Laboratory animals

A total of 240 specific pathogen-free male Sprague-Dawley (SD) rats (8–10 weeks old, 250 ± 30 g) were obtained from Jinan Pengyue Animal Breeding Co., Ltd. The rats were kept under specific conditions (23 ±2°C, 55 ±5% humidity) with a 12-hour light/dark cycle and had unrestricted access to food and water. In order to minimize experimental bias and ensure the scientific validity and reliability of the results, all rats were assigned to different experimental groups or control groups according to the needs of the study by third-party staff unrelated to the subsequent operations using the random number table method prior to the experiment. At the same time, in order to prevent subjective interference and information bias, the experimental operators and data collectors were kept in ignorance of the information on grouping until after all operations and data recording had been completed, and then the grouping was blinded and statistically analyzed by a third party. All animal experiments were conducted in accordance with the National Institutes of Health (NIH) guidelines and were approved by the Animal Care and Use Committee of our Hospital (Approval Number: SDTHEC).

Rat MCAO/R model

An MCAO/R rat model was established using a surgical procedure as described previously [27]. Briefly, rats were anesthetized via intraperitoneal injection of 3% (w/v) pentobarbital sodium (40 mg/kg). The left internal carotid artery was exposed, and a 4/0 surgical nylon monofilament was inserted to occlude blood flow in the middle cerebral artery, internal carotid artery, anterior cerebral artery, and posterior cerebral artery. After 2 h of occlusion, the filament was withdrawn to allow reperfusion for 24 h. Body temperature was maintained at approximately 37°C throughout the procedure. No instances of post-anesthesia peritonitis or intestinal obstruction were observed. Rats in the sham group underwent the same surgical procedure without filament insertion.

Rat treatments

SD rats were randomly assigned to six groups (n = 6 per group), with PPF dosing adjusted based on body weight [28]:

Figure 1

PPF attenuates oxidative stress injury in MCAO/R rats. A – Chemical structure diagram of PPF. Effects of PPF on neurological function (B), cerebral edema (C), infarction area (D). n = 6; *p < 0.05 Cerebral cortex histopathology (E), morphology of cerebral cortical neurons (F), apoptosis of rat cerebral cortex (G). n = 6; *p < 0.05 Inflammatory indicators (H, I), and oxidative stress indices (J–L). n = 6; *p < 0.05

Additional groups included combinations of PPF with antagomirs and lentiviral vectors:

Using a microsyringe, 50 µl of lentivirus was slowly injected into the right cerebral cortex of the rats. AgomiR-NC, agomiR-miR-6838-5p, lentiviral vectors for AQP11 (LV-AQP11), and non-targeting plasmid (LV-NC) were obtained from RIBO-BIO (Guangzhou, China). From a total of 65 rats, 59 were treated with MCAO/R, and 6 underwent sham operations. The study excluded five rats that died during the MCAO/R treatment and intraperitoneal injection. No mortality was observed in the sham group. Following the modeling, rats were euthanized by cervical dislocation, and brain tissues were extracted.

Neurological deficit score

At 24 h after reperfusion, neurological deficits were evaluated using a standardized scoring system: 0 = no deficit; 1 = failure to fully extend the contralateral forepaw; 2 = incomplete forepaw extension; 3 = mild contralateral rotation; 4 = vigorous contralateral rotation; 5 = falling to the contralateral side. All assessments were conducted by a blinded investigator [31].

Measurement of brain edema

The measurement of cerebral edema was conducted by assessing the water content in the brain. To determine the wet weight (WW), the cerebral hemispheres were weighed and then dried at 105°C for 24 h to obtain the dry weight (DW). The formula for determining brain water content was: Brain water content = (WW – DW)/WW × 100% [32].

2,3,5-Triphenyl tetrazolium chloride (TTC) staining

Brain tissues were incubated on ice for 24 h, sectioned into continuous coronal slices, and placed in 2% TTC staining solution at 37°C for 20 min. TTC staining was halted by washing with phosphate-buffered saline (PBS), and tissues were fixed in 4% paraformaldehyde for 2 h. Infarcted regions appeared unstained and were quantified as infarct volume relative to total brain volume using Image Pro Plus 6.0 software. Infarct volume (%) was calculated as the total infarct volume divided by the total brain volume, multiplied by 100%.

