The Methanol Extract of Allium cepa L. Protects Inflammatory Markers in LPS-Induced BV-2 Microglial Cells and Upregulates the Antiapoptotic Gene and Antioxidant Enzymes in N27-A Cells

Neuroinflammation, apoptosis, and oxidative stress are connected to the pathogenesis of neurodegenerative diseases (NDDs). Targeting these three factors, the intervention of neuroprotective agents may have great potential in the treatment of NDDs. In the current study, the anti-inflammatory effects of the methanol extract of Allium cepa (MEAC) in lipopolysaccharide (LPS)-induced BV-2 microglial cells were investigated. MEAC has been studied in regard to the regulation of the antiapoptotic gene (Bcl-2) and various antioxidant enzyme (HO-1, NQO-1, and catalase) expressions in N27-A cells. Additionally, the protective action of MEAC has also been studied against MPP+-induced death in N27-A cells. The results suggest that MEAC is significantly protected from NO release and increase iNOS expression at the mRNA and protein levels in LPS-stimulated BV-2 microglial cells. MEAC treatment also protects COX-2 expression at the mRNA and protein levels. Furthermore, MEAC treatment prevents LPS-stimulated increases of proinflammatory cytokines, including TNF-α, IL-6, and IL-1β. In N27-A cells, MEAC treatment significantly upregulates antiapoptotic gene (Bcl-2) and antioxidant enzyme (HO-1, NQO1, and catalase) expressions. Moreover, MEAC treatment protects against MPP+-induced death in N27-A cells. To conclude, A cepa extract takes protective action against LPS and MPP+, and upregulates the antioxidant enzymes that could potentially be used in the therapy of NDDs.


Introduction
Progressive dysfunction and toxicities within neurons characterize the debilitating multifactorial disorders known as neurodegenerative diseases (NDDs). These diseases lead to a slow but irreversible weakening of all brain function. Around 30 million individuals worldwide are affected by NDDs. Although some symptomatic treatments are available, specific treatments have not yet been discovered [1]. Neurons and microglial cells are considered to be key parts of the central nervous system (CNS), and microglial cells occupy 5-10% of brain cells [2]. In a healthy brain, microglia play a vital role in neuroprotection, and phagocytes remove cell debris and damaged neurons [3,4]. On the other hand, the neuroinflammatory and neurotoxic responses are accelerated from activated microglia and astrocytes by releasing numerous inflammatory markers, includingcyclooxygenase-2 (COX-2),

Cell Viability and Nitrite Assay
The BV-2 microglial cells were seeded at a density of 2.5 × 10 5 cells/mL and were co-treated with various concentrations of MEAC (50 µg/mL, 250 µg/mL, and 500 µg/mL) and LPS (200 ng/mL) for 24 h. The viability of the cells was measured according to the method of Park et al. [19]. The nitrite assay procedure was followed according to the method of Cho et al. [2]. The BV-2 microglial cells were seeded at a density of 2.5 × 10 5 cells/mL and then treated with various concentrations of MEAC (50 µg/mL, 250 µg/mL, and 500 µg/mL) and LPS (200 ng/mL) for 24 h for the nitrite assay. To check the protective action of MEAC against MPP + -induction, the N27-A cells were pretreated with several concentrations of MEAC and, 2 h later, MPP + was treated and harvested for 24 h and cell viability was calculated by following the protocol of Park et al. [19].

Western Blot Analysis
The cells were placed at 4 • C upon the 2-time washing with PBS and lysed for 10 min in a lysis buffer (1× RIPA lysis buffer, protease-inhibitor cocktail, and phosphatase-inhibitor cocktail). The lysates were centrifuged at 14,000 rpm at 4 • C, and the collected supernatants were collected for further investigation. Equal amounts of protein (20 µg or 40 µg) were separated electrophoretically using a 10% sodium dodecyl sulfate-polyacrylamide electrophoresis, and the resolved proteins were transferred to polyvinylidene-difluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated for 1 h with 3% bovine serum albumin in Tris-buffered saline; a 0.1% Tween 20 buffer was used to prevent nonspecific binding.

Statistical Analysis
A Graph Pad Prism ver. 5.01 (Graph Pad, Inc., La Jolla, CA, USA) was employed to analyze experimental data. All of the data is expressed as the mean ± standard error of at least three independent experiments that were performed in triplicate. A one-way variance analysis and Tukey's multiplecomparison test were employed for the statistical analysis; p < 0.05 is considered statistically significant.

