Tetramethylpyrazine promotes remyelination by conversing M1 to M2 polarization of microglia via JAK2-STAT1/3 and GSK3-NFκB signaling pathways in ischemic stroke

doi:10.21203/rs.3.rs-2725089/v1

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Tetramethylpyrazine promotes remyelination by conversing M1 to M2 polarization of microglia via JAK2-STAT1/3 and GSK3-NFκB signaling pathways in ischemic stroke

https://doi.org/10.21203/rs.3.rs-2725089/v1

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Ischemic stroke results in demyelination that underlies neurological disfunction.Promoting oligodendrogenesis will rescue the injured axons and accelerate remyelination after stroke. Microglia react to stroke and polarize to M1/M2 phenotypes. M1 microglia secrete proinflammatory factors to inhibit oligodendrocyte precursor cell (OPC)proliferation and differentiation, inversely, M2 microglia favor the remyelinating process. Tetramethylpyrazine (TMP) has been routinely used in treating cerebrovascular disorders, whereas the role of TMP-mediated microglial polarization on remyelination and the underlying mechanisms remain unknown. In this study, magnetic resonance imaging (MRI) and histopathological evaluation were performed to characterize TMP’s efficacy on remyelinated axon preservation and oligodendrogenesis, particularly, TMP inhibited M1 and enhanced M2 polarization of microglia in cerebral ischemic rats. Moreover, we firstly demonstrated that TMP reversed M1/M2 phenotype via JAK2-STAT1/3 and GSK3-NFκB pathway in lipopolysaccharide (LPS) plus interferon-γ (IFN-γ)-stimulated BV2 microglia. Blocking the crucial target JAK2 will counteract TMP’s effect on mediating M2 polarization of microglia. This study uncovers that TMP’s facilitation on remyelination warrants promising targets for stroke therapy.

Tetramethylpyrazine

ischemic stroke

remyelination

microglia

polarization

JAK-STAT

GSK3-NFκB

Ischemic stroke results in demyelination and remains the leading cause of long-term neurological dysfunction worldwide [1, 2]. Efficient remyelination provides an essential therapeutic strategy to support dyspraxia recovery in patients with demyelination [3].

Tetramethylpyrazine (TMP) is an alkaloid extracted from the herbal medicine chuanxiong and has been extensively used in cardiovascular and cerebrovascular disease treatment [4].Our previous research based on MRI analysis has revealed TMP’s effect on white matter remodeling of cerebral ischemic rats [5]. Nevertheless, the mechanism of TMP promoting the recovery of neurological function after stroke needs to be further clarified.

Poststroke enhancement of oligodendrogenesis is a promising strategy for functional recovery that oligodendrocyte precursor cells (OPC) proliferate and differentiate into oligodendrocytes to accomplish remyelination at sites of ischemic cerebral injury [2, 6, 7]. Microglia are required for the maintenance of OPC development, oligodendrocyte maturation and subsequent physiological myelinogenesis [8, 9]. Under ischemic stimulation, microglia rapidly polarize to classical M1 and alternative M2 phenotypes [10]. M1 microglia not merely phagocytize intact myelin sheath but also secrete proinflammatory cytokines to inhibit OPC and oligodendrocyte survival after ischemia [11]. On the contrary, M2 microglia express antiinflammatory and neurotrophic factors to favor the endogenous oligodendrogenesis [12–14], and promote the repair and regeneration of ischemic brain tissues [15]. TMP is reported to inhibit microglial activation in rats that contributes to neuroprotection against acute cerebral ischemia [16–18], and shift M1 to M2 phenotype of microglia in experimental autoimmune encephalomyelitis mice [19, 20]. Therefore, we hypothesized that TMP’s regulation on microglial polarization might be the critical link of oligodendrogenesis and remyelination poststroke.

Janus protein tyrosine kinase (JAK) is a class of intracellular soluble tyrosine protein kinase including JAK1-3 and TYK2 [21]. JAK2 is especially expressed in microglia and involved in inflammation, tissue repair and neurological recovery poststroke [22, 23]. Phosphorylation of JAK2 provides the docking sites for signal transducer and activator of transcription (STAT), specially, the activation of STAT1 and STAT3 occurs in parallel with microglial M1 and M2 polarization respectively after cerebral ischemia [24, 25]. Besides, JAK2 works in concert with the downstream nuclear transcription factor κB (NFκB) whose activation induces the M1 polarization in CoCl₂-induced BV2 microglia [26]. Therefore, JAK2-manipulated STAT1/3 and NFκB transcription might be involved in microglia-mediated remyelination after stroke under TMP’s intervention.

Glycogen synthase kinase 3 (GSK3) also plays a critical role in initiating microglial M1 polarization via activating NFκB [27, 28]. GSK3 is an evolutionary conserved serine-threonine kinase that consists of α and β isoforms [29]. Phosphorylation of Tyr279/216 on GSK3α/β inhibits its activity while Ser21/9 phosphorylation activates GSK3α/β [29, 30]. Activated GSK3 could regulate JAK/STAT pathway and then participate in glial proliferation and inflammatory response [31], and bioinformatic analysis has revealed there is a relatively conserved STAT-binding site in GSK3β [32]. However, whether JAK2 could dominate GSK3 to mediate microglial polarization is worth in-depth study.

In the current study, magnetic resonance imaging (MRI) and histopathological evaluation were conducted to characterize TMP’s efficacy on remyelination in rats with stroke. To investigate the regulation of TMP on JAK2-STAT1/3 and GSK3-NFκB pathways, we utilized permanent middle cerebral artery (MCAO) rats and LPS plus IFN-γ-stimulated BV2 microglia, and observed TMP’s effects on the phenotypic transformation of microglia.

2.1. Animals and Drugs

Seventy-six male Sprague Dawley rats weighing 300 ± 20 g (aged two months) were purchased from Vital River Laboratory Animal Technology Co., Ltd. and raised in a specific pathogen-free animal research center in Capital Medical University (SYXK [jing] 2018-0003). Animal care and experimental protocols were followed with the guidelines set by the National Institute of Health Guide for the Care and Use of Laboratory Animals, and approved by Capital Medical University Animal Ethics Committee (Permit Number: AEEI-2018-052). TMP hydrochloride injection (HPLC, > 98%) was purchased from Harbin Medisan Pharmaceutical Co., Ltd., (Lot No. 090923A, Harbin, Heilongjiang, China).

2.2. Permanent ischemic model and experimental groups

Focal cerebral ischemia was induced by intraluminal occlusion of the right middle cerebral artery (MCAO) in line with our previous recordation [5]. The successful model was identified that rats could circle or walk to the left were involved in the present experiment [33]. After eliminating the rats died after surgery and without neurological symptoms, MCAO rats were randomly divided into the model group (n = 18) and TMP (10, 20, 40 mg/kg) groups (n = 14 per group). Sham-operated rats were grouped into the sham group (n = 10). TMP dissolved in saline was intraperitoneally injected to rats at 4 h after MCAO and once daily for successive two weeks (1 mL/kg/day). Rats in the sham and model groups were injected with the same volume of saline. Rats from the model group (n = 12), TMP 40 mg/kg group (n = 12), TMP 20 mg/kg group (n = 11), TMP 10 mg/kg group (n = 11) and all sham-operated rats survived.

