Male C57BL/6J mice (6–8 weeks old, weighing 20–25 g, Beijing LiHua Experimental Animal Technology Co., Ltd., Beijing, China) were randomly divided into four groups (n = 6 per group): (1) Control group (n = 6): Mice underwent sham surgery without middle cerebral artery occlusion (MCAO) and received no treatment; (2) MCAO group (n = 6): Mice underwent MCAO surgery without any additional treatment; (3) MCAO+DMSO group (n = 6): Mice underwent MCAO surgery and received vehicle solution (0.5% CMC-Na containing 1% DMSO, 10 mL/kg) by oral gavage once daily; (4) MCAO+Formononetin group (n = 6): Mice underwent MCAO surgery and received Formononetin (30 mg/kg, dissolved in 0.5% CMC-Na containing 1% DMSO, 10 mL/kg, Cat. No. F805983, Macklin, Suzhou, China) by oral gavage once daily. All mice were housed under specific pathogen-free (SPF) conditions with controlled temperature (22 $\pm$ 2 °C), humidity (55 $\pm$ 5%), and a 12-hour light/dark cycle, with free access to standard laboratory chow and water. Drug administration began immediately after MCAO reperfusion and continued for the duration of the experiment. The MCAO technique was executed following established protocols cited in the literature [18]. Mice were anesthetized with sodium pentobarbital (1.5%, 40 mg/kg, intraperitoneal injection). After reperfusion, Formononetin (dissolved in 0.5% CMC-Na containing 1% DMSO) was administered by oral gavage at a dose of 30 mg/kg body weight once daily, with the dosing volume maintained at 10 mL/kg. The control group received an equal volume of vehicle solution (0.5% CMC-Na containing 1% DMSO) by the same route. Subsequent neurological deficit evaluations were carried out to assess the degree of neurological impairment, using the previously outlined methods [18]. At the end of the experiment, mice were deeply anesthetized with sodium pentobarbital (3.0%, 150 mg/kg, intraperitoneal injection) and euthanized by cervical dislocation. Brain tissues were immediately collected for subsequent analyses. All experimental procedures were approved by the Animal Ethics Committee of Jiangsu Kanion Pharmaceutical Co., Ltd. (Experimental Animal Ethics Number: 20240312) and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Three mice were randomly selected from each group to measure the infarct volume. The remaining mice had their brain tissues divided into two parts: one part was used for histopathological analysis, and the other part was used for molecular biology analysis.

The infarct volume in 1 mm sections of brain was assessed by 2,3,5-tribenzotetrazole staining. All infarcts extended into both cortical and subcortical areas. Hematoxylin and Eosin (H&E) staining was used to assess brain tissue pathology. Tissue sections, embedded in paraffin and sliced to 5 micrometers, were stained and examined histologically. TUNEL assay was performed with an In Situ Cell Death Detection Kit (Cat. No. 11684817910, Roche, Germany) to identify apoptotic neurons at injury sites. Fluorescence microscopy (BX53, Olympus, Tokyo, Japan) was utilized to visualize and capture images of TUNEL-positive cells. Paraffin-embedded sections were subjected to a xylene dewaxing process for five minutes and subsequently stained with Nissl stain. The sections were then immersed in 95% ethanol until the Nissl substance appeared dark blue against a colorless or pale blue background. Nissl bodies within the cortical neurons were visualized under a microscope, providing insights into cellular morphology and structure.

The SH-SY5Y cell line was purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China) and confirmed by Short Tandem Repeat (STR) for purity. Mycoplasma, bacteria, and fungi were all negative. The cells were cultured in DMEM/F12 medium with 10% fetal bovine serum and antibiotics at 37 °C with 5% CO₂. Before the oxygen-glucose deprivation (OGD) procedure [19, 20], cells were pretreated with Formononetin (10 or 50 mmol/L) or stattic (10 µmol/L) for one hour. OGD involved transferring cells to glucose-free DMEM and exposing them to 95% N₂ and 5% CO₂ at 37 °C for four hours. After OGD, cells were returned to standard culture conditions for a twelve-hour reoxygenation period.

