Skip to main content
NCBI home page
Lippincott Open Access logoLink to Lippincott Open Access
. 2025 Jul 11;36(13):737–745. doi: 10.1097/WNR.0000000000002196

Panax notoginseng saponins regulate the polarization of microglia by inhibiting the hematopoietic progenitor kinase 1 signaling pathway

Dan Li 1, Jiaqi Yin 1, Yan Zhang 1, Yujie Zhang 1, Xin Liu 1, Jia Liu 1,✉
PMCID: PMC12316117  PMID: 40736327

Abstract

Objective

Microglia can be polarized into a proinflammatory M1 phenotype or an anti-inflammatory M2 phenotype. An excess of the M1 phenotype and a deficiency of the M2 phenotype are crucial to the pathological process of ischemic stroke, but the molecular mechanism is still unclear. Although several studies have confirmed the therapeutic effects of PNS (Panax notoginseng saponins) on ischemic stroke, the precise molecular mechanisms of these effects remain poorly understood. The aim of this study was to investigate the molecular mechanism of PNS influencing microglia polarization via hematopoietic progenitor kinase 1 (HPK1) signaling pathway regulation.

Methods

BV2 cells were pretreated with PNS or GNE-1858 (HPK1 inhibitor) and then polarization into M1- and M2-like phenotypes via lipopolysaccharide + interferon-gamma or interleukin (IL)-4, respectively. Detection of M1- and M2-like phenotypes by flow cytometry. The mRNA levels of tumor necrosis factor-alpha, L-1β, Arg1, and IL-10 were measured by real-time PCR. The phosphorylation levels of HPK1, nuclear factor kappa-B (NF-κB), and c-Jun N-terminal kinase (JNK) were detected by western blot.

Results

The phosphorylation levels of HPK1, NF-κB, and JNK gradually increased under the M1 polarization condition. Under the M2 polarization condition, the phosphorylation levels of HPK1, NF-κB, and JNK gradually decreased. Inhibition of HPK1 activity effectively inhibited the activation of NF-κB and JNK during M1 polarization. PNS can inhibit the activation of JNK and NF-κB by inhibiting the activity of HPK1, thereby inhibiting the polarization of M1-like phenotype and promoting the polarization of M2-like phenotype.

Conclusions

This research confirmed that PNS effectively inhibits M1 microglial polarization while stimulating M2 microglial polarization via HPK1, JNK, and NF-κB signaling pathway suppression.

Keywords: c-Jun N-terminal kinase, hematopoietic progenitor kinase 1, microglia, nuclear factor kappa-B, Panax notoginseng saponins

Introduction

Ischemic stroke is a severe cerebrovascular disease with high morbidity and mortality rates [1]. Ischemic and necrotic cerebral tissues trigger the release of numerous inflammatory mediators that lead to the activation and aggregation of microglia. Activated microglia mediate inflammatory cascade effects that either accelerate and exacerbate or decelerate and reduce cerebral ischemic damage [2].

Microglia, which are analogous to macrophages, are important immune cells in the central nervous system and regulate homeostasis of the central nervous system microenvironment [3]. Microglia can be polarized into a proinflammatory M1-like phenotype or an anti-inflammatory M2-like phenotype, which are closely associated with secondary brain damage or repair, respectively [4–6]. Microglia with the M1-like phenotype can produce proinflammatory cytokines such as Interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α), causing extensive inflammatory damage to neurons in the ischemic area. Conversely, cells with the M2-like phenotype can secrete anti-inflammatory cytokines, such as IL-4, IL-10, and TGF-β, aiding in neuronal debris clearance and tissue repair [7–9]. An excess of the M1-like phenotype and a deficiency of the M2-like phenotype are crucial to the pathological process of stroke [3,8,10]. Activation of c-Jun N-terminal kinase (JNK) and nuclear factor kappa-B (NF-κB) signaling pathways can promote differentiation of macrophage/microglia into an M1-like phenotype and mediate the occurrence and development of inflammatory responses [11–14]. Consequently, suppression of JNK and NF-κB signaling pathways can disrupt microglia polarization towards M1-like phenotype and encourage polarization towards M2-like phenotype, promoting recovery from ischemic stroke-induced damage to the brain.

