Ginsenoside Rh2-Modified Liposomes for Targeted Delivery of Puerarin Alleviate Brain Ischemia-Reperfusion Injury

Ginsenoside Rh2-Modified Liposomes for Targeted Delivery of Puerarin Alleviate Brain Ischemia-Reperfusion Injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ginsenoside Rh2-Modified Liposomes for Targeted Delivery of Puerarin Alleviate Brain Ischemia-Reperfusion Injury Meiyan Wei, Wei Han, Jinglan Wu, Zhe Li, Mengbin Tian, Jian Xu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8550026/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Apr, 2026 Read the published version in Drug Delivery and Translational Research → Version 1 posted 11 You are reading this latest preprint version Abstract Brain Ischemia poses a significant unmet medical need, demanding novel therapeutic approaches. Puerarin (Pue), despite its potential for treating brain disorders, suffers from poor blood-brain barrier (BBB) permeability due to its low oil/water partition coefficient. To overcome this, we developed a novel ginsenoside Rh2-based liposome formulation (Rh2-Pue-LP) to enhance Pue delivery to the ischemic brain. A rat model of middle cerebral artery occlusion-reperfusion (MCAO/R) was established. Neurological deficits were evaluated using the Longa scoring system 24 hours post-MCAO. After seven days of tail-vein administration of Rh2-Pue-LP, the following analyses were performed: TTC staining to assess cerebral infarct volume, HE and TUNEL staining to examine hippocampal histopathology, ELISA to quantify serum levels of IL-1β, TNF-α, and IL-6, immunofluorescence to detect NLRP3 expression, and immunohistochemistry to evaluate the activation of JAK2-STAT3 and expression of inflammatory cytokines. Additionally, this study was conducted to further verify the targeting ability and safety of the formulation. Our results showed Rh2-Pue-LP treatment reduced infarct volume, improved neurological function, and decreased serum levels of inflammatory cytokines (IL-1β, TNF-α, IL-6). Histological examination revealed better-preserved hippocampal neurons. Rh2-Pue-LP inhibited the JAK2-STAT3 signaling pathway and NLRP3 inflammasome expression, suppressing microglial activation and neuronal apoptosis. Additionally, Rh2-Pue-LP exhibited stronger brain targeting ability with no significant biotoxicity in vivo. Rh2-Pue-LP represents a promising strategy for treating ischemic stroke by enhancing Pue delivery and exerting potent neuroprotective effects. Ginsenoside Rh2-modified liposomes Puerarin Brain Ischemia JAK2-STAT3 signaling pathway Middle cerebral artery occlusion-reperfusion (MCAO/R) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Brain Ischemia, a major global health threat, has become one of the leading causes of death and long-term disability. The latest World Health Organization data shows that approximately 13.7 million people worldwide suffer from stroke annually, with ischemic stroke accounting for up to 87%[1,2]. Although reperfusion therapies such as intravenous thrombolysis and mechanical thrombectomy have significantly improved outcomes for some patients, their therapeutic time window is strictly limited to 4.5 to 6 hours, leaving most patients with irreversible neurological damage due to missed optimal treatment opportunities[3]. Studies indicate that during ischemia-reperfusion injury, inflammatory responses and oxidative stress are intricately intertwined, jointly driving disease progression[4]. Abnormal activation of the JAK2-STAT3 signaling pathway can induce the assembly and activation of the NLRP3 inflammasome, leading to massive release of pro-inflammatory cytokines such as IL-1 β , IL-6, and TNF- α , exacerbating neuronal apoptosis and disrupting the integrity of the blood-brain barrier (BBB)[5,6]. Therefore, developing novel therapeutic strategies that can precisely regulate the inflammatory cascade has become an urgent priority. As a classic nanodrug delivery system, liposomes have gained significant attention in biomedicine due to their good biocompatibility, diverse drug-loading capacity, and ability to improve drug pharmacokinetics[7,8]. In recent years, innovative research replacing traditional liposomal excipients with natural active ingredients has emerged as a hotspot. Ginsenoside Rh2, a key active component extracted from ginseng, is a tetracyclic triterpenoid compound that not only possesses multiple biological activities such as anti-inflammation, anti-oxidation, and anti-apoptosis but also features a unique amphiphilic structure, effectively regulating liposomal membrane fluidity and stability[9,10]. Existing studies have confirmed that ginsenoside Rh2-modified liposomes can significantly enhance drug targeted delivery, showing promising applications in cancer therapy and other fields[11]. However, the application of ginsenoside Rh2 liposomes in the treatment of ischemic stroke and the exploration of their regulatory mechanisms on the JAK2-STAT3/NLRP3 signaling pathway remain uncharted. Puerarin, an isoflavone compound extracted from the traditional Chinese herb Pueraria, exhibits multi-target protective potential in cerebral ischemia treatment[12,13]. Basic research has shown that puerarin can effectively reduce neuroinflammatory responses and exert neuroprotective effects by inhibiting JAK2/STAT3 signaling pathway activation[14]. However, due to its low oil-water partition coefficient, puerarin has an oral bioavailability of less than 5 % and difficulty penetrating the intact BBB, which greatly limits its clinical application[15]. Meanwhile, constrained by delivery efficiency, the regulatory effect of puerarin on the NLRP3 inflammasome has not been fully validated. This study aims to develop a ginsenoside Rh2-modified puerarin liposome (Rh2-Pue-LP), we systematically evaluate the protective effect of Rh2-Pue-LP against cerebral ischemia-reperfusion injury and deeply analyze its molecular mechanism of inhibiting inflammation and promoting neural repair by regulating the JAK2-STAT3/NLRP3 signaling pathway, with the aim of providing new drug delivery strategies and theoretical basis for the treatment of ischemic stroke. ment of ischemic stroke. Materials and Methods Animals Adult male Sprague-Dawley (SD) rats (220±20 g) were purchased from Henan Sikebeisi Biotechnology Co. Ltd. The experimental license number is SCXK (Yu) 2020-0005.The license number for the use is SCXK (Qian) 2021-0005. They were raised at a temperature of 25±2 ℃, a relative humidity of 60±10 %, and a natural light-dark cycle. All animal experiments were conducted in accordance with the regulations of the Laboratory Animal Research Institute of Guizhou University of Traditional Chinese Medicine.. It has been reviewed and met the requirements by the Experimental Animal Ethics Review Committee of Guizhou University of Traditional Chinese Medicine(Animal Ethics Review:20250605003). Regents Ginsenoside Rh2 (Item No. DR0019) was purchased from Chengdu Desiter Biotechnology Co., Ltd. Puerarin reference substance (Item No. SP8690, purity ≥ 98 %) was obtained from Beijing Solarbio Science & Technology Co., Ltd. Puerarin bulk drug (Item No. S30646, purity ≥ 98 %) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Cholesterol (Item No. C804517) was obtained from Shanghai Macklin Biochemical Co., Ltd. Egg yolk lecithin (Item No. L8260) and Dialysis membranes (Item No. YA1078) were purchased from Beijing Solarbio Science & Technology Co., Ltd. Rat IL-1β, IL-6, and TNF-α ELISA kits (Item Nos. YJ16733, YJ064292, YJ002859) Purchased from Shanghai Yuanju Biotechnology Center. IL-1β antibody (Item No. WL02257), IL-6 antibody (Item No. WL02841), p-JAK2 antibody (Item No. WLH3592), p-STAT3 antibody (Item No. WLP2412), and NLRP3 antibody (Item No. WL02635) were purchased from Shenyang Wanlei Biotechnology Co., Ltd. TNF-α antibody (Item No. GB11188), DAB (SA-HRP) Tunel Cell Apoptosis Detection Kit (Item No. G1507), TTC staining solution (Item No. G1017), HE staining solution (Item No. G1005), and Alexa Fluor 594-conjugated goat anti-rabbit IgG secondary antibody (Item No. GB28301) were obtained from Wuhan Servicebio Technology Co., Ltd. Preparation of Rh2-Pue-LP The Rh2-Pue-LP was prepared by the thin-film dispersion method[16]. Briefly, 3.80 mg of ginsenoside Rh2, 1.80 mg of puerarin, and 53.5 mg of egg yolk lecithin were precisely weighed and placed in a centrifuge tube. Dichloromethane and methanol were added for ultrasonic dissolution. The solvent was removed by rotary evaporation at room temperature for 30 min to form a uniform thin film. Distilled water was then added, and the mixture was hydrated in a water bath at 50 °C for 30 min. The liposomes were placed in ice water, sonicated for 5 min using an ultrasonic cell disruptor, and filtered through a 0.45 µm microporous membrane to obtain Rh2-Pue-LP, which was stored at 4 °C for later use. For comparison, a conventional liposome (C-Pue-LP) was prepared by replacing ginsenoside Rh2 with an equal amount of cholesterol using the same method. Characterization of Rh2-Pue-LP The particle size, size distribution, and zeta potential of liposomes were measured using a nanosize analyzer (Beckman Coulter, Brea， CA， USA). The surface morphology and nanoparticle shape of liposomes were observed by transmission electron microscopy (TEM) (JEOL, Tokyo, Japan). The encapsulation efficiency and drug loading of Rh2-Pue-LP were determined by high-performance liquid chromatography (HPLC) (Shimadzu， CA, Japan). Chromatographic conditions: The chromatographic column is Diamonsil 5 µ m C18 (250 × 4.6 mm); The mobile phase is methanol: 0.1% citric acid, with a ratio of 25:75; Detection wavelength is 254 nm; column temperature is 30 °C; flow rate is 1 mL/min; Inject 10 µL.The encapsulation efficiency (EE%) and drug loading (LE%) were calculated accordingly. EE (%) = (W Total −W Free )/W Total ×100 % LE (%) =( W Total −W Free )/W All ×100 % W Total represents the amount of puerarin after liposome demulsification, W Free represents the amount of puerarin that has not been encapsulated into the liposome, and W All represents all the excipients added. In vitro release The in vitro release of Rh2-Pue-LP was determined by the dialysis method[17]. Briefly, Rh2-Pue-LP was sealed in a dialysis bag and placed into a beaker containing 10 mL of PBS. The beaker was then shaken in a thermostatic shaker at 37 °C and 60 rpm. PBS samples were collected at 0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 12.0 h time points, with an equal volume of fresh PBS solution replenished simultaneously. The concentration of puerarin released at each time point was detected by HPLC, and the cumulative release rate was calculated. Construct the middle cerebral artery occlusion (MCAO) model and treatment The MCAO model was established as previously described[18]. Briefly, rats anesthetized with sodium pentobarbital underwent cervical incision to expose the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). Proximal ligation of CCA/ECA and temporary clamping of ICA were performed, followed by insertion of a nylon suture into the ICA to occlude the MCA for 2 h. Reperfusion was induced by suture withdrawal, and incisions were closed. Sham controls underwent the same surgery without MCA occlusion. Animals were randomized into 5 groups (n=10): Sham, Model, Puerarin, C-Pue-LP, and Rh2-Pue-LP. At 24 h post-MCAO, Sham and Model groups received intravenous saline, while treatment groups were administered 7.5mg/kg of respective formulations daily for 7 d. Ex Vivo Tissue Imaging To verify the targeting ability of Pue, C-Pue-LP, and Rh2-Pue-LP in middle cerebral artery occlusion (MCAO) model rats, Pue, C-Pue-LP, and Rh2-Pue-LP were labeled with DiR. MCAO rats were divided into three administration groups: DiR, C-DiR-LP, and Rh2-DiR-LP (all at a concentration of 40 μg/mL), with 3 rats per group. Additionally, sham-operated rats injected with normal saline (NS) via the tail vein served as the control group. At 2 h after tail vein injection of the drugs, the rats were anesthetized and sacrificed. The brain, heart, liver, spleen, lung, and kidney were harvested, and ex vivo imaging was performed using a small animal imaging system to observe the fluorescence distribution in the isolated tissues of each group. Neurological evaluation and infarct analysis Neurological function was evaluated 24 h post-MCAO using the Longa scale , assessing limb function and locomotion[19]. After evaluation, rats were euthanized, and brains were harvested for TTC staining[20]. Coronal slices were incubated in 2% TTC at 37 °C for 30 min, with viable tissue stained red and infarcted areas white. Infarct volume was quantified using Image J and expressed as a percentage of total brain volume to determine ischemic injury severity. Histopathology and Tunel analysis Brain tissues were fixed in 4 % paraformaldehyde for 24 h, dehydrated, and embedded in paraffin. 