Oxymatrine Alleviates Cerebral Ischemia/Reperfusion Injury By Targeting HDAC1 to Regulate Mitochondria-Related Autophagy and Oxidative Stress

In this study, we employed 164 male C57BL/6 mice, aged between 6 and 8 weeks. The experimental procedures received approval from the Institutional Animal Care and Use Committee and the Ethics Review Committee of Shandong Second Medical University (2021SDL481). The animals were housed in the Laboratory Animal Center of Shandong Second Medical University under controlled conditions: a temperature range of 20 − 23 °C, humidity levels of 40 − 70%, and a 12-h light/dark cycle. The mice were randomly assigned to three groups: the Sham group, the I/R group, the I/R+OMT group (administered oxymatrine intraperitoneally at a dosage of 100 mg/kg/day for one week before MCAO and during the reperfusion period), and the I/R+OMT + Vorinostat group, treated with oxymatrine (as above) combined with Vorinostat (SAHA, MCE, China,100 mg/kg in 200 μl PBS, administered intraperitoneally once daily for 3 weeks) [22].

The cerebral I/R injury model was established using the MCAO method as traditional. Briefly, mice were anesthetized with 2% isoflurane for induction, maintaining anesthesia with 1.5% isoflurane during the procedure. A monofilament (Doccol Corporation, USA) was inserted via the common carotid artery to obstruct the middle cerebral artery. The filament was removed to restore blood flow after 60 min of occlusion. Neurological function was assessed 24 h after reperfusion using the modified neurological severity score (mNSS) system.

HT22 cells were cultured in DMEM/F12 medium (HyClone Laboratories, Waltham, MA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco, USA) under standard conditions (37 °C, 5% CO₂, 90% humidity), with medium replacement every 24 h. To induce oxidative injury, cells were treated with glutamate at graded concentrations (2, 4, 6, 8, or 10 μM) for 24 h. Concurrently, oxymatrine (5, 15, 35, or 45 μM) was administered to assess neuroprotective effects. Control groups included untreated cells (fresh medium only) and vehicle controls (equivalent volumes of PBS for glutamate or ≤0.1% DMSO for OMT). HT-22 cells were seeded in 96-well plates at a density of 5 × 10³ cells/well and allowed to adhere for 24 h under standard culture conditions (37 °C, 5% CO₂). After treatment, 10% (v/v) CCK-8 reagent (BS350B, Biosharp, China) was added to each well, followed by incubation at 37 °C for 3 h. Absorbance was measured at 450 nm using a Multiskan FC microplate reader (Thermo Fisher Scientific, Shanghai, China).

The expression of manganese superoxide dismutase (MnSOD) and intracellular superoxide levels in HT-22 cells was analyzed via Western blot and DHE fluorescence staining, respectively. For MnSOD detection, cells were lysed in RIPA buffer (P0013, Beyotime, China) containing protease inhibitors (A32963, Thermo Fisher, USA). Protein concentrations were quantified using a BCA assay kit (BL521A, Biosharp, China), and 30 μg of total protein per sample was separated by 12% SDS-PAGE. Membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with a primary antibody against MnSOD (1:1000, ab13533, Abcam, UK), followed by HRP-conjugated secondary antibody (1:5000, SA00001-2, Proteintech, China) at room temperature for 1 h. Signals were visualized using an ECL substrate (BL520A, Biosharp, China) and quantified via ImageJ.

For superoxide detection, cells were incubated with 10 μM dihydroethidium (DHE, S0063, Beyotime, China) in serum-free medium at 37 °C for 30 min in the dark. After three PBS washes, fluorescence images were captured using a confocal microscope (Zeiss LSM 880, 543 nm excitation/590 nm emission) at 400 × magnification. Fluorescence intensity was normalized to cell count using ImageJ.

Locomotor activity and anxiety-like behavior were evaluated using an open field apparatus (40 × 40 × 30 cm, white acrylic chamber; RWD, China). Mice were individually placed in the center and allowed to freely explore for 10 min under dim light. Movement trajectories were recorded by an overhead camera and analyzed via ANY-maze software (Smart 3.0, RWD, China). Total distance traveled (mm) was quantified. The apparatus was cleaned with 70% ethanol between trials to eliminate odor cues.

Spatial novelty exploration was evaluated using a Y-maze composed of three identical arms (35 cm length × 10 cm width × 15 cm height; 120° inter-arm angles; Shanghai Jiliang, China). During the training phase, mice were permitted to explore two arms for 10 min, while the third arm (novel arm) was temporarily blocked. After a 24-h interval, the test phase was conducted by unblocking the novel arm and allowing free exploration of all three arms for 8 min. Arm entries were defined as the complete entry of all four paws into an arm. Exploration behavior was analyzed for three parameters: (1) novel arm entry preference (percentage of entries into the novel arm relative to total entries), (2) time spent in the novel arm (seconds), and (3) latency to first novel arm entry (time delay in seconds). Trials with fewer than 5 total arm entries were excluded to ensure behavioral validity. The maze was rigorously cleaned with 70% ethanol between trials to eliminate residual olfactory cues.

