Electroacupuncture Alleviates Post-stroke Cognitive Impairment Through Inhibiting miR-135a-5p/mTOR/NLRP3 Axis-mediated Autophagy

—Post-stroke cognitive impairment is a signiﬁcant challenge with limited treatment options. Electroacupuncture (EA) has shown promise in improving cognitive function after stroke. Our study explores the underlying mechanism of EA in alleviating cognitive impairment through the inhibition of autophagy. We utilized a rat model of stroke induced by middle cerebral artery occlusion (MCAO) to evaluate the eﬃcacy of EA. Treatment with EA was observed to markedly improve cognitive function and reduce inﬂammation in MCAO rats, as evidenced by decreased neurological deﬁcit scores, shorter latencies in the water maze test, and diminished infarct volumes. EA also attenuated tissue damage in the hippocampus and lowered the levels of pro-inﬂammatory cytokines and oxidative stress markers. Although autophagy was upregulated in MCAO rats, EA treatment suppressed this process, indicated by a reduction in autophagosome formation and alteration of autophagy-related protein expression. The protective eﬀects of EA were reversed by the autophagy activator rapamycin. EA treatment elevated the levels of microRNA (miR)-135a-5p expression, and suppression of this elevation attenuated the remedial eﬃcacy of EA in addressing cognitive impairment and inﬂammation. MiR-135a-5p targeted mammalian target of rapamycin (mTOR)/NOD-like receptor protein 3 (NLRP3) signaling to repress autophagy. EA treatment inhibits autophagy and alleviates cognitive impairment in post-stroke rats. It exerts its bene-ﬁcial eﬀects by upregulating miR-135a-5p and targeting the mTOR/NLRP3 axis. (cid:1) 2024 The Author(s). Published by Elsevier Inc. on behalf of IBRO. This is an open access article under the CC BY license (http://creativecommons.org/licenses/ by/4.0/).