4 μm thick coronal sections were prepared and stained with hematoxylin-eosin to observe morphological changes in the hippocampus and peri-ischemic regions under light microscopy. For TUNEL analysis, paraffin sections were deparaffinized, hydrated, and subjected to antigen retrieval. TdT enzyme reaction mixture was applied for 60 min at 37 °C to label DNA strand breaks, followed by sequential incubation with biotinylated anti-digoxigenin antibody and streptavidin-HRP. Color development was performed using DAB, and apoptotic cells were quantified by Image J software. Immunohistochemical assay Paraffin-embedded brain sections were deparaffinized in xylene and rehydrated through graded ethanol. Antigen retrieval was performed via heat-induced epitope retrieval in pH 6.0 citrate buffer. Endogenous peroxidase activity was blocked with 3% H 2 O 2 for 15 min, followed by blocking with 5% BSA in PBS for 1 h. Sections were incubated overnight at 4 °C with primary antibodies (1:200). After washing, biotinylated secondary antibodies (1:200) were applied for 1 h at room temperature, followed by streptavidin-HRP complex for 30 min. Color development used DAB, terminated with distilled water. Sections were counterstained with hematoxylin, dehydrated, cleared in xylene, and mounted. Protein expression was quantified by optical density analysis of positive staining using Image J software. Immunofluorescence assay Paraffin-embedded brain tissues were sectioned into 4 μm slices, deparaffinized in xylene, and rehydrated with graded ethanol. Antigen retrieval was performed using high-temperature pressure in pH 6.0 citrate buffer, followed by permeabilization with 0.3 % Triton X-100 in PBS for 15 min and blocking with 5 % BSA for 1 h at room temperature. Sections were incubated overnight at 4°C with primary antibodies (1:200). After washing, Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:500) was applied in the dark for 1 h at room temperature. Nuclei were counterstained with DAPI using an antifade mounting medium. Fluorescent images were captured by laser confocal microscopy , with NLRP3 signals (red) and nuclei (blue). ELISA assay Serum were analyzed using rat IL-1 β , IL-6, and TNF- α ELISA kits according to the manufacturer’s instructions. Samples, standards, and detection antibodies were sequentially added, incubated at room temperature, and washed. HRP-conjugated secondary antibodies were then applied, followed by color development and reaction termination. Absorbance at 450 nm was measured using a microplate reader, and cytokine concentrations were calculated from standard curves. In Vivo Safety Evaluation via Histopathological Analysis To verify the in vivo safety of Pue, C-Pue-LP, and Rh2-Pue-LP, normal rats were selected and subjected to tail vein injection of Pue, C-Pue-LP, or Rh2-Pue-LP once daily for 7 consecutive days. Rats in the blank control group received an equal volume of normal saline (NS) via the same route. After the 7-day treatment period, all rats were anesthetized and sacrificed. The brain, heart, liver, spleen, lung, and kidney were harvested, followed by hematoxylin-eosin (HE) staining. The morphological changes of the above tissues in each group were observed under a microscope. Statistical analysis Statistical analyses were conducted using SPSS 20.0 software, while graphical representations were generated with Origin 2024. All data were presented as mean ± standard deviation. one-way analysis of variance (ANOVA) was applied for intergroup comparisons. Statistical significance was defined as P < 0.05. Results Characterization of Liposome Particle Size Distribution, Transmission Electron Microscopy (TEM) Microscopic Morphology, and Content Analysis This study replaced cholesterol in traditional liposomes with ginsenoside Rh2, successfully prepared Rh2-Pue-LP and C-Pue-LP using the thin-film dispersion method. Both liposomes presented milky-white translucent aqueous suspensions (Fig. 1a), indicating a stable colloidal dispersion system formed under optimal thin-film sonication conditions. TEM results (Fig. 1a) showed round or elliptical morphologies for both, confirming Rh2 did not disrupt liposomes’ typical microstructure. Particle size and zeta potential analyses (Fig. 1a, Table 1) revealed their average particle sizes (93.10 nm) were within the ideal 50-200 nm range, with no significant differences in PDI or zeta potential. This particle size facilitates stable circulation after rat tail vein injection, reduces embolization risk, and enhances tissue uptake via the EPR effect[21]. Rh2, with an amphiphilic structure similar to cholesterol, maintains liposome physical stability comparable to cholesterol. As a functional component, it has anti-inflammatory, anti-oxidant, and anti-apoptotic effects[22,23]; co-loading with puerarin may enhance cerebral ischemic region enrichment. Its natural origin offers superior biocompatibility and safety over synthetic cholesterol, providing a new clinical direction for liposomes[24]. In vitro release study of Rh2-Pue-LP. To investigate the drug release characteristics of Rh2-Pue-LP, an in vitro release kinetics study was performed and compared with free puerarin (Pue). The results showed that free Pue achieved 96.2 % cumulative release within 4 hours, presenting a rapid and nearly complete release pattern. In contrast, Rh2-Pue-LP exhibited significantly delayed release, with approximately 58 % cumulative release at 4 hours and increasing to around 60% after 12 hours (Fig. 1b), indicating good sustained-release effects. This property is attributed to the unique bilayer membrane structure of liposomes, which encapsulates puerarin in hydrophobic or hydrophilic regions to form a physical barrier slowing drug diffusion, and the synergistic effect of ginsenoside Rh2, which regulates membrane fluidity and compactness to further restrict release. Critical for cerebral ischemia treatment-where post-stroke excitotoxicity, oxidative stress, and inflammation persist for hours to days causing secondary brain injury[25], Rh2-Pue-LP maintains stable drug supply in ischemic areas, avoids the difficulty of free Pue in sustaining an effective therapeutic window and potential systemic adverse reactions, and confirms its value as a structurally a stable controlled-release novel drug delivery system. Neuroprotective of Rh2-Pue-LP in MCAO in rats In the focal cerebral ischemia-reperfusion model of MCAO rats, neurological deficits were evaluated using the Longa scale (0-4 points); rats with scores of 2-3 (contralateral forelimb flexion, circling, falling) were selected, excluding those with severe injury (score 4) or unsuccessful modeling (score <2). TTC staining (Fig. 1c) showed no obvious white infarcted areas in the sham-operated group, while the model group’s infarct volume was (37.38±1.14)%. All treatment groups significantly reduced infarct volume: Pue group (30.88±0.61)%, C-Pue-LP group (24.59±1.20)%, and Rh2-Pue-LP group (12.95±0.83%, 24% reduction compared with model group, P <0.01). In vitro studies confirmed the sustained release property of Rh2-Pue-LP, which matching pathological process after ischemia, avoids the "peak-valley effect" of free drugs, and thereby durably inhibits the nerve cell damage cascade. Ginsenoside Rh2 itself exerts anti-inflammatory effects (inhibiting the activation of the NLRP3 inflammasome), antioxidant effects (increasing SOD activity), and anti-apoptotic effects (downregulating Caspase-3 expression)[26]. After embedding into the liposomal membrane, it not only maintains structural stability but also enhances neuroprotection through multi-target mechanisms. Rh2 may inhibit the excessive activation of microglia by regulating the JAK2/STAT3 signaling pathway, thereby reducing apoptosis of neurons in the peri-ischemic area. Results and Mechanism Analysis of Targeting Evaluation In this study, liposomes were labeled with fluorescein DiR to enable visual tracking of drug distribution via targeted administration in rats. As shown in (Fig. 2), no fluorescent intensity was detected in the isolated tissues of rats in the control group. In contrast, the drugs in the free DiR group, C-DiR-LP group, and Rh2-DiR-LP group were distributed to varying degrees in organs including the brain, heart, liver, spleen, lung, and kidney, with the fluorescent intensity in the brain being particularly prominent. Further comparison revealed that the brain fluorescent intensity of the Rh2-DiR-LP group was significantly higher than that of the C-DiR-LP group and the free DiR group, confirming that Rh2-DiR-LP can achieve targeted drug delivery, effectively cross the blood-brain barrier (BBB), reach brain injury sites, and increase puerarin accumulation in the brain. Free DiR, a small-molecule substance, distributes widely via passive diffusion; C-DiR-LP is easily cleared by the reticuloendothelial system (RES), accumulating in non-target organs and reducing brain delivery efficiency. Ginsenoside Rh2 modification plays a critical regulatory role: Rh2 can bind to specific receptors (as GLUT1, LDLR) highly expressed on BBB endothelial cells and promote liposome transcytosis across the BBB via "receptor-mediated endocytosis." This active targeting mechanism, absent in C-DiR-LP and free DiR, is the core reason for the efficient brain accumulation of Rh2-DiR-LP [27]. Hematoxylin and Eosin (HE) Staining and TUNEL Apoptotic Analysis The results of HE staining (Fig. 3a) and TUNEL apoptosis analysis (Fig. 3b) directly demonstrated that brain cells in the sham-operated group had normal morphology, while the model group showed extensive necrosis, neuron loss, and a significant increase in cell apoptosis, confirming the successful establishment of the MCAO model. Although the Pue group alleviated tissue damage, notable cell shrinkage and glial hyperplasia remained. Compared with the Pue group, the C-Pue-LP group further reduced pathological changes but did not show a significant advantage in inhibiting apoptosis. The Rh2-Pue-LP group not only minimized neuron shrinkage and glial hyperplasia but also effectively suppressed apoptosis by significantly reducing the TUNEL-positive rate. This effect is attributed to ginsenoside Rh2: it modulates blood-brain barrier permeability to facilitate efficient liposome delivery to ischemic regions, activates the anti-apoptotic PI3K/Akt pathway to intervene in the apoptosis cascade and reduce