Tissue of ischemic penumbra of the cerebral cortex or cell samples were lysed, and protein concentrations were determined using the BCA assay. Protein extracts were separated on 10%–12% SDS-PAGE gels, transferred to PVDF membranes (Millipore), and probed with the following primary antibodies: Apaf-1 (1:1000, ABclonal, Wuhan, China), cleaved-caspase3 (1:500, Proteintech, Wuhan, China), Fis1 (1:2000, Proteintech, Wuhan, China), NBR1 (1:1000, Proteintech, Wuhan, China), Mfn2 (1:4000, Proteintech, Wuhan, China), LC3 (1:1000, ABclonal, Wuhan, China), HDAC1(1:2000, ABclonal, Wuhan, China), PINK1 (1:800, ABclonal, Wuhan, China), Parkin (1:2000, Proteintech, Wuhan, China), Beclin1 (1:2000, Proteintech, Wuhan, China), p62 (1:2000,Proteintech, Wuhan, China). And GAPDH (1:4000, Proteintech, Wuhan, China) was used as a loading control. The secondary antibody was incubated for 1 h at room temperature.

Brain tissue was quickly removed and frozen at −20 °C for 10 min; 1-mm-thick coronal sections were placed in 1.5% TTC and incubated at 37 °C for 15 min. Infarct volume was quantified using ImageJ software. Relative infarct volume was calculated as (contralateral volume minus ipsilateral non-infarct volume) divided by contralateral volume.

Paraffin-embedded brain sections (Ischemic penumbra of the cerebral cortex) were dehydrated, hydrated, and fixed according to the instructions of the TUNEL assay kit (KeyGEN BioTECH, China). TUNEL-positive cells were stained green, while normal cell nuclei were stained blue. Sections were observed under a fluorescence microscope.

Following deep anesthesia, mice were euthanized via transcardial perfusion with PBS. Ischemic penumbra of the cerebral cortex tissues was then collected, fixed in 4% paraformaldehyde, dehydrated in graded ethanol, and embedded in paraffin for sectioning (5 μm thickness). Coronal sections underwent antigen retrieval using citrate buffer (Beyotime, Jiangsu, China), followed by blocking with 10% goat serum in PBS for 1 h at room temperature. Sections were incubated overnight at 4 °C with the following primary antibodies diluted in PBS:LC3 and PINK1 (1:200, ABclonal, Wuhan, China); Beclin1, Apaf1, NeuN, P62 and NBR1 (1:100, Proteintech, Wuhan, China).After three 5-min washes with PBS, sections were incubated with Alexa Fluor-conjugated secondary antibodies (1:500) at 37 °C for 1 h. Nuclei were counterstained with DAPI (Solarbio, Beijing, China) for 10 min at room temperature. All steps included three PBS washes (5 min each). Images were acquired using a fluorescence microscope (400× magnification, Olympus).

The targets of Oxymatrine were screened using the Swiss Target Prediction database (http://www.swisstargetprediction.ch/↗). All screened targets were converted into the UniProt ID format using the UniProt database (https://www.uniprot.org/↗).

We searched for "Autophagy" and "Cerebral ischemia–reperfusion" related targets in the GeneCardsdatabase (https://www.genecards.org/↗). The UniProt database (https://www.uniprot.org/↗) was used to convert all targets obtained from the above screening into the UniProt ID format.

By identifying the common targets shared by autophagy and cerebral ischemia–reperfusion targets and the predicted Oxymatrine targets, a visual representation was shown in a Venn diagram using the VENN Website (http://bioinformatics.psb.ugent.be/webtools/Venn/↗).

The crystal structures of the key targets were downloaded from the AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/↗) and saved in PDB format. In AutoDockTools, water molecules are removed from the protein crystal structure, polar hydrogen atoms are added, and charges are added. Chemical constituents are in PDBQT format. In the grid box module, the distance of X, Y and Z coordinates is set to determine the active site of the protein. Molecular docking was performed in AutoDock Vina (https://autodock.scripps.edu/↗) to obtain the docking affinity (affinity, kcal/mol) between the active ingredient and the key target. The visualization of the molecular docking and its interactions was displayed in PyMOL (https://pymol.org/2/↗).