Hematopoietic progenitor kinase 1 (HPK1) plays an important role in immune responses and inflammatory signaling pathways, as well as in the stress response, proliferation, and apoptosis of hematopoietic cells [15–17]. Currently, research on the relationship between HPK1 and immune cells is mostly focused on T cells, and it is unclear whether HPK1 participates in the differentiation and maturation of microglia. Panax notoginseng saponins (PNS), the key active components of Panax notoginseng, primarily consist of notoginsenoside R1 and ginsenosides Rb1, Rd, Re, Rf, and Rg1, similar in chemical structure to triterpenoid saponin. The analysis revealed PNS to comprise 9.76% notoginsenoside R1, 35.1% Rg1, and 38.98% Rb1. In China, PNS effectively manages cardiovascular and cerebrovascular diseases, especially strokes [18–20]. However, the precise molecular mechanism remains unknown. Whether PNS resists ischemic injury via microglial polarization requires further exploration. Through cellular experimentation, we assessed the regulation of microglia polarization by PNS via the HPK1 signaling pathway, thereby elucidating the mechanism underlying the use of PNS in treating ischemic stroke.

Materials and methods

Cell culture and polarization

Murine microglial BV2 cells were cultured in a 37 °C incubator with 5% CO2 in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. BV2 cells were induced to differentiate into an M1-like phenotype using 100 ng/ml lipopolysaccharide (LPS) + 20 ng/ml Interferon-gamma and were induced to differentiate into an M2-like phenotype using 20 ng/ml IL-4 [21]. The cells were harvested after 48 h. LPS and all cytokines were purchased from eBioscience (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

To observe the effect of PNS or the HPK1 inhibitor GNE-1858 (MedChemExpress, South Brunswick Township, New Jersey, USA) on BV2 cell polarization, BV2 cells were pretreated with PNS or GNE-1858 for 6 h and then polarization into M1- and M2-like phenotypes was induced. GNE-1858 was used at a concentration of 1.9 nM [22].

Cell survival assay

To ascertain optimal PNS dose and circumvent the detrimental effects of excessive concentration on BV2 cellular viability, we utilized cell counting kit-8 reagent (MedChemExpress, South Brunswick Township, New Jersey, USA) to assess the survival rate of BV2 cells exposed to various PNS concentrations. CCK-8 is a reagent containing the chemical WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]. WST-8 is bioreduced by cellular dehydrogenases to an orange formazan product that is soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of living cells. Briefly, BV2 cells were inoculated in 96-well plates and, after 4 h in culture, were treated with 0, 10, 50, 100, or 1000 μM PNS for 48 h. Then, 10 µl of CCK-8 was added to each well, and the plate was incubated at 37 °C for 1 h. Absorbance was measured at 450 nm using a microplate reader.

Flow cytometry detection of M1- and M2-like phenotypes

Culture suspension (inclusive of BV2 cells) was transferred from the cell culture flask into a sterile centrifuge tube. The flask was flushed with 1 ml PBS, followed by transfer of PBS into the same tube. Trypsin solution (1 ml) was added to the flask, incubated at 37 °C for 2 min, supplemented by 3 ml of serum-containing culture medium to terminate digestion. Gentle mixing was performed with a micropipette to equally distribute the cells. They were transferred to the sterile centrifuge tube, rinsed with PBS, and labeled with antibodies. Each sample was labeled with anti-CD86-FITC (Merck Millipore, Burlington, Massachusetts, USA) and anti-CD206-APC (Merck Millipore, Burlington, Massachusetts, USA) antibodies to detect M1- and M2-like phenotypes, respectively. As a control, cells were simultaneously stained with appropriate isotype-matched mAbs. Poststaining, cells were washed twice in cold FACS buffer and analyzed via BD FACScan (BD Biosciences, New Jersey, USA).

