Introduction
Ischemic stroke, reduced cerebral blood flow caused by an arterial thrombus, which afflicts approximately 795, 000 individuals worldwide each year, and the number of patients suffering stroke is rising [
1,
2]. However, the therapeutic options for stroke are desperately limited. Slow and incomplete recovery is compounded by limited drug treatments that facilitate the recovery process. Acute care mainly depends on thrombolytic treatment by administrating tissue plasminogen activator (tPA), although the narrow therapeutic time window of within 6 h ensures that only a small fraction of patients benefit [
3]. Furthermore, reperfusion of the ischemic brain is considered a secondary injury, and the efficacy of tPA treatment is inconsistent among individuals. Consequently, novel and effective drug treatments that improve the symptoms and sequelae of stroke, especially in acute phases, are urgently needed [
4].
Inflammation is a critical component of the secondary injury resolution process under ischemic brain insult. In the event of stroke, microglia residing in the central nervous system (CNS) are the first responders to cerebral ischemia, and they are activated within several minutes [
5,
6]. Subsequently, infiltrating immune cells, including monocytes, macrophages, neutrophils, lymphocytes, and natural killer (NK) cells, pass through the disrupted blood-brain barrier (BBB) and secrete a plethora of cytokines to promote the progression of inflammation [
7,
8]. Therefore, understanding the contribution of those immune cells to the immunomodulation reaction is a prerequisite for therapeutic intervention. In particular, resident microglia, as well as invading macrophages, are commonly recognized as vital contributors to inflammatory circumstances under the pathophysiology of ischemic stroke [
9]. Morphological transformation and common antigens expressed on both cell types give them certain overlapping functions, such as phagocytosis and analogous polarization abilities toward M1- or M2-like phenotypes [
6,
10]. The diverse phenotypes distinctively impact the expression of inflammatory cytokines, which are correlated with neuronal functions. Generally, the M1 microglia/macrophages, marked CD16/32 and CD68, are commonly characterized by pro-inflammatory effects accompanied by the release of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and interleukin-6 (IL-6), whereas microglia with the M2 phenotype (marked by CD206) secrete transforming growth factor beta (TGF-β), insulin-like growth factor 1 (IGF-1), interleukin (IL)-10, and IL-4 to rescue local inflammation and favor tissue repair [
11,
12]. Furthermore, the M2 phenotype is divided into three subsets: M2a (marked by CD206 and arginase-1) is associated with anti-inflammation and immunity against parasites, M2b (marked by CD86 and SOCS3) is related to adaptive immunity, and M2c (marked by TGF-β and IL-10) facilitates tissue regeneration [
13]. Notably, the unique temporal and spatial changes in microglia and macrophages under pathophysiological conditions indicate that each cell type has indispensable and complementary roles in the context of ischemic stroke. A growing number of studies have focused on the phenotypic moderation of microglia/macrophages rather than the exclusive suppression of their activation. However, the participation of invading immune cells is usually obscured.
Fibroblast growth factor 21 (FGF21), as a novel and potent regulator of glucose uptake and lipid metabolism, is predominantly expressed in both the rodent and human liver and thymus [
14]. Compared with other FGFs, FGF21 scarcely has mitogenic effects and may be the only FGF that can cross the BBB due to its weak binding affinity with heparin [
15,
16]. To date, accumulating evidence has indicated that FGF21 exhibited therapeutic effects in multiple disease models, such as atherosclerosis [
17], diabetic cardiomyopathy [
18], age-related disorders [
19], and enhanced neurite outgrowth [
20]. Although the mechanisms underlying its pharmacologic actions remain elusive, the therapeutic mechanism of FGF21 primarily involves anti-inflammation [
21], energy metabolism and vascular homeostasis [
22], oxidative stress [
23], and tissue repair [
24]. FGF21 mediates these effects by interacting with FGF receptors (mainly FGFR1 and FGFR2) via binding a cofactor, β-klotho, a single-pass transmembrane protein from the klotho family [
25]. FGFR1 and its coreceptor β-klotho have also been reported to be widely observed in brain tissue, including microglia [
26]. Therefore, FGF21 may have a potential impact on microglia. Additionally, our previous study demonstrated that FGF21 effectively upregulated the downstream effector peroxisome proliferator-activated receptor (PPAR)-γ in human bone marrow endothelial cells [
27] and activated PPAR-γ was closely associated with microglial phenotype and inflammatory regulation in the CNS [
28]. Moreover, a recent study confirmed that FGF21 suppressed macrophage-mediated inflammation by nuclear factor-erythroid 2-related factor 2 (Nrf2) and the NF-κB signaling pathway in a collagen-induced arthritis model [
29].