2.3. MRI scanning and analysis

MRI experiments were utilized with a 7.0 T animal MRI scanner (Bruker PharmaScan, Germany) on the 15th day posttreated with TMP. During MRI scanning, rats (n = 6) were anesthetized with isoflurane and settled in a feedback-controlled system to keep the rectal temperature at 37 ± 0.5℃. Diffusion tensor imaging (DTI) was conducted to detect the microstructural changes of myelinated axons [34], and fractional anisotropy (FA) images were reconstructed following with our previous research [5]. ROIs were delineated in the periinfarct cortex and striatum on DTI parametric maps to obtain FA values [35]. The relative FA (rFA) values = ipsilateral / contralateral FA values [36].

2.4. Tissue processing and LFB staining

After MRI experiments, rats were anesthetized for histopathological evaluation (n = 4). Sample handling of brains was processed as the previous method [37]. Luxol fast blue (LFB) staining was conducted to evaluate the histological injury as previously described [38]. Three nonoverlapping microscopic areas of the periinfarct cortex and striatum were randomly captured and the integrated optical density (IOD) was analyzed with NIS Elements Basic Research Image Collection Analysis system (Nikon, Japan). Relative IOD = ipsilateral / contralateral IOD [39].

2.5. Immunofluorescence staining

Double-label immunofluorescence staining was conducted following previous method [40]. For remyelination assays, OPC proliferation was visualized with Ki67 (labelled proliferative cells)/NG2 (labelled OPC), and oligodendrocyte maturation was determined with Ki67/CNPase (labelled oligodendrocytes) [39]. For microglial polarization examination, M1/M2 microglia were severally double labeled by Iba1(labelled active microglia)/CD16 (labelled M1 phenotype) and Iba1/Arg-1(labelled M2 phenotype) [41]. Briefly, sections were stained with primary antibodies against rabbit anti-Ki67 (1:100; GTX16667, GeneTex) in combination with mouse anti-NG2 (1:100; ab50009, Abcam) or mouse anti-CNPase (1:300; ab6319, Abcam), and mouse anti-Iba1 (1:300; GTX632426, GeneTex) in combination with rabbit anti-CD16 (1:200; ab198507, Abcam) or rabbit anti-Arg-1 (1:200; GTX109242, GeneTex), followed by incubation in species- and isotype-appropriate Alexa Fluor 488 and 594 secondary antibodies. For quantitative analysis, Ki67⁺/NG2⁺, Ki67⁺/CNPase⁺, Iba1⁺/CD16⁺ and Iba1⁺/Arg-1⁺ cells were counted in three randomly selected regions from the periinfarct cortex and striatum. Data were expressed by the average cells/mm² [42]. Images were visualized with a digital camera connected to a fluorescence microscope (Nikon 80i, Nikon, Tokyo, Japan) and blindly analyzed by an investigator to the experimental groups (n = 4).

2.6. BV2 microglia culture and treatment

BV2 microglia were purchased from the Cell Resource Center, Peking Union Medical College and maintained at 37°C in a humidified incubator with 5% CO₂. The components of the culture medium contained Dulbecco’s Modified Eagle medium (DMEM) (10-013-CVR, Corning, USA), 10% fetal bovine serum (FBS) (35-081-cv, Corning, USA) and antibiotics (100IU/mL penicillin and 100mg/mL streptomycin), (Keygen Biotech, Jiangsu, China). Microglia were respectively seeded in 96-well plates for cell viability determination, 24-well plates for Griess assay, and 6-well plates for protein extraction and western blotting. The inoculation density was 1 × 10⁵ cells/mL.

For M1 polarization induction, LPS (100 ng/mL, L2880, Sigma-Aldrich, USA) and IFN-γ (20 ng/mL, HY-P7071, MedChemExpress, USA) were added to microglial cultures [43]. For TMP treatment, microglia were treated with TMP (100, 200, 400, 600 µg/mL) for 24 h ahead and then stimulated with LPS and IFN-γ for another 1 h or 24 h. To block JAK2 pathway [44], microglia were pretreated with AG490 (10, 20, 40, 80 µM) for 1 h ahead and then incubated with TMP for 24 h, followed by LPS and IFN-γ stimulation for another 1 h or 24h.

2.7. Cell viability determination

The potential cytotoxicity of TMP on BV2 microglia was measured by a cell counting kit-8 (CCK-8, DOJINDO, CK04-3000) assay. Briefly, BV2 microglia (1×10⁵ cells/mL) were grown in 96-well plates for 24 h and then treated with TMP (100, 200, 400, 600 µg/mL) or PBS for an additional 24 h. TMP was dissolved in PBS. BV2 microglia were then incubated with CCK-8 reagent, which was dissolved in fresh culture medium for 4 h. The absorbance was determined at 450 nm and the cell viability was calculated as the percentage of surviving cells compared to the control group.

2.8. Nitrate assay

NO production was quantified based on the accumulation of nitrite, a stable metabolite of NO and molecular oxygen [20]. The BV2 microglia were incubated with TMP (100, 200, 400, 600 µg/mL) for 24 h followed by LPS + IFN-γ stimulation for another 24 h. The supernatants were collected and mixed with Griess reagent (1% sulfanilamide and 0.1% N-naphthylethyl-ethylenediamine dihydrochloride in 5% phosphoric acid) (Applygen Technologies Inc., Beijing, China) for 10 min at room temperature. Then, the absorbance was determined under 540 nm and sodium nitrite was used as a standard curve. The production of NO was determined according to the manufacturer’s protocol.

2.9. Western blot analysis

In vivo, periinfarct cortices of rats (n = 4) were quickly dissected and protein detection was followed with previous procedures [37]. In vitro, BV2 microglia were collected and lysed with RIPA buffer (C1053, Applygen, China). Western blot analyses were performed as previously describe [5]. The following primary antibodies were used for western blotting: rabbit anti-CD16 (1:2000; ab198507, Abcam), anti-arginase-1 (Arg-1, 1:8000; GTX109242, GeneTex), anti-IL-6 (1:8000; GTX110527, GeneTex), anti-IL-10 (1:4000; ab9969, Abcam), anti-TGFβ1 (1:4000; ab92486, Abcam), anti-p-GSK3β(Ser9) (1:8000; 9336s, Cell Signaling Technology), anti-p-GSK3(α + β)(Tyr279 + Tyr216) (1:8000; ab68476, Abcam), anti-GSK3β (1:10000; GTX111192, GeneTex), anti-GSK3α (1:8000; ab40870, Abcam), anti-p-JAK2(Tyr1007/1008) (1:1000; ab32101, Abcam), anti-JAK2(1:40000; ab108596,Abcam), anti-p-STAT1(Tyr701) (1:4000; 7649, Cell Signaling Technology), anti-STAT1 (1:4000; 14994, Cell Signaling Technology), anti-p-STAT3(Tyr705) (1:2000; 9145, Cell Signaling Technology), anti-p-NFκB p65 (Ser536) (1:10000; 3033, Cell Signaling Technology), anti-NFκB p65 (1:20000; 8242, Cell Signaling Technology); mouse anti-STAT3 (1:10000; 9139, Cell Signaling Technology) and anti-GAPDH (1:160000; GTX627408, GeneTex). After rewarming and washing with 1×TBST, membranes were incubated with secondary antibodies anti-rabbit (1:20000; C1309, Applygen) or anti-mouse (1:20000; ANM02-1, NeoBioscience) IgG(H + L)-HRP. Immunoreactive protein bands were visualized with ECL plus kit (P1050, Applygen, China) and chemiluminescent Imager (Vilber, USA). Intensities of proteins were acquired quantitatively with ImageJ Software.

