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Introduction

Stroke is the leading cause of mortality and disability worldwide (1). According to the World Health Organization, stroke causes ~5.9 million deaths annually, with ischemic stroke accounting for ~87% of these cases (2). Mechanical thrombectomy and early pharmacological thrombolysis using tissue plasminogen activator are clinically effective interventions for restoring cerebral blood flow (reperfusion) in ischemic stroke (3). However, reperfusion can trigger cerebral ischemia-reperfusion injury (CIRI), a form of secondary damage that critically affects clinical outcomes (4–6). CIRI is a complex pathological process characterized by excitotoxicity, oxidative stress, inflammation and mitochondrial dysfunction, eventually triggering neuronal apoptosis (7). As such, there is an urgent need for effective therapeutic strategies for CIRI.

Mitochondria maintain cellular homeostasis through the induction of dynamic processes, including fission, fusion, autophagy and regeneration (8). Disturbances in these processes, particularly mitochondrial fission, facilitate neuronal apoptosis (9). Excessive mitochondrial fission is a prominent early upstream event that drives neuronal apoptosis during CIRI (10). Dynamin-related protein 1 (Drp1), a critical regulator of mitochondrial fission, is primarily activated by the phosphorylation of serine 616, which promotes its translocation from the cytoplasm to the mitochondrial membrane, triggering mitochondrial fission (11). Inhibition of Drp1 phosphorylation by pharmacological inhibition or Drp1 knockdown has been shown to alleviate CIRI by reducing infarct size and neuronal apoptosis (12,13).

Drp1-mediated excessive fission not only causes mitochondrial fragmentation, reactive oxygen species (ROS) production, reduced mitochondrial membrane potential (MMP) and increased cytochrome c release, but also leads to the overactivation of mitophagy (14–17). Although previous studies have shown that mitophagy exerts neuroprotective effects and improves the prognosis of CIRI (18,19), several studies have demonstrated that type II programmed cell death, induced by overactivated mitophagy, aggravates brain damage (20–22). Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a multifunctional co-transcription factor involved in mitochondrial biosynthesis and function (23), which negatively regulates Drp1 expression by directly binding to the Drp1 promoter, thereby preventing excessive mitochondrial fission (24). As a direct upstream regulator of PGC-1α, sirtuin 1 (SIRT1) serves a crucial role in maintaining mitochondrial function by regulating its expression (25). Previous studies have demonstrated that activation of the SIRT1/PGC-1α pathway can inhibit excessive mitochondrial fission and attenuate the development of diabetes-induced cardiac dysfunction (24,26). Moreover, the inhibition of Drp1-mediated excessive mitochondrial fission has been shown to mitigate mitophagy, thereby offering protection against CIRI (27).

Astragali Radix (Huangqi) is a widely used traditional Chinese medicine, which exhibits potent pharmacological effects on cardiovascular diseases and immune regulation (28). Calycosin-7-O-β-D-glucoside (CG), a major bioactive ingredient of Astragali Radix, has further been reported to exert neuroprotective effects through anti-inflammatory, antioxidant and anti-apoptotic mechanisms (29–31). Although our previous study showed that CG can alleviate oxygen-glucose deprivation/reperfusion (OGD/R)-induced HT22 cell damage (32), whether this action is related to the prevention of excessive mitochondrial fission and mitophagy overactivation remains unclear. Therefore, the present study investigated the protective effects of CG against OGD/R-induced injury by focusing on the inhibition of Drp1-mediated mitochondrial fission. Additionally, the study investigated whether CG could inhibit the overactivation of mitophagy.