Hematoxylin and eosin (HE) staining

Rat brain tissues were fixed in 4% paraformaldehyde (Sigma-Aldrich, Merck KGaA), and subsequently hydrated and permeabilized. Tissues were then embedded in paraffin, sectioned into 4 µm slices, and subjected to HE staining. Each slice was examined under a light microscope at 200 × magnification in three fields.

Nissl staining

Paraffin-embedded brain sections were dehydrated and stained with cresyl violet solution for 1 h. Following staining, slices were rinsed sequentially with 70%, 80%, and 95% ethanol (10 s each), dehydrated with anhydrous ethanol (5 min per step, twice), cleared with xylene (5 min each step, twice), mounted with neutral gum, and observed under a microscope (Nikon).

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining

TUNEL staining was performed using a TUNEL kit (Qiming Bio Engineering Co., Ltd., Shanghai, China; OX02752). Sections of rat brain tissue underwent fixation using 4% paraformaldehyde for 20 min, were permeabilized using 0.1% Triton X-100 for 5 min, and then subjected to incubation with a TUNEL reaction blend (in darkness at 37°C for 1.5 h). The samples underwent counterstaining using 4,6-diamidino-2-phenylindole (DAPI). Apoptotic cells testing positive for TUNEL were measured using fluorescence microscopy [33].

Enzyme-linked immunosorbent assay (ELISA)

Rat brain tissues were homogenized in PBS on ice and centrifuged at 3500 rpm for 15 min. The supernatant was collected, and levels of tumor necrosis factor-α (TNF-α; orb79138, Biorbyt) and interleukin-1β (IL-1β; orb79117, Biorbyt) were measured using respective ELISA kits. Absorbance was read at 450 nm using a microplate reader (RT-6100, LeiDu).

Determination of oxidative stress indicators

Malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) in rat brain tissues and PC12 cells were quantified using specific detection kits (JK-(a)-2197, JK-(a)-2396, JK-(a)-2293; Jingkang Bioengineering Co., Ltd., Shanghai, China).

Cell culture

PC12 cells, sourced from the China Center for Type Culture Collection in Wuhan, China, were grown in Dulbecco’s Modified Eagle Medium (DMEM), enriched with 10% fetal bovine serum (Beyotime, Shanghai, China), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Invitrogen), in an environment at 37°C and 5% CO₂. To create the H/R injury model, PC12 cells were categorized into three distinct groups:

Cell viability

PC12 cells (2 × 10³ cells/well) were seeded into 96-well plates and incubated at 37°C in 5% CO₂ for 72 h. Cell viability was assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay kit (Sigma-Aldrich). Briefly, 20 µl of MTT solution was added to each well and incubated at 37°C for 4 h. Subsequently, 150 µl of dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to dissolve the formazan crystals. Optical density was measured at 570 nm using a Bio-Tek microplate reader (Hopkinton, MA, USA).

Cell apoptosis

The measurement of apoptosis was conducted with the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit (Sangon Biotech, Shanghai, China). PC12 cells, totaling 5 × 10⁵ cells per well, were cultured in 6-well plates and subjected to a 5-minute incubation with 5 µl of Annexin V-FITC and 10 µl of PI at ambient temperature in darkness. Flow cytometry was performed using a FACS Verse flow cytometer (BD Biosciences), and the data were examined with FlowJo software (version 10; TreeStar) [35].

Quantitative analysis of mRNA expression

RNA was extracted from brain tissues and PC12 cells using Trizol reagent (Invitrogen). For miRNA analysis, cDNA was synthesized using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) with specific stem-loop RT primers. For mRNA analysis, cDNA was synthesized using the PrimeScript RT kit (Takara). Expression levels of miR-6838-5p and AQP11 were quantified using SYBR Green Real-Time PCR Master Mix (Takara) on a 7500 Real-Time PCR System (Applied Biosystems). The internal control for AQP11 was β-actin, and miR-6838-5p was referenced using U6. Primer sequences are listed in Table I.

