ApTOLL ameliorates cognitive dysfunction and brain injury in ischemic stroke by regulating the miR-335-5p/IRAK1 axis

Abstract

ApTOLL, a promising therapeutic agent, has demonstrated effectiveness in mitigating stroke-induced neurological damage in clinical settings. Despite this, the detailed molecular mechanisms by which ApTOLL impacts ischemic stroke remain inadequately understood. In the present study, we explored how ApTOLL modulates downstream microRNAs (miRNAs) to alleviate brain damage and cognitive dysfunction associated with ischemic stroke. We established a rat model of ischemic stroke. Administration of ApTOLL upregulated miR-335-5p and suppressed IRAK1 expression in the ischemic brain. ApTOLL treatment significantly reduced infarct size, diminished neuronal apoptosis, and attenuated pathological damage in the brain. Additionally, ApTOLL led to the inhibition of inflammation and oxidative damage while enhancing autophagy. Similar effects were observed when miR-335-5p was overexpressed or IRAK1 was knocked down. Conversely, the beneficial impacts of ApTOLL were negated by miR-335-5p antagomir or IRAK1 overexpression, suggesting that ApTOLL's neuroprotective effects are mediated by the miR-335-5p/IRAK1 pathway. Mechanistically, ApTOLL exerted its protective role by promoting the expression of miR-335-5p, thereby reducing IRAK1 levels, leading to amelioration of ischemic brain damage. ApTOLL effectively mitigates ischemic stroke-induced neuronal damage by modulating the miR-335-5p/IRAK1 axis. These findings reveal a novel mechanistic pathway for ApTOLL's therapeutic effects and highlight its potential as a promising treatment strategy for ischemic stroke.

Introduction

Ischemic stroke is the most prevalent form of cerebrovascular disease, accounting for roughly 80 ​%–85 ​% of all stroke cases [1]. The underlying causes include arterial occlusion due to atherosclerosis, thrombus formation, and emboli originating from the heart or other locations, as well as cerebral microvascular disease affecting small vessels [2,3]. These lesions eventually lead to disruption of cerebral blood flow, depriving brain tissue of vital oxygen and nutrients, leading to neuronal damage and cell death [4]. Clinically, ischemic stroke presents with characteristic symptoms, such as weakness or numbness in the face, arms, or legs, slurred speech, visual disturbances, severe headaches, and coordination or balance issues [5]. Long-term effects of stroke may include persistent cognitive dysfunction, including memory loss, impaired executive function, decreased attention span, slower processing speed, and language and communication difficulties [6]. These deficits are reflective of both structural and functional changes in the post-stroke brain, highlighting the necessity of rapid treatment. Current interventions—such as thrombolytic therapy, mechanical thrombectomy, and preventive measures—are aimed at reducing cerebral damage and supporting functional recovery through rehabilitation [7,8]. However, their efficacy is largely limited to a narrow early window after stroke onset, and these treatments are not universally applicable. Additionally, the existing therapeutic approaches fail to adequately address the long-term neurological and cognitive impairments induced by stroke [9]. As such, there is an urgent need for innovative drug-based and targeted therapeutic options that extend the treatment window, reduce adverse effects, and promote neuroprotection and recovery. These approaches can greatly improve the overall efficacy and accessibility of stroke treatment, improve patients' quality of life, and mitigate the socioeconomic impact associated with the disease.

ApTOLL is an experimental pharmacological compound that has been developed for the treatment of acute ischemic stroke. ApTOLL acts as a specific antagonist for Toll-like receptor 4 (TLR4), and works by inhibiting TLR4-mediated immune responses, thereby reducing inflammation and promoting tissue repair and neuroprotection after ischemic injury [10,11]. This compound is currently being evaluated in clinical trials to determine its safety, effectiveness, and optimal dosage [12]. If clinical trials prove successful, ApTOLL could become an important therapeutic option for ischemic stroke, particularly in terms of reducing long-term disability and improving the quality of life of survivors [13]. However, further investigation into its underlying mechanisms of action in ischemic stroke using animal models is required. Such studies will provide a scientific foundation for its clinical development, ensuring both the safety and the broad applicability of ApTOLL as a novel therapeutic.

MicroRNAs (miRNAs) are a class of non-coding RNA molecules that regulate gene expression at the post-transcriptional level by binding to the 3′ untranslated region (3′UTR) of target mRNA, thus influencing protein synthesis [14,15]. In ischemic stroke, miRNAs play pivotal roles in modulating pathways linked to cell death, inflammation, angiogenesis, and neuroprotection [[16], [17], [18]]. This makes miRNAs valuable not only for unraveling the molecular mechanisms underpinning ischemic brain injury but also for identifying potential targets for innovative therapeutic and diagnostic strategies. MiR-335-5p exerts neuroprotective effects in ischemic stroke by attenuating neuronal injury [19]. In addition, overexpression of miR-335-5p has been shown to alleviate hypoxia/reoxygenation-induced myocardial injury [20]. Our preliminary experiments have indicated that ApTOLL may upregulate miR-335-5p expression in transient middle cerebral artery occlusion (tMCAO) rat models, leading us to hypothesize that ApTOLL may exert its therapeutic effect on ischemic stroke through miR-335-5p modulation.