neuronal death, and its low immunogenicity decreases liposome-induced immune rejection, safeguarding the repair of the brain tissue microenvironment and providing new strategies for ischemic stroke treatment. Immunohistochemical analysis of TNF-α, IL-1 β , IL-6, p-JAK2, and p-STAT3 protein expression Immunohistochemical analysis (Fig. 4) revealed that in the sham-operated group, the expression levels of pro-inflammatory cytokines (TNF- α , IL-1 β , IL-6) and p-JAK2/p-STAT3 proteins were extremely low, confirming the low inflammatory baseline of normal brain tissue. In contrast, the model group exhibited intense positive staining for these proteins, indicating a close association between the post-stroke inflammatory cascade and the excessive activation of the signaling pathway. Although the Pue group partially inhibited the release of pro-inflammatory cytokines and the activation of JAK2/STAT3, residual positive staining indicated limited regulatory capacity. The C-Pue-LP group, leveraging the advantages of liposomal delivery, further enhanced the suppression of inflammatory signals, highlighting the role of the carrier system in improving drug efficacy. Notably, the expression levels of inflammatory cytokines and phosphorylated proteins in the Rh2-Pue-LP group were comparable to those in the sham-operated group. Its unique superiority may stem from the multi-target properties of ginsenoside Rh2. In addition to synergizing with puerarin to inhibit the synthesis of inflammatory mediators, Rh2 may directly target the active site of JAK2 kinase, blocking signal transduction mediated by receptor tyrosine kinases and thus more effectively suppressing the excessive activation of microglia and astrocytes. Furthermore, the lipophilic structure of Rh2 may enhance the fusion efficiency of liposomes with cell membranes, promoting drug enrichment in the ischemic area. Immunofluorescence analysis of NLRP3 protein expression Immunofluorescence analysis (Fig. 5a) demonstrated that in the sham-operated group, only weak fluorescence of NLRP3 protein was observed, confirming its low expression level in normal brain tissue. In contrast, the intense enhancement of fluorescence signals in the model group revealed that the NLRP3-mediated inflammatory cascade was acutely activated after stroke and was closely associated with the progression of ischemic brain injury. Although both the Pue group and the C-Pue-LP group could reduce the fluorescence intensity of NLRP3 in a dose-dependent manner, the fluorescence level of the Rh2-Pue-LP group almost returned to that of the sham-operated group, indicating a more thorough anti-inflammatory effect. This discrepancy may be attributed to the dual characteristics of Rh2-Pue-LP. On one hand, ginsenoside Rh2 can directly target the key assembly sites of NLRP3, block the oligomerization of ASC and the activation of Caspase-1, and inhibit the maturation and release of inflammatory cytokines such as IL-1β and IL-18 at the source. On the other hand, the unique molecular structure of Rh2 may enhance the fusion efficiency of liposomes with cell membranes, promote the efficient enrichment of drugs in the ischemic area, and synergize with puerarin to form a multiple protection mechanism of "precise targeting-signal blocking-sustained drug release". In addition, the profound inhibition of NLRP3 by Rh2-Pue-LP may further mitigate blood-brain barrier disruption and neuronal pyroptosis caused by inflammation. Inflammation Analysis of TNF- α , IL-1 β , IL-6 ELISA results (Fig. 5b) showed that serum IL-1 β , IL-6, and TNF- α levels increased significantly in the model group, confirming systemic inflammatory responses after cerebral ischemia. The Pue and C-Pue-LP groups reduced these factors, but the Rh2-Pue-LP group exhibited a more remarkable inhibitory effect, with levels approaching those of the sham operation group. This superiority is attributed to its multi-dimensional regulatory effects: ginsenoside Rh2 regulates immune cell activity, inhibits monocyte and neutrophil chemotaxis, and reduces inflammatory factor release at the source [28]; the liposomal carrier prolongs drug circulation, ensuring a continuous anti-inflammatory effect. Additionally, the synergy between Rh2 and puerarin enhances anti-inflammatory capacity. These results confirm its inhibitory effect on intracerebral inflammation and reveal its potential in regulating systemic inflammation. In summary, this study confirms that Rh2-Pue-LP, liposomes with ginsenoside Rh2 replacing cholesterol, exhibits significant advantages in the treatment of ischemic stroke. It forms a stable colloidal dispersion, similar to C-Pue-LP, with no significant differences in particle size, zeta potential, or microstructure. In vitro, it releases 60 % of the drug within 12 hours, which matches the pathological needs after ischemia. In the MCAO rat model, it reduces the infarct volume by 24.43 %, improves neurological deficit scores, and HE staining and TUNEL analysis show that it alleviates brain tissue necrosis, neuronal shrinkage, and apoptosis. It strongly inhibits the release of pro-inflammatory factors, blocks the JAK2/STAT3 pathway, and suppresses the activation of the NLRP3 inflammasome. ELISA results confirm that it has a better regulatory effect on systemic inflammation than free drugs and C-Pue-LP. This study demonstrates that Rh2-Pue-LP is a safe and efficient drug delivery system for cerebral ischemia. Future research needs to further explore its in vivo pharmacokinetic characteristics, long-term safety, and clinical transformation potential, thereby laying the foundation for the application of natural components as substitutes for traditional liposomal excipients. In Vivo Safety Study Results As shown in (Fig. 6), hematoxylin-eosin (HE) staining was used to observe the histological morphology of the brain, heart, liver, spleen, lung, and kidney in rats from the blank control group and each administration group. The results showed no obvious histological abnormalities in all organs of rats after tail vein injection of Pue, C-Pue-LP, or Rh2-Pue-LP compared with the blank control group, with intact organ structures, normal parenchymal cells, and no pathological damage. These findings indicate that the three formulations have no obvious biotoxicity to the major organs of normal rats and possess good biocompatibility. It should be noted that this study only verified the safety under short-term (7-day) administration and specific dosages; the safety of Rh2-Pue-LP under long-term administration or at higher dosages still requires further experimental verification. Discussion Ischemic stroke, a cerebrovascular disorder with high disability and mortality rates, poses a severe threat to human health [29,30]. Clarifying its pathogenesis and achieving efficient drug delivery to lesions are crucial. Puerarin, derived from Pueraria lobata, exerts neuroprotective effects against cerebral ischemia [12], but its efficacy is limited by the blood-brain barrier (BBB), making the development of brain-targeted delivery systems an urgent challenge. The BBB is the primary physiological barrier restricting drug penetration into the brain parenchyma [31]. Liposomes, lipid bilayer microvesicles ("artificial biomembranes") that can encapsulate diverse cargoes, possess biocompatibility and tunable targeting capabilities, enhancing drug efficacy while reducing toxicity and thus serving as ideal carriers [32,34]. Ginsenoside Rh2, a saponin constituent derived from medicinal plants such as Panax ginseng and Panax notoginseng, exhibits multiple bioactivities including anti-inflammatory and anti-tumor effects, and has been validated as an effective agent for mitigating middle cerebral artery occlusion (MCAO)-induced injury [11]. Given its amphiphilic nature, substituting cholesterol with ginsenoside Rh2 to fabricate novel functional liposomal drug delivery systems holds significant translational potential. In the present study, ginsenoside Rh2 was used as a cholesterol replacement for liposome modification. Notably, ex vivo tissue distribution assays revealed that ginsenoside Rh2-modified liposomes significantly enhanced puerarin accumulation in the brain compared to cholesterol-modified liposomes and free puerarin. Furthermore, Rh2-Pue-LP treatment led to a marked reduction in cerebral infarct volume and a significant amelioration of neurological deficits. These findings confirm that Rh2-Pue-LP improves therapeutic outcomes in ischemic stroke, supporting ginsenoside Rh2-modified liposomes as a viable strategy for targeted drug delivery. The JAK2/STAT3 signaling pathway is implicated in a spectrum of physiological processes, including cell proliferation, differentiation, apoptosis, and immune regulation [35,36]. Sustained activation of this pathway is closely associated with the initiation and progression of various inflammation-driven diseases. The NLRP3 inflammasome, a key effector of the innate immune system, mediates caspase-1 activation, which in turn promotes the maturation and secretion of pro-inflammatory cytokines (such as TNF- α , IL-1 β , IL-6) and triggers a robust inflammatory cascade. Importantly, inhibition of the JAK2/STAT3 pathway—whether under basal conditions or in NLRP3 activator-induced inflammatory states—effectively attenuates NLRP3 inflammasome activity and suppresses the production and release of pro-inflammatory cytokines [37,38]. In our animal MCAO model, Rh2-Pue-LP intervention significantly inhibited JAK2/STAT3 pathway activation, downregulated NLRP3 expression, and reduced the levels of IL-1 β , IL-6, TNF- α , p-JAK2, and p-STAT3. These results provide valuable mechanistic insights into inflammation-related diseases and highlight the JAK2/STAT3 pathway as a potential therapeutic target. This observation is consistent with the findings of Wu et al., who demonstrated that reducing JAK2 and STAT3 phosphorylation levels inhibits downstream inflammatory responses, thereby alleviating disease pathology. This study has some limitations. We have not conducted cellular-level mechanism research. However, our data clearly indicate that ginsenoside Rh2 modification can enhance the brain accumulation of puerarin, reduce nerve damage, and alleviate inflammatory responses. This study provides a promising strategy for improving the efficacy of puerarin in treating brain diseases. Importantly, this delivery system does not have adverse effects on the brain or other major organ systems. Overall, our research results indicate that the ginsenoside Rh2-modified liposome system can target puerarin delivery to the brain, significantly improving its therapeutic efficacy in ischemic stroke. Conclusion This study demonstrates that Rh2-Pue-LP, a liposomal formulation with ginsenoside Rh2 replacing cholesterol, exhibits enhanced therapeutic efficacy in ischemic stroke. Compared to free drugs and conventional liposomes, it reduces brain injury, alleviates neurological deficits, and suppresses inflammation by inhibiting the JAK2/STAT3 pathway and NLRP3 inflammasome. The formulation combines sustained release with multi-target regulation, offering a promising strategy for ischemic stroke treatment. Future research should explore its pharmacokinetics, long-term safety, and clinical translation potential. Declarations Author Contributions WMY: conducted research, analyzed data. HW: wrote original draft, analyzed data. LZ, TMB: conducted animal experiments. XJ, CXL, ZYP: participated in the preparation of the formulation. WJL: designed research and reviewed & edited article. All authors read and approved the final manuscript. Fundind This research was supported by General Project of Basic Research Plan of Guizhou Provincial Department of Science Technology (grant no. Qiankehe Jichu ZK[2024] General 361)，National Natural Science Foundation of China project(grant no. 82560779), General Project of the Basic Research Program of Guizhou Provincial Department of Science and Technology (grant no. Qiankehe Jichu-ZK [2022] General 472) and Guizhou Provincial Science and Technology Projects (grant no. Qiankehe Pingtai ZSYS[2025]016). Data availability All data associated with this study can be obtained by reaching out to the corresponding author of this manuscript Ethical Approval and Informed Consent: The animal experiments in this study have been approved by the local ethics committee of Guizhou University of Chinese Medicine (approval number: 20250605003), and all ARRIVE standards have been followed . Adult male Sprague-Dawley (SD) rats (220±20 g) were purchased from Henan Sikebeisi Biotechnology Co. Ltd. The experimental license number is SCXK (Yu) 2020-0005, The license number for the use is SCXK (Qian) 2021-0005. They were raised at a temperature of 25±2 °C, a relative humidity of 60±10 %, and a natural light-dark cycle.For at least seven days, they were given access to water and given certifed standard meals. Consent for publication All participants provided written informed consent prior to the publication of related data. Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships. References Aloizou AM, Siokas V, Pateraki G, Liampas I, Bakirtzis C, Tsouris Z, et al. Thinking outside the Ischemia Box: Advancements in the Use of Multiple Sclerosis Drugs in Ischemic Stroke. J Clin Med. 2021 ;10:630. https://doi:10.3390/jcm10040630 Feigin VL, Brainin M, Norrving B, Martins S, Sacco RL, Hacke W, et al. World Stroke Organization (WSO): Global Stroke Fact Sheet 2022. Int J Stroke. 2022 ;17:18-29. https://doi:10.1177/17474930211065917 Jolugbo P, Ariëns RAS. Thrombus Composition and Efficacy of Thrombolysis and Thrombectomy in Acute Ischemic Stroke. Stroke. 2021 ;52:1131-1142 https://doi:10.1161/STROKEAHA.120.032810 Koutsaliaris IK, Moschonas IC, Pechlivani LM, Tsouka AN, Tselepis AD. 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Acta Pharm Sin B. 2021 ;11:1767-1788. https://doi:10.1016/j.apsb.2020.11.019 Qi Z, Yan Z, Wang Y, Ji N, Yang X, Zhang A, et al. Ginsenoside Rh2 Inhibits NLRP3 Inflammasome Activation and Improves Exosomes to Alleviate Hypoxia-Induced Myocardial Injury. Front Immunol. 2022 ;13:883946. https://doi:10.3389/fimmu.2022.883946 Xia J, Ma S, Zhu X, Chen C, Zhang R, Cao Z, et al. Versatile ginsenoside Rg3 liposomes inhibit tumor metastasis by capturing circulating tumor cells and destroying metastatic niches. Sci Adv. 2022 ;8:eabj1262. https://doi: 10.1126/sciadv.abj1262. Im DS. Pro-Resolving Effect of Ginsenosides as an Anti-Inflammatory Mechanism of Panax ginseng. Biomolecules. 2020 ;10:444. https://doi:10.3390/biom10030444 Zhao Z, Pan Z, Zhang S, Ma G, Zhang W, Song J, et al. Neutrophil extracellular traps: A novel target for the treatment of stroke. Pharmacol Ther. 2023 ;241:108328. https://doi: 10.1016/j.pharmthera. Tu WJ, Wang LD; Special Writing Group of China Stroke Surveillance Report. China stroke surveillance report 2021. Mil Med Res. 2023 ;10:33. https://doi: 10.1186/s40779-023-00463-x. . Yang Y, Torbey MT. Angiogenesis and Blood-Brain Barrier Permeability in Vascular Remodeling after Stroke. Curr Neuropharmacol. 2020;18:1250-1265. https://doi: 10.2174/1570159X18666200720173316. Chang HI, Yeh MK. Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy. Int J Nanomedicine. 2012;7:49-60. https://doi: 10.2147/IJN.S26766. Epub 2011 Dec 30. Dadwal A, Baldi A, Kumar Narang R. Nanoparticles as carriers for drug delivery in cancer. Artif Cells Nanomed Biotechnol. 2018;46:295-305. https://doi: 10.1080/21691401.2018.1457039. Ahmed KS, Changling S, Shan X, Mao J, Qiu L, Chen J. Liposome-based codelivery of celecoxib and doxorubicin hydrochloride as a synergistic dual-drug delivery system for enhancing the anticancer effect. J Liposome Res. 2020 ;30:285-296. https://doi: 10.1080/08982104.2019.1634724. Yu L, Zhang Y, Chen Q, He Y, Zhou H, Wan H, et al. Formononetin protects against inflammation associated with cerebral ischemia-reperfusion injury in rats by targeting the JAK2/STAT3 signaling pathway. Biomed Pharmacother. 2022 ;149:112836. https://doi: 10.1016/j.biopha.2022.112836. Lu M, Yin J, Xu T, Dai X, Liu T, Zhang Y, Wang S, Liu Y, Shi H, Zhang Y, Mo F, Sukhorukov V, Orekhov AN, Gao S, Wang L, Zhang D. Fuling-Zexie formula attenuates hyperuricemia-induced nephropathy and inhibits JAK2/STAT3 signaling and NLRP3 inflammasome activation in mice. J Ethnopharmacol 319 (2024) 117262, https://doi: 10.1016/j.jep.2023.117262. Zhu H, Jian Z, Zhong Y, Ye Y, Zhang Y, Hu X, et al. Janus Kinase Inhibition Ameliorates Ischemic Stroke Injury and Neuroinflammation Through Reducing NLRP3 Inflammasome Activation via JAK2/STAT3 Pathway Inhibition. Front Immunol. 2021 ;12:714943. https://doi: 10.3389/fimmu.2021.714943. Ji N, Wu L, Shi H, Li Q, Yu A, Yang Z. VSIG4 Attenuates NLRP3 and Ameliorates Neuroinflammation via JAK2-STAT3-A20 Pathway after Intracerebral Hemorrhage in Mice. Neurotox Res. 2022 ;40:78-88. https://doi: 10.1007/s12640-021-00456-5. Tables Table 1 The physicochemical properties of liposomes Formulation Particle size (nm) Polydispersity Index Zeta potential (mV) Encapsulation efficiency (%) Drug loading (%) C-Pue-LP 93.53±0.01 0.252±0.01 -25.03±1.17 61.38±0.20 1.85±0.01 Rh2-Pue-LP 93.10±0.60 0.282±0.01 -24.02±1.42 60.99±0.17 1.85±0.02 C-Pue-LP is a liposome loaded with cholesterol, and Rh2-Pue-LP is a liposome loaded with ginsenoside Rh2. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.tif Cite Share Download PDF Status: Published Journal Publication published 19 Apr, 2026 Read the published version in Drug Delivery and Translational Research → Version 1 posted Editorial decision: Revision requested 07 Mar, 2026 Reviews received at journal 26 Feb, 2026 Reviews received at journal 21 Feb, 2026 Reviewers agreed at journal 14 Feb, 2026 Reviewers agreed at journal 13 Feb, 2026 Reviewers agreed at journal 13 Feb, 2026 Reviewers agreed at journal 12 Feb, 2026 Reviewers invited by journal 19 Jan, 2026 Editor assigned by journal 09 Jan, 2026 Submission checks completed at journal 09 Jan, 2026 First submitted to journal 08 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8550026","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":576897461,"identity":"b3969725-bd3a-4387-803b-baa7393fe707","order_by":0,"name":"Meiyan Wei","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Meiyan","middleName":"","lastName":"Wei","suffix":""},{"id":576897462,"identity":"071267b4-1642-4699-92dc-94eed0bfe6e0","order_by":1,"name":"Wei Han","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Han","suffix":""},{"id":576897465,"identity":"34140003-9436-45fa-adf8-cfe09e7b479e","order_by":2,"name":"Jinglan Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIie3NP0vDQBjH8d9RaJZzf4pI30JKoFaoBN/JHYFOLQiCUykHhbrEuuq7iIvgdnBwU9Q1g2BddHEouISSwdQ/Y2JGwfsu99xxHx7A5fqbCQ0MyvPhNV9/vfhNCAEsReeyIdlWklaKXd6E+Jl4Nscb6u6373Qw3Myk8uY3hOljHRHmakm92/heRJNlWypuTwn2pZL0t2QnJpZk2jeTmEtF4z4xZX4lYfK06s0PYpKq+9aA8JxkotOghdwvt/B6EqarcouiKNF2xM6VCBZ8dDIQtpp0zsbROy+Gh4k2Fnkx27vwzHW2nlYTgAuwxfdcDkefs6gBgKeB4udSIKz97HK5XP+yD1AOYKtqtEadAAAAAElFTkSuQmCC","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Jinglan","middleName":"","lastName":"Wu","suffix":""},{"id":576897467,"identity":"51459110-9f34-4dbc-9566-848461c4ea71","order_by":3,"name":"Zhe Li","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Li","suffix":""},{"id":576897469,"identity":"ccc9e428-6141-49e1-854a-fa3f3472db00","order_by":4,"name":"Mengbin Tian","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mengbin","middleName":"","lastName":"Tian","suffix":""},{"id":576897470,"identity":"c6526006-72c4-48df-8e7a-c6fd8390f3b0","order_by":5,"name":"Jian Xu","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Xu","suffix":""},{"id":576897473,"identity":"6aabc984-03a4-4df8-8c7f-17840ac0042b","order_by":6,"name":"Xiaolan Chen","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaolan","middleName":"","lastName":"Chen","suffix":""},{"id":576897475,"identity":"09064c58-b704-4f24-85f2-8669c4c84aba","order_by":7,"name":"Yongping Zhang","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yongping","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2026-01-08 10:08:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8550026/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8550026/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13346-026-02123-8","type":"published","date":"2026-04-19T15:58:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":100819388,"identity":"7c35a051-a1b9-4835-8dab-a5c22ca2c42e","added_by":"auto","created_at":"2026-01-21 17:25:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":531901,"visible":true,"origin":"","legend":"\u003cp\u003e(a)Liposome characterization. (b)Cumulative release curve of drug. (C)he neuroprotective effect of drugs on MCAO in rats.Representative coronal sections of each group of brains. The infarct size of each group. Data are presented as mean ± S.D. (n=3 per group) \u003csup\u003e##\u003c/sup\u003evs the blank group, \u003csup\u003e**\u003c/sup\u003ethe model group showed a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) and the treatment group showed a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01) compared to the MCAO group.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8550026/v1/88bbb9e4a91af94590a30d13.png"},{"id":100819389,"identity":"80610f4f-eaf6-4481-96c8-a67e711e722f","added_by":"auto","created_at":"2026-01-21 17:25:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":301328,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution map of DiR fluorescent probe in isolated tissues of rats.(n=6 per group)\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8550026/v1/50f11d5b3ecec688151db3ac.png"},{"id":100819385,"identity":"ce801506-874b-4741-8c2f-e8805e481b88","added_by":"auto","created_at":"2026-01-21 17:25:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":953860,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Representative histopathological images of rat brain tissue. Showed normal neuronal morphology in the control group, severe neuronal shrinkage and nuclear pyknosis in the model group, and alleviated pathological changes in the Pue, C-Pue-LP, Rh2-Pue-LP group (200× magnification). (b)The effect of drugs on neuronal apoptosis detected by TUNEL assay.(200× magnification).Data are presented as mean ± S.D. (n=3 per group).\u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. sham group; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. MCAO group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e: Purple arrow: Moderate-range necrosis is visible in the cerebral cortex within the visual field; Red arrow: Nuclear fragmentation, loose structure, pale staining, and abundant gliosis are observed; Orange arrow: A small number of irregularly shaped vacuoles are visible; Black arrow: Abundant neuronal shrinkage is observed in the necrotic area and its periphery; Yellow arrow: Reduced cell volume, deepened staining, increased basophilia, obscured boundary between cytoplasm and nucleus, and a small number of neuronal degenerations are noted.