Nimodipine was purchased from Hunan Baicao Pharmaceutical Co., Ltd. (Hunan, China). Chloroquine (CQ) was purchased from MCE (Shanghai, China). αAsarone was purchased from Yuanye biological company (Shanghai, China), with purity ≥98%. SYBR Green Real-Time PCR Master Mix and gDNA remover ReverTra qPCR RT Master Mix were provided by Toyota CO., Ltd., located in Toyota, Japan. Meanwhile, the BCA Protein Assay Kit was sourced from Biosharp Labgic Technology CO., Ltd., based in Hefei, China. Antibodies for ATG5, LC3I, and LC3II were obtained from Proteintech. The PDCD4, LAMP1, goat anti-mouse and goat anti-rabbit were offered by Chengdu Zhengneng Biology Co., Ltd. (Chengdu, China).

The HT22 cell line was perserved in DMEM media enriched with 1% penicillin-streptomycin and 10% fetal bovine serum, incubated at 37°C in a 5% CO2 atmosphere, change cell culture medium daily. When the cells reached a confluence level of 60%–70%, they were subjected to hypoxic conditions by being rinsed with phosphate-buffered saline (PBS) and then underwent OGD for a duration of 6 h. Sugar-free medium was then replaced with complete culture medium and cells were reoxygenated for 24 h under standard oxygen levels. The HT22 cells were subsequently categorized into 6 different groups: Control, OGD/R group, OGD/R + Nimodipine group, and three groups with αAsarone (5, 10, and 20 nM). Therefore, 5, 10, and 20 nM were chosen as Low (L), Medium (M), High (H) concentrations in the flowing experiments. The mechanism part was divided into 8 groups: Control, OGD/R, OGD/R + αAsarone (20 nM), Inhibit-Control, Inhibit-miR-499-5p, Inhibit-miR-499-5p + αAsarone (20 nM), Mimic-Control and Mimic-miR-499-5p.

HT22 cells were inoculated at a density of 1 × 10⁷cells/cm² into 96-well plates. Following a 6-h interval of OGD, the cells received treatment with αAsarone after 12 h. The evaluation of cell viability using the non-radioactive CCK-8 assay (Biosharp, Jiangsu, China), adhering to the manufacturer’s guidelines, with absorbance levels recorded at 450 nm. Each experiment was executed in triplicate.

HT22 cells were seeded on 35 mm dishes featuring a glass bottom at a density of 1 × 10⁷ cells and subsequently transfected with mRFP-GFP-LC3 tandem fluorescent lentivirus. The next day, replace the medium with 2 mL of fresh medium. After 72 h post-transfection, the neurons were subjected to OGD/R for a duration of 6 h, with or without the addition of PNU282987 (100 μM). Additionally, Autophagosomes (GFP-positive, mRFP-positive) and autolysosomes (GFP-negative, mRFP-positive) within the neurons were visualized using confocal microscopy (Olympus, Tokyo, Japan). The Spots (>1 µm) per cell were conducted.

Luciferase reporter vectors, namely miR-499-5p-wt and PDCD4-wt, were constructed by Hanheng in Hunan, China. Mutant vectors, miR-499-5p-mut and PDCD4-mut, were generated by modifying the gene sequences at the predicted binding sites. Cells were plated in 12-well plates at a suitable density. Following this, all plasmids were introduced into the cells and allowed to incubate for 24 h. Upon completion of the transfection period, the cells were collected, and the activity of firefly luciferase was assessed utilizing a dual-luciferase reporter assay system. Subsequently, the firefly luciferase activity was normalized against the activity of renilla luciferase.

Adult male specific pathogen-free (SPF) Sprague-Dawley mice, weighing between 20 and 24 g, were obtained from GemPharmatech Co., Ltd. The mice were housed under SPF-grade conditions for 1 week, during which they had unrestricted access to food and water. All experiments involving animals were performed following the guidelines set forth by the National Institutes of Health regarding the management and utilization of laboratory animals (NIH Publications No. 8023, revised 1978). The Animal Ethics Committee at Anhui University of Traditional Chinese Medicine (Hefei, Anhui, China) provided approval for these studies (ethics number: 2023149), which aimed to reduce pain, suffering, and distress. Before the experiments commenced, the mice experienced a fasting duration of 12 h.

