Catalpalactone protects rats nerve function from hypoxic lesion by polarizing microglial cells toward M2 phenotype

Abstract

Background

Ischemic brain injury results in high disability due to neuroinflammation and oxidative stress, and M1/M2 polarization of glial cells plays a key role in neuroinflammation. This research explored the protective effect of Catalpalactone on middle cerebral artery occlusion (MCAO)-induced brain injury and its underlying regulation mechanism in rats.

Methods

The ischemic lesions were induced by the MCAO, and the oxygen and glucose deprivation/reoxygenation (OGD/R) was used for BV2 microglial cell induction. The polarization of glial cells was determined via immunohistochemistry staining assessment. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) assays were used for the glycolysis and oxidative phosphorylation test. After that, the cell counting kit-8 (CCK-8) for cell viability test and flow cytometry for apoptosis and phosphorylation analysis were performed. Furthermore, a co-culture model of BV2 and PC12 cells was used for the purpose of exploring the effects of Catalpalactone on the interaction and of microglia and neurons in ischemic brain injury. Finally, the Modified Neurological Severity Score (mNSS) analysis was used for the analysis on the neurological function.

Results

After MCAO induction, the infiltration of microglial cells were significantly increased in the injury area, and its M1 phenotype was enhanced (up-regulated Cd86). In vitro, the OGD/R-induced BV2 microglial cell also exhibited the increasing M1 phenotype with higher glycolysis activity, but lower oxidative phosphorylation through the activating JAK-SATA signaling pathway. Finally, we determined that 15 μM Catalpalactone optimally induces M2 microglial polarization with increased cell viability and decreased apoptosis in the OGD/R-induced BV2 cell model, while also reducing mNSS scores and improving neurological function in the MCAO rat model.

Conclusion

We clarified the underlying mechanism of Catalpalactone treatment for ischemic lesions through promoting M2 microglial cells phenotype.

Introduction

Ischemic brain injury is a potentially fatal disease with extremely high rates of incidence, disability, and recurrence around the world and across all ages, hence posing a serious health threating and heavy economic burden on society globally [1]. Meanwhile, it is also a primary contributor of ischemic stroke, which was characterized by the obstruction of blood flow to the brain that caused by the broken or blocked blood vessels, starving the tissue of glucose and oxygen [2, 3]. The ischemic stroke accounts for more than 85% of all the stroke cases, affecting about 14 million people annually [4]. The mortality of ischemic stroke has decreased in the past 40 years, but the disability rates remain high [5], which may be associated with the nerve injury and the secondary of intracerebral hemorrhage, the neuronal loss caused by the inflammatory response and excessive oxidative stress is the main factor [6]. Inflammation represents a protective immune response that occurs in the damage or infected site, mounting evidences supported crucial roles of neuroinflammation and immune response in the pathophysiological processes of ischemic stroke [7, 8], which results from a rapid and consistent increase in the production of reactive oxygen species (ROS) in the brain that causes neuronal death and secondary neurological impairment [9, 10]. Currently, the therapeutic strategies of ischemic stroke are lacking, and the common and effective treatment method in clinical practice is the t-PA thrombolysis that alleviates the neuronal injury and restores the blood supply [11]. However, post-ischemia–reperfusion injury repair and prevention of stroke remains an urgent need in current medical practice.

Microglia cells are the core resident immune cells of brain that regulate neurological repair, brain development, and maintenance of neuronal networks in the central nervous system (CNS). Several studies have demonstrated that the microglia cells are the core cells that induce neuroinflammatory response [12] or inhibit oxidative stress via scavenge ROS [13], and can be an important therapeutic target for manipulating neuroinflammatory response in neural repair. Microglia cells serve as a specific brain macrophage that are different from other tissue macrophages due to their unique immune niche and homeostatic phenotype, which was tightly regulated by the CNS microenvironment to maintain the homeostasis and normal brain function [14]. They are responsible for the elimination of the redundant synapses, dead cells, protein aggregates, microbes, and other large particulate and soluble antigens that may damage the CNS [15]. Moreover, the microglia cells, as the main source of proinflammatory cytokines, are pivotal mediators of neuroinflammation and regulate a widespread spectrum of cellular responses [16]. Two polarization phenotypes decided their different inflammatory response function, in which the M1 microglia cells are pro-neuroinflammatory, and aggravate the neuronal and synaptic damage via releasing proinflammatory cytokines and oxidative stress, while the M2 microglia cells inhibit the neuroinflammatory response through the releasing anti-inflammatory cytokine clearing cell debris and misfolded proteins for neuro-regeneration [17]. Their polarized phenotypes determine the neuroinflammation direction, which is closely associated with the pathology of ischemic stroke. Thus, the regulation of microglia cells polarization is a crucial mechanism affecting the neuroinflammatory response that promotes or inhibits the deterioration of ischemic stroke.

