Protective Effect of Acmella Ciliata Extract and Spilanthol in Streptozotocin-induced Sporadic Alzheimer's Disease Mouse Model: Possible Involvement of the of Decreased Oxidative Stress by Activating TRPV1 Receptors

This study investigated the effect of Acmella ciliata extract on memory impairment and oxidative stress dysfunction in the brain after intracerebral (i.c.v.) administration of 0.5 mg/kg streptozotocin (STZ) twice within 48 h in mice. Animals received orally treatment with ethanolic extract (50–150 mg/kg), hexane fraction (100 mg/kg) galantamine (40 mg/kg) and spilanthol (10 mg/kg), an alkamide isolated from the hexane fraction for 15 days. They were subjected to memory tests, inhibitory prevention (IAT) and the new object recognition tests (NOR). Experiments were performed with the animals' brains to evaluate the activity of catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH), glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione-S-transferase (GST) enzymes and to estimate malondialdehyde (MDA) concentration. We found that the treatments prevented STZ-induced memory loss, assessed by the IAT and NOR. Biochemical analysis revealed that STZ signicantly increased levels of MDA and depleted (GSH) in mice’s brain. The extract decreased oxidation, as evidenced by a signicant decrease in MDA and an increase in the antioxidant markers levels in mice treated with STZ. The positive effects of plant extract and fraction on STZ-induced memory decits appear to be related to spilanthol and its activity on TRPV1 receptors, since pretreatment with capsazapine promotes the reversal of these effects. The results together demonstrate the benecial effects of A. ciliata and spilanthol in preventing memory impairment, oxidative stress and dysfunction caused by STZ in mice. Therefore, there is a potential of this plant and its main compound spilanthol in the treatment of neurodegenerative disorders.


Introduction
Alzheimer's disease (AD) is a neurodegenerative disease related to aging, characterized by slow and progressive loss of neurons in different regions of the central nervous system (CNS) and consequent de cits in memory and cognition. The main cause forthis disease is still unknown, but it occurs in the brain of patients, with accumulation of beta-amyloid peptides (Aβ) and hyperphosphorylation of the tau protein, which are respectively responsible for the formation of the two histopathological ndings of AD, the senile plaques and the neuro brillary tangles (Kozlov et  For the research of pharmacological targets for AD many animal models are used (Drummond and Wisniewski 2017). The intracerebroventricular (i.c.v.) streptozotocin (STZ) is a well-established animal model that has been widely used for investigating this pathology ( Berté et al. 2018). STZ is a glucosamine-nitrosourea compound biosynthesized by Streptomycetes achromogenes, which is commonly used in the systemic induction of diabetes due to its ability to damage the pancreatic β cells and to induce insulin resistance. Furthermore, its administration also decreases cerebral glucoseuptake and produces effects related to AD, such as reduced cognition and increased cerebral aggregated Aβ fragments, tau protein, and Aβ deposits (Grieb 2016;Drummond and Wisniewski 2017). TRPV1, formerly known as vanilloid receptor 1 (VR1), is a nonselective cation channel with high Ca 2+ permeability, that is expressed in primary sensory neurons as well as in the brain. It is activated by heat (> 42°C) and phytochemicals such as the alkamide capsaicin (Crouzin et al. 2010;Nomura et al. 2013;Premkumar 2014). TRPV1 has clinical relevance to neurodegenerative disorders as its activation can decrease neuroin ammation, production of cytokines (Jayant et al. 2016), cellular injury, and oxidative stress, prevents hyperphosphorylation of AD-associated tau protein (Xu et al. 2017), and also participates in cognition, ischemic damage and neuroprotection (Gupta et al. 2014).
Acmella ciliata, one of the plants popularly known as jambu in Brazil, is an herb used in typical northern Brazil cuisine and folk medicine. Jambu is famous for its sensorial effects, such as anesthetic, numbing, tingling and cooling feeling, due to the well-known phytochemical alkamide spilanthol [(2E,6E,8E)-N- Considering the background, the purpose of the present work was to evaluate the protective effect of A. ciliata extract and spilanthol in an experimental model of AD induced by STZ in mice.

