Baicalin Mitigates the Neuroinflammation through the TLR4/MyD88/NF-κB and MAPK Pathways in LPS-Stimulated BV-2 Microglia

Baicalin (BA) is a major flavone from Scutellaria baicalensis Georgi and has showed significant curative effects in Parkinson's and Alzheimer's diseases. In the present study, we investigated the effects of BA on antineuroinflammation and related signaling cascade in lipopolysaccharide- (LPS-) induced BV-2 microglial model. The results showed that BA significantly attenuated inflammatory mediators (NO, iNOS, IL-1β, COX-2, and PGE2) and suppressed the expression of miR-155. More crucially, BA could regulate the expression of related proteins in Toll-like receptor 4 (TLR4)/myeloid differentiation protein 88 (MyD88)/nuclear factor κB (NF-κB) pathway and suppress the phosphorylation of mitogen-activated protein kinase (MAPK) family. In addition, molecular docking analysis indicated that BA binds to the amino acids Lie 63 and Tyr 65 of TLR4 by π-σ and π-π T-shaped interaction. Thus, BA suppressed the LPS-stimulated neuroinflammation in BV-2 microglia by blocking the TLR4-mediated signal transduction through TLR4/MyD88/NF-κB and MAPK pathways and inhibiting the miR-155 expression. Our findings demonstrated that BA could be a valuable therapeutic for the treatment of neuroinflammation and neurodegenerative diseases.


Introduction
Neurodegenerative disease characterized by progressive neuronal death is a kind of chronic disease of the central nervous system (CNS) including Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington disease (HD) [1]. Studies have shown that chronic inflammation in the brain may be one of the essential pathological features of neurodegenerative diseases [2]. Microglial cells are resident immunocompetent cells of CNS and play a crucial role in the brain injury and development of neurodegenerative diseases. Under normal physiological conditions, resting microglia are pivotal players in nourishing, supporting, and protecting neurons [3]. However, when there is a neuronal injury or other stimuli, such as lipopolysaccharide (LPS), microglia will be activated to secrete massive proinflammatory and cytotoxic factors, including nitric oxide (NO), prostaglandin E2 (PGE2), interleukin 1β (IL-1β), and peroxides, which ultimately leads to the cell death [4]. Excessive activation of microglia can damage the surrounding normal neural tissue, and the inflammatory factors secreted by the dying neurons in turn aggravate the chronic activation of microglia resulting in the gradual loss of neurons. Such case was observed in AD, PD, and HD [5,6]. Studies have shown that overactivated microglia also highly express Toll-like receptor 4 (TLR4) and miR-155 [7]. LPS binds and activates TLR4, leading to the sequential recruitment/activation of the myeloid differentiation protein 88 (MyD88) and interleukin-1 receptor-associated kinases (IRAKs). Stimulation of TLR4 also activates mitogen-activated protein kinase (MAPK) and the nuclear factor-κB (NF-κB) pathways, which then evokes the inflammatory response in microglia [8]. Thus, control of LPS-induced microglia activation, suppression of the production of neurotoxic proinflammatory mediators and cytokines, and downregulation of proteins involved in TLR4-mediated signaling pathways would be effective therapeutic strategies for neuroinflammatory diseases.
Baicalin (baicalein 7-O-β-D-glucopyranosiduronate, BA, chemical structure shown in Figure 1(a)) is a major active flavone isolated from the Radix of Scutellaria baicalensis Georgi. It has obvious curative effect in CNS diseases. Studies have shown that BA displays neuroprotective effect on permanent cerebral ischemia injury in rats through downregulating the expression of inducible nitric oxide synthase (iNOS) mRNA, COX-2 mRNA, and cleaved caspase-3 protein [9]. BA can also reduce amyloid β protein-induced neurotoxicity in PC12 cells [10]. In addition, BA exerts anti-inflammatory effect by inhibiting the NF-κB signaling cascade in Helicobacter pylori-induced gastritis model [11]. These researches indicate that BA plays a key role in the treatment of neuroinflammatory and neurodegenerative diseases. However, the pharmacological effects of BA on activated microglia have not been elucidated. Thus, our study evaluated the neuroinflammatory-modulatory effects and the underlying mechanisms of BA in LPSstimulated microglia.