Catalpalactone is an iridoid glycoside that can extract from the Catalpa ovata (Bignoniaceae), and can significantly inhibit the excessive NO production that can cause inflammatory disorders and reduce the proinflammatory cytokine levels, such as the tumor necrosis factor-α and IL6 [18, 19], and exerted a cytoprotective effects against H₂O₂-mediated oxidative injury in the hepatocellular carcinoma epithelial cell (HepG2)[20]. The underlying anti-inflammatory protective mechanism of Catalpalactone includes the suppression of janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling via inhibiting the expression of STAT-1 protein, interferon-β (IFN-β) production, and the activation of nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) [21]. However, the anti-inflammatory protective function of Catalpalactone in the ischemic stroke progression is rarely reported. Hence, the purpose of this study was to investigate the anti-inflammatory and protective effects of Catalpalactone in ischemic brain injury and its potential molecular mechanisms for achieving neuroprotection.

Material and methods

Middle cerebral artery occlusion (MCAO) model

The clear male C57BL/6 Sprague–Dawley (SD) rats weighing about 20 g (specific pathogen-free grade) in this study were provided by the Slack Experimental Animal Company (Shanghai, China), and were cared for according to the guidelines of National Institutes of Health. These rats were housed in a suitable cages of 50–60% relative humidity at 25 ± 1 °C, and with free access to water and food, and randomly and blindly divided into 6 rats /group under a 12-h light/dark cycle condition for 7 days pretreatment, including the normal and MCAO group with reasonable technique and sample repetition. The rats were anesthetized with 4% pentobarbital sodium (100 mg/kg) and subjected to the 90 min transient MCAO through the intraluminal vascular occlusion method [22], resulting in the unilateral repetitive ischemic lesions in the striatum and cortex. In short, a surgical neck midline incision was carefully performed to fully expose the internal carotid artery (ICA), common carotid artery (CCA), and external carotid artery (ECA), and the proximal ends of CCA and ECA were ligated with the 6–0 nylon suture. Then, the ICA was temporarily clamped with a microsurgical clamp, and a silicon tip of nylon suture was inserted into the ICA through the ECA stump to block the origin of the middle cerebral artery, the suture around the CCA was tighten, and the skin incision was sutured carefully. After the 90 min MCAO, the suture was withdrawn from ICA to recover blood flow perfusion, and the rats were maintained on a heating pad that kept the body temperature at 37 °C during the operation. Rats of sham operation underwent the same procedure without inserting the thread, and rats that died during surgery were excluded. Notably, the normal group, which did not undergo any surgical operation, was used to assess neurological function and tissue properties in the normal physiological state, and the MCAO group was used to simulate the pathological state of ischemic brain injury.

In addition, rats received the first intraperitoneal injection of Catalpalactone at a dose of 15 mg/kg within 2 h after MCAO (MCAO + CATA). Subsequently, the injections were repeated every 24 h for 7 consecutive days in order to observe the protective effects on neurological function and brain damage. To ensure freshness of the drug, all solutions were freshly prepared before use and stored under light protected conditions to prevent degradation. After the experiment, the surviving rats were euthanized with 4% pentobarbital sodium (150 mg/kg), and the brain tissue was harvested for subsequent analyses. All protocols were approved by Animal Ethical Care Committee of the Third Affiliated Hospital of Qiqihar Medical University (approval No. [2022] 006 (AECC-2022-006)).

