Neuroprotective Effects of Ecklonia cava in a Chronic Neuroinflammatory Disease Model

Inflammation is a natural defense mechanism against noxious stimuli, but chronic inflammation can lead to various chronic diseases. Neuroinflammation in the central nervous system plays an important role in the development and progression of neurodegenerative diseases. Polyphenol-rich natural products, such as Ecklonia cava (E. cava), are known to have anti-inflammatory and antioxidant properties and can provide treatment strategies for neurodegenerative diseases by controlling neuroinflammation. We investigated the effects of an E. cava extract on neuroinflammation and neurodegeneration under chronic inflammatory conditions. Mice were pretreated with E. cava extract for 19 days and then exposed to E. cava with lipopolysaccharide (LPS) for 1 week. We monitored pro-inflammatory cytokines levels in the serum, inflammation-related markers, and neurodegenerative markers using Western blotting and qRT-PCR in the mouse cerebrum and hippocampus. E. cava reduced pro-inflammatory cytokine levels in the blood and brain of mice with LPS-induced chronic inflammation. We also measured the activity of genes related to neuroinflammation and neurodegeneration. Surprisingly, E. cava decreased the activity of markers associated with inflammation (NF-kB and STAT3) and a neurodegenerative disease marker (glial fibrillary acidic protein, beta-amyloid) in the cerebrum and hippocampus of mice. We suggest that E. cava extract has the potential as a protective agent against neuroinflammation and neurodegenerative diseases.


Introduction
Inflammation is a biological defense mechanism against harmful stimuli from injuries or infections [1]. Inflammation can be triggered by various factors, including infections, environmental factors, lifestyle choices, and underlying genetic predispositions [2]. The function of inflammation is to prevent the initial cause of cell damage, remove destroyed tissue and necrotic cells from the wound, and repair the tissue [3]. Inflammation can be acute or chronic. Acute inflammation causes immediate injury lasting from a few days to a few weeks [4]. Cytokines and chemokines promote the migration of neutrophils and macrophages to areas of inflammation. Persistent acute inflammation can lead to chronic inflammation [5].
Chronic inflammation can last months or years [1,5]. Chronic inflammation is caused by the persistence of a harmful trigger and a continuous inflammatory response [1]. Chronic inflammation releases pro-inflammatory cytokines and other signaling molecules that disrupt normal physiological functions of the immune system [1]. The constant occurrence of these inflammatory processes can lead to the activation of immune cells such as macrophages and T cells, which can increase the inflammatory response and lead to tissue damage and dysfunction [6]. Based on this biological damage, chronic inflammation is

Total RNA Extraction and Real-Time Quantitative PCR
Using TRIzol reagents (15596-026, Life technologies, Carlsbad, CA, USA), total RNA was extracted in the cerebrum and hippocampus. cDNA was generated according to the protocol of the reverse transcription kit (SG-cDNAS100, Smartgene, Daejeon, Republic of Korea). Quantitative PCR (Stratagene Mx3000P, Agilent Technologies, Santa Clara, CA, USA) was performed using SYBR green SG-SYBR-500, Smartgene, Raleigh, NC, USA). The transcripts levels were calculated based on the cycle threshold and monitored for the amplification curve. RPLP0 was used for internal control. The primer sequences are listed in Table 2.

Immunohistochemistry
For immunohistochemistry staining, paraffin-embedded tissues were cut by 4µm and attached to silane coated slide. With serial hydration step in xylene, EtOH, and distilled water, and then progressed antigen retrieval step. The tissue slides were blocked with 3%  After 3 times washing with TBS-T, the Alexa-Fluor secondary  antibodies (#A21471, #A21207, Thermo Fisher Scientific Inc., Waltham, MA, USA) were incubated at room temperature. After 3 times washing, the slides were mounted in ProLong Gold antifade reagent (P36935, Thermo Fisher Scientific Inc., Waltham, MA, USA). The slides were examined using a DMi8 microscope (Leica Microsystems, Wetzlar, Germany).