The primary hypothesis of this study is that ApTOLL improves neurological outcomes in ischemic stroke by modulating miR-335-5p expression. Specifically, we aim to explore the therapeutic mechanisms of ApTOLL in an ischemic stroke animal model, focusing on the involvement of miRNA-mediated pathways in mitigating brain injury and promoting neuroprotection.

Materials and methods

Establishment of tMCAO Rat model

A total of 50 male sprague-dawley rats (8 weeks old, 210 ​± ​10 ​g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. All experimental procedures were approved by the Animal Ethics Committee of Chengdu Pidu District People's Hospital, and followed the guidelines for animal experiments. The model of tMCAO was set up as previously described [21,22]. Briefly, under anesthesia, rats were subjected to surgery on a heating blanket that maintained their rectal temperature at 37 ​°C. The common carotid artery, external carotid artery, and internal carotid artery were exposed on one side. The distal end of the common carotid artery was ligated with a nylon suture, as were two points on the external carotid artery: one near its terminus and the other near the bifurcation with the internal carotid artery. A small incision was made between these two ligatures, and a nylon filament was gently inserted through the external carotid artery stump into the internal carotid artery, advancing until resistance was felt. The filament was left in place for 2 ​h to induce ischemia and then withdrawn to allow reperfusion. The surgical area was treated with antibiotics to prevent infection. Sham-operated rats, used as controls, underwent the same surgical procedure except for filament insertion. Immediately after reperfusion, ApTOLL was administered via tail vein injection at doses of 1.2 ​mg/kg and 6 ​mg/kg.

Selection of ApTOLL Dose: 0.2 ​mg/kg of ApTOLL significantly improved ischemic damage and was proven to be safe for human use [13]. The human equivalent dose is derived by adjusting the animal dose based on body surface area differences. Therefore, doses equivalent to 1 ​× ​(1.2 ​mg/kg) and 5 ​× ​(6 ​mg/kg) of the recommended animal dose were selected.

Three days before tMCAO, agomir and adeno-associated viral (AAV) vectors were administered, following previously described methods [23]. Rats were anesthetized and placed prone in a stereotaxic apparatus (RWD Life Science, China). A midline incision was made along the scalp, and a burr hole was drilled in the skull, 0.5 ​mm posterior and 1.0 ​mm lateral to the bregma on the right side. Through this hole, miR-335-5p agomir (0.8 ​nmol, RiboBio, China), AAV-sh-IRAK1, AAV-oe-IRAK1 (15 ​μL, 1.3 ​× ​10¹² vg/mL, VectorBuilder, China), or the corresponding control vector was injected into the right lateral ventricle at a vertical depth of 2.5 ​mm using a Hamilton syringe. The injection was performed at a rate of 0.2 ​μL/min using a microsyringe pump (KDS 310, KD Scientific). Following injection, the needle was kept in place for 5 ​min, then slowly withdrawn over 4 ​min. The hole was sealed with bone wax, and the incision was sutured. Animals were allowed to recover afterward [24,25].

After motor function assessments, rats were euthanized using overdosed CO₂ inhalation. Brain tissues were collected and preserved either in 4 ​% paraformaldehyde or stored at −80 ​°C for subsequent analyses.

Neurological function assessment

Neurological function was assessed 6 ​h after reperfusion using the Zea-Longa scoring system [26]. This scoring system assigns points from 0 to 5 as follows: 0 – no observable neurological deficit, normal behavior; 1 – mild deficit with reduced left forelimb extension; 2 – moderate deficit where the rat cannot walk in a straight line and instead circles consistently; 3 – severe deficit with the rat unable to stand independently, tending to fall; 4 – inability to walk or loss of consciousness; 5 – death.

2,3,5-Triphenyltetrazolium chloride (TTC) staining

Twenty-four hours after reperfusion, brains from three randomly chosen rats per group were collected and sliced into five coronal sections of 2 ​mm thickness each. The sections were stained in a 1 ​% TTC (Sigma-Aldrich, Australia) at 37 ​°C for 30 ​min in the dark. Non-infarct tissue appeared red, whereas infarct tissue remained white. The stained slices were subsequently fixed in 2 ​% paraformaldehyde and photographed using a digital camera (Canon IXUS175, Tokyo, Japan). Infarct volume percentages were calculated using ImageJ software.

Morris water maze (MWM) test

MWM test was conducted to evaluate spatial learning and memory, following previously described protocols [27]. The test involved five days of training sessions, followed by a probe trial on the sixth day. Rats were introduced into the water pool from four different starting points (North, South, East, and West) in a randomized order each day. During each daily training session, rats performed four trials to locate a hidden platform, with a maximum allowed time of 120 ​s. If unsuccessful, rats were guided to the platform. Once located, rats remained on the platform for 30 ​s. The time taken to reach the platform, recorded as latency, was used to measure spatial learning abilities. A single probe trial, conducted on the day following the last training session, assessed spatial memory. In this trial, the platform was removed, and rats were released from a random starting point, swimming freely for 120 ​s. Their swimming paths were tracked using a video camera and analyzed with EthoVision software (Noldus), focusing particularly on the number of times they crossed the target quadrant, where the platform was previously located.