Plant extraction and spilanthol isolation
A. ciliata aerial parts were collected at the garden of the Associação dos Funcionários Fiscais do Estado de Santa Catarina (AFFESC, Florianópolis, Santa Catarina, Brazil) in October, 2012. The ethanol extract, hexane fraction and spilanthol were obtained as previously described (Silveira et al. 2016). HR-MS spectrum and chromatogram can be found as supplementary material.

Drugs, Reagents and doses
The following substances were used: STZ, capsazepine (TRPA1 channel antagonist) and galantamine (commonly used drug in the treatment of AD) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
The reagents (ethanol and hexane) used to obtain the spilanthol were commercial grade, purchased from VETEC (RJ, BR). Ketamine (Vetbrands, FL, USA), xylazine (Agener União, SP, BR), lidocaine with epinephrine 2% (Cristália, SP, BR). All drugs were dissolved in saline [except STZ which was dissolved in arti cial cerebrospinal uid prepared as described by Jayant et al. (2016) and were infused at room temperature]. The dose of galantamine used in this study as positive control in memory experiments was determined from a preliminary study based on the doses used by Berté et al (2018). The other doses used were determined based on pilot experiments. Due to the low amount of the compound, capsazepine was used only acutely administered 15 minutes before the administration of spilanthol in the inhibitory avoidance experiment.

Animals and Ethical statement
Male swiss albino mice (25 -30 g), approximately 90 days old, were used for the study. The animals were obtained from the Universidade do Vale do Itajaí (UNIVALI). They were kept at 22 ± 2 °C with free access to food and water, under a 12:12 h natural light (sunlight) and dark cycle, except during pharmacological assays. This study followed the guidelines established by the Research Ethics Committee of UNIVALI, and the Brazilian Law on Animal Experimentation and was approved by the Research Ethics Committee of UNIVALI (CEUA/UNIVALI), protocol 11/18.

Experimental groups
Mice were randomly divided into nine groups, each containing 8 -10 mice: Group 1 (n = 10) regards the sham-operated animals that did not receive STZ nor oral treatment; however, received the same amount of the vehicle used to solubilize STZ (arti cial liquor), 2 μl i.c.v. This group is important to show that i.c.v. does not cause hippocampal lesion interfering with behavioral and biochemical assays.
Group 2 (n = 9) is the negative control, injected with STZ (2 µL of 2.5 mg/ml; i.c.v.) and treated with a vehicle per os (p.o.) route in which extract and spilanthol were solubilized (DMSO 2% and distillated water).
Neurobehavioral tests were carried out within 24 h after the 15 th day of treatment (Fig. 1).
Experimental model of SAD induced by STZ SAD was induced by the freehand i.c.v. procedure previously reported (Pinton et al. 2010) with slightly modi cations. Brie y, mice were anesthetized with xylazine/ketamine, than they were submitted to a minor surgery to remove the cutaneous tissue aiming at the exposure of the skull. After cleaning and asepsis of the cranial region, the animals still under anesthesia received STZ (2 µL of 2.5 mg/mL solution; i.c.v.) through a hypodermic needle attached to a cannula, which was linked to a 5 μL Hamilton syringe. After 48 h, each mouse received a second injection of STZ (same way as the rst one). Drug treatment started after the second dose of STZ and lasted for more than 15 days, including the days of the behavioral tests. Treatment was done orally (p.o.) and experiments were carried out 1 hour after them.