Statistical Analysis
Data are reported as an average ± SEM. The differences between means were obtained through one-way ANOVA, and then a Tukey post-analysis was performed using Graph Pad Software (GraphPad Inc., San Diego, CA, USA).

E. cava Reduces Pro-Inflammatory Cytokines Induced by Lipopolysaccharide
ICR mice were exposed to E. cava for 25 days and then exposed to lipopolysaccharide (LPS) for 7 days. The mouse brain tissue was sampled and divided into the cerebrum and hippocampus for analysis ( Figure 1A). The body weights of the mice were monitored before euthanasia ( Figure 1B). Compared with the control group, the LPS group (88%), LPS + E. cava 10 mg/kg group (87%), LPS + E. cava 50 mg/kg group (85%), and LPS + E. cava 100 mg/kg group (87%) showed decreased weight (p < 0.05, Figure 1B). However, no differences were observed between the groups treated with LPS. When we measured pro-inflammatory cytokines, we found that E. cava regulated chronic inflammation induced by LPS. The levels of serum interleukin-1 beta (IL-1β) increased significantly (p < 0.001, 2.75-fold) in the LPS group compared to the control group ( Figure 1C). The LPS + E. cava 10 mg/kg group did not differ from the LPS group. However, the LPS + E. cava 50 mg/kg and LPS + E. cava 100 mg/kg groups showed a significant decrease (p < 0.001, 93%, and 80%, respectively) compared to the LPS group ( Figure 1C). Furthermore, we measured IL-1β and interleukin-6 (IL-6) mRNA levels in the cerebrum and hippocampus. In the cerebrum, IL-1β mRNA levels increased significantly (p < 0.001, 13.5-fold) in the LPS group compared to the control group, and the LPS + E. cava 10 mg/kg group (41%), the LPS + E. cava 50 mg/kg group (8%) and the LPS + E. cava 100 mg/kg group (14%) showed a significant decrease (p < 0.001) compared to the LPS group ( Figure 1D). IL-6 mRNA levels increased significantly (p < 0.001, 14.5-fold) in the LPS group compared to the control group, and the LPS + E. cava 10 mg/kg (50%), LPS + E. cava 50 mg/kg (39%), and LPS + E. cava 100 mg/kg (26%) groups showed a dose-dependent decrease (p < 0.001) compared to the LPS group ( Figure 1D). In the hippocampus, IL-1β mRNA levels increased significantly (p < 0.001, 14.9-fold) in the LPS group compared to the control group and the LPS + E. cava 10 mg/kg (76%), the LPS + E. cava 50 mg/kg (14%) and the LPS + E. cava 100 mg/kg (36%) groups showed a significant decrease (p < 0.001) compared to the LPS group ( Figure 1E). At the same time, the differences in IL-6 mRNA levels were not significant in all groups.