2.10. Statistical analysis

Data were presented as mean ± standard error of the mean (SEM) and analyzed with SPSS 26.0 software. Data of the beam walking test were analyzed by Student’s t-test. Other experimental data were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. Significance was defined as P < 0.05.

3.1. TMP promoted remyelination of ischemic rats

DTI-derived parameter FA was conducted to analyze the axonal microstructures (Figure.1A). Quantitative data showed the rFA was clearly decreased in the model periinfarct cortex and striatum of MCAO rats compared with the sham group (p < 0.01). TMP (20, 40 mg/kg) treatment significantly upregulated rFA of the periinfarct cortex of rats (p < 0.01) (Figure.1E).

LFB staining was utilized to exhibit axons and myelin sheath (Figure.1B-D). The relative IOD of periinfarct cortex and striatum of model rats were decreased compared to sham rats (p < 0.01), which were upregulated in TMP (20, 40 mg/kg) groups (p < 0.05 or p < 0.01). TMP (10 mg/kg) increased the relative IOD of the periinfarct cortex of MCAO rats (p < 0.05) (Figure.1F). To sum up, TMP played a prominent role in preserving myelinated axons and alleviating tissue injury in cerebral ischemic rats.

3.2. TMP facilitated endogenous oligodendrogenesis in ischemic rats

To verify whether remyelination is paralleled with cerebral tissue repair, we characterized the OPC proliferation (Ki67/NG2) and maturation (Ki67/CNPase) by immunofluorescence staining at the 15th day post ischemia (Figure.2A-B). Compared with the model group, TMP (10, 20, 40 mg/kg) notably increased Ki67⁺/NG2⁺ and Ki67⁺/CNPase⁺ cells in in periinfarct cortex and striatum (p < 0.05 or p < 0.01) (Figure.2C-D). These results proved TMP intervention boosted the generation of newborn myelinating oligodendrocytes.

3.3. TMP switched M1 to M2 polarization of microglia in ischemic rats and LPS + IFN-γ-stimulated BV2 microglia

M1/M2 polarization of microglia were severally double labeled with Iba1/CD16 and Iba1/Arg-1 by immunofluorescence. Microglia were significantly activated after MCAO, characterized by hypertrophic morphology, with thickened and retracted processes. In contrast, TMP (10, 20, 40 mg/kg)-treated rats showed fewer Iba1⁺/CD16⁺ cells displaying smaller cell bodies and thinner processes (p < 0.01), while significantly more Iba1⁺/CD16⁺ cells existed in TMP (40 mg/kg) group in periinfarct cortex and striatum (p < 0.05 or p < 0.01) (Figure.3A(a)-(d)).

To further verify TMP’s effect on M1 (IL-6) and M2 (IL-10 and TGF-β1) markers expression induced by ischemia, western blot was conducted and revealed IL-6 in the periinfarct cortex was distinctly increased in model rats (p < 0.05), which was reversed by TMP (10, 20, 40 mg/kg) (p < 0.01). Compared with the model group, the levels of IL-10 in TMP (20, 40 mg/kg) group and TGF-β1 in TMP (10, 20, 40 mg/kg) group were respectively upregulated (p < 0.05 or p < 0.01) (Figure.3A(e)-(h)). Proofs indicated that TMP may attenuate neuroinflammation post-stroke by suppressing M1 and activating M2 polarization of microglia in peri-lesions.

CCK-8 assay results proved that TMP (100, 200, 400, 600 µg/mL) had no inhibitory effect on the cell viability of BV2 microglia (Supplementary Figure.1). Griess assay showed LPS + IFN-γ induced an obvious NO production from microglial cells, which was significantly reversed by TMP (100, 200, 400, 600 µg/mL) pretreatment in a dose-dependent manner. Western blot revealed that M1 markers (CD16 and IL-6) were significantly increased and M2 markers (Arg-1, IL-10, TGF-β1) were greatly suppressed by LPS + IFN-γ stimulation. These alterations in M1 and M2 markers were significantly reversed by TMP (200, 400, 600 µg/mL) treatment (Figure.3B), indicating its direct modulation in driving LPS + IFN-γ-induced pro-inflammatory toward anti-inflammatory action.

3.4. TMP regulated JAK2-STAT1/3 and GSK3-NFκB pathway in ischemic rats

Western blot showed that MCAO for 15 days resulted in the significant overexpressions of p-JAK2 (Tyr1007/1008), p-STAT1 (Tyr701) and p-STAT3 (Tyr705) in periinfarct cortex of rats (p < 0.05 or p < 0.01). Compared with the model group, the levels of p-JAK2 in TMP (10, 20, 40 mg/kg) and p-STAT3 in TMP (20, 40 mg/kg) groups were further improved (p < 0.05 or p < 0.01), whereas p-STAT1 was suppressed by TMP (10, 20, 40 mg/kg) (p < 0.01). These findings revealed that TMP activated JAK2-STAT3 while inhibiting STAT3 signaling in ischemic rats (Figure.4A-D).

The distinct lower p-GSK3β (Ser9) level and higher p-NFκB p65 (Ser536) level in the periinfarct cortex of model rats compared with the sham group (p < 0.01). In comparison with the model group, TMP (10, 20, 40 mg/kg) clearly increased p-GSK3β (Ser9), while decreased p-NFκB p65 (Ser536) expression (p < 0.05 or p < 0.01) (Figure.4E-F), suggesting GSK3 activity was decreased and then the downstream NFκB p65 transcription was blocked by TMP treatment.

3.5. TMP regulated JAK2-STAT1/3 and GSK3-NFκB pathway in LPS + IFN-γ-stimulated BV2 microglia

JAK2-STAT signals are closely related to immune response and LPS/IFN-γ induced inflammation [43]. To examine whether TMP could regulate JAK2-STAT1/3 signaling, BV2 microglia were incubated with TMP (200, 400, 600 µg/mL) for 24 h followed by LPS (100 ng/mL) + IFN-γ (20 ng/mL) stimulation for 1 h, and western blotting results showed the exposure to LPS + IFN-γ significantly increased the levels of p-JAK2 (Tyr1007/1008), p-STAT1 (Tyr701) and p-STAT3 (Tyr705) (p < 0.01). Compared to LPS + IFN-γ group, TMP (400, 600 µg/mL) significantly downregulated p-JAK2, p-STAT1, and p-STAT3 levels (p < 0.05 or p < 0.01), in addition, TMP (200 µg/mL) reduced p-STAT1 expression stimulated by LPS + IFN-γ (p < 0.01) (Figure.5A-D).

