ABSTRACT
Objectives: To explore the beneficial effects and mechanisms of Poloxamer 188 (P188) in mitigating cerebral ischemia-reperfusion (I/R) injury in mice.
Methods: This study was conducted from 2020 to 2022. Neurological function, brain water content, and infarct size were assessed in mice 24 h after I/R injury. Iridium-labeled Poloxamer 188 (Ir-P188) was characterized using 1H-NMR, UV-Vis spectroscopy, and fluorescence emission analysis. Immunofluorescence was used to evaluate intracellular distribution of Ir-P188 in OGD/R-induced HT22 cells in vitro and ischemic mice in vivo. 24 h after reperfusion, the levels of ROS and inflammation in ischemic brain were measured, along with the protein levels of mitochondrial, lysosomal, and cytoplasmic fractions. Additionally, the protective effects of p188 and Ginkgolide B, both as single agents and in combination, against I/R were compared.
Results: P188 intravenous administration could significantly reduce the infarct brain areas, improved neurological deficit, and decreased brain water content in mice after I/R injury. The accumulation of Ir-P188 was observed in OGD/R-induced HT22 cells and ischemic brain in mice. P188 suppressed ROS, inflammatory factors (NF-kB, IL-6, TNF-a), and inhibiting mitochondrial cytochrome C release and lysosomal protease translocation to the cytoplasm.
Conclusion: P188 can penetrate intracellular compartments and effectively protect mice against I/R injury. The underlying mechanism may involve inhibiting ROS generation, mitigating inflammatory responses, and alleviating mitochondrial dysfunction and lysosomal damage.
Stroke has a major impact on public health in every country. Ischemic strokes, which account for over 80% of all stroke events.1,2 Ischemia-reperfusion (I/R) injury occurs when blood flow is reperfused after a certain period of cerebral ischemia, exacerbating ischemic damage to brain tissue cells, mainly in the form of neural cell damage and apoptosis.
Reperfusion after ischemia leads to cellular alterations, such as organelle damage and the buildup of misfolded proteins, and particularly, obvious mitochondrial dysfunction occurs, including mitochondrial morphological damage, mitochondrial permeability-transition pore opening, Ca2+-induced mitochondrial swelling, and the release of mitochondrial Cytochrome C (Cyt C) into the cytoplasm.3,4 Targeted pharmacological interventions could potentially interrupt neurodegeneration pathways, offering promising therapeutic strategies. Yet, despite advancing scientific insights into neuronal damage mechanisms, effective clinical treatments remain challenging to develop.5
Poloxamer is a class of non-ionic polymer surfactants formed by polyoxyethylene and polyoxypropylene.6 It is mainly used as emulsifier and solubilizer in pharmaceutical formulations. Poloxamer 188 (P188) is a non-ionic, linear copolymer that is marketed under several trade names, including FLOCOR, PLURONIC F68, and RheothRx.7 This multifaceted molecule has received FDA approval for more than fifty years, principally employed as a therapeutic treatment to diminish blood viscosity during transfusions, due to its distinctive physical and chemical properties. Recent research has shown that P188 may offer neuroprotective effects against glutamate-induced damage in rat brains and mechanical stress. Animal studies also showed that P188 had protective effects on spinal cord compression, excitotoxic injury and cerebral hemorrhage.8 Moreover, P188 can increase local blood flow after cerebral ischemia in rabbits.9 In our previous research, we showed that P188 could mitigate I/R injury, with the mechanism involving the repair of the outer cell membrane and a decrease in blood-brain barrier (BBB) permeability.10 Here, we further examined the effects of P188 on ischemic injury and intracellular membranes following cerebral ischemia in mice. This was achieved by investigating the distribution of P188 within cells and assessing the release of contents from mitochondria and lysosomes.
Methods
Animals and cells
This study was conducted from 2020 to 2022. Male ICR mice (18-25 g, SPF) were obtained from Nantong University (License No. S20230615-012). All procedures involving animals were conducted following the institutional guidelines for animal care and use, with approval from the Ethical Committee of Nantong University. The HT22 cell line, a gift from the School of Pharmacy (Soochow University), was maintained in our laboratory.