E. cava Decreases LPS-Induced NF-κB and STAT3 Activation in Mouse Cerebrum
Based on previous results, pro-inflammatory cytokines have been shown to be induced by LPS. Furthermore, E. cava decreases the secretion of pro-inflammatory cytokines. Accordingly, we measured the activation of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) and signal transducer and activator of transcription 3 (STAT3) genes, which regulate the immune response and inflammation. In the cerebrum, the phospho-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) protein level increased significantly (p < 0.001, 2.89-fold) in the LPS group compared to the control group. The LPS + E. cava 50 mg/kg group (65%) and the LPS + E. cava 100 mg/kg group (48%) showed a significant decrease (p < 0.001) in the phospho-IκBα protein level compared to the LPS group ( Figure 2). IκBα protein levels did not differ significantly among all groups. However, the pIκBα/IκBα ratio increased significantly (0.001, 3.11-fold) in the LPS group compared to the control group. The LPS + E. cava  50 mg/kg group (55%) and the LPS + E. cava 100 mg/kg group (43%) showed a significant decrease compared to the LPS group ( Figure 2). Next, the phospho-NF-κB protein level increased significantly (p < 0.001, 3.28-fold) in the LPS group compared to the control group. The LPS + E. cava 50 mg/kg group (78%) and the LPS + E. cava 100 mg/kg group (48%) showed a significant decrease (p < 0.001) in the phospho-NF-κB protein level compared to the LPS group ( Figure 2). NF-κB protein levels did not differ significantly among all groups. However, the pNF-κB/NF-κB ratio increased significantly (p < 0.001, 3.73-fold) in the LPS group compared to the control group. The LPS + E. cava 50 mg/kg group (p < 0.05, 74%) and the LPS + E. cava 100 mg/kg group (p < 0.001, 36%) showed a significant decrease compared to the LPS group ( Figure 2). The phospho-STAT3 protein level increased significantly (p < 0.05, 1.45-fold) in the LPS group compared to the control group. The LPS + E. cava 10 mg/kg (p < 0.05, 82%), LPS + E. cava 50 mg/kg (p < 0.001, 65%), and LPS + E. cava 100 mg/kg (p < 0.001, 55%) groups showed a significant decrease in phospho-STAT3 protein levels compared to the LPS group ( Figure 2). The STAT3 protein levels did not differ significantly between the control and LPS groups. However, the LPS + E. cava 10 mg/kg (1.42-fold), LPS + E. cava 50 mg/kg (1.6-fold), and LPS + E. cava 100 mg/kg (1.68-fold) groups showed a dose-dependent increase (p < 0.001) compared to the LPS group ( Figure 2). Additionally, pSTAT3/STAT3 levels increased significantly (p < 0.001, 1.31-fold) in the LPS group compared to those in the control group. The LPS + E. cava 10 mg/kg (58%), LPS + E. cava 50 mg/kg (41%), and LPS + E. cava 100 mg/kg (34%) groups showed a dose-dependent decrease (p < 0.001) compared to the LPS group ( Figure 2).

E. cava Decreases LPS-Induced NF-κB and STAT3 Activation in Mouse Cerebrum
Based on previous results, pro-inflammatory cytokines have been shown to be induced by LPS. Furthermore, E. cava decreases the secretion of pro-inflammatory cytokines. Accordingly, we measured the activation of the nuclear factor kappa-lightchain-enhancer of activated B-cells (NF-κB) and signal transducer and activator of transcription 3 (STAT3) genes, which regulate the immune response and inflammation. In the cerebrum, the phospho-nuclear factor of kappa light polypeptide gene enhancer in Bcells inhibitor, alpha (IκBα) protein level increased significantly (p < 0.001, 2.89-fold) in the LPS group compared to the control group. The LPS + E. cava 50 mg/kg group (65%) and the LPS + E. cava 100 mg/kg group (48%) showed a significant decrease (p < 0.001) in the phospho-IκBα protein level compared to the LPS group ( Figure 2). IκBα protein levels did The mice (n = 25) were divided into five groups (n = 5 control group, n = 5 Lipopolysaccharide (LPS) group, n = 5 LPS + E. cava 10 mg/kg group, n = 5 LPS + E. cava 50 mg/kg group, n = 5 LPS + E. cava 100 mg/kg group). For 25 days, they were injected orally, and the control group was treated with the same capacity of distilled water, which was used for the E. cava extract treatment. On day 19, LPS was treated at 750 ug/kg/body weight for a week. The mice were sacrificed before 8 h LPS treatment at 1.5 mg/kg/bodyweight, and their organs were isolated. (B) Change of mouse body weight (C) Serum blood IL-1β level measured after sacrifice. (D) QRT-PCR measured IL-1β and IL-16 genes mRNA levels in the cerebrum of each group of male mice. (E) QRT-PCR measured IL-1β and IL-16 genes mRNA levels in the hippocampus. RPLP0 was used for internal control. The points on the graph are represented by different shapes, with a white circle indicating the control group, a red square indicating the LPS group, a triangle representing the LPS + E. cava 10 mg/kg group, an inverted triangle indicating the LPS + E. cava 50 mg/kg group, and a rhombus representing the LPS + E. cava 100 mg/kg group. The differences between means were obtained through one-way ANOVA, and then a Tukey post-analysis. The values represent means ± S.D. # p < 0.001 vs. control group and *** p < 0.01 vs. LPS group.