Novel object recognition (NOR) test
The NOR test is used to evaluate long-term memory and also cognition. It is based on the principle that, in a familiar environment, laboratory animals show an instinctive a nity for novelty, or preference for exploring a new object rather than a familiar (Ennaceur 2010). To this end, animals from Group 1 to 6 were evaluated.
The test consisted of three different phases (habituation phase, training phase, and test phase) conducted on three successive days and followed the protocol described by Myskiw et al. (2008) with slightly modi cations. Brie y, in the rst phase, mice were exposed to an open eld apparatus consisted of a wooden box of 40 x 60 x 50 cm dimensions and were left for 15 min in the absence of stimulus objects to adapt to the apparatus. On the next day animals were placed on the apparatus for 10 min in the presence of two identical in shape, color, and size objects put in opposite corners of the wooden box for familiarization. On day three, mice were tested by placing them again in the open eld apparatus but now in the presence of a familiar and a novel object (different in shape, color and size from the other) and were left to explore the objects for 10 min. The time taken by each mouse to explore the two objects during the acquisition and retention phases of the test were recorded manually and separately with two stop watches (by a trained observer). Its Discrimination Index (DI) wasthen calculated for the retention trial as DI=B-A1/B+A1 (B=novelobject, A1 = familiar object).

Effect of treatments on the locomotor activity of animals (Exploratory behavior) -Open Field Test (OFT)
Locomotor activity was assessed using the same open eld apparatus used in the NOR test. In this test, during animals' habituation phase, the exploratory behavior of them was evaluated during the rst six minutes. Mice were placed individually in the center of the open eld and crossing movements were recorded during this time.

Inhibitory avoidance test (IAT)
This experiment is used to measure long-term memory. In the present study, all nine group of animals were submitted to an IAT as previously performed (Izquierdo and Dias 1983) with some modi cations. Mice were trained and tested in an apparatus consisted of a 50 cm long, 25 cm wide, 25 cm high plywood box with a glass frontal wall and oor made of steel bars stainless steel parallel with caliper of 0.1 cm and to 1 cm of distance of each other, with a 2.5 cm high, 7.0 cm wide, 2.5 long platform. A 15 W lamp lit up the apparatus, while the room remains dark. On the training trial, animals were gently placed and held on the platform facing the rear left corner. Their latency to step down (placing the four paws on the grid) was timed, and immediately after a 0.4 mA foot shock was delivered for 2 s.The test session was conducted 24 h later. The procedure was identical to training session with omission of the foot shock.
Their latency to step down was also measured up to a limit of 180 s.

Biochemical analysis
Preparation of brain homogenate After completion of experiments animals were killed and, whole intact brain was carefully removed and rinsed with 0.9% NaCl solution for cleaning, and weighed. Braintissue was homogenized in a phosphate buffer (pH 7.6), centrifuged at 20,000×g, at 4 °C for 10 min. An aliquot of supernatant was collected and stored at -20 °C for furtherbiochemical tests. Protein concentrations were determined by the Bradford assay with Bovine serum albumin as standard (0.05-1.00 mg/mL).