INTRODUCTION
Post-stroke cognitive impairment (PSCI) represents a frequent and debilitating outcome of stroke, manifesting as deficiencies in memory, attention, and executive function (Rost et al., 2022).PSCI diagnosis requires at least six months, severely impacting the quality of life for stroke survivors and posing a significant challenge for healthcare providers (Mijajlovic´et al., 2017).PSCI involves targeted drug treatments, lifestyle modifications, rehabilitative therapies, and management of concurrent health issues, primarily to prevent stroke recurrence (Sun et al., 2014).However, despite progress in stroke treatment, effective PSCI therapies are scarce.
Recent studies indicate that autophagy, a cellular mechanism responsible for the breakdown and reuse of cellular constituents, holds a pivotal role in neurodegenerative diseases and cognitive dysfunction (Mizushima & Levine, 2020;Zhang et al., 2021).The mammalian target of rapamycin (mTOR)/NOD-like receptor protein 3 (NLRP3) pathway is critical in controlling autophagy, vital for cellular balance (Sun et al., 2021).mTOR, a paramount regulator of cellular growth and metabolism, exerts a negative regulation on autophagy.Concurrently, NLRP3, an integral component of the inflammatory response complex, engages with the autophagy mechanism in complex manners (Shi et al., 2019;Biasizzo & Kopitar-Jerala, 2020).Recent findings suggest https://doi.org/10.1016/j.neuroscience.2024.03.008 0306-4522/Ó 2024 The Author(s).Published by Elsevier Inc. on behalf of IBRO.This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).that mTOR signaling controls autophagy to improve outcomes in acute ischemic stroke during neuronal injury (Deng et al., 2022).The NLRP3 inflammasome also serves as a principal mediator in the neuroinflammation and subsequent cerebral damage induced by ischemic stroke (Z.Huang et al., 2021b).Therefore, the mTOR/ NLRP3 axis may offer a promising target for autophagy modulation in PSCI.Additionally, microRNAs (miRNAs) are acknowledged as pivotal modulators of autophagy, linked to various neurological conditions (Chen et al., 2021;Ghafouri-Fard et al., 2022).Of particular interest is miR-135a-5p, found to be imbalanced in brain injury due to hypoxia/reoxygenation (Chen & Li, 2019).MiR-135a-5p has been found to protect neuronal cell death in Parkinson's disease through suppressing mTOR pathway-mediated autophagy (Qin et al., 2022).Moreover, miR-135a-5p can dampen the NLRP3 inflammasome activation, reducing neuronal autophagy and ischemic brain injury (Liu et al., 2021).Therefore, miR-135a-5p may regulate PSCI progression through its influence on mTOR/NLRP3-driven autophagy, suggesting its potential as a therapeutic target for PSCI.
Electroacupuncture (EA), a therapeutic technique combining acupuncture and electrical stimulation, has emerged as a potential intervention for improving cognitive function after stroke (Hu et al., 2023).Previous studies have demonstrated its beneficial effects in alleviating cognitive impairment and enhancing neurological recovery in stroke patients (Zhou et al., 2020;Li et al., 2022).In a study on acupuncture for brain-related cognitive issues, researchers targeted the bilateral ''Fengchi" (GB20) points believed to boost mental function and cognition (L.Huang et al., 2021a).Zhang et al. found that EA at the ''Fengfu" (GV16) point lowered levels of specific proteins linked to cell death, protecting brain cells and improving learning and memory in mice with Alzheimer's-like symptoms (Zhang et al., 2020).Yang et al. indicated that early electroacupuncture at ''Dazhui" (GV14) improved brain health and cognitive function in aging-prone SAMP8 mice (Yang et al., 2020).Consequently, we chose ''Fengchi" (GB20), ''Fengfu" (GV16), and ''Dazhui" (GV14) points for administering EA on PSCI to assess its therapeutic effectiveness.
EA is suggested to influence autophagy, providing potential therapeutic implications for various diseases.A previous study indicates that EA pretreatment may protect the brain from ischemia/reperfusion injury by inhibiting autophagy (Mei et al., 2020).Notably, EA modulates autophagy through activation of the mTOR signaling pathway in cerebral ischemia/reperfusion injury (Huang et al., 2019).Nonetheless, it remains to be seen whether EA mitigates PSCI by modulating autophagy.
In summary, this study aims to explore the potential neuroprotective effects of EA on PSCI, particularly focusing on its ability to modulate the miR-135a-5p/ mTOR/NLRP3 axis-mediated autophagy.Using the rat middle cerebral artery occlusion (MCAO) model, we investigate the mechanisms by which EA influences autophagy and cognitive functions, potentially contributing to the development of novel therapeutic approaches for PSCI.