Assessment of motor function

Psychomotor coordination and limb strength were evaluated through an accelerated rotarod test and forelimb grip strength measurement, respectively [24]. In the rotating stick test, a 600-mm-long wooden rotarod was elevated and rotated at 10, 20, or 40 ​revolutions per minute. The rats were trained to run on the rotarod at the corresponding speed to avoid falling. Prior to surgery, the animals were trained daily for three consecutive days, starting at 10 ​revolutions per minute and gradually increasing to 40 ​revolutions per minute over a 5-min period. After two weeks post-tMCAO, animals were tested at 40 ​rpm, and their performance was assessed based on the time they remained on the rotarod. To assess forelimb strength, rats were allowed to grasp a grid with either their healthy or impaired forelimbs and pulled backward. The maximum force was recorded, and the average force from three trials was calculated.

Hematoxylin and eosin (HE) staining

HE staining was performed to observe neuronal morphology. After paraffin-embedding, sections were deparaffinized and rehydrated before being stained with hematoxylin for 5 ​min. Sections were differentiated in 0.6 ​% hydrochloric acid ethanol for 30 ​s and counterstained with eosin (pH 4.2) for 3 ​min, followed by dehydration. Tissue pathology was examined under a Nikon Eclipse C1 microscope.

TUNEL staining for apoptosis

The One-Step TUNEL Apoptosis Assay kit (Roche) was used to identify apoptotic cells in the brain, following protocols described previously [28]. Paraffin sections were deparaffinized and rehydrated, treated with proteinase K for 20 ​min, and incubated with fluorescent dUTP and TdT enzyme mixture at 37 ​°C for 1 ​h. Positive control sections were incubated with DNase I at room temperature for 10 ​min before labeling, while negative controls were incubated with dUTP. After labeling, the samples were treated with diaminobenzidine, counterstained with DAPI (Sigma-Aldrich), dehydrated, cleared with xylene, and mounted with neutral balsam. The images were captured using a Carl Zeiss LSM700 fluorescence microscope.

Immunofluorescence (IF) staining

IF staining was performed according to established methods. Brain tissues fixed in 4 ​% paraformaldehyde were sectioned into 10 ​μm coronal slices using a cryostat. Sections were blocked with 10 ​% goat serum for 1 ​h and incubated overnight at 4 ​°C with primary antibodies: ATG5 (Cell Signaling Technology, 12994), p62 (Cell Signaling Technology, 5114), cleaved caspase-3 (Cell Signaling Technology, 9661), and phospho-p65 (Cell Signaling Technology, 3033). After washing with PBS (pH 7.4), sections were incubated with the appropriate secondary antibodies. Fluorescence was detected using an Olympus BX51 microscope, and images were acquired using Nikon NIS-Elements software.

Quantitative Real-time PCR (RT-qPCR)

RNA was extracted from brain tissue using TRIzol reagent (Invitrogen, 15596026). For mature microRNA, cDNA synthesis was performed using the HiScript Q Select RT SuperMix for qPCR Kit (Vazyme, R133-01) with stem-loop primers (RiboBio). For mRNA quantification, the HiScript Q RT SuperMix for qPCR Kit (Vazyme, R123-01) was used, and SYBR Green Real-time PCR Master Mix (Vazyme, R141-02) was used for amplification. GAPDH and U6 were used as internal controls for mRNA and miRNA, respectively.

Western blotting

Protein extraction from brain tissue was performed using RIPA lysis buffer (Beyotime, P0013B). The protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, Frederick, MD, USA). Proteins were separated on 10 ​% and 12 ​% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were incubated with primary antibodies overnight at 4 ​°C, followed by incubation with HRP-conjugated goat anti-mouse IgG secondary antibody (Cell Signaling Technology, 7076P2). Immunoreactive bands were visualized using the ECL detection system (Thermo Fisher Scientific). The following primary antibodies were used: ATG5 (Cell Signaling Technology, 12994), p62 (Cell Signaling Technology, 5114), cleaved caspase-3 (Cell Signaling Technology, 9661), Bax (Abcam, ab32503), Bcl-2 (Proteintech, 12789-1-AP), p65 (Cell Signaling Technology, 8242), and phospho-p65 (Cell Signaling Technology, 3033).

Fluorescence In Situ hybridization (FISH)

FISH was performed to detect miR-355-5p expression in cultured neurons. Neurons were fixed with 4 ​% paraformaldehyde, permeabilized with 0.25 ​% Triton X-100, and incubated with hybridization buffer containing a digoxigenin-labeled miR-355-5p probe (Invitrogen) overnight at 37 ​°C. After washing with TBS, signal amplification was carried out using the TSA Cy5 Kit (PerkinElmer, NEL745001KT). The sections were mounted with Prolong Gold Antifade reagent containing DAPI. Images were obtained using a Carl Zeiss LSM700 fluorescence microscope.