Assessment of brain antioxidant markers
Catalase (CAT) activity Superoxide dismutase (SOD) activity In this experiment, Kakkar et al. (1984) method was utilized. Buffers were exploited for the assessment of SOD activity. Centrifugation of tissue homogenate was done at 1500×g for 10 min and then at 10,000×g for 15 min. Supernatant was collected and 150 μL of it was added to the aliquot containing 600 μL of 0.052 mM sodium pyrophosphate buffer (pH 7.0) and 186 mM of phenazine methosulphate (50 μL). 100 μL of 780 μM NADH was added to initiate enzymatic reaction. After 1 min, glacial acetic acid (500 μL) was added to stop the reaction. At 560 nm optical density was determined to enumerate the color intensity. Results were evaluated in units/mg protein.
Glutathione-S-transferase (GST) assay Scheme of Habig et al. (1974) protocol was strictly followed for the estimation of GST potency. 150 μL aliquot of tissue homogenate was added to 720 μL of sodium phosphate buffer together with 100 μL of reduced glutathione (1 mM) and 12.5 μL of 1-chloro-2,4-dinitrobenzene (CDNB) 1 mM. Changes in absorbance were recorded at 340 nm and enzymes activity was calculated as nmol CDNB conjugate formed/min/mg protein using a molar extinction coe cient of 9.6 × 103 M-1 cm-1.
Glutathione reductase (GR) assay GR activity in tissue samples was analyzed as described by Carlberg eand Mannervik (1975)  This assay was carried out following the previous protocol (Wills 1966) with minor modi cations. MDA is one of lipid peroxidation product that can be used as a marker of oxidative stress.The reaction mixture in a total volume of 1.0 mL contained 0.58 mL phosphate buffer (0.1 mol; pH 7.4), 0.2 mL homogenate sample, 0.2 mL ascorbic acid (100 mmoL), and 0.02 mL ferric chloride (100 mmol). The reaction mixture was incubated at 37 °C in a shaking water bath for 1 h. The reaction was stopped by addition of 1.0 mL 10% trichloroacetic acid. Following addition of 1.0 mL 0.67% thiobarbituric acid, all tubes were placed in boiling water bath for 20 min and then shifted to ice bath before centrifuging at 2500×g for 10 min. The amount of thiobarbituric acid reactive substances (TBARS) formed in each of the samples was assessed by measuring optical density of the supernatant at 535 nm using a spectrophotometer against a reagent blank. The results were expressed as nmol TBARS/min/mg tissue at 37 °C using a molar extinction coe cient of 1.56 × 105 M-1 cm-1.

Statistical analysis
The results were submitted to one-way analysis of variance (ANOVA), followed by multiplepost hoc comparison test using the software GraphPad InStat ® . The results are expressed as mean ± standard error values of the mean and p lower than 0.05 (p <0.05) were considered statistically signi cant.

Effect of treatments on the locomotor activity of animals
Statistical analysis of the data obtained in the OFT revealed no statistical difference between the groups treated in the behavioral parameters recorded in this experiment. Our data show that the administration of treatments does not change the number of animal passages, indicating that it probably does not change the mobility of these animals in the memory tests (Results not shown).

Effect of STZ and A. ciliata extract on exploring the old and new object discrimination index
The NOR test was performed 16 days after STZ-induced AD. As can be seen by the statistical analysis, the discrimination index of objects for Group 2 (negative control) is signi cantly lower than of mice treated with the positive control galantamine (Group 3) and 100 mg/kg of A. ciliate extract (Group 5) (p < 0.05) and greater than sham-operated animals (Group 1) and treated with 50 and 150 mg/kg of A. ciliata extract (groups 4 and 6, respectively) (p < 0.01) (Fig. 2). These results indicate that treatment with A. ciliata extract increased discrimination index compared with negative group.

Inhibitory avoidance test
This assay investigated if mice developed accurate memory after they received footshock in inhibitory avoidance training trial. Therefore, mice were trained on the one-trial inhibitory avoidance task. As shown in Fig. 3B and 4B, retention latencies of mice were signi cantly longer in the test session compared to training trial, except for animals from Group 2 (negative group). Bene cial effects of treatments on cognitive de cits induced by STZ are best seen in panels C of Fig. 3 and Fig. 4 where we represent only the inhibitory avoidance test sessions. Signi cantly treatments with the extract, hexane fraction, spilanthol and galantamine produced an increase in memory of the inhibitory avoidance when compared with the vehicle group. Thus, these ndings indicate that treatment of mice with A. ciliata extract (groups 4 to 6), hexane fraction (group 7) and spilanthol (group 8) increased accurate memory similar to treatment with galantamine. It was also demonstrated in this experiment that the pretreatment of the animals with capsazepine, an TRPV1 receptor antagonist blocked the nootropic effects of spilanthol when compared to the group that received the vehicle (Fig. 4 C).

