Scutellarin Acts via MAPKs Pathway to Promote M2 Polarization of Microglial Cells

Scutellarin, an herbal agent, is known to possess anti-oxidant and anti-inflammatory properties. In activated microglia, it has been reported that this is achieved through acting on the MAPKs, a key pathway that regulates microglia activation. This study sought to determine if scutellarin would affect the commonly described microglia phenotypes, namely, M1 and M2, thought to contribute to pro- and anti-inflammatory roles, respectively. This is in consideration of its potential effect on the polarization of microglia phenotypes that are featured prominently in cerebral ischemia. For this purpose, we have used an experimentally induced cerebral ischemia rat model and LPS-stimulated BV-2 cell model. Thus, by Western blot and immunofluorescence, we show here a noticeable increase in expression of M2 microglia markers, namely, CD206, Arg1, YM1/2, IL-4 and IL-10 in activated microglia both in vivo and in vitro. Besides, we have confirmed that Scutellarin upregulated expression of Arg1, IL-10 and IL-4 in medium supernatants of BV-2 microglia. Remarkably, scutellarin treatment markedly augmented the increased expression of the respective markers in activated microglia. It is therefore suggested scutellarin can exert the polarization of activated microglia from M1 to M2 phenotype. Because M1 microglia are commonly known to be proinflammatory, while M2 microglia are anti-inflammatory and neuroprotective effect, it stands to reason therefore that with the increase of M2 microglia which became predominant by scutellarin, the local inflammatory response is ameliorated. More importantly, we have found that scutellarin promotes the M2 polarization through inhibiting the JNK and p38 signaling pathways, and concomitantly augmenting the ERK1/2 signaling pathway. This lends its strong support from observations in LPS activated BV-2 microglia treated with p38 and JNK inhibitors in which expression of M2 markers was increased; on the other hand, in cells subjected to ERK1/2 inhibitor treatment, the expression was suppressed. In light of the above, MAPKs pathway is deemed to be a potential therapeutic target of scutellarin in mitigating microglia mediated neuroinflammation in activated microglia.


Introduction
Ischemic stroke is one of the most common life-threatening and debilitating neurological diseases affecting many world-wide. Stroke brings severe neurological impairments which may lead to perpetual disability [1]. Following a stroke, sequential pathological changes occur in the damaged brain areas. In view of this, many therapeutic strategies have been designed targeting to modify the acute pathological changes in the ischemic tissue at the cellular and molecular levels with the aim to restore brain homeostasis and functions. In the event of an ischemic stroke, microglia which act as a neuropathology sensor are readily activated [2]. It is well recognized that in response to ischemia, activated microglia undergo a series of phenotypic transformation and functional alterations, including proliferation and migration [3]. At the site of ischemic infarct and penumbral legion, activated microglia are admixed with infiltrated macrophage [4,5] described as M1 and M2 phenotypes [6]. Polarization of activated microglia is a two-edge sword, which involves the classical M1 and alternative M2 phenotype [7]. In the ischemic stroke environment, microglia act swiftly to form of an active immune defence. They migrate readily to the site of infarct to perform phagocytic clearance of cellular debris thus serving a protective function at the early onset of the ischemic damage [8]. Additionally, in response to neuronal injury, activated microglia primarily of M1 phenotype release a variety of proinflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), nitric oxide (NO) and reactive oxygen species (ROS) etc. [9]. On the other hand, in adverse conditions, some activated microglia maybe polarized to M2 phenotype which is known to produce large amounts of anti-inflammatory and neurotrophic mediators, such as interleukin-10 (IL-10), interleukin-4 (IL-4), CD206 and arginase-1 (Arg-1) etc., that help mitigate the inflammatory reaction M2 microglia phenotype is regarded to be neuroprotective as they foster the progressive neural tissue reconstruction and restoration of neurological function [10]. Therefore, suppressing M1 microglia activation has been widely considered a therapeutic option to attenuate or arrest undesirable neuronal damage and promote activated micrglia polarization toward M2 phenotype. In consideration of this, it is therefore essential to fully define the underlying mechanisms of key signaling pathways that regulate microglia activation. Therefore, suppressing M1 microglia activation has been widely considered a therapeutic option to attenuate or arrest undesirable neuronal damage and promote activated micrglia polarization toward M2 phenotype. In consideration of this, it is therefore essential to fully define the underlying mechanisms of key signaling pathways that regulate microglia activation [11]. The activated P38 MAPK and JNK signaling pathways are closely related to apoptosis; however, activated ERK1/2 acts on cell proliferation [12]. According to our previous study and recent studies, MAPKs signaling pathway is a major regulator of proinflammatory cytokines in lipopolysaccharide (LPS)stimulated microglia [13,14]. MAPKs signaling pathway is well known to be a major regulator of proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated microglia [15]. Among them, p38 MAPK and ERK appear to be primarily involved in the production of proinflammatory mediators in activated microglia. We have reported previously that scutellarin, a herbal compound, acts via MAPKs pathway to regulate the expression of iNOS, TNF-α and IL-1β in activated microglia, however, it remained uncertain whether scutellarin is able to promote M2 microglia polarization via MAPKs pathway [16].
Scutellarin, 5,6,4-Trihydroxyflavone-7-O-glucoronide is a flavone and the major active component of Erigeron breviscapus (Vant.) Hand-Mazz. Breviscapus is one of the most commonly used Chinese herbal medicines in Yunnan, accounting for 95 percent of the country's total production [17]. Increasing reports in recent years have shown the beneficial effects of scutellarin in different experimental animal and cell models owing to its anti-oxidant [18], anti-inflammatory [19], and calcium channel antagonist properties [20]. It has been reported that scutellarin can attenuate ischemic brain injury in patients and animal models. More specifically, it has been shown to significantly reduce the infarct size as well as improve neurological scores [3]. Additionally, we have shown that scutellarin was able to attenuate the expression of different proinflammatory mediators by activated microglia through MAPKs pathway, such as TNF-α, IL-1β and iNOS which are markers of M1 microglia [21]. However, It remains obscure whether scutellarin would act to promote M2 polarization to exert its anti-inflammatory function. The objective of this study was to explore if scutellarin would exert its anti-inflammatory effect though M2 polarization via regulating the MAPKs pathway in an experimentally induced stroke model as well in BV-2 microglial cells challenged with LPS. More importantly, the study may turn up the realities that the MAPKs pathway might serve as a therapeutic target to promote M2 microglia polarization for functional recovery following an ischemic stroke.