Enzyme-linked immunosorbent assay (ELISA)

The levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in brain tissues were quantified using commercial ELISA kits, according to the manufacturer's instructions (Abcam).

Assessment of oxidative stress

To evaluate oxidative stress markers in brain tissues, the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and CAT were determined using commercially available kits, following the manufacturer's protocols. The kits were obtained from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Dual-luciferase reporter assay

The 3′-UTR of IRAK1, containing predicted binding sites for miR-335-5p, was amplified by PCR from human genomic DNA. The resulting DNA fragment was cloned into the pmiR-RB-REPORT vector (RiboBio), generating the wild-type (WT) IRAK1 construct. To assess the interaction of miR-335-5p with IRAK1, point mutations were introduced in the miR-335-5p binding sites within the IRAK1 3′-UTR to generate the mutant luciferase reporter vector. HEK293T cells were co-transfected with the WT or mutant constructs and miR-335-5p mimic or mimic NC using Lipofectamine 3000 (Invitrogen, USA), as per the manufacturer's instructions. Luciferase activity was measured 24 ​h post-transfection, and the data were normalized by comparing the Renilla luciferase activity to the firefly luciferase activity. The results were expressed as a percentage relative to the control group, following the protocol provided by Promega (E2920).

RNA immunoprecipitation (RIP) assay

For the RIP assay, cell lysates were incubated with RIP buffer, which was supplemented with magnetic beads conjugated to either anti-Ago2 antibody or mouse IgG. After complex formation, the RNA-protein complexes were captured using proteinase K digestion and isolated for RNA extraction. The concentration and quality of the isolated RNA were determined using a spectrophotometer (NanoDrop, Thermo Scientific). The presence of target binding was verified by RT-qPCR.

Results

ApTOLL Improves Cognitive Impairment and neuron damage in tMCAO rats

To investigate the underlying molecular mechanisms of ApTOLL treatment in ischemic stroke, we established a tMCAO rat model and administered low and high doses of ApTOLL. Cognitive impairment was first assessed using the MWM test. Compared to the Sham group, rats in the tMCAO group showed significantly increased latency in locating the hidden platform and a marked reduction in the number of target quadrant crossings. Both of these impairments were notably improved following ApTOLL treatment (Fig. 1A–C). Grip strength and rotarod tests,∖ also showed significant improvements with ApTOLL treatment in the tMCAO rats (Fig. 1D and E). Neurological function was scored to assess the extent of damage. As shown in Fig. 1F, tMCAO rats had higher neurological scores, which were significantly reduced by both doses of ApTOLL. To assess brain tissue damage, we performed TTC staining. Fig. 1G illustrates a marked increase in infarct brain tissues in tMCAO rats compared to Sham controls, with a noticeable reduction in infarct area following ApTOLL administration. Further examination of neuronal damage using HE and TUNEL staining revealed pronounced nuclear shrinkage and cytoplasmic disappearance in tMCAO rats, along with a significant increase in TUNEL-positive cells, both of which were significantly ameliorated by ApTOLL (Fig. 1H and I). Western blot and immunofluorescence analyses corroborated these findings. The expression of cleaved caspase-3 and Bax was elevated, while Bcl-2 levels were reduced in tMCAO rats. ApTOLL treatment effectively reversed these alterations (Fig. 1J and K). Next, we evaluated autophagic activity in brain tissues. tMCAO rats showed a decreased LC3II/LC3I ratio and reduced ATG5 expression, alongside elevated p62 levels. These changes were reversed by ApTOLL administration (Fig. 1J and K). We also assessed the levels of inflammatory cytokines and oxidative stress markers. As shown in Fig. 1L-M, the levels of TNF-α, IL-1β, IL-6, and MDA were significantly elevated in tMCAO rats, while SOD and CAT levels were diminished. ApTOLL treatment effectively attenuated these alterations, highlighting its potential to mitigate inflammation and oxidative stress. Collectively, these data demonstrate that ApTOLL treatment significantly ameliorates cognitive and motor dysfunction, reduces neurodegeneration and infarct size, and positively influences apoptosis and autophagy in the brain. Additionally, ApTOLL mitigates inflammation and oxidative stress, offering substantial neuroprotection against ischemic injury.

Fig. 1.

ApTOLL Improves Cognitive Impairment and Neuroinjury in tMCAO Rats. The tMCAO rat model was established and treated with low and high doses of ApTOLL. A–C: Morris water maze tests to assess cognitive impairment in rats. D and E: Accelerating rotarod and forelimb strength tests to evaluate motor dysfunction. F: Zea-Longa score for each group of rats. G: TTC staining to assess infarct area in the brain. H: HE staining to evaluate neuronal pathological damage in the brain tissue. I: TUNEL staining to detect apoptosis in the brain tissue. J: IF staining to examine the expression of cleaved caspase-3, ATG5, and p62 in the brain tissue. K: Western blot analysis of cleaved caspase-3, ATG5, p62, Bcl-2, and Bax expression in the brain. L: ELISA detection of TNF-α, IL-1β, and IL-6 levels in the brain tissue. M: Commercial kit assays for MDA, SOD, and CAT levels in the brain tissue. Data are presented as mean ​± ​SD (n ​= ​7). ∗P ​< ​0.05.