Immunohistochemistry staining

Briefly, the brain samples in striatum and cortex of rats were made into 10 μm coronal sections with liquid nitrogen treatment and were collected onto a glass slide coated with poly-l-lysine [23]. Then, 4% paraformaldehyde (Beyotime, P0099-100 ml) was applied to fix the cells for 10 min, and the brain slices were washed with phosphate buffered solution (PBS) for 3 times blocked with 10% goat serum in PBST (0.01 M PBS containing 0.05% Tween 20) for 1 h, and rinsed in PBS. After that, the primary antibodies against Iba1 (1:500, 019-19741, Wako, Osaka, Japan), CD86 (1:500, ab119857, Abcam, Cambridge, UK), and CD206 (1:500, 60143–1-Ig, Proteintech, Chicago, IL, USA) were added for 1 h incubation at 37 °C and the sections were rinsed with PBS. The corresponding secondary antibodies, including the goat anti-rabbit IgG conjugated with Alexa Fluor™ 594 (A-11037, Invitrogen, Carlsbad, CA, USA) and Goat anti-Mouse IgG conjugated with Fluor™ 488 (A11008, Invitrogen, USA), were added dropwise and incubated for 2 h at room temperature. Lastly, following the rinse in PBS, the slices were stained with the DAPI (4′,6-diamidino-2-phenylindole, Vector Laboratories Inc., USA) and washed in PBS again to remove excessive dyeing solution. Images and positive cell count were captured and performed under a fluorescence microscope with ImageJ software (Zeiss Axio Imager, Germany); Iba1, CD86, and CD206 indicates microglial cell, M1-, and M2-type microglial cell, respectively [23].

Real-time qPCR analysis

Using the TRIzol reagent (15596026, Invitrogen, USA), total RNA of the cultured cells was extracted for the cDNA synthesis with the PrimeScript RT reagent Kit (RR037A, TaKaRa, Shiga, Japan) following the manufacturer’s instructions. The quantitative PCR was performed by using the LightCycler 480 (Roche, Basel, Switzerland) on a CFX 96 real-time PCR system (Bio-Rad, Hercules, CA, USA) as described [24]. The expression levels of Cd80, Cd86, Cd163, Cd206, Nos2, and Arg1 and the cytokines (IL1B, IL6, IL4 and IL10) were detected by 2^−ΔΔCt method with β-actin as the housekeeping gene [25]. The specific primer are listed in Table 1, and each sample has three biological and technique replicates.

Table 1 The specific primer of target genes

Cell culture and oxygen glucose deprivation/re-oxygenation (OGD/R) assay

The PC12 rat neuronal cells and BV2 microglial cells were obtained from the Hycyte Biotech corporation (Suzhou, China) and cultured in DMEM/F12 culture medium that was supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin and penicillin in a temperature incubator with 5% CO₂ atmosphere, at 37 °C [26]. In vitro experiments were performed using the OGD/R method to simulate the ischemia/reperfusion (I/R) process, in which 1 × 10⁵/well PC12 and BV2 cells were placed in a hypoxia chamber (0.2% O₂, 5% CO₂) for 4 h. Then, the reperfusion process was simulated in vivo by placing the cells in normoxia (21% O₂, 5% CO₂, and normal glucose) for 8 h. The cells in control groups (CON) were cultured in DMEM without oxygen deprivation and their culture medium were updated at the same time as the experimental group [27].

Detection of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) for glycolysis and oxidative phosphorylation evaluation

We used the Seahorse XF24 Extracellular Flux analyzer (Seahorse Bioscience, North Billerica, MA, USA) to assess the ability of cell glycolysis and mitochondrial oxidative phosphorylation through measuring the ECAR and OCR as previously described [28]. The BV2 microglial cells (1 × 10⁴ cells/ well) were seeded in the cell culture microplates of Seahorse XF96 with complete medium at 37 °C one day before the test. After 24 h, the medium was replaced by the pre-warmed XF-base DMEM medium containing phenol red, 2 mM sodium pyruvate, 2 mM glutamine, and 25 mM glucose (Agilent Technologies) and the Seahorse assay in the Seahorse XF24 analyzer was conducted. The Seahorse XF Glycolysis Stress Test Kit (Agilent Technologies) was adopted for glycolysis test, and 180 μL XF-base medium with 2 mM L-Glutamine was added to the cells after 1 h incubation with non-CO₂ at 37 °C; then the cells were loaded into the Agilent Seahorse Analyzer and sequentially treated with 10 mM glucose, 1 μM oligomycin, and 50 mM 2-deoxyglucose (2-DG) for baseline measurements with the methodology of mixture for 3 min, measured for 3 min and repeated three cycles. Following the mitochondrial stress test, 180 μL XF-base medium supplemented with 2 mM L-Glutamine, 1 mM pyruvate, and 10 mM glucose were added to each well; then the baseline was measured after the sequential treatments of 1.5 μM oligomycin, 1 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and a combination of 0.5 μM rotenone/antimycin A, the measurement methodology is the same as above [29].