Ethics Statement
This study was performed in line with the principles of the Declaration of Helsinkik. In the handling and use of rats, ethical guidelines as described in the National Institutes of Health Guidelines for The Care and Use of Laboratory Animals were followed. All experimental protocols and use of animals were approved by Kunming Medical University and all efforts were made to minimize the number of rats used and their suffering. Ethics number: KMMU20220066.

Animals and Experimental Groups
Seventy-eight adult male SD rats, weighing 250-280 g, were provided by experimental Animal Center of Kunming Medical University. All animals were cared for in accordance with the National Institutes of Health Guidelines for The Care and Use of Laboratory Animals. The animals were randomly divided into sham-operated + saline (sham), middle cerebral artery occlusion (MCAO) + saline (MCAO), and MCAO + scutellarin(100 mg/kg) groups [22][23][24] (Table 1).
The rats in the MCAO group were anesthetized with pentobarbital sodium administered intraperitoneally. The middle cerebral artery was cauterized or permanently occluded in rats. To do this, a dental drill is used to drill a round hole 3 mm in diameter into the parietal bone(as reported previously by as [25]). The main middle cerebral artery(MCA) was exposed and occluded. In sham rats, the same procedure was performed, but the MCA from the group was not severed. If the rats died during or after surgery, they were excluded.

Injection of Scutellarin
The rats in MCAO + scutellarin groups were given an intraperitoneal injection of scutellarin (100 mg/kg dissolved in saline [22]; Cat. No. 131021, Shanghai Winherb Medical Technology, China) at 2 h before MCAO and at 12, 24, 48 and 60 h after MCAO. The rats were sacrificed at 1, 3 and 7d (n = 5.for each time point) after MCAO; for sham, n = 3 for each time point. In our preliminary study, we found that microglia response to ischemic cerebral cortex was strongest at 3d compared to other time points. In view of this, both Western blot and immunofluorescence labeling results in this study focused on this time point.

BV-2 Microglia Cell Culture and Treatment
BV-2 microglia (ATCC, USA) were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS) in a moist incubator with 5% CO 2 at 37℃. The cells were divided into control, LPS induced, inhibitor treatment, LPS + scutellarin, LPS + inhibitor, LPS + scutellarin + inhibitor groups. Inhibitors of MAPKs pathway includes p38 inhibitor(America, APExBIO, Cat. SB203580), JNK inhibitor(America, MCE, Cat.HY-12041) and ERK inhibitor(America, MCE, Cat.HY-112287). In all of the experiments, the cells were seeded at a density of 6 × 10 5 cells/well in six-well plates. The cells were pretreated with inhibitor(p38 inhibitor at a dosage of 10 μM; JNK inhibitor at dosage of 25 μM; ERK inhibitor at dosage of 7.5 μM as recommended by the manufacturer's instructions) for 1 h followed by washing thoroughly with phosphate buffered saline (PBS). After that, the cells were pretreated with scutellarin (0.54 μM [22][23][24]) for 1 h at 37 °C in a humidified incubator under 5% CO 2 . After incubation, the culture medium was discarded, the cells were washed with PBS and then incubated with LPS (1 mg/ml, Sigma-Aldrich, MO, USA) for 3 h. The medium was replaced with basic DMEM before treatment. As a control, the medium was replaced by basic DMEM cultured in 95% air and 5% CO 2 . Finally, medium supernatants were collected and kept at − 86 °C for ELISA and proteins were extracted from the cells for Western blot analysis.

Cell Migration Assay
The effect of scutellarin on BV-2 microglia cell migration was assessed using Transwell™ permeable support polycarbonate membrane inserts (8.0 µm pore size; Costar, USA), placed in a 24-well plate. BV-2 microglia cells were divided into three groups (Control, LPS, LPS-activated and treatment with scutellarin).After treatment with scutellarin at 0.54 mM concentration for 3 h, the cells were washed with PBS, trypsinized, and resuspended in basic medium. 200 µl cell suspension without FCS (3 × 10 4 cells) was seeded into the inside of the insert, and 600 µl DMEM with 20% FCS was added into each well of the 24-well plate. After incubation at 37 °C with 5% CO2 for 24 h, the medium was discarded and the cells remaining inside the insert were carefully removed. Cells were then fixed with 100% methanol for 15 min at room temperature and were stained with 0.5% of crystal violet at room temperature for 30 min. The cells were rinsed with distilled water until excess dye was removed. The images of migrated cells on the lower surface of the insert were captured at a magnification of 100x. Cells in five visual fields were counted on each insert using Image J software. Results are expressed as mean (± SD) of the number of cells per visual field.