Upregulation of miR-335-5p ameliorates cognitive decline and brain injury in tMCAO rats

In the present study, we observed a reduction in the expression of miR-335-5p in tMCAO rats, which was restored following ApTOLL treatment (Fig. 2A). This led us to hypothesize that ApTOLL's therapeutic benefits may be linked to its influence on miR-335-5p expression. To explore this hypothesis, we utilized miR-335-5p agomir to enhance its expression in tMCAO rats (Fig. 2B). Overexpression of miR-335-5p significantly reduced the time required for the rats to locate the hidden platform and increased the frequency of target quadrant crossings (Fig. 2C–E), demonstrating a marked improvement in cognitive abilities. Additionally, miR-335-5p overexpression effectively restored motor function in the tMCAO rats (Fig. 2F and G). The neurological deficit score further confirmed that miR-335-5p overexpression resulted in a substantial reduction in neurofunctional impairments (Fig. 2H). Furthermore, miR-335-5p overexpression led to a significant reduction in infarct size (Fig. 2I). Histological analyses revealed that this treatment alleviated neuronal damage, including the reversal of nuclear condensation and cytoplasmic degeneration, as well as a reduction in TUNEL-positive cells (Fig. 2J and K). Protein analysis supported these findings, showing that miR-335-5p overexpression suppressed cleaved caspase-3 and Bax levels, while promoting Bcl-2 expression (Fig. 2L-M), indicating its role in modulating apoptotic pathways to confer neuroprotection. In addition, autophagy-related protein assays revealed that miR-335-5p overexpression restored the LC3II/LC3I ratio, upregulated ATG5 expression, and downregulated p62, suggesting its involvement in regulating autophagic flux (Fig. 2L-M). Moreover, miR-335-5p overexpression was found to suppress inflammatory cytokines (such as TNF-α, IL-1β, and IL-6) and oxidative stress markers (such as MDA), while simultaneously enhancing the activity of antioxidant enzymes, including SOD and CAT (Fig. 2N-O), further underscoring its potent role in controlling inflammation and oxidative stress. In summary, our data not only reveal that ApTOLL exerts neuroprotective effects by modulating miR-335-5p expression, but also provide compelling evidence for the therapeutic potential of miR-335-5p in treating ischemic brain injury.

Fig. 2.

Overexpression of miR-335-5p Improves Cognitive Impairment and Brain Injury in tMCAO Rats. A-B: RT-qPCR detection of miR-335-5p expression in the brain tissue of each group. C–E: Morris water maze tests to assess cognitive impairment in rats. F–G: Accelerating rotarod and forelimb strength tests to evaluate motor dysfunction. H: Zea-Longa score for each group of rats. I: TTC staining to assess infarct area in the brain. J: HE staining to observe neuronal pathological damage in the brain tissue. K: TUNEL staining to detect apoptosis in the brain tissue. L: IF staining to examine cleaved caspase-3, ATG5, and p62 expression in the brain tissue. M: Western blot analysis of cleaved caspase-3, ATG5, p62, Bcl-2, and Bax expression in the brain. N: ELISA detection of TNF-α, IL-1β, and IL-6 levels in the brain tissue. O: Commercial kit assays for MDA, SOD, and CAT levels in the brain tissue. Data are presented as mean ​± ​SD (n ​= ​7). ∗P ​< ​0.05.

ApTOLL mediates therapeutic effects on tMCAO-Induced brain injury via miR-335-5p regulation

To further explore whether ApTOLL's therapeutic effects on tMCAO-induced brain injury are mediated through the regulation of miR-335-5p expression, we administered miR-335-5p antagomir alongside ApTOLL treatment to inhibit its expression. As shown in Fig. 3A, the beneficial effect of ApTOLL in promoting miR-335-5p expression was successfully reversed by miR-335-5p antagomir. ApTOLL significantly reduced the latency to find the hidden platform and increased the number of crossings in the target quadrant. However, these improvements were reversed upon miR-335-5p knockdown (Fig. 3B–D). Similarly, the enhancement of motor coordination and limb function by ApTOLL was compromised by the downregulation of miR-335-5p (Fig. 3E and F). The neurological deficit score further validated the neuroprotective effects of ApTOLL, which were significantly diminished when miR-335-5p expression was suppressed (Fig. 3G). Furthermore, the reduction in infarct size observed with ApTOLL treatment was abolished by the inhibition of miR-335-5p (Fig. 3H). Pathological staining showed a similar trend, where the protective effects of ApTOLL on nuclear shrinkage and cytoplasmic loss were reversed after miR-335-5p knockdown, and the reduction in TUNEL-positive cells was also attenuated (Fig. 3I and J). At the protein level, ApTOLL promoted Bcl-2 and inhibited cleaved caspase-3 and Bax levels, an effect that was neutralized upon miR-335-5p inhibition (Fig. 3K-L). Autophagy marker analysis further supported these findings, showing that ApTOLL restored the LC3II/LC3I ratio, increased ATG5 levels, and reduced p62 expression, which were all reversed by miR-335-5p knockdown (Fig. 3K-L). Additionally, biochemical assays revealed that ApTOLL suppressed the expression of inflammatory cytokines and oxidative stress markers, while increasing antioxidant enzyme activity. These beneficial changes were prevented by miR-335-5p inhibition (Fig. 3M ​− ​N). Together, these results suggest that the therapeutic effects of ApTOLL on cognitive dysfunction and brain injury in tMCAO rats are primarily mediated through the upregulation of miR-335-5p.