Cells were transfected with IL-10 siRNA or scramble control siRNA (GenePharma, Shanghai, China). siRNA sequences were: IL-10 siRNA sense: 5^′-UAAGCUCCAAGAGAAAGGCdTdT-3^′, anti-sense: 5^′-GCCUUUCUCUUGGAGCUUAdTdT-3^′; scramble siRNA sense: 5^′-AACAGUCGCGUUUGCGACUGGdTdT-3^′, anti-sense: 5^′-CCAGUCGCAAACGCGACUGUUAdTdT-3^′. c-Fos siRNA: sense: 5^′-GCAAGGUGGAACAGUUAUC dTdT-3^′ and anti-sense: 3^′-dTdT CGUUCCACCUUGUCAAUAG-5^′. Cells were seeded in 6-well plates (2 $\times$ 10⁵ cells/well) and grown to 60–70% confluence. For transfection, 50 nM siRNA and 5 µL Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) were separately diluted in 125 µL Opti-MEM (Gibco, Carlsbad, CA, USA), combined, and incubated for 15 minutes at room temperature. The mixture was added to cells in antibiotic-free medium, and replaced with complete medium after 6 hours. After 48 hours of incubation (37 °C, 5% CO₂), knockdown efficiency was verified by Western blot. Transfected cells then underwent OGD treatment as described previously.

SH-SY5Y cells and mice brain tissues were homogenized in cold PBS and centrifuged at 14,000 rpm. IL-10 (Cat. No. ab255729), IL-1$\beta$ (Cat. No. ab197742), IL-6 (Cat. No. ab222503), and tumor necrosis factor-$\alpha$ (TNF-$\alpha$) (Cat. No. ab208348) levels were quantified using specific ELISA kits (Abcam, Cambridge, UK) according to the manufacturer’s instructions.

Total RNA was extracted from SH-SY5Y cells and peripheral mouse brain tissue near the infarct area using TRIzol reagent (Cat. No. 15596026, Invitrogen, USA). SYBR Green (Cat. No. RR420A, Takara, Ostu, Japan) was used for real-time PCR, with GAPDH serving as the normalization reference. The relative mRNA expression was calculated using the 2^{-Δ⁢Δ⁢Ct} method. Each sample was analyzed in triplicate, and the results are presented as mean $\pm$ standard deviation (SD). The primer sequence list is shown in Supplementary Tables 1,2.

Cell samples and brain tissues were lysed in Radioimmunoprecipitation Assay buffer (RIPA) buffer supplemented with phosphatase and protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was determined using the BCA Protein Assay Kit (Cat. No. 23225, Thermo Fisher Scientific, USA). Equal amounts of protein (30 µg) were separated by 10% Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Burlington, MA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies against: IL-1$\beta$ (1:1000, Cell Signaling Technology, Danvers, MA, USA, #12242); TNF-$\alpha$ (1:1000, Cell Signaling Technology, #3707); IL-6 (1:1000, Abcam, ab229381); IL-10 (1:1000, Abcam, ab133575); p-STAT3 (1:1000, Abcam, ab267373); STAT3 (1:1000, Abcam, ab68153); c-Fos (1:1000, Abcam, ab222699); GAPDH (1:5000, Cell Signaling Technology, #5174); Histone H3 (1:1000, Cell Signaling Technology, #4499). After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies (goat anti-rabbit IgG, 1:5000, #7074; goat anti-mouse IgG, 1:5000, #7076; Cell Signaling Technology) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Bio-Rad, Hercules, CA, USA) and captured using the ChemiDoc XRS+imaging system (Bio-Rad). Band intensities were quantified using Image Lab software (Version 6.0, Bio-Rad, Hercules, CA, USA). GAPDH was used as the loading control for cytoplasmic proteins, while Histone H3 served as the loading control for nuclear proteins. The relative protein expression was calculated as the gray value ratio of target protein to their respective loading controls, and results were expressed as fold change relative to control. Each experiment was performed in triplicate.

