**4. Discussion**

In the present work, we investigated whether treatment with EC is beneficial in combating the toxic effects of Aβ25–35 in the Hp. The results show an improvement in the spatial memory task, as well as a reduction in markers of oxidative stress and inflammation. Additionally, a decrease in the immunoreactivity of HSP-60, HSP-70, and HSP-90 was detected, strongly suggesting a positive regulating of the processes of protein aggregation and neurodegeneration. Therefore, the treatment with EC reduces the hippocampal death events induced by the injection of Aβ25–35.

Catechins are a group of polyphenolic compounds belonging to the flavonoid class, present in high concentrations in a variety of plant-based fruits, vegetables, and beverages [25–27]. Particularly, EC shows protection against dementia, as well as diseases in which oxidative stress seems to be highly prevalent [13]. Oxidative stress is considered a key factor in the AD pathogenesis [28]. Aβ is responsible for promoting the generation of ROS, the oxidation of lipids to destabilize the neuronal membrane, and decreasing the activity of antioxidant enzymes such as SOD and catalase, which encourage the necrosis and apoptosis states [29]. Reports indicate that Aβ25–35 interacts with the cell membrane, altering the structure and function of the lipid bilayer and augmenting the concentration of ROS and

lipid peroxidation, as shown in our results. In addition, the injection of Aβ25–35 into the Hp of rats produces a decrease in activity of SOD and catalase, indicating that the Hp is a brain region susceptible to oxidative and neuronal damage [30].

The interactions that Aβ25–35 has with the cell membrane induces ion channel dysfunction, which is associated with dysregulation of neuronal Ca2+. The intracellular increase of Ca2+ in neurons provokes vulnerability to the apoptotic processes [31]. In other reports, neuronal death is mainly associated with the generation of peroxidation products induced by Aβ25–35, such as 4-hydroxy-nonenal (4-HNE), which alters the glutamate transporter (GLT1). This causes the excessive entry of Ca2+ to activate the nitric oxide neuronal synthase (nNOS) responsible for catalyzing the formation of nitric oxide (NO) in neurons. The interaction of NO with the superoxide ion (O<sup>2</sup>−) promotes the formation of peroxynitrite (ONOO−) [7]. This reactive species alters the structure and function of proteins of the cytoskeleton and the mitochondria by means of a nitration or nitrosylation reaction, structures considered as cell damage markers which have been reported on several occasions by our research team [6]. The inflammatory response is a complementary mechanism of Aβ25–35 toxicity. The inflammation is mediated by the production and excessive release of proinflammatory cytokines, such as IL-1β and TNFα. These cytokines are considered regulators of the intensity and duration of the inflammatory processes. TNF-induced cell death signaling is carried out by the TNF receptor (TNFR) [32]. Essentially, TNFR permits the recruitment of intracellular "death signaling inducing signaling complex" (DISC) proteins [33,34]. These proteins generate a sca ffolding, so that recruitment is favored and caspase-8 is activated [35]. The freed, active caspase-8 then enzymatically processes procaspase-3, converting this executioner procaspase into active enzyme [35]. The activation of caspase-3 is essential for TNF-induced cell death, as it targets a latent DNase that degrades genomic DNA, thus causing apoptotic cell death, as was observed in the Aβ25–35 group [6,36]. On the other hand, IL-1 is a pivotal mediator of the inflammation that is expressed rapidly in response to neuronal injury, predominantly by microglia, and elevated levels markedly exacerbate the injury [37,38]. The associated mechanism to IL-1β release is the increase of proinflammatory molecules that results in rapidly recruited neutrophils, which cause tissue damage through the generation of free radicals, proteolytic enzymes, and more proinflammatory cytokines such as IL-1β and TNFα [39]. The activation of both Nuclear Factor kappa B (NF-κB) and TNFα triggers the activation of Mitogen-Activated Protein Kinase (MAPK)-related stress-activated Jun kinase (JNK) that induces cell death [40,41]. Studies have shown that increases in both IL-1β and TNFα produce a ROS accumulation and cell death as we have observed in our results, where the intrahippocampal injection of Aβ25–35 showed a significant increase in the concentration of these cytokines compared to the control group [42]. Therefore, it is suggested that Aβ25–35 promotes an inflammatory response mediated by the production of proinflammatory cytokines, which could trigger the generation of ROS and the proliferation of glial cells, leading to reactive gliosis [43]. All these mechanisms of neurodegeneration cause damage to the structure and function of the limbic system, particularly in the Hp, a brain region that has a key role in the development of spatial memory, in such a way that a characteristic of the neurotoxicity of Aβ25–35 is the deterioration in learning and spatial memory, which is supported by a large number of publications [17,18,24,28,29].

On the other hand, unregulated ROS levels can activate oxidant stress sensor proteins, particularly the heat shock factor (HSF), a transcription factor responsible for increasing heat shock proteins (HSPs) because they react at the cellular level in response to environmental changes [44,45]. Under physiological conditions, HSPs act as molecular chaperones, preventing changes in the functional folding of proteins, while under stress conditions, they prevent protein aggregation [46]. Likewise, HSPs promote the regulation of specific signaling pathways that respond to inhibit apoptosis [47]. The neurotoxic changes caused by Aβ25–35 provoke conformational changes in HSF and induce the expression and synthesis of HSPs [48]. It is proposed that these proteins are activated as a neuroprotective mechanism. In this work, it is demonstrated that the increase in ROS and lipid peroxidation in response to the toxicity of Aβ25–35 promotes the increase in the immunoreactivity of HSP-60, HSP-70, and HSP-90 at 30 days postinjection with respect to the control group but does not prevent neuronal death.