EXPERIMENTAL PROCEDURES Experimental animals and groups
A cohort of 42 male Sprague-Dawley rats, aged 10-12 weeks and weighing 250-280 g, was obtained from the College of Veterinary Medicine, Yangzhou University (Institute of Comparative Medicine) for this study.The rats were housed under controlled conditions at a temperature of 25 ± 2 °C with a 12-h light/dark cycle and had free access to both food and water.An acclimatization period of 7 days was allotted before beginning the experiments.
We established the MCAO model in 36 randomly selected rats.These rats were anesthetized with isoflurane (5% for induction, and 0.5% for maintenance).The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were subsequently exposed and isolated.After ligating the CCA and ECA, a 4-0 monofilament nylon suture, marked at 20 mm from its tip, was inserted into the Middle Cerebral Artery (MCA) to obstruct the blood flow.The insertion was halted when the marked point reached the entry site, ensuring the suture was positioned at a depth of 18.0 ± 0.5 mm.This was accompanied by a slight resistance, indicating the correct depth for occluding the artery.Following 2 h of ischemia, the suture was gently retracted to allow reperfusion.In the sham group, the same anesthesia procedure was performed on six rats, including the exposure and isolation of the CCA, ECA, and ICA, but without suture insertion.All other procedures were identical to the MCAO model.Rats subjected to MCAO were then divided randomly into six distinct groups: MCAO, MCAO + RAPA (rapamycin, an inducer of autophagy), MCAO + EA, MCAO + RAPA + EA, MCAO + EA + miR-NC, and MCAO + EA + miR-135 a-5p-antagomir, with each group comprising six rats.
Rats in the MCAO + RAPA group were given intraperitoneal injections of 3 mg/kg RAPA for three consecutive days before MCAO surgery.In the MCAO + EA group, EA treatment commenced 2 h following the MCAO procedure.Acupuncture needles, measuring 0.3 mm in diameter, were inserted to a depth of 2-3 mm into the Fengchi, Fengfu, and Dazhui acupoints on the head, angled at 45°.The ''Fengchi" (GB20) point is located at the nape, below the occipital bone, level with ''Fengfu", nestled in the depression between the upper part of the trapezius muscle and the sternocleidomastoid (Du et al., 2020).The ''Fengfu" (GV16) point resides on the dorsal midline within the Du Mai (Governor Vessel), situated in the indentation between the occipital bone and the first cervical vertebra (atlas) (Kwon et al., 2016).The ''Dazhui" (GV14) is located on the Governor Vessel, right below the seventh neck vertebra's protrusion, serving as a junction for various Yang pathways (Kwon et al., 2016).Stimulation was set to parameters that included an expanding wave frequency of 1-20 Hz (calibrated to the muscle twitching threshold), a peak voltage of 6 V, and an intensity set at 2 mA.The procedure was carried out for 30 min daily over a continuous 8-day period.In the MCAO + RAPA + EA group, rats were pre-treated with intraperitoneal injections of 3 mg/kg RAPA for three days before MCAO surgery.Two hours post-MCAO surgery, EA treatment was commenced, delivering combined electrical acupuncture stimulation to the rats for 30 min daily over a continuous 8-day period.For the MCAO + EA + miR-NC and MCAO + EA + miR-135a-5p-antagomir groups, MCAO rats received EA intervention for 30 min daily for a continuous period of eight days.Four hours post-MCAO model construction, the animals were stereotactically injected with corresponding reagents into the paretic side of the brain based on the groupings.Rats were anesthetized using an intraperitoneal injection of pentobarbital sodium (40 mg/kg).Subsequently, an incision approximately 8 mm in length was made along the sagittal plane, positioned at the midpoint of a line connecting the posterior margins of both ears.After identifying the bregma, a small hole, roughly 1 mm in diameter, was drilled into the skull 1 mm to the right and 1 mm posterior to it.Utilizing a 5 lL syringe, either the plasmid containing miR-NC or miR-135a-5p-antagomir (2.5 nmoL) was injected to a depth of about 3 mm at a consistent rate of 0.2 lL/min.After the injection, the needle was maintained in position for an additional 10 min before its removal.
The schematic representation of the animal experimental procedures is depicted in Fig. 1.Pathological assessments were performed after the water maze experiment.At the end of the study, rats were humanely euthanized with an intraperitoneal injection of 2% sodium pentobarbital at a dose of 40 mg/kg, after completing all sampling procedures.All animal experiments received approval from the Experimental Animal Ethics Committee of Yangzhou University (Approval No. 202212010).

Neurological deficit assessment (Zea-Longa)
The neurological deficits at 2 h, 1 day, 3 days, and 8 days after MCAO and RAPA/EA treatment in rats were assessed using the Zea-Longa neurological deficit scoring system (Li et al., 2023).This scale, with a total of five points, evaluates motor function, sensory perception, reflexes, and balance.A score of 0 is considered to represent normal function, with higher scores indicating increased severity of neurological injury.The Zea-Longa score is a 5-point scale used to evaluate the extent of neurological deficits following MCAO, with the following criteria: 0 points: No observable neurological deficit.Successful modeling of MCAO is typically confirmed when mice exhibit a score of 1-3 immediately after MCAO, implying that the procedure has induced reproducible neurological deficits.

Morris water maze (MWM) testing
The Morris water maze tests were conducted using a Flyde-A device with a stainless-steel tank, 150 cm in diameter and 60 cm high, heated to a steady 22-26 °C with precise temperature control.Rats were randomly introduced into the water with their faces oriented towards the pool wall, starting from one of the four cardinal directions (east, west, south, north).The time each rat took to locate the submerged platform was recorded in seconds.During initial training sessions, if a rat could not locate the platform within a 120-second timeframe, it was gently directed to it.Once on the platform, the rat was allowed to remain for 10 s.Subsequently, the rat was removed from the water, dried it with a towel, and, if needed, placed it under a 150 W incandescent lamp for a duration of 5 min prior to being returned to its housing cage.Each rat underwent this procedure four times daily, with 15-20min intervals between sessions, across five consecutive days.On day eight, subsequent to the conclusion of the final acquisition training session, the platform was subsequently removed.Rats were introduced into the pool from a location diametrically opposed to the quadrant previously occupied by the platform.The time taken and the number of times each rat crossed the previous platform location were documented.