Western Blot Analysis for Mcao Tissues and BV-2 Cells
A total of 39 rats were used for Western blot analysis. The sham-operated (n = 3) and MCAO rats given saline or scutellarin injections (n = 5), respectively, were sacrificed at 1d, 3d and 7d. The control or ischemic cortex was dissected and immersed in lysis buffer (1 × RIPA lysis buffer, proteaseinhibitor cocktail, and phosphatase-inhibitor cocktail) (Cell Signaling Technology) for 10 min on ice cubes. Tissue samples from various groups were homogenized with protein extraction reagent (Pierce, IL, USA) containing protease inhibitors.

Enzyme-linked Immunosorbent Assay (ELISA) for BV-2 Cells
For BV-2 culture, IL-10, IL-4 and Arg1 levels in supernatants were determined using commercially available ELISA kits according to the manufacturer's protocol (Abcam, CA, USA. Cat.ab255729 for IL-10, Cat.ab222274 for IL-6 and Cat.ab269541 for Arg1). Treatment concentrations were selected according to the cytotoxicity results, considering the pharmaceutical use. Sample supernatants were halfdiluted with sample diluent and the ELISA was carried out according to the manufacturers'recommendations. Briefly, diluted samples and standards were added to the wells and incubated at 37 °C for 90 min. Then, the plates were washed and detection antibody was added and incubated for 1 h at 37 °C. The plates were washed again

Double Immunofluorescence Labeling of Microglia in MCAO and BV-2 Cells
Thirty-nine rats in all groups were labeled with double immunofluorescence. After deep anesthesia with 3% pentobarbital sodium administered intraperitoneally, the rats were infused with 2% paraformaldehyde in 0.1 M phosphate buffer. The brain was removed and paraffin embedded. Coronal brain sections were cut at 8 μm thickness on a microtome. Sections were rinsed with PBS. To block non-specific binding proteins, tissue sections were incubated at room temperature (22-24 ℃) in 5% normal goat serum diluted with PBS for 1 h. After discarding the serum, the sections were incubated in a humidified chamber with primary polyclonal antibodies against CD206 ( After 3 rinses with PBS, the sections were mounted with a fluorescent mounting medium containing 4', 6-diamidino-2-phenylindole (DAPI). Colocalization was detected by confocal microscopy (Laser confocal scanning microscope, Leica SP5). BV-2 microglia were fixed with 4% paraformaldehyde 0.1 M PBS for 20 min. After washing with PBS, immunofluorescence staining was performed on the cover slips with adherent microglia. In each group, BV-2 microglia were incubated with the primary antibody at 4℃ overnight. Finally, the cells were incubated in FITC (1:200; SIGMA; Cat. No. SLPB1894V) /Cy3(1:200; Jackson Immuno-Research; Cat. NO. 125364)-conjugated secondary antibodies for 1 h at room temperature. After washing, the coverslips were mounted with a fluorescent mounting medium with DAPI. Colocalization was visualized by immunofluorescence microscopy (Zeiss cell observer, Zeiss CaT #1,026,470,987). The quantification of immunofluorescence intensity in the images was expressed as integrated density, which was quantified using Image J software; changes in intensity were then plotted as bar graphs (Table 2).

Statistical Analyses
The data were presented as mean standard deviation (M ± SD). First of all, the data was tested for homogeneity of variance. If the variance was uniform, one-way analysis of variance (ANOVA) was used to assess the statistical significance. If the variance was uneven, nonparametric test, such as H test, was used. When p < 0.05, the difference was considered statistically significant. Prism 5.0 statistical software was used to analyze all data.

Scutellarin Induced Immunofluorescence of M2 Markers CD206, YM, Arg1, IL-10 and IL-4 in Activated Microglia in MCAO Rats Sacrificed at 1d, 3d and 7d
Consistent with Western blot analysis, immunofluorescence labeling showed increased CD206, YM, Arg1, IL-10 and IL-4 immunoceactivity in activated microglia double labeled with lectin in MCAO rats. The immunofluorescence of all biomarkers was further enhanced in MCAO rats given scutellarin treatment. Of note, the immunofluorescence of all M2 microglia markers was most conspicuous when MCAO rats were pretreated with scutellarin and sacrificed at 3d (Fig. 2).

Scutellarin Inhibited Migration of Microglia in LPS-activated BV-2 Microglia
Transwell migration experiment in vitro was performed to assess whether scutellarin influences the motility of microglia. Migration of BV-2 microglia towards the lower compartment treated with scutellarin was significantly decreased compared with the LPS group; Migration of BV-2 microglia of LPS group towards the lower compartment was significantly increased compared with Control group (Fig. 3).

