Abstract

Background

   Ischemic stroke (IS) remains a leading cause of long-term disability.
   Neurovascular regeneration and remodeling of the corticospinal tracts
   are essential for neurological functional recovery. Zuogui pill (ZGP)
   has good efficacy in treating cerebral ischemia, but the mechanism
   remains unclear.

Purpose

   To investigate the effects of ZGP on angiogenesis, neurogenesis,
   corticospinal tract (CST) remodeling, and further evaluate its
   mechanisms of action in mice with ischemic stroke.

Methods

   Network pharmacology was used to analyze the active components, related
   targets, and mechanisms of ZGP’s action in promoting neurovascular
   regeneration after ischemic stroke. Using a photothrombotic (PT) stroke
   mouse model, ZGP’s effects on neurological recovery were assessed using
   behavioral tests. Angiogenesis and neurogenesis were evaluated by
   immunofluorescence of glucose transporter type 1(Glut-1)
   +/5-bromo-20-deoxyuridine (BrdU) + vessels and doublecortin
   (DCX)+/BrdU+ cells. CST remodeling was evaluated through diffusion
   tensor imaging (DTI). The levels of vascular endothelial growth factor
   (VEGF), brain-derived neurotrophic factor (BDNF), and mammalian target
   of rapamycin (mTOR) expression were tested by Western blot.

Results

   Network pharmacology identified 94 active ingredients and 83
   overlapping targets related to IS and neurovascular regeneration. mTOR
   was identified as one of the core targets. Behavioral tests
   demonstrated ZGP significantly reduced error rates in irregular ladder
   walking (ZGP-H vs Stroke: p=0.003) and shortened sticker removal time
   (ZGP-H vs Stroke: p=0.003). Immunofluorescence revealed ZGP enhanced
   angiogenesis (Glut-1+/BrdU+ vessels: ZGP-H vs Stroke, p=0.018), neural
   progenitor cell proliferation and migration (BrdU+/DCX+ cells: ZGP-H vs
   Stroke: p=0.014). DTI showed increased fractional anisotropy (FA) in
   ipsilateral CST regions (ZGP-H vs Stroke: 0.001<p<0.05). Western blot
   confirmed elevated VEGF (p=0.002), BDNF (p=0.002), and p-mTOR/mTOR
   ratio (p<0.001) in peri-infarct tissues.

Conclusion

   ZGP promotes neurovascular regeneration, CST remodeling, and
   neurological function recovery after ischemic stroke. The positive
   impacts of ZGP are linked to heightened VEGF and BDNF expression and
   the activation of the mTOR pathway.

   Keywords: zuogui pill, ischemic stroke, angiogenesis, neurogenesis,
   corticospinal tract remodeling, mTOR

Graphical Abstract

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Video Abstract

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Introduction

   Ischemic stroke, accounting for 87% of the 14 million stroke cases
   worldwide annually according to the World Health Organization (WHO),
   remains a leading cause of severe neurological dysfunction.[48]1
   Patients who fail to receive vascular recanalization therapy within the
   critical 4.5–6hour window after symptom onset face high risks of
   permanent neurological deficits, including hemiparesis, cognitive
   impairment, speech disorders, and dysphagia. These consequences impose
   substantial burdens on individuals, families, and society. In
   developing countries, limited access to timely interventions due to
   restricted therapeutic windows and medical resources often results in
   lifelong disability for early-onset stroke patients.[49]2 Following
   ischemic brain injury, persistent inflammatory responses and
   degenerative changes hinder the neurovascular system’s ability to
   restore normal function, leading to irreversible deficits.[50]3

   Neurovascular regeneration and CST remodeling are critical for
   post-stroke neurological recovery.[51]4 Mirzahosseini et al[52]5
   demonstrated that vascular endothelial growth factor (VEGF) and
   brain-derived neurotrophic factor (BDNF) serve as pivotal signaling
   molecules in these processes. Exogenous administration of VEGF and BDNF
   has been shown to activate regenerative pathways, mediating
   angiogenesis, neurogenesis, synaptic remodeling, and functional
   restoration in experimental stroke models.[53]6 Central to these
   mechanisms is the mTOR signaling pathway, which regulates cells’
   inherent capacity for growth and repair.[54]7,[55]8 Evidence suggests
   that crosstalk between BDNF and VEGF activates mTOR signaling,
   synergistically promoting angiogenesis and neurogenesis.[56]9 Thus,
   therapies that elevate BDNF/VEGF levels and stimulate mTOR signaling
   hold promise for enhancing brain regeneration and neurological recovery
   after ischemic stroke.

   In traditional Chinese medicine (TCM), ischemic stroke pathogenesis is
   attributed to an imbalance of yin and yang, coupled with kidney essence
   deficiency, which manifests as cerebral marrow depletion during the
   recovery phase. Insufficient kidney essence leads to the loss and
   difficulty in repairing the brain marrow. New blood vessels are created
   when the kidneys are nourished and the essence is filled, which
   nourishes the brain marrow and promote its repair.[57]10 ZGP, a
   classical TCM formula, is renowned for its ability to tonify kidney yin
   and generate marrow. Clinical studies have confirmed ZGP’s efficacy in
   improving neurological outcomes in ischemic stroke patients.[58]11
   Preclinical research further supports its neuroprotective properties,
   promoting axonal regeneration, and anti-inflammatory
   effects.[59]12,[60]13 However, whether ZGP promotes angiogenesis,
   neurogenesis, and CST remodeling—and the underlying mechanisms—remains
   unclear. In this study, we employed network pharmacology to identify
   potential targets and pathways of ZGP in treating ischemic stroke. The
   effects of ZGP on angiogenesis, neurogenesis, CST remodeling,
   neurological recovery, and the underlying mechanisms were also verified
   by using a mouse model of ischemic stroke.