GSK3β/NFκB signals play a vital role in the process of microglial polarization and neuroinflammation [45, 46]. To further explore the mechanisms of TMP transferring M1 to M2 polarization of microglia, western blot analyses were performed and p-GSK3α (Tyr279) level in TMP (600 µg/mL) group was proved to increase and decrease respectively compared to the control group (p < 0.01), indicating TMP inactivated GSK3 signal in microglia without stimulation. Following LPS + IFN-γ exposure for 1 h, p-GSK3β (Ser9), p-GSK3β (Tyr216) and p-NFκB p65 (Ser536) levels were all upregulated (p < 0.05 or p < 0.01). Interestingly, TMP (200, 400, 600 µg/mL) not merely improved p-GSK3β (Ser9) but also downregulated p-GSK3α (Tyr279) and p-GSK3β (Tyr216) levels in LPS + IFN-γ-stimulated microglia (p < 0.05 or p < 0.01), suggesting TMP bore the activity of inhibiting GSK3 activity. Eventually, the transcription of p-NFκB p65 was effectively blocked by TMP (200, 400, 600 µg/mL) compared to LPS + IFN-γ group (p < 0.05 or p < 0.01) (Figure.5E-H).

3.6. TMP facilitated BV2 microglia toward M2 polarization via JAK2 pathway

To further explore TMP’s effect on microglial M1/M2 polarization when blocking JAK2 signal, JAK2 inhibitor AG490 was used and CCK-8 assay proved that AG490 (10, 20, 40, 80 µM) had no inhibitory effect on the cell viability of BV2 microglia (Supplementary Figure.2). Griess assay showed NO production from LPS + IFN-γ-stimulated microglia was clearly decreased by AG490 (20, 40, 80 µM) (Supplementary Figure.3). Then western blotting showed that AG490 decreased p-JAK2 (Tyr1007/1008), p-STAT3 (Tyr705) and p-NFκB p65 (Ser536) levels in LPS + IFN-γ-induced microglia. AG490 (80 µM) with the most significant inhibitory effect on suppressing JAK2-STAT3-GSK3β-NFκB p65 activation was chosen for the subsequent experiments (Supplementary Figure.4).

Pretreating BV2 microglia with TMP (600 µg/mL)/PBS for 24 h and AG490 (80 µM)/PBS for 1 h followed by LPS (100 ng/mL) + IFN-γ (20 ng/mL) stimulation for 24 h, then M1/M2 polarization was quantified with western blot (Figure.6A(a)). Results showed the IL-6 level was decreased by TMP and TMP + AG490 in microglia without LPS + IFN-γ stimulation compared with the control group (p < 0.01). LPS + IFN-γ-induced the robust higher expression of M1 markers (CD16 and IL-6) were inversed by TMP, AG490 and TMP + AG490 pretreatment (p < 0.01). Moreover, under LPS + IFN-γ induction, CD16 and IL-6 expression in TMP + AG490 group was significantly lower than in AG490 group, demonstrating the effect on suppressing M1 polarization with TMP was strengthened when blocking JAK2 signal (Figure.6A(b)-(c)).

In microglial M2 polarization examination, AG490 and TMP + AG490 were found to decrease Arg-1 and TGF-β1 expressions compared to the control group (p < 0.01). Arg-1, TGF-β1 and IL-10 levels distinctly downregulated by LPS + IFN-γ stimulation were reversed by TMP (p < 0.01), and IL-10 level was improved by AG490 (p < 0.01). However, AG490 and TMP + AG490 evidently inhibited Arg-1 and TGF-β1 expression in stimulated microglia compared with LPS + IFN-γ group (p < 0.01), proving that JAK2 blockage suppressed both M1 and M2 polarization of microglia. After LPS + IFN-γ stimulation, Arg-1, TGF-β1 and IL-10 levels were lower in TMP + AG490 than AG490 group (Figure.6A(d)-(f)), underscoring that TMP could no longer motivate M2 polarization after JAK2 signal was blocked.

3.7. TMP further suppressed STAT1/3-GSK3-NFκB pathway in BV2 microglia via JAK2 signal

Western blotting was conducted to further verify TMP’s effect on JAK2-STAT1/3-GSK3-NFκB p65 pathway after blocking the critical signal JAK2 with AG490 (Figure.6B). BV2 microglial cells were treated with/without TMP (600 µg/mL) for 24 h, AG490 (80 µM) for 1 h, and LPS (100 ng/mL) + IFN-γ (20 ng/mL) for 1 h in sequence. In microglia without LPS + IFN-γ stimulation, compared with the control group, the expressions of p-JAK2 (Tyr1007/1008) in AG490 and TMP + AG490 groups, p-STAT1 (Tyr701) in TMP and TMP + AG490 groups, p-GSK3β (Ser9) in TMP + AG490 group, and p-NFκB p65 (Ser536) in TMP, AG490 and TMP + AG490 groups were decreased respectively (p < 0.05 or p < 0.01). After stimulated by LPS + IFN-γ, the overexpressed p-JAK2, p-STAT1, p-STAT3 and p-NFκB p65 were downregulated by TMP and AG490 (p < 0.05 or p < 0.01). Notably, p-GSK3β (Ser9) level was improved by LPS + IFN-γ (p < 0.01), while TMP and AG490 not only increased p-GSK3β (Ser9) but also decreased p-GSK3α/β(Tyr279/216) levels (p < 0.05 or p < 0.01).

When blocking JAK2 with AG490, TMP significantly strengthened the promoting effect on p-GSK3β (Ser9) expression (p < 0.01), suggesting TMP further suppressed GSK3 activity. Meanwhile, p-JAK2 (Tyr1007/1008), p-STAT1 (Tyr701), p-STAT3 (Tyr705), p-GSK3α (Tyr279), p-GSK3β (Tyr216) and p-NFκB p65 (Ser536) levels showed a trend of decreasing in TMP group. These results revealed TMP enhanced inhibitory effect on JAK2-STAT1/3-GSK3-NFκB p65 activation of microglia after blocking JAK2 signal. Schematic representation of the molecular mechanism that TMP regulates microglial M1/M2 polarization via JAK2-STAT1/3-GSK3-NFκB pathway was showed in Figure.7.

The present study showed TMP notably attenuated brain histological injury, protected axonal microstructure, and promoted endogenous oligodendrogenesis of rats in the convalescence of ischemic stroke. The underlying mechanism for promoting remyelination by TMP was attributed to shift microglial M1 toward M2 phenotype.

To illustrate whether TMP contributed to brain tissue repair and oligodendrogenesis in rats with stroke, we visualized microstructures of axons and myelin with noninvasive MRI-DTI. The decreased rFA in the ischemic cortex and striatum was reversed by TMP, which represents the alleviated axonal injury and demyelination post-stroke [47, 48]. LFB staining showed the improved myelinated axons and cerebral tissue injury, and immunofluorescence staining of Ki67/NG2 and Ki67/CNPase proved the enhanced OPC proliferation and maturation in periinfarction of MCAO rats with TMP treatment.