Drug administration and cell viability detection

After 4 h OGD induction, the STAT1 phosphorylation inhibitor Catalpalactone (CATA, MCE, HY-N3548) as treatment drug was added to the BV2 microglial cell for phenotypic therapy, with 0, 5, 10, 15, 20, 25, and 30 µM concentrations in each well for 8 h incubation. After 8 h, the mediums were replaced with the normal medium to explore the optimal concentration of Catalpalactone for treatment in vitro (OGD/R + CATA). Cell Counting Kit-8 (CCK-8) is a fast and highly sensitive cell proliferation and cytotoxicity detection kit based on the WST-8 reagent, which can be reduced by the dehydrogenases within the mitochondria to produce orange-yellow formazan, the darker color indicates the faster cell proliferation. The CCK-8 kit (BS350B, Biosharp, Hefei, China) was used for the cell viability assessment based on the manufacturer’s instructions. Briefly, 100 μL (7 × 10³) cell suspension/ well treated by drugs was seeded into 96-well plates for 24 h incubation; then the 10 μL/cell CCK-8 solution was added into the cell suspension for 1–4 h incubation at 37 °C. Lastly, the absorbance at 450 nm was measured by a microplate reader to assess cell viability.

Flow cytometry for phosphorylation and apoptosis detection

The cultured cell suspension was filtered by 40 μm filter (BD Biosciences, Franklin Lakes, NJ, USA) to remove cell masses and large particles and centrifuged (1000rpm) for 10 min. Then, the pre-cold PBS containing 1% Bovine Serum Albumin (BSA) was used to wash the cell precipitate, which was further centrifuged (1000rpm) for 10 min to retain precipitate. The permeabilization wash buffer (Yeasen, shanghai) was added for 15 min incubation and centrifugation (1000rpm, 5 min), and the cells were resuspended with the permeabilization wash buffer and incubated with the antibodies (Invitrogen, USA) of P-JAK1 (MA5-36891, 1: 500), P-JAK2 (MA5-37197, 1: 500), P-STAT1 (MA5-37039, 1: 500), and P-STAT6 (MA5-36908, 1: 500) on ice for 30 min. Lastly, LSRFortessa™ Cell Analyzer (BD Biosciences, USA) was used for data acquisition and FlowJo V10.0 (BD Biosciences, USA) for apoptosis data analysis and visualization. The apoptosis was analyzed by the flow cytometry and FITC-Annexin V/PI apoptosis kit (C1062S, Beyotime, Shanghai, China). In briefly, 1 × 10⁵ PC12 cells/well were co-cultured with the OGD/R-induced BV2 cells treated with Catalpalactone or not; then the collected cells are washed with pre-cooled PBS before it was harvested with trypsin by centrifugation (1000rpm, 5 min), and then the collected cells of sediment were fully resuspended in the pre-cooled (4 °C) D-Hanks suspension and centrifuged (1000rpm) for the cell sediment [26]. After that, 1 × binding buffer was added for cell resuspension at 1–5 × 10⁶/mL, and the cell resuspension (100 µL) was mixed with 5 µL Annexin V-APC and 5 µL PI for 15 min incubation in the dark, followed by the addition of 400 µL 1 × Annexin binding buffer for flow cytometry detection. Each experiment has three technique repeats.

Neurological scores

Modified Neurological Severity score (mNSS) was calculated to assess the neurological impairments after modeling in animals as previously described [30]. The mNSS includes 5 categories (motor, reflexes, sensory systems, balance, and abnormal motion), with the final score ranging from 0 to 18, in which the minimal score (0) indicated the absence of impairment, while the maximum score (18) indicated the severe neurological impairment. In the present study, the evaluation of mNSS score was performed by three independent experimenters using a blinded assessment based on the observation on the rats’ behavior and responses. Eventually, the average value was taken as the final score to ensure the objectivity and reliability of the results.

Statistical analysis

All statistical analysis and visualization were performed by the GraphPad software (version 8.0) and the data were presented as mean ± standard deviation. Normal distribution of continuous variables was assessed by the Shapiro–Wilk test, and the non-parametric equivalence analysis was performed for data that did not show normal/Gaussian distributions. The Mann–Whitney U test was used to determine the significant difference between the two groups of continuous variables and Kruskal–Wallis univariate variance analysis was used to evaluate the difference among the three groups with multiple comparisons. p < 0.05 was considered statistically significant. (*p < 0.05, **p < 0.01, ***p < 0.001).