The levels of total antioxidant capacity (TAC), superoxide dismutase (SOD), and reduced/oxidized glutathione (GSH/GSSG) in brain tissues were measured using the following commercial assay kits: SOD Activity Kit (S0101S, Beyotime, China), TAC Detection Kit (BC1315, Solarbio, China), and GSH/GSSG Ratio Assay Kit (S0053, Beyotime, China). Briefly, the injured ischemic penumbra of the cerebral cortex was homogenized in ice-cold RIPA lysis buffer (P0013, Beyotime, China), and total protein concentrations were quantified via a BCA protein assay (BL521A, Biosharp, China) to normalize results to tissue weight (μg/mg). Tissue lysates were then incubated with kit-specific working solutions under manufacturer-specified conditions, followed by absorbance measurements at designated wavelengths using a BioTek Synergy H1 microplate reader. Final concentrations of MDA, TAC, SOD, and GSH/GSSG were calculated based on standard curves generated for each assay.

Data was collected from at least three independent experiments. Statistical analyses were performed using GraphPad Prism version 9.0 software (GraphPad, USA). Data were analyzed by one-way ANOVA with Tukey's post hoc test for multi-group comparisons. Intergroup differences were assessed using unpaired Student's t tests where appropriate. All values are expressed as mean ± SD. Statistical significance was set at p < 0.05.

To investigate the protective effect of oxymatrine on cerebral I/R injury, cerebral infarct volume, edema volume, and neurological deficit scores were measured. As anticipated, the I/R+OMT group exhibited reductions in cerebral infarct volume, edema volume, and deficit scores compared with the I/R group. The percentage of brain infarct volume in the I/R+OMT group significantly decreased from 30.08 ± 2.11% to 15.37 ± 1.48% (p < 0.05). The relative edema volume significantly decreased after oxymatrine treatment (Fig. 1C–E, J). Additionally, the mice's motor and cognitive impairments were significantly improved after oxymatrine administration (Fig. 1F–I). The deficit scores also decreased after oxymatrine treatment (Fig. 1J). These results demonstrate that oxymatrine ameliorates cerebral I/R injury.

TUNEL staining was used to evaluate the effect of oxymatrine on apoptosis. As shown in Fig. 2B, E, I/R injury significantly increased the number of positive cells in the I/R group. Oxymatrine treatment significantly reduced the number of positive cells in the I/R+OMT group compared with the I/R group. Quantitative analysis showed that pre-treatment with oxymatrine reduced apoptotic cells by 43.37% (p < 0.05). In conclusion, oxymatrine can alleviate neuronal cell damage and cell apoptosis caused by brain I/R injury in mice.

The effect of oxymatrine on the autophagy signaling pathway after I/R injury was further investigated, and the changes in the levels of related proteins were detected by Western blotting and immunofluorescence. Compared with the sham group, the LC3-II/I ratio and the expression of PINK1, Parkin, Beclin-1, and NBR1 in the I/R group increased, while the level of P62 decreased. In contrast, the I/R+OMT group showed the opposite trend. Compared with the I/R group, the levels of key proteins promoting autophagy in the I/R+OMT group decreased (Fig. 3A–G). In contrast, the I/R+OMT group demonstrated a reversal of these trends. Specifically, oxymatrine treatment in the I/R+OMT group led to a decrease in the levels of key autophagy-promoting proteins. Moreover, the P62 protein, which inhibits autophagy, showed an increased level in the I/R+OMT group, suggesting that oxymatrine influenced the regulation of autophagy. Immunofluorescence analysis further confirmed these results, indicating that oxymatrine treatment reduced the expression of autophagy-related proteins induced by cerebral I/R injury. Immunofluorescence co-localization studies revealed that LC3, PINK1, Beclin-1, and NBR1 were co-expressed with the neuronal marker NeuN (Fig. 3H–K). This suggests that I/R injury induces excessive autophagy in neurons, and oxymatrine alleviates this excessive autophagy via the PINK1/Parkin pathway in mouse neurons.

Next, to determine the optimal protective concentration of oxymatrine, HT22 cells were co-treated with 6 μM glutamate and varying concentrations of oxymatrine (5–45 μM). The greatest protective effect was observed at 25 μM, which was therefore selected as the working concentration for all following analyses (Fig. 4B). Morphological observations under microscopy further confirmed that oxymatrine treatment alleviated the characteristic cellular damage caused by glutamate exposure (Fig. 4C).

To explore whether oxymatrine attenuates mitochondrial apoptosis, we examined the expression and cellular localization of apoptotic protease activating factor-1 (Apaf-1). Immunofluorescence analysis revealed minimal Apaf-1-positive cells in the control group, while a marked increase was observed following 24-h glutamate exposure (Fig. 4D). Oxymatrine treatment significantly reduced the number of Apaf-1-positive cells in glutamate-challenged cultures. These findings were corroborated by Western blot results, which showed increased Apaf-1 protein levels after glutamate treatment and a marked reduction upon oxymatrine intervention. Additionally, we assessed caspase-3 activation as a downstream marker of mitochondria-mediated apoptosis. Glutamate exposure led to a robust increase in cleaved caspase-3 expression, without affecting total caspase-3 levels (Fig. 4G–I). Treatment with oxymatrine significantly downregulated cleaved caspase-3, further suggesting that oxymatrine mitigates glutamate-induced neuronal apoptosis via the mitochondrial pathway.