Neuroinflammation response initiated by microglia affects the demyelinating pathogenesis of ischemic stroke [49]. In periinfarction of ischemic rats, TMP decreased M1 microglia marker CD16⁺/Iba1⁺ cells and proinflammatory mediator IL-6 expression, while upregulating M2 microglia marker Arg-1⁺/Iba1⁺ cells and antiinflammatory IL-10 and TGF-β1 levels. Moreover, the same tendency that switching M1 towards M2 phenotype with TMP pretreatment was detected in BV2 microglia stimulated by LPS plus IFN-γ, accompanied by the decreased IL-6 and increased IL-10 and TGF-β expression. TMP’s efficacy on regulating microglial polarization might provide a suitable microenvironment for oligodendrogenesis after ischemic injury. However, its intricate mechanism requires further investigation.

Results presented here demonstrated the importance of JAK2-STAT1/3 pathway in TMP-afforded microglial M1/M2 polarization against cerebral ischemia. JAK2, STAT1 and STAT3 phosphorylation were upregulated following stroke, which were consistent with previous studies [50, 51]. TMP further activated JAK2 and STAT3 while inhibited STAT1 activation in the periinfarct cortex of MCAO rats. Microglia could be polarizated to the M1 phenotype via STAT1 phosphorylated at Tyr701 in rodents with stroke [52, 53], and STAT1 knockout mice shows smaller cerebral infarction and neurological recovery [54]. Conversely, STAT3 phosphorylated at Tyr705 facilitates not merely microglial M2 polarization [43], but also neuronal survival, oligodendrogenesis and white matter repair following cerebral ischemia [23, 55, 56]. Therefore, under TMP’s treatment, inhibiting STAT1 as well as enhancing STAT3 activity is value-oriented for microglial polarization and brain remodeling after stroke.

Moreover, GSK3-NFκB signaling axis is vital in microglia-mediated neuroinflammation and tissue repair after cerebral ischemia [57, 58]. We found TMP suppressed GSK3β and NFκB p65 activation in the periinfarct cortex of MCAO rats. Accumulating evidence has demonstrated that inhibition on GSK3β activity is capable of attenuating M1 and enhancing M2 phenotype of microglia in cerebral ischemic rats [29, 59]. Besides, OPC maturation and differentiation were promoted via inactiving GSK3β in multiple sclerosis model [3, 60–62], and demyelination and proinflammation were alleviated by inhibiting NFκB in cuprizone-induced mice [63, 64]. GSK3β blocker LiCl could also suppress NFκB activation and microglial M1 polarization in rats with postoperative cognitive dysfunction [27]. In consequence, TMP’s restriction on GSK3-NFκB provides a rational direction for boosting the remyelination process poststroke.

In LPS plus IFN-γ-induced BV2 microglia in vitro, the activated JAK2-STAT1/3 signals were suppressed by TMP pretreatment. LPS and IFN-γ synergistically raise the transcription efficiency of STAT and eventually initiate microglial M1 polarization [65, 66]. Previous studies have suggested that inactivating STAT1 or STAT3 could decrease IL-6, IL-1β and TNF-α expression in IFN-γ-stimulated microglia [67, 68], and the inhibition on JAK2-STAT3 pathway distinctly promoted the M2 polarization in LPS-induced microglia [69]. These findings were in consistent with our in vitro results, and TMP’s restriction on JAK2-STAT1/3 activation laid a foundation for reversing M1 to M2 polarization of microglia.

We further found the activated GSK3α/β and the downstream NFκB p65 in BV2 microglia stimulated by LPS plus IFN-γ were distinctly inhibited by TMP. Active GSK3 has been proved to induce LPS-stimulated microglia to release IL-1β, IL-6 and TNF-α [70]. By contrast, suppressing GSK3 is beneficial for LPS or Aβ-induced M1 microglia shifting to the M2 phenotype [27, 59, 71], and endowing OPC with proliferation capacity under pharmacological intervention in vitro [3]. It is well known that NFκB is activated by LPS binding to toll-like receptor 4 (TLR4) located on glial membranes [65], and IFN-γ activates both JAK/STAT and NFκB signals to synergistically polarize M1 microglial [66, 72]. Downregulation of NFκB is classical in promoting microglial polarization to the M2 phenotype and attenuating inflammatory response [73]. Combined with our in vivo findings, inactivated GSK3-NFκB pathway with TMP intervention provides a new therapeutic avenue for the treatment of ischemic stroke associated with microglial polarization.

Innovatively, in order to explore whether the crucial target JAK had the possible regulation on GSK3 and NFκB signals in TMP-mediated microglial polarization, we conducted the specific inhibitor AG490 to block JAK2. Then we found AG490 inactivated GSK3α/β and NFκB p65 transcription besides JAK2-STAT1/3 signals. Of interest, when inactivating JAK2 with AG490, TMP further inhibited the downstream STAT1/3-GSK3-NFκB p65 activation and M1 polarization, whereas TMP’s promotion on M2 polarization was vanished in BV2 microglia induced by LPS plus IFN-γ.

In conclusion, we elucidated for the first time that TMP promoted remyelination by inhibiting M1 and boosting M2 polarization of microglia through activating JAK2-STAT3 and suppressing STAT1-GSK3β-NFκB pathway in ischemic stroke. On the strength of BV2 microglia stimulated by LPS plus IFN-γ, TMP shifted M1 towards M2 polarization via restraining JAK2-mediated STAT1/3-GSK3α/β-NFκB signaling pathway. This study provides a theoretical basis that TMP facilitates remyelination relying on microglial polarization to treat ischemic stroke.

Availability of data and materials

All data generated and/or analyzed during this study are included in this article.

Ethics approval

All animal experiments were performed in accordance with approved animal protocols and guidelines established by the National Institute of Health Guide for the Care and Use of Laboratory Animals, and approved by Capital Medical University Animal Ethics Committee (Permit Number: AEEI-2018-052).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Authors’ Contributions

Xuefeng Feng performed the experiments, analyzed the data, and finished the manuscript. Email: [email protected]

Mingcong Li collected behavioral data. Email: [email protected]

Ziyue Lin fed rats. Email: [email protected]

Yun Lu performed the behavioral examination. Email: [email protected]

Yuming Zhuang fed rats. Email: [email protected]

Jianfeng Lei prepared for MRI examination. Email: [email protected]

Lei Wang provided experimental suggestions. Email: [email protected]

Hui Zhao programmed the whole work and modified the final manuscript. Email: [email protected]

Funding

This research was supported by Beijing Municipal Natural Science Foundation (Grant No.7212161).