Scutellarin Upregulated Expression of Arg1, IL-10 and IL-4 in Medium Supernatants of BV-2 Microglia
By ELISA, with respect to IL-10, the anti-inflammatory effects were observed with LPS group given scutellarin pretreatment (5560.8 ± 268.1 pg/mL); with respect to IL-4, the anti-inflammatory effects were observed with LPS group given scutellarin pretreatment (2921 ± 139.4 pg/mL); with respect to Arg1, the anti-inflammatory effects were observed with LPS group given scutellarin pretreatment (2574 ± 239.4 pg/mL). The expression of Arg1, IL-10 and IL-4 in medium supernatants of LPS group given scutellarin pretreatment was significantly increased compared with LPS group. The results were consistent with the expression of intracellular protein detected by Western blot (Fig. 5).

Effects of Scutellarin on Arg1 and MAPKs Signaling Pathway Proteins P-JNK and P-P38 in Microglia
Double immunofluorescence staining with M2 microglia marker Arg1(green) and activated MAPKs pathway proteins p-JNK and p-P38 (red). The results revealed small numbers of inactive cells in the control group. Microglia with many cell processes hardly expressed M2 microglia markers (Arg1, green), while sedentary microglia almost did not express activated MAPKs pathway proteins (p-JNK and p-P38, red). No co-expression of M2 microglia marker and activated MAPKs pathway proteins was observed. In the LPS group, there were many activated microglia with few cell processes. The number of Arg1 immunopositive   Arg1(B, b), IL-10 (C, c), YM1/2 (D, d) and IL-4 (E, e) immunofluorescence (red) in lectin positive microglia (green) of MCAO 3d rats and MCAO 3d rats given scutellarin treatment. Note the marked increase in M2 microglia markers immunofluorescence in the activated microglia (b2-b3) in MCAO 3d rat; furthermore, it was noticeably augmented in activated microglia(c2-c3) by following scutel-larin treatment. Bar graph shows increased immunofluorescence in MCAO 3d rats by scutellarin. DAPI-blue. Scale bar = 75 μm. * and # represent significant differences. (P < 0.05); * when sham group is compared with MCAO3d; # when MCAO 3d group is compared with MCAO + S 3d group. The values represent the mean ± SD in triplicate. Characteristic cells are zoomed and indicated by red arrow cells of M2 microglia increased, but immunopositive cells of activated MAPKs pathway proteins p-JNK and p-P38 was increased, some exhibited Arg1 co-expression (yellow). After pretreatment with scutellarin, Arg1 expression was significantly increased. The expression of p-JNK and p-P38 was significantly decreased, which was statistically different from that in LPS group (Figs. 7, 8). Transwell migration assay shows that scutellarin reduces the migration of BV-2 microglia. Light microscopy images of LPS-activated BV-2 cells treated with scutellarin, LPSactivated BV-2 cells and control are shown. The quantitative analysis reveals a significant decrease in the migration of microglia with scutellarin treatment compared wtih LPS group. The quantitative analysis reveals a decrease in the migration of LPS-activated BV-2 cells compared with control. * and # represent significant differences in protein levels, P < 0.05; * when control group is compared with LPS group; # when LPS group is compared with the corresponding LPS + S group. The values represent the mean ± SD in triplicate. Scale bar = 100 µm Fig. 4 The CD206, YM, Arg1, IL-10 and IL-4 expression in BV-2 microglia was increased significantly when compared with the control group.The expression of the above markers was substantially increased significantly following pretreatment with scutellarin when compared with the LPS group. * and # represent significant differ-ences in protein levels, P < 0.05; * when control group is compared with LPS group; # when LPS group is compared with the corresponding LPS + S group. The values represent the mean ± SD in triplicate

Effects of Scutellarin on Arg1 and MAPKs Signaling Pathway Proteins p-ERK1/2 in Microglia
Double immunofluorescence staining with M2 microglia marker Arg1(green) and activated MAPKs pathway proteins p-ERK1/2 (red). The results revealed small numbers of inactive cells in the control group. Microglia with many cell processes hardly expressed M2 microglia markers (Arg1, green), while sedentary microglia almost did not express activated MAPKs pathway protein (p-ERK1/2, red). No coexpression of M2 microglia marker and activated MAPKs pathway proteins was observed. In the LPS group, there were many activated microglia with few cell processes. The number of Arg1 immunopositive cells of M2 microglia increased, and immunopositive cells of activated MAPKs pathway protein p-ERK1/2 was decreased, some exhibited Arg1 co-expression (yellow). After pretreatment with scutellarin, Arg1 expression was significantly increased. The expression of p-ERK1/2 was significantly decreased, which was statistically different from that in LPS group (Fig. 9).

Discussion
Cerebrovascular diseases have surpassed cancer as the main risk factor endangering human health and life safety; indeed, they account for a large proportion of morbidity and mortality [1]. In cerebral ischemia, neuronal death and apoptosis, glial activation and glia-mediated inflammatory response are featured prominently in the core area and penumbra of ischemia [26].