2,3,5-Triphenyltetrazolium chloride (TTC) staining
The hippocampal CA1 region in the ischemic penumbra of brain tissues was extracted for histopathological examination.Prior to brain tissue extraction, rats underwent humane euthanasia with an intraperitoneal injection of 2% sodium pentobarbital, administered at a dose of 40 mg/kg.Immediately after removal, the brain tissue was frozen at À20 for a duration of 20 min.Cerebral infarction was examined using the TTC staining method.After the excision of the olfactory bulb, cerebellum, and lower brainstem, the tissue was sectioned coronally into five distinct slices.The brain was cut at various landmarks with each slice being approximately 2 mm thick.Slices were immersed in an opaque container containing 2% TTC stain (Solarbio, China).The container was covered with aluminum foil to preclude light exposure.The container was incubated at 37 in a water bath for 30 min, and we periodically inverted it for even staining.After staining, slices were gently rinsed with distilled water and subsequently fixed in 4% paraformaldehyde (Beyotime, China).The staining was observed and documented with photography.The cerebral infarct volume was measured using Image J software.

Hematoxylin and eosin (H&E) staining
For histopathological examination, we performed H&E staining using the Hematoxylin and Eosin Staining Kit (Beyotime, China).Specimens were initially preserved in 4% formaldehyde for a duration of 24 h.Subsequent to this fixation, samples were rinsed to eliminate any residual fixative.This was followed by a sequential dehydration process utilizing a graded series of ethanol concentrations: 50%, 70%, 85%, 95%, and culminating with absolute ethanol, with each step spanning 2 h.Transparency was achieved by two sequential 2-h xylene baths, followed by impregnation in a mixture of melted paraffin and xylene for 2 h, then in two separate paraffin baths for about 3 h each.Tissues were embedded in paraffin blocks and froze at À20 for at least 30 min prior to slicing.The sections, 4-7 mm thick, were cut using a microtome with a 5°angle between the knife blade and the tissue surface.Subsequently, sections were secured to slides and subjected to heating at a temperature of 65 for a duration of 30 min.The sections were then subjected to two rounds of deparaffinization in xylene, each lasting 15 min.For rehydration, a graded series of ethanol was employed, and the sections were stained with hematoxylin for 5 min.This was followed by differentiation using ammonia water and a thorough rinse in tap water for a span of 15 min.Eosin staining was subsequently conducted for a period of 2 min, which was then followed by dehydration in absolute ethanol.After dehydration, sections underwent two clearance processes in xylene, each lasting 3 min, and were ultimately mounted using neutral balsam.The sections were then baked at 65 for 15 min.Images of the relevant areas were captured and analyzed under a microscope (Olympus, Japan).

Analysis of inflammatory cytokines and oxidative stress markers
Blood samples were drawn via ocular extraction and left to coagulate at room temperature for 2 h.The coagulated blood was subjected to centrifugation at 3,000 rpm for a period of 10 min to obtain the serum.The levels of TNF-a, TGF-b, IL-6, and IL-10 were quantified with corresponding ELISA kits: Rat TNF-a ELISA Kit (Elabscience, Chian), TGF-beta 1 ELISA Kit (R & D Systems, USA), Rat IL-6 ELISA Kit (Elabscience), and Rat IL-10 ELISA Kit (Elabscience).Superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels were determined using the Total Superoxide Dismutase (T-SOD) assay kit (Hydroxylamine method) and the MDA assay kit (TBA method), respectively, both from Nanjing Jiancheng Bioengineering Institute, China.