RNA extraction and real-time PCR

Total RNA was isolated from BV2 cells using the TRIzol Reagent (Invitrogen, Carlsbad, California, USA). The RNA was subsequently reverse transcribed into cDNA using random primers and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions. Then, the cDNA was amplified by real-time PCR with fluorescent SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, Massachusetts, USA) using an ABI Prism 7500 system (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The housekeeping gene human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control for sample normalization. Data analysis was performed using the 2−ΔΔCt method. The primers are listed in Table 1.

Table 1.

Primer sequences for real-time PCR

Forward primer Reverse primer
TNF-α 5’-CAGGCGGTGCCTATGTCTC-3’ 5’-CGATCACCCCGAAGTTCAGTAG-3’
IL-1β 5’-TGTCTTTCCCGTGGACCTT-3’ 5’-TCATCTCGGAGCCTGTAGTG-3’
Arg1 5’-CAGAAGAATGGAAGAGTCAG-3’ 5’-CAGATATGCAGGGAGTCACC-3’
IL-10 5’-TGGCCCAGAAATCAAGGAGG-3’ 5’-CAGCAGACTCAATACACACT-3’
GAPDH 5’-CTCAAGATCATCAGCAATG-3’ 5’-GTCATGAGTCCTTCCACG-3’
Open in a new tab

GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Western blot analysis

BV2 cells were lysed in ice-cold lysis buffer containing 1% phosphatase inhibitor (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 1% phenylmethylsulfonyl fluoride (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Lysates were centrifuged for 15 min at 12 000 g at 4 °C, and the protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Then, proteins were separated using FuturePAGE 4–12% Gels (ACE Biotechnology, Nanjing, China) and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, Massachusetts, USA). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 01% Tween-20 buffer for 1 h at room temperature and immunoblotted with the primary antibodies at 4 °C overnight. The primary antibodies included anti-HPK1 (1:1000), anti-pHPK1 (1:1000), anti-JNK (1:1000), anti-pJNK (1:1000), anti-NF-κB p65 (1:1000), anti-pNF-κB p65 (1:1000), and anti-GAPDH (1:2000). Anti-HPK1 and anti-pHPK1 antibodies were purchased from Abnova (Taipei, China), and the other antibodies were obtained from Cell Signaling Technology (Danvers, Massachusetts, USA). After three rinses, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. The band intensity was quantified using Quantity One software (Bio-Rad, Berkeley, California, USA).

Statistical analysis

All data are presented as means and standard deviations and were statistically analyzed using SPSS 25.0 software. Data were analyzed using one-way analysis of variance followed by a post hoc least significant difference test. P-values of less than 0.05 were considered statistically significant.

Results

Enhancement in hematopoietic progenitor kinase 1 expression and activity during the M1 polarization

To observe the relationships between the HPK1 and microglial polarization, BV2 cells were polarized into M1- and M2-like phenotypes, and HPK1 protein expression and phosphorylation were detected in each group by western blot at 0, 24, and 48 h after induction of polarization. The percentage of the M1-like phenotype (Fig. 1a), the phosphorylation level of HPK1 (Figs. 1b and c) gradually increased under the M1 polarization condition. Under the M2 polarization condition, the percentage of the M2-like phenotype gradually increased (Fig. 1a), and the phosphorylation level of HPK1 gradually decreased (Figs. 1b and d). No significant alterations occurred in HPK1 total protein expression during M1 and M2 polarization (Figs. 1e and f). These results suggest that upregulated HPK1 phosphorylation is closely related to the polarization of M1-like phenotypes.

Fig. 1.