Therefore, FGF21 is likely to regulate the stroke-induced immune-inflammatory response by modulating microglia and macrophages both in the brain and in peripheral tissue in favor of functional recovery. Recently, recombinant human FGF21 (rhFGF21) has been reported to modulate the shift of microglia from M1 activation to M2 activation at the subacute and chronic stages of db/db mice with middle cerebral artery occlusion (MCAO), which is accompanied by the activation of PPAR-γ in the peri-infarct area [
30]. However, the potential mechanism by which FGF21 acts on microglia/macrophages and the dynamic alteration of microglia/macrophages and their phenotypes have not been elucidated. In the current study, we investigated the neuroprotective effect and promising mechanism by which FGF21 ameliorates inflammatory responses and microglia/macrophage polarization in a mouse model of MCAO.
Materials and methods
Reagents and antibodies
rhFGF21 was supported by the laboratory of Biotechnology Pharmaceutical Engineering at Wenzhou Medical University and synthesized on the basis of the study previously reported [
31]. Antibodies in flow cytometry analysis involving CD3-PE (17A2, 100206), CD3-PerCP-Cy5.5 (17A2, 100217), CD8-FITC (53-5.8, 140404), CD4-APC (GK1.5, 100412), F4/80-FITC (BM8, 123108), NK1.1-APC (PK136, 180710), Ly6G-PE (1A8, 127608), Ly6C-APC (HK1.4, 128016), CD45-APC (103112), CD11b-PE (101208), CD206-APC (C068C2, 141708), CD206-FITC (C068C2, 141704), CD68-PerCP-Cy5.5 (FA-11, 137014), CD86-PE (GL-1, 105008), CD45-PE/Cy7 (30-F11, 103114), CD11b-PE/Cy7 (M1/70, 101216), and CD11b-PerCP-Cy5.5 (M1/70, 101230) were purchased from BD Biosciences (San Jose, CA, USA) and CD45-APC (OX33, 17046280), CD11b-PE (OX42, 12011080), and CD86-FITC (24F, 11086081) were purchased from eBioscience (San Diego, CA, USA).
Antibodies applied in immunofluorescence including CD16/32(AF1460) and CD206 (AF2535) were purchased from R&D Systems (Minneapolis, MN, USA) and Iba1 (019-19741) purchased from Wako pure chemical corporation (Tokyo, Japan).
The primary antibodies applied in western blot including anti-NF-κB (3033T), anti-FGFR1 (ab824), anti-p-FGFR1 (ab59194), anti-PPAR-γ (ab28364), and anti-β-Actin (ab8227) were purchased from Cell Signaling Technology (Danvers, MA, USA) or Abcam (Cambridge, MA, USA). The secondary antibodies used were donkey anti-rabbit IgG H&L (HRP) (ab150075) or goat anti-mouse IgG H&L (HRP) (ab150115), which were commercially purchased from Abcam (Cambridge, MA, USA).
Corresponding reagent or kit applied in this study include trizol reagent (Qiagen, Duesseldorf, Germany), PrimeScriptTM RT Reagent Kit (TaKaRa, Shiga, Japan), iQTM SYBR Green supermix (Bio-Rad, Hercules, CA, USA), miRNeasy Micro Kit (Qiagen, Duesseldorf, Germany), QuantiTect Reverse Transcription kit (Qiagen, Duesseldorf, Germany), TaqMan® Gene Expression Assays (ThermoFisher Scientific, Fremont, CA, USA), Neural Tissue Dissociation Kits (Miltenyi Biotech, Bergisch Gladbach Germany), and Fluoroshield mounting medium with DIPI (Abcam, Cambridge, MA, USA).