The establishment of a stroke model in mices was conducted using an altered method of middle cerebral artery occlusion (MCAO), utilizing 3% isoflurane for anesthesia in adult male SPF mice. During the surgical procedure, the body temperature was maintained at 37°C. An incision along the midline of the neck was created to gain access to and isolate the common carotid artery, the external carotid artery, and the internal carotid artery. Nylon sutures were then placed approximately 10 mm deep from the common carotid artery to the internal carotid artery. After a duration of ischemia lasting 1 h, the nylon suture was taken out to promote reperfusion. In the sham group, the identical surgical procedure was carried out, but the nylon suture was not inserted. After 1 h of stroke, behavioral assessments were conducted using the Zealonga scoring method (Longa et al., 1989). The evaluation criteria are as follows. 0 points: There are no symptoms of neurological disorder and the patient’s functions are normal. 1: Unable to fully extend the contralateral forepaw. 2: Contralateral hemiplegia with crawling and turning. 3: The body leans toward the hemiplegic side when walking. 4: Unable to walk alone, unconscious; 5: death. The mice were subsequently randomized into seven groups: Sham, Model, αAsarone (10 and 40 mg/kg), Nimodipine (1.6 mg/kg), CQ (80 mg/kg), and CQ combined with αAsarone (40 mg/kg). Each drug was injected intraperitoneally into mice once for 3 consecutive days.

The assessment of the brain infarct volume was conducted with the use of 2,3,5-triphenyltetrazolium chloride (TTC) staining technique. Five coronal slices of the brain, each measuring 1 mm in thickness, were treated with 1.5% TTC solution. For analyzing the volume of the infarct, ImageJ software was utilized.

Following fixation, dehydration, and embedding in transparent paraffin wax, brain tissue specimens were sectioned to a thickness of approximately 4 µm. Hematoxylinand eosin (H&E) staining was then performed, allowing for the examination of pathological changes in the CA1 region of the hippocampus under a 400-fold magnification. The tissue sections were then immersed in Nissl solution at 50°C for a duration of 10 min, followed by immersion in 95% alcohol. After rinsing with distilled water, the slices were transferred to 70% alcohol. Finally, xylene was added before sealing the container.

After fixation, embedding, preparation, permeabilization, and blocking, brain specimens underwent treatment with primary antibodies (MAP2 and LC3) at a temperature of 4°C overnight. Following three rinses in 0.02 M PBS, each lasting 10 min, the specimens the specimens were exposed to the secondary antibody at 37°C for a duration of 1 h. The samples were then rinsed again three times for 10 min with 0.02 M PBS, after which the nuclei were then labeled with DAPI for 2 h at 37°C, confocal laser scanning microscopy (Nikon, Tokyo, Japan) was utilized to scan the sections and dishes.

In vitro, HT22 cells underwent incubation with primary antibodies (LAMP1 and LC3), and the remaining steps were performed as previously outlined.

Ultrastructural changes in brain tissues and HT22 cells were evaluated using TEM. The hippocampi were fixed overnight at 4°C in a 2.5% glutaraldehyde solution. The ultra-thin sections were then subjected to staining with lead citrate and uranyl acetate before being observed and imaged with atransmission electron microscope (TEM; Tokyo, Japan).