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8550026/v1/8cfc9acd65ec4213d675b5e4.png"},{"id":100819383,"identity":"102ac8ed-b3cf-4595-a697-e8938d599393","added_by":"auto","created_at":"2026-01-21 17:25:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":899913,"visible":true,"origin":"","legend":"\u003cp\u003ePositive expression of TNF-α, IL-1\u003cem\u003eβ\u003c/em\u003e, IL-6, p-JAK2, and p-STAT3 in the brain tissue of MCAO rats. Protein levels in brain tissues.Data are presented as mean ± S.D. (n=3 per group)\u003csup\u003e.##\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 vs. Sham group;\u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. MCAO group.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8550026/v1/f897184d1a577cbfbb09a694.png"},{"id":100819390,"identity":"d6389be8-0f16-4e19-b959-1cca0f283056","added_by":"auto","created_at":"2026-01-21 17:25:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":289923,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Fluorescence intensity of NLRP3 in the brain tissue of MCAO rats.(200× magnification). (b)The effect of the drug on inflammatory factor levels in MCAO Rat serum. Data are presented as mean ± S.D. (n=6 per group).\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. Sham group; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. MCAO group.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8550026/v1/f3243422dff97669c1ee6fff.png"},{"id":100819386,"identity":"57d317ee-d403-4b03-829e-ffa6f6eb874a","added_by":"auto","created_at":"2026-01-21 17:25:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1773723,"visible":true,"origin":"","legend":"\u003cp\u003eHE-stained sections of heart, liver, spleen, lung, kidney and brain tissues from rats in the different treatment groups Data are presented. (n=6 per group).\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-8550026/v1/6c95120224e7bc85f4575daf.png"},{"id":107352866,"identity":"f2f1bbaa-486e-483a-8f66-2677cdcac67b","added_by":"auto","created_at":"2026-04-20 16:17:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5070085,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8550026/v1/623dd75b-ed59-4b48-be48-3f1df8cbe058.pdf"},{"id":100819391,"identity":"89d6bc6a-d17f-44ca-bbe0-764b8f69efc8","added_by":"auto","created_at":"2026-01-21 17:25:27","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7739576,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-8550026/v1/9b0869fd5265b187fa2b5f69.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ginsenoside Rh2-Modified Liposomes for Targeted Delivery of Puerarin Alleviate Brain Ischemia-Reperfusion Injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBrain Ischemia, a major global health threat, has become one of the leading causes of death and long-term disability. The latest World Health Organization data shows that approximately 13.7 million people worldwide suffer from stroke annually, with ischemic stroke accounting for up to 87%[1,2]. Although reperfusion therapies such as intravenous thrombolysis and mechanical thrombectomy have significantly improved outcomes for some patients, their therapeutic time window is strictly limited to 4.5 to 6 hours, leaving most patients with irreversible neurological damage due to missed optimal treatment opportunities[3]. Studies indicate that during ischemia-reperfusion injury, inflammatory responses and oxidative stress are intricately intertwined, jointly driving disease progression[4]. Abnormal activation of the JAK2-STAT3 signaling pathway can induce the assembly and activation of the NLRP3 inflammasome, leading to massive release of pro-inflammatory cytokines such as IL-1\u003cem\u003e\u0026beta;\u003c/em\u003e, IL-6, and TNF-\u003cem\u003e\u0026alpha;\u003c/em\u003e, exacerbating neuronal apoptosis and disrupting the integrity of the blood-brain barrier (BBB)[5,6]. Therefore, developing novel therapeutic strategies that can precisely regulate the inflammatory cascade has become an urgent priority.\u003c/p\u003e\n\u003cp\u003eAs a classic nanodrug delivery system, liposomes have gained significant attention in biomedicine due to their good biocompatibility, diverse drug-loading capacity, and ability to improve drug pharmacokinetics[7,8]. In recent years, innovative research replacing traditional liposomal excipients with natural active ingredients has emerged as a hotspot. Ginsenoside Rh2, a key active component extracted from ginseng, is a tetracyclic triterpenoid compound that not only possesses multiple biological activities such as anti-inflammation, anti-oxidation, and anti-apoptosis but also features a unique amphiphilic structure, effectively regulating liposomal membrane fluidity and stability[9,10]. Existing studies have confirmed that ginsenoside Rh2-modified liposomes can significantly enhance drug targeted delivery, showing promising applications in cancer therapy and other fields[11]. However, the application of ginsenoside Rh2 liposomes in the treatment of ischemic stroke and the exploration of their regulatory mechanisms on the JAK2-STAT3/NLRP3 signaling pathway remain uncharted.\u003c/p\u003e\n\u003cp\u003ePuerarin, an isoflavone compound extracted from the traditional Chinese herb Pueraria, exhibits multi-target protective potential in cerebral ischemia treatment[12,13]. Basic research has shown that puerarin can effectively reduce neuroinflammatory responses and exert neuroprotective effects by inhibiting JAK2/STAT3 signaling pathway activation[14]. However, due to its low oil-water partition coefficient, puerarin has an oral bioavailability of less than 5 % and difficulty penetrating the intact BBB, which greatly limits its clinical application[15]. Meanwhile, constrained by delivery efficiency, the regulatory effect of puerarin on the NLRP3 inflammasome has not been fully validated. This study aims to develop a ginsenoside Rh2-modified puerarin liposome (Rh2-Pue-LP), we systematically evaluate the protective effect of Rh2-Pue-LP against cerebral ischemia-reperfusion injury and deeply analyze its molecular mechanism of inhibiting inflammation and promoting neural repair by regulating the JAK2-STAT3/NLRP3 signaling pathway, with the aim of providing new drug delivery strategies and theoretical basis for the treatment of ischemic stroke.\u003c/p\u003ement of ischemic stroke.\n"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdult male Sprague-Dawley (SD) rats (220\u0026plusmn;20 g) were purchased from Henan Sikebeisi Biotechnology Co. Ltd. The experimental license number is SCXK (Yu) 2020-0005.The license number for the use is SCXK (Qian) 2021-0005. They were raised at a temperature of 25\u0026plusmn;2 ℃, a relative humidity of 60\u0026plusmn;10 %, and a natural light-dark cycle. All animal experiments were conducted in accordance with the regulations of the Laboratory Animal Research Institute of Guizhou University of Traditional Chinese Medicine.. It has been reviewed and met the requirements by the Experimental Animal Ethics Review Committee of Guizhou University of Traditional Chinese Medicine(Animal Ethics Review:20250605003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRegents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGinsenoside Rh2 (Item No. DR0019) was purchased from Chengdu Desiter Biotechnology Co., Ltd. Puerarin reference substance (Item No. SP8690, purity \u0026ge; 98 %) was obtained from Beijing Solarbio Science \u0026amp; Technology Co., Ltd. Puerarin bulk drug (Item No. S30646, purity \u0026ge; 98 %) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Cholesterol (Item No. C804517) was obtained from Shanghai Macklin Biochemical Co., Ltd. Egg yolk lecithin (Item No. L8260) and Dialysis membranes (Item No.\u0026nbsp;YA1078) were purchased from Beijing Solarbio Science \u0026amp; Technology Co., Ltd. Rat IL-1\u0026beta;, IL-6, and TNF-\u0026alpha; ELISA kits (Item Nos. YJ16733, YJ064292, YJ002859) Purchased from Shanghai Yuanju Biotechnology Center. IL-1\u0026beta; antibody (Item No. WL02257), IL-6 antibody (Item No. WL02841), p-JAK2 antibody (Item No. WLH3592), p-STAT3 antibody (Item No. WLP2412), and NLRP3 antibody (Item No. WL02635) were purchased from Shenyang Wanlei Biotechnology Co., Ltd. TNF-\u0026alpha; antibody (Item No. GB11188), DAB (SA-HRP) Tunel Cell Apoptosis Detection Kit (Item No. G1507), TTC staining solution (Item No. G1017), HE staining solution (Item No. G1005), and Alexa Fluor 594-conjugated goat anti-rabbit IgG secondary antibody (Item No. GB28301) were obtained from Wuhan Servicebio Technology Co., Ltd.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of Rh2-Pue-LP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Rh2-Pue-LP was prepared by the thin-film dispersion method[16]. Briefly,\u0026nbsp;3.80\u0026nbsp;mg of ginsenoside Rh2, 1.80\u0026nbsp;mg of puerarin, and\u0026nbsp;53.5\u0026nbsp;mg of egg yolk lecithin were precisely weighed and placed in a centrifuge tube. Dichloromethane and methanol were added for ultrasonic dissolution. The solvent was removed by rotary evaporation at room temperature for 30 min to form a uniform thin film. Distilled water was then added, and the mixture was hydrated in a water bath at 50 \u0026deg;C for 30 min. The liposomes were placed in ice water, sonicated for 5 min using an ultrasonic cell disruptor, and filtered through a 0.45 \u0026micro;m microporous membrane to obtain Rh2-Pue-LP, which was stored at 4 \u0026deg;C for later use. For comparison, a conventional liposome (C-Pue-LP) was prepared by replacing ginsenoside Rh2 with an equal amount of cholesterol using the same method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of Rh2-Pue-LP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe particle size, size distribution, and zeta potential of liposomes were measured using a nanosize analyzer (Beckman Coulter, Brea，\u0026nbsp;CA，\u0026nbsp;USA). The surface morphology and nanoparticle shape of liposomes were observed by transmission electron microscopy (TEM) (JEOL, Tokyo, Japan). The encapsulation efficiency and drug loading of Rh2-Pue-LP were determined by high-performance liquid chromatography (HPLC) (Shimadzu，\u0026nbsp;CA, Japan). Chromatographic conditions: The chromatographic column is Diamonsil 5 \u0026micro; m C18 (250 \u0026times; 4.6 mm); The mobile phase is methanol: 0.1% citric acid, with a ratio of 25:75; Detection wavelength is 254 nm; column temperature is 30 \u0026deg;C; flow rate is 1 mL/min; Inject 10 \u0026micro;L.The encapsulation efficiency (EE%) and drug loading (LE%) were calculated accordingly.\u003c/p\u003e\n\u003cp\u003eEE (%) = (W\u003csub\u003eTotal\u003c/sub\u003e\u0026minus;W\u003csub\u003eFree\u003c/sub\u003e)/W\u003csub\u003eTotal\u003c/sub\u003e\u0026times;100 %\u003c/p\u003e\n\u003cp\u003eLE (%) =( W\u003csub\u003eTotal\u003c/sub\u003e\u0026minus;W\u003csub\u003eFree\u003c/sub\u003e)/W\u003csub\u003eAll\u003c/sub\u003e\u0026times;100 %\u003c/p\u003e\n\u003cp\u003eW\u003csub\u003eTotal\u003c/sub\u003e represents the amount of puerarin after liposome demulsification, W\u003csub\u003eFree\u003c/sub\u003e represents the amount of puerarin that has not been encapsulated into the liposome, and W\u003csub\u003eAll\u003c/sub\u003e represents all the excipients added.