Effect of A. ciliata extract on brain oxidative status of mice
The i.c.v. application of STZ signi cantly decreased the activity of antioxidant enzymes in the brain tissue of mice (control). On the other hand, in animals that received STZ, the treatment with ethanolic extract (100 mg/kg) signi cantly improved (p <0.0001; p <0.001, p <0.05) the activity of CAT, SOD, GSH, GR, GST, and GPx enzymes in brain tissue (Fig. 5). In addition, animals' treatment with plant's ethanolic extract was able to establish the activity of antioxidant enzymes to patterns similar to the SHAM animals which did not receive treatment with STZ and did not have cognitive de cits. Our results also demonstrate that STZ signi cantly increased oxidative stress markers, such as lipid peroxidation (TBARS content) (Fig. 6).

Discussion
Currently, AD represents one of the main health problems among other disorders of the CNS worldwide. Because it is a neurodegenerative pathological process in which the etiology is not fully established and the treatment is palliative, execution of adequate animal models is essential forunderstanding of its neurobiological bases and facilitates the approaches for the discovery of new therapeutic targets. . Firstly, the model was developed for rats, but it was also standardized for mice (Ravelli et al. 2017). Glucose, its metabolites and energy products metabolism (like ATP) are brain's main energy source and, therefore, normal glucose metabolism is fundamental for correct brain functions such as protein synthesis and cellular functions and molecular processes (Dienel 2019). It has been widely reported in the literature that the i.c.v. STZ model of memory de cit shows an impaired glucose metabolism. In addition, various pathological aspects of AD like impaired brain glucose and energy metabolism are closely mimicked in animals after sub diabetogenic i.c.v. injection of STZ (Mayer et al. 1990;Lannert and Hoyer 1998), which leads to progressive de cits in learning and memory in rats and mice (Grieb 2016 It has also been reported in this model thatthe decrease in the levels of choline acetyltransferase in the hippocampus, leading to a decrease in acetylcholine (Costa et al. 2016), and that the septo-hippocampus system is also damaged which is drastic, since such brain structures are essential in the process of memory consolidation (Thomas 2015).
In the present work, A. ciliata extract was studied and itreversed de cit of memory and oxidative stress in STZ (i.c.v.) induced model of dementia. In this AD animal model, as expected, STZ caused a persistent memory de cit, as evidenced by the non-alteration in the behavior of animals in the inhibitory avoidance comparing the training and test sessions, as well as by the decrease in the index of recognition in the object recognition test. This memory de cit was reversed by treating the animals with different doses of the plant extract.
Oxidative stress might be the underlying cause related to the pathophysiology of AD or associated behavioral changes. In the present study, administration i.c.v. of STZ produced oxidative stress as evidenced by the signi cant increase in the level of TBARs and decrease in the GSH level, associated with decreased enzymes such as CAT, SOD, GST, GR and GPX. This increase in oxidative stress may be due to an increase in the level of glucose in the brain after STZ infusion. In a beautiful experiment, Pathan and colleagues demonstrated that brain slices from rats that received an STZ infusion showed reduced glucose consumption in the incubation medium compared to control mice with a hyperglycemic condition in the brain (Pathan et al. 2006). In addition, it has also been reported that oxidative stress, due to a hyperglycemic condition (Taïlé et al. 2020) or direct effect of STZ, can cause endothelial dysfunction. So, in this model, impaired glucose metabolism and oxidative stress may be responsible for an endothelial dysfunction in animals' brains. Impaired endothelial function is accompanied by decreased cerebral perfusion that has recently been associated with dementia (Wolters et al. 2017). Our results clearly demonstrated that treatment of animals with the extract of A. ciliata decreased oxidative stress and, consequently, the cognitive de cit caused by the i.c.v infusion of STZ.