Fig. 1

Open in a new tab

Enhancement in HPK1 expression and activity during the M1 polarization. (a) Flow cytometry results of the proportion of M1- and M2-like phenotypes at each time point of M1 and M2 polarization. (b) Representative western blot results for HPK1 expression and phosphorylation levels in cells with M1- and M2-like phenotypes at each time point of M1 and M2 polarization. Expression levels were normalized to that of GAPDH. (c and e) Quantitative analysis of band intensities for the M1-like phenotype. (d and f) Quantitative analysis of band intensities for the M2-like phenotype. Data represent the means of three independent experiments (**P < 0.01). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPK1, hematopoietic progenitor kinase 1.

Inhibition of hematopoietic progenitor kinase 1 activity prevents microglial polarization toward the M1 phenotype

To confirm the effect of HPK1 inhibition on microglial polarization, BV2 cells were treated with the HPK1 inhibitor GNE-1858 or vehicle, and the cells in each group were simultaneously cultured under M1 and M2 polarization conditions. GNE-1858, an ATP-competitive HPK1 inhibitor, attenuates HPK1 activation through direct binding to residue Glu92 at the HPK1 hinge [22]. In the GNE-1858 group, the proportion of the M1-like phenotype was significantly lower than that in the vehicle group (Figs. 2a and b), while the proportion of the M2-like phenotype was significantly higher than that in the vehicle group (Figs. 2a and b). As shown in Fig. 2c, the mRNA levels of the typical M1gene markers TNF-α and IL-1β were lower in the GNE-1858 group than those in the vehicle group. However, the mRNA levels of the typical M2 gene markers Arg1 and IL-10 were higher in the GNE-1858 group than those in the vehicle group (Fig. 2d). These findings suggest that suppressing HPK1 activity can impede M1 and promote M2 polarization.

Fig. 2.

Fig. 2

Open in a new tab

Inhibition of HPK1 activity prevents microglial polarization towards the M1 phenotype. (a) Flow cytometry results of the proportion of M1- and M2-like phenotypes in the GNE-1858 and vehicle groups. (b) Statistical analysis of flow cytometry data. (c) Relative TNF-α and IL-1β mRNA levels in both the GNE-1858 and vehicle groups. (d) Relative Arg1 and IL-10 mRNA levels in both the GNE-1858 and vehicle groups. Data represent the means of three independent experiments (**P < 0.01). HPK1, hematopoietic progenitor kinase 1.

Hematopoietic progenitor kinase 1 promotes polarization of microglia toward the M1 phenotype by upregulating the c-Jun N-terminal kinase and nuclear factor kappa-B signaling pathways

BV2 cells were polarized into M1- and M2-like phenotypes, NF-κB, and JNK phosphorylation were detected in each group at 0, 24, and 48 h after induction of polarization. The phosphorylation levels of NF-κB p65, and JNK gradually increased under the M1 polarization condition (Figs. 3a and b). Under the M2 polarization condition, the phosphorylation levels of NF-κB p65, and JNK gradually decreased (Figs. 3a and c). Furthermore, to investigate the relationship between the upregulation of NF-κB and JNK phosphorylation and the activation of HPK1 during the polarization of M1-like phenotype, we polarized M1-like phenotype after treating BV2 cells with GNE-1858 or vehicle. Western blot results showed that the phosphorylation levels of HPK1, NF-κB p65, and JNK in the GNE-1858 group were significantly lower than those in the vehicle group (Figs. 3d and e). These findings indicate that the activation of NF-κB and JNK in M1 polarization hinges upon the activation of HPK1.

Fig. 3.

Fig. 3

Open in a new tab

HPK1 promotes polarization of microglia toward the M1 phenotype by upregulating the JNK and NF-κB signaling pathways. (a) Representative western blot results for the NF-κB p65, and JNK phosphorylation levels at each time point of M1 and M2 polarization. (b, c) Quantitative analysis of band intensities. (d) Representative western blot results depicting the HPK1/NF-κB/JNK phosphorylation levels in cells with M1-like phenotypes observed in both the GNE-1858 and vehicle groups. (e) Quantitative analysis of band intensities. Data represent the means of three independent experiments (**P < 0.01). HPK1, hematopoietic progenitor kinase 1; JNK, c-Jun N-terminal kinase; NF-κB, nuclear factor kappa-B.