Materials and methods

Materials

The following reagents were acquired for use in the present study: CG (Fig. 1A; purity >98%; Shanghai Aladdin Biochemical Technology Co., Ltd.); edaravone (EDA; purity >99%; MilliporeSigma); Cell Counting Kit-8 (CCK-8; Abbkine Scientific Co., Ltd.); lactate dehydrogenase (LDH) kit (Nanjing Jiancheng Bioengineering Institute); MitoTracker™ Orange CMTMRos kit (Thermo Fisher Scientific, Inc.); MitoSOX™ kit (Thermo Fisher Scientific, Inc.); JC-1, MitoTracker Green kits and Hoechst 33342 (Beyotime Institute of Biotechnology); BCA kit (Beijing Solarbio Science & Technology Co., Ltd.); Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin (HyClone; Cytiva); and glucose-free DMEM (Gibco; Thermo Fisher Scientific, Inc.). The following antibodies were also purchased: SQSTM1/p62 (cat. no. P0067; MilliporeSigma); LC3B (cat. no. A19665), Bcl-2 (cat. no. A19693), Drp1 (cat. no. A21968) and HRP-conjugated secondary antibody (cat. no. AS014) (all from ABclonal Biotech Co., Ltd.); translocase of outer mitochondrial membrane 20 (TOM20; cat. no. 42406S), phosphorylated p-Drp1 (Ser616) (cat. no. 4494S) and caspase-3 (cat. no. 14220S) (all from Cell Signaling Technology, Inc.); PGC-1α (cat. no. ab313559), Bax (cat. no. ab32503) and SIRT1 (cat. no. ab12193) (all from Abcam); β-actin (cat. no. K101527P; Beijing Solarbio Science & Technology Co., Ltd.).

Figure 1. CG protects against OGD/R-induced neuronal damage in HT22 cells. (A) Chemical structure of CG. (B) Cell viability. HT22 cells were subjected to OGD/R, and treated with different concentrations of CG (20, 40 and 80 µM) and EDA (50, 100 and 200 µM). n=6. (C) Cell morphology. Scale bar, 50 µm. (D) LDH content in the cell supernatants. n=6. Data are presented as the mean ± SEM, and were analyzed by one-way ANOVA and Tukey's post hoc test. **P<0.01 vs. control group; ##P<0.01 vs. model group. CG, calycosin-7-O-β-D-glucoside; EDA, edaravone; LDH, lactate dehydrogenase; OGD/R, oxygen-glucose deprivation/reperfusion.

OGD/R model and drug treatment

HT22 mouse hippocampal neurons were purchased from Pricella (cat. no. CL-0697), and were cultured in DMEM supplemented with 10% FBS and antibiotics (penicillin, 100 IU/ml; streptomycin, 100 µg/ml) at 37°C in an incubator under 5% CO2. To construct the OGD/R model, cells were cultured in glucose-free DMEM and then transferred to a hypoxia incubator chamber (MIC-101; Embrient, Inc.) containing 5% CO2 and 95% N2 at 37°C. After 10 h, the cells were transferred to an incubator with 5% CO2 and 95% air, and the culture medium was replaced with standard DMEM for 6 h at 37°C (reperfusion). CG (20, 40 and 80 µM) and EDA (50, 100 and 200 µM) were added for 6 h during the reperfusion phase. The control group was maintained in standard culture medium without OGD. The concentration of CG was selected based on a previous study (31).

Cell viability assay

HT22 cells were cultured in 96-well plates at a density of 5×103 cells/well. After OGD/R modelling and drug treatment, images were captured under an optical microscope (Nikon Corporation), after which the medium was discarded, 10 µl CCK-8 solution was added, and the cells were cultured for 30 min in a regular incubator. The absorbance was measured at 450 nm using a microplate reader (BioTek; Agilent Technologies, Inc.).

Measurement of LDH activity

The LDH activity in the supernatant was measured using an LDH assay kit, according to the manufacturer's instructions. After OGD/R modelling and drug treatment, the cell medium was collected and mixed with substrate solution at 37°C for 15 min. The mixture was subsequently incubated with 2,4-dinitrophenylhydrazine for 15 min at 37°C and the absorbance was measured at 450 nm using a microplate reader (BioTek; Agilent Technologies, Inc.).

Cell apoptosis assay

Cell apoptosis was determined using an Annexin V-FITC/PI kit (Beijing Solarbio Science & Technology Co., Ltd.), according to the manufacturer's protocol. After treatment, the cells were harvested by centrifugation at 100 × g for 5 min at 37°C, followed by trypsin digestion. The cells were then resuspended in binding buffer mixed with 5 µl Annexin V-FITC and were incubated in the dark for 5 min at 37°C. Subsequently, 1 µl PI was added and incubated for another 5 min at 37°C. The apoptosis rate in each group was analyzed using flow cytometry (CytoFLEX V2-B2-R0; Beckman Coulter, Inc.).