Animal groups and drug administration
C57BL/6 mice (20–25 g) were purchased from the Animal Center of the Chinese Academy of Science (Beijing, China), and all surgical procedures and experimental protocols were approved by the Animal Care and Use Committee of Wenzhou Medical University. All animals were randomly assigned to the following three groups by a randomized block design: sham group, MCAO group, and MCAO+rhFGF21 group. In the sham group, mice were subjected to the same anesthesia and surgical procedures as the other groups but the filament was not inserted. In the MCAO+rhFGF21 group, the mice were intraperitoneally injected with rhFGF21 once per day at a dose of 1.5 mg/kg for 7 consecutive days beginning at 6 h after reperfusion.
Transient focal cerebral ischemia and reperfusion model preparation
The surgical procedures to establish the MCAO model were based on the intraluminal filament technique [
32]. Briefly, the mice were anesthetized by isoflurane and placed on a heating blanket to maintain body temperature at 37 ± 0.5 °C. A midline incision was made to expose the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The CCA was temporarily closed and a monofilament (0.18 ± 0.01 mm, Jialing Biotechnology Company, Guangdong, China) was inserted into the ICA through the ECA until it reached the middle cerebral artery, and it was left for 60 min. Laser Doppler flowmetry (model P10, Moor Instruments, Wilmington, DE, USA) was used to monitor whether cerebral flow dropped to lower than 20% of the pre-ischemic level. The occluding filament was returned to the ICA to achieve reperfusion after 60 min of occlusion. In the MCAO model, the mortality rate was 9.3% (23 of total 246) and exclusion rate was 10.9% (11 of the total 246 experienced inadequate reperfusion, and 16 of the total 246 reached the criteria limitations set for the modified Neurological Severity Score (mNSS) scoring system, i.e., mNSS scores < 6 or > 13 at 24 h after MCAO were excluded).
Neurological function assessment
The mNSS, rotarod test, corner-turning test, and adhesive removal test were performed to assess neurodeficits, motor coordination, sensorimotor asymmetry, and feeling functions at 1, 3, 7, and 14 days after surgery. All animals received training for 3 consecutive days before suffering ischemia-reperfusion injury. Behavior data were recorded as preoperative data at the second day after training. Subsequently, a transient focal cerebral ischemia and reperfusion model about MCAO was performed on the following day. The assessment procedure was performed by the same investigator who was blinded to the group identity of each mouse.
Quantitative real-time PCR
Total mRNA was isolated from the cortex samples around the infarcted zone using trizol reagent according to the manufacturer’s instructions. The cDNA was synthesized by the PrimeScript
TM RT Reagent Kit (TaKaRa, Shiga, Japan) following the manufacturer’s protocol. PCR assays were performed on a CFX Connect Real-time System (Bio-Rad, Hercules, CA, USA) using SYBR Green. The primers used in this study are shown in Table
1. Additionally, total RNA was extracted from the sorted microglia using the miRNeasy Micro Kit according to the manufacturer’s protocol. cDNA was transcribed with a Reverse Transcription kit and amplified in step one using Gene Expression Assays for TNF-α, IL-6, IL-1β, and TGF-β. The reaction volume was set to 20 μl and performed at 50 °C for 2 min, 95 °C for 20 s, followed by 40 cycles of 1 s at 95 °C and 20 s at 60 °C. Data were analyzed using the 2
-∆∆Ct method, and the expression level of relative mRNA was then reported as the fold difference.