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro release\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe in vitro release of Rh2-Pue-LP was determined by the dialysis method[17]. Briefly, Rh2-Pue-LP was sealed in a dialysis bag and placed into a beaker containing 10 mL of PBS. The beaker was then shaken in a thermostatic shaker at 37 \u0026deg;C and 60 rpm. PBS samples were collected at 0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 12.0 h time points, with an equal volume of fresh PBS solution replenished simultaneously. The concentration of puerarin released at each time point was detected by HPLC, and the cumulative release rate was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruct the middle cerebral artery occlusion (MCAO) model\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MCAO model was established as previously described[18]. Briefly, rats anesthetized with sodium pentobarbital underwent cervical incision to expose the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). Proximal ligation of CCA/ECA and temporary clamping of ICA were performed, followed by insertion of a nylon suture into the ICA to occlude the MCA for 2 h. Reperfusion was induced by suture withdrawal, and incisions were closed. Sham controls underwent the same surgery without MCA occlusion. Animals were randomized into 5 groups (n=10): Sham, Model, Puerarin, C-Pue-LP, and Rh2-Pue-LP. At 24 h post-MCAO, Sham and Model groups received intravenous saline, while treatment groups were administered 7.5mg/kg of respective formulations daily for 7 d.\u003c/p\u003e\n\u003cp\u003eEx Vivo Tissue Imaging\u003c/p\u003e\n\u003cp\u003eTo verify the targeting ability of Pue, C-Pue-LP, and Rh2-Pue-LP in middle cerebral artery occlusion (MCAO) model rats, Pue, C-Pue-LP, and Rh2-Pue-LP were labeled with DiR. MCAO rats were divided into three administration groups: DiR, C-DiR-LP, and Rh2-DiR-LP (all at a concentration of 40 \u0026mu;g/mL), with 3 rats per group. Additionally, sham-operated rats injected with normal saline (NS) via the tail vein served as the control group. At 2 h after tail vein injection of the drugs, the rats were anesthetized and sacrificed. The brain, heart, liver, spleen, lung, and kidney were harvested, and ex vivo imaging was performed using a small animal imaging system to observe the fluorescence distribution in the isolated tissues of each group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeurological evaluation and infarct analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNeurological function was evaluated 24 h post-MCAO using the Longa scale , assessing limb function and locomotion[19]. After evaluation, rats were euthanized, and brains were harvested for TTC staining[20]. Coronal slices were incubated in 2% TTC at 37 \u0026deg;C for 30 min, with viable tissue stained red and infarcted areas white. Infarct volume was quantified using Image J and expressed as a percentage of total brain volume to determine ischemic injury severity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathology and Tunel analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrain tissues were fixed in 4 % paraformaldehyde for 24 h, dehydrated, and embedded in paraffin. 4 \u0026mu;m thick coronal sections were prepared and stained with hematoxylin-eosin to observe morphological changes in the hippocampus and peri-ischemic regions under light microscopy.\u003c/p\u003e\n\u003cp\u003eFor TUNEL analysis, paraffin sections were deparaffinized, hydrated, and subjected to antigen retrieval. TdT enzyme reaction mixture was applied for 60 min at 37 \u0026deg;C to label DNA strand breaks, followed by sequential incubation with biotinylated anti-digoxigenin antibody and streptavidin-HRP. Color development was performed using DAB, and apoptotic cells were quantified by Image J software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParaffin-embedded brain sections were deparaffinized in xylene and rehydrated through graded ethanol. Antigen retrieval was performed via heat-induced epitope retrieval in pH 6.0 citrate buffer. Endogenous peroxidase activity was blocked with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 15 min, followed by blocking with 5% BSA in PBS for 1 h. Sections were incubated overnight at 4 \u0026deg;C with primary antibodies (1:200). After washing, biotinylated secondary antibodies (1:200) were applied for 1 h at room temperature, followed by streptavidin-HRP complex for 30 min. Color development used DAB, terminated with distilled water. Sections were counterstained with hematoxylin, dehydrated, cleared in xylene, and mounted. Protein expression was quantified by optical density analysis of positive staining using Image J software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParaffin-embedded brain tissues were sectioned into 4 \u0026mu;m slices, deparaffinized in xylene, and rehydrated with graded ethanol. Antigen retrieval was performed using high-temperature pressure in pH 6.0 citrate buffer, followed by permeabilization with 0.3 % Triton X-100 in PBS for 15 min and blocking with 5 % BSA for 1 h at room temperature. Sections were incubated overnight at 4\u0026deg;C with primary antibodies (1:200). After washing, Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:500) was applied in the dark for 1 h at room temperature. Nuclei were counterstained with DAPI using an antifade mounting medium. Fluorescent images were captured by laser confocal microscopy , with NLRP3 signals (red) and nuclei (blue).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum were analyzed using rat IL-1\u003cem\u003e\u0026beta;\u003c/em\u003e, IL-6, and TNF-\u003cem\u003e\u0026alpha;\u003c/em\u003e ELISA kits according to the manufacturer\u0026rsquo;s instructions. Samples, standards, and detection antibodies were sequentially added, incubated at room temperature, and washed. HRP-conjugated secondary antibodies were then applied, followed by color development and reaction termination. Absorbance at 450 nm was measured using a microplate reader, and cytokine concentrations were calculated from standard curves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn Vivo Safety Evaluation via Histopathological Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo verify the in vivo safety of Pue, C-Pue-LP, and Rh2-Pue-LP, normal rats were selected and subjected to tail vein injection of Pue, C-Pue-LP, or Rh2-Pue-LP once daily for 7 consecutive days. Rats in the blank control group received an equal volume of normal saline (NS) via the same route. After the 7-day treatment period, all rats were anesthetized and sacrificed. The brain, heart, liver, spleen, lung, and kidney were harvested, followed by hematoxylin-eosin (HE) staining. The morphological changes of the above tissues in each group were observed under a microscope.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were conducted using SPSS 20.0 software, while graphical representations were generated with Origin 2024. All data were presented as mean \u0026plusmn; standard deviation. one-way analysis of variance (ANOVA) was applied for intergroup comparisons. Statistical significance was defined as \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCharacterization of Liposome Particle Size Distribution, Transmission Electron Microscopy (TEM) Microscopic Morphology, and Content Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study replaced cholesterol in traditional liposomes with ginsenoside Rh2, successfully prepared Rh2-Pue-LP and C-Pue-LP using the thin-film dispersion method. Both liposomes presented milky-white translucent aqueous suspensions\u0026nbsp;(Fig. 1a), indicating a stable colloidal dispersion system formed under optimal thin-film sonication conditions. TEM results\u0026nbsp;(Fig. 1a)\u0026nbsp;showed round or elliptical morphologies for both, confirming Rh2 did not disrupt liposomes\u0026rsquo; typical microstructure. Particle size and zeta potential analyses\u0026nbsp;(Fig. 1a, Table 1)\u0026nbsp;revealed their average particle sizes (93.10 nm) were within the ideal 50-200 nm range, with no significant differences in PDI or zeta potential. This particle size facilitates stable circulation after rat tail vein injection, reduces embolization risk, and enhances tissue uptake via the EPR effect[21]. Rh2, with an amphiphilic structure similar to cholesterol, maintains liposome physical stability comparable to cholesterol. As a functional component, it has anti-inflammatory, anti-oxidant, and anti-apoptotic effects[22,23]; co-loading with puerarin may enhance cerebral ischemic region enrichment. Its natural origin offers superior biocompatibility and safety over synthetic cholesterol, providing a new clinical direction for liposomes[24].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro release study of Rh2-Pue-LP.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the drug release characteristics of Rh2-Pue-LP, an in vitro release kinetics study was performed and compared with free puerarin (Pue). The results showed that free Pue achieved 96.2 % cumulative release within 4 hours, presenting a rapid and nearly complete release pattern. In contrast, Rh2-Pue-LP exhibited significantly delayed release, with approximately 58 % cumulative release at 4 hours and increasing to around 60% after 12 hours\u0026nbsp;(Fig. 1b), indicating good sustained-release effects. This property is attributed to the unique bilayer membrane structure of liposomes, which encapsulates puerarin in hydrophobic or hydrophilic regions to form a physical barrier slowing drug diffusion, and the synergistic effect of ginsenoside Rh2, which regulates membrane fluidity and compactness to further restrict release. Critical for cerebral ischemia treatment-where post-stroke excitotoxicity, oxidative stress, and inflammation persist for hours to days causing secondary brain injury[25], Rh2-Pue-LP maintains stable drug supply in ischemic areas, avoids the difficulty of free Pue in sustaining an effective therapeutic window and potential systemic adverse reactions, and confirms its value as a structurally a stable controlled-release novel drug delivery system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeuroprotective of Rh2-Pue-LP in MCAO in rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the focal cerebral ischemia-reperfusion model of MCAO rats, neurological deficits were evaluated using the\u0026nbsp;Longa scale (0-4 points); rats with scores\u0026nbsp;of\u0026nbsp;2-3 (contralateral forelimb flexion, circling, falling) were selected, excluding those with severe injury (score 4) or unsuccessful modeling (score \u0026lt;2). TTC staining\u0026nbsp;(Fig.\u0026nbsp;1c)\u0026nbsp;showed no obvious white infarcted areas in the sham-operated group, while the model group\u0026rsquo;s infarct volume was (37.38\u0026plusmn;1.14)%. All treatment groups significantly reduced infarct volume: Pue group (30.88\u0026plusmn;0.61)%, C-Pue-LP group (24.59\u0026plusmn;1.20)%, and\u0026nbsp;Rh2-Pue-LP group (12.95\u0026plusmn;0.83%, 24% reduction compared with model group, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). In vitro studies confirmed the sustained release property of Rh2-Pue-LP, which matching pathological process after ischemia, avoids the \u0026quot;peak-valley effect\u0026quot; of free drugs, and thereby durably inhibits the nerve cell damage cascade.\u0026nbsp;Ginsenoside Rh2 itself\u0026nbsp;exerts\u0026nbsp;anti-inflammatory effects (inhibiting the activation of the NLRP3 inflammasome), antioxidant effects (increasing SOD activity), and anti-apoptotic effects (downregulating Caspase-3 expression)[26]. After embedding into the liposomal membrane, it not only maintains structural stability but also enhances neuroprotection through multi-target mechanisms. Rh2 may inhibit the excessive activation of microglia by regulating the JAK2/STAT3 signaling pathway,\u0026nbsp;thereby\u0026nbsp;reducing apoptosis of neurons in the peri-ischemic area.