Results

The M1-type microglial cells were significantly infiltrated in ischemic lesions

After MCAO induction, we characterized the ischemic lesions area in the striatum and cortex of MCAO rats (n = 6) by using the immunohistochemistry staining and found that the infiltration levels of microglial cells were increased in the injured area. The fluorescence intensity of Iba1 (marker of microglial cells) was obviously enhanced (Fig. 1A), and the statistical analysis showed that the numbers of Iba1⁺ cells were significantly increased in the MCAO group (p < 0.05, Fig. 1B). In addition, the expression levels of Cd86 and Nos2 were increased (Fig. 1C, D), while the expression of Arg1 (M1 microglial cells) was inhibited and Cd206 has no significant difference in the MCAO groups (Fig. 1E, F), indicating the M1-type microglial cells (Cd86) were significantly infiltrated after cerebral ischemia in the injured area.

Fig. 1

The change of microglial cells in the injure area after MCAO induction. A The immunohistochemistry staining assay for microglial cells infiltration analysis. B Differences in the proportion of Iba1 + cells in the control and MCAO groups. C–F Differential analysis of the mRNA expression of Cd86 (C), Nos2 (D), Cd206 (E), and Arg1 (F) in the control and MCAO groups. The number of Sprague-Dawley rats used in each group were six each (n = 6). (**p < 0.01, ****p < 0.0001 and ns stands for non-significant difference)

OGD/R promotes M1 microglial cells polarization

Subsequently, we detected the phenotypic change of microglial cells in the ischemia/reperfusion (I/R) process by the OGD/R assay. After 4 h OGD injury induction, the mean fluorescence intensity (MFI) of CD86 in the BV2 microglial cells was significantly enhanced (p < 0.05, Fig. 2A, B), but the MFI of CD206 was not significantly increased in comparison with that of the control group (Fig. 2C, D). Meanwhile, the Cd80 and Nos2 were significantly overexpressed in the OGD/R group (p < 0.05, Fig. 2E, F), while the expression of Cd163 has no significant change (Fig. 2G), and Arg1 expression was inhibited in the OGD/R group (p < 0.05, Fig. 2H). These results are consistent with those in the MCAO treatment group. In other words, the M1-type microglial cells were polarized following OGD/R modeling.

Fig. 2

The change of microglial cells in the OGD/R model. A The immunohistochemistry staining assay for M1 microglial cells infiltration analysis. B The mean fluorescence intensity (MFI) of M1 microglial cells in control and OGD/R groups. C The immunohistochemistry staining assay for M2 microglial cells infiltration analysis. D The MFI of M2 microglial cells in control and OGD/R groups. E–H The expression difference of Cd86 (E), Nos2 (F), Cd163 (G), and Arg1 (H) gene in the control and OGD/R groups. (**p < 0.01 and ns stands for non-significant difference)

The OGD-induced microglial cells with enhanced glycolysis-supported proinflammatory response

We analyzed the glycolysis and mitochondrial oxidative phosphorylation activity by the ECAR and OCR method to explore the metabolic difference of BV2 microglial cells after OGD/R induction. The results showed that the levels of ECAR are increased (Fig. 3A), while the levels of OCR are decreased in the OGD/R-induced group (Fig. 3B), indicating the glycolytic capacity was enhanced in the injured microglial cells. The proinflammatory factors such as the IL1b and IL6 expression levels were significantly up-regulated (p < 0.05, Fig. 3C, D), while the anti-inflammatory factors including the IL4 and IL10 expression levels were significantly decreased in the OGD group (Fig. 3E, F), suggesting that the microglial cells with the enhanced glycolysis ability could promote the proinflammatory response after hypoxia injury induction.

Fig. 3

Analysis of metabolic characteristics of microglia cells. A The extracellular acidification rate (ECAR) difference of microglial cells in the control and OGD/R groups. B The oxygen consumption rate (OCR) difference of microglial cells in the control and OGD/R groups. C The expression difference of IL1b in the control and OGD/R groups. D The expression difference of IL6 in the control and OGD/R groups. E The expression difference of IL4 in the control and OGD/R groups. F The expression difference of IL10 in the control and OGD/R groups. (**p < 0.01 and ***p < 0.001)

JAK-STAT signaling involved in the phenotypic imbalance of microglial cells

To elucidate the underlying downstream pathways that mediated the phenotypic imbalance of microglial cells after hypoxia injury induction, we measured the change of JAK-STAT signaling in the OGD and control groups. After the OGD/R modeling, the JAK-STAT signaling was significantly activated compared with that in the isotype and normal control, in which the phosphorylation level of JAK proteins, such as JAK1 (Fig. 4A, B) and JAK2 (Fig. 4C, D), and the STAT proteins, including STAT1 (Fig. 4E, F) and STAT6 (Fig. 4G, H), was significantly (p < 0.05) enhanced in the OGD/R group. These results demonstrated that the activated JAK-STAT signaling pathway plays a crucial role in the phenotypic imbalance of microglial cells.