For the immunofluorescence study, procedures from prior reports were adapted [19, 20]. Initially, the brain samples were promptly rinsed with cold PBS and then fixed using a 4% paraformaldehyde solution for 15 minutes at room temperature. After this stabilization step, the samples underwent a 3–5-minutes rinse in PBS. Subsequently, the brain sections were treated for 30 minutes at room temperature with a blocking solution made up of 0.2% powdered milk, 2% normal goat serum (Merck Millipore, NS20L), 0.1 M glycine, 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA, A7906), and 0.01% Triton X-100. For brain tissue immunofluorescence: primary antibody IL-10 (1:200, Abcam, ab133575) for 90 minutes at room temperature in a humid chamber. After that, the brain sections were washed three times in PBS and then incubated with fluorescent secondary antibodies (Alexa Fluor 647-conjugated goat anti-rabbit IgG (1:500, Invitrogen, A21245)) for 45 minutes at room temperature. The nuclei were highlighted by staining the sections with DAPI (4,6-diamidino-2-phenylindole). A laser scanning confocal microscope (TCS SP8, Leica Microsystems, Wetzlar, Germany) was utilized to capture and document the resulting fluorescence.

The intracellular ROS levels were measured using 2^′,7^′-dichlorofluorescin diacetate (DCFH-DA) fluorescent probe (Sigma-Aldrich, D6883). SH-SY5Y cells were seeded in 24-well plates at a density of 1 $\times$ 10⁵ cells/well and divided into four groups: Control, OGD/R, OGD/R+DMSO, and OGD/R+Formononetin (20 µM). After respective treatments, cells were washed twice with PBS and incubated with DAPI for 12 h, following 10 µM DCFH-DA in serum-free medium for 30 minutes at 37 °C in the dark. The cells were then washed three times with PBS to remove excess probe. Fluorescence images were captured using a fluorescence microscope (Olympus IX73, Japan) with excitation at 488 nm and emission at 525 nm. The fluorescence intensity was quantified using ImageJ software (1.52U, National Institutes of Health, Bethesda, MD, USA) and normalized to the control group.

The data involved in this study are all continuous variables. A normality test was performed first with Shapiro-Wilk methods and data that followed a normal distribution were presented as mean $\pm$ SD, then, differences between two groups were assessed using the Student’s t-test, while comparisons among more than two groups were made using analysis of variance (ANOVA) following post Bonferroni test. A p-value of less than 0.05 was considered statistically significant.

The structural depiction of Formononetin is illustrated in Fig. 1A. To determine the therapeutic potential of Formononetin, the study implemented the MCAO model in mice, administering Formononetin at a concentration of 30 mg/kg. 2,3,5-tribenzotetrazole staining assay conducted 24 hours after MCAO induction revealed that Formononetin markedly decreased the brain infarct volumes in mice (Fig. 1B,C, p 0.001).

The effect of Formononetin on neuronal damage in mouse brain tissues after MCAO was assessed. Post-I/R, extensive neuronal damage was observed in the ischemic penumbra of the cerebral cortex, as demonstrated by H&E staining (Fig. 2A). In the sham operation group, the neuronal cells were arranged regularly, the structure was clear, and the nuclei were round and regular. In contrast, most of the neurons in MACO are disorganized and severely atrophy. Conversely, Formononetin notably ameliorated these detrimental effects. Nissl staining showed that compared with the sham operation group, the number of Nissl bodies decreased significantly in the drug-loading group and increased significantly after Formononetin treatment (Fig. 2B,C, p 0.001). Apoptosis within the brain tissues was quantified using the TUNEL assay, which suggests that neuronal apoptosis was markedly elevated in the MCAO group compared to the control group, whereas Formononetin application reduced apoptosis levels, depicted in Fig. 2D,E (p 0.001). Furthermore, Formononetin reduced the MCAO-induced increase in the pro-apoptotic gene Bcl-2-associated X protein (BAX) and the anti-apoptotic protein B-cell lymphoma 2 (BCL-2), as demonstrated through RT-qPCR analyses (Fig. 2F,G, p 0.001). Collectively, these results demonstrate that Formononetin significantly attenuated neuronal damage and brain cell apoptosis in mice subjected to MCAO.

Fig. 2.