Quantitative analysis of protein expression

Proteins were harvested from rat brain mixtures using a radioimmunoprecipitation assay lysis buffer (Beyotime). Protein concentrations were measured with a bicinchoninic acid protein assay kit. The proteins were denatured by boiling, separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a polyvinylidene fluoride membrane, which was blocked using 5% skim milk and subjected to overnight incubation at 4°C with primary antibodies targeting AQP11 (1 : 100; ab122821, Abcam), AQP1 (1 : 1000; AB2219, MilliporeSigma), AQP4 (1 : 1000; AB3594, MilliporeSigma), AQP9 (1 : 1000; sc-74409, Santa Cruz Biotechnology), and β-actin (1 : 1000; ab8277, Abcam). The membrane was incubated with a horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (1 : 10,000; AB175781, Abcam) at ambient temperature for an 1 h. Protein bands were visualized using an enhanced chemiluminescence agent. Bands intensities were measured with the ImagePro Plus 6.0 software.

Luciferase reporter gene assay

TargetScan software predicted that miR-6838-5p targets AQP11. Wild-type (AQP11-WT) and mutant (AQP11-MUT) fragments containing potential miR-6838-5p binding sites were cloned into the pMIR-REPORT plasmid vector (Promega). The luciferase reporter constructs were co-transfected with miR-6838-5p mimic into PC12 cells using Lipofectamine 2000 reagent (Invitrogen). After 24 h, luciferase activity was measured using a luciferase assay kit (Promega) [36].

RNA immunoprecipitation (RIP) assay

RIP assays were conducted using a RIP kit (Millipore). Cells were lysed with RIP lysis buffer, and the lysate was incubated with RIP buffer containing human anti-AGO2-conjugated magnetic beads. The immunoprecipitated complexes were treated with protease K to digest proteins, and the associated RNA was isolated. The presence of miR-6838-5p and AQP11 in the precipitated RNA was determined by RT-qPCR [37].

Statistical analysis

Data were analyzed using SPSS version 21.0 software. Normality was assessed using the Kolmogorov-Smirnov test. Results are presented as mean ± standard deviation. For comparisons involving multiple groups, one-way analysis of variance followed by the least significant difference post hoc test was used. Categorical data (rates or percentages) were compared using the χ² test. All statistical analyses were two-tailed, with a P-value under 0.05 deemed statistically significant.

PPF mitigates oxidative stress injury in MCAO/R rats

To investigate the role of PPF in CI/RI, an MCAO/R rat model was established, and PPF treatment (10 mg/kg) was administered. Previous studies have determined the LD₅₀ of PPF in rats to be 22.6 mg/kg, which is significantly higher than the 10 mg/kg used in this study, confirming its safety [38]. Neurological function scores revealed that MCAO/R rats exhibited severe neuronal damage, which was alleviated by PPF treatment (Figure 1 B, p < 0.05). Cerebral edema was assessed by measuring brain water content, which was elevated in MCAO/R rats but significantly reduced following PPF administration (Figure 1 C, p < 0.05). TTC staining demonstrated substantial infarct areas in MCAO/R rats, whereas PPF treatment resulted in a notable reduction in infarct size (Figure 1 D, p < 0.05). HE staining indicated that sham-operated rats had clear and intact tissue structures with dense cell distribution, normal nuclear morphology, and no evidence of tissue hydrolase release. In contrast, MCAO/R rats exhibited loose cell arrangement, neuronal degeneration and necrosis, nuclear shrinkage, obscured nucleoli, and large intercellular voids. PPF treatment significantly attenuated cerebral cortical injury in MCAO/R rats (Figure 1 E). Nissl staining revealed severe neuronal loss in MCAO/R rats, which was markedly reduced by PPF treatment (Figure 1 F, p < 0.05). TUNEL staining showed a significant increase in apoptotic cells in the cerebral cortex of MCAO/R rats, which was reduced by PPF treatment (Figure 1 G, p < 0.05). Inflammatory and oxidative stress markers, which are central to CI/RI pathophysiology, were elevated in MCAO/R rats. PPF intervention resulted in decreased TNF-α, IL-1β, and MDA, while promoting GSH-Px and SOD activities (Figures 1 H–L, p < 0.05).