E. cava Reduces Apoptosis Caused by Neuroinflammation
We observed that factors related to inflammatory control increased in response to LPS and that these factors are known to be involved in apoptosis [33]. In addition, apoptosis mediated by caspase-3 is linked to brain damage [34]. We expected that apoptosis would decrease because E. cava extract reduces inflammation.
In the cerebrum, the cleaved caspase-3 protein level increased significantly (p < 0.001, 2.52-fold) in the LPS group compared to the control group. The LPS + E. cava 50 mg/kg (58%) and LPS + E. cava 100 mg/kg (51%) groups showed a significant decrease (p < 0.001) in cleaved caspase-3 protein level compared with the LPS group ( Figure 4A). The caspase-3 protein levels did not differ significantly between the groups. However, the cleaved caspase-3 to caspase-3 ratio was increased significantly (p < 0.001, 2.82-fold) in the LPS group compared to those in the control group. Additionally, The LPS + E. cava 50 mg/kg (56%) and LPS + E. cava 100 mg/kg (46%) groups showed a significant decrease (p < 0.001) compared to the LPS group ( Figure 4A). significant decrease in cleaved caspase-3 protein level compared with the LPS group ( Figure 4B). The caspase-3 protein levels did not differ significantly between the groups. However, the cleaved caspase-3 to caspase-3 ratio was increased significantly (p < 0.001, 3.47-fold) in the LPS group compared to the control group. Additionally, The LPS + E. cava 50 mg/kg (90%) and LPS + E. cava 100 mg/kg (90%) groups showed a significant decrease (p < 0.05) compared to the LPS group (Figure 3).  In the hippocampus, the cleaved caspase-3 protein level increased significantly (p < 0.001, 3.39-fold) in the LPS group compared to the control group. The LPS + E. cava 50 mg/kg (p < 0.01, 86%) and LPS + E. cava 100 mg/kg (p < 0.001, 81%) groups showed a significant decrease in cleaved caspase-3 protein level compared with the LPS group ( Figure 4B). The caspase-3 protein levels did not differ significantly between the groups. However, the cleaved caspase-3 to caspase-3 ratio was increased significantly (p < 0.001, 3.47-fold) in the LPS group compared to the control group. Additionally, The LPS + E. cava 50 mg/kg (90%) and LPS + E. cava 100 mg/kg (90%) groups showed a significant decrease (p < 0.05) compared to the LPS group (Figure 3).