Li S, Rao JH, Lan XY, Li X, Chu CY, Liang Y, et al. White matter demyelination predates axonal injury after ischemic stroke in cynomolgus monkeys. Exp Neurol. 2021;340:113655.
Franklin RJM, Simons M. CNS remyelination and inflammation: From basic mechanisms to therapeutic opportunities. Neuron. 2022;110(21):3549-65.
Liu L, Du X, Yang Q, Li M, Ran Q, Liu Q, et al. Ginsenoside Rg1 promotes remyelination and functional recovery in demyelinating disease by enhancing oligodendrocyte precursor cells-mediated myelin repair. Phytomedicine. 2022;106:154309.
Zhao Y, Liu Y, Chen K. Mechanisms and Clinical Application of Tetramethylpyrazine (an Interesting Natural Compound Isolated from Ligusticum Wallichii): Current Status and Perspective. Oxid Med Cell Longev. 2016;2016:2124638.
Feng XF, Lei JF, Li MZ, Zhan Y, Yang L, Lu Y, et al. Magnetic Resonance Imaging Investigation of Neuroplasticity After Ischemic Stroke in Tetramethylpyrazine-Treated Rats. Front Pharmacol. 2022;13:851746.
Orthmann-Murphy J, Call CL, Molina-Castro GC, Hsieh YC, Rasband MN, Calabresi PA, et al. Remyelination alters the pattern of myelin in the cerebral cortex. Elife. 2020;9.
Shibahara T, Ago T, Nakamura K, Tachibana M, Yoshikawa Y, Komori M, et al. Pericyte-Mediated Tissue Repair through PDGFRbeta Promotes Peri-Infarct Astrogliosis, Oligodendrogenesis, and Functional Recovery after Acute Ischemic Stroke. eNeuro. 2020;7(2).
Hagemeyer N, Hanft KM, Akriditou MA, Unger N, Park ES, Stanley ER, et al. Microglia contribute to normal myelinogenesis and to oligodendrocyte progenitor maintenance during adulthood. Acta Neuropathol. 2017;134(3):441-58.
Zhang W, Tian T, Gong SX, Huang WQ, Zhou QY, Wang AP, et al. Microglia-associated neuroinflammation is a potential therapeutic target for ischemic stroke. Neural Regen Res. 2021;16(1):6-11.
Franco R, Fernandez-Suarez D. Alternatively activated microglia and macrophages in the central nervous system. Prog Neurobiol. 2015;131:65-86.
Shi H, Hu X, Leak RK, Shi Y, An C, Suenaga J, et al. Demyelination as a rational therapeutic target for ischemic or traumatic brain injury. Exp Neurol. 2015;272:17-25.
Ma Y, Wang J, Wang Y, Yang GY. The biphasic function of microglia in ischemic stroke. Prog Neurobiol. 2017;157:247-72.
Garcia-Martin G, Alcover-Sanchez B, Wandosell F, Cubelos B. Pathways Involved in Remyelination after Cerebral Ischemia. Curr Neuropharmacol. 2022;20(4):751-65.
Miron VE, Boyd A, Zhao JW, Yuen TJ, Ruckh JM, Shadrach JL, et al. M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination. Nat Neurosci. 2013;16(9):1211-8.
Wang J, Xing H, Wan L, Jiang X, Wang C, Wu Y. Treatment targets for M2 microglia polarization in ischemic stroke. Biomed Pharmacother. 2018;105:518-25.
Chang CY, Kao TK, Chen WY, Ou YC, Li JR, Liao SL, et al. Tetramethylpyrazine inhibits neutrophil activation following permanent cerebral ischemia in rats. Biochem Biophys Res Commun. 2015;463(3):421-7.
Kao TK, Chang CY, Ou YC, Chen WY, Kuan YH, Pan HC, et al. Tetramethylpyrazine reduces cellular inflammatory response following permanent focal cerebral ischemia in rats. Exp Neurol. 2013;247:188-201.
Liao SL, Kao TK, Chen WY, Lin YS, Chen SY, Raung SL, et al. Tetramethylpyrazine reduces ischemic brain injury in rats. Neurosci Lett. 2004;372(1-2):40-5.
Zhang L, Lu X, Gong L, Cui L, Zhang H, Zhao W, et al. Tetramethylpyrazine Protects Blood-Spinal Cord Barrier Integrity by Modulating Microglia Polarization Through Activation of STAT3/SOCS3 and Inhibition of NF-small ka, CyrillicB Signaling Pathways in Experimental Autoimmune Encephalomyelitis Mice. Cell Mol Neurobiol. 2021;41(4):717-31.
Liu HT, Du YG, He JL, Chen WJ, Li WM, Yang Z, et al. Tetramethylpyrazine inhibits production of nitric oxide and inducible nitric oxide synthase in lipopolysaccharide-induced N9 microglial cells through blockade of MAPK and PI3K/Akt signaling pathways, and suppression of intracellular reactive oxygen species. J Ethnopharmacol. 2010;129(3):335-43.
Brooks AJ, Dai W, O'Mara ML, Abankwa D, Chhabra Y, Pelekanos RA, et al. Mechanism of Activation of Protein Kinase JAK2 by the Growth Hormone Receptor. Science. 2014;344(6185):710-+.
De-Fraja C, Conti L, Magrassi L, Govoni S, Cattaneo E. Members of the JAK/STAT proteins are expressed and regulated during development in the mammalian forebrain. J Neurosci Res. 1998;54(3):320-30.
Li Y, Zhang X, Cui L, Chen R, Zhang Y, Zhang C, et al. Salvianolic acids enhance cerebral angiogenesis and neurological recovery by activating JAK2/STAT3 signaling pathway after ischemic stroke in mice. J Neurochem. 2017;143(1):87-99.
Hu GQ, Du X, Li YJ, Gao XQ, Chen BQ, Yu L. Inhibition of cerebral ischemia/reperfusion injury-induced apoptosis: nicotiflorin and JAK2/STAT3 pathway. Neural Regen Res. 2017;12(1):96-102.
Ding Y, Qian J, Li H, Shen H, Li X, Kong Y, et al. Effects of SC99 on cerebral ischemia-perfusion injury in rats: Selective modulation of microglia polarization to M2 phenotype via inhibiting JAK2-STAT3 pathway. Neurosci Res. 2019;142:58-68.
Tang L, Zhang C, Lu L, Tian H, Liu K, Luo D, et al. Melatonin Maintains Inner Blood-Retinal Barrier by Regulating Microglia via Inhibition of PI3K/Akt/Stat3/NF-kappaB Signaling Pathways in Experimental Diabetic Retinopathy. Front Immunol. 2022;13:831660.
Li J, Shi C, Ding Z, Jin W. Glycogen Synthase Kinase 3beta Promotes Postoperative Cognitive Dysfunction by Inducing the M1 Polarization and Migration of Microglia. Mediators Inflamm. 2020;2020:7860829.
Cohen P, Frame S. The renaissance of GSK3. Nat Rev Mol Cell Biol. 2001;2(10):769-76.
Rana AK, Singh D. Targeting glycogen synthase kinase-3 for oxidative stress and neuroinflammation: Opportunities, challenges and future directions for cerebral stroke management. Neuropharmacology. 2018;139:124-36.
Patel S, Woodgett J. Glycogen synthase kinase-3 and cancer: good cop, bad cop? Cancer Cell. 2008;14(5):351-3.
Beurel E, Jope RS. Differential regulation of STAT family members by glycogen synthase kinase-3. J Biol Chem. 2008;283(32):21934-44.
Moh A, Zhang W, Yu S, Wang J, Xu X, Li J, et al. STAT3 sensitizes insulin signaling by negatively regulating glycogen synthase kinase-3 beta. Diabetes. 2008;57(5):1227-35.
Yu K, Wu Y, Hu Y, Zhang Q, Xie H, Liu G, et al. Prior exposure to enriched environment reduces nitric oxide synthase after transient MCAO in rats. Neurotoxicology. 2013;39:146-52.
Zhang J, Zou H, Zhang Q, Wang L, Lei J, Wang Y, et al. Effects of Xiaoshuan enteric-coated capsule on neurovascular functions assessed by quantitative multiparametric MRI in a rat model of permanent cerebral ischemia. BMC Complement Altern Med. 2016;16:198.
Schober W. The rat cortex in stereotaxic coordinates. J Hirnforsch. 1986;27(2):121-43.
Guo JA, Zheng HB, Duan JC, He L, Chen N, Gong QY, et al. Diffusion tensor MRI for the assessment of cerebral ischemia/reperfusion injury in the penumbra of non-human primate stroke model. Neurological Research. 2011;33(1):108-12.
Zhan Y, Li MZ, Yang L, Feng XF, Lei JF, Zhang N, et al. The three-phase enriched environment paradigm promotes neurovascular restorative and prevents learning impairment after ischemic stroke in rats. Neurobiol Dis. 2020;146:105091.
Shibata M, Ohtani R, Ihara M, Tomimoto H. White matter lesions and glial activation in a novel mouse model of chronic cerebral hypoperfusion. Stroke. 2004;35(11):2598-603.
Li MZ, Zhan Y, Yang L, Feng XF, Zou HY, Lei JF, et al. MRI Evaluation of Axonal Remodeling After Combination Treatment With Xiaoshuan Enteric-Coated Capsule and Enriched Environment in Rats After Ischemic Stroke. Front Physiol. 2019;10:1528.
Zarruk JG, Greenhalgh AD, David S. Microglia and macrophages differ in their inflammatory profile after permanent brain ischemia. Exp Neurol. 2018;301(Pt B):120-32.
Yang X, Xu S, Qian Y, Xiao Q. Resveratrol regulates microglia M1/M2 polarization via PGC-1alpha in conditions of neuroinflammatory injury. Brain Behav Immun. 2017;64:162-72.
Haldorsen IS, Stefansson I, Gruner R, Husby JA, Magnussen IJ, Werner HMJ, et al. Increased microvascular proliferation is negatively correlated to tumour blood flow and is associated with unfavourable outcome in endometrial carcinomas. Brit J Cancer. 2014;110(1):107-14.
Qin C, Fan WH, Liu Q, Shang K, Murugan M, Wu LJ, et al. Fingolimod Protects Against Ischemic White Matter Damage by Modulating Microglia Toward M2 Polarization via STAT3 Pathway. Stroke. 2017;48(12):3336-46.
Fan Z, Zhang W, Cao Q, Zou L, Fan X, Qi C, et al. JAK2/STAT3 pathway regulates microglia polarization involved in hippocampal inflammatory damage due to acute paraquat exposure. Ecotoxicol Environ Saf. 2022;234:113372.
Zu HB, Liu XY, Yao K. DHCR24 overexpression modulates microglia polarization and inflammatory response via Akt/GSK3beta signaling in Abeta25-35 treated BV-2 cells. Life Sci. 2020;260:118470.
Huang B, Liu J, Meng T, Li Y, He D, Ran X, et al. Polydatin Prevents Lipopolysaccharide (LPS)-Induced Parkinson's Disease via Regulation of the AKT/GSK3beta-Nrf2/NF-kappaB Signaling Axis. Front Immunol. 2018;9:2527.
Jung WB, Han YH, Chung JJ, Chae SY, Lee SH, Im GH, et al. Spatiotemporal microstructural white matter changes in diffusion tensor imaging after transient focal ischemic stroke in rats. Nmr Biomed. 2017;30(6).
Mailhard P, Carmichael O, Harvey D, Fletcher E, Reed B, Mungas D, et al. FLAIR and Diffusion MRI Signals Are Independent Predictors of White Matter Hyperintensities. Am J Neuroradiol. 2013;34(1):54-61.
Verden D, Macklin WB. Neuroprotection by central nervous system remyelination: Molecular, cellular, and functional considerations. J Neurosci Res. 2016;94(12):1411-20.
Suzuki S, Tanaka K, Nogawa S, Dembo T, Kosakai A, Fukuuchi Y. Phosphorylation of signal transducer and activator of transcription-3 (Stat3) after focal cerebral ischemia in rats. Exp Neurol. 2001;170(1):63-71.
Yu L, Zhang Y, Chen Q, He Y, Zhou H, Wan H, et al. Formononetin protects against inflammation associated with cerebral ischemia-reperfusion injury in rats by targeting the JAK2/STAT3 signaling pathway. Biomed Pharmacother. 2022;149:112836.
Lu Y, Zhou M, Li Y, Li Y, Hua Y, Fan Y. Minocycline promotes functional recovery in ischemic stroke by modulating microglia polarization through STAT1/STAT6 pathways. Biochem Pharmacol. 2021;186:114464.
Liu H, Zhang Z, Zang C, Wang L, Yang H, Sheng C, et al. GJ-4 ameliorates memory impairment in focal cerebral ischemia/reperfusion of rats via inhibiting JAK2/STAT1-mediated neuroinflammation. J Ethnopharmacol. 2021;267:113491.
Takagi Y, Harada J, Chiarugi A, Moskowitz MA. STAT1 is activated in neurons after ischemia and contributes to ischemic brain injury. J Cereb Blood Flow Metab. 2002;22(11):1311-8.
Zhu H, Zou L, Tian J, Du G, Gao Y. SMND-309, a novel derivative of salvianolic acid B, protects rat brains ischemia and reperfusion injury by targeting the JAK2/STAT3 pathway. Eur J Pharmacol. 2013;714(1-3):23-31.
Wang R, Zhang S, Yang Z, Zheng Y, Yan F, Tao Z, et al. Mutant erythropoietin enhances white matter repair via the JAK2/STAT3 and C/EBPbeta pathway in middle-aged mice following cerebral ischemia and reperfusion. Exp Neurol. 2021;337:113553.
Mostafa DG, Satti HH, Khaleel EF, Badi RM. A high-fat diet rich in corn oil exaggerates the infarct size and memory impairment in rats with cerebral ischemia and is associated with suppressing osteopontin and Akt, and activating GS3Kbeta, iNOS, and NF-kappaB. J Physiol Biochem. 2020;76(3):393-406.
Liao S, Wu J, Liu R, Wang S, Luo J, Yang Y, et al. A novel compound DBZ ameliorates neuroinflammation in LPS-stimulated microglia and ischemic stroke rats: Role of Akt(Ser473)/GSK3beta(Ser9)-mediated Nrf2 activation. Redox Biol. 2020;36:101644.
Cai M, Sun S, Wang J, Dong B, Yang Q, Tian L, et al. Sevoflurane preconditioning protects experimental ischemic stroke by enhancing anti-inflammatory microglia/macrophages phenotype polarization through GSK-3beta/Nrf2 pathway. CNS Neurosci Ther. 2021;27(11):1348-65.
Tian J, Li X, Zhao L, Shen P, Wang Z, Zhu L, et al. Glycyrrhizic acid promotes neural repair by directly driving functional remyelination. Food Funct. 2020;11(1):992-1005.
Benitez-Fernandez R, Gil C, Guaza C, Mestre L, Martinez A. The Dual PDE7-GSK3beta Inhibitor, VP3.15, as Neuroprotective Disease-Modifying Treatment in a Model of Primary Progressive Multiple Sclerosis. Int J Mol Sci. 2022;23(22).
Dohare P, Cheng B, Ahmed E, Yadala V, Singla P, Thomas S, et al. Glycogen synthase kinase-3beta inhibition enhances myelination in preterm newborns with intraventricular hemorrhage, but not recombinant Wnt3A. Neurobiol Dis. 2018;118:22-39.
Bruck W, Pfortner R, Pham T, Zhang J, Hayardeny L, Piryatinsky V, et al. Reduced astrocytic NF-kappaB activation by laquinimod protects from cuprizone-induced demyelination. Acta Neuropathol. 2012;124(3):411-24.
Abdi M, Pasbakhsh P, Shabani M, Nekoonam S, Sadeghi A, Fathi F, et al. Metformin Therapy Attenuates Pro-inflammatory Microglia by Inhibiting NF-kappaB in Cuprizone Demyelinating Mouse Model of Multiple Sclerosis. Neurotox Res. 2021;39(6):1732-46.
Pawate S, Shen Q, Fan F, Bhat NR. Redox regulation of glial inflammatory response to lipopolysaccharide and interferongamma. J Neurosci Res. 2004;77(4):540-51.
Rauch I, Muller M, Decker T. The regulation of inflammation by interferons and their STATs. JAKSTAT. 2013;2(1):e23820.
Gan ZS, Wang QQ, Li JH, Wang XL, Wang YZ, Du HH. Iron Reduces M1 Macrophage Polarization in RAW264.7 Macrophages Associated with Inhibition of STAT1. Mediators Inflamm. 2017;2017:8570818.
Weng L, Zhang H, Li X, Zhan H, Chen F, Han L, et al. Ampelopsin attenuates lipopolysaccharide-induced inflammatory response through the inhibition of the NF-kappaB and JAK2/STAT3 signaling pathways in microglia. Int Immunopharmacol. 2017;44:1-8.
Ryu KY, Lee HJ, Woo H, Kang RJ, Han KM, Park H, et al. Dasatinib regulates LPS-induced microglial and astrocytic neuroinflammatory responses by inhibiting AKT/STAT3 signaling. J Neuroinflammation. 2019;16(1):190.
Huang WC, Lin YS, Wang CY, Tsai CC, Tseng HC, Chen CL, et al. Glycogen synthase kinase-3 negatively regulates anti-inflammatory interleukin-10 for lipopolysaccharide-induced iNOS/NO biosynthesis and RANTES production in microglial cells. Immunology. 2009;128(1 Suppl):e275-86.
Zu HB, Liu XY, Yao K. DHCR24 overexpression modulates microglia polarization and inflammatory response via Akt/GSK3beta signaling in Abeta(25)(-)(35) treated BV-2 cells. Life Sci. 2020;260:118470.
O'Connell D, Bouazza B, Kokalari B, Amrani Y, Khatib A, Ganther JD, et al. IFN-gamma-induced JAK/STAT, but not NF-kappaB, signaling pathway is insensitive to glucocorticoid in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2015;309(4):L348-59.
Wang XL, Chen F, Shi H, Zhang M, Yan L, Pei XY, et al. Oxymatrine inhibits neuroinflammation byRegulating M1/M2 polarization in N9 microglia through the TLR4/NF-kappaB pathway. Int Immunopharmacol. 2021;100:108139.