miR-6838-5p enhances anti-inflammatory and antioxidant responses in MCAO/R rats

In the MCAO/R model, the expression of miR-6838-5p was significantly downregulated, as demonstrated by quantitative RT-PCR analysis (Figure 2 A, p < 0.05). To investigate the functional role of miR-6838-5p in this ischemic injury model, we employed lentiviral vectors to overexpress miR-6838-5p in MCAO/R rats prior to the induction of ischemia–reperfusion. Successful overexpression was confirmed by a substantial increase in post-injection miR-6838-5p levels (Figure 2 B, p < 0.05). Histological examination revealed that miR-6838-5p overexpression markedly mitigated neuronal damage, as evidenced by reduced neuronal loss and structural preservation in brain tissue sections (Figure 2 C, p < 0.05). Additionally, rats with elevated miR-6838-5p exhibited a significant decrease in brain water content, indicating reduced cerebral edema (Figure 2 D, p < 0.05), and a notable reduction in infarct size, assessed through infarct volume measurements (Figure 2 E, p < 0.05). Further pathological assessment using HE staining demonstrated that miR-6838-5p overexpression led to improved brain tissue morphology, with fewer signs of necrosis and inflammation compared to control groups (Figure 2 F). Apoptotic analysis via TUNEL staining revealed a significant decrease in neuronal apoptosis in the miR-6838-5p overexpressing group (Figures 2 G, H, p < 0.05). At the molecular level, overexpression of miR-6838-5p was associated with suppressed inflammatory responses, as indicated by reduced TNF-α and IL-1β (Figures 2 I, J, p < 0.05). Concurrently, markers of oxidative stress were ameliorated; MDA levels were significantly decreased (Figure 2 K, p < 0.05), while the activities of GSH-Px and SOD were markedly increased (Figures 2 L, M, p < 0.05). Collectively, these results suggest that miR-6838-5p plays a pivotal role in enhancing anti-inflammatory and antioxidant defenses, thereby conferring neuroprotection in MCAO/R-induced ischemic injury.

Figure 2

miR-6838-5p inhibits inflammation and oxidative stress in MCAO/R rats. A – CAO/R-induced change in miR-6838-5p expression in rats. Effects of miR-6838-5p lentivirus on miR-6838-5p expression in rat brain tissue (B), neurological function (C), cerebral edema (D), infarction area (E). n = 6; *p < 0.05 Cerebral cortex histopathology (F), morphology of cerebral cortical neurons (G), apoptosis of rat cerebral cortex (H). n = 6; *p < 0.05 Inflammatory indicators (I, J), and oxidative stress indices (K–M). n = 6; *p < 0.05

Inhibition of miR-6838-5p diminishes the therapeutic efficacy of PPF in MCAO/R rats

Treatment with PPF was found to restore the suppressed expression of miR-6838-5p in MCAO/R rats, as evidenced by increased miR-6838-5p levels after PPF administration (Figure 3 A, p < 0.05). To delineate the interaction between PPF and miR-6838-5p, a miR-6838-5p antagonist was administered alongside PPF treatment to effectively inhibit miR-6838-5p expression, which was confirmed by RT-qPCR (Figure 3 B, p < 0.05). The inhibition of miR-6838-5p significantly abrogated the neuroprotective effects of PPF in MCAO/R rats. Specifically, neuronal injury scores were elevated (Figure 3 C, p < 0.05), indicating increased neuronal damage. Additionally, brain water content was significantly higher in the miR-6838-5p-inhibited group, suggesting exacerbated cerebral edema (Figure 3 D, p < 0.05). Infarct size measurements revealed an enlargement of the infarct area upon miR-6838-5p inhibition (Figure 3 E, p < 0.05). Histopathological analysis using HE staining showed aggravated brain tissue pathology, characterized by increased necrosis and inflammation (Figure 3 F). TUNEL assays further confirmed an increase in neuronal apoptosis in the miR-6838-5p-inhibited group (Figures 3 G, H, p < 0.05). At the molecular level, inhibition of miR-6838-5p led to elevated TNF-α and IL-1β (Figures 3 I, J, p < 0.05), as well as increased MDA levels (Figure 3 K, p < 0.05), indicating heightened oxidative stress. Conversely, the activities of antioxidant enzymes GSH-Px and SOD were significantly suppressed (Figures 3 L, M, p < 0.05). These findings collectively demonstrate that miR-6838-5p is indispensable for the therapeutic efficacy of PPF, as its inhibition negates the anti-inflammatory and antioxidant benefits conferred by PPF treatment in MCAO/R-induced ischemic injury.

