Neuroprotective Effect of α-Lipoic Acid against Aβ25–35-Induced Damage in BV2 Cells

The prevalence of Alzheimer’s disease (AD) is significantly increasing due to the aging world population, and the currently available drug treatments cannot cure or even slow its progression. α-lipoic acid (LA) is a biological factor widely found in spinach and meat and can dissolve in both lipid and aqueous phases. In medicine, LA has been shown to reduce the symptoms of diabetic polyneuropathy, acute kidney injury, cancers, and some metabolism-related diseases. This study to proves that α-lipoic acid (LA) can stabilize the cognitive function of patients with Alzheimer’s disease (AD). BV2 cells were divided into control, LA, Aβ25–35, and LA + Aβ25–35 groups. Cell growth; IL-6, IL-1β, TNF-α, IFN-γ, SOD, GPx, CAT, ROS, NO, and iNOS secretion; Wnt-related proteins; cell apoptosis; and cell activation were examined. Here, we found that LA could effectively repress apoptosis and changes in the morphology of microglia BV2 cells activated by Aβ25–35, accompanied by the inhibition of the inflammatory response induced by Aβ25–35. The Wnt/β-catenin pathway is also involved in preventing Aβ25–35-induced cytotoxicity in microglia by LA. We found an inhibitory effect of LA on microglia toxicity induced by Aβ25–35, suggesting that a combination of anti-inflammatory and antioxidant substances may offer a promising approach to the treatment of AD.


Introduction
Alzheimer's disease (AD) is the most common neurodegenerative disease and seems to be one of the major healthcare challenges of the present century [1]. Approximately 50 million people worldwide had AD in 2018, and this number is expected to increase to 152 million by 2050 [2]. AD is the most common type of dementia, resulting in memory impairment and behavioral disorders [3]. It is a chronic lethal disease with a complicated pathogenesis. The two major hallmarks of AD are the formation of amyloid-β (Aβ) plaques and neurofibrillary tangles, primarily comprising the hyperphosphorylated Tau protein [4,5]. Aβ is produced and secreted by neurons in response to synaptic activity under physiological conditions. Once secreted in an extracellular environment, it is degraded by glial cells. The mechanism that causes the transition from normal physiological function to pathological Aβ accumulation is still unknown [6]. Since the currently available drug treatments cannot cure or even slow its progression [7], patients are left to rely solely on supportive care from family and other caregivers. Therefore, extensive research is necessary to investigate the molecular mechanisms of AD pathogenesis and uncover new treatment options.
α-lipoic acid (LA), an organosulfur medium-chain fatty acid ( Figure 1) that was first discovered in 1951 as a catalytic agent for the oxidative decarboxylation of pyruvate and Microglia are an indispensable component of the central nervous system and play an important role in the nutrition, protection, and repair of neurons [15,16]. Studies have shown that microglia and AD are closely associated [17,18]. In the pathogenesis of AD, Aβ can activate microglial cells, causing them to overexpress interleukin-1, tissue growth factor (TGF)-β, and tumor necrosis factor (TNF)-α through different signal transduction pathways, as well as mediating inflammatory injury [18][19][20]. The Wnt pathways play important roles in cell activities, and Wnt dysregulation is known to be involved in Tau hyperphosphorylation and the loss of synapses [21] and neuroinflammation [22].
Most studies primarily focus on LA's neuroprotective effects on neurons [18], while little is known about microglial cells. This study aimed to investigate LA's role in Aβ25-35induced microglial BV2 cell toxicity and Wnt/β-catenin signaling pathway activation.

LA Improves Aβ25-35-Induced Morphology Changes and Activation in BV2 Cells
We examined the cell morphology in BV2 cells after treatment with Aβ25-35 to investigate whether Aβ25-35 treatment could induce cytotoxicity in these cells. We observed changes in cell morphology in the treated cells compared with controls, including larger cell bodies, cell aggregation, fusiform shape, multiple dendrites on the surface, and protrusions connecting the cells. We added LA to the BV2 medium before Aβ25-35 treatment and observed that LA could protect the cells from morphological changes induced by Aβ25-35. In addition, we compared these cells with Aβ25-35-treated cells. Decreased cell surface dendrites and changes in the shape of the cells were found in LA + Aβ25-35-treated cells (Figure 2a).  Microglia are an indispensable component of the central nervous system and play an important role in the nutrition, protection, and repair of neurons [15,16]. Studies have shown that microglia and AD are closely associated [17,18]. In the pathogenesis of AD, Aβ can activate microglial cells, causing them to overexpress interleukin-1, tissue growth factor (TGF)-β, and tumor necrosis factor (TNF)-α through different signal transduction pathways, as well as mediating inflammatory injury [18][19][20]. The Wnt pathways play important roles in cell activities, and Wnt dysregulation is known to be involved in Tau hyperphosphorylation and the loss of synapses [21] and neuroinflammation [22].
Most studies primarily focus on LA's neuroprotective effects on neurons [18], while little is known about microglial cells. This study aimed to investigate LA's role in Aβ 25-35induced microglial BV2 cell toxicity and Wnt/β-catenin signaling pathway activation.