Materials and Methods

Prediction and Identification of Active Components and Target Genes of ZGP

   Cervi Cornus Colla, Testudinis Carapacis ET Plastri Colla, Rehmanniae
   Radix Praeparata, Dioscoreae Rhizoma, Corni Fructus, Lycii Fructus,
   Achyranthis Bidentatae Radix, and Cuscutae Semen are the principal
   constituents of ZGP. The TCMSP database was used to obtain and validate
   the primary chemical constituents of the nine medicinal materials that
   make up ZGP. Oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥
   0.18 are the screening parameters used to find the active ingredients
   in the Chinese drug composite. Searches were conducted using the HERB
   and SymMap databases to find medications not listed in the TCMSP
   database. Targets that were pertinent to drug-related components were
   found using the SwissTargetPrediction database.

Disease Target Screening

   Disease-related targets for ischemic stroke (IS) and neurovascular
   regeneration were retrieved from the OMIM, GeneCards, and TTD databases
   using the keywords “ischemic stroke” and “neurovascular regeneration”.

Common Targets of ZGP Against Ischemic Stroke and Neurovascular Regeneration

   A method of crossing the predicted targets with those of ischemic
   stroke and neurovascular regeneration was used to identify potential
   ZGP targets for the treatment of ischemic stroke and the promotion of
   neurovascular regeneration.

Construction “Herb- Ingredients – Targets” Network Diagram

   The drug-disease common targets and potentially active ingredients of
   ZGP were entered into the Cytoscape3.9.1 program, and the separated
   components which did not intersect with the targets were eliminated,
   resulting in the creation of a network diagram of “ Herb - Ingredients
   - Targets diseases”.

Protein-Protein Interaction (PPI) Network Analysis

   Choose “Homo sapiens” as the protein species and 0.7 as the minimum
   interaction threshold when searching the above-mentioned STRING
   database targets. The minimum interaction threshold of 0.7 in the
   STRING database was selected to ensure high-confidence protein-protein
   interactions. This threshold balances specificity and biological
   relevance, avoiding overly dense networks while retaining functionally
   significant interactions critical for identifying core targets. By
   removing isolated nodes, PPI network was produced.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
Enrichment Analysis

   The GO and KEGG enrichment analyses on common targets were carried out
   using the David database, and the R programming language was utilized
   to visualize the enrichment outcomes.

Animals and Photothrombotic Focal Stroke Model

   The research involved adult male C57BL/6J mice aged 8 to 10 weeks, with
   weights ranging from 25 to 30 grams and ages between 2.5 and 3 months.
   The Ethics Committee of the Affiliated Hospital of Nanjing University
   of Chinese Medicine sanctioned all animal studies (2021 DW-04-02,
   February 9, 2021). All animal procedures were conducted in accordance
   with the National Institutes of Health (NIH) Guide for the Care and Use
   of Laboratory Animals (8th edition, 2011) and the ARRIVE Guidelines
   2.0. A photothrombotic stroke model was induced following the method
   described by Liu et al with minor modifications.[61]14 Briefly, mice
   received a tail vein injection of Rose Bengal (10 mg/kg body weight, 5
   mg/mL in saline) five minutes before focal illumination of the intact
   skull. A 4-mm-diameter cold light source (Zeiss CL1500HAL, 150 W,
   3000K) was positioned 2.5 mm lateral to Bregma to target the right
   sensorimotor cortex. Illumination lasted five minutes. Successful
   induction of the photothrombotic stroke model was confirmed 72 hours
   post-stroke through two independent validation criteria: MRI
   T2-weighted imaging demonstrated hyperintense signal by revealing ≥4
   distinct ischemic lesions in the right sensorimotor cortex, while the
   irregular ladder walking test corroborated functional impairment by
   showing an error rate ≥50% in the injured forelimb compared to
   pre-stroke baseline performance. These stringent multimodal criteria
   ensured both structural consistency of ischemic damage and reproducible
   sensorimotor deficits across experimental cohorts.

Experimental Design

   The outline and flow of the experiment are shown in [62]Figure 1.
   Cystamine (cystamine dihydrochloride, Sigma, USA) has been shown to
   enhance axon remodeling, neuronal progenitor cell proliferation, and
   functional recovery after ischemic stroke.[63]15 It was selected as a
   positive control drug in this study. Seventy-two hours post-ischemic
   stroke induction, the test subjects (40 male C57BL/6J mice in total)
   were randomized into four groups: Stroke group (Stroke, n=10), low-dose
   ZGP group (ZGP-L, 3.7 g/kg, n=10), high-dose ZGP group (ZGP-H, 7.4
   g/kg, n=10), and Cystamine group (100 mg/kg, intraperitoneally, n=10).
   The low and high doses of ZGP (3.7 g/kg and 7.4 g/kg, respectively)
   were selected based on our previous published studies demonstrating
   efficacy and safety of ZGP in ischemic stroke models.[64]13 Initially,
   the human equivalent dose (36.2 g per formulation) was converted to a
   mouse-equivalent dose using body surface area normalization, multiplied
   by a safety factor of 3, to establish the highest tested dose (14.8
   g/kg for the high-dose group). Subsequent sequential halving generated
   intermediate and low doses of 7.4 g/kg and 3.7 g/kg, respectively.
   However, preliminary experiments revealed that 14.8 g/kg induced
   toxicity and reduced efficacy. Subsequent dose-response evaluations
   confirmed that 7.4 g/kg and 3.7 g/kg achieved optimal therapeutic
   effects without adverse effects, justifying their selection for this
   study. The ZGP solution was prepared in saline and administered via
   oral gavage once daily. The stroke group received an equivalent volume
   of saline alone using the same regimen. Cystamine was dissolved in
   0.09% saline and given intraperitoneally every day. To identify
   proliferating cells, each animal was administered intraperitoneal
   injections of BrdU (Sigma, USA) dissolved in 1×PBS buffer at a daily
   dose of 50 mg/kg. All interventions started on day 3 and continued to
   day 30 days after ischemia induction. Behavioral tests were performed
   pre-stroke and on days 3, 7, 14, and 30 post-stroke to assess motor and
   sensory functions of mice. On day 14 of ischemia induction, Nissl and
   immunofluorescence staining were used to observe histopathological
   changes, angiogenesis, proliferation and migration of neural progenitor
   cells. On day 30 of ischemia induction, 7.0T MRI diffusion tensor
   imaging (DTI) was performed to visualize the remodeling of CST in mice,
   and BDNF, VEGF, and mTOR protein expression in peri-infarct tissues was
   examined by protein immunoblotting.