Figure 3

Inhibiting miR-6838-5p reverses PPF treatment efficacy in MCAO/R rats. A – Effects of PPF on miR-6838-5p expression in rat brain tissue. Effect of combined miR-6838-5p antagonist + PPF on miR-6838-5p expression in rat brain tissue (B), neurological function (C), cerebral edema (D), infarction area (E). n = 6; *p < 0.05 Cerebral cortex histopathology (F), morphology of cerebral cortical neurons (G), apoptosis of rat cerebral cortex (H). n = 6; *p < 0.05 Inflammatory indicators (I, J), and oxidative stress indices (K–M). n = 6; *p < 0.05

miR-6838-5p regulates AQP11 expression

We identified potential downstream targets using bioinformatics prediction tools (Figure 4 A). AQP11 emerged as a promising candidate target of miR-6838-5p. Dual luciferase reporter assays were conducted to validate this interaction. The results demonstrated that miR-6838-5p mimics significantly inhibited the luciferase activity of the AQP11-WT but had no effect on the AQP11-MUT, confirming a direct binding interaction between miR-6838-5p and the 3′ UTR of AQP11 mRNA (Figure 4 B, p < 0.05). Further validation was performed using RIP assays, which revealed that miR-6838-5p was significantly enriched in complexes containing AQP11, thereby corroborating the direct interaction between the two molecules (Figure 4 C, p < 0.05). Subsequent analysis of protein expression levels in brain tissues of MCAO/R rats overexpressing miR-6838-5p showed a significant reduction in AQP11 expression (Figure 4 D, p < 0.05). Importantly, the expression levels of other aquaporin family members, including AQP1, AQP4, and AQP9, remained unchanged, indicating the specificity of miR-6838-5p in targeting AQP11 (Figure 4 E, p < 0.05). These results collectively establish AQP11 as a direct and specific target of miR-6838-5p, suggesting that the regulatory axis miR-6838-5p/AQP11 plays a crucial role in mediating the anti-inflammatory and antioxidant responses in ischemic injury.

Figure 4

miR-6838-5p targets AQP11. A – Binding site of miR-6838-5p to AQP11. B, C – Confirmation of the binding relationship between miR-6838-5p and AQP11. D, E – Expression levels of AQP1/AQP4/AQP9/AQP11 in rat brain tissue overexpressing miR-6838-5p. *P < 0.05