Reagent
P188 and GB were purchased from Sigma-Aldrich (St. Louis, MO, USA). P188 labeled with iridium complex (Ir-P188) was synthesized by Suzhou Nakai Technology Co., Ltd. (Suzhou, Nanjing Province, China). TTC (F20060313) was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China), and diluted with PBS at a concentration of 1%, and stored in the dark. Cyt c (4272), Cytochrome c oxidase (COX, 4850), Cleaved Caspase-3 (9654), Phospho-NF-kB p65 (3033) and NF-kb (8242) were sourced from Cell Signaling Technology (CST, Beverly, MA, USA). Cathepsin (Cathepsin L, ab6314), lysosome associated membrane protein 2 (LAMP2, ab37024) were purchased from Abcam (Cambridge, MA, USA). Mitochondrial isolation kit (C3606) and ROS assay kit (S0033M) were purchased from Beyotime Biotechnology (Shanghai, China). ELISA kits (IL-6 and TNF-a) were purchased from R&D Systems Inc. (Minneapolis, MN, United States).
Experimental group design
MCAO model and brain tissue dissection
Experimental grouping: mice were categorized into three groups, 8 or 6 mice/group, named Sham, Saline, P188 group (0.4 g/kg). P188 or Saline was administered to mice intravenously (i.v.) via tail vein 5 min before reperfusion. The dose of P188 was chosen by previous studies.10
Mice were anesthetized intraperitoneally with a combined anesthesia (5 mL/kg). The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated under a stereo microscope. A nylon suture (6023, Doccol Corporation, Redlands, USA) was inserted into ICA via the ECA until the MCA. After 2 h, the suture was withdrawn to restore perfusion. Mice in the Sham group underwent similar surgery without the suture insertion. The room temperature was kept at 22~25°C during the whole operation. After the operation, animals were free to water and food.
The mice were sacrificed after 24 h reperfusion following 2 h MCAO. The mouse brain was removed and immediately incubated in ice-cold PBS for 3 min. Then, the brain was placed on a pre-cold glass board on wet ice. To avoid the tissue selection bias, the TTC staining area was referred as shown in Figure 1. Immediately freeze the brain tissue samples section “a” in dry ice and then stored at -80°C for the western blot experiment.
- Tissues for TCC staining and Western blotting.
Determination of cerebral infarct volume
After 24 h, the brains were frozen at -20°C, stained with 1% TTC at 37°C with occasional shaking, fixed in 4% paraformaldehyde after 20 min, and stored in the dark at 4°C for further analysis.11
Neurological deficit score
Neurological deficits were assessed 24 h post-I/R by a blinded observer using a standardized scoring system: 0=no deficit; 1=partial left forepaw extension impairment; 2=left-sided circling; 3=left-side falling; 4=reduced consciousness and spontaneous movement.12 Mice without immediate post-reperfusion behavioral deficits were excluded from the analysis.
Brain water content
Wet and dry weights of brain matter were measured 24 h after reperfusion. The method used to find the water content after drying at 105°C for 48 h was Water Content (%)=(wet weight - dried weight) / wet weight × 100%.
Western blot
After homogenizing brain tissues, the protein concentration was measured. Protein (20 µg) was used for SDS-PAGE to transfer the membranes with the power (1A, 1 h). After blocking, the nitrocellulose membrane was placed in a plastic bag and incubated with primary antibody (0.1 mL/cm²) overnight at 4°C. Transferred the nitrocellulose membranes into 1xTBST solution and washed for 3 times (15 min each time). The samples were incubated with secondary antibody for 2 h at room temperature. Visualization was performed using X-ray film (Hyperfilm ECL, GE Healthcare, USA), and the blots were analyzed with the Odyssey V3.0 imaging system (LI-COR Biosciences, Lincoln, NE, USA).
PI staining
24 h after reperfusion, 3 µL mixture of PI and Ir-P188 (0.25 µg PI dissolved in 1 µL 10% Ir-p188) was intracerebroventricularly (i.c.v) injected into the lateral ventricle of mice. After 30 min, brain samples were rapidly cooled to -80°C for 20 min. Thin 12 µm slices were prepared and sequentially washed with PBS and absolute ethanol. Nuclear staining was performed using DAPI for 10 min. The fluorescent mounting solution (F4680, Sigma-aldrich) was used to cover with the cover glass.
Cell treatment with oxygen-glucose deprivation and reoxygenation (OGD/R)
Cultured in DMEM at 37°C and 5% CO2, HT22 cells were used to establish the OGD/R model by placing them in glucose-deprived DMEM within an anaerobic chamber (94% N2, 5% CO2, 1% O2) for 4 h, followed by 24 h reoxygenation in complete medium under normoxic conditions. Ir-P188 (10-5 M) was then applied for 1 h.