Figure 1.

   [65]Figure 1
   [66]Open in a new tab

   Experimental design timeline for evaluating ZGP’s effects in ischemic
   stroke mice. Mice underwent photothrombotic (PT) stroke induction on
   Day 0. ZGP (low/high dose), Brdu, and cystamine treatments began on Day
   3 and continued until Day 30. Behavioral tests (irregular ladder
   walking, sticker removing) for evaluating functional recovery were
   performed pre-stroke (baseline) and post-stroke (Days 3, 7, 14, 30).
   Histological (Nissl, immunofluorescence) analyses evaluating
   angiogenesis and neurogenesis were performed at Days 14, and imaging
   (MRI, DTI) analyses for evaluating CST remodeling were conducted at
   Days 3 and 30. Western blot analysis for elucidating the mechanisms
   underlying ZGP’s effects was performed at Days 30.

ZGP Preparation

   A single dose of ZGP consists of a blend of 8 traditional Chinese herbs
   in the proportions of 8: 4: 4: 4: 3: 4: 4: 4, which includes 24g of
   Rehmanniae Radix Praeparata, 12g of Dioscoreae Rhizoma, 12g of Corni
   Fructus, 12g of Lycii Fructus, 9g of Achyranthis Bidentatae Radix, 12g
   of Cervi Cornus Colla, 12g of Testudinis Carapacis ET Plastri Colla,
   and 12g of Cuscutae Semen. All the herbs originated in China. The ZGP,
   created by the pharmacy department at Jiangsu Provincial Hospital of
   Chinese Medicine, was validated using the 2015 edition of the Chinese
   Pharmacopoeia. The pharmacy at Jiangsu Provincial Hospital of Chinese
   Medicine obtained a reference sample (2023–0615). ZGP was prepared
   according to the method described in the previous
   study.[67]13,[68]16,[69]17 30 prescriptions (3150g) were made in total,
   and 1086g of ZGP-dried powder were produced. The substance was stored
   in portions at −20°C. ZGP has been analyzed by UPLC-QTOF-MS assay, and
   detailed information can be obtained by referring to our previously
   published article.[70]16

Irregular Ladder Walking Test

   The capacity for ladder walking was evaluated in various mouse groups
   employing a method detailed in earlier studies.[71]13,[72]14 Proper
   movements (Hit) occur when the palm’s center is accurately positioned
   on the rung for both front and back limbs, with the fingers of the
   front limbs closed. The leftover instances were classified as errors,
   divided into two types: 1) Miss: When a mouse is moving along the
   ladder, its forelimb or hindlimb either completely misses the rung or
   touches it with the wrist or heel instead of the paw; 2) Slip: When a
   mouse uses only a few fingers instead of the paw to grip the rungs,
   leading to a subsequent slide. The results were presented using the
   (Miss + Slip) / (Hit + Miss + Slip).

Sticker Removing Test

   Prepare stickers with a size of 3mm × 3mm, apply them to the center of
   the mouse’s forelimb palm with ophthalmic forceps, and place the mouse
   in a transparent glass device. Record the time it takes for the mouse
   to perceive and remove the sticker, with a maximum recording time of 2
   minutes. All experimental animals were tested on one side of the
   forelimb palm and then the other side. Each mouse was tested 3 times
   before the induction of ischemia, and the average of the 3 tests was
   used as the baseline.

Triphenyl Tetrazolium Chloride (TTC) Staining

   On the third day after focal cerebral ischemia, the brains of mice were
   cut into 1mm thick coronal slices from front to back in a mouse brain
   mold. Brain sections were immersed in PBS solution with 1% TTC and kept
   at 37°C for 15 minutes. The brain sections, which were stained, were
   submerged in a 4% paraformaldehyde (PFA) phosphate buffer solution and
   left to be fixed for a day.

Immunofluorescence Staining

   Two weeks post-stroke, brains preserved in paraffin were cut into 4μm
   thick coronal sections, and immunohistochemistry was conducted
   following the methods outlined by Li et al.[73]18,[74]19 The primary
   antibodies employed for single or double staining included rat
   anti-BrdU (1:400; Abcam) for cell proliferation, goat anti-doublecortin
   (DCX; 1:200; Santa Cruz Biotechnology) for migrating neuroblasts, and
   rabbit anti-Glut-1 (1:1000; Chemicon, Temecula, CA) for endothelial
   cells. Secondary antibodies used were Alexa Fluor 488 anti-rat,
   anti-goat, anti-rabbit IgG (1:200; Invitrogen, Carlsbad, CA), or
   Cy3-conjugated anti-rat IgG (1:1000; Invitrogen). The staining was
   observed using fluorescent and confocal microscopy (BX61; Olympus,
   Tokyo, Japan). For each mouse, three coronal brain sections were
   selected for cell counting, with each slice separated by 300μm within
   the same target area. In the ischemic border area, four fields per
   brain slice were randomly selected for multistage random sampling.
   Every counting assay was carried out under blind conditions.

Nissl Staining

   After paraffin-embedded brain slices were deparaffinized, the slices
   were immersed in cresyl violet for 2–3 minutes. The excess staining
   solution was rinsed off with water, and the sections were
   differentiated with glacial acetic acid and observed under the
   microscope. Differentiation was continued until the background turned
   light blue and the nidus turned dark blue. The differentiation process
   was stopped and the neuronal cells morphology was observed under the
   microscope in randomly selected fields around the infarct area.

Western Blot Analysis

   According to our earlier study,[75]18 the peri-infarct region was
   defined by a 500μm boundary stretching from the medial, lateral, and
   infarct edges toward the infarct. Proteins were isolated from tissue
   specimens of the peri-infarct region through homogenization in a
   protein lysis solution. The amount of protein was measured through the
   BCA method. The SDS-PAGE gel electrophoresis was performed at 80 volts
   for half an hour, followed by 120 volts for one hour. To convert the
   membrane, a steady current was applied of 250 mA for 90 minutes, then
   cool it on ice, and finally, seal it with skimmed-milk powder for an
   hour at 25°C. The membranes were stored at 4°C overnight with one of
   these primary antibodies: mouse anti-mTOR (1:5000), rabbit anti-p-mTOR,
   rabbit anti-VEGF, mouse anti-BDNF, or mouse anti-β-actin. Following a
   one-hour incubation with secondary antibodies (goat anti-rabbit IgG at
   1:5000 or goat anti-mouse IgG at 1:2000) and a 30-second to two-minute
   exposure to ECL reagents, the membranes were placed on Kodak film
   (Japan). Use ImageJ program to measure and evaluate IOD ratios to
   display the data.