Fig. 4

The JAK-STAT signaling pathway activation difference. A Flow cytometry for the phosphorylation analysis of JAK1 in the control and OGD/R groups. B The percentage of P-JAK1-positive cells was analyzed for differences between the two groups. C Flow cytometry for the phosphorylation analysis of JAK2 in the control and OGD/R groups. D The percentage of P-JAK2-positive cells was analyzed for differences between the two groups. E Flow cytometry for the phosphorylation analysis of STAT1 in the control and OGD/R groups. F The percentage of P-STAT1-positive cells was analyzed for differences between the two groups. G Flow cytometry for the phosphorylation analysis of STAT6 in the control and OGD/R groups. H The percentage of P-STAT6-positive cells was analyzed for differences between the two groups. (*p < 0.05, **p < 0.01 and ns stands for non-significant difference)

Catalpalactone attenuates the apoptosis of neuronal cells through inhibiting M1 microglial cells

The phosphorylation level of JAK1 and STAT1 was significantly enhanced in the microglial cells treated by the OGD/R, and we aimed to explore the therapeutic effect of STAT1 phosphorylation inhibitor (Catalpalactone) in the OGD/R-treated microglial cells. First, we detected the cell viability of the OGD/R-treated microglial cells under different concentrations of Catalpalactone and observed that the cell viability increased with the drug concentration and peaked at the 15 μM (p < 0.05, Fig. 5A). After 15 μM of Catalpalactone treatment, the marker of M1 microglial cells, such as Cd86 expression, was significantly inhibited (p < 0.05, Fig. 5B), while the Cd206 (as marker of M2 microglial cells) was significantly overexpressed in the OGD/R + CATA group compared to the untreated group (p < 0.05, Fig. 5C), indicating that the Catalpalactone treatment can promote the generation of damage repair M2 microglial cells phenotype. In addition, similar results were found in inflammatory factors expression. In other words, the proinflammatory cytokines including the IL1β and IL6 expression were inhibited (Fig. 5D, E) and the anti-inflammatory cytokines of IL4 and IL10 were up-regulated significantly (p < 0.05) in the OGD/R + CATA group compared to the OGD/R group (Fig. 5F, G). To illuminate the effect of microglial polarization to neuronal apoptosis, we performed the co-culture analysis of OG-induced BV2 and PC12 cells treated with Catalpalactone or not (Fig. 5H). Accordingly, the data of flow cytometry revealed that the proportion of normal living cells in the CON, OGD/R, and OGD/R + CATA groups is 92.6, 82.8, and 90.4%, respectively (Fig. 5I), and the apoptosis rate of PC12 cells is the highest in the OGD/R group and was significantly decreased in the OGD/R + CATA group (p < 0.05, Fig. 5J), indicating the inflammatory microglia cells can promote the apoptosis of neuronal cells, while the treatment of Catalpalactone can attenuate the inflammatory apoptosis of neuronal cells.

Fig. 5

The therapeutic effect analysis of Catalpalactone in OGD/R model. A The cell viability analysis under different Catalpalactone concentrations. B The Cd86 expression difference in OGD/R and OGD/R + CATA groups. C The Cd206 expression difference in OGD/R and OGD/R + CATA groups. D The IL1B expression difference in OGD/R and OGD/R + CATA groups. E The IL6 expression difference in OGD/R and OGD/R + CATA groups. F The IL4 expression difference in OGD/R and OGD/R + CATA groups. G The IL10 expression difference in OGD/R and OGD/R + CATA groups. H The co-culture of BV2 and PC12 system. I Flow cytometry to demonstrate apoptosis (%) in CON, OGD/R, and OGD/R + CATA groups, respectively. J The apoptosis ratio in the BV2 and PC12 co-culture system. (**p < 0.01, ***p < 0.001, ****p < 0.001, and ns stands for non-significant difference)

Catalpalactone promotes the polarization M2 microglial cells with inhibited glycolysis

Subsequently, we detected the phosphorylation level of JAK-STAT signaling in the OGD/R-induced microglial cells (BV2) treated by the Catalpalactone or not. The results showed that the phosphorylation of JAK1 and STAT1 was significantly inhibited, while the phosphorylation of JAK2 and STAT6 was significantly increased in the OGD/R + CATA group compared to the OGD/R group (p < 0.05, Fig. 6A–H), in which the phosphorylation activation of JAK1 and STAT1 is an indicator of the downstream signal event of M1 microglial cells and the phosphorylation activation of JAK2 and STAT6 is an indicator of M2 downstream signal events. These results suggested that Catalpalactone treatment promotes the polarization of M1 microglial cells to the M2 anti-inflammatory phenotype. In addition, the treatment of Catalpalactone also change the metabolic characteristic of microglial cells. Concretely, the ECAR was decreased (Fig. 6I) and the OCR was increased (Fig. 6G) in the OGD/R + CATA group, indicating the microglial cells have the enhanced oxidative phosphorylation and the decreased glycolysis activity.