Mitochondrial isolation
100 mg of cortex tissue from the ischemic brain was taken and separation reagent A (protease inhibitor PMSF was added just before use) was added. The supernatant obtained after centrifuging at 600 g for 5 min was further processed. Mitochondrial isolation was achieved through centrifugation at 11,000 g for 10 min, while subsequent centrifugation of the remaining supernatant at 12,000 g for 10 min yielded mitochondrial-free cytoplasmic proteins.
Lysosomal isolation
100 mg of brain tissue were taken and homogenized buffer was added and gently homogenized for 5-10 times. The homogenate was centrifuged at 750 g for 10 min (4°C), after which the pellet was re-homogenized and recentrifuged under identical conditions. To extract the cytoplasmic fraction, the combined supernatants were sequentially centrifuged at 20,000 g for 10 min and 105,000 g for 1 h (4°C). The pellet was resuspended and mixed with 27% (v/v) Percoll (Pharmacia Inc, Piscataway, NJ, USA), then centrifuged at 20,000 g for 1 h. The upper layer was collected as lysosome and used for the following analysis.
Statistical analysis
SigmaScan Pro 5 and GraphPad Prism 5 software were used for statistical analysis, with data shown as mean ± SEM. One-way ANOVA followed by Dunnett’s t-test was used for intergroup comparisons, considering p<0.05 as statistically significant.
Results
P188 reduced cerebral I/R injury in mice
TTC staining at 24 h post-reperfusion demonstrated that P188 markedly reduced cerebral infarct volume compared to saline controls (p<0.05, Figure 2A and 2B). Additionally, P188 improved neurological function and attenuated brain water content in I/R-injured mice (p<0.05, Figure 2C and 2D), demonstrating its neuroprotective effects on I/R mice.
- P188 reduced cerebral I/R injury in mice. P188 (0.4 g/kg) or Saline was administered to mice intravenously (i.v.) via tail vein 5 min before reperfusion. 24 h after reperfusion, mice were used for TTC staining, motor deficits and water content analysis. (A) Representative images of TTC staining. Infarct regions of brain displayed white after TTC staining. n=8 in each group. (B) Quantitative analysis of brain infarct volume, which was calculated as percentage of ipsilateral hemisphere. n=8 in each group. (C) Motor deficits of mice 24 h after reperfusion. n=8 in each group. (D) Quantitative analysis of water content. n=8 in each group. *p<0.05 vs. Saline; #p<0.05 vs. sham. Data were presented as mean±SEM.
Identification and analysis of P188 labeled by iridium complex
Iridium complexes have excellent photophysical and photochemical properties and are widely used in biological imaging (Figure 3A). In this study, Iridium complexes-labeled P188 (Ir-P188) was synthesized, and its structure was shown in Figure 3B. Ir-P188 was then identified by 1H-NMR (Figure 3C). Besides, ultraviolet-visible spectrum results showed that Ir-P188 had a strong absorption around 275 nm (Figure 3D). Fluorescence emission spectrum analysis demonstrated that Ir-P188 had a fluorescence emission at 525 nm-575 nm with yellow-green fluorescence (Figure 3E).
- Identification and analysis of P188 labeled by iridium complex. The structure of Iridium complexes (A) and Ir-P188 (B). (C) 1H-NMR spectroscopy of P188 and Ir-P188. The UV-Vis absorption (D) and photoluminescence spectra (E) of Ir-P188 in CHCl3.
Dual Localization of Ir-P188: Cellular Distribution in OGD/R-Induced HT-22 Cells and Intracerebral Accumulation Post-I/R Injury in Mice
Ir-P188 (10-5 M) was added to the OGD/R–induced HT22 cell culture medium, and after 10 min, the distribution of Ir-P188 inside and outside the cells was observed with fluorescence microscopy. The results revealed that in OGD/R-induced HT22 cells, the cytoplasm was a pale yellow-green, and the nucleus had a deep stain. The distribution characteristics of this fluorescence suggested that Ir-P188 could enter the cell (Figure 4A, B).