Mitochondrial ROS (mtROS) detection

According to the manufacturer's instructions, following treatment, the supernatants were discarded, and the cells were incubated with MitoSOX™ (3 µM) for 30 min at 37°C, followed by Hoechst staining for 15 min at 37°C to label the nuclei. The cells were then washed three times with PBS and observed under an inverted fluorescence microscope (TS-2; Nikon Corporation).

MMP detection

JC-1 is a widely used probe for monitoring MMP. In healthy cells with normal MMP levels, JC-1 forms J-aggregates that emit intense red fluorescence. By contrast, in apoptotic or unhealthy cells with a low MMP, JC-1 remains in its monomeric form and emits green fluorescence. Thus, mitochondrial depolarization can be measured as a decrease in the red/green fluorescence intensity ratio. According to the manufacturer's protocol, following treatment, the supernatants were discarded, and cells were incubated with 1 ml JC-1 solution at 37°C for 50 min. The JC-1 solution was then aspirated and the cells were washed twice with dye buffer. The fluorescence intensity of the JC-1 aggregate/monomer was subsequently observed under an inverted fluorescence microscope (TS-2; Nikon Corporation).

Mitochondrial morphology observation

MitoTracker Green, a live cell mitochondria-specific fluorescent dye, was applied to distinguish changes in mitochondrial morphology. Briefly, HT22 cells were incubated with 100 nM MitoTracker Green for 15 min at 37°C. The staining solution was subsequently removed and the cells were washed twice with HBSS (Beyotime Institute of Biotechnology). Images were then captured using a laser confocal microscope (Leica Microsystems GmbH) and the length of the mitochondria was measured using Image-Pro Plus 6.0 (Media Cybernetics, Inc.).

Immunofluorescence

For immunofluorescence detection, HT22 cells were first stained with MitoTracker™ Orange CMTMRos (200 nM) at 37°C for 10 min, and then fixed with 4% paraformaldehyde for 15 min at 37°C. After three washes with PBS, the cells were permeabilized with 0.3% Triton X-100 in PBS for 15 min at 37°C. The cells were then blocked with 5% BSA (Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at 37°C, and incubated overnight at 4°C with primary antibodies against LC3 (1:500). The next day, after washing three times with PBS, the samples were incubated with a Alexa Fluor™ 488-conjugated goat anti-rabbit IgG secondary antibody (1:500; cat. no. A11008; Thermo Fisher Scientific, Inc.) for 1 h at room temperature in the dark, followed by DAPI staining for 30 min at 37°C to label the nuclei. Finally, the fluorescence intensity was analyzed using an inverted fluorescence microscope (TS-2; Nikon Corporation).

Molecular docking

The 3D structure of SIRT1 [Protein Data Bank (PDB) ID: 4KXQ] was downloaded from the PDB (https://www.rcsb.org/), and protein structure was optimized to ensure it can be used for molecular docking using the Protein Preparation Wizard module in Schrödinger 12.9 (Schrödinger, Inc.). The 3D structure of CG was obtained from the PubChem database (compound CID: 5318267; http://pubchem.ncbi.nlm.nih.gov/compound/5318267). Molecular docking between CG and SIRT1 was performed using the Glide module (Schrödinger, Inc.), and the molecular mechanics generalized Born surface area (MM/GBSA) was calculated. The binding sites were visualized using PyMOL 3.0 (Schrödinger, Inc.).

Western blotting

Total proteins were extracted from the cells using RIPA lysis buffer (cat. no. CW2333S; CWBio), whereas protein concentration was detected using a BCA kit. Proteins (10 µg) were separated by SDS-PAGE on 10 or 12% gels (the percentage gel used depended on the molecular weight of the targeted protein), were transferred to polyvinylidene difluoride membranes and were blocked for 2 h with 5% skim milk in PBS containing 0.1% Tween-20 (PBST) at 37°C. The membranes were subsequently incubated with primary antibodies against the following proteins at 4°C overnight: Bax (1:2,000), Bcl-2 (1:500), caspase-3 (1:4,000), TOM20 (1:4,000), p62 (1:4,000), LC3 (1:1,500), Drp1 (1:1,000), p-Drp1 (1:1,000), PGC-1α (1:1,000), SIRT1 (1:1,000) and β-actin (1:5,000). After washing three times with PBST, the membranes were incubated with an HRP-conjugated secondary antibody for 50 min at room temperature. The membranes were then scanned using an imaging system (Bio-Rad Laboratories, Inc.), and Image-Pro Plus 6.0 (Media Cybernetics, Inc.) was used to analyze the optical density of the bands. The relative protein expression levels were normalized to β-actin.