Studies indicate that HSPs can be activated directly or indirectly. The ROS oxidize the thiol groups of proteins and cause the conformational change that activates the HSPs [49,50]. The in vitro and in vivo models indicate that each HSP performs specific functions in cells under conditions of oxidative stress. The HSP-90, being a folding chaperone, prevents the aggregation of the Aβ peptide (a necessary condition to cause neurotoxicity) and promotes its degradation. The increase in the expression of HSP-70 favors interaction with the intracellular Aβ, suggesting the sequestering of the peptide to avoid its interaction with other proteins and thus inhibiting its toxicity [9]. HSP-70 also activates microglia to synthesize and release cytokines and stimulate phagocytosis. There is evidence that HSP-70 promotes cell survival and reduces neuronal death [51]. It is also proposed that the overexpression of HSP-70 participates in inflammatory processes by reducing the levels of Cyclooxygenase (COX)-2 and the production of NO. In the same way, HSP-60, a mitochondrial chaperonin that is typically held responsible for the transportation and refolding of proteins from the cytoplasm into the mitochondrial matrix, also prevents the release of cytochrome-C in the mitochondria, the formation of apoptosome, and the activation of caspases, and therefore inhibits neuronal death. Reports indicate that Aβ impairs mitochondrial activity, however, HSP-60 prevents this damage and thus reduces the formation of ROS and promotes the native and functional conformation of SOD [52]. Our results indicate that all these chaperones act as damage biomarkers or neurosensors, suggesting their neuroprotective activity when increasing their immunoreactivity. These are active in di fferent cellular compartments and interfere with oxidative, inflammatory processes and in the apoptotic signaling cascade that induces the toxicity of Aβ25–35 in the rat Hp.

The administration of EC increased spatial memory, decreased ROS concentrations and peroxidation levels, increased the enzymatic activity of SOD and catalase, and decreased IL-1β and TNFα levels. It is proposed that EC captures oxidizing species and prevents lipid peroxidation, which results in blocking the neurotoxicity caused by Aβ25–35, therefore, the treatment of EC shows an improvement in spatial memory. It is also suggested that the catechol group in EC confers the ability to donate a hydrogen atom and therefore stabilizes the activity of free radicals, favoring antioxidant enzymes to help maintain redox balance [53]. Catechin treatments have demonstrated that they inactivate the Signal Transducer and Activator of Transcription 3 (STAT3) pathway, which plays a critical role in inflammation promotion, as well as being able to bind the p65 subunit of the transcription factor NF-κB and to inhibit cytokine and chemokines transcription stimulated by IL-1β and TNFα. EC has also been shown to inhibit the aggregation of amyloidogenic proteins, including Aβ, present in AD [54]. Interestingly, in the presence of EC, it has been observed that the Aβ monomers adopt a new conformational form with an increased inter-center-of-mass distance with reduced β-sheet content [55,56]. This thereby a ffects its inclination to form fibril-prone states that otherwise increase the severity of Alzheimer's disease [56]. At a peripheral level, EC at concentrations of 0.1–1 mM was found to inhibit nitrite formation and the IL-1β-induced expression of iNOS by blocking the nuclear localization of the p65 subunit of NF-κB [57]. Therefore, it is reasonable to hypothesize that at the central level and, particularly, in the CA1 hippocampal region, EC can use similar pathways.

In addition, EC possesses the ability to directly or indirectly scavenge ROS by chemically reacting with ROS or by modulating pathways that regulate ROS scavenging compounds and enzymes, respectively [18]. EC can directly reduce ROS levels because the inhibitory strength of this compound is in the number of hydroxyl groups present [17]. In support of this idea, a [51] recent study confirmed that the o-catechol moiety of EC is essential for the direct detoxifying e ffects of the reaction with superoxide and hydrogen peroxide, or by blocking ROS generation [57–59]. EC also modulates the signaling of nuclear factor erythroid 2p45-related factor-2 (Nrf2), an important factor in cellular detoxification, proliferation, survival, and di fferentiation [60] that increases the synthesis and activity of antioxidant enzymes, such as SOD y catalase, correlating with our results. Likewise, chronic degenerative diseases have shown a ffectation on the oxidative phosphorylation machinery. Cytochrome c (Cyt-c) and cytochrome c oxidase (COX) catalyze the terminal and proposed rate-limiting step in the mitochondrial electron transport chain [60], thus their correct function is decisive in the dysregulations in mitochondrial

respiration and cell signaling [51]. Since the oxidative phosphorylation machinery is suppressed in most of the degenerative diseases, it can be speculated that reactivation of mitochondrial function might be a strategy that interferes with the progress of the degenerative process. In this sense, EC stimulates mitochondrial respiration [51], significantly stimulating the expression of oxidative phosphorylation protein complexes. Also, EC can inhibit the decrease in mitochondrial succinate dehydrogenase activity, cytochrome c release, mitochondrial fragmentation, and cytochrome c oxidase protein levels [61], and thus inhibit the activation of ERK, caspase-3, and JNK, and the release of cytochrome c [51], diminishing cell death, as was observed. In the same way, oxidative and inflammation changes caused by the Aβ peptide can induce the unfolded, mitochondrial protein stress response. However, the expression of the nuclear-encoded HSP-60 mitochondrial chaperones prevents mitochondrial damage and reduces cell death [61]. It is suggested that EC modulates the cellular signaling cascade through the activation of di fferent pathways, such as serine/threonine kinase 1 (AKT), PKC (protein kinase-C), and MAP kinases. All these pathways participate in the processes that mediate the activation of transcription factors and antiapoptotic signaling, promoting the regulation of the heat shock factor and consequently the downregulation of the HSPs. As far as we know, there are no reports that indicate the potential role of EC on the activity of HSP under conditions of cell stress.