Magnetic Resonance Imaging (MRI)

   Thirty days post-stroke, MRI scans were conducted on mice utilizing a
   7.0 Tesla small animal MRI system (Bruker PharmaScan, Ettlingen,
   Germany) as described in a prior study.[76]15 The ImageJ software
   (National Institutes of Health, Bethesda, MD, USA) was utilized to
   determine the infarct volume by subtracting the volume of the uninjured
   ipsilateral hemisphere from that of the contralateral hemisphere in
   T2-weighted images. Subsequently, a computerized analysis was performed
   to express the infarct volume as a percentage, calculated by dividing
   the infarct volume by the volume of the contralateral hemisphere.
   Diffusion tensor imaging (DTI) revealed injury to the corticospinal
   tract within the sensorimotor cortex and internal capsule. An
   echo-planar imaging (EPI) sequence was parameterized with 30 distinct
   diffusion directions and 5 reference images. Paravision 5.0 software
   (Bruker Biospin, Ettlingen, Germany) was employed to compute fractional
   anisotropy (FA), focusing on the anatomical regions of the corpus
   callosum and hippocampus for localization. Areas of interest (ROIs)
   were outlined on FA maps within the sensory-motor cortex and corpus
   callosum across five brain image planes called Genu, Body, Dorsal
   fornix, Dorsal syndesis, and Dentate gyrus. Fiber bundle tracts were
   reconstructed, visualized, and examined with Diffusion Toolkit
   (v0.6.2.1) and TrackVis (v0.5.2.1) software. Regions of interest (ROIs)
   were chosen for the motor-sensory cortex at the corpus callosum’s knee
   and for the internal capsule structures at the hippocampus dentate
   gyrus level.

Statistics

   The data are presented as the average value plus or minus the standard
   error of the mean. Each experiment was performed as a separate,
   independent experiment. To minimize bias, all behavioral assessments,
   histological analyses, and data quantification were conducted by
   investigators blinded to experimental group assignments. Group labels
   (eg, Stroke, ZGP-L, ZGP-H, Cystamine) were anonymized during data
   collection and analysis. Quantitative and statistical analyses of
   imaging, molecular biology, histological regional results, and
   immunofluorescence staining cell count results were performed using
   ImageJ software. The software GraphPad Prism 9, based in La Jolla,
   California, USA, was utilized for creating plots. To evaluate group
   differences, we employed one-way Analysis of Variance (ANOVA), the
   Brown-Forsythe test, multiple-comparison methods, and Tukey’s multiple
   comparisons test. A P-value below 0.05 suggests a meaningful
   statistical outcome.

Results

Construction of “Herb- Ingredients – Targets” Network Diagram

   After filtering and removing duplicates, 94 potentially active
   ingredients were identified from ZGP, including 4 from Achyranthis
   Bidentatae Radix, 45 from Lycii Fructus, 6 from Testudinis Carapacis ET
   Plastri Colla, 5 from Cervi Cornus Colla, 16 from Dioscoreae Rhizoma,
   20 from Corni Fructus, 2 from Rehmanniae Radix Praeparata, 11 from
   Cuscutae Semen. Using the SwissTargetPrediction database, 711 potential
   drug targets associated with these ingredients were screened.
   Concurrently, disease-related targets for ischemic stroke (IS) and
   neurovascular regeneration were retrieved from the OMIM, GeneCards, and
   TTD databases, yielding 7,424 unique ischemic stroke targets and 444
   neurovascular regeneration targets after deduplication. A Venn diagram
   intersecting ZGP targets (711), ischemic stroke targets (7,424), and
   neurovascular regeneration targets (444) revealed 83 common targets
   ([77]Figure 2A). These overlapping targets were further visualized in a
   “Herb-Ingredients-Targets” network to integrate active ingredients,
   their herbal sources, and shared disease-related mechanisms ([78]Figure
   2B). This network highlights ZGP’s multi-component, multi-target
   therapeutic profile, with node sizes reflecting interaction degrees to
   prioritize core targets for subsequent analysis.

Figure 2.

   [79]Figure 2
   [80]Open in a new tab

   Network pharmacology analysis of ZGP’s active components and targets.
   (A) Venn diagram illustrating the intersection of ZGP-related targets
   (711), ischemic stroke-related targets (7,424), and neurovascular
   regeneration-related targets (444), identifying 83 common therapeutic
   targets. (B) The network diagram of “Herb-Ingredients-Targets”. The
   hexagons in the graph represent the drugs, the circles represent the
   respective unique components of each drug, the triangles represent the
   common components of the drugs, the diamonds represent the 83 common
   targets, and the node sizes vary depending on the size of the node
   degree value. There are 162 nodes and 574 edges in the graph. The
   network highlights ZGP’s multi-component, multi-target therapeutic
   profile, with core targets prioritized for mechanistic investigation.

PPI Network Construction and Core Target Analysis

   The 83 drug-disease common targets were imported into the STRING
   database to create a PPI network of protein interactions ([81]Figure
   3A). To map the network of protein interactions, the resulting network
   data was imported into the Cytoscape program to import the PPI network
   ([82]Figure 3B). NetworkAnalyzer tool was used to perform a topology
   analysis and find core targets according to degree values. The top 20
   targets ranked by degree values were subsequently plotted as a bar
   graph using R 4.2.1 ([83]Figure 3C), highlighting key nodes such as
   tumor necrosis factor (TNF), signal transducers and activators of
   transcription 3 (STAT3), mTOR, and matrix metalloproteinase 9 (MMP9),
   which exhibited the highest connectivity and are critically implicated
   in neurovascular regeneration pathways.