Table 1
PPrimers sequences for qRT-PCR
β-Actin | Forward CACTGCAAACGGGGAAATGG |
Reverse TGAGATGGACTGTCGGATGG |
IL-1β | Forward GCG CTG CTC AAC TTC ATC TTG Reverse GTG ACA CAT TAA GCG GCT TCA C |
IL-6 | Forward CTC CCA ACA GAC CTG TCT ATA C |
Reverse CCA TTG CAC AAC TCT TTT CTC A |
TNF-α | Forward GTG ACA AGC CTG TAG CCC A |
Reverse ACT CGG CAA AGT CGA GAT AG |
Cox-2 | Forward CCCTTGGGTGTCAAAGGTAA |
Reverse GCCCTCGCTTATGATCTGTC |
MCP-1 | Forward ATAGCAGCCACCTTCATTCC Reverse TTCCCCAAGTCTCTGTATCT |
CXCL1 | Forward ACC GAA GTC ATA GCC ACA CCTC AAG Reverse TTG TCA GAA GCC AGC GTT CAC C |
IL-10 | Forward TTC TTT CAA ACA AAG GAC CAG C |
Reverse GCA ACC CAA GTA ACC CTT AAA G |
TGF-β | Forward TTGCTTGAGCTCCACAGAGA |
Reverse TGGTTGTAGAGGGCAAGGAC |
Flow cytometry
After the mice were euthanized, fresh brain, spleen, and blood tissues were harvested for single-cell suspension preparation for subsequent single-cell analysis using fluorochrome-conjugated antibodies. Spleen and blood tissues were dissociated into single-cell suspensions as previously described [
33,
34]. Splenocytes were dissociated by sieving through a 70-μm filter, and then lysing solution (BD Bioscience, CA, USA) was used to deplete red blood cells in the spleen and blood. Brain mononuclear cells were prepared by Neural Tissue Dissociation Kits (Miltenyi Biotech, Bergisch Gladbach Germany) according to its protocol. Briefly, the ischemic hemisphere of the brain was collected and dissected into small pieces. The pieces were pipetted back into an appropriate-sized conical tube, rinsed with cold Hank’s balanced salt solution (HBSS), and then centrifuged (300
g, 2 min) at room temperature. After the supernatant was carefully aspirated, preheated enzyme mix1 (37 °C, 10 min) in a Neural Tissue Dissociation Kit was added to digest tissue pieces for 15 min, and then preheated enzyme mix 2 (37 °C, 10 min) was added to the tissue sample for 10 min. Subsequently, HBSS was used and single pellets were isolated by passing through a 30-μm cell strainer. Cell pellets obtained from the spleen, blood, and brain were washed and incubated with antibodies targeting CD3, CD8, CD4, F4/80, NK1.1, Ly6G, Ly6C, CD45, CD11b, CD206, CD68, and CD86, and tagged with phycoerythrin (PE), fluorescein isothiocyanate (FTIC), allophycocyanin (APC), PerCP-Cy5.5, or PE-Cy7. Antibody staining was performed following the manufacturer’s protocol. Fluorescence-minus-one (FMO) controls were used to determine the gate of each antibody. Flow cytometry analysis was conducted using a FACS Aria flow cytometer (BD Bioscience, CA, USA), and data were analyzed by FlowJo software (Informer Technologies, USA).
Sorting of microglia and macrophages
Microglia of the mouse brain tissue were sorted by magnetic cell sorting (MACS) in combination with fluorescence-activated cell sorting (FACS). Single-cell suspensions of the brain tissue were prepared as described above (“Flow cytometry” section). Cells were stained with APC-conjugated anti-mouse CD45 antibody and PE-conjugated anti-mouse CD11b for 30 min at 4 °C. Unstained antibody was washed in PBS and cells were incubated with anti-PE microbeads at 4 °C for 15 min. HBSS was used to wash off unlabeled microbeads. Cells labeled with primary antibody conjugated to PE were enriched by using MACS columns (Miltenyi Biotech, Bergisch Gladbach Germany) according to the explanatory memorandum, and then targeted microglia were gathered using the FACS Aria cell sorting system. Resident microglia were identified as the CD45intCD11b+ population, whereas infiltrated macrophages in the CNS were identified as the CD11b+CD45highF4/80+ population. In addition, cell sorting of macrophages from the spleen was performed following the method in the “Flow cytometry” section to obtain the cell suspensions of the spleen. Then, cells were stained with APC-conjugated anti-mouse CD45 antibody, PE-conjugated anti-mouse CD11b, and FITC-conjugated anti-mouse F4/80 30 min at 4 °C. Unstained antibody was washed in PBS, and then targeted macrophages defined as the CD45highCD11b+ F4/80+ population were gathered using the FACS Aria cell sorting system. Isolated cells were collected in trizol reagent, vortexed, and kept at − 80 °C for further experiments.