Transmission electron microscopy (TEM)
The degree of mitochondrial damage in brain tissues was assessed using TEM.Brain tissue samples (1 mm 3 ) from each rat group were fixed in 2.5% glutaraldehyde for 48 h and rinsed six times with phosphate-buffered saline (PBS; Beyotime, China).The tissues were further fixed with 1% osmium tetroxide and rinsed again.Room temperature dehydration was performed using ascending acetone concentrations, with subsequent infiltration and embedding in EPON812.After baking, the hardened blocks were trimmed and sectioned using an ultramicrotome (Leica Biosystems RM2245, Germany).Semi-thin sections underwent staining with toluidine blue, while ultra-thin sections were procured from delineated regions.Selected areas were sectioned into ultra-thin slices, placed on copper grids with support films, and stained with uranyl acetate and lead citrate.The prepared samples were finally imaged using TEM (JEM-1400Flash Electron Microscope, USA) at 80 kV.

Dual-luciferase reporter assay
HEK293T cells were propagated in MEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Ephraim, China) and 1% penicillin-streptomycin (Hyclone, USA), maintaining the conditions at 37 °C with 5% CO 2 .For the execution of the dual-luciferase reporter assay, as guided by the Pierce TM Cypridina-Firefly Luciferase Dual Assay Kit (Thermo, USA), the cells underwent co-transfection with plasmids harboring either the wild-type (WT) or mutant (MUT) mTOR 3 0 UTR in conjunction with the miR-135a-5p mimic or a negative control (NC).After 48 h, cells were lysed and processed for luciferase activity measurement.

Statistical analysis
All experiments were conducted at least three times, with data expressed as mean ± SD.In the box plots, the horizontal lines inside the boxes indicate the mean values, while the whiskers extend to data points outside the 25th-75th percentile range.Group differences were assessed using one-way analysis of variance (ANOVA).Post-ANOVA, Tukey's test was employed for pairwise comparisons to identify significant differences between individual groups.The statistical analyses were conducted using GraphPad Prism 7.0, and a Pvalue less than 0.05 was considered statistically significant.

EA alleviates cognitive impairment and inflammation of MCAO rats
To investigate the impact of EA on autophagy and cognitive impairment following stroke, we established a MCAO rat model and subsequently treated it with RAPA (an activator of autophagy) or/and EA.We evaluated the neurological deficit scores at 2 h, 1 day, 3 days, and 8 days after MCAO and RAPA/EA treatment in rats.Rats with MCAO displayed significantly higher neurological deficit scores than sham-operated rats (P < 0.01).There was no significant difference between MCAO and RAPA/EA treatment groups at the 2 h, 1st day, and 3rd day.EA treatment significantly lowered these scores in MCAO rats at the 8th day (P < 0.01; Fig. 2(A)).We also performed the Morris water maze (MWM) test to assess spatial learning and memory.The MWM test showed a significant increase in escape latency for MCAO rats (P < 0.01).However, EA treatment markedly reduced the latency to escape in MCAO rats (P < 0.01).Furthermore, EA-treated MCAO rats crossed the platform significantly more often than their untreated counterparts (P < 0.01; Fig. 2(B)).In addition, TTC staining demonstrated that the MCAO rats exhibited a significantly higher infarct volume of 44.1 ± 2.49% compared to the sham group (P < 0.01).However, EA treatment significantly reduced the cerebral infarct volume in the MCAO rats to 10.21 ± 1.21% (P < 0.01; Fig. 3(A)).H&E staining of the hippocampus revealed significant tissue damage in MCAO rats as compared to sham rats, manifested as disorganized hippocampal neurons, edema, increased intercellular spaces, vacuolar degeneration, nuclear condensation, and dissolution, as well as extensive  cellular necrosis.EA administration reduced this tissue damage with a well-organized arrangement of hippocampal neurons (Fig. 3(B)).Furthermore, ELISA quantified the serum levels of pro-inflammatory cytokines (TNF-a and IL-6), anti-inflammatory cytokines (TGF-b and IL-10), and oxidative stress markers (SOD and MDA) in the rats.As shown in Fig. 3(C), TNF-a, IL-6, and MDA levels significantly increased, while IL-10 and SOD levels decreased in MCAO rats compared to sham rats (P < 0.01).EA treatment alleviated the elevated inflammation and oxidative stress in MCAO rats (P < 0.05; Fig. 3(C)).