Characteristics of Activated Microglia Polarization and Its Mediated Inflammatory Response
In ischemic stroke, microglia retracted their branching processes and assumed an amoeboidic phenotype with a large round cell body. Meanwhile, the expression of CD11b protein in activated microglia is increased [27]. Activated microglia in cerebral ischemia have opposing functions. Activated microglia are engaged in phagocytosis in damaged area and penumbra zone; at the same time, they also release ELISA shows the expression level of Arg1, IL-10 and IL-4 in the cerebral cortex in medium supernatants of LPS group was increased when compared with the Control group; the expression was further increased following treatment with scutellarin when compared with LPS group. * and # represent significant differences. (P < 0.01); * when LPS group is compared with control; # when LPS-mediated BV-2 pretreatment with scutellarin group is compared with LPS group. The values represent the mean ± SD in triplicate  Arg1(B, b), IL-10 (C, c), YM1/2 (D, d) and IL-4 (E, e) expression was nepreguladed in LPSactivated BV-2 microglia in comparison to control (a2-a3). Scutellarin pretreatment led to further increase in expression of M2 microglia markers (c2-c3) in LPS-activated BV-2 microglia. The expression changes of markers were represented by the respective bar graphs. DAPI-blue. Scale bar = 50 μm. * and # represent significant differences in protein levels, P < 0.05; * when control group is compared with LPS group; # when LPS group is compared with the corresponding LPS + S group. The values represent the mean ± SD in triplicate. Characteristic cells are zoomed indicated by red arrow different neurotrophic and inflammatory factors so that the microenvironment in damaged area and penumbra zone is effectively maintained for tissue restoration [28]. In the early stage of ischemic stroke, activated microglia were of M2 phenotype. With the progress of the disease, they gradually transformed into M1 phenotype causing inflammation and cell toxicity [29]. Therefore, microglia are activated and are functional immediately after acute ischemic stroke. They then transform into M1 phenotype within 7 days [29]. If there is no proper or adequate therapeutic interference at this stage for ischemic stroke, M1 microglia would release inflammatory factors and cytotoxic factors, such as TNF-ɑ, iNOS and IL-1β, which can further activate a complex signaling cascade. Excessive inflammatory reaction can further activate microglia and astrocytes which would accelerate the glial cells to clean up cellular debris in damage and penumbra areas and aggravate the damage of neurons or apoptosis [30,31]. However, excessive inflammatory response can else lead to apoptosis or death of neurons in the penumbral area that still possess the ability to recover. This would aggravate the cerebral infarct damage caused by ischemic stroke thus making the recovery after ischemic stroke more difficult [32,33]. However, M2 microglia release anti-inflammatory factors, such as IL-10, TGF-β and IGF-1, which can attenuate inflammatory reaction, and effectively promote recovery in the penumbrae area and reduce the brain infarction volume after ischemic stroke. It would appear that IL-10 and TGF-β mRNA expression reaches peak after stroke peaking at 2-6 days; thereafter, the expression level was decreased. TGF-β secreted by activated microglia not only can it protect the damage of the nervous system, but also promote proliferation of glial cells and strengthen the biological function of microglia [34]. Therefore, the present study was aimed to identify an effective therapeutic strategy to promote polarization of M1 microglia into M2 microglia considered to be neuroprotective. It has been shown in many studies that scutellarin has the effect of inhibiting neuronal death or apoptosis. In addition, studies have shown that the cerebral infarct volume of MCAO rats treated with scutellarin is significantly reduced compared with that of sham group by TTC staining. In this study, we found that the expressions of neuroinflammatory factors, TNF-ɑ, iNOS and interleukin-1β in activated microglia were significantly reduced after scutellarin intervention, so that scutellarin could fully inhibit the excessive neuroinflammatory response in the brain and play a role of neuroprotective effect [3,[35][36][37][38][39][40]. Scutellarin pretreatment further increased the expression of Arg1(c2-c3), but the expression of p-JNK(c2-c3) was decreased in LPS-activated BV-2 microglia. The expression changes of p-JNK and Arg1 were represented by the respective bar graphs. DAPI-blue. Scale bar = 50 μm. * and # represent significant differences in protein levels, P < 0.05; * when control group is compared with LPS group; # when LPS group is compared with the corresponding LPS + S group. The values represent the mean ± SD in triplicate. Characteristic cells are indicated by red arrow