- Distribution of Ir-P188 in the OGD/R–induced HT-22 cell and in brain from mice after ischemic injury. (A, B) Ir-P188 (10-5 M) was added to the OGD/R–induced HT22 cell culture medium, and after 10 min, the distribution of Ir-P188 inside and outside the cells was observed by fluorescence microscope. Distribution of Ir-P188 in the OGD/R–induced HT22 cell. Scale bar=50 µm. Mice was subjected to MCAO for 2 h and 24 h reperfusion, and administered (i.c.v.) with 3µL mixture of PI and Ir-P188 (0.25µg PI dissolved in 1µL 10% Ir-p188). 30 min later the brain was used for fluorescence analysis. (C) Distribution of Ir-P188 in the brain from mice after I/R. Scale bar=50 µm. (D and E) The images were high-magnification for boxes in The images were highmagnification for boxes in (C). Scale bar=20 µm. n=8 in each group.
Furthermore, the distribution of Ir-P188 in mice with cerebral ischemia model was observed. A mixture of PI and Ir-P188 was injected into the lateral ventricle after 24 h of I/R in mice, and a frozen section of the brain was taken 0.5 h later for observation. The results showed that in the striatum, most cells produced significant yellow-green fluorescence due to the staining of Ir-P188 (Figure 4C). These cells include PI staining positive and negative cells, indicating that after cerebral ischemia injury, Ir-P188 can not only enter cells with outer cell membrane damage (PI staining positive cells, Figure 4D), but also enter cells with relatively intact cell membranes (PI staining negative, Figure 4E). The above in vivo results indicated the accumulation of Ir-P188, which might have a repairing effect on the inner cell organelle membrane in I/R mice.
Effects of P188 on inflammatory markers, oxidative stress, and organelle membrane integrity in mice following cerebral I/R injury
The impact of Ir-P188 on the release of substances in mitochondria and lysosomes was examined to further validate the reparative action of P188 on the cellular inner membrane. To evaluate Ir-P188’s membrane repair capacity, we analyzed the effects on mitochondrial and lysosomal integrity by measuring Cyt Cand Cathepsin L release in brain tissues. Following 24 h of I/R injury, significant Cyt C leakage from mitochondria to cytoplasm was observed, which was substantially reduced by Ir-P188 treatment (Figure 5A), suggesting its mitochondrial membrane-stabilizing properties.
In addition, the lysosome and cytoplasm were also separated to determine the release of the protease Cathepsin L in the lysosomes. Similar to Cyt C released by mitochondria, Ir-P188 effectively decreased Cathepsin L release from lysosomes to cytoplasm (Figure 5B), indicating its protective effect on lysosomal membranes. In addition, we also observed a significant reduction in protein expression levels of C-Caspase 3, NF-kB, ROS, TNF-a and IL-6 in the P188-treated group (Figure 5C-G), suggesting that P188 may modulate these key inflammatory and oxidative stress pathways to stabilize mitochondrial and lysosomal membranes during I/R injury.
- Effects of P188 on inflammatory markers, oxidative stress, and organelle membrane integrity in mice following cerebral I/R injury. (A) 24 h after I/R injury, ischemic cortex was used for western blotting analysis. Cyt C, COX in the mitochondrial and cytosolic fractions were determined. M: mitochondrial fraction; C: cytosol fraction. (B) Cathepsin L, LAMP2 in the lysosome and cytosolic fractions of ischemic cortex were analyzed with western blotting. L: lysosome fraction; C: cytosol fraction. n=8 in each group. (C) Cleaved Caspase 3 expression. (D) NF-κB phosphorylation. n=6 in each group. (E) ROS quantification. (F) TNF-α level (G) IL-6 level. n=3 in each group. *p<0.05, **p<0.01, ***p<0.001 vs. Sham Group; #p<0.05, ##p<0.01, ###p<0.001 vs. Saline Group. Data were presented as mean ± SEM.
Comparative Effects of P188 and Ginkgolide B (GB) Combination Therapy Versus Monotherapy on cerebral I/R injury
As an excipient widely used in pharmaceutical formulations, P188 possesses inherent advantages for combination with various neuroprotective agents. Guided by this rationale, we conducted preliminary exploration of its combined application with GB, a classical neuroprotective drug. The combined treatment of P188 and GB significantly reduced cerebral infarct area (p<0.01), demonstrating superior efficacy in mitigating infarct size compared to equivalent monotherapy doses of P188 (0.4 g/kg) and GB (20 mg/kg) administered separately (p<0.05). However, while the combination solution significantly decreased brain water content and alleviated neurological deficits, these therapeutic effects showed no statistically significant difference relative to individual administration of either P188 or GB (Figure 6).