Fig. 6

Metabolic change analysis after Catalpalactone treatment in OGD/R model. A Flow cytometry for the phosphorylation analysis of JAK1 after Catalpalactone treatment. B The MFI of JAK1 after Catalpalactone treatment. C Flow cytometry for the phosphorylation analysis of JAK2 after Catalpalactone treatment. D The MFI of JAK2 after Catalpalactone treatment. E Flow cytometry for the phosphorylation analysis of STAT1 after Catalpalactone treatment. F The MFI of STAT1 after Catalpalactone treatment. G Flow cytometry for the phosphorylation analysis of STAT6 after Catalpalactone treatment. H The MFI of STAT6 after Catalpalactone treatment. I The ECAR analysis in the OGD/R + CATA group. J The OCR analysis in the OGD/R + CATA group. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001, and ns stands for non-significant difference)

CATA treatment protects rats nerve function by polarizing M2 microglial cell

As shown in Fig. 7A, we present the timeline of the experimental design, which clearly indicates that after Transient Brain Ischemia (TBI), Catalpalactone treatment was given, followed by assessment of neurological function by behavioral tests, and finally immunohistochemical analysis. Subsequently, we used mNSS scores to assess neurological deficits in rats in the MCAO model and found that the mNSS scores of the MCAO + CATA group were significantly lower than those of the MCAO group on days 3 and 7. This suggests that Catalpalactone treatment has a potential role in improving neurological deficits caused by ischemic brain injury (p < 0.05, Fig. 7B). After Catalpalactone treatment, the number of proinflammatory M1 microglia cells was decreased (Fig. 7C) and the number of anti-inflammatory M1 microglia cells was increased (Fig. 7D) in the OGD/R group, the marker of M1 microglia cells (Cd86) expression was down-regulated (Fig. 7E), and the marker of M2 microglia cells (Cd206) was overexpressed (Fig. 7F) in the OGD/R + CATA group compared to the OGD/R group significantly (p < 0.05), suggesting that the treatment of Catalpalactone protects the neuronal cells from the hypoxic lesion through polarizing the microglial cell toward M2 phenotype.

Fig. 7

The therapeutic effect of Catalpalactone in the MCAO model. A The therapeutic process of Catalpalactone in the MCAO model. B The mNSS score in the MCAO + CATA group. C The immunohistochemistry staining assay for M1 microglial cells infiltration analysis after Catalpalactone. D The immunohistochemistry staining assay for M2 microglial cells infiltration analysis after Catalpalactone. E The expression difference of CD86 in the MCAO + CATA group. F The expression difference of CD206 in the MCAO + CATA group. Traumatic Brain Injury (TBI) indicates transient cerebral ischemia stage induced by the MCAO model in rats. Immunohistochemistry (IHC) indicates the experimental stage at the end of the experiment when brain tissue was analyzed by immunohistochemical staining (*p<0.05, ***p < 0.001 and ****p < 0.0001)

Discussion

The ischemic cerebrovascular disease has become a severe threat to health due to its high morbidity and mortality worldwide [31]. Catalpalactone is an iridoid glycoside isolated from Chinese herb (Bignoniaceae), and has antioxidant, anti-tumor, and anti-inflammatory effects [20]. As previously reported, Catalpalactone can inhibit the NO synthase (iNOS) expression and the production of NO, IL6 and tumor necrosis factor-α in the lipopolysaccharide-induced macrophage, and regulate the anti-inflammatory signaling, such as inhibiting STAT-1, IFN-β and IRF3 activation, to play a cytoprotecting role [21]. Meanwhile, the neuroinflammation plays an extremely crucial role in the ischemic stroke process [7]. Combined with these cues, we speculate that the Catalpalactone may alleviate the MCAO-induced ischemic brain injury and neurological deficits.