Fig. 3.

ApTOLL Ameliorates Brain Injury in tMCAO Rats through miR-335-5p Regulation. A: RT-qPCR detection of miR-335-5p expression in the brain tissue of each group. B–D: Morris water maze tests to assess cognitive impairment in rats. E–F: Accelerating rotarod and forelimb strength tests to evaluate motor dysfunction. G: Zea-Longa score for each group of rats. H: TTC staining to assess infarct area in the brain. I: HE staining to evaluate neuronal pathological damage in the brain tissue. J: TUNEL staining to detect apoptosis in the brain tissue. K: IF staining to examine cleaved caspase-3, ATG5, and p62 expression in the brain tissue. L: Western blot analysis of cleaved caspase-3, ATG5, p62, Bcl-2, and Bax expression in the brain. M: ELISA detection of TNF-α, IL-1β, and IL-6 levels in the brain tissue. N: Commercial kit assays for MDA, SOD, and CAT levels in the brain tissue. Data are presented as mean ​± ​SD (n ​= ​7). ∗P ​< ​0.05.

miR-335-5p targets the IRAK1 mRNA 3′ UTR to regulate its expression

To explore the downstream targets of miR-335-5p, we employed bioinformatics tools to identify the top 10 candidate genes most strongly supported by AGO Clip-seq data. Among these, RT-qPCR analysis revealed that only IRAK1 mRNA was significantly downregulated by miR-335-5p in the brain tissue of tMCAO rats (Fig. 4A). Subsequent RIP assays confirmed the binding interaction between miR-335-5p and IRAK1 mRNA, with both molecules showing high enrichment on Ago2 beads (Fig. 4B). Based on predicted binding sites, we generated wild-type and mutated IRAK1 3′ UTR constructs (Fig. 4C). Dual-luciferase reporter assays demonstrated that co-transfection of miR-335-5p mimic and the wild-type IRAK1 3′ UTR led to a significant reduction in luciferase activity (Fig. 4D). Moreover, elevated IRAK1 expression was observed in the brains of tMCAO rats (Fig. 4E). Collectively, these data indicate that IRAK1 mRNA is a direct target of miR-335-5p and is regulated by its expression in the context of ischemic brain injury.

Fig. 4.

miR-335-5p Targets IRAK1. A: RT-qPCR detection of potential downstream mRNA targets of miR-335-5p. B: RIP assay to examine the enrichment of miR-335-5p and IRAK1 in Ago2 magnetic beads. C: Potential target relationship between miR-335-5p and IRAK1. D: Dual-luciferase reporter assay to verify the targeting relationship between miR-335-5p and IRAK1. E: Western blot analysis of IRAK1 expression in tMCAO rats. Data are presented as mean ​± ​SD (n ​= ​7). ∗P ​< ​0.05.

Knockdown of IRAK1 Improves Cognitive Impairment and brain injury in tMCAO rats

To elucidate the role of IRAK1 in ischemic brain injury, we performed IRAK1 knockdown in tMCAO rats using AAV-mediated shRNA technology (Fig. 5A). Cognitive performance demonstrated that silencing IRAK1 significantly reduced the time taken to locate the hidden platform and increased the number of target quadrant crossings, suggesting a marked improvement in cognitive function (Fig. 5B–D). Additionally, motor function was improved in the tMCAO rats, as evidenced by enhanced performance in the grip strength and rotarod tests following IRAK1 knockdown (Fig. 5E and F). Neurological deficit scores further supported these findings, showing that IRAK1 silencing reduced neurological damage in these animals (Fig. 5G). Consistent with these results, a significant reduction in infarct size was observed in IRAK1-knockdown rats (Fig. 5H). Histological analysis revealed that IRAK1 silencing mitigated nuclear shrinkage and cytoplasmic loss, alongside a marked reduction in TUNEL-positive cell numbers (Fig. 5I and J). Protein expression analysis showed that IRAK1 knockdown decreased cleaved caspase-3 and Bax levels while increasing Bcl-2 expression, indicating a protective effect on apoptosis (Fig. 5K-L). Furthermore, autophagic activity was also normalized, as evidenced by restored LC3II/LC3I ratio, increased ATG5 expression, and decreased p62 levels following IRAK1 knockdown (Fig. 5K-L). Additionally, IRAK1 silencing attenuated the expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and oxidative stress markers (MDA), while enhancing antioxidant enzyme activity (SOD and CAT) (Fig. 5M ​− ​N), further suggesting that IRAK1 knockdown mitigates both inflammation and oxidative damage. Collectively, these findings indicate that IRAK1 silencing ameliorates cognitive deficits, reduces neuroinflammation, oxidative stress, and apoptosis, and restores autophagic function in the tMCAO rat model.