LA Improves Aβ 25-35 -Induced Morphology Changes and Activation in BV2 Cells
We examined the cell morphology in BV2 cells after treatment with Aβ 25-35 to investigate whether Aβ 25-35 treatment could induce cytotoxicity in these cells. We observed changes in cell morphology in the treated cells compared with controls, including larger cell bodies, cell aggregation, fusiform shape, multiple dendrites on the surface, and protrusions connecting the cells. We added LA to the BV2 medium before Aβ 25-35 treatment and observed that LA could protect the cells from morphological changes induced by Aβ [25][26][27][28][29][30][31][32][33][34][35] . In addition, we compared these cells with Aβ 25-35 -treated cells. Decreased cell surface dendrites and changes in the shape of the cells were found in LA + Aβ 25-35 -treated cells (Figure 2a).

LA and Aβ25-35 Do Not Affect BV2 Cell Viability
The cells were treated with Aβ25-35, which is a toxic fragment of full-length Aβ1-42, to investigate whether Aβ could affect the cell proliferation of BV2 cells. After 48 h, cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The results show that the amyloid peptide had little effect on BV2 cell

LA rescues Cell Apoptosis Promoted by Aβ 25-35
Considering that Aβ 25-35 treatment could induce BV2 cell morphology changes, using flow cytometry, we next investigated whether Aβ 25-35 treatment promoted BV2 cell apoptosis. As shown in Figure   We also found increased protein levels of the apoptosis-related protein caspase-3, while the anti-apoptosis protein Bcl-2 was downregulated after Aβ 25-35 treatment (Figure 3d-f) (p < 0.05). The expression of the Bcl-2 protein after Aβ 25-35 treatment significantly decreased compared to the control group (p < 0.05). In addition, the Bax/Bcl-2 ratio of the Aβ 25-35treated group was significantly different from that of the control group (p < 0.05). After LA treatment, the abnormal expression of protein caspase-3 and Bcl-2, which were induced by Aβ 25-35 treatment, changed. Compared with the Aβ 25-35 -treated group, the protein expression level of caspase-3 significantly decreased (p < 0.05), and Bcl-2 significantly increased (p < 0.05). In addition, the Bax/Bcl-2 ratio of the LA + Aβ 25-35 -treated group significantly decreased compared to the Aβ 25-35 -treated group (p < 0.05). The LA treatment alone has no significant effect on protein expression. [25][26][27][28][29][30][31][32][33][34][35] AD pathophysiological events are usually accompanied by neuroinflammation, which is a defensive mechanism for pathogen clearance and maintenance of tissue homeostasis [23]. It has been reported that LA could reduce NF-κB activity in vitro in cells stimulated with TNF-α in a dose-dependent manner [24]. We wondered if Aβ [25][26][27][28][29][30][31][32][33][34][35]   Inducible nitric oxide synthase (iNOS) is an important catalytic enzyme in organisms, which plays a biological role by catalyzing the production of nitric oxide (NO) by the substrate arginine. Innumerable studies have shown that iNOS is closely related to inflammation, and bacteria, viruses, and a variety of inflammatory factors can induce its expression to produce endogenous NO, which, in turn, plays an important biological role. Therefore, we next measured nitric oxide (NO) and inducible nitric oxide synthase (iNOS) levels in BV2 cells after Aβ25-35 treatment and found that NO and iNOS were increased. When we added LA to BV2 cells before Aβ25-35 treatment, the levels of NO and iNOS induced by Aβ25-35 treatment were repressed (Figure 5a,b). Inducible nitric oxide synthase (iNOS) is an important catalytic enzyme in organisms, which plays a biological role by catalyzing the production of nitric oxide (NO) by the substrate arginine. Innumerable studies have shown that iNOS is closely related to inflammation, and bacteria, viruses, and a variety of inflammatory factors can induce its expression to produce endogenous NO, which, in turn, plays an important biological role. Therefore, we next measured nitric oxide (NO) and inducible nitric oxide synthase (iNOS) levels in BV2 cells after Aβ 25-35 treatment and found that NO and iNOS were increased. When we added LA to BV2 cells before Aβ 25-35 treatment, the levels of NO and iNOS induced by Aβ 25-35 treatment were repressed (Figure 5a,b). Inducible nitric oxide synthase (iNOS) is an important catalytic enzyme in organisms, which plays a biological role by catalyzing the production of nitric oxide (NO) by the substrate arginine. Innumerable studies have shown that iNOS is closely related to inflammation, and bacteria, viruses, and a variety of inflammatory factors can induce its expression to produce endogenous NO, which, in turn, plays an important biological role. Therefore, we next measured nitric oxide (NO) and inducible nitric oxide synthase (iNOS) levels in BV2 cells after Aβ25-35 treatment and found that NO and iNOS were increased. When we added LA to BV2 cells before Aβ25-35 treatment, the levels of NO and iNOS induced by Aβ25-35 treatment were repressed (Figure 5a,b).