Panax notoginseng saponins inhibited the polarization of microglia toward M1 and promoted their polarization toward M2

CCK-8 analysis indicated that BV2 cell viability remained unaltered at PNS concentrations of 10 and 50 μM compared to controls (Fig. 4a). However, at PNS concentrations exceeding 50 μM (e.g. 100 and 1000 μM), there was a notable decrease in BV2 cell viability (Fig. 4a), potentially due to PNS’s cytotoxic effects. To optimally stimulate cells while preserving cell viability, we opted for 50 μM as the optimal PNS treatment concentration for BV2 cells.

Fig. 4.

Fig. 4

Open in a new tab

PNS inhibited the polarization of microglia towards M1 and promoted their polarization toward M2. (a) A cell counting kit-8 assay was used to detect the effect of PNS on the viability of BV2 cells. (b) Flow cytometry results of the proportion of M1- and M2-like phenotypes in the PNS and vehicle groups. (c) Statistical analysis of flow cytometry data. (d) Relative TNF-α and IL-1β mRNA levels in the PNS and vehicle groups. (e) Relative Arg1 and IL-10 mRNA levels in the PNS and vehicle groups. Data represent the means of three independent experiments (**P < 0.01). PNS, Panax notoginseng saponins.

To confirm the effect of PNS on microglial polarization, BV2 cells were treated with PNS or vehicle, and the cells in each group were simultaneously cultured under M1 and M2 polarization conditions. In the PNS group, the proportion of the M1-like phenotype was significantly lower than that in the vehicle group, while the proportion of the M2-like phenotype was significantly higher than that in the vehicle group (Figs. 4b and c). The expression levels of typical M1 or M2 gene markers in each group were detected by real-time PCR. As shown in Fig. 4d, the expression levels of the typical M1 gene markers TNF-α and IL-1β were lower in the PNS group than those in the vehicle group. However, the expression levels of the typical M2 gene markers Arg1 and IL-10 were higher in the PNS group than those in the vehicle group (Fig. 4e).

Panax notoginseng saponins inhibits the activation of hematopoietic progenitor kinase 1/nuclear factor kappa-B/c-Jun N-terminal kinase signaling pathways in microglia toward M1 phenotype polarization

To determine whether PNS played a role in regulating M1/M2 polarization by regulating the HPK1, NF-κB, and JNK pathways, the phosphorylation levels of HPK1, NF-κB p65, and JNK in each cell group were detected by western blot. Among cells with the M1-like phenotype, the phosphorylation levels of HPK1, NF-κB p65, and JNK were significantly lower in the PNS group than those in the vehicle group (Figs. 5a and b). However, among cells with the M2-like phenotype, there was no statistically significant difference in the phosphorylation levels of these proteins between the PNS group and the vehicle group (Fig. 5c). These results indicate that PNS inhibits M1-like phenotype polarization by inhibiting the HPK1/NF-κ B/JNK signaling pathway.

Fig. 5.

Fig. 5

Open in a new tab

PNS inhibits the activation of HPK1/NF-Κb/JNK signaling pathways in microglia toward M1 phenotype polarization. (a) Representative western blot results depicting the HPK1, NF-κB p65, and JNK phosphorylation levels in cells with M1- and M2-like phenotypes observed in both the PNS and vehicle groups. (b) Quantitative analysis of band intensities for the M1-like phenotypes. (c) Quantitative analysis of band intensities for the M2-like phenotypes. Data represent the means of three independent experiments (**P < 0.01). HPK1, hematopoietic progenitor kinase 1; JNK, c-Jun N-terminal kinase; NF-κB, nuclear factor kappa-B; PNS, Panax notoginseng saponins.