Hippocampal cells were homogenized in cold RIPA buffer with PMDF for a duration of 10 min, both in vivo and in vitro, and total protein was extracted through high-speed centrifugation. After quantifying the protein using the BCA assay, 30 μg of the extract was loaded onto 10% or 12% (w/v) SDS-PAGE gels for 1.5 h of electrophoresis, after which the proteins were conveyed onto a polyvinylidene fluoride membrane for about 1.5 h. The membranes were incubated in a solution of 5% skim milk for 1.5 h at a temperature of 37°C. Afte three washes of 10 min each using Tris-buffered saline containing Tween 20. The membranes were then treated with primary antibodies LC3 (1:2,500), LAMP1 (1:1,000), PDCD4 (1:1,000), ATG5 (1:1,000), and GAPDH (1:1,000) for a period of 8 h at 4°C. After this incubation, the membranes were washed three more times before being exposed to either goat anti-rabbit IgG (1:10,000) or goat anti-mouse IgG (1:10,000) in Tris-buffered saline containing Tween (TBST) at room temperature for a duration of 2 h. Once more, three additional washes with TBST were carried out, each lasting 10 min, after which enhanced chemiluminescence solution and the ImagerJ software were employed to visualize protein expression on the membrane.

Isolate RNA from ischemic brain homogenate or HT22 cells using RNA extraction reagents. Primers for miR-499-5p, PDCD4, ATG5, LC3I, LC3II, LAMP1, and β-Actin were designed by Sangon Biotech Co., Ltd. (Shanghai, China), and the sequences of these primers can be found in Table 1. The RT-qPCR process followed the protocols described in earlier studies (Ponsford et al., 2021).

Data are presented as the mean ± standard deviation (SD) derived from a minimum of three independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by the least significant difference post hoc test for multiple comparisons, utilizing GraphPad Prism version 9.0 software. A significance level of P < 0.05 set to determine statistically significant differences.

To establish the safe and protective concentrations of αAsarone, HT22 cells received treatment across various concentrations: 1, 5, 10, 20, 25, 40, and 100 nM. We observed an increase in proliferative ability at 5, 10, and 20 nM (p < 0.01) (Figure 1A), Four time points (2 h, 4 h, 6 h, and 8 h after OGD/R) were selected for observation. We found that after 6 h of OGD/R, which was statistically significant when compared to the control group, the cell survival rate was approximately 50%, (p < 0.01) (Figure 1B), indicating substantial toxicity to cell growth. Therefore, 6 h of OGD/R was selected as the modeling condition. Subsequently, treatment of HT22 cells with αAsarone for 12 h revealed that 5, 10, and 20 nM enhanced cell survival rates compared to the model group (p < 0.05) (Figure 1C). The protective effects of αAsarone on OGD/R-induced HT22 cells were evident, with the most pronounced effects noted at dose-dependent increases at 5, 10, and 20 nM. Therefore, 5, 10, and 20 nM were chosen as Low (L), Medium (M), High (H) concentrations in the flowing experiments. Additionally, After the OGD/R cells were treated with αAsarone, their morphology improved and the number of adherent cells increased (Figure 1D). Additional examination of cellular apoptosis through flow cytometry revealed a significant increase in neuronal apoptosis post-OGD/R, which was effectively reversed by αAsarone at concentrations of 5, 10, and 20 nM (Figures 1E, F). These findings suggest that αAsarone alleviates cell injury induced by OGD/R.

To explore the effect of αAsarone on neuronal autophagy, we evaluated its effects on the expression levels of crucial proteins and genes, including LC3 and LAMP1, usin western blotting, RT-qPCR and fluorescent staining techniques. As shown in Figures 2A, B, double immunofluorescence staining was conducted for LAMP1, a marker of lysosomes, and LC3, an autophagy marker, to assess the potential effect of αAsarone on autophagy activation (measured by the percentage of LAMP1 that colocalizes with LC3) (Matarrese et al., 2014). The findings demonstrated that αAsarone markedly enhanced the colocalization of LAMP1 and LC3, indicating an increased rate of autophagy in the αAsarone-treated group. Furthermore, western blot and RT-qPCR analyses corroborated these findings, revealing elevated ratios of LC3-II/LC3-I and LAMP1 in the presence of αAsarone following OGD/R (Figures 2C–G), thereby suggesting that αAsarone activates autophagy.