Statistical analysis

The data are presented as the mean ± SEM and were analyzed using SPSS 27.0 (IBM Corporation). Figures were drawn using GraphPad Prism 8.0 (Dotmatics). One-way ANOVA followed by Tukey's post hoc test was used for data analysis. P<0.05 was considered to indicate a statistically significant difference.

Results

CG ameliorates damage to HT22 cells induced by OGD/R challenge

To investigate the protective effects of CG against OGD/R-induced HT22 cell injury, the viability of HT22 cells was measured using the CCK-8 assay. The results showed that cell viability was significantly decreased in the OGD/R group compared with that in the control group, but it was significantly increased by CG (40 µM) and EDA (100 and 200 µM) treatment (Fig. 1B); therefore, 40 µM CG and 100 µM EDA were selected for subsequent experiments. The morphological characteristics of the OGD/R group included irregular shrinkage, unclear boundaries and floating (Fig. 1C). Additionally, the release of LDH was markedly increased in the OGD/R group compared with that in the control group (Fig. 1D). However, these trends were markedly reversed by CG and EDA.

CG alleviates the OGD/R-induced apoptosis of HT22 cells

Neuronal apoptosis is the final event of CIRI (33). To assess the effects of CG on the apoptosis of HT22 cells, flow cytometry and western blot analysis were performed. The results revealed a significant increase in the percentage of apoptotic cells and caspase-3 expression, accompanied by a marked decrease in the Bcl-2/Bax ratio in the OGD/R group compared with those in the control group (Fig. 2). However, these changes were reversed by the CG and EDA treatment. These findings confirmed the anti-apoptotic effect of CG and indicated that CG ameliorated OGD/R by reducing neuronal apoptosis.

Figure 2. CG alleviates the OGD/R-induced apoptosis of HT22 cells. HT22 cells were subjected to OGD/R, and treated with 40 µM CG or 100 µM EDA. (A) Representative flow cytometry plots from the apoptosis analysis. (B) Quantification of apoptosis rate. n=3. (C) Representative western blots, and semi-quantitative analysis of the apoptosis markers (D) Bcl-2/Bax and (E) caspase-3. n=3. Data are presented as the mean ± SEM, and were analyzed by one-way ANOVA and Tukey's post hoc test. **P<0.01 vs. control group; #P<0.05, ##P<0.01 vs. model group. CG, calycosin-7-O-β-D-glucoside; EDA, edaravone; OGD/R, oxygen-glucose deprivation/reperfusion.

CG improves the OGD/R-induced mitochondrial dysfunction of HT22 cells

Mitochondrial dysfunction is an important characteristic of CIRI (22,34). To investigate whether CG could restore the mitochondrial dysfunction caused by OGD/R, mtROS levels were analyzed using MitoSOX Red. As shown in Fig. 3A and B, mtROS levels were significantly increased in the OGD/R group compared with those in the control group, but were notably reduced following CG and EDA treatment. By contrast, MMP, a hallmark of mitochondrial integrity, was significantly elevated by CG and EDA treatment compared with that in the OGD/R group (Fig. 3C and D). Collectively, these results indicated that CG may protect cells from OGD/R-induced mitochondrial dysfunction.