Given the role of reactive oxygen species (ROS) in mitochondrial dysfunction and apoptosis, we next evaluated ROS accumulation using the DHE fluorescent probe. Glutamate markedly elevated intracellular ROS levels compared to control, whereas oxymatrine treatment significantly suppressed this increase (Fig. 4E). To further elucidate oxymatrine's antioxidative mechanism, we examined the expression of manganese superoxide dismutase (MnSOD), a key mitochondrial antioxidant enzyme. Immunofluorescence analysis demonstrated that glutamate reduced MnSOD-positive cells, while oxymatrine restored MnSOD immunoreactivity. Western blotting provided consistent results, showing a significant increase in MnSOD expression in the oxymatrine-treated group compared to glutamate alone (Fig. 4F). These findings suggest that oxymatrine's neuroprotective effects are mediated, at least in part, through the attenuation of oxidative stress and enhancement of intrinsic antioxidant defenses.

Mitochondrial dynamics, defined by the balance between fission and fusion processes, are critical for maintaining cellular homeostasis and function. To assess whether oxymatrine influences mitochondrial dynamics in the context of glutamate-induced neuronal injury, we analyzed the expression of key regulatory proteins by western blotting in HT22 cells. Glutamate exposure significantly elevated the levels of the mitochondrial fission protein Fis1, while simultaneously reducing the expression of the fusion-related protein Mfn2, indicating a shift toward mitochondrial fragmentation. Interestingly, co-treatment with oxymatrine reversed this imbalance. Specifically, oxymatrine markedly suppressed the glutamate-induced increase in Fis1 levels, while restoring Mfn2 expression to levels closer to those observed in control cells (Fig. 5A–C). These findings suggest that oxymatrine mitigates glutamate-induced mitochondrial fragmentation and preserves mitochondrial dynamics, thereby contributing to cellular protection.

To further elucidate the role of oxymatrine in modulating autophagy, particularly through the PINK1/Parkin pathway, we examined the expression levels of multiple autophagy-related proteins using Immunofluorescence (Fig. 5D) and Western blotting (Fig. 5E–K). Following glutamate treatment, HT22 cells exhibited a substantial increase in the LC3-II/I ratio and elevated expression of PINK1, Parkin, Beclin-1, and NBR1, coupled with a notable reduction in P62 levels. These alterations are indicative of enhanced autophagy activity triggered by glutamate-induced mitochondrial stress.

Notably, oxymatrine intervention significantly reversed these glutamate-induced changes. Oxymatrine treatment reduced the LC3-II/I-a ratio and the expression of PINK1, Parkin, Beclin-1, and NBR1, while simultaneously restoring P62 levels. These results strongly support the notion that oxymatrine suppresses excessive autophagy via downregulation of the PINK1/Parkin signaling axis in glutamate-injured HT22 cells. Taken together, the data indicates that oxymatrine confers neuroprotective effects against glutamate-induced cytotoxicity by restoring mitochondrial dynamic balance and inhibiting pathologically elevated autophagy through modulation of the PINK1/Parkin pathway.

Furthermore, the ways by which Oxymatrine might mediate cerebral ischemia–reperfusion protein targets were simulated by AutoDock vina software. The results showed that the binding energy of Oxymatrine to HDAC1 was the lowest (Fig. 6D). As for HDAC1, Oxymatrine occupies the active site of HDAC1 by forming a hydrogen bond with HIS57 and it has hydrophobic interactions with VAL122, LYS123, and ILE389 (Fig. 6E).

To assess the role of oxidative stress, we measured key antioxidant markers including the GSH/GSSG ratio, superoxide dismutase (SOD), and total antioxidant capacity (TAC). As shown in Fig. 7M–O, oxymatrine treatment increased the levels of GSH/GSSG, SOD, and TAC compared to the I/R group. However, SAHA co-treatment reduced the levels of these antioxidants, further supporting the hypothesis that HDAC1 inhibition impairs the antioxidative effects of OMT. Behavioral recovery was evaluated using the open field test, where Fig. 7P–Q shows the tracking and total distance traveled of mice in the sham, I/R, I/R+OMT, and I/R+OMT+SAHA groups. The I/R group exhibited significant deficits in locomotion and anxiety behavior, while OMT treatment improved both parameters. However, the combined treatment with SAHA reversed these behavioral improvements, suggesting that HDAC1 inhibition impedes the neuroprotective effects of OMT on functional recovery.