Isolation of primary microglia
Primary rat microglia culture was isolated as previously reported [
35]. In brief, the cerebral cortices separated from neonatally 1-day-old rats and meninges were removed. Trypsinization was used to digest the striped cortical tissues for 30 min, and 70-μm nylon mesh cell strainer was used to obtain the mixed cortical cells. Cells were maintained in DMEM/F12 with fetal bovine serum (FBS), penicillin, and streptomycin (Gibco, Grand Island, NY, USA). Culture media were changed every 3 days until achieving a confluent monolayer at approximately 15 days. For the isolation of primary microglia, mild trypsinization was added to isolate microglia from the mixed glial cells. Purified microglia were cultured at 37 °C under atmosphere condition for further experiments.
Oxygen-glucose deprivation (OGD)
To establish an ischemic-like condition in vitro, primary microglia were subjected to OGD as previously reported [
36]. Briefly, microglia were cultured with serum-glucose-deprived cultures and placed in a hypoxic chamber with 95% nitrogen and 5% CO
2 for 5 min and sealed tightly. Subsequently, the chamber moved to an incubator under 5% CO
2/37 °C for 3 h. After the OGD treatment, serum and glucose-free medium were exchanged by glucose-containing medium with or without rhFGF21, which was followed by incubating with 95% air and 5% CO
2 for 5 h and then analysis by qRT-PCR.
Cell culture and treatment
Primary cultured microglia and the BV2 cell line were used to characterize the effect of rhFGF21 on microglial polarization, inflammation cytokine release, and NF-κB and PPAR-γ signaling activation. Cells were exposed to lipopolysaccharide (LPS) to induce polarized microglia and inflammatory secretion [
37]. Briefly, microglia were treated with LPS (250 ng/mL) in the presence and absence of rhFGF21 (100 nM) or PD173074 (10 μM) for 4 h. Gene assays (involving IL-1β, iNOS, TNF-α, IL-6, CD86, CD206, Arg-1, IGF-1, and IL-10) were then detected in LPS-stimulated or OGD-treated primary microglia by qRT-PCR, and the effects of rhFGF21 on transcriptional activity of NF-κB in primary microglia were detected using immunofluorescence. Additionally, the polarization of microglia was analyzed by assessing the expression of the M1 marker CD86, which was identified by FACS staining.
Western blot
Total proteins of LPS-treated BV2 cells were purified by RIPA lysis supplemented with a protease and phosphatase inhibitor mixture. Protein concentrations were measured with a Bradford Protein Detection Kit. Then, 60 μg of proteins from the samples and positive controls were separated on sodium dodecyl sulfate (SDS) polyacrylamide gels by electrophoresis. Subsequently, proteins were transferred onto PVDF membranes followed by blocking with primary antibodies FGFR1 (1:1000), p-FGFR1 (1:1000), NF-κB (1:1000), PPAR-γ (1:400), and β-Actin (1:500) overnight at 4 °C. Then, the membranes were incubated with secondary antibody donkey anti-rabbit IgG or goat anti-mouse IgG at a 1:10,000 dilution for 1 h at room temperature. Finally, the protein bands were detected with Image Lab software using Gel Doc Imager (Bio-Rad, Hercules, CA, USA) and the expression of target proteins was normalized against β-Actin.
Immunofluorescence analysis
Immunofluorescence staining was performed on paraffin brain sections as previously described [
38]. Briefly, non-specific binding of antibodies was blocked with 5% BAS for 1 h at 37 °C and the sections were then incubated with one or more primary antibodies against CD16/32, CD206, Iba1, or NF-κB in a dilution following the manufacturer’s instruction at 4 °C overnight. After washing, secondary antibodies conjugated with adequate fluorochrome were added to visualize the expression of corresponding proteins and DAPI was used to stain the nuclei. Images of the penumbra of the infarct cortex were captured using a confocal laser scanning microscope (Laika, Japan). Data were analyzed with ImageJ (NIH Image, Bethesda, MD, USA) to calculate the fluorescence intensity or counting number of recognized cells per field.