Cell Viability
Assay. Cell viability was determined by the MTT assay. Briefly, BV-2 (5 × 10 4 /well) was inoculated into 96-well plates and cultured for 24 h. Then, various concentrations of BA (0.8-67.5 μM) were added into the wells for 24 h. Following the incubation, the supernatant was substituted for the same volume of fresh serum-free RPMI-1640 for 24 h. The cells were then cultured with 0.5 mg/mL MTT for another 4 h. Subsequently, 150 μL DMSO was added and incubated for 10 min. The absorbance was measured with a spectrophotometer at 570 nm.
2.4. NO and iNOS Assays. The content of NO and iNOS secreted by microglia was measured using NO assay kit and iNOS assay kit, respectively. Briefly, BV-2 (5 × 10 5 cells/mL) was cultured in 24-well plates overnight and pretreated with BA at the concentration of 2.5, 7.5, and 22.5 μM for 1 h. The cells were then incubated with LPS (0.1 μg/mL) for 12 h. Indomethacin (INDO, 10 μM) and aminoguanidine (AG, 1 mM) were added as positive control. The supernatant was collected; the absorbance of NO and iNOS was measured at 550 nm and 530 nm.
2.5. Measurement of IL-1β and PGE2 Production. The levels of the PGE2 and IL-1β in culture medium were assessed using ELISA assay kits. BV-2 (5 × 10 5 cells/mL) was cultured in 24-well plates for overnight and pretreated with BA at the concentration of 2.5, 7.5, and 22.5 μM for 1 h. The cells were incubated with LPS (0.1 μg/mL) for 24 h. Dexamethasone (DEX, 10 μM) and indomethacin (INDO, 10 μM) were added as positive control. Absorbance was measured at 450 nm using a spectrometer.
2.6. Real-Time PCR Analysis. Real-time PCR was carried out to analyze the mRNA level of miR-155. BV-2 (5 × 10 5 cells/ mL) was cultured in 6-well plates overnight and pretreated with BA (2.5, 7.5, and 22.5 μM) for 1 h. The cells were incubated with LPS (0.1 μg/mL) for 12 h. The supernatant was removed, and total RNA was extracted by TRIzol reagent following the manufacturer's instructions. The total RNA was quantitatively analyzed and then primed with mmu-miR-155-5p-real-time-F: 5 ′ -CGGCGGTTAATGCTAATTG TGAT-3 ′ and mmu-miR-155-5p-real-time-R: 5 ′ -GTGCAG GGTCCGAGGT-3 ′ to synthesize cDNA using M-MLV reverse transcriptase. PCR was performed by 40 cycles of denaturation at 95°C for 3 min, annealing at 62°C for 50 s, and extension at 95°C for 50 s. Indomethacin (INDO, 10 μM) and dexamethasone (DEX, 10 μM) were added as positive control. Membranes were then incubated with secondary antibodies combined to horseradish peroxidase for appropriate time.
After washing three times in Tris-buffered saline with Tween 20 (TBST), the protein bands were visualized using the enhanced chemiluminescence reagents.
2.8. Molecular Docking Analysis. The crystal structure of TLR4-myeloid differential protein-2 (MD2) complex (PDB ID 3FXI, chain A (605 residues), and C (142 residues)) was obtained from the Protein Data Bank (PDB) (http://www .wwpdb.org/). The structure of BA was sketched in Chem-Draw 20.0 and saved as Mol2 format. The TLR4-MD2 crystal structure was optimized by Pymol software following the removal of water molecules and ligands, the addition of missing residues [12]. Molecular docking scores and poses were calculated in Autodock Tools 1.5.6 [13]. All rotatable bonds in ligand remained flexible, while the protein structure kept rigid. In the docking setup, binding site center was set up to x = 20:000, y = −14:111, and z = 12:000; the spacing box was 0.369 Å; and the number of points in dimension was set up to x = 88, y = 86, and z = 92. Molecular interactions of TLR4-MD2 ligand complexes were identified with Discovery Studio 2016 software [14]. The interaction energy of the TLR4-MD2 ligand complex was calculated, 3D and 2D diagrams of interaction were created, and their interactions include van der Waals force, hydrogen bond, and π-σ interaction.
2.9. Statistical Analysis. Results were analyzed by GraphPad Prism 6.0 software (GraphPad, United States). The data were expressed as means ± SD of three independent experiments performed in triplicate measurements. Statistical analysis was performed using ANOVA followed by Tukey's test. P < 0:05 was considered as statistical significance.