Discussion

The enhanced phosphorylation of HPK1 in the hippocampal CA1 region of rats subjected to tMCAO is strongly associated with Src-PTK activation. Activation of Src-PTK triggered by cerebral ischemia results in tyrosine phosphorylation in HPK1, which then activates the MLK3-MKK7-JNK3 pathway to induce inflammatory damage in brain tissue [11]. In tMCAO rats, intraventricular infusion of an HPK1 antisense oligonucleotide attenuated increases in HPK1, MLK3, MKK7, and JNK3 phosphorylation and increased the number of surviving neurons [12]. These results suggest that HPK1 activation is an important factor that mediates ischemic brain injury, and this process may be achieved through activation of the JNK signaling pathway.

Blocking of the NF-κB signaling pathway can inhibit LPS-induced differentiation of macrophages into the M1-like phenotype, promote polarization of macrophages into the M2-like phenotype, and thus inhibit the inflammatory response [13]. Azelastine inhibits LPS-induced phosphorylation of JNK in microglial BV2 cells, thereby inhibiting LPS-induced production of proinflammatory mediators, including IL-6, TNF-α, and nitric oxide [14]. Dicalcium silicate particles induced high TLR-2 levels to activate the NF-κB and JNK pathways and then induced TNF-α, IL-1β, and IL-6 expression in RAW 264.7 macrophages [23]. These results suggest that JNK and NF-κB activation an important conditions to promote M1-like phenotype polarization and mediate the inflammatory response.

Our findings demonstrate that HPK1 phosphorylation was markedly upregulated during microglial polarization toward the M1-like phenotype but conversely regulated during polarization toward the M2-like phenotype. Furthermore, we found that inhibiting the activity of HPK1 in microglia significantly impedes the activation of JNK and NF-κB signaling pathways, thereby inhibiting their polarization toward the M1-like phenotype while promoting their polarization toward the M2-like phenotype. This result indicates that the activation of JNK and NF-κB signaling pathways during the polarization of microglia towards the M1-like phenotype depends on the activation of HPK1. Based on our experimental results, we hypothesized that after ischemic stroke, damaged and necrotic cells in the ischemic region mediated the occurrence of inflammatory response and the activation of microglia. The overactivation of HPK1 in activated microglia subsequently upregulates downstream JNK and NF-κB signaling pathways, causing microglia to polarize toward M1-like phenotype and participate in and amplify inflammatory responses. Therefore, controlling the overactivation of HPK1 in ischemic microglia is the key to inhibiting the inflammatory injury of ischemic stroke.

Studies have shown that PNS can increase grip strength, alleviate neurological deficits, improve regional cerebral blood flow, and reduce pathological damage in the cerebral cortex and hippocampus in tMCAO rats. The pharmacological mechanism may be related to thrombolysis, antihypoxia effects, alleviating intracellular Ca2+ overload, and brain energy metabolism [24]. In addition, PNS can down-regulate the HIF-1α/PKM2/STAT3 signaling pathway by inhibiting PKM2 expression in microglia, thereby inhibiting microglial activation and the inflammatory response [25]. However, the effect of PNS on microglia after stroke has not been fully elucidated. In particular, the effect of PNS on microglial polarization after stroke is rarely reported. Our study confirmed that PNS could inhibit JNK and NF-κB signaling pathway activation by down-regulating HPK1 phosphorylation in microglia, effectively inhibit M1 microglial polarization and promote M2 microglial polarization.

Conclusion

Our results elucidate the important role of HPK1 in microglial polarization and confirm that PNS regulates microglial polarization by inhibiting HPK1 activation. These findings provide a reasonable explanation for the therapeutic effects of PNS.