Statistical analysis
All statistical analyses of the data were processed with Prism 7.0 software (GraphPad, San Diego, CA, USA) in a blinded manner. Data from individual groups were expressed as mean ± SEM and characterized by a one-way ANOVA for multiple comparisons or Student’s t test (and nonparametric tests). Behavioral data were statistically analyzed by a two-way ANOVA for multiple comparisons. Statistical significance was considered at P < 0.05 level.
Discussion
The inflammatory response evoked by focal ischemia stroke is a complex and pleiotropic process [
13]. The complexity is emphasized by the immunomodulation progression associated with multiple immune cells—resident microglia and an influx of hematogenous cells, that changes based on the time, space, and stage-specific milieu. The heterogeneity is highlighted by detrimental and protective immune effects mediated by those immune cells. Regulation of neuroinflammation has been recognized as an attractive approach for promising therapies in stroke. rhFGF21 is a safe and effective endocrine regulator that has been demonstrated to have strong anti-inflammatory effects, and it represents a promising candidate for microglia/macrophage-based therapy in acute stroke.
In the current study, we demonstrated the neuroprotective effects of rhFGF21 and highlighted its immunomodulatory effects by regulating resident microglia and hematogenous macrophages in acute ischemic stroke. Although this study is not the first to show that rhFGF21 may protect against cerebral ischemic injury in rats [
39], it confirmed that rhFGF21 significantly reduced the infarct size and ameliorated the neurological deficit in mice affected by stroke through a set of experiments (including TTC and behavior assessment). Moreover, our study also revealed that rhFGF21 remarkably dampened the upregulation of pro-inflammatory gene expression. Therefore, our study provides evidence that these outcomes associated with the neuroprotective effects of rhFGF21 are tightly linked with its anti-inflammatory function.
Post-ischemic inflammation is a hallmark of ischemic stroke pathology, which plays critical roles in acute brain damage and profoundly affects long-term recovery [
40,
41]. Inflammation-associated conditions regulated by pro-inflammatory and anti-inflammatory cytokines are associated with impaired neurogenesis and neuronal survival [
42]. Our findings are consistent with a previous report [
28] that showed that almost all inflammatory cytokines were upregulated immediately following stroke, with levels peaking at 24 or 48 h after stroke. However, the increase in IL-10 expression appears strongly but transiently as early as 6 h post-stroke. Among the cytokines detected, TNF-α has both neurotoxic and neuroprotective effects, while IL-1β have characteristically neurotoxic effects. Both TNF-α and IL-1β are mainly produced by microglia and macrophages [
43] and synthesized by segregated subsets [
44]. The cellular source of IL-6 remains controversial, although it is likely predominantly expressed in threatened neurons and activated microglia around the infarct region and targeted at neurons or microglia, and it contributes to both the damage and repair processes [
45]. TGF-β, as an anti-inflammatory cytokine, is associated with tissue repair. We next investigated the effect of rhFGF21 on inflammatory gene (TNF-α, IL-1β, IL-6, TGF-β) expression in microglia and invading peripheral macrophages, which are defined as the major cellular contributors to neuroinflammation [
46,
47]. Although there are analogous phenotypic and functional characteristics among these inflammatory genes, numerous studies have revealed that they may play unique roles under pathological conditions [
48]. Zarruk et al. [
49] detected higher expression of TNF-α in microglia than in macrophages of LysM-EGFP knock-in mice and higher expression of IL-1β and Arg-1 in macrophages than in macroglia in a permanent MCAO model. In our model, rhFGF21 significantly reduced the level of IL-1β expression not only in microglia and infiltrated macrophages but also in splenic macrophages. Surprisingly, the mRNA level of IL-6 in resident microglia isolated from MCAO mice was far lower than that in sham mice. Although we have no suitable explanation for this phenomenon, investigating the temporal pattern of the source of IL-6 may provide a reasonable explanation.