Effects of Scutellarin on Polarization of Microglia
Activated microglia are classified into classical activated state (M1) and alternative activated state (M2) [13]. In cerebral ischemia, M1 microglia are activated and show increased expression of toll-like receptors. The cell body appear to be more rounded and hypertrophic. Microglia reduced their processes and produce different proinflammatory factors, such as IL-1β, IL-6 and TNF-ɑ, as well as CCL2 and among other cytokines and chemokines. All that can cause excessive inflammatory reaction and have toxic effects on neurons, and tissue damage [41].
M2 microglia are divided into the M2a, M2b, M2c and M2a sub-phenotypes and are induced by IL-4 and IL-13. They exhibit smaller and few protrusions, which produce high levels of anti-inflammatory factors such as IL-4, IL-13 and transforming growth factor beta (TGF-β). This is accompanied by reduced expression of interleukin 12 (IL-12) but a high expression of IL-10. M2 phenotype inhibits excessive inflammatory reaction and promotes tissue repair and neuron regeneration, which can prevent excessive inflammatory reaction mediated by M1 microglia. Markers of M2 phenotype include arginase-1 (Arg1), and CD206 [42]. When IL-1β and LPS are applied at the same time, or when exposed to IgA immune complexes, microglia can activate to become M2b phenotype with immune function. The markers are shared by both M1 microglia and M2a microglia. Meanwhile,the expression of signal transduction and transcription activating factor 1 (STAT1), and nuclear transcription factor κB (NF-κB) is increased. Thus, M2b phenotype has the dual functional roles of pro-inflammatory/antiinflammatory. After phagocytosis of apoptotic cells, microglia showed an activated anti-inflammatory M2c phenotype. M2c microglia can help tissue remodeling and cell regeneration after the down-regulation of inflammatory response, and its markers such as CD16, CD206 and TGF-β [43].
In this study, Western blot results showed that scutellarin effectively promoted expression of M2 microglia markers CD206, IL-10, IL-4 YM1/2 and Arg1 in LPS-activated BV2 microglia. Furthermore, by immunofluorescence staining, LPS-activated microglia increased production of more neurotrophic factors (YM1/2 and Arg1) and neuroprotective factors (IL-10 and IL-4). Of note, the expression of M2 microglia markers was increased after pretreating the cells with scutellarin. Along with BV-2 microglia, we investigated the experimental results by using a rat MCAO model [44,45]. In agreement with our previous study, we found that scutellarin can significantly reduce the cerebral infarction volume Fig. 8 p-p38 and Arg1 immunoreactivit is upregulated (b2-b3) in LPSactivated BV-2 microglia in comparison to control (a2-a3). Scutellarin pretreatment further increased the expression of Arg1(c2-c3), but the expression of p-p38(c2-c3) was decreased in LPS-activated BV-2 microglia. The expression changes of p-p38 and Arg1 were represented by the respective bar graphs. DAPI-blue. Scale bar = 50 μm. * and # represent significant differences in protein levels, P < 0.05; * when control group is compared with LPS group; # when LPS group is compared with the corresponding LPS + S group. The values represent the mean ± SD in triplicate. Characteristic cells are indicated by red arrow of MCAO rat model. Additionally, scutellarin inhibited effectively inflammatory cytokines and cytotoxic factors secreted by M1 microglia [25]. The present study has shown that scutellarin, not only can it effectively inhibit the M1 microglia phenotype to reduce and excessive microglia-induced inflammatory reaction, but also promote M2 microglia polarization. In the latter, it is conceivable that large amounts of neurotrophic and neuroprotective factors would be produced to promote the neural functions. Already, many studies have shown that microglia polarization to M2 phenotype is pivotal to the functional recovery after ischemic stroke.