LA Downregulates ROS Levels Induced by Aβ25-35
Neurodegenerative disorders such as AD are associated with oxidative damage [4]. In order to investigate whether LA could modify the Aβ25-35-induced ROS increase, we treated BV2 cells with LA and Aβ25-35 to observe the activity of SOD, GPx, CAT, and ROS in BV2 cells induced by Aβ25-35 and LA. We found that after LA treatment, the enzyme activities of SOD, GPx, and CAT increased (Figure 6a-c), while ROS levels were significantly repressed (Figure 6d) compared to treatment with Aβ25-35 alone. The results demonstrate that LA could reduce the Aβ25-35-induced ROS levels in mouse microglia BV2 cells.

LA-regulated Wnt Pathway-Specific Protein Expression in Aβ25-35-Treated BV2 cells
It has been reported that Wnt signaling inactivation promotes the neurotoxicity of Aβ [25,26]. In order to determine whether the Wnt pathway participated in the neuroprotective role of LA, we analyzed the cellular localization and expression of GSK3β and βcatenin after Aβ25-35 treatment or treatment with both Aβ25-35 and LA (Figure 7a). The expression of GSK3β increased after Aβ25-35 treatment while β-catenin decreased. In addition, LA treatment upregulated β-catenin expression and inhibited the expression of GSK3β induced by Aβ25-35. Western blot indicated that the expression of phosphorylated GSK3β (p-GSK3β), Frizzled2, and β-catenin was downregulated, while phosphorylated β-catenin (p-βcatenin) was upregulated in BV2 cells after Aβ25-35 treatment (Figure 7b,c). When BV2 cells were treated with both LA and Aβ25-35, the inactivated Wnt pathway was re-activated, and the associated proteins were recovered. Specifically, after Aβ25-35 treatment, the expressions of Frizzled2, GSK3β, p-GSK3β, β-catenin, and p-β-catenin were significantly different compared to the control group, and the expressions of Frizzled2, p-GSK3β, and β-