Limitations

This study was exclusively performed in vitro, however, the activation of microglia by single-factor artificial stimulation in vitro is significantly different from the activation of microglia in vivo. In vivo, microglial activation during inflammation engages numerous cytokines and signaling cascades. For instance, microglia transition into M1- or M2-like phenotypes due to interferons, LPS, damage-associated molecular patterns, TLR4 signaling, or IL-4/IL-13; activation of trigger receptors expressed on myeloid cells 2 enhances the phagocytic capacity of microglia [3,26]. In vitro experimental results may not fully illuminate the therapeutic efficacy of PNS. Furthermore, the BV2 cell line is murine, and there exist discrepancies in activated phenotypes between mouse and human microglia, as such, conclusions derived from mouse cells may not wholly translate to human microglia.

Acknowledgements

We thank Lisa Kreiner, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn), for editing the English text of a draft of this manuscript.

This work was funded by the National Natural Science Foundation of China (No. 81960792).

J.L. and D.L. contributed to the design and planning of the experiments. D.L., J.Y., Y.Z., and Y.J.Z. conducted the animal experimentation and the laboratory work. J.L. and D.L. carried out the data collection and analysis. J.L. contributed to the writing of the manuscript. All authors critically revised the manuscript and gave final approval of the version to be submitted.

Conflicts of interest

There are no conflicts of interest.

