and memory as previously described . The detailed procedures can be reviewed in the Supplementary Information. Ymitest. e Y Th -maze included remarkable intra-maze cues (Ymi). In this prompted version, one vast wooded, painted object (unique in relation to those utilized as a part of the object recognition test and sufficiently heavy to keep the mice from moving it) was set toward the end of every arm. The detailed procedures can be reviewed in the Supplementary Information. ELISA measurement of amyloid β-42. e m Th ean of the Aβ-42 concentration present in mouse brains was calcu- lated by using a mouse Aβ-42 ELISA kit (Anaspec, Germany) . The detailed procedures can be reviewed in the Supplementary Information. Estimation of gamma-secretase in the brain homogenate. Quantitative determination of the mouse gamma secretase subunit APH-1a concentration in the brain homogenate was done using a mouse gamma-secretase subunit APH-1a ELISA kit (Catalog No: E0406m, EIAab, China).The detailed procedures can be reviewed in the Supplementary Information. Estimation of alpha-secretase in the brain homogenate. e t Th otal activity of α-secretase or tumor necrosis factor alpha-converting enzyme (TACE) present in mouse brain homogenates was determined using a commercially available Sensolyte520 TACE (α-secretase) activity kit (Anaspec, CA, USA), according to the provided instruc- tions. The detailed procedures can be reviewed in the Supplementary Information. Estimation of beta-secretase in the brain homogenate. In this assay, a beta-secretase-specific peptide was used and conjugated to two reporter molecules, EDANS and DABCYL. Then, the fluorescent emissions from EDANS were quenched by the physical proximity of the DABCYL moiety in the un-cleaved form. The detailed procedures can be reviewed in the Supplementary Information. Immunohistochemistry for COX-2 deposition in the brain. The immunohistochemistry procedures for COX-2 deposition in the brain were performed according to Ramos-Vara et al. . 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Invest. 20, 393–413 (2008). Acknowledgements This project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant no. (250/166/1434). The authors, therefore, acknowledge with thanks DSR technical and financial support. Author Contributions A.M.E., H.M.A. and R.S.E. all performed the isolation and identification of gingerol and shared in designing the experiments and editing the manuscript. N.S.E. also helped to design the experiments, performed the pharmacological study, carried out the data analysis and graphical presentation, and shared in editing the manuscript. Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-02961-0 Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 10 www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2017 Scientific Repo R ts | 7: 2902 | DOI:10.1038/s41598-017-02961-0 11

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