Action of MEAC on LPS-Induced iNOS Expressions in BV-2 Microglial Cells at mRNA and Protein Levels
As the MEAC treatment reduced the NO concentration, iNOS-mRNA and -protein expressions were evaluated further. The stimulation (200 ng/mL) of LPS noticeably upregulated the iNOS-mRNA level to 4 . 23 ± 0. 18 folds after 6 h in comparison with the control group ( Figure 2A). In treated to microglial cells for 24 h. LPS induction markedly elevated NO production (18.8 ± 0.55 µM) compared to the control (2.52 ± 0.12 µM). As per results, the treatment with MEAC notably prevented the NO production in a concentration-dependent manner to 14.12 ± 0.21 µM, 10.71 ± 0.10 µM, and 6.03 ± 0.04 µM, respectively, in the LPS-stimulated BV-2 cells ( Figure 1A).

Action of MEAC on LPS-Induced iNOS Expressions in BV-2 Microglial Cells at mRNA and Protein Levels
As the MEAC treatment reduced the NO concentration, iNOS-mRNA and -protein expressions were evaluated further. The stimulation (200 ng/mL) of LPS noticeably upregulated the iNOS-mRNA level to 4.23 ± 0.18 folds after 6 h in comparison with the control group (Figure 2A). In a concentration-dependent manner, MEAC pretreatment prevented the iNOS-mRNA in the LPS-induced BV-2 microglial cells. The western blot analysis evaluated the iNOS expression at the protein level. At 18 h after the LPS stimulation, the iNOS-protein level was noticeably elevated to 4.95 ± 0.056 folds compared to the control group. Meanwhile, the MEAC treatment significantly prevented iNOS expression at the protein level ( Figure 2B).  -mediated  cell  viability  was  evaluated  using  the  3 -(4 , 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The results present as a percentage of the control samples. Data are presented as the mean ± SEM. ### p < 0.001 versus untreated group; *** p < 0.001 versus LPS group (n = 3 ).

Action of MEAC on LPS-Induced iNOS Expressions in BV-2 Microglial Cells at mRNA and Protein Levels
As the MEAC treatment reduced the NO concentration, iNOS-mRNA and -protein expressions were evaluated further. The stimulation (200 ng/mL) of LPS noticeably upregulated the iNOS-mRNA level to 4 . 23 ± 0. 18 folds after 6 h in comparison with the control group (Figure 2A). In a concentration-dependent manner, MEAC pretreatment prevented the iNOS-mRNA in the LPS-induced BV-2 microglial cells. The western blot analysis evaluated the iNOS expression at the protein level. At 18 h after the LPS stimulation, the iNOS-protein level was noticeably elevated to 4 .95 ± 0.056 folds compared to the control group. Meanwhile, the MEAC treatment significantly prevented iNOS expression at the protein level ( Figure 2B).

Action of MEAC on LPS-Induced COX-2 Expressions in BV-2 Microglial Cells at mRNA and Protein Levels
BV-2 microglial cells were treated with LPS in the absence or presence of MEAC for 6 h or 18 h to investigate COX-2 expression at the mRNA and protein levels. Stimulation with LPS for 6 h significantly elevated the COX-2 gene expression compared with that of the control group; however, the pretreatment with MEAC 1 h before LPS stimulation significantly prevented the COX-2-mRNA expression in a dose-dependent manner ( Figure 3A). In addition, LPS treatment for 18 h also dose-dependently increased COX-2 expression at the protein level, while MEAC pretreatment prevented LPS-induced increase COX-2 expression ( Figure 3B).
to investigate COX-2 expression at the mRNA and protein levels. Stimulation with LPS for 6 h significantly elevated the COX-2 gene expression compared with that of the control group; however, the pretreatment with MEAC 1 h before LPS stimulation significantly prevented the COX-2-mRNA expression in a dose-dependent manner (Figure 3 A). In addition, LPS treatment for 18 h also dose-dependently increased COX-2 expression at the protein level, while MEAC pretreatment prevented LPS-induced increase COX-2 expression (Figure 3 B).

Action of MEAC on LPS-Induced Expressions of Inflammatory Cytokines in BV-2 Microglial Cells at mRNA Level
MEAC treatments suppressed the LPS-induced expression of IL-6, TNF-α, and IL-1β in BV-2 microglial cells as per the RT-PCR analysis. The induction of LPS significantly elevated proinflammatory cytokine (IL-6, TNF-α, and IL-1β) expressions, while the pretreatment with MEAC 1 h before LPS treatment prevented an increase in expression of those markers (Figure 4 A-D).

Action of MEAC on LPS-Induced Expressions of Inflammatory Cytokines in BV-2 Microglial Cells at mRNA Level
MEAC treatments suppressed the LPS-induced expression of IL-6, TNF-α, and IL-1β in BV-2 microglial cells as per the RT-PCR analysis. The induction of LPS significantly elevated proinflammatory cytokine (IL-6, TNF-α, and IL-1β) expressions, while the pretreatment with MEAC 1 h before LPS treatment prevented an increase in expression of those markers ( Figure 4A-D).

Protective Activity of MEAC Against MPP + -Induced N27-A Cells
Various concentrations of MEAC (25, 50, 100, 250, and 500 μg/mL) were treated in N27-A cells to investigate whether the treatment causes mortality. The results indicate that being treated with MEAC for 24 h does not cause any significant cytotoxicity in N27-A cells ( Figure 5A). Indeed, the

Protective Activity of MEAC Against MPP + -Induced N27-A Cells
Various concentrations of MEAC (25,50,100,250, and 500 μg/mL) were treated in N27-A cells to investigate whether the treatment causes mortality. The results indicate that being treated with MEAC for 24 h does not cause any significant cytotoxicity in N27-A cells ( Figure 5A). Indeed, the MEAC treatment (500 μg/mL) significantly protects N27-A cells from MPP + -induced cell death ( Figure 5B).

Regulatory Effect of MEAC on Bcl-2, HO-1, NQO1, and Catalase Expression in N27-A Cells
The activity of MEAC treatment on the regulation of mRNA expressions of Bcl-2, HO-1, NQO1, and catalase is displayed in Figure 6A. To investigate the regulatory action of MEAC on Bcl-2 expression, N27-A cells were treated with MEAC (500 µg/mL) for 18 h. The treatment significantly upregulated Bcl-2 expression to 1.48 ± 10 folds compared to the control group ( Figure 6B). N27-A cells were treated by MEAC (500 µg/mL) to evaluate the regulatory effect on HO-1 and NQO1 expressions. The treatment with MEAC for 18 h significantly upregulated expression of HO-1 to 1.85 ± 0.04 folds compared to the control group ( Figure 6C). In comparison with the control group, MEAC extract significantly upregulated NQO1 expression to 2.72 ± 0.25 ( Figure 6D). Apart from HO-1 and NQO1, N27-A cells were treated with MEAC to check its regulatory role on catalase expression. An 18-h MEAC (500 µg/mL) treatment significantly upregulated catalase expression to 1.78 ± 0.04 folds in comparison with the control group ( Figure 6E). ± 0.04 folds compared to the control group ( Figure 6C). In comparison with the control group, MEAC extract significantly upregulated NQO1 expression to 2.72 ± 0.25 ( Figure 6D). Apart from HO-1 and NQO1, N27-A cells were treated with MEAC to check its regulatory role on catalase expression. An 18-h MEAC (500 μg/mL) treatment significantly upregulated catalase expression to 1.78 ± 0.04 folds in comparison with the control group ( Figure 6E).

Discussion
The elucidation of the mechanism of progression and therapeutics of NDDs remains a significant challenge [ 20] . Recently, however, several natural products produced protective actions in the model of NDDs [21][22][23][24][25] . The natural product-derived intervention of therapeutics could have potential in the therapy of NDDs [ 21] . The design of studies focused on toxic agent-intoxicated neuronal injury may aid to find possible therapeutics for treating NDDs [ 20] . LPS is a well-known cell wall component of gram-negative bacteria, which has the ability to trigger the microglia. LPS-induced activation of microglia leads to inflammatory responses that initiate the progression of disease in models of neurodegeneration. LPS interacts with Toll-like receptors (TLRs), such as TLR4 , on the surface of the microglia. LPS and TLR4 interaction activate several downstream signaling cascades. The activation of TLR4 signaling influences NF-κB and/or other transcription factors in the nucleus and triggers the

Discussion
The elucidation of the mechanism of progression and therapeutics of NDDs remains a significant challenge [20]. Recently, however, several natural products produced protective actions in the model of NDDs [21][22][23][24][25]. The natural product-derived intervention of therapeutics could have potential in the therapy of NDDs [21].
The design of studies focused on toxic agent-intoxicated neuronal injury may aid to find possible therapeutics for treating NDDs [20]. LPS is a well-known cell wall component of gram-negative bacteria, which has the ability to trigger the microglia. LPS-induced activation of microglia leads to inflammatory responses that initiate the progression of disease in models of neurodegeneration. LPS interacts with Toll-like receptors (TLRs), such as TLR4, on the surface of the microglia. LPS and TLR4 interaction activate several downstream signaling cascades. The activation of TLR4 signaling influences NF-κB and/or other transcription factors in the nucleus and triggers the proinflammatory cytokine release [3]. Thus, for preventing/treating neuroinflammation-related diseases, the modulation of LPS and TLR interaction and/or activation has a potential value.
In a mouse MCAO model, AC extract prevents brain edema, hyperpermeability of blood-brain barrier, and disruption of tight junction proteins, probably through its antioxidant effects. AC extract may be a potential nutrient in the prevention of blood-brain barrier function during brain ischemia [26]. This study investigated the actions of MEAC extracts against LPS-stimulated increased expressions of inflammatory markers. According to this study, the treatment with MEAC protected from the LPS-induced NO release in BV-2 microglial cells. In the brain, prostaglandins are crucial when neuroinflammation occurs. The synthesizing enzymes COX-2 and iNOS are some of the most-referred proinflammatory mediators [2]. As per this study, MEAC also prevents the expression of COX-2 and iNOS at the mRNA and protein levels. In addition, in the BV-2 microglial cells, the elevation of the expression of inflammatory cytokines such as IL-6, TNF-α, and IL-1β is a pointer of neuronal cell damage [27]. Various concentrations of MEAC were treated to reveal the action against inflammatory cytokine expressions. The present study has found that pretreatment with MEAC exhibits preventive actions against LPS-stimulated increase mRNA expressions of IL-6, TNF-α, and IL-1β in BV-2 microglial cells. However, further investigations are needed to confirm MEAC action on cytokine expressions at the protein level.
In the progression of NDDs, apoptosis plays a critical role, according to several animal and cellular model studies. However, research on human brains has demonstrated diverse outcomes. In the brains of NDD patients, much-died neurons are detected and these conditions often accompany significant cell loss as a result of distinctive morphological characteristics of apoptosis, including DNA fragmentation, chromatin condensation, and cysteine-proteases, caspases activation. NDDs and cell death are associated with oxidative stress [28,29].
As an antiapoptotic marker against acute neural injury, Bcl-2 belongs to the Bcl-2 family of apoptotic regulators and is vital for cell survival. The downregulation of the Bcl-2 protein often precedes apoptosis. On the contrary, other members of the Bcl-2 family, for example, Bcl-2 antagonist killer 1 (Bak) and Bcl-2 Associated X (Bax) promote apoptosis by increasing the release of cytochrome c from the mitochondria and activating caspases. Bcl-2 can avert the activation of Bax via heterodimerization. For the cell survival, the interplay of antiapoptotic and proapoptotic regulators is crucial, and Bax/Bcl-2 ratio constitutes an important factor of the apoptotic status of the cell. Pro and antiapoptotic members of the Bcl-2 family are extensively expressed throughout the CNS. Thus, the upregulation of Bcl-2 expression may improve neuroprotective activity [30][31][32]. Based on the results, MEAC treatment upregulates Bcl-2 expression.
Several endogenous and exogenous antioxidant enzymes have been found to play a neuroprotective role in the model of NDDs [33]. Nrf2 and its downstream phase II genes (for instance, HO-1 and NQO1) have potent capability to detoxify harmful compounds, combat reactive oxygen species, and directly or indirectly modulate the BBB permeability, immune system, and inflammatory response. Thus, they are considered as the emerging targets for the treatment of neurological diseases. Indeed, various inducers of Nrf2 have been proven effective in familiar neurological disease models [34]. According to the study results, MEAC treatment upregulates the expression of antioxidant enzymes, including HO-1, NQO1, and catalase.
MPP + is one of the agents that induced cytotoxicity in the experimental model; it causes oxidative stress-mediated cell death [35]. The protective role of MEAC against MPP + induction was also studied in this study. The treatment with MEAC for 24 h protects from MPP + -induced N27-A cell death. The stability of MEAC in plasma could be studied further based on the data on both cell lines.

Conclusions
MEAC suppresses the LPS-induced production of proinflammatory mediators and cytokines in BV-2 microglial cells. MEAC extract also upregulates the antiapoptotic gene (Bcl-2) and antioxidant enzyme (HO-1 and NQO1) expressions in N27-A cells. Therefore, our study results support the previously published outcomes of A. cepa extract against stroke and metalloid-induced neurotoxicity in rodents. Future research should examine the A. cepa action on NDDs model. This study promotes further investigation into the role of A. cepa extract against neurodegenerative disease.

Conflicts of Interest:
The authors declare that they have no conflicts of interest.