Our results showed that the infiltration of microglial cells is obviously increased after MCAO induction in the ischemic lesions area, especially M1 microglial cells phenotype. It is well known that the microglial cell/macrophages and neurons play a crucial role for the proinflammatory cytokines production in the inflammatory cascades response [32]. Previous studies have shown that the cerebral ischemia resulted in cell death, such as neurons, which generate ROS, and inflammatory cytokines, leading to the activation and recruitment of microglia/macrophages in the ischemia and penumbra regions [33]. Furthermore, the microglia/macrophages can be directly activated by the neuronal death, and the activated microglia produce more inflammatory mediators, including the inflammasome of nucleotide-binding oligomerization domain-like receptor protein 3 (NLPR3), causing the blood–brain barrier damage, brain edema and hemorrhage, and more neuronal death [34]. Therefore, various anti-inflammatory agents exhibited great potential in alleviating ischemic injury via inhibiting inflammation and rescuing neuronal cells.

The M1 microglial cells express M1-specific markers CD86, CD80, and Nos2 and produce proinflammatory cytokines, such as IL-6, IL-1β and TNF-α, while M2 microglial cells express M2-specific markers CD206, CD163, and Arg1 and release the anti-inflammatory cytokines, such as IL-4 and IL-10 [35]. Hypoxia–ischemia stimulation can lead to the M1 microglial cells polarization; the similar result in the MCAO model was also observed in the OGD/R-induced BV2 model, the M1 phenotype contributes to oxidative stress, neurotoxicity, and neuronal and synaptic damage. A study of neurodegenerative disease (Alzheimer) reported that the clearance and surveillance functions of microglial cells are gradually lost with age or disease progression [36] and inflammation may be involved in this dysfunction [37], in which the microglial cells switch the mitochondrial oxidative phosphorylation (OXPHOS) to the enhanced glycolysis under stress conditions [38]. A consequence of the metabolic alteration is that the massive ATP and glycolytic metabolites were generated in the microglia rapidly, importantly the increasing lactate can directly promote the release of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) from the microglia [39], contributing to the immune functions of microglia, but its phagocytosis and clearing are compromised [40]. In contrast, inhibition of glycolysis in microglia can ameliorate the several inflammation-related diseases, including the multiple sclerosis, ischemic stroke, and sepsis [41, 42], thus reprogramming of microglial glycolysis may be a promising strategy of ameliorating neuroinflammation progression. In our results, the OGD/R-induced microglial cells are prone to the activation of M1 proinflammatory phenotype with the enhanced glycolysis activity, whereas the microglial cells treated with the Catalpalactone are polarized toward the M2 anti-inflammatory phenotype with the inhibited glycolysis activity and the immensely activated JAK2/SATA6 signaling that is a classic M2 downstream feature [43], and the apoptosis rate of neurons was significantly decreased in the co-culture system of BV2 and PC12 cells. Another study reported that the IL-4 treated BV2 cells presented higher M2 proportion and vastly decreased PC12 pyroptosis rate [44]. In the MCAO model, the neurological defects of rats were alleviated after Catalpalactone treatment and the ischemic lesions area had higher M2 microglia infiltration. Overall, these results proven that the neuroprotective effect of Catalpalactone on the neurons from the overactivated M1 microglia in pathological conditions.

However, there are some limitations to our study. First, the BV2 cells used in the in vitro experiments were immortalized cell lines of mouse microglia, which may have some differences in their biological properties. In future studies, we plan to use primary microglia models to better mimic the in vivo microenvironment. Second, this study mainly used a rat MCAO model to simulate ischemic brain injury, and although this model is widely used in neurological injury research, it does not fully reflect the complex pathological process of ischemic stroke in humans. Therefore, the role and mechanism of Catalpalactone can be further validated in future by introducing large animal models or patient-derived brain tissue samples. In addition, this study has only explored the mechanism of action of Catalpalactone at the molecular and cellular levels, but has not deeply analyzed its targets and the upstream and downstream regulatory networks of related signaling pathways. In this regard, we plan to combine gene editing technology and multi-omics analysis to comprehensively explore the mechanism of Catalpalactone’s action and screen potential drug targets, so as to provide a more powerful basis for the clinical treatment of ischemic brain injury.

Conclusion

Our results indicated that Catalpalactone (15 µM) exhibited a neuroprotective effect against the apoptosis of neurons by relieving neuroinflammation via polarizing the M1 microglia toward the M2 phenotype and inhibiting the JAK1/STAT1 pathway in the M1 microglia. Hence, Catalpalactone could be a potential anti-inflammatory agent for ischemic stroke treatment.