Scutellarin ActsviaMAPKs Pathway to Promote M2 Polarization
MAPKs signaling pathway plays an important role in the development of nervous system, and mediates neuroinflammatory response. Recent studies have shown that MAPKs signaling pathway is involved in central neuropathy, such as ischemic stroke and Alzheimer's disease, additionally, they play an important role in the pathophysiological processes such as apoptosis and inflammatory response [46]. As a classic signaling pathway of the nervous system, many studies have shown that the expression of various components of MAPKs signaling pathway is significantly altered during the ischemic stroke. It has been reported that this may be involved in the regulation of cell damage or repair [47][48][49][50]. Immediately after the cerebral ischemia, the G protein-coupled receptor activates protein kinase C (PKC) pathway, adenylate cyclase (AC) pathway, phosphatidylinositol kinase (P13-K) pathway, tyrosine receptor pathway and calmodulin (Ca 2+ CaM) pathway among others, may activate the MAPKs signaling pathway, followed by the next cascade reaction [51]. Studies have found that MAPKs signaling pathway plays an important biological role in both transient and permanent ischemia in ischemic encephalopathy. Many signaling cascades have been reported at different sites of ischemia and hypoxia injury. If the neurons and glial cells in the central brain region were damaged first, the activation sequence of signal transduction pathway was ERK (1 min to 2 h after injury, mainly neurons) [47,48], c-jun (30 min to 6 h after injury, mainly neurons) [52] and p38 (2d to 4d after injury, mainly glial cells) [53,54]. It is described that the activation of the pathways above can promote the neuron death. In the rat MCAO model, the expression of ERK, JNK and p38 was significantly increased at different time points after ischemia, thus confirming that cerebral ischemia can readily Fig. 9 p-ERK1/2 and Arg1 immunoreactivit is upregulated (b2-b3) in LPS-activated BV-2 microglia in comparison to control (a2-a3). Scutellarin pretreatment further increased the expression of Arg1 andp-ERK1/2(c2-c3) in LPS-activated BV-2 microglia. The expression changes of p-ERK1/2 and Arg1 were represented by the respective bar graphs. DAPI-blue. Scale bar = 50 μm. * and # represent significant differences in protein levels, P < 0.05; * when control group is compared with LPS group; # when LPS group is compared with the corresponding LPS + S group. The values represent the mean ± SD in triplicate. Characteristic cells are indicated by red arrow trigger the phosphorylation of MAPKs. The results suggested that MAPKs signaling pathway is involved in the signal transduction of the early pathophysiological mechanism of cerebral ischemia [55]. Recently, it has been reported that the expressions changes of signal cascade reaction in the damaged core area and the surrounding penumbral area were not completely consistent. The phosphorylated JNK was significantly increased in the central area, while the p38 activity was significantly increased in the penumbral area [56]. Wang et al. showed that the expression of phosphorylated ERK1/2 was increased in the ischemic core and peripheral areas after 30 min in MCAO mouse models [52]. In our previous studies both in vivo and in vitro, Western blot results showed that in the rat MCAO model, the expression of p-p38 p-JNK and p-ERK in the cerebral cortex was significantly increased. Additionally, the MAPKs signaling pathway protein p38 was phosphorylated and increased in the LPS-activated BV-2 microglia. Concomitantly, the expression of p-JNK and p-ERK was significantly increased. Immunofluorescence staining showed that p-p38 p-JNK and p-ERK expression was highly expressed in the rat MCAO model and LPS-activated microglia [25]. All this further demonstrated that MAPKs signaling pathway is involved in a series of pathophysiological changes after cerebral ischemia. Separately, Su et al. reported that MAPKs signaling pathway was closely related to the polarization of M2 macrophages/microglia. When p38 and JNK inhibitors were used, we expected the expression of p-p38 and p-JNK was significantly down-regulated. The expression of CD206 and Arg1, markers of M2 macrophage/microglias was increased. In other words, when p38 and JNK were inhibited, macrophages/microglia would polarize to the M2 macrophage/microglia. However, when ERK protein was inhibited, the expression of CD206 and Arg1 markers for M2 macrophage/microglia was significantly reduced, indicating that the number of M2 macrophage/microglia is reduced when ERK protein was inhibited [57]. Together the results suggest that MAPKs signaling pathway is closely related to not only the occurrence and development of M1 microglia mediated inflammatory response, but also exerts neurotrophic and neuroprotective effects mediated by M2 microglia in cerebral ischemia injury. Scutellarin possesses anti-oxidative and anti-inflammatory properties. It acts against anti-neuronal apoptosis, thus exerting neuroprotective effects. Presently, different preparations of scutellarin are widely used in the clinical treatment of cardiovascular diseases such as hypertension, angina pectoris, coronary heart disease, stroke etc. However, its complex mechanism of action remains to be fully explored. Arising from the many studies using different experimental paradigms, it is evident that the pharmacological activity of scutellarin involves multiple signaling pathways. Adding to this conundrum is the precise target of pharmacological action of scutellarin which remains obscure. In study by Tang H, et al., in the ischemia reperfusion injury in rats, it was reported that the expression of glycine γ-aminobutyric acid (GABA) and Tau protein (Tau) improved the activities of Ca2 + -ATP kinase, Na + , K + -ATP kinase and neuron injury [38]. The present results have shown that scutellarin not only inhibited inflammatory response through MAPKs signaling pathway, but also promoted the polarization of M2 microglia through MAPKs signaling pathway, thus showing its effect of neuroprotection and nerve repair. In vivo, it was found that the expression of specific markers in activated M2 microglia was increased after the treatment of rat MCAO model with scutellarin. This was also evident by immunofluorescence staining, which showed that M2 marker-positive cells co-expressed with microglia.
The present results have shown that in LPS stimulated BV-2 microglia pretreated with scutellarin, the expression of the M2 markers was significantly increased. Of note in BV-2 microglia pretreated with p38 inhibitor and JNK inhibitor, the M2 microglia marker expression was significantly increased; on the other hand, with ERK1/2 inhibitor, the expression of M2 markers was decreased. By double immunofluorescence staining, it was found that the number of M2 microglia markerpositive cells was increased. The number of p-p38 and p-JNK positive cells was decreased, whereas that of p-ERK1/2 positive cells was increased after scutellarin pretreatment. The present study showed that the p38 pathway is the most important member of the MAPKs family to control the inflammatory response. It can promote the activation and aggregation of white blood cells, and regulate the activity of transcription factors and inflammatory factors, thus playing a key role in the regulation of inflammatory response. In microglia activation, MAPKK is activated through some intermediate link. Activated-MAPKK regulates the activity of p38 phosphorylase, leading to phosphorylation of p38 and p-p38 is then translocated to the nucleus to activate inflammatory factors such as TNF-α, iNOS and etc. [37,47,58]. Concurrently, p38 and JNK regulate the M1/M2 polarization through the cytomembrane-5, -nucleotide enzyme, shifting the activated microglia polarization toward the M1 microglia and showing pro-inflammatory Note scutellarin (s) alone not only suppressed the expression level of p-p38,but also promoted M2 microglia markers. * and # denote significant difference, P < 0.05; * when LPS + S group is compared with the LPS group; # when LPS + I + S compared with LPS + S group. The values represent the mean ± SD in triplicates effects [59]. Zhang, et al. found in their study in LPS-activated microglia that 4,5, 7-trihydroxy flavonone is dependent on MAPKs signaling pathway to promote the polarization of M2 microglia, especially the p38 and JNK signaling pathways. When p38 and JNK signaling pathway proteins are inhibited, leading to signaling pathway inactivation, the selective activators of p38 and JNK inhibit 4,5, 7-trihydroxy flavonone promote the polarization of microglia to M2 microglia [60]. Qu, et al. found that dexmedetomidine can effectively promote M2 polarization of activated microglia by inhibiting p38 and JNK, thereby reducing the inflammatory response in Parkinson's and Alzheimer's rat models [61]. The above results suggest that p38 and JNK signaling pathways can inhibit the polarization of M2 microglia, thus inhibiting the neurotrophic and neurorepair functions of activated microglia. This would ultimately aggravate the neuroinflammatory response and cause greater neural damage. As an effective inhibitor of p38 and JNK signaling pathways, we showed here that scutellarin can significantly promote the polarization of M2 microglia. Therefore, inhibiting p38 and JNK signaling pathways by scutellarin may prove to be a potential therapeutic strategy to foster the M2 polarization that is neuroprotective essential for tissue repair. ERK1/2, the first member of MAPKs signaling pathway family, is closely related to cell survival and proliferation [62]. ERK can regulate cell biological functions such as cell growth, proliferation, differentiation and apoptosis, and may play an important role in cerebral neuropathy and regulate the development of the disease. However, the exact role of ERK 1/2 in the death or survival of nerve cells is still controversial. Su et al. reported that high glucose environment and TGF-β could effectively promote the polarization of activated microglia to M2 microglia. However, when Su et al. inhibited ERK protein, the expression of markers CD206 and Arg1 of M2 macrophages/microglia was significantly reduced. This indicated that when ERK was inhibited, M2 macrophages/ microglia were significantly decreased [57]. Separately, it was found that apoptotic SKOV3 cells stimulated the polarization of M0 macrophages to M2 macrophages by activating * and # denote significant difference, P < 0.05; * when LPS + S group is compared with the LPS group; # when LPS + I + S compared with LPS + S group. The values represent the mean ± SD in triplicates the ERK signaling pathway, and promoted the proliferation and migration of ovarian cancer cells by Zhang et al. When the ERK1/2 signaling pathway was inhibited, the number of M2 macrophages decreased, and the proliferation and migration ability of ovarian cancer cells also decreased [63]. In this study, we confirmed that scutellarin can inhibit migration of LPS-activated BV-2 microglia. Cao demonstrated that hypoxia of tumor cells selectively promoted the polarization of macrophages to M2 microglia through the activation of ERK signal, thereby enhancing the metastasis of non-small Fig. 13 A schematic diagram displays modulatory mechanisms of microglia polarization following pretreatment with scutellarin after ischemic stroke. scutellarin binds to membrane receptors, such as the cytokine receptors EGF, TGF-β and IGF, to trigger pro-inflammatory and anti-inflammatory cellular signaling pathways. These pathways include the PI3K/PKC/ERK1/2, HPK1/p38, and JNK pathways. The downstream targets JNK and p38 are released and translocated from the cytoplasm to the nucleus following the phosphorylation, where they initiate the transcription of proinflammatory genes. M2 microglia can be activated by scutellarin, which promotes ERK1/2 mobilization from the cytoplasm to the nucleus following the phosphorylation. Subsequently, ERK1/2 inhibits transcription of pro-inflammation genes and promotes the transcription of antiinflammatory genes. Additionally, JNK and p38 inhibit transcription of anti-inflammation gene and promotes the transcription of pro-inflammatory genes. Furthermore, scutellarin not only inhibits the activation of JNK and P38, but also promotes the activation of ERK1/2. Thus, scutellarin promotes the expression of IL-10, IL-4, Arg1, YM1/2, but induces the expression of iNOS, TNF-ɑ and IL-1β. In short, scutellarin acts via MAPKs pathway to promote M2 polarization of microglial cells cell lung cancer [64]. These studies confirmed that ERK signaling pathway plays a crucial role in the process of M2 polarization of microglia/macrophages. We have shown in the present study that scutellarin could promote the expression of M2 microglia markers in LPS-activated BV-2 microglia. More importantly, we have shown that scutellarin also promoted the expression of p-ERK1/2. In LPS stimulated BV-2 microglia pretreted with the ERK1/2 inhibitor HY-112287, the expression of p-ERK1/2 was decreased, and remarkably, expression of M2 microglia markers was also down-regulated. This is contrary to the findings of Li, Zhang and Shan, who reported activation of ERK1/2 signaling pathway can inhibit the polarization of microglia/macrophages to M2; and conversely, inhibition of ERK1/2 signaling pathway can promote the polarization of microglia/macrophages to M2 [36,61,65,66]. The discrepancy in results may be attributed to the difference between experimental conditions and detection indicators, but the underlying mechanism requires further investigation.