BA Does Not Affect Cell Viability in Microglia.
MTT assay showed that BA had no obvious effect on the viability of BV-2 cells in concentration range from 0.8 μM to 67.5 μM (Figure 1(b)). Therefore, we used BA at concentration range from 2.5 to 22.5 μM for subsequent studies.

BA Inhibits the Production of Proinflammatory
Cytokines IL-1β and PGE2 in LPS-Induced Microglia. IL-1β and PGE2 are important proinflammatory cytokines. Production of IL-1β in the microglia is the initiating step of nerve inflammation, which drives the amplification of inflammatory cascade and pathogenesis of neurodegenerative disease. PGE2 is the metabolite of arachidonic acid generated by rate-limiting enzyme cyclooxygenase-2 (COX-2), and it is another important indicator of varied inflammatory diseases [15]. The inhibitory effects of BA on proinflammatory cytokines IL-1β and PGE2 were determined using ELISA. As expected, treatment of BV-2 with LPS resulted in a significant increase of IL-1β and PGE2 release compared    ). Then, the cells were stimulated with or without LPS (0.1 μg/mL); the production of NO and iNOS (incubated for 12 h) and IL-1β and PGE2 (incubated for 24 h) was determined by ELISA kit. MiR-155 expression (e) was detected by miRNA real-time PCR kit, BV-2 was pretreated with BA (2.5-22.5 μM) for 1 h followed by LPS stimulation for 12 h, and then, RT-PCR was performed. Results were presented as mean ± SD (n = 3). ### P < 0:001 vs. control and * P < 0:05, * * P < 0:01, and * * * P < 0:001 vs. LPS-treated cells.  [16,17]. We evaluated the effects of BA on the expression of miR-155 by real-time PCR. As expected, treatment of BV-2 cells with LPS resulted in a significant increase of miR-155 level compared with control. The level of miR-155 in LPSinduced microglia treated with 2.5, 7.5, and 22.5 μM of BA was reduced by 11.84%, 28.90%, and 33.55%, respectively ( Figure 2(e)). These results indicated that BA downregulates the expression of miR-155 in LPS-stimulated microglia in a dose-dependent manner.   Figures S7-S8  These results indicate that BA can inhibit the phosphorylation of proteins involved in MAPK signaling in a dose-dependent manner.

Molecular Docking Analysis Reveals the Binding Mode
between BA and TLR4-MD2 Complex. BA showed a favorable conformation at the active site of TLR4-MD2 with the energy of −13.94 kJ/mol. The aromatic ring of BA is connected with amino acids Lie 63 of TLR4-MD2 by π-σ interaction (bonding distances of 3.93 Å) and connected with amino acids Tyr 65 of TLR4-MD2 by forming π-π Tshaped interaction (bonding distances of 5.33 Å). BA also could bind to Leu 61 of TLR4-MD2 by π-alkyl interaction, bonding distances of 5.24 Å (Figures 7(a) and 7(b)). INDO, as TLR4 antagonist, also adopted optimal conformation at the active site of TLR4-MD2 with the minimum energy of