Following OGD/R injury, the expression levels of miR-499-5p in HT22 cells was observed to decrease after 6 h, Further, it was found that αAsarone enhanced the expression level of miR-499-5p in a dose-dependent manner. (Figure 3A, P < 0.01). Luciferase reporter gene assays demonstrated a reduction in luciferase activity in HT22 cells that were co-transfected with miR-499-5p mimics and the wild-type PDCD4 plasmid. Conversely, co-transfection with the mutant PDCD4 plasmid did not yield a significant effect, suggesting a direct physical interaction between PDCD4 and miR-499-5p (Figure 3B). Subsequent transfected with miR-499-5p inhibitors and mimics led to a significant alteration in miR-499-5p expression within HT22 cells and transfection efficiency reached 70% at a concentration of 50 nM, which was therefore selected as the concentration for subsequent transfections (Figures 3C, D). Western blot analysis indicated that PDCD4 was upregulated in HT22 cells transfected with the miR-499-5p inhibitor, whereas it was downregulated in cells transfected with the miR-499-5p mimic (Figures 3E, F).

To investigate the role of αAsarone in the activation of autophagy via the miR-499-5p/PDCD4/ATG5 pathway, miR-499-5p mimics and inhibitors were employed. The results consistently demonstrated that treatment with αAsarone enhanced the autophagy flue, the colocalization of LAMP1 and LC3, and the accumulation of autophagosome. Interestingly, increased autophagy by αAsarone was partially inhibited by the inhibit-miR-499-5p. This inhibition resulted in a reduction in autophagy flue, both colocalization and autophagic body accumulation (Figures 4A–E). Furthermore, αAsarone was found to downregulate the protein and mRNA expression of miR-499-5p, ATG5, and the LC3-II/LC3-I ratio, while simultaneously upregulating PDCD4 expression (Figures 5A–J). These findings indicate that αAsarone enhances autophagy in neurons in vitro through a mechanism dependent on miR-499-5p.

Here we investigated the protective effects of αAsarone against MACO-induced neuronal injury and examined the mechanisms through which autophagy was caused by αAsarone. The survival rate of mice subjected to MCAO modeling ranged from 60% to 70%, with no significant differences observed in the survival curves among the various treatment groups while there was a significant difference between the normal group and MCAO group (P < 0.05) (Figure 6A). In comparison to the model group, both neurological scores and brain edema were reduced in the treatment groups (Figures 6B, C). Infarct size was assessed using vital staining with TTC, and representative images of TTC-stained ischemic brain infarctions are presented (Figure 6D). Normal brain tissue exhibited a deep red color, while infarcted tissue remained unstained. Each treatment group demonstrated a significant reduction in infarct volume compared to the model group (Figure 6E). Laser speckle contrast imaging (LSCI) corroborated the findings from the TTC staining, blood flow increased after αAsarone (Figure 6F). Additionally, examination of H&E and Nissl staining revealed a reduction in neuron numbers within the model group, which was marked by darkened and wrinkled staining of the cells. In comparison, the αAsarone groups exhibited notable enhancements, including a rise in neuron count, clearer cytoplasmic details, and more robust cellular morphology (Figure 6G). Furthermore, as an autophagy inhibitor, CQ was found to block the protective effect of αAsarone.

To investigate the impact of αAsarone on the neuronal autophagy through miR-499-5p/PDCD4/ATG5 pathway, we performed immunofluorescence staining and electron microscopy in mice after MCAO. Treatment with αAsarone significantly increased the colocalization of MAP2 and LC3, indicating enhanced autophagy. In contrast, Pretreated with CQ reduced this colocalization, suggesting that CQ inhibited the effects of αAsarone (Figures 7A, B). Furthermore, post-MCAO administration of αAsarone resulted in a significant increase in the number of autophagosomes, which was partially reversed by CQ pretreatment (Figure 7C). Western blot and RT-qPCR analyses demonstrated that αAsarone decreased PDCD4 levels while increasing the ratios of LC3-II/LC3-I, LAMP1, miR-499-5p, PDCD4, and ATG5. Conversely, CQ administration reduced the expression of autophagy-related proteins. However, the combination of CQ and αAsarone resulted in an increased expression level of these proteins (Figures 8A–J). Overall, our findings suggest that αAsarone promotes autophagy, thereby providing neuroprotection through the miR-499-5p/PDCD4/ATG5 pathway.