Figure 3.

   [84]Figure 3

   Continued.

Figure 3.

   [85]Figure 3

   PPI network construction and core target analysis. (A): PPI network of
   83 common targets related to ZGP, ischemic stroke, and neurovascular
   regeneration, generated using the STRING database. Node sizes (69 nodes
   and 187 edges) and color intensity reflect degree values (larger/darker
   nodes = higher connectivity). (B): Refined network visualized in
   Cytoscape, highlighting densely connected modules. (C): Top 20 core
   targets ranked by degree centrality. Key targets such as TNF, STAT3,
   mTOR, and MMP9 exhibit high degrees, indicating their pivotal roles in
   neurovascular regeneration and ischemic stroke recovery.
   [86]Open in a new tab

Analysis of GO and KEGG Enrichment

   According to the results of GO enrichment analyses, a total of 387
   biological processes (BPs), 52 cellular components (CCs), and 93
   molecular functions (MFs) were enriched for overlapping genes. The top
   10 significantly enriched terms (ranked by P-value) for each category
   were visualized as bar and bubble charts ([87]Figure 4A and [88]B). For
   biological processes (BPs), the most enriched terms included response
   to xenobiotic stimulus, positive regulation of phosphatidylinositol
   3-kinase/protein kinase B signaling, and transport across the
   blood-brain barrier. Key cellular components (CCs) comprised the plasma
   membrane, receptor complexes, and membrane rafts, while predominant
   molecular functions (MFs) involved identical protein binding, nuclear
   receptor activity, and peptidase activity. KEGG pathway analysis using
   the DAVID database identified 115 enriched pathways. The top 20
   pathways with the lowest P-values were visualized in bar and bubble
   charts ([89]Figure 5A and [90]B), where warmer colors (eg, red)
   indicated higher significance (-log10[P-value]). Major signaling
   pathways included proteoglycans in cancer, pathways in cancer, insulin
   resistance, calcium signaling, HIF-1 signaling, neuroactive
   ligand-receptor interaction, diabetic cardiomyopathy, IL-17 signaling,
   and relaxin signaling.

Figure 4.

   [91]Figure 4
   [92]Open in a new tab

   GO enrichment analysis of ZGP targets. (A): Bubble chart displaying the
   top 10 enriched GO terms (Biological Process, BP; Cellular Component,
   CC; Molecular Function, MF). Bubble size represents gene count; color
   intensity reflects −log10(p-value). Key processes include “response to
   xenobiotic stimulus” (BP), “plasma membrane” (CC), and “identical
   protein binding” (MF). (B): Bar graph summarizing the top 10 enriched
   GO terms across all categories, ranked by statistical significance.
   Together, these results highlight ZGP’s multi-faceted roles in
   neurovascular regeneration, spanning cellular localization, molecular
   interactions, and signaling pathway modulation.

Figure 5.

   [93]Figure 5
   [94]Open in a new tab

   KEGG pathway enrichment analysis of ZGP targets. (A): Bubble chart of
   the top 20 enriched KEGG pathways ranked by statistical significance.
   Bubble size represents the number of genes associated with each
   pathway, and color intensity reflects the −log10(P-value) (redder hues
   indicate higher significance). Key pathways include HIF-1 signaling,
   neuroactive ligand-receptor interaction, calcium signaling, and IL-17
   signaling. (B): Bar graph summarizing the same top 20 pathways, ranked
   by −log10 (P-value).

ZGP’s Influence on Infarct Size in Mice After Ischemic Stroke

   A reproducible cortical infarction was established using the method of
   photochemical thrombosis, which unilaterally destroys the sensory motor
   cortex ([95]Figure 6A and [96]B). On day 3 after cerebral ischemia, TTC
   staining showed complete cell necrosis in the right sensorimotor cortex
   region, which appeared white ([97]Figure 6A). The other red areas were
   normal brain tissue with active cells. MRI T2-weighted images showed
   significant high signal in the right sensorimotor cortex, suggesting
   acute infarction ([98]Figure 6B). This indicates that the
   photothrombotic focal stroke model has good consistency and
   reproducibility. On day 30 after cerebral ischemia, MRI T2-weighted
   images showed tissue necrosis and liquefaction at the infarct site,
   forming a cystic cavity ([99]Figure 6B). There were no significant
   variations in infarct size among the stroke, ZGP-treated, and
   cysteamine-treated groups (p =0.536, [100]Figure 6B and [101]C).

Figure 6.

   [102]Figure 6
   [103]Open in a new tab

   Photothrombotic (PT) stroke model and the impact of ZGP on functional
   recovery following stroke. (A): Representative distinct pale infarct
   area was observed in TTC staining 3 days after focal stroke induced by
   photothrombotic method. (B): Representative focal infarcts in the right
   sensorimotor cortex were demonstrated by T2-weighted images at 3 days
   and 30 days after photothrombotic focal stroke. (C): Measurement of
   infarct volume at 30 days. (D) Functional recovery was evaluated
   through irregular ladder walking and sticker removing experiments. Data
   are expressed as the mean ± SEM (n=5). * p<0.05, ** p<0.01, ***
   p<0.001, relative to the stroke cohort.

ZGP Promotes Functional Recovery in Mice Following Ischemic Stroke

   To evaluate ZGP’s therapeutic effects on post-stroke functional
   recovery, motor and sensory functions were assessed using irregular
   ladder walking and sticker removing tests. In the irregular ladder
   walking test, the error rate of the injured forelimb increased to
   approximately 50% across all groups by day 3 post-stroke, significantly
   higher than pre-stroke baselines. From days 14 to 30, the cysteamine
   group (positive control) showed a markedly lower error rate compared to
   the stroke group (p = 0.006, [104]Figure 6D). By day 30, both ZGP-L
   (3.7 g/kg) and ZGP-H (7.4 g/kg) groups achieved error rates comparable
   to the cysteamine group (p = 0.682 and p = 0.962, respectively), with
   both doses demonstrating significant improvement over the stroke group
   (p = 0.013 and p = 0.003, respectively, [105]Figure 6D). In the
   uninjured forelimb, no significant differences were observed among
   groups (p > 0.05, [106]Figure 6D), except that the cysteamine group
   exhibited a significantly lower error rate compared to the stroke group
   (p = 0.031, [107]Figure 6D). Similar to the results of irregular ladder
   walking, the sticker removal test revealed that ZGP-L, ZGP-H, and
   cysteamine groups exhibited significantly shorter removal times for the
   injured forelimb compared to the stroke group at day 30 (p = 0.006, p =
   0.003, and p < 0.001, respectively, [108]Figure 6D). There was no
   significant difference in the removal times of the uninjured forelimb
   among the groups (p>0.05, [109]Figure 6D). These data collectively
   demonstrate that ZGP enhances sensorimotor recovery in ischemic stroke
   mice, with efficacy comparable to the positive control cysteamine.