Overexpression of AQP11 impairs the therapeutic effects of PPF in MCAO/R rats

In the MCAO/R rat model, both AQP11 and AQP4 expression levels were significantly elevated, as determined by RT-qPCR and Western blot analyses (Figures 5 A, B, p < 0.05). PPF treatment effectively reduced the expression of these aquaporins, suggesting a modulatory effect of PPF on their expression. To further investigate the specific role of AQP11 in mediating the therapeutic effects of PPF, AQP11 was overexpressed in MCAO/R rats prior to administering PPF, which was confirmed by RT-qPCR and Western blot (Figure 5 C, p < 0.05). Overexpression of AQP11 in MCAO/R rats undergoing PPF treatment resulted in a significant increase in neuronal injury scores, indicating exacerbated neuronal damage despite PPF administration (Figure 5 D, p < 0.05). Additionally, brain water content was markedly elevated in the AQP11-overexpressing group (Figure 5 E, p < 0.05), suggesting aggravated cerebral edema. Infarct size measurements revealed that AQP11 overexpression led to significantly larger infarct areas in PPF-treated MCAO/R rats (Figure 5 F, p < 0.05). Histopathological analysis using HE staining showed that AQP11 overexpression aggravated brain tissue damage, characterized by increased necrosis and inflammatory cell infiltration (Figure 5 G). TUNEL assays confirmed a significant increase in neuronal apoptosis in the AQP11-overexpressing group (Figures 5 H, I, p < 0.05). Elevating AQP11 elevated TNF-α and IL-1β (Figures 5 J, K, p < 0.05), as well as increasing MDA levels (Figure 5 L, p < 0.05). Concurrently, the activities of GSH-Px and SOD were significantly reduced (Figures 5 M, N, p < 0.05). These findings demonstrate that elevating AQP11 undermines the neuroprotective effects of PPF, thereby highlighting the critical role of the miR-6838-5p/AQP11 axis in mediating the therapeutic actions of PPF in MCAO/R-induced ischemic injury.

Figure 5

Overexpression of AQP11 reduces the therapeutic effect of PPF on MCAO/R rats. A, B – MCAO/R-induced change in AQP11 and AQP4 expression in rat brain tissue and effect of PPF on AQP11 and AQP4 expression in MCAO/R rats. Effect of combined lV-AQP11 + PPF on AQP11 expression in rat brain tissue (C). n = 6; *P < 0.05 Neurological function (D), cerebral edema (E), infarction area (F), cerebral cortex histopathology (G), morphology of cerebral cortical neurons (H), apoptosis of rat cerebral cortex (I), inflammatory indicators (J, K), and oxidative stress indices (L–N). n = 6; *P < 0.05 Apoptosis of rat cerebral cortex (I), inflammatory indicators (J, K), and oxidative stress indices (L–N). n = 6; *P < 0.05

PPF alleviates H/R injury in PC12 cells via the miR-6838-5p/AQP11 axis

To translate our in vivo findings to an in vitro context, we used a PC12 cell model subjected to H/R injury. In this model, miR-6838-5p expression was markedly decreased, while AQP11 expression was significantly increased, mirroring the in vivo MCAO/R findings (Figures 6 A–C, p < 0.05). Pretreatment with PPF effectively reversed these expression changes, resulting in elevated miR-6838-5p levels and reduced AQP11 expression (Figures 6 A–C, p < 0.05), suggesting that PPF modulates the miR-6838-5p/AQP11 axis in vitro. Functional assays further elucidated the protective effects of PPF in PC12 cells subjected to H/R injury. The MTT assay revealed that H/R significantly reduced cell viability (Figure 6 D, p < 0.05), whereas PPF treatment substantially enhanced cell viability, approaching baseline levels. Flow cytometry analysis demonstrated that H/R induced a significant increase in apoptotic cells (Figure 6 E, p < 0.05), which was effectively mitigated by PPF treatment, indicating its anti-apoptotic properties. Moreover, oxidative stress markers were assessed to evaluate the antioxidant effects of PPF. H/R resulted in a significant increase in MDA levels and a concomitant decrease in the activities of GSH-Px and SOD in PC12 cells (Figures 6 F–H, p < 0.05). PPF treatment effectively reversed these alterations by reducing MDA levels and restoring GSH-Px and SOD activities. These in vitro results corroborate the in vivo data, demonstrating that PPF alleviates H/R-induced cellular injury through the modulation of the miR-6838-5p/AQP11 axis, thereby exerting anti-inflammatory and antioxidant effects.

Figure 6

PPF attenuates H/R injury in PC12 cells by targeting AQP11 by upregulating miR-6838-5p. Effects of H/R and PPF on miR-6838-5p and AQP11 expression in cells (A–C), viability (D). n = 6, *p < 0.05 Apoptosis (E), and oxidative stress indicators (F–H). n = 6, *p < 0.05