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults and Mechanism Analysis of Targeting Evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, liposomes were labeled with fluorescein DiR to enable visual tracking of drug distribution via targeted administration in rats. As shown in\u0026nbsp;(Fig. 2), no fluorescent intensity was detected in the isolated tissues of rats in the control group. In contrast, the drugs in the free DiR group, C-DiR-LP group, and Rh2-DiR-LP group were distributed to varying degrees in organs including the brain, heart, liver, spleen, lung, and kidney, with the fluorescent intensity in the brain being particularly prominent. Further comparison revealed that the brain fluorescent intensity of the Rh2-DiR-LP group was significantly higher than that of the C-DiR-LP group and the free DiR group, confirming that Rh2-DiR-LP can achieve targeted drug delivery, effectively cross the blood-brain barrier (BBB), reach brain injury sites, and increase puerarin accumulation in the brain. Free DiR, a small-molecule substance, distributes widely via passive diffusion; C-DiR-LP is easily cleared by the reticuloendothelial system (RES), accumulating in non-target organs and reducing brain delivery efficiency. Ginsenoside Rh2 modification plays a critical regulatory role: Rh2 can bind to specific receptors (as\u0026nbsp;GLUT1, LDLR) highly expressed on BBB endothelial cells and promote liposome transcytosis across the BBB via \u0026quot;receptor-mediated endocytosis.\u0026quot; This active targeting mechanism, absent in C-DiR-LP and free DiR, is the core reason for the efficient brain accumulation of Rh2-DiR-LP\u0026nbsp;[27].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematoxylin and Eosin (HE) Staining and TUNEL Apoptotic Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of HE staining\u0026nbsp;(Fig. 3a)\u0026nbsp;and TUNEL apoptosis analysis\u0026nbsp;(Fig.\u0026nbsp;3b)\u0026nbsp;directly demonstrated that brain cells in the sham-operated group had normal morphology, while the model group showed extensive necrosis, neuron loss, and a significant increase in cell apoptosis, confirming the successful establishment of the MCAO model. Although the Pue group alleviated tissue damage, notable cell shrinkage and glial hyperplasia remained. Compared with the Pue group, the C-Pue-LP group further reduced pathological changes but did not show a significant advantage in inhibiting apoptosis. The Rh2-Pue-LP group not only minimized neuron shrinkage and glial hyperplasia but also effectively suppressed apoptosis by significantly reducing the TUNEL-positive rate. This effect is attributed to ginsenoside Rh2: it modulates blood-brain barrier permeability to facilitate efficient liposome delivery to ischemic regions, activates the anti-apoptotic PI3K/Akt pathway to intervene in the apoptosis cascade and reduce neuronal death, and its low immunogenicity decreases liposome-induced immune rejection, safeguarding the repair of the brain tissue microenvironment and providing new strategies for ischemic stroke treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical analysis of TNF-\u0026alpha;, IL-1\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026beta;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e, IL-6, p-JAK2, and p-STAT3 protein expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical analysis\u0026nbsp;(Fig. 4)\u0026nbsp;revealed that in the sham-operated group, the expression levels of pro-inflammatory cytokines (TNF-\u003cem\u003e\u0026alpha;\u003c/em\u003e, IL-1\u003cem\u003e\u0026beta;\u003c/em\u003e, IL-6) and p-JAK2/p-STAT3 proteins were extremely low, confirming the low inflammatory baseline of normal brain tissue. In contrast, the model group exhibited intense positive staining for these proteins, indicating a close association between the post-stroke inflammatory cascade and the excessive activation of the signaling pathway.\u003c/p\u003e\n\u003cp\u003eAlthough the Pue group partially inhibited the release of pro-inflammatory cytokines and the activation of JAK2/STAT3, residual positive staining indicated limited regulatory capacity. The C-Pue-LP group, leveraging the advantages of liposomal delivery, further enhanced the suppression of inflammatory signals, highlighting the role of the carrier system in improving drug efficacy. Notably, the expression levels of inflammatory cytokines and phosphorylated proteins in the Rh2-Pue-LP group were comparable to those in the sham-operated group. Its unique superiority may stem from the multi-target properties of ginsenoside Rh2. In addition to synergizing with puerarin to inhibit the synthesis of inflammatory mediators, Rh2 may directly target the active site of JAK2 kinase, blocking signal transduction mediated by receptor tyrosine kinases and thus more effectively suppressing the excessive activation of microglia and astrocytes. Furthermore, the lipophilic structure of Rh2 may enhance the fusion efficiency of liposomes with cell membranes, promoting drug enrichment in the ischemic area.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence analysis of NLRP3 protein expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescence analysis\u0026nbsp;(Fig. 5a)\u0026nbsp;demonstrated that in the sham-operated group, only weak fluorescence of NLRP3 protein was observed, confirming its low expression level in normal brain tissue. In contrast, the intense enhancement of fluorescence signals in the model group revealed that the NLRP3-mediated inflammatory cascade was acutely activated after stroke and was closely associated with the progression of ischemic brain injury.\u003c/p\u003e\n\u003cp\u003eAlthough both the Pue group and the C-Pue-LP group could reduce the fluorescence intensity of NLRP3 in a dose-dependent manner, the fluorescence level of the Rh2-Pue-LP group almost returned to that of the sham-operated group, indicating a more thorough anti-inflammatory effect. This discrepancy may be attributed to the dual characteristics of Rh2-Pue-LP. On one hand, ginsenoside Rh2 can directly target the key assembly sites of NLRP3, block the oligomerization of ASC and the activation of Caspase-1, and inhibit the maturation and release of inflammatory cytokines such as IL-1\u0026beta; and IL-18 at the source. On the other hand, the unique molecular structure of Rh2 may enhance the fusion efficiency of liposomes with cell membranes, promote the efficient enrichment of drugs in the ischemic area, and synergize with puerarin to form a multiple protection mechanism of \u0026quot;precise targeting-signal blocking-sustained drug release\u0026quot;. In addition, the profound inhibition of NLRP3 by Rh2-Pue-LP may further mitigate blood-brain barrier disruption and neuronal pyroptosis caused by inflammation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInflammation Analysis of TNF-\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026alpha;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e, IL-1\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026beta;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e, IL-6\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eELISA results\u0026nbsp;(Fig. 5b)\u0026nbsp;showed that serum IL-1\u003cem\u003e\u0026beta;\u003c/em\u003e, IL-6, and TNF-\u003cem\u003e\u0026alpha;\u003c/em\u003e levels increased significantly in the model group, confirming systemic inflammatory responses after cerebral ischemia. The Pue and C-Pue-LP groups reduced these factors, but the Rh2-Pue-LP group exhibited a more remarkable inhibitory effect, with levels approaching those of the sham operation group. This superiority is attributed to its multi-dimensional regulatory effects: ginsenoside Rh2 regulates immune cell activity, inhibits monocyte and neutrophil chemotaxis, and reduces inflammatory factor release at the source\u0026nbsp;[28]; the liposomal carrier prolongs drug circulation, ensuring a continuous anti-inflammatory effect. Additionally, the synergy between Rh2 and puerarin enhances anti-inflammatory capacity. These results confirm its inhibitory effect on intracerebral inflammation and reveal its potential in regulating systemic inflammation.\u003c/p\u003e\n\u003cp\u003eIn summary, this study confirms that Rh2-Pue-LP, liposomes with ginsenoside Rh2 replacing cholesterol, exhibits significant advantages in the treatment of ischemic stroke. It forms a stable colloidal dispersion, similar to C-Pue-LP, with no significant differences in particle size, zeta potential, or microstructure. In vitro, it releases 60 % of the drug within 12 hours, which matches the pathological needs after ischemia. In the MCAO rat model, it reduces the infarct volume by 24.43 %, improves neurological deficit scores, and HE staining and TUNEL analysis show that it alleviates brain tissue necrosis, neuronal shrinkage, and apoptosis. It strongly inhibits the release of pro-inflammatory factors, blocks the JAK2/STAT3 pathway, and suppresses the activation of the NLRP3 inflammasome. ELISA results confirm that it has a better regulatory effect on systemic inflammation than free drugs and C-Pue-LP. This study demonstrates that Rh2-Pue-LP is a safe and efficient drug delivery system for cerebral ischemia. Future research needs to further explore its in vivo pharmacokinetic characteristics, long-term safety, and clinical transformation potential, thereby laying the foundation for the application of natural components as substitutes for traditional liposomal excipients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn Vivo Safety Study Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in (Fig. 6), hematoxylin-eosin (HE) staining was used to observe the histological morphology of the brain, heart, liver, spleen, lung, and kidney in rats from the blank control group and each administration group. The results showed no obvious histological abnormalities in all organs of rats after tail vein injection of Pue, C-Pue-LP, or Rh2-Pue-LP compared with the blank control group, with intact organ structures, normal parenchymal cells, and no pathological damage. These findings indicate that the three formulations have no obvious biotoxicity to the major organs of normal rats and possess good biocompatibility. It should be noted that this study only verified the safety under short-term (7-day) administration and specific dosages; the safety of Rh2-Pue-LP under long-term administration or at higher dosages still requires further experimental verification.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIschemic stroke, a cerebrovascular disorder with high disability and mortality rates, poses a severe threat to human health\u0026nbsp;[29,30]. Clarifying its pathogenesis and achieving efficient drug delivery to lesions are crucial. Puerarin, derived from Pueraria lobata, exerts neuroprotective effects against cerebral ischemia\u0026nbsp;[12], but its efficacy is limited by the blood-brain barrier (BBB), making the development of brain-targeted delivery systems an urgent challenge.\u003c/p\u003e\n\u003cp\u003eThe BBB is the primary physiological barrier restricting drug penetration into the brain parenchyma\u0026nbsp;[31]. Liposomes, lipid bilayer microvesicles (\u0026quot;artificial biomembranes\u0026quot;) that can encapsulate diverse cargoes, possess biocompatibility and tunable targeting capabilities, enhancing drug efficacy while reducing toxicity and thus serving as ideal carriers\u0026nbsp;[32,34]. Ginsenoside Rh2, a saponin constituent derived from medicinal plants such as Panax ginseng and Panax notoginseng, exhibits multiple bioactivities including anti-inflammatory and anti-tumor effects, and has been validated as an effective agent for mitigating middle cerebral artery occlusion (MCAO)-induced injury\u0026nbsp;[11]. Given its amphiphilic nature, substituting cholesterol with ginsenoside Rh2 to fabricate novel functional liposomal drug delivery systems holds significant translational potential. In the present study, ginsenoside Rh2 was used as a cholesterol replacement for liposome modification. Notably, ex vivo tissue distribution assays revealed that ginsenoside Rh2-modified liposomes significantly enhanced puerarin accumulation in the brain compared to cholesterol-modified liposomes and free puerarin. Furthermore, Rh2-Pue-LP treatment led to a marked reduction in cerebral infarct volume and a significant amelioration of neurological deficits. These findings confirm that Rh2-Pue-LP improves therapeutic outcomes in ischemic stroke, supporting ginsenoside Rh2-modified liposomes as a viable strategy for targeted drug delivery.