Conclusion
All in all, the present results have shown that LPS-activated BV-2 microglia and rat cerebral ischemia-activated microglia over-expressed M2 microglia markers CD206, Arg1, YM1/2, IL-4 and IL-10 and inhibit migration of LPS-activated BV-2 microglia. However, in MCAO and BV-2 microglia given scutellarin pretreatment the expression of M2 microglia markers was increased. Therefore, It is justified to suggest that this would reduce the microglia mediated inflammatory response, because M2 microglia are known to exert neuroprotective effect and promote tissue repair. The detection of MAPKs signaling pathway along with the pretreatment of p38 inhibitors (SB203580), JNK inhibitors (HY-12041) and ERK1/2 inhibitors (HY-112287) confirmed that scutellarin may directly promote the polarization of M2 microglia and its expression of neurotrophic and protective mediators by inhibiting the JNK and p38 signaling pathways. Moreover, scutellarin promotes the polarization of M2 microglia by augmenting the ERK1/2 signaling pathway (Fig. 13).

Supplementary Information
The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s12035-023-03338-3. Availability of data and material The date and materials supporting the conclusions of this study are available from the corresponding author on reasonable request.

Declarations
Ethics statement All experiments were approved by the Experimental Animal Care and Use Committee of Kunming Medical University, and were in agreement with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

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