- Neuroprotective effects of combined GB and P188 in mouse model of I/R. P188 (0.4g/kg), GB (20 mg/kg) and mixture were administered after reperfusion. (A) Representative TTC staining sections. (B) Quantitation of infarct volumes. (C) Cerebral water content and (D) Neurological deficits. Data are expressed as mean ± SEM (n=10). *p<0.05, **p<0.01 compared to the saline-treated group, #p<0.05, ##p<0.01 compared to p188+GB group in (B) and compared to Sham group in (C).
Discussion
This study demonstrates P188’s neuroprotective effects against ischemic stroke through plasma membrane repair-mediated mechanisms. The copolymer suppresses ROS/inflammatory pathways, penetrates cells to protect lysosomal-mitochondrial integrity by containing Cathepsin L and cytochrome C leakage, and exhibits excipient properties enabling synergistic neuroprotective combinations. These findings position P188 as a multipotent therapeutic candidate for clinical stroke intervention strategies.
P188 protect peripheral tissues such as myocardium, testis and skeletal muscle from I/R injury.13 Pharmacokinetic studies have found that P188 could enter brain tissue through the BBB.14 In recent years, in terms of the nervous system, animal experiments have demonstrated that P188 protects against spinal cord compression and cerebral hemorrhage and other nervous system injuries.8,13 In the late 1980s, Colbassani et al. used a rabbit model and found that P188 can reduce the mutual adhesion of proteins in the blood and increase the blood flow in the ischemic area.9 A further study demonstrated that P188 diminished autophagy activation in neuronal cells subjected to OGD in vitro and, when administered intravenously, decreased infarct size and enhanced neurological and motor function in a live mouse model of cerebral ischemia.15
The molecular structure of P188 is composed of a hydrophobic polyoxypropylene chain at the centre and polyoxyethylene chains at both ends. This structural feature resembles the cell membrane’s lipid bilayer, and studies suggest that P188 can directly repair the cell membrane following electroporation. This repair effect is similar to that of a “band-aid”, which can be selectively inserted into the damaged cell membrane site for repair. The targeting of the cell membrane has been a hot topic of research in the investigation of the biological mechanism of action of P188.
Building upon our previous findings demonstrating P188’s protective effects against IRI through BBB stabilization, MMP-9 inhibition, and membrane repair mechanisms, this study systematically investigated its subcellular distribution and organelle-specific therapeutic actions.10 We first labelled P188 with an iridium complex (which produces yellow-green fluorescence) and studied its distribution in OGD/R-induced HT22 cells and mice after I/R injury to study its impact on intracellular membranes. We separated lysosomes, mitochondria, and cytoplasmic fractions from brain I/R tissues after demonstrating P188 integrates into cell membranes. The effects of P188 on ROS generation, inflammation, and Cyt c and Cathepsin L release from mitochondria or lysosomes were then examined.
Mitochondria are important organelles of eukaryotic cells and the main place for animal cells to produce ATP.16 In apoptosis, mitochondria are vital, as indicated by a decrease in inner mitochondrial membrane permeability and transmembrane potential, causing Cyt C to be released into the cytoplasm.17 The release is essential as it permits Apaf-1 to trigger Caspase 9, which subsequently cleaves and activates Caspase 3, resulting in cell apoptosis. The mitochondria and cytoplasm from the ischemic brain tissue were separated, and the distribution of Cyt C after I/R were analyzed after P188 treatment. P188 greatly prevented Cyt C leaking from mitochondria into the cytoplasm. P188 suppressed Caspase 3 activation, suggesting that P188 inhibited cell apoptosis caused by cerebral I/R by targeting mitochondria. Consistent with our results, P188 application could inhibit apoptosis in cells and animal models of brain trauma. Its molecular mechanism involves the inhibition of p38 MAPK and caspase-3 activation.