Figure 3. CG improves OGD/R-induced mitochondrial dysfunction of HT22 cells. HT22 cells were subjected to OGD/R, and treated with 40 µM CG or 100 µM EDA. (A) mtROS detection. Scale bar, 25 µm. (B) Quantification of fluorescence density of mtROS (four images were randomly selected in each group). (C) MMP was monitored using the JC-1 probe. Red, JC-1 aggregates; green, JC-1 monomers. Scale bar, 25 µm. (D) Ratio of aggregates/monomers (four images were randomly selected in each group). Data are presented as the mean ± SEM, and were analyzed by one-way ANOVA and Tukey's post hoc test. *P<0.05 vs. control group; #P<0.05, ##P<0.01 vs. model group. CG, calycosin-7-O-β-D-glucoside; EDA, edaravone; mtROS, mitochondrial reactive oxygen species; OGD/R, oxygen-glucose deprivation/reperfusion.

CG prevents OGD/R-induced excessive mitochondrial fission in HT22 cells

Mitochondria are highly dynamic organelles that undergo fission and fusion, both of which are processes closely related to mitochondrial function (8). The MitoTracker Green probe was used to examine the effects of CG on mitochondrial morphology in OGD/R-treated HT22 cells. As shown in Fig. 4A and B, the average length of mitochondria in OGD/R-treated cells was significantly shorter than that in control cells, indicating a marked decrease in the linear shape and diffusely interspersed fragmented or dotted mitochondria. As hypothesized, CG and EDA mitigated mitochondrial fragmentation in OGD/R-treated HT22 cells. Mitochondrial fission is mediated by p-Drp1 (Ser616); to confirm whether CG regulated mitochondrial fission, the expression levels of this marker of mitochondrial fission were examined. As shown in Fig. 4C and D, an increased p-Drp1/Drp1 ratio was observed in the OGD/R group compared with that in the control group, which was effectively interrupted by CG and EDA treatment. These results indicated the protective effect of CG against OGD/R-induced mitochondrial fission.

Figure 4. CG treatment prevents OGD/R-induced excessive mitochondrial fission. (A) Representative images of mitochondrial morphology detected by MitoTracker Green probe. Scale bars, 20 µm (upper images) and 10 µm (lower images). (B) Average length of mitochondrial branches (20 mitochondria were randomly selected in each group). (C) Representative western blots and (D) semi-quantitative analysis of the mitochondrial fission marker p-Drp1. n=3. Data are presented as the mean ± SEM, and were analyzed by one-way ANOVA and Tukey's post hoc test. *P<0.05, **P<0.01 vs. control group; #P<0.05, ##P<0.01 vs. model group. CG, calycosin-7-O-β-D-glucoside; EDA, edaravone; Drp1, dynamin-related protein 1; OGD/R, oxygen-glucose deprivation/reperfusion; p-, phosphorylated.

CG downregulates OGD/R-induced mitophagy overactivation

To investigate whether CG can affect mitophagy, the expression levels of the mitophagy-related proteins LC3, p62 and TOM20 were investigated (Fig. 5A). The results showed an increased LC3II/LC3I ratio in the OGD/R group compared with that in the control group, and, as expected, this increase was markedly inhibited by CG treatment (Fig. 5D). By contrast, a significant increase in the expression levels of p62 and TOM20 were observed following CG and EDA administration, indicating that CG may reduce the overactivation of mitophagy induced by OGD/R (Fig. 5B and C). To further observe overactivated mitophagy, mitochondria and autophagosomes were colocalized using immunofluorescence staining. As presented in Fig. 5E and F, the ratio of colocalization of mitochondria stained with LC3 was greatly increased in the OGD/R group compared with that in the control group, and was markedly abrogated by treatment with CG and EDA. Taken together, these results indicated that CG downregulated OGD/R-induced mitophagy overactivation.

Figure 5. CG treatment inhibits OGD/R-induced mitophagy overactivation. (A) Representative western blots, and semi-quantitative analysis of (B) p62, (C) TOM20 and (D) LC3 protein expression. n=3. (E) Representative immunofluorescence images of MitoTracker™ Orange CMTMRos (red) costaining with LC3B (green). Scale bar, 20 µm. (F) Quantification of the ratio of LC3B colocalization with mitochondria (20 cells were randomly selected in each group). Data are presented as the mean ± SEM, and were analyzed by one-way ANOVA and Tukey's post hoc test. **P<0.01 vs. control group; #P<0.05, ##P<0.01 vs. model group. CG, calycosin-7-O-β-D-glucoside; EDA, edaravone; TOM20, translocase of outer mitochondrial membrane 20.