EA inhibits autophagy in MCAO rats
Furthermore, we investigated the role of autophagy in cognitive impairment mitigation after stroke with EA intervention.Initially, we examined the presence of autophagosomes in the cerebral cortex of rats using TEM.The results revealed a significant increase in the number of autophagosomes in the cerebral cortex of MCAO rats.This number decreased notably post-EA treatment (Fig. 4(A)).Additionally, western blotting analysis was conducted to evaluate the expression levels of several autophagy-related proteins, including LC3-II/I, caspase-3, Beclin1, Bax, Bcl-2, p62, mTOR, and NLRP3, in the brain tissues of rats.The results demonstrated significantly higher expression levels of LC3-II/I, caspase-3, Beclin1, Bax, and NLRP3 in MCAO rats compared to the sham group, while Bcl-2, p62, and mTOR expression levels were significantly lower (P < 0.01).EA treatment significantly alleviated autophagy markers in MCAO rats (P < 0.05; Fig. 4(B)).
Notably, RAPA treatment had a detrimental effect of EA by exacerbating cognitive impairment and promoting autophagy in MCAO rats (P < 0.05).In addition, the addition of RAPA reversed the protective effects of EA on MCAO rats (P < 0.05; Figs.2-4).These findings imply that EA mitigates cognitive impairment after stroke by inhibiting autophagy.

EA inhibits inflammation by targeting miR-135a-5p in MCAO rats
MiRNAs have garnered considerable attention as potential biomarkers for diagnosis, prognosis, and brain injury assessment in ischemic stroke.RT-qPCR analysis showed that miR-135a-5p was significantly downregulated in MCAO rats compared to controls (P < 0.01).EA reversed this trend, increasing miR-135a-5p levels, although co-treatment with RAPA diminished this effect (P < 0.05; Fig. 5(A)).These results suggest that EA may suppress autophagy in MCAO rats, potentially via the upregulation of miR-135a-5p expression.Confirming this, MCAO rats treated with a miR-135a-5p-antagomir experienced an increased infarct size when subjected to EA (P < 0.01; Fig. 5(B)).H&E staining showed that miR-135a-5pantagomir negated the protective effect of EA on tissue damage in post-stroke hippocampus (Fig. 5(C)).Moreover, as depicted in Fig. 5(D), miR-135a-5pantagomir treatment reversed the inhibitory effect of EA on the levels of TNF-a, IL-6, and IL-10, while also attenuating the stimulatory effect of EA on TGF-b levels in MCAO rats (P < 0.01).