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SupplementaryFig.1.tif
Supplementary Figure.1. TMP had no inhibitory effect on cell viability of BV2 microglial cells. BV2 microglial cells were pretreated with different concentrations of TMP (100, 200, 400, 600 μg/mL) for 24 h. Cell viability of BV2 microglial cells was detected by the CCK-8 assay and shown as the percentage of surviving cells compared to control group (n = 6).
SupplementaryFig.2.tif
Supplementary Figure.2. AG490 (10, 20, 40, 80 μM) had no inhibitory effect on cell viability of BV2 microglia. BV2 microglial cells were pretreated with different concentrations of AG490 (10, 20, 40, 80, 100, and 150 μM) for 24 h. Cell viability of BV2 microglial cells was detected by the CCK-8 assay and shown as the percentage of surviving cells compared to Control group (n = 6). ^**P < 0.01 vs. Control group.
SupplementaryFig.3.tif
Supplementary Figure.3. AG490 (20, 40, 80 μM) decreased NO production from LPS + IFN-γ-stimulated BV2 microglia. NO concentration released by BV2 microglial cells was quantitatively analyzed with Nitric Oxide assay followed by AG490 (20, 40, 80 μM) incubation for 24 h and LPS (100 ng/ml) + IFN-γ (20 ng/ml) stimulation for 24 h (n = 6). ^##P < 0.01 vs. Control group. ^**P < 0.01 vs. LPS + IFN-γ group.
SupplementaryFig.4.tif
Supplementary Figure.4. AG490 suppressed STAT3-GSK3β-NFκBp65 activation of BV2 microglia. (A) BV2 microglial cells were incubated with AG490 (20, 40, 80 μM) for 1 h followed by LPS (100 ng/mL) + IFN-γ (20 ng/mL) stimulation for 1 h. Cell lysates were collected and the expression of phosphorylated and total proteins of (B) JAK2, (C) STAT3, (D) GSK3β, and (E) NFκB p65 were detected with western blot (n = 4).^#P < 0.05 and ^##P < 0.01 vs. Control group. ^*P < 0.05 and ^**P < 0.01 vs. LPS + IFN-γ group.
SupplementaryFiguresWesternblot.zip