E. cava Reduces Glial Fibrillary Acidic Protein (GFAP) Expression in Mouse Brain
Astrocytes are known to regulate the immune response in the damaged CNS, and brain damage, such as injury, inflammation, and disease, increases the expression of GFAP proteins in astrocytes [35]. Therefore, we measured the expression of GFAP, a known astrocyte marker. When astrocytes in the cerebrum were stained with the GFAP antibody, positive signals were higher (p < 0.001, 1.70-fold) in the LPS group than in the control group. Additionally, a concentration-dependent decrease was observed in the LPS + E. cava 10 mg/kg (p < 0.001, 78%), LPS + E. cava 50 mg/kg (p < 0.001, 63%), and LPS + E. cava 100 mg/kg (p < 0.001, 55%) groups compared to that in the LPS group ( Figure 5A). Furthermore, GFAP protein levels increased significantly (p < 0.001, 1.78-fold) in the LPS group compared to the control group, the LPS + E. cava 10 mg/kg (p < 0.05, 86%), LPS + E. cava 50 mg/kg (p < 0.05, 87%) and LPS + E. cava 100 mg/kg (p < 0.001, 64%) groups showed a significant decrease in GFAP protein levels compared to the LPS group ( Figure 5B). fold) in the LPS group than in the control group. The LPS + E. cava 50 mg/kg (72%) and LPS + E. cava 100 mg/kg (65%) groups showed a significant decrease (p < 0.001) in GFAP quantitative level compared with the LPS group ( Figure 5C). GFAP protein levels increased significantly (p < 0.001, 1.65-fold) in the LPS group compared to the control group, the LPS + E. cava 50 mg/kg (p < 0.001 48%) and LPS + E. cava 100 mg/kg (p < 0.001, 50%) groups showed a significant decrease in GFAP protein levels compared to the LPS group ( Figure 5D). Alpha-Tubulin was used for internal control. (C) Representative immunohistochemistry (IHC) of hippocampus astrocytes in mouse brain indicates GFAP (red) and dapi (blue). Quantification of GFAP staining was analyzed by Image J as setting white holes for positive standards. (D) Western blot analysis and quantification of GFAP were evaluated in the hippocampus of each group of male mice. Alpha-Tubulin was used for internal control. The points on the graph are represented by different shapes, with a white circle indicating the control group, a red square indicating the LPS group, a triangle representing the LPS + E. cava 10 mg/kg group, an inverted triangle indicating the LPS + E. cava 50 mg/kg group, and a rhombus representing the LPS + E. cava 100 mg/kg group. The differences between means were obtained through one-way ANOVA, and then a Tukey post-analysis. The values represent means ± S.D. # p < 0.001 vs. control group and * p < 0.05, *** p < 0.01 vs. LPS group.
In the hippocampus, GFAP antibody, positive signals were higher (p < 0.001, 1.50-fold) in the LPS group than in the control group. The LPS + E. cava 50 mg/kg (72%) and LPS + E. cava 100 mg/kg (65%) groups showed a significant decrease (p < 0.001) in GFAP quantitative level compared with the LPS group ( Figure 5C). GFAP protein levels increased significantly (p < 0.001, 1.65-fold) in the LPS group compared to the control group, the LPS + E. cava 50 mg/kg (p < 0.001 48%) and LPS + E. cava 100 mg/kg (p < 0.001, 50%) groups showed a significant decrease in GFAP protein levels compared to the LPS group ( Figure 5D).

E. cava Weakens Alzheimer's Markers by Chronic Neuroinflammation
Based on the previous results, we observed that chronic inflammatory responses lead to NI. NI is known to induce neurodegeneration [18,19]. Therefore, we measured markers related to Alzheimer's disease. In the cerebrum, APP and tau protein levels did not differ significantly among the groups. While beta-amyloid protein levels increased significantly (p < 0.001, 3.07-fold) in the LPS group compared to the control group, the LPS + E. cava 50 mg/kg (p < 0.001, 53%) and LPS + E. cava 100 mg/kg (p < 0.001, 55%) groups showed a significant decrease in beta-amyloid protein levels compared to the LPS group ( Figure 6A). In the hippocampus, tau protein levels did not differ significantly between groups. However, APP protein levels increased significantly (p < 0.001, 1.48-fold) in the LPS group compared to the control group, whereas the LPS + E. cava 50 mg/kg (p < 0.001, 54%) and LPS + E. cava 100 mg/kg (p < 0.001, 48%) groups showed a significant decrease in APP protein levels compared to the LPS group ( Figure 6B). Additionally, beta-amyloid protein levels increased significantly (p < 0.001, 2.16-fold) in the LPS group compared to the control group, while the LPS + E. cava 50 mg/kg group (p < 0.01, 80%) and the LPS + E. cava 100 mg/kg (p < 0.001, 68%) group showed a significant decrease in beta-Amyloid protein levels compared to the LPS group ( Figure 6B).

E. cava Weakens Alzheimer's Markers by Chronic Neuroinflammation
Based on the previous results, we observed that chronic inflammatory responses lead to NI. NI is known to induce neurodegeneration [18,19]. Therefore, we measured markers related to Alzheimer's disease. In the cerebrum, APP and tau protein levels did not differ significantly among the groups. While beta-amyloid protein levels increased significantly (p < 0.001, 3.07-fold) in the LPS group compared to the control group, the LPS + E. cava 50 mg/kg (p < 0.001, 53%) and LPS + E. cava 100 mg/kg (p < 0.001, 55%) groups showed a significant decrease in beta-amyloid protein levels compared to the LPS group ( Figure  6A). In the hippocampus, tau protein levels did not differ significantly between groups. However, APP protein levels increased significantly (p < 0.001, 1.48-fold) in the LPS group compared to the control group, whereas the LPS + E. cava 50 mg/kg (p < 0.001, 54%) and LPS + E. cava 100 mg/kg (p < 0.001, 48%) groups showed a significant decrease in APP protein levels compared to the LPS group ( Figure 6B). Additionally, beta-amyloid protein levels increased significantly (p < 0.001, 2.16-fold) in the LPS group compared to the control group, while the LPS + E. cava 50 mg/kg group (p < 0.01, 80%) and the LPS + E. cava 100 mg/kg (p < 0.001, 68%) group showed a significant decrease in beta-Amyloid protein levels compared to the LPS group ( Figure 6B).