CG upregulates the expression of SIRT1 and PGC-1α after OGD/R

The SIRT1/PGC-1α signaling pathway serves an essential role in mitochondrial protection (24). Thus, it was hypothesized that the SIRT1/PGC-1α pathway may be involved in the protective effect of CG against neuronal apoptosis by inhibiting mitophagy overactivation. To assess whether SIRT1 is a target of CG, the affinity between CG and SIRT1 was analyzed using molecular docking. As presented in Fig. 6A, CG can bind well with amino acids in SIRT1, including Asn465, Glu467, Gly263, Arg274 and Phe273, and the docking score of CG and SIRT1 was −8.02 kcal/mol. Furthermore, the effects of CG on the protein expression levels of SIRT1 and PGC-1α were examined, with results showing that the levels of SIRT1 and PGC-1α were markedly reduced in the OGD/R group compared with those in the control group, while CG and EDA treatment upregulated the expression of SIRT1 and PGC-1α (Fig. 6B-D). These results indicated that CG may attenuate mitochondrial dysfunction through the expression of SIRT1 and PGC-1α.

Figure 6. CG promotes PGC-1α and SIRT1 expression in HT22 cells subjected to OGD/R. (A) Molecular docking of CG with SIRT1. The 2D structure (left) and 3D structure (right) of the docking interaction between CG and the predicted human SIRT1 structure are shown. (B) Representative western blots, and semi-quantitative analysis of (C) PGC-1α and (D) SIRT1 protein expression. n=3. Data are presented as the mean ± SEM, and were analyzed by one-way ANOVA and Tukey's post hoc test. *P<0.05 vs. control group; #P<0.05, ##P<0.01 vs. model group. CG, calycosin-7-O-β-D-glucoside; EDA, edaravone; PGC-1α, peroxisome proliferator-activated receptor γ coactivator-1α; SIRT1, sirtuin 1.

Discussion

High rates of disability and recurrence are characteristic of ischemic stroke, a common cerebral disease that represents a serious global public health concern (35). According to our previous study, CG may mitigate OGD/R-induced injury by improving mitochondrial function (32). Notably, abundant evidence has demonstrated that mitophagy overactivation, triggered by excessive mitochondrial fission, is central to neuronal apoptosis following cerebral ischemia/reperfusion (36,37). However, whether the protective effects of CG against OGD/R-induced injury are related to the modulation of mitochondrial fission and mitophagy has not yet been investigated, to the best of our knowledge.

The results of the present study indicated that CG notably enhanced cell viability, increased the Bcl-2/Bax ratio, and decreased caspase-3 expression and LDH release following OGD/R, thus validating the protective effects of CG against OGD/R-induced neuronal apoptosis. It has previously been well established that mitochondrial dysfunction damages neurons and is a major factor in the etiology of CIRI (22). One of the main causes of CIRI is the overproduction of mtROS (6). This process contributes to and permeates all aspects of CIRI, including oxidative stress, mitochondrial swelling, membrane instability and reduced MMP (38). Furthermore, cerebral ischemia-reperfusion causes mitochondrial morphological fragmentation, which ultimately triggers neurological injury (39,40). Improving mitochondrial morphology has been shown to effectively reduce the damage caused by CIRI (39,41). EDA was first approved for the treatment of ischemic stroke in Japan in 2001 (42). Notably, ~50% of EDA is present in the body as the EDA anion, which transfers electrons to remove different types of radicals, such as H2O2 and O-2. The produced EDA radical at that time then combines with the oxygen molecules in the reaction system to form EDA peroxyradical, and ultimately 2-oxo-(phenylhydrazono)-butanoic acid, a reaction product unrelated to radicals (43–45). Thus, in order to assess the effectiveness and mechanism of CG, EDA was selected as a positive control medication. The present study verified that CG protected the mitochondria of OGD/R-induced HT22 cells, as demonstrated by decreased mtROS, increased MMP and improved mitochondrial morphology. The present data further suggested that CG may reduce OGD/R-induced damage by improving mitochondrial dysfunction.