Formononetin therapy reduced nerve injury and brain cell death in MCAO mice. (A) Hematoxylin and eosin staining experiment. Scale bar, 100 µm. Red arrows: indicate the lesion area or structural changes. (B,C) Nissl staining assay. Scale bar, 100 µm. Red arrows: indicate the neuronal damage and structural destruction. (D,E) Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling (TUNEL) assay, Scale bar, 100 µm. (F,G) Bcl-2-associated X protein (BAX) and B-cell lymphoma 2 (BCL-2) mRNA levels were determined by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). ***p 0.001. n = 3, data are represented as mean $\pm$ SD. H&E, Hematoxylin and Eosin.

In the present study, mRNA expression conducted 24 hours post-reperfusion revealed a notable elevation in pro-inflammatory cytokine levels due to MCAO, which Formononetin treatment significantly attenuated (Fig. 3A–C, p 0.05, or p 0.01). ELISA results confirmed a significant elevation in the expressions of IL-1$\beta$, TNF-$\alpha$, and IL-6 in MCAO mice (Fig. 3D–F, p 0.001), and in OGD/R-treated SH-SY5Y cells (Fig. 3G–I, p 0.001). Formononetin effectively mitigated these changes, restoring cytokine levels to their baseline. To investigate the effect of Formononetin on ROS generation during OGD/R injury, we performed DCFH-DA fluorescence staining (Fig. 3J). The results showed that OGD/R treatment significantly increased intracellular ROS levels compared to the control group (p 0.001). The OGD/R+DMSO group showed similar elevated ROS levels to the OGD/R group, indicating that the vehicle had no significant effect. Notably, treatment with Formononetin (20 µM) markedly attenuated the OGD/R-induced ROS accumulation (p 0.001 vs. OGD/R+DMSO group). The fluorescence intensity in the Formononetin-treated group was reduced by approximately 60% compared to the OGD/R+DMSO group, suggesting that Formononetin effectively suppressed oxidative stress in SH-SY5Y cells during OGD/R injury.

Fig. 3.

Formononetin therapy decreased inflammatory response. (A–C) The mRNA expression of pro-inflammatory cytokines was determined by RT-qPCR assay in MCAO mice. (D–F) The levels of pro-inflammatory cytokines were measured using Enzyme-Linked Immunosorbent Assay (ELISA) in MCAO mice. (G–I) The levels of pro-inflammatory cytokines were measured using ELISA in oxygen-glucose deprivation/reperfusion (OGD/R)-treated SH-SY5Y cells. (J) Representative fluorescence images showing intracellular reactive oxygen species (ROS) levels detected by 2^′,7^′-dichlorofluorescin diacetate (DCFH-DA) staining in SH-SY5Y cells under different treatments, Scale bar, 100 µm. *p 0.05, **p 0.01, ***p 0.001. n = 3, data are represented as mean $\pm$ SD.

Formononetin administration was shown to increase IL-10 production, enhancing survival in mice compared to non-treated controls. This underscores IL-10’s vital role in modulating immune responses, suggesting that Formononetin’s neuroprotective effects against cerebral I/R injury may involve increased IL-10 levels. Fig. 4A–C shows that after Formononetin treatment, IL-10 levels and STAT3 phosphorylation levels were significantly increased (p 0.001). Fig. 4D–F also showed that IL-10 increased in the MCAO+Formononetin group (p 0.001). The effect of Formononetin was further validated in SH-SY5Y cells in vitro (Fig. 4G–K, p 0.05, or p 0.01, or p 0.001). However, using stattic, a STAT3 inhibitor, negated Formononetin’s protective effects, and potentially reverses the suppression of pro-inflammatory cytokines IL-1$\beta$, TNF-$\alpha$, and IL-6, as well as apoptosis (Fig. 5A–H, p 0.05, or p 0.01, or p 0.001). Collectively, these results indicate Formononetin’s protective effects against OGD/R-induced inflammation are mediated via the IL-10/STAT3 pathway.

Fig. 4.