We also found that the lysosomal enzyme cathepsin L leaked into the cytoplasm after the application of P188 in the lysosomal and cytoplasmic parts of ischemic brain tissue was significantly reduced. Lysosomes are another important organelle of eukaryotic cells. Lysosomes contain a variety of proteolytic enzymes that are essential for the removal of intracellular material and cell suicide.18 Cathepsin within the lysosome is crucial for the control of apoptosis. Cathepsin B cannot directly activate caspase after release, but it can cleave Bid, which then moves to the mitochondria, leading to Cyt C release and apoptosis.19
The dual assault of ROS-driven lipid peroxidation and calcium-mediated phospholipase activation during cerebral I/R injury synergistically disrupts plasma membrane integrity, triggering downstream inflammatory cascades. Mechanistic studies identified P188’s dual functionality: its membrane-stabilizing action physically limits ROS propagation between cellular compartments, while structural analysis suggests the polymer’s ethylene oxide domains may scavenge hydroxyl radicals. Importantly, P188 suppressed NF-kB nuclear translocation and subsequent pro-inflammatory gene expression, indicating cross-talk between membrane repair and inflammatory signaling pathways. These findings position P188 as a multimodal therapeutic agent that concurrently addresses membrane integrity, oxidative stress, and neuroinflammation in I/R injury.20
In a rabbit model of localized cerebral ischemia, P188 enhanced blood circulation by minimizing adhesive interactions among blood cells, vessel walls, and fibrin-fibrinogen within the microcirculation.9 It was also discovered that P188 could save growing hippocampal neurons after they had been damaged by excitotoxicity and oxidative stress, which are two main ways that hypoxia can cause neurodegeneration.21 Here, the underlying mechanism is related to the repairment of mitochondrial and lysosome membranes. Consistent with these observations, P188 mitigated the permeabilization of mitochondrial and lysosomal membranes due to traumatic brain injury in cultured primary neurons.22 It also plays a role in maintaining lysosomal membrane integrity in models of Parkinson’s disease.23 In addition, it alleviates cerebral hypoxia/ischemia injury by inhibiting mitochondrial membrane permeabilization and modulating autophagy.24 Pille et al20 also highlight P188’s potential in protecting rat brain mitochondria from oxidative stress. It seems that P188 may act as a potential drug against mitochondrial and lysosome damage, which needs further investigation.
Current neuroprotective drug development encounters significant translational challenges, as single-target agents that demonstrate preclinical efficacy frequently fail in clinical trials due to the multifactorial nature of stroke pathology.25 P188, a widely utilized emulsifier and solubilizer in pharmaceutical formulations, has exhibited protective effects against I/R injury in our studies, with a distinct membrane-stabilizing mechanism. Consequently, combining P188 with other neuroprotective agents acting via alternative mechanisms presents inherent advantages. Our previous in vivo and in vitro studies have demonstrated that GB (20 mg/kg) significantly reduces cerebral infarct volume, alleviates brain edema, and improves neurological deficits. Its neuroprotective effects are mediated through the inhibition of NF-kB-driven neuroinflammation and microglial activation, as well as modulation of the Bax/Bcl-2 apoptotic pathway.26 In this context, we investigated the combination of P188 with the classical neuroprotective agent GB to elucidate the differences between combination therapy and monotherapy. Experimental findings revealed that the combination of P188 and GB significantly reduced infarct areas in MCAO mice compared to the same doses of P188 (0.4 g/kg) or GB (20 mg/kg) administered individually. These results provide crucial preclinical evidence supporting the translational potential of P188 in combinatorial stroke treatment strategies.
Although P188 shows promising neuroprotective effects in preclinical models, several limitations must be considered before clinical translation. One significant challenge is its bioavailability, as P188 may not efficiently cross the BBB in sufficient quantities. Additionally, the potential for toxicity, particularly with prolonged use or at higher doses, remains a concern. These issues highlight the need for further studies to assess the safety profile and optimize delivery strategies for P188 in clinical settings. This experiment provides valuable preclinical data supporting the clinical translation of P188 for ischemic stroke treatment. Furthermore, we acknowledge the limitation of not including minocycline or edaravone in the current study and plan to incorporate this in future research.
Conclusion
P188 (0.4 g/kg) could be transported into the cells and played a protective effect on I/R injury in mice. The proposed mechanism for its action involves minimizing mitochondrial and lysosomal damage following I/R. While P188 shows potential for treating cerebral I/R injury, concerns regarding its long-term toxicity and bioavailability in certain situations remain.
Acknowledgments
We would like to acknowledge Editage (Editage.com) for the English language editing.
Footnotes
Disclosure. Authors have no conflict of interests, and the work was not supported or funded by any drug company. This work was supported by the National Natural Science Foundation of China (Grant No. 81870941), the Scientific Research Project of Health Commission of Jiangsu Province (Grant No. MQ2024057), the Science and Technology Project of Nantong City (Grant No. JC2024028), and the Nantong University Special Research Fund for Clinical Medicine (Grant No. 2024JY045).
- Received February 26, 2025.
- Accepted May 17, 2025.
- Copyright: © Neurosciences
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