Effects of ZGP on Brain Tissue Surrounding Infarct Lesion

   Nissl staining revealed distinct histopathological differences between
   the contralateral normal cortex and peri-infarct regions. In the
   contralateral normal cortex, neurons were densely packed, uniformly
   stained, and exhibited well-organized laminar architecture with clearly
   defined cellular boundaries ([110]Figure 7A). In contrast, the
   peri-infarct tissue of stroke mice displayed marked structural
   degradation, including neuronal loss, cytoplasmic-nuclear boundary
   blurring, vacuolation, connective tissue hyperplasia at lesion margins,
   and fragmented or faintly stained Nissl bodies. Compared to the stroke
   group, mice treated with ZGP-L, ZGP-H, and cysteamine showed attenuated
   tissue damage, characterized by reduced neuronal loss, fewer vacuoles,
   preserved cellular morphology with sharp cytoplasmic boundaries, and
   abundant Nissl substance ([111]Figure 7A). These findings suggest that
   ZGP mitigates post-stroke histopathological degeneration in
   peri-infarct regions.

Figure 7.

   [112]Figure 7
   [113]Open in a new tab

   Effects of ZGP on histopathology and angiogenesis in the sensory motor
   cortex at 14 days after stroke. (A): Typical pictures of Nissl staining
   in the area surrounding the infarct of the sensory motor cortex of mice
   in different groups. (B): Immunostaining for the endothelial cell
   marker Glut-1 and the proliferation marker BrdU was used to identify
   angiogenesis and vessel density in the area surrounding the infarct.
   Dual-positive staining for Glut-1 (green) and BrdU (red), along with
   the combined Glut-1/BrdU image from mice across various groups. (C):
   Quantification of vessels double-positive for Glut-1/BrdU was
   demonstrated. (D): Quantification of cells positive for BrdU was
   demonstrated. (E) The measurement of vessels positive for Glut-1 was
   demonstrated. Data are expressed as the mean ± SEM (n=5). * p<0.05, **
   p<0.01, *** p<0.001, in comparison to the stroke cohort. Scale bars in
   (A) = 40 µm; Scale bars in (B) =100µm.

ZGP Promotes Angiogenesis and Vascularization in the Sensory Motor Cortex
After Stroke

   To determine the potential role of ZGP in vascular regeneration, Glut-1
   and BrdU-labeled vessels were detected by immunofluorescence staining
   to assess angiogenesis and vascularization 14 days after cerebral
   ischemia. As seen in [114]Figure 7B, the peri-infarct regions of ZGP-L,
   ZGP-H, and cysteamine-treated mice exhibited significantly higher
   counts of BrdU-positive cells (ZGP-L vs Stroke: p = 0.049; ZGP-H vs
   Stroke: p = 0.030; Cysteamine vs Stroke: p < 0.001), Glut-1/BrdU
   double-labeled vessels (ZGP-L vs Stroke: p = 0.003; ZGP-H vs Stroke: p
   = 0.018; Cysteamine vs Stroke: p = 0.001), and Glut-1-positive vessels
   (ZGP-L vs Stroke: p = 0.007; ZGP-H vs Stroke: p = 0.048; Cysteamine vs
   Stroke: p = 0.044) compared to the stroke group ([115]Figure 7B–E). No
   significant differences were observed among the ZGP-L, ZGP-H, and
   cysteamine groups in BrdU-positive cells, Glut-1/BrdU double-labeled
   vessels, or Glut-1 labeled vessels (p > 0.05, [116]Figure 7B–E). These
   results demonstrate that ZGP successfully enhanced angiogenesis and
   increased Glut-1-positive vessel density in post-stroke brain tissue,
   suggesting its potent pro-angiogenic effects.

ZGP Promotes Neurogenesis and Neuroblast Migration in Mice After Ischemic
Stroke

   Following focal ischemia, neuroblasts originating from the
   subventricular zone (SVZ) of the lateral ventricles proliferate and
   migrate toward the ischemic lesion. These migrating neuroblasts can be
   identified by their expression of doublecortin (DCX). To evaluate ZGP’s
   effects on neuroblast proliferation and migration, immunofluorescence
   double staining for BrdU (proliferation marker) and DCX was performed
   at 14 days post-stroke. As shown in [117]Figure 8A, a substantial
   number of BrdU- and DCX-positive cells were observed in the SVZ of
   stroke mice. Compared to the stroke-only group, both ZGP-L and ZGP-H
   treatments significantly increased the number of BrdU-positive cells
   (ZGP-L vs Stroke: p = 0.009; ZGP-H vs Stroke: p = 0.032), DCX-positive
   cells (ZGP-L vs Stroke: p = 0.023; ZGP-H vs Stroke: p = 0.035), and
   BrdU/DCX double-positive cells (ZGP-L vs Stroke: p = 0.017; ZGP-H vs
   Stroke: p = 0.014) ([118]Figure 8A–D). No significant differences were
   observed between ZGP-treated groups and the cysteamine positive control
   (p > 0.05, [119]Figure 8A–D). Furthermore, neuroblast migration was
   assessed in the white matter tract between the infarcted cortex and
   ipsilateral SVZ ([120]Figure 8E and [121]F). ZGP-L, ZGP-H, and
   cysteamine treatments significantly elevated DCX-positive cell density
   in this region (ZGP-L vs Stroke: p = 0.015; ZGP-H vs Stroke: p = 0.021;
   Cysteamine vs Stroke: p = 0.009) ([122]Figure 8F and [123]G). Notably,
   DCX-positive cells in ZGP-treated mice migrated farther from the SVZ
   compared to the stroke-only group. These results indicate that ZGP
   enhances both the proliferation of neural progenitor cells in the SVZ
   and their directed migration toward the ischemic lesion.