Discussion
Microglial cells are the resident immuno competent cells in CNS, and inflammation mediated by microglia is closely associated with neurological disorders [18]. Antiinflammatory treatment which targeted microglia could be a promising therapy for multiple neurodegenerative conditions [19]. BA, an anti-inflammatory flavonoid from the Radix of S. baicalensis, is excellent for the prevention of neurodegenerative diseases by its potent neuroprotective effects [20]. BA also makes a show of therapeutic effect on liver inflammation by blocking NF-κB signal transduction [21]. In addition, BA can alleviate oxygen-glucose-deprived chal-lenged microglia injury in ischemic diseases by attenuating expression of inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8 through TLR4 pathway [22]. Furthermore, Aβinduced microglia activation is inhibited by BA through the JAK2/STAT3 signaling [23]. BA could also suppress the polarization and inflammatory injury of microglia by suppressing the 5-LOX/LTB4 pathway in BV-2 [24]. BA has showed potential therapeutic effect for depression by reducing the levels of proinflammatory factor IL-1β possibly through regulation of SIRT1-NF-κB pathway in BV-2 microglia [25]. Further, BA improves neuroinflammationinduced depression-like behavior by inhibiting the expression of TLR4 through PI3K/Akt/FoxO1 signals [26]. Acute neurocognitive impairment and neuroinflammation in LPS-challenged mice are ameliorated by BA via a SIRT1/ HMGB1-dependent pathway [27]. In the current study, we investigated the antineuroinflammatory effects of BA in microglia and further explored the effect of BA on TLR4/ MyD88/NF-κB and MAPK pathways. Present results indicate that BA can inhibit the production of proinflammatory The neuroprotective effect of BA is closely associated with the downregulation of the expression of iNOS and COX-2 [9,11]. Therefore, we first assessed the effects of BA on the representative proinflammatory mediators and cytokines in LPS-stimulated BV-2 cells and found that BA can inhibit the release of NO and PGE2 by downregulating the levels of iNOS and COX-2. NO is a key regulatory molecule involved in a train of physiological and pathological processes, e.g., inflammatory and neurodegenerative diseases. INOS is responsible for the production of NO in different cells by converting L-arginine to L-citrulline [28,29]. COX-2 can promote the release of arachidonic acid IRAK4 (e, f), and IRAK1 (g, h) were analyzed by Western blot. BV-2 was pretreated with BA (2.5-22.5 μM) for 1 h before being stimulated with 0.1 μg/mL LPS for different times (12 h for TLR4 and MyD88 and 20 min for IRAK4 and IRAK1). Results were presented as mean ± SD (n = 3). ### P < 0:001 vs. control; * P < 0:05, * * P < 0:01, and * * * P < 0:001 vs. LPS. 8 BioMed Research International (AA) from membrane glycerol phospholipids and oxidized AA to proinflammatory eicosenoic acid (PGE2, leukotrienes, and thromboxanes), aggravating neuroinflammatory events in the brain. Overproduction of PGE2 can bring about the upregulation of cytokines, growth factors, and proinflammatory molecules [15,30]. Similarly, IL-1β is another neuroimmune mediator that participates directly in neurodegeneration [31]. Recent research has been 11 BioMed Research International suggested that people carrying IL-1β (1473C/G) genotype are more vulnerable to neuroinflammation, and further develop to AD [32]. In addition, binding of IL-1β with TLR can lead to the overexpression of transcription factors and long-term chronic inflammation in the brain [33]. Our data showed that BA can inhibit IL-1β expression in LPS-stimulated microglia. Multiple studies have shown that PGE2, COX-2, and IL-1β expression levels are significantly increased in AD patients' brain [34,35]. Our results showed that BA can significantly reduce the release of NO, IL-1β, and PGE2 by downregulating the iNOS and COX-2 at the kinase activity or protein levels in LPSinduced microglia. These results indicate that BA could be potentially used for the treatment of neurodegenerative diseases. miR-155 shows a critical role in the progression of neurodegenerative diseases. According to one study, several TLR4 ligands could increase miR-155 expression via either MyD88-dependent or MyD88-independent signaling pathways [16]. Our result showed that pretreatment with BA decreases the expression of miR-155 in LPS-stimulated microglia.
TLR4 signaling-mediated neuroinflammation leads to secondary brain damage in ischemic stroke. TLR4 is expressed on the surface of BV-2 microglia. LPS is a common ligand of TLR4, which can activate TLR4 in microglia via MyD88-dependent and independent patterns. In MyD88-dependent manner, recruitment of MyD88 to TLR complexes contributes to the activation of IRAKs, including IRAK1 and IRAK4 [36]. Our present study showed that BA inhibits the expression of TLR4, MyD88, IRAK4, and IRAK1 in a dose-dependent manner, indicating that BA can target TLR4-mediated MyD88-dependent pathway. Furthermore, the molecular docking analysis indicated that BA could stably bind TLR4-MD2 via π-bond. As a downstream factor of TLR4/MyD88/IRAK, NF-κB also shows a pivotal role in inflammation meditated by microglia. Blockade of NF-κB transcription in the microglial nucleus leads to the downregulation of NO, PGE2, and related proinflammatory cytokines [37][38][39]. In the cytoplasm, phosphorylation of IκBα results in its further ubiquitination and degradation, and consequently, NF-κB dimers are released and translocated to the nucleus to activate the expression of target genes [40]. Our result showed that BA can inhibit NF-κB upregulation and IκBα degradation and increase the phosphorylation of IκBα in LPS-stimulated BV-2 cells, indicating that BA can also target NF-κB signaling pathway to inhibit inflammatory response in microglia. MAPKs including ERK, JNK, and P38 subfamilies play crucial roles in inflammatory response in microglia [41]. MAPKs are also involved in the LPS-stimulated overproduction of COX-2 and iNOS [41,42]. We showed that BA can inhibit LPS-induced phosphorylation of P38, ERK, and JNK, indicating that MAPK can also be targeted by BA.
Indomethacin (INDO) is a kind of nonsteroidal antiinflammatory drugs (NSAIDs). Studies have confirmed that NSAIDs can effectively reduce incidence rate and risk of AD and delay the disease progression [43,44]. In this study, we used INDO as a positive control. Interestingly, our study showed that BA may play a similar role as NSAIDs in LPS-stimulated BV-2. Our results showed that INDO significantly reduced the release of inflammatory mediators and cytokines and the expression of miR-155 and multiple