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Tetramethylpyrazine promotes remyelination by conversing M1 to M2 polarization of microglia via JAK2-STAT1/3 and GSK3-NFκB signaling pathways in ischemic stroke

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Animals and Drugs

2.2. Permanent ischemic model and experimental groups

2.3. MRI scanning and analysis

2.4. Tissue processing and LFB staining

2.5. Immunofluorescence staining

2.6. BV2 microglia culture and treatment

2.7. Cell viability determination

2.8. Nitrate assay

2.9. Western blot analysis

2.10. Statistical analysis

3. Results

3.1. TMP promoted remyelination of ischemic rats

3.2. TMP facilitated endogenous oligodendrogenesis in ischemic rats

3.3. TMP switched M1 to M2 polarization of microglia in ischemic rats and LPS + IFN-γ-stimulated BV2 microglia

3.4. TMP regulated JAK2-STAT1/3 and GSK3-NFκB pathway in ischemic rats

3.5. TMP regulated JAK2-STAT1/3 and GSK3-NFκB pathway in LPS + IFN-γ-stimulated BV2 microglia

3.6. TMP facilitated BV2 microglia toward M2 polarization via JAK2 pathway

3.7. TMP further suppressed STAT1/3-GSK3-NFκB pathway in BV2 microglia via JAK2 signal

4. Discussion

Declarations

Availability of data and materials

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Consent for publication

Competing interests

Authors’ Contributions

Funding

References

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