\u003c/p\u003e\n\u003cp\u003eThe JAK2/STAT3 signaling pathway is implicated in a spectrum of physiological processes, including cell proliferation, differentiation, apoptosis, and immune regulation\u0026nbsp;[35,36]. Sustained activation of this pathway is closely associated with the initiation and progression of various inflammation-driven diseases. The NLRP3 inflammasome, a key effector of the innate immune system, mediates caspase-1 activation, which in turn promotes the maturation and secretion of pro-inflammatory cytokines (such as TNF-\u003cem\u003e\u0026alpha;\u003c/em\u003e, IL-1\u003cem\u003e\u0026beta;\u003c/em\u003e, IL-6) and triggers a robust inflammatory cascade. Importantly, inhibition of the JAK2/STAT3 pathway\u0026mdash;whether under basal conditions or in NLRP3 activator-induced inflammatory states\u0026mdash;effectively attenuates NLRP3 inflammasome activity and suppresses the production and release of pro-inflammatory cytokines\u0026nbsp;[37,38]. In our animal MCAO model, Rh2-Pue-LP intervention significantly inhibited JAK2/STAT3 pathway activation, downregulated NLRP3 expression, and reduced the levels of IL-1\u003cem\u003e\u0026beta;\u003c/em\u003e, IL-6, TNF-\u003cem\u003e\u0026alpha;\u003c/em\u003e, p-JAK2, and p-STAT3. These results provide valuable mechanistic insights into inflammation-related diseases and highlight the JAK2/STAT3 pathway as a potential therapeutic target. This observation is consistent with the findings of Wu et al., who demonstrated that reducing JAK2 and STAT3 phosphorylation levels inhibits downstream inflammatory responses, thereby alleviating disease pathology.\u003c/p\u003e\n\u003cp\u003eThis study has some limitations. We have not conducted cellular-level mechanism research. However, our data clearly indicate that ginsenoside Rh2 modification can enhance the brain accumulation of puerarin, reduce nerve damage, and alleviate inflammatory responses. This study provides a promising strategy for improving the efficacy of puerarin in treating brain diseases. Importantly, this delivery system does not have adverse effects on the brain or other major organ systems. Overall, our research results indicate that the ginsenoside Rh2-modified liposome system can target puerarin delivery to the brain, significantly improving its therapeutic efficacy in ischemic stroke.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that Rh2-Pue-LP, a liposomal formulation with ginsenoside Rh2 replacing cholesterol, exhibits enhanced therapeutic efficacy in ischemic stroke. Compared to free drugs and conventional liposomes, it reduces brain injury, alleviates neurological deficits, and suppresses inflammation by inhibiting the JAK2/STAT3 pathway and NLRP3 inflammasome. The formulation combines sustained release with multi-target regulation, offering a promising strategy for ischemic stroke treatment. Future research should explore its pharmacokinetics, long-term safety, and clinical translation potential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWMY: conducted research, analyzed data. HW: wrote original draft, analyzed data. LZ, TMB: conducted animal experiments. XJ, CXL, ZYP: participated in the preparation of the formulation. WJL: designed research and reviewed \u0026amp; edited article. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundind\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by General Project of Basic Research Plan of Guizhou Provincial Department of Science Technology (grant no. Qiankehe Jichu ZK[2024] General 361)，National Natural Science Foundation of China project(grant no. 82560779), General Project of the Basic Research Program of Guizhou Provincial Department of Science and Technology (grant no. Qiankehe Jichu-ZK [2022] General 472) and Guizhou Provincial Science and Technology Projects (grant no. Qiankehe Pingtai ZSYS[2025]016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data associated with this study can be obtained by reaching out to the corresponding author of this manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and Informed Consent: \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiments in this study have been approved by the local ethics committee of Guizhou University of Chinese Medicine (approval number: 20250605003), and all ARRIVE standards have been followed\u003cstrong\u003e.\u003c/strong\u003eAdult male Sprague-Dawley (SD) rats (220\u0026plusmn;20 g) were purchased from Henan Sikebeisi Biotechnology Co. Ltd. The experimental license number is SCXK (Yu) 2020-0005, The license number for the use is SCXK (Qian) 2021-0005. They were raised at a temperature of 25\u0026plusmn;2 \u0026deg;C, a relative humidity of 60\u0026plusmn;10 %, and a natural light-dark cycle.For at least seven days, they were given access to water and given certifed standard meals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants provided written informed consent prior to the publication of related data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing financial interests or personal relationships.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAloizou AM, Siokas V, Pateraki G, Liampas I, Bakirtzis C, Tsouris Z, et al. Thinking outside the Ischemia Box: Advancements in the Use of Multiple Sclerosis Drugs in Ischemic Stroke. 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Neurotox Res. 2022 ;40:78-88. https://doi: 10.1007/s12640-021-00456-5.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e \u003cstrong\u003eThe physicochemical properties of liposomes\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eFormulation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eParticle size (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003ePolydispersity Index\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eZeta potential (mV)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eEncapsulation efficiency (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003eDrug loading (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eC-Pue-LP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e93.53\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e0.252\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e-25.03\u0026plusmn;1.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e61.38\u0026plusmn;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e1.85\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eRh2-Pue-LP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e93.10\u0026plusmn;0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e0.282\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e-24.02\u0026plusmn;1.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e60.99\u0026plusmn;0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e1.85\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eC-Pue-LP is a liposome loaded with cholesterol, and Rh2-Pue-LP is a liposome loaded with ginsenoside Rh2.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ginsenoside Rh2-modified liposomes, Puerarin, Brain Ischemia, JAK2-STAT3 signaling pathway, Middle cerebral artery occlusion-reperfusion (MCAO/R)","lastPublishedDoi":"10.21203/rs.3.rs-8550026/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8550026/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Brain Ischemia poses a significant unmet medical need, demanding novel therapeutic approaches. Puerarin (Pue), despite its potential for treating brain disorders, suffers from poor blood-brain barrier (BBB) permeability due to its low oil/water partition coefficient. To overcome this, we developed a novel ginsenoside Rh2-based liposome formulation (Rh2-Pue-LP) to enhance Pue delivery to the ischemic brain. A rat model of middle cerebral artery occlusion-reperfusion (MCAO/R) was established. Neurological deficits were evaluated using the Longa scoring system 24 hours post-MCAO. After seven days of tail-vein administration of Rh2-Pue-LP, the following analyses were performed: TTC staining to assess cerebral infarct volume, HE and TUNEL staining to examine hippocampal histopathology, ELISA to quantify serum levels of IL-1β, TNF-α, and IL-6, immunofluorescence to detect NLRP3 expression, and immunohistochemistry to evaluate the activation of JAK2-STAT3 and expression of inflammatory cytokines. Additionally, this study was conducted to further verify the targeting ability and safety of the formulation. Our results showed Rh2-Pue-LP treatment reduced infarct volume, improved neurological function, and decreased serum levels of inflammatory cytokines (IL-1β, TNF-α, IL-6). Histological examination revealed better-preserved hippocampal neurons. Rh2-Pue-LP inhibited the JAK2-STAT3 signaling pathway and NLRP3 inflammasome expression, suppressing microglial activation and neuronal apoptosis. Additionally, Rh2-Pue-LP exhibited stronger brain targeting ability with no significant biotoxicity in vivo. Rh2-Pue-LP represents a promising strategy for treating ischemic stroke by enhancing Pue delivery and exerting potent neuroprotective effects.","manuscriptTitle":"Ginsenoside Rh2-Modified Liposomes for Targeted Delivery of Puerarin Alleviate Brain Ischemia-Reperfusion Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 17:24:37","doi":"10.21203/rs.3.rs-8550026/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-08T02:23:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-26T21:32:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-21T14:13:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15368647025556049204628885803439929356","date":"2026-02-14T22:12:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156831434765478500917204067386695750186","date":"2026-02-14T01:01:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110487476918469213632782089590448592019","date":"2026-02-13T09:58:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"121946184658784306858594372647263801406","date":"2026-02-13T00:05:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-19T13:56:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-09T11:52:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-09T11:52:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Drug Delivery and Translational Research","date":"2026-01-08T09:47:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6cffb64a-d26f-4e17-a724-d6c309990f4c","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T16:17:48+00:00","versionOfRecord":{"articleIdentity":"rs-8550026","link":"https://doi.org/10.1007/s13346-026-02123-8","journal":{"identity":"drug-delivery-and-translational-research","isVorOnly":false,"title":"Drug Delivery and Translational Research"},"publishedOn":"2026-04-19 15:58:16","publishedOnDateReadable":"April 19th, 2026"},"versionCreatedAt":"2026-01-21 17:24:37","video":"","vorDoi":"10.1007/s13346-026-02123-8","vorDoiUrl":"https://doi.org/10.1007/s13346-026-02123-8","workflowStages":[]},"version":"v1","identity":"rs-8550026","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8550026","identity":"rs-8550026","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}