Figure 8.

   [124]Figure 8
   [125]Open in a new tab

   Effects of ZGP on neurogenesis and neuroblast migration at 14 days
   after stroke. (A): Typical dual immunofluorescent staining pictures of
   BrdU (red), DCX (green) and merged pictures in the same side of SVZ in
   different groups. (B): The count of BrdU/DCX double-labeled cells in
   the SVZ was displayed. (C): The number of BrdU positive cells in SVZ
   was shown. (D): The quantity of DCX-positive cells in the SVZ was
   displayed. (E): The study focused on the migration of neuronal cells
   within the white matter (boxed area) located between the damaged lesion
   (white) and the same side SVZ. (F): Typical immunofluorescent staining
   pictures of DCX (green) in the white matter between the ischemic cortex
   and ipsilateral SVZ. (G): Quantification of DCX labeled cells was shown
   between the ischemic cortex and ipsilateral SVZ. Data are expressed as
   the mean ± SEM (n=5). * p<0.05, ** p<0.01, compared to the stroke
   group. Scale bars = 100 µm.

ZGP Promotes Corticospinal Tract Axon Remodeling After Stroke

   The aforementioned results demonstrated that ZGP enhanced neurological
   recovery, angiogenesis, and neurogenesis in cerebral ischemia mice. To
   further evaluate its long-term effects on axonal remodeling, in vivo
   DTI was performed 30 days post-stroke, with fractional anisotropy
   (FA)—a key DTI parameter reflecting white matter integrity—analyzed
   across five corticospinal tract (CST) regions: Genu, Body, Dorsal
   fornix, Dorsal syndesis, and Dentate gyrus ([126]Figure 9A). On the
   ipsilateral side of injury, ZGP-L, ZGP-H and cysteamine treatments
   significantly increased FA values in at least four CST regions compared
   to the stroke group (0.001<p<0.05), with no significant differences
   between ZGP and cysteamine groups (p > 0.05) ([127]Figure 9B).
   Contralaterally, FA values showed no group differences except in the
   Genu, where cysteamine exhibited higher values than the stroke group (p
   = 0.015) ([128]Figure 9C). Fiber tracking further revealed that ZGP and
   cysteamine significantly increased ipsilateral fiber density at the
   Genu (sensorimotor cortex) and Dentate gyrus (internal capsule)
   compared to the stroke group (0.001<p<0.01, [129]Figure 10A and
   [130]B). No differences were observed among treated groups (p > 0.05),
   while contralateral fiber density showed a non-significant upward trend
   (p > 0.05, [131]Figure 10A–C). Collectively, these findings indicate
   that ZGP promotes CST axon remodeling, particularly in ipsilateral
   regions, aligning with its functional recovery benefits observed in
   behavioral and histological assays.

Figure 9.

   [132]Figure 9
   [133]Open in a new tab

   Effect of ZGP on corticospinal tract axon remodeling. (A): FA color
   maps of representative orientation coding of typical brains from
   different groups of mice in five different levels from the dentate
   gyrus of hippocampus to the genu. Orientation of fiber trajectories is
   color-coded, with green showing the anterior and posterior directions,
   blue demonstrating the superior and inferior directions, and red
   indicating the left and right directions. (B): FA values measured in
   the levels of Genu, Body, Dorsal fornix, Dorsal syndesis, and Dentate
   gyrus in the ipsilateral side of the injury from different groups of
   mice. (C): FA values quantified in the contralateral side of the injury
   at the same locations as in (B). Data are expressed as the mean ± SEM
   (n=5). * p<0.05, ** p<0.01, *** p<0.001, in contrast to the stroke
   cohort.

Figure 10.

   [134]Figure 10
   [135]Open in a new tab

   The role of ZGP in the sensorimotor fiber tracking. (A): Typical photos
   of multi-dimensional fiber tracing of ROI at the levels of genu and
   dentate gyrus in different groups of mice 30 days after stroke. The ROI
   radius is 0.468 mm. Color coding is employed to show the fiber tracing
   direction: green for anterior and posterior, red for left and right,
   and blue for superior and inferior. (B): Quantitative assessment of
   fiber tracking density within the regions of interest at the genu and
   dentate gyrus levels in various groups of mice in the ipsilateral side
   of the injury. (C): Quantitative analysis of fiber tracking intensity
   of ROI at levels of genu and dentate gyrus in different groups of mice
   in the contralateral side of the injury. Data are expressed as the mean
   ± SEM (n=5). ** p<0.01, *** p<0.001, compared to the stroke cohort.

ZGP Enhances the Expression of VEGF, BDNF and p-mTOR Proteins

   To elucidate the mechanisms underlying ZGP’s therapeutic effects,
   Western blot analysis was performed on peri-infarct tissues 30 days
   post-stroke to quantify levels of mTOR, p-mTOR, and neurovascular
   regeneration markers VEGF and BDNF ([136]Figure 11A). Compared to the
   stroke group, the ZGP-H and cysteamine groups exhibited significantly
   elevated VEGF (ZGP-H vs Stroke: p = 0.002; Cysteamine vs Stroke: p =
   0.010), BDNF (ZGP-H vs Stroke: p = 0.002; Cysteamine vs Stroke: p =
   0.050), and p-mTOR/mTOR ratios (ZGP-H vs Stroke: p < 0.001; Cysteamine
   vs Stroke: p = 0.031) ([137]Figure 11A–D). Compared to the stroke
   group, the ZGP-L group exhibited elevated VEGF(p=0.723), BDNF(p=0.306),
   and p-mTOR/mTOR ratio levels, with the p-mTOR/mTOR ratio showing a
   statistically significant difference (p=0.008, [138]Figure 11B–D). Full
   original Western blot images corresponding to all experimental groups
   are provided in [139]Figure S1.The above findings indicate that the
   promotion of neurovascular regeneration and axonal remodeling by ZGP
   may be associated with the upregulation of VEGF and BDNF expression and
   activation of mTOR signaling pathway.