Phytochemically, from the hexane fraction of A.ciliata ethanol extract 10 alkamides were identi ed: spilanthol, (2E,7Z)- 6,9- N-isobutyl-7-trideca-10,12-diinamide. In our studies, both hexane fraction and isolated spilanthol were administered to animals with STZ-induced AD and evaluated in the inhibitory avoidance test. Our results showed that both treatments reduced animals' cognitive de cits by improving the memory of inhibitory avoidance.
As already reported, alkamides represent a class of natural compounds that are highly active in the CNS as they produce antinociceptive (Rios et al. 2006;Gertsch et al. 2008 The relationship between alkamides and TRPV1 receptors, and among them and AD made us think that the mechanism of action of the nootropic property found in the extract and hexane fraction of A. ciliata, as well as spilanthol could also be related to the activation of receptors TRPV1. So, we decided to check if the pretreatment of animals with the TRPV1 receptor antagonist, capsazapine, could reverse the effect of spilanthol in animals tested in the inhibitory avoidance. Our results showed that the nootropic effect of spilanthol may be related to the TRPV1 receptors since the bene cial effect of this alkamide on the animals' memory is not observed with the pretreatment of the animals with capsazepine. Our results corroborate other results in the literature (Jayant et al. 2016;Xu et al. 2017) and point out that positive pharmacological modulation of TRPV1 channels may be a potential research target for mitigating AD. Also indirectly, it can be veri ed that the reduction of oxidative stress in animals with STZ infusion and treated with the plant extract as observed in this study, can be related to the presence of the alkamides present in the plant which would be modulating the TRPV1 receptors, however more experiments would be necessary to ascertain such a hypothesis.
In STZ induced memory de cit, there is a decreased activity of glycolytic enzymes resulting in a reduction in acetylcholine level (Sorial and Sayed 2017) which is intricately associated with cognition (Záborszky et al. 2018). Acetylcholine is degraded by AChE whose inhibition by AChE inhibitors is the most effective pharmacological approach for the symptomatic treatment of AD (Sorial and Sayed 2017; Weller 2018; Záborszky et al. 2018;Zhang et al. 2019). In the current study, the effect of treatments on AChE activity was not evaluated but we do not rule out the possibility that spilanthol, the compound we consider responsible for the plant's nootropic effects, has AChE inhibiting activity since it is reported that alkamides have such a property (Tu et al. 2016).
In AD the most important diagnostic symptom is the loss of memory and throughout the course of the in addition to the biochemical changes produced in the brain of animals, induces important cognitive de cits such as the loss of different types of memory (Halawany et al. 2017;Ravelli et al. 2017;Berté et al. 2018). In the present study, to assess the effects of treatments on cognitive de cits induced by SZT, we used two memory tests that are well-known in the literature and widely used in the screening of substances with anti-AD potential (Crystal 2016): the inhibitory avoidance test and the object recognition test. The object recognition task is a behavioral test used to access declarative memory in rodents, which is based on animal's natural tendency to explore more the new object in detriment of the familiar, in a known context (Myskiw et al. 2008;Ennaceur 2010). In inhibitory avoidance, the type of memory evaluated is emotional memory. In this test, the animal learns to associate the context of the apparatus in which it nds itself, initially not aversive to receiving an electric shock in its paws (aversive) when it descends from a platform to explore the environment. Shocks occur in the training session. In the test session animals are replaced in the apparatus (on the platform) and the latency of descending the platform is timed. The difference between the latencies of descent between the training and test sessions are considered indices of memory (Izquierdo and Dias 1983). Our results showed in both memory tests

Conclusion
In conclusion, A. ciliata demonstrated a promising therapeutic effect in the treatment of STZ-induced Alzheimer's. The plant's effect appears to be mediated by one of its phytochemicals, spilanthol. Our results together also allow us to conclude that the therapeutic effects of the plant involve inhibition of oxidative stress, which is involved in the pathogenesis of Alzheimer's and also activate TRPV1 type receptors.

Declarations
Ethical approval