Microglia are major cellular contributor to post-injury inflammation and have the potential to act as a key factor for disease onset and progression and contribute to the neurological outcome of acute brain injury [
50]. Under pathological conditions, microglia are rapidly activated and undergo dramatic morphological and phenotypic changes accompanied by the induction of inflammatory cytokines. The classical activation phenotype (M1) is an inflammatory phenotype that produces pro-inflammatory cytokines, while the alternative activation phenotype (M2) is an anti-inflammatory phenotype that is characterized by the secretion of anti-inflammatory cytokines [
51]. Our findings further demonstrated that rhFGF21 attenuated the polarization of microglia toward M1 but have no effect on the M2 phenotype in the acute phase of the MCAO model. Consistently, our in vitro results demonstrated that rhFGF21 hampered the expression of pro-inflammatory cytokines in LPS- or OGD-treated microglia but had no influence on anti-inflammatory genes (IL-10, CD206, and IGF-1). Concomitantly, mice that underwent experimental MCAO exhibited a gradual decrease in the ischemic hemisphere from day 1 to 3 after reperfusion, and the level subsequently returned to preinjury levels by day 7. A similar phenomenon was also described by a previous study [
52] in which microglia from the ischemic hemisphere were remarkably reduced at 3 days after stroke. Furthermore, our results revealed the temporal profile of microglial polarization. In accordance with previous research [
53], we observed that the levels of the M1-type marker (CD68) significantly increased beginning from day 1 onward. Notably, the expression of the M2 marker CD206 was also increased at 1 day after MCAO, although this increase was no longer observed within 7 days of MCAO injury, which is consistent with the findings of Perego et al. [
54]. Taking into consideration the earlier upregulation of IL-10 gene expression, which mediates the shift of microglia to the M2 phenotype, there may be a temporary increase in M2-like microglia at 1 to 3 days post-stroke, and this notion is supported by the results of Kanazawa et al. [
4].
Moreover, our study focused on the temporal and spatial presence of migrated immune cells in the acute phase of stroke and highlighted the participation of peripheral macrophages. A previous study [
55] showed that the different immune cell types in the post-ischemic area had distinct temporal profiles while the majority of immune cells dramatically accumulated in the ischemic hemisphere at 3 days after stroke. Similarly, in our findings, a massive accumulation of immune cells occurred at 3 days after reperfusion, and the levels were restored back to baseline by day 7. Importantly, rhFGF21 effectively eliminated the invasion of peripheral immune cells. Additionally, rhFGF21 suppressed the activation of peripheral macrophages in the spleen and blood of mice subjected to MCAO, which is consistent with previous reports showing that FGF21 reduced macrophage-mediated inflammation by NF-κB in RWA264.7 macrophages [
29]. These findings suggest that the anti-inflammatory effects of rhFGF21 are mediated not only by resident microglia in the brain but also by hematogenous macrophages.
NF-κB is a key transcription factor in the progression of inflammation, and its activation is accompanied by the release of a panel of inflammatory cytokines and chemokines, such as TNF-α, IL-1β, IL-6, and Cox-2 [
56,
57]. Indeed, microglia polarization has been proposed to induce multiple mechanisms, including NF-κB signaling pathways [
58]. Previous literature demonstrated that the beneficial effects of rhFGF21 on macrophages occur through the inhibition of NF-κB, and our study further validated that rhFGF21 suppresses the activity of NF-κB via FGFR1 in LPS-stimulated murine microglia. In addition, PPAR-γ is a nuclear transcriptional factor [
59], and its activation affects not only peripheral systems in ischemia-reperfusion-induced kidney injury and trinitrobenzenesulfonic acid (TNBS)-induced inflammatory bowel disease but also the CNS due to its anti-inflammatory ability [
28]. In the present study, rhFGF21 significantly elevated the transcriptional activities of PPAR-γ in LPS-stimulated BV2 cells, which further contributed to anti-inflammation. To recapitulate, rhFGF21, through its actions on FGFR1, suppresses the inflammatory response by modulating the activation of microglia via inhibiting NF-κB and elevating PPAR-γ.
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