Under normal physiological conditions, Drp1 is typically found in the cytoplasm and is an essential regulator of mitochondrial fission. Upon stimulation, Drp1 is recruited to the mitochondrial surface to induce fission (11). Previous studies have shown that when Drp1 is translocated to the mitochondria, it releases Beclin1 and interacts with LC3 to activate autophagy (16,46). Excessive mitochondrial fission during cerebral ischemia-reperfusion triggers mitophagy, a crucial process in maintaining mitochondrial quality control (47,48). Although mitophagy is essential for cellular homeostasis, it is a double-edged sword and its overactivation can lead to apoptosis (49). Prior research has shown that preventing mitophagy overactivation caused by excessive mitochondrial fission can alleviate CIRI (39,40,50). In the present study, excessive mitochondrial fission and mitophagy overactivation were detected following OGD/R, as evidenced by the upregulation of p-Drp1/Drp1 and LC3II/LC3I, and decreased p62 and TOM20 expression. As expected, treatment with CG reversed the changes induced by OGD/R.

SIRT1 is the most researched member of the SIRT family (51), with numerous studies showing that SIRT1 serves a role in autophagy and apoptosis following cerebral ischemia (52–54). PGC-1α, a transcription factor downstream of SIRT1, is crucial for controlling mitochondria, and may be used as a therapeutic target for a variety of neurodegenerative illnesses (23). To explore whether the downregulation of p-Drp1 by CG is related to the expression of SIRT1 and PGC-1α, the binding ability of CG to SIRT1 was simulated through molecular docking, and the protein expression levels of SIRT1 and PGC-1α were detected. The results of these analyses demonstrated that CG has the ability to directly bind to SIRT1, and regulate the expression of SIRT1 and PGC-1α. These results indicated that CG may reduce Drp1 phosphorylation via regulation of the expression of SIRT1 and PGC-1α, thereby preventing the overactivation of mitophagy induced by excessive mitochondrial fission.

In conclusion, the present study provided compelling evidence that CG alleviated OGD/R-induced HT22 cell apoptosis by preventing the overactivation of mitophagy induced by excessive mitochondrial fission by regulating the expression of SIRT1 and PGC-1α (Fig. 7). These findings provide a theoretical basis for the development of clinical strategies for the use of CG in the management of CIRI. However, the current study only focused on the protective effects of CG in vitro; the precise mechanism by which CG protects against CIRI through the regulation of mitochondrial fission and mitophagy, and how excessive mitochondrial fission leads to mitophagy overactivation, still requires further elucidation in vivo. Furthermore, establishing the pharmacokinetics and ideal dosage of CG in the treatment of ischemic stroke is essential for its possible integration into clinical practice.

Figure 7. Schematic diagram showing that CG inhibits the overactivated mitophagy induced by excessive mitochondrial fission through regulating the expression of SIRT1 and PGC-1α, and then alleviates apoptosis. In OGD/R-induced HT22 cells, excessive mitochondrial fission led to mitophagy overactivation, which resulted in apoptosis. CG inhibited excessive mitochondrial fission by regulating the expression of SIRT1 and PGC-1α, and then inhibited the overactivated mitophagy to alleviate apoptosis. CG, calycosin-7-O-β-D-glucoside; Drp1, dynamin-related protein 1; mtROS, mitochondrial reactive oxygen species; PGC-1α, peroxisome proliferator-activated receptor γ coactivator-1α; SIRT1, sirtuin 1.

Acknowledgements

The authors would like to thank Dr Yong Yuan (Academy of Chinese Medicine of Henan University of Chinese Medicine) for their technological help with laser confocal microscopy.

Funding

This study was supported by the National Natural Science Foundation of China (grant nos. 82304771 and 82274496), the Joint Fund of Science and Technology Research and Development Project of Henan Province (grant no. 232301420018) and the Science and Technology Research Project of Henan Province (grant nos. 232102311193 and 232102310419).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

XY designed the experiments. SQ and RG performed the experiments. XY and SQ wrote the original draft. ZL, MB and BW analyzed the data. PS analyzed the data and confirmed the version to be published. EX and YL designed the experiments, drafted the manuscript and confirm the authenticity of all the raw data. All authors contributed to the critical revision of the manuscript. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References