Fig. 5.

Knockdown of IRAK1 Improves Cognitive Impairment and Brain Injury in tMCAO Rats. A: Western blot detection of IRAK1 expression in the brain tissue of each group. B–D: Morris water maze tests to assess cognitive impairment in rats. E–F: Accelerating rotarod and forelimb strength tests to evaluate motor dysfunction. G: Zea-Longa score for each group of rats. H: TTC staining to assess infarct area in the brain. I: HE staining to evaluate neuronal pathological damage in the brain tissue. J: TUNEL staining to detect apoptosis in the brain tissue. K: IF staining to examine cleaved caspase-3, ATG5, and p62 expression in the brain tissue. L: Western blot analysis of cleaved caspase-3, ATG5, p62, Bcl-2, and Bax expression in the brain. M: ELISA detection of TNF-α, IL-1β, and IL-6 levels in the brain tissue. N: Commercial kit assays for MDA, SOD, and CAT levels in the brain tissue. Data are presented as mean ​± ​SD (n ​= ​7). ∗P ​< ​0.05.

ApTOLL modulates IRAK1 expression to ameliorate brain injury in tMCAO rats

In this study, we sought to explore whether the therapeutic effects of ApTOLL on brain injury in the tMCAO rat model are mediated through regulation of IRAK1 expression. To test this hypothesis, we co-administered ApTOLL with AAV-oe-IRAK1, which induced overexpression of IRAK1. As shown in Fig. 6A, the ApTOLL-mediated reduction in IRAK1 expression was effectively reversed by IRAK1 overexpression. Cognitive assessments indicated that while ApTOLL treatment alone reduced the latency to locate the hidden platform and increased the number of target quadrant crossings, but these beneficial effects were negated by IRAK1 overexpression (Fig. 6B–D). In parallel, improvements in motor coordination and strength, as assessed by grip strength and rotarod tests, were also abrogated by the overexpression of IRAK1 (Fig. 6E and F). Neurological scores further supported the findings, demonstrating that ApTOLL significantly alleviated neurological deficits in tMCAO rats, yet this recovery was reversed by the increased expression of IRAK1 (Fig. 6G). Histological analyses showed that ApTOLL reduced brain infarct size (Fig. 6H), while IRAK1 overexpression reversed this effect. Additionally, ApTOLL treatment inhibited cell nucleus shrinkage and cytoplasmic loss, alongside a reduction in TUNEL-positive cells. However, these improvements were reversed in the presence of IRAK1 overexpression (Fig. 6I and J). At the protein level, ApTOLL suppressed cleaved caspase-3 and Bax expression, while enhancing Bcl-2 levels in the brain tissues of tMCAO rats. However, these changes were reversed when IRAK1 was overexpressed (Fig. 6K-L). Furthermore, ApTOLL restored the LC3II/LC3I ratio, increased ATG5 expression, and reduced p62 levels, but these alterations were reversed by IRAK1 overexpression (Fig. 6K-L). Finally, biochemical analysis revealed that ApTOLL treatment decreased the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and oxidative stress markers (MDA), while boosting antioxidant enzyme activities (SOD and CAT). These biochemical effects were also reversed upon overexpression of IRAK1 (Fig. 6M ​− ​N). Collectively, these findings strongly suggest that ApTOLL's therapeutic action in the tMCAO rat model is primarily driven by its ability to downregulate IRAK1 expression, thereby improving cognitive function and mitigating brain damage.

Fig. 6.

ApTOLL Ameliorates Brain Injury in tMCAO Rats through miR-335-5p Regulation. A: Western blot detection of IRAK1 expression in the brain tissue of each group. B–D: Morris water maze tests to assess cognitive impairment in rats. E–F: Accelerating rotarod and forelimb strength tests to evaluate motor dysfunction. G: Zea-Longa score for each group of rats. H: TTC staining to assess infarct area in the brain. I: HE staining to evaluate neuronal pathological damage in the brain tissue. J: TUNEL staining to detect apoptosis in the brain tissue. K: IF staining to examine cleaved caspase-3, ATG5, and p62 expression in the brain tissue. L: Western blot analysis of cleaved caspase-3, ATG5, p62, Bcl-2, and Bax expression in the brain. M: ELISA detection of TNF-α, IL-1β, and IL-6 levels in the brain tissue. N: Commercial kit assays for MDA, SOD, and CAT levels in the brain tissue. Data are presented as mean ​± ​SD (n ​= ​7). ∗P ​< ​0.05.