Figure 11.

   [140]Figure 11
   [141]Open in a new tab

   Impact of ZGP on BDNF, VEGF, mTOR, and p-mTOR proteins expression. (A):
   Typical photographs of Western blot for BDNF, VEGF, mTOR, and p-mTOR in
   ischemic brain 30 days post-stroke in various groups. (B): The
   p-mTOR/mTOR ratio in ischemic brain tissues was quantified by
   normalization to β-actin. (C): Quantitative expression of VEGF in
   tissues around the infarct lesion. (D) Quantitative expression of BDNF
   in tissues around the infarct lesion. Data are expressed as the mean ±
   SEM (n=3). * p<0.05, ** p<0.01, *** p<0.001, compared to the stroke
   cohort.

Discussion

   While ischemic stroke induces massive cell death within the ischemic
   lesion, it also induces neurovascular regeneration responses in
   peri-infarct and more distant regions of the ischemic lesion.[142]20
   These responses involve two interconnected processes: angiogenesis and
   neuroregeneration, the latter comprising neurogenesis and axonal
   remodeling.[143]21 However, the regenerative and reparative capacity of
   adult neural tissue is severely limited, and the extent of spontaneous
   regenerative repair induced by cerebral ischemia falls far short of
   clinical rehabilitation demands.[144]22 Consequently, exploring
   pharmacological agents and therapeutic approaches to promote
   neurovascular regeneration and repair represents a pivotal strategy for
   enhancing functional recovery in ischemic stroke.

   Recent advances in neurovascular regeneration highlight the potential
   of multi-target strategies to address post-stroke recovery.
   Cutting-edge approaches, such as exosome-mediated BDNF delivery[145]23
   and immune-neurovascular crosstalk regulated by immunomodulatory
   hydrogel microspheres,[146]24 enhance angiogenesis and axonal
   remodeling but face challenges in specificity and clinical translation.
   In contrast, TCM compound preparations, characterized by
   multi-component synergy, multi-pathway modulation, and low toxicity,
   offer unique advantages for ischemic stroke treatment.[147]25,[148]26
   For instance, Naotaifang alleviates ischemic stroke-induced brain
   injury by modulating microglial polarization to suppress
   neuroinflammation and ferroptosis,[149]27,[150]28 while also mitigating
   neuronal damage through restoring mitochondrial fission/fusion
   homeostasis.[151]29 Despite these advances, research on the therapeutic
   effects of TCM in promoting neurovascular regeneration and repair after
   cerebral ischemia remains limited, underscoring the need for further
   mechanistic exploration. As a representative TCM formula, Zuogui Pill
   (ZGP) has been used since the Ming Dynasty to nourish kidney yin and
   replenish marrow. Our prior studies identified 76 bioactive compounds
   in ZGP via UPLC-QTOF-MS, including 14 high-abundance components.[152]16
   These active ingredients have various biological effects, for example,
   anti-oxidant and anti-inflammatory, immune enhancement,
   neuroprotection, promotion of axonal regeneration and neurite’s
   outgrowth, and improvement of cognition and memory.[153]16 Our clinical
   trials confirm ZGP’s efficacy in improving neurological outcomes in
   stroke patients.[154]11 The basic researches have shown that ZGP can
   enhance axonal regeneration and crossing ability of neural cells in
   vitro, promote axonal growth and restore neural function in ischemic
   stroke mice.[155]13,[156]16,[157]17 Moreover, research by Liu et
   al[158]30 indicates that ZGP can suppress inflammation, safeguard
   neurons, and improve cognitive function. However, whether ZGP promotes
   angiogenesis and neurogenesis post-stroke has not been systematically
   investigated until this study.

   In this study, we employed network pharmacology to elucidate the
   mechanisms underlying ZGP’s therapeutic effects on ischemic stroke,
   with a focus on neurovascular regeneration. Through systematic
   screening, we identified quercetin, beta-sitosterol, isorhamnetin,
   hydroxygenkwanin, and kaempferol as ZGP’s core components promoting
   post-stroke neurovascular repair. These compounds have been extensively
   documented to enhance synaptic plasticity, stimulate vascular
   endothelial cell proliferation/differentiation, and improve cognitive
   function.[159]31–33 Mechanistically, they activate the Cyclic AMP
   Response Element-Binding Protein (CREB) /BDNF signaling pathway and
   modulate Mitogen-Activated Protein Kinase (MAPK), mTOR, and VEGF
   cascades.[160]33,[161]34 PPI network analysis prioritized 20
   high-confidence targets for ZGP, with mTOR emerging as a central node.
   mTOR is a crucial signaling molecule that regulates the inherent
   capacity of neurovascular cells to develop. Our team’s previous
   research has found that the mTOR pathway is inhibited in the adult
   nervous system.[162]35 By reactivating the mTOR pathway, it was able to
   promote the regeneration of optic nerve axons and recovery of vision
   after optic nerve injury, and significant regeneration of CST axons as
   well as recovery of neurological function after spinal cord injury and
   cerebral ischemia.[163]14,[164]36 Therefore, activating the mTOR
   pathway can stimulate intrinsic growth ability and promote the
   regeneration and repair of brain tissue. In addition, there is evidence
   to suggest that BDNF can promote neuronal differentiation and
   facilitate axonal growth through activating the mTOR signaling
   pathway.[165]37 Furthermore, VEGF/VEGF receptor 2 interaction triggers
   mTOR signaling to drive endothelial proliferation and vascular lumen
   formation,[166]9,[167]38 synergistically enhancing ZGP’s neurovascular
   regenerative capacity.

   Employing a photothrombotic focal cerebral ischemia rodent model, we
   demonstrated that ZGP promotes angiogenesis, neurogenesis, and function
   recovery post-stroke. To evaluate its therapeutic effects during the
   subacute recovery phase, ZGP treatment was initiated on day 3
   post-ischemia. Angiogenesis