Formononetin’s protective effect against I/R damage was associated with the activation of Interleukin-10 (IL-10)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling. (A–C) protein expression of IL-10 and phosphorylated-STAT3 (p-STAT3)/STAT3 in the indicated groups in the MCAO model. (D) IL-10 levels were measured using an ELISA kit in the in vivo. (E,F) Immunofluorescence staining of IL-10 pro-inflammatory cytokines levels was measured in vivo. Scale bar, 100 µm. (G–I) protein expression of IL-10 and p-STAT3/STAT3 in SH-SY5Y cells in the indicated groups. (J,K) Quantification of TUNEL-positive cells in SH-SY5Y cells in the indicated groups (TUNEL, green; DAPI, blue). Scale bar, 50 µm. *p 0.05, **p 0.01, ***p 0.001. n = 3, data are represented as mean $\pm$ SD.

Fig. 5.

Reducing STAT3 diminished Formononetin’s protective effect. (A–C) mRNA expression of pro-inflammatory cytokines measured by RT-qPCR assay. (D–F) Pro-inflammatory cytokines levels were measured using an ELISA kit. (G,H) Quantification of TUNEL-positive cells in SH-SY5Y cells in the indicated groups (TUNEL, green; DAPI, blue). Scale bar, 100 µm. *p 0.05, **p 0.01, ***p 0.001. n = 3, data are represented as mean $\pm$ SD.

To elucidate IL-10’s contribution to Formononetin’s modulation of SH-SY5Y cellular responses, IL-10-targeted siRNAs were utilized to suppress IL-10 expression prior to subjecting cells to 24 hours of OGD/R treatment. WB verified the successful reduction of IL-10 (Fig. 6A–C, p 0.01). Inhibition of IL-10 expression notably compromised Formononetin’s ability to mitigate expression of IL-10, STAT3, and the diminution of inflammatory markers (Fig. 6D–I, p 0.01, or p 0.001). These outcomes highlight IL-10’s pivotal function in facilitating Formononetin’s neuroprotective effects in SH-SY5Y cells following OGD/R insult.

Fig. 6.

Inhibiting IL-10 greatly reduced the protective effect of Formononetin. (A–C) RT-qPCR and Western Blot (WB) experiments were conducted to verify the efficiency of gene interference. (D–F) Protein expression of IL-10 and p-STAT3/STAT3 were detected by WB experiments. (G–I) The pro-inflammatory cytokines were measured using ELISA. **p 0.01, ***p 0.001. n = 3, data are represented as mean $\pm$ SD.

The Activator Protein-1 (AP-1) transcription factor complex, consisting of Fos and Jun family members, boosts IL-10 transcription. Given this, it’s suggested that Formononetin’s neuroprotective effects against cerebral I/R injury may involve the modulation of c-Fos [21, 22]. In our study, we observed a significant decrease in c-Fos expression in the nucleus of the OGD/R group. However, Formononetin reversed this effect, promoting the nuclear translocation of c-Fos (Fig. 7A–C, p 0.001). To further explore the function and role of c-Fos, we suppressed its expression through siRNA interference experiments, with the efficiency of interference evaluated by mRNA and protein results (Fig. 7D–F, p 0.001). The research results demonstrated that inhibiting c-Fos markedly suppressed the activation of IL-10 and STAT3 expression induced by Formononetin (Fig. 7G–J, p 0.05, or p 0.01, or p 0.001). Further investigation revealed that knockdown of c-Fos significantly reversed the activation of inflammatory factor expression induced by Formononetin (Fig. 7K–M, p 0.001). Collectively, these results suggested that Inhibited c-Fos greatly reduced the expression of the IL-10 /STAT3 pathway and the protective effect of Formononetin.

Fig. 7.

Inhibited c-Fos greatly reduced expression of IL-10/STAT3 pathway and the protective effect of Formononetin. (A–C) Protein and mRNA expression of cy-c-Fos and n-c-Fos expression. (D–F) Knockout efficiency of c-Fos in sic-Fos knockdown experiment. (G–J) Protein expression of c-Fos/IL10/STAT3 in sic-Fos knockdown experiment. (K–M) The pro-inflammatory cytokines were measured using ELISA. *p 0.05, **p 0.01, ***p 0.001. n = 3, data are represented as mean $\pm$ SD.