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BioMed Research International signaling proteins. In addition, our results showed that both BA and INDO can interact with TLR4-MD2 complex in molecular docking analysis. Therefore, TLR4 pathway may be the target of both NSAIDs and BA in microglia. BA may be a valuable therapeutic candidate for the treatment of neuroinflammation and neurodegenerative diseases. In current research, microglia generated abundant inflammatory meditators, NO, IL-1β, and PGE2 after being activated by LPS. The abnormal production and release of neuroimmune factors NO, PGE2, and IL-1β further aggravated the damage of neurons. While microglia are exposed to BA (1.5-22.5 μM), the levels of inflammatory factors and the expression of related metabolic enzymes iNOS, COX-2, and miR-155 were reduced in a dose-dependent manner. As deeper research, the above changes were closely related with TLR4/MyD88/NF-κB and MAPK pathway. After being combined with TLR4, BA suppressed the upregulation of TLR4, MyD88, IRAK4, IRAK1, P-IκBα, and NF-κB P65 and the downregulation of IκBα in TLR4/MyD88/NF-κB pathway and further inhibited the phosphorylation of ERK, JNK, and P38 of MAPK family ( Figure 8). Finally, BA showed markedly antineuroinflammation effects in LPS-stimulated BV-2 microglia, which was similar to NSAID INDO.
In conclusion, this study demonstrated that BA exhibits anti-inflammatory function in LPS-stimulated BV-2 through inhibiting TLR4/NF-κB and MAPK activation and may reduce the expression of neuroimmune mediator NO, PGE2, and IL-1β. BA may be a potential therapeutic drug for neuroinflammation-associated disorders in the future.

Data Availability
The data presented in this study are available upon request from the corresponding author.