Discussion
Chronic inflammation is increasing worldwide, largely due to aging, lifestyle, and environmental factors [2]. Chronic inflammation can lead to chronic diseases, such as diabetes, obesity, cardiovascular diseases, and neurodegenerative diseases [2]. NDs are known to be mostly caused by inflammation. NI is considered chronic rather than acute [16,36]. NI is associated with ND [18,20,37] and reducing inflammation is expected to weaken the progression of ND. Strikingly, several studies have reported that inhibition of inflammatory cytokines reduces nerve loss in ND [38][39][40].
NI drugs include nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), and corticosteroids. However, they have various side effects, such as gastrointestinal disorders, kidney damage, and osteoporosis [41][42][43]. Therefore, safe and effective treatment options must be identified. Recently, numerous studies have investigated the protective effects of natural products and bioactive compounds against neurological diseases [21,22,44]. Flavonoids, phlorotannins, curcumin, resveratrol, and genistein are among the compounds that have shown potential for treating and preventing NI [23][24][25][26][27]. Inflammation and oxidative stress play a role in neurodegenerative diseases. In vitro and in vivo studies have demonstrated that antioxidant plant polyphenols, such as resveratrol and curcumin, can reduce the deposition of amyloid-beta in Alzheimer's disease, inhibit ROS production and cell death through their antioxidant effects, and demonstrate neuroprotective effects through anti-inflammatory and antioxidant mechanisms in Parkinson's disease. It is expected that the neuroprotective actions of polyphenols in neurodegenerative diseases involve the downregulation of pro-inflammatory transcription factors, such as NF-kB, through their anti-inflammatory effects in the brain [45,46]. Phlorotannin is a polyphenol that is specifically present in brown algae and in the natural product E. cava [28]. Some studies have suggested that E. cava has antioxidant, anti-inflammatory, immunomodulatory, and neuroprotective effects [29][30][31]. Based on this evidence, we observed that E. cava reduced NI and neurodegeneration in mice with LPS-induced chronic inflammation.
Chronic inflammation is known to result in the secretion of cytokines that regulate the immune response and hematopoietic action by acting on cells involved in host defense and damage healing [1,5,47]. We measured IL-1β, the brain's main inflammatory cytokine [11,48], in the serum. The LPS group showed an increase in IL-1β levels compared to the control group, and the groups that were administered E. cava showed a decrease in IL-1β levels in both the LPS + E. cava 50 mg/mL group and the LPS + E. cava 100 mg/mL group compared to the LPS group. In addition, we measured mRNA levels of IL-1β and IL-6 in the cerebrum and hippocampus. In the cerebrum, the LPS group showed increased levels of both IL-1β and IL-6 mRNA compared to the control group, while all groups administered LPS + E. cava showed lower levels than the LPS group. In the hippocampus, the LPS group showed increased levels of IL-1β mRNA compared to the control group, and LPS + E. cava groups administered E. cava showed lower levels than the LPS group. Overall, our results suggest that chronic inflammation induces NI through LPS and that E. cava may regulate cytokines through its anti-inflammatory properties.
Furthermore, many studies have shown that interleukin (IL) 1 beta and IL-6 signaling is activated by NF-kB and STAT3 when inflammation occurs through LPS, and NF-kB and STAT3 are also known to interact with cytokines and are involved in regulating the immune response and inflammation [49][50][51]. Therefore, we measured the activation of NF-κB and STAT3 to investigate the potential mechanisms underlying the anti-inflammatory effects of E. cava. In the cerebrum, LPS increased levels of pIκBα, pNF-κB, and pSTAT3 proteins compared to the control group. In groups administered LPS + E. cava, pIκBα and pNF-κB levels were decreased compared to the