DISCUSSION
Autophagy is a vital cellular process responsible for the degradation and recycling of damaged or unnecessary proteins and organelles to maintain cellular homeostasis (Levine & Kroemer, 2019).Dysfunctional autophagy has been implicated in numerous pathological conditions, including neurodegenerative diseases and stroke (Liu et al., 2019;Ajoolabady et al., 2021).Emerging studies indicate a link between autophagy and PSCI, a common and debilitating sequela of stroke (Ding et al., 2022).The mTOR pathway, a key regulator of autophagy, has been implicated in the pathophysiology of PSCI.A previous study suggests that hyperactive mTOR signaling and the consequent inhibition of autophagy contribute to cognitive deficits following stroke (Tang et al., 2013).Furthermore, inflammation plays a critical role in PSCI, with pro-inflammatory cytokines such as IL-1b and TNF-a intensifying neuronal damage (Kim et al., 2020).Emerging evidence suggests a complex interplay between autophagy and inflammation in stroke outcomes (Mo et al., 2020).The NLRP3 inflammasome, an intracellular multiprotein complex responsible to produce proinflammatory cytokines, is regulated by autophagy.Dysregulated autophagy can result in excessive NLRP3 inflammasome activation, leading to an inflammatory response that aggravates ischemic injury and exacerbates cognitive deficits (Heneka et al., 2013).Therefore, the mTOR/NLRP3-mediated autophagy axis may offer a potential therapeutic target for PSCI.
In this study, we demonstrated that EA, a variation of traditional acupuncture therapy, mitigates cognitive impairment and inflammation in MCAO rat model.Crucially, we revealed that the mechanism of EA treating PSCI by involving the suppression of mTOR/ NLRP3-mediated autophagy.EA treatment promotes the protein expression of p62 and mTOR and inhibits the expression of NLRP3.These protein expression alterations correlate with the inhibition of autophagy.The induction of autophagy exacerbated neurological deficit scores, cerebral infarction, and inflammatory factor levels in MCAO rats.Also, autophagy induction negated the beneficial effects of EA on cognitive impairment and neuroinflammation after stroke.
EA therapy, a form of traditional Chinese medicine physiotherapy, has been shown to attenuate the inflammatory response (Zhang et al., 2022).Prior studies have indicated that autophagy serves as a therapeutic target for electroacupuncture treatment against ischemic cerebrovascular diseases (Huang et al., 2019).Notably, EA treatment has been demonstrated to offer neuroprotection against cerebral ischemia/reperfusion injury by suppressing autophagy (Mei et al., 2020).In this study, we corroborated the cognitive enhancement imparted by EA in a MCAO rat model, achieved through the suppression of mTOR/NLRP3-mediated autophagy.
Recent research has highlighted miRNAs as novel and potent regulators of autophagy (Li et al., 2019).MiR-135a-5p, a microRNA expressed across diverse cell types and tissues, regulates critical biological processes such as autophagy (Qin et al., 2022).Research has revealed a dichotomous role for miR-135a-5p in autophagy; upregulation inhibits, while downregulation promotes this process (Qin et al., 2022).Specifically, miR-135a-5p can modulate the activity of autophagy by targeting and regulating the expression of autophagyrelated genes and signaling pathways.For instance, miR-135a-5p influences autophagy through the modulation of the mTOR signaling pathway, a crucial inhibitor of autophagy initiation (Qin et al., 2022).A study has indicated that miR-135a-5p overexpression could improve the cerebral hypoxia/reoxygenation injury (Chen & Li, 2019).Our study provides novel insights, revealing a significant downregulation of miR-135a-5p expression in MCAO rats, which EA treatment effectively reversed.Moreover, we found that miR-135a-5p-antagomir addition reversed the inhibitory effects of EA on cognitive impairment, inflammation, and autophagy in MCAO rats.These results strongly imply that miR-135a-5p functions as an upstream regulator of autophagy under the influence of EA treatment in the context of PSCI.Noteworthily, our data showed a direct interaction between miR-135a-5p and mTOR, indicating that miR-135a-5p regulates mTOR/NLRP3-mediated autophagy in PSCI.Therefore, we conclude that EA alleviates post-stroke cognitive impairment through inhibiting miR-135a-5p/mTOR/ NLRP3 axis-mediated autophagy.
The current study revealed that EA treatment significantly mitigated cognitive impairment, inflammation, and oxidative stress in MCAO rats, underscoring its neuroprotective effects.Our results further revealed that the neuroprotective effects of EA were mediated through the inhibition of the miR-135a-5p/mTOR/NLRP3 axis-induced autophagy.The study highlights the potential of EA as a non-invasive therapeutic approach for PSCI through the regulation of autophagy.The present study has some limitations, including the use of an animal model, which calls for cautious interpretation when extrapolating the findings to human subjects.To fully elucidate the underlying mechanisms and to validate the therapeutic efficacy of EA in clinical settings, more comprehensive and indepth studies involving human subjects are warranted.
1 point: Failure to extend left paw fully, indicating a mild focal neurological deficit. 2 points: Circling to the left, suggesting a moderate focal deficit.3 points: Falling to the left, denoting a severe focal deficit.4 points: No spontaneous walking with a depressed level of consciousness.

Fig. 2 .
Fig. 2. EA alleviates neurological deficits and enhances spatial learning and memory in MCAO rats.(A) The neurological deficit scores were evaluated at 2 h, 1 day, 3 days, and 8 days after MCAO and RAPA/EA treatment in rats.(B) The time taken to cross the platform and the escape latency of rats in the MWM task.**P < 0.01.EA: electroacupuncture; MCAO: middle cerebral artery occlusion; RAPA: rapamycin; MWM: morris water maze.