ApTOLL inhibits p65 pathway activation through modulation of the miR-335-5p/IRAK1 axis

NF-κB plays a central role in regulating inflammatory responses and immune reactions. IRAK1, by activating the IκB kinase complex, induces phosphorylation and degradation of IκB, leading to the phosphorylation of p65. To investigate whether ApTOLL exerts its effects via the miR-335-5p/IRAK1 axis to inhibit p65 activation, we assessed the expression of p-p65 in treated rats. Western blotting revealed that ApTOLL administration significantly reduced p-p65 levels. However, this inhibitory effect was reversed by treatment with miR-335-5p antagomir and AAV-oe-IRAK1, which restored p-p65 expression (Fig. 7A and B). These results suggest that ApTOLL effectively suppresses p65 pathway activation, and this effect is mediated through the regulation of the miR-335-5p/IRAK1 axis.

Fig. 7.

ApTOLL Inhibits p65 Pathway Activation via miR-335-5p/IRAK1 Axis. A-B: Western blot detection of p-p65 and p65 protein expression in the brain tissue. Data are presented as mean ​± ​SD (n ​= ​3). ∗P ​< ​0.05.

Discussion

ApTOLL, an experimental TLR4 antagonist, has been shown in clinical studies to offer promising therapeutic effects in ischemic stroke [11,29], effectively reducing both mortality and infarct size. However, the precise molecular mechanisms underlying its therapeutic potential remain unclear. An in-depth understanding of these mechanisms can optimize the application of ApTOLL, including its dosage and potential combination strategies, thereby improving efficacy and safety. In this study, we explored the multifaceted effects of ApTOLL on brain injury in tMCAO rats, particularly focusing on inflammation, oxidative stress, neuronal apoptosis, and autophagy. We identified the miR-335-5p/IRAK1 axis as a crucial signaling pathway mediating these therapeutic effects.

Previous studies in rodent models have confirmed ApTOLL's ability to mitigate inflammation and reduce brain injury under blinded experimental conditions [12,13]. Our findings aligned with this conclusion, showing that ApTOLL effectively decreased the levels of pro-inflammatory cytokines and inhibited the activation of the p65 pathway. Additionally, we discovered that ApTOLL administration reduced oxidative stress markers in the brain tissue. Autophagy levels were significantly reduced in tMCAO rats following brain injury. Notably, autophagy typically increases during the early stages of ischemic injury as a stress response to ischemia [30]. However, during prolonged ischemia-reperfusion injury, reduced autophagic activity likely results from excessive cellular damage [31,32]. ApTOLL's ability to restore autophagic function in the recovery phase likely aids cellular stress responses, enabling the removal of damaged organelles and proteins, which helps maintain cell survival and function. We speculate that this restoration of autophagy contributes to reduced ROS production, decreased inflammatory cytokine release, and inhibition of downstream p65 signaling activation. ELISA and commercial kit results further support this hypothesis.

At the molecular level, we demonstrated that ApTOLL exerted its effects primarily by modulating the miR-335-5p/IRAK1 axis. ApTOLL enhanced the expression of miR-335-5p in the brain tissue of tMCAO rats. miR-335-5p has been previously identified as an anti-inflammatory molecule in various disease contexts. For example, miR-335-5p reduces inflammatory cytokine levels in periodontal disease and inhibits TLR4 expression [33]. Additionally, exosomes derived from human umbilical mesenchymal stem cells, which carry miR-335-5p, attenuate inflammation and epithelial-to-myofibroblast transdifferentiation in renal tubular cells by downregulating ADAM19 [34]. In the current study, we found that miR-335-5p alleviated inflammation, oxidative stress, and neuronal apoptosis in tMCAO rats. The critical mechanism involved miR-335-5p targeting IRAK1 mRNA, reducing its translation and limiting its activation. IRAK1, a key signaling molecule in immune responses, plays a pivotal role in the TLR-mediated inflammatory pathway. Upon recognition of pathogens by TLRs, IRAK1 is activated via the MyD88-dependent pathway, which subsequently phosphorylates and activates downstream molecules, including TRAF6, leading to the activation of NF-κB and MAPK signaling pathways. By reducing IRAK1 expression, miR-335-5p significantly inhibits the phosphorylation of p65, thereby dampening inflammatory responses.

In conclusion, ApTOLL exerts its therapeutic effects by upregulating miR-335-5p expression, which in turn inhibits IRAK1 activity and suppresses the activation of inflammatory signaling pathways. This mechanism reveals how ApTOLL exerts its anti-inflammatory and neuroprotective effects at the molecular level, offering a promising strategy for treating ischemic stroke. This action not only alleviates inflammation but also reduces oxidative stress and neuronal apoptosis, thereby improving recovery following stroke.

Author's Contribution

FengQin Qin and Xiang Feng designed the research study. FengQin Qin and Xiang Feng performed the research. HongFu Yang and Hao Liu provided help and advice. Wei Yuan analyzed the data. FengQin Qin and Xiang Feng wrote the manuscript. Wei Yuan reviewed and edited the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Ethics approval

All animal experiments were complied with the ARRIVE guidelines and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experiments were approved by the Institutional Animal Care and Use Committee of Chengdu Pidu District People's Hospital (No.2021-CD125).

Funding

Not applicable.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.