LPS group at concentrations of 50 mg/mL and 100 mg/mL, and the level of pSTAT3 protein decreased in a concentration-dependent manner compared to the LPS group. Furthermore, in the hippocampus, the LPS group had increased levels of pIκBα, pNF-κB, and pSTAT3 proteins compared to the control group. In groups administered LPS + E. cava, pNF-κB levels were decreased compared to the LPS group at concentrations of 50 mg/mL and 100 mg/mL, and the levels of pIκBα and pSTAT3 proteins decreased in a concentration-dependent manner compared to the LPS group. Our results suggest that E. cava treatment downregulates the phosphorylation of IκBα, NF-κB, and STAT3 in NI brains, demonstrating anti-inflammatory effects.
Our results have shown that cytokines and inflammatory response-related proteins are increased in NI states and that E. cava decreases levels of cytokines and inflammatory regulatory proteins. In addition, chronic inflammatory animal models are known to display persistent inflammatory reactions [52]. Continuous inflammatory reactions promote apoptosis [1], and activation of NF-κB [53][54][55][56] and STAT3 [57][58][59][60] is known to induce apoptosis in various organs as well as the brain. Therefore, we measured the capase-3 protein, an indicator of apoptosis, in a chronic NI state. Interestingly, the group treated with LPS showed a higher ratio of cleaved caspase-3 to capase-3 in the cerebrum and hippocampus than the control group. Moreover, the LPS + E. cava 50 and 100 mg/mL groups showed decreased protein levels of cleaved caspase-3 compared to the LPS group. We suggest that E. cava might exert anti-apoptotic effects that protect nerve cells from death. Neuronal cell death is one of the main pathological features of ND. Therefore, we expect E. cava to help with NI as well as ND.
Since E. cava has shown a protective effect against NI and apoptosis, and given that NI and ND are interrelated processes, we measured glial fibrillary acidic protein (GFAP), a biomarker used to diagnose Alzheimer's disease [61]. Quantitative levels of GFAP in the cerebrum were higher in the LPS group than in the control group. Interestingly, GFAP IHC staining decreased in a concentration-dependent manner in the LPS + E. cava group. Additionally, we measured the levels of Alzheimer's disease-related markers. Interestingly, in the LPS group, amyloid-beta protein levels increased in the cerebrum, whereas both amyloid-beta and APP protein levels increased in the hippocampus, compared to those in the control group. In the LPS group, amyloid-beta levels increased in the cerebrum, while both amyloid-beta and APP protein levels increased in the hippocampus compared to the control group, and compared to the LPS group, it was reduced in LPS + E. cava 50 mg/mL and 100 mg/mL groups. These results suggested that E. cava reduces the risk of developing ND caused by chronic NI. Thus, E.cava was considered to improve NI and ND. In summary, we have shown that E. cava dampen the LPS-mediated NF-kB and STAT3 activation, leading to the signaling related to inflammation and immune responses in the brain. As a natural product, E. cava contains phlorotannins which are polyphenols of marine algae. Phlorotannin-rich E. cava has the potential for functional foods with antioxidant and anti-inflammatory activities [62]. One of the major polyphenolic compounds in E. cava, Dieckol, may also play a key role in allergic inflammatory reactions [63]. By reducing NF-kB and STAT3 activation, we suggest that E. cava could protect against apoptosis and neurodegeneration. Based on this rationale, this study provides evidence that E. cava reduce neuroinflammation and play a substance in neurodegenerative disease therapeutics.

Conclusions
To summarize our results, we induced chronic inflammation using LPS to activate cytokine-and inflammation-related genes, leading to NI. We showed that E. cava has anti-inflammatory and anti-apoptotic effects under NI conditions. Moreover, E. cava may exert protective effects against ND. These findings suggest that E. cava can be used to treat chronic NI and ND.