References

  • 1.Saini V, Guada L, Yavagal DR. Global epidemiology of stroke and access to acute ischemic stroke interventions. Neurology. 2021; 97(20 Suppl 2):S6–S16. [DOI] [PubMed] [Google Scholar]
  • 2.Guruswamy R, ElAli A. Complex roles of microglial cells in ischemic stroke pathobiology: new insights and future directions. Int J Mol Sci. 2017; 18:496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Kanazawa M, Ninomiya I, Hatakeyama M, Takahashi T, Shimohata T. Microglia and monocytes/macrophages polarization reveal novel therapeutic mechanism against stroke. Int J Mol Sci. 2017; 18:2135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Qin C, Zhou LQ, Ma XT, Hu Z-W, Yang S, Chen M, et al. Dual functions of microglia in ischemic stroke. Neurosci Bull. 2019; 35:921–933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Jiang CT, Wu WF, Deng YH, Ge J-W. Modulators of microglia activation and polarization in ischemic stroke (Review). Mol Med Rep. 2020; 21:2006–2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Kim JY, Kim N, Yenari MA. Mechanisms and potential therapeutic applications of microglial activation after brain injury. CNS Neurosci Ther. 2015; 21:309–319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Chen J, Leak RK, Yang GY. Perspective for stroke and brain injury research: mechanisms and potential therapeutic targets. CNS Neurosci Ther. 2015; 21:301–303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Ascoli BM, Gea LP, Colombo R, Barbé-Tuana FM, Kapczinski F, Rosa AR. The role of macrophage polarization on bipolar disorder: identifying new therapeutic targets. Aust N Z J Psychiatry. 2016; 50:618–630. [DOI] [PubMed] [Google Scholar]
  • 9.Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG. Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci. 2009; 29:13435–13444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Dong R, Huang R, Wang J, Liu H, Xu Z. Effects of microglial activation and polarization on brain injury after stroke. Front Neurol. 2021; 12:620948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Li T, Yu XJ, Zhang GY. Tyrosine phosphorylation of HPK1 by activated Src promotes ischemic brain injury in rat hippocampal CA1 region. FEBS Lett. 2008; 582:1894–1900. [DOI] [PubMed] [Google Scholar]
  • 12.Li T, Han D, Chen J, Yu X-J, Zhang G-Y. Neuroprotection against ischemic brain injury by knockdown of hematopietic progenitor kinase 1 expression. Neuroreport. 2008; 19:647–651. [DOI] [PubMed] [Google Scholar]
  • 13.Kang J, Tae N, Min BS, Choe J, Lee J-H. Malabaricone C suppresses lipopolysaccharide-induced inflammatory responses via inhibiting ROS-mediated Akt/IKK/NF-kappaB signaling in murine macrophages. Int Immunopharmacol. 2012; 14:302–310. [DOI] [PubMed] [Google Scholar]
  • 14.Nguyen PL, Bui BP, Duong MTH, Lee K, Ahn H-C, Cho J. Suppression of LPS-induced inflammation and cell migration by azelastine through inhibition of JNK/NF-κB pathway in BV2 microglial cells. Int J Mol Sci. 2021; 22:9061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zhou L, Wang T, Zhang K, Zhang X, Jiang S. The development of small-molecule inhibitors targeting HPK1. Eur J Med Chem. 2022; 244:114819. [DOI] [PubMed] [Google Scholar]
  • 16.Chuang HC, Wang X, Tan TH. map4k family kinases in immunity and inflammation. Adv Immunol. 2016; 129:277–314. [DOI] [PubMed] [Google Scholar]
  • 17.Zhang Q, Ding S, Zhang H. Interactions between hematopoietic progenitor kinase 1 and its adaptor proteins (Review). Mol Med Rep. 2017; 16:6472–6482. [DOI] [PubMed] [Google Scholar]
  • 18.Chen J, Xue R, Li L, Xiao LL, Shangguan J, Zhang W, et al. Panax notoginseng saponins protect cardiac myocytes against endoplasmic reticulum stress and associated apoptosis through mediation of intracellular calcium homeostasis. Front Pharmacol. 2019; 10:1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Ning K, Jiang L, Hu T, Wang X, Liu A, Bao Y. ATP-sensitive potassium channels mediate the cardioprotective effect of panax notoginseng saponins against myocardial ischaemia-reperfusion injury and inflammatory reaction. Biomed Res Int. 2020; 2020:3039184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Yang X, Xiong X, Wang H, Wang J. Protective effects of panax notoginseng saponins on cardiovascular diseases: a comprehensive overview of experimental studies. Evid Based Complement Alternat Med. 2014; 2014:204840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Li Y, Yin C, Jiang J, Yang H, Zhang F, Xing Y, et al. Tumor necrosis factor α-induced protein 8-like-2 controls microglia phenotype via metabolic reprogramming in BV2 microglial cells and responses to neuropathic pain. Int J Biochem Cell Biol. 2024; 169:106541. [DOI] [PubMed] [Google Scholar]
  • 22.Wu P, Sneeringer CJ, Pitts KE, Day ES, Chan BK, Wei B, et al. Hematopoietic progenitor kinase-1 structure in a domain-swapped dimer. Structure. 2019; 27:125–133.e4. [DOI] [PubMed] [Google Scholar]
  • 23.Lai S, Chen L, Cao W, Cui S, Li X, Zhong W, et al. Dicalcium silicate induced proinflammatory responses through TLR2-mediated NF-κB and JNK pathways in the murine RAW 264.7 macrophage cell line. Mediators Inflamm. 2018; 2018:8167932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Dong C, Li J, Zhao M, Chen L, Zhai X, Song L, et al. Pharmacological effect of panax notoginseng saponins on cerebral ischemia in animal models. Biomed Res Int. 2022; 2022:4281483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Gao J, Yao M, Zhang W, Yang B, Yuan G, Liu J-X, Zhang Y. Panax notoginseng saponins alleviates inflammation induced by microglial activation and protects against ischemic brain injury via inhibiting HIF-1α/PKM2/STAT3 signaling. Biomed Pharmacother. 2022; 155:113479. [DOI] [PubMed] [Google Scholar]
  • 26.Chen Z, Jalabi W, Shpargel KB, Farabaugh KT, Dutta R, Yin X, et al. Lipopolysaccharide-induced microglial activation and neuroprotection against experimental brain injury is independent of hematogenous TLR4. J Neurosci. 2012; 32:11706–11715. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Neuroreport are provided here courtesy of Wolters Kluwer Health

RESOURCES