Discussion
The pathogenesis of AD is complicated, and the underlying mechanisms are not fully understood. Accumulating evidence shows that inflammation plays an important role in AD's pathogenesis, and the deposition of Aβ can activate brain inflammation, resulting in nervous system damage [27][28][29]. Microglia, the central nervous system's immune cells, are widely distributed in the central nervous system. Their activation promotes inflammatory responses in the brain, increasing the progression of AD [30]. The results show that BV2 cells were activated and morphologically changed after treatment with Aβ. The number of OX-42-positive cells also increased after Aβ 25-35 treatment, indicating an increase in activated microglia. After LA intervention, the cell morphology was improved compared with the Aβ 25-35 treatment alone. The results demonstrate that LA can effectively inhibit the Aβ-induced apoptosis of glial cells, which might be one of the important mechanisms of LA neuroprotection.
Previous studies have reported that the Wnt/β-catenin pathway is involved in AD's pathogenesis, although most studies were mainly focused on neurons and less on glial cells [25,33]. Here, we found that the expression of some Wnt/β-catenin pathway proteins such as Frizzled2, GSK3β, p-GSK3β, β-catenin, and p-β-catenin was altered in the glia after Aβ 25-35 treatment, suggesting that the Wnt pathway was also involved in Aβ 25-35induced glial cytotoxicity. The Wnt pathways are known to play important roles in cell activities, and Wnt dysregulation is known to be involved in Tau hyperphosphorylation, the loss of synapses [21], and neuroinflammation [22]. The already known effect of Aβ on Wnt pathways has two aspects. One is that Aβ and the amyloid precursor protein (APP) promote β-catenin phosphorylation and degradation, thus inhibiting the canonical Wnt pathway [21,34]. The tau protein is believed to stabilize b-catenin so that it can resist degradation, and the abnormal modification of tau can also cause damage to the canonical Wnt pathway [35]. The dysregulated expression of these proteins was rescued following LA intervention. The results suggest that the Wnt pathway genes are involved in LA's neuroprotection potential in Aβ 25-35 -treated microglia cells. Further studies are needed to elucidate how LA plays its protective role through this pathway.
Taken together, the effects of LA observed here are consistent with its effects in various chronic diseases [10][11][12], as well as in nerve cells [13,14]. Of note, LA had no cytotoxicity effects on BV6, suggesting that it is not toxic for these cells. These effects seen at the microglia levels are supported by clinical observations that LA can improve patients' outcomes with AD [36][37][38]. Nevertheless, the effects of LA in AD are controversial [39,40] and might depend upon the model used. The present study shows that the effect of LA on microglia was consistent with effects that should slow down AD's progression, but in vivo studies remain necessary.

Cell Culture
BV2 cells were purchased from Peking Union Medical College, Chinese Academy of Medical Sciences, School of Basic Medicine Cell Center (Beijing, China). These cells were then cultured in DMEM (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA) and supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin at 37 • C in 5% CO 2 . The medium was replaced every 2-3 days.

Cell Growth Assays
Cell viability was measured using an MTT assay, as previously described [41]. The cells were seeded into 96-well plates and maintained in culture. After treatments according to grouping, the cells were further incubated for 48 h, washed twice with PBS, and incubated with 100 µL MTT (5 g/L) for 4 h at 37 • C. The optical densities of the solutions were measured at 570 nm. Duplicate measurements were performed in three independent wells at each time point. For the LA + Aβ 25-35 treatment group, 100 µmol/L LA was added to the plates and incubated for 2 h before Aβ 25-35 treatment.

Detection of SOD, GPx, CAT, ROS, NO, and iNOS
Cells in the logarithmic growth phase were adjusted to 5 × 10 5 /mL and seeded into 24-well plates. The cells were treated according to their grouping. The supernatant was collected for SOD, GPx, CAT, ROS, NO, and iNOS detection, according to the manufacturer's instructions (Jiancheng Institute of Biotechnology, Nanjing, China).

Western Blot
The cells were washed with PBS and lysed on ice for 30 min with RIPA (Applygen Technologies Inc., Beijing, China) containing a protease inhibitor mixture (Fermentas, Burlington, ON, Canada). The total protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was transferred to nitrocellulose membranes (Millipore Corp., Billerica, MA, USA). After blocking in 5% non-fat dry milk in TBST, the membranes were incubated with primary antibodies overnight at 4 • C. The membranes were washed three times with TBST and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Proteins were visualized using a chemiluminescent substrate (Millipore Corp., Billerica, MA, USA) according to the manufacturer's instructions.

Cell Apoptosis and Activation by Flow Cytometry
For the detection of cell activation, the cells were harvested and washed with PBS. The OX-42 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added, and the cells were incubated overnight at 4 • C. The next day, a secondary FITC-conjugated antibody (Zhongshan Biotechnologies Inc., Zhongshan, China) was added and incubated for 30 min. The cells were washed with PBS and analyzed. For the apoptosis analysis, the Annexin V/propidium iodide (PI) staining kit was used according to the manufacturer's instructions (BioLegend, San Diego, CA, USA), and the cells were stained with FITC-conjugated with Annexin V and PI. Stained cells were examined using a FACSCanto II (FACSAria, BD Biosciences, Franklin Lake, NJ, USA). The data were analyzed using the FlowJo software 10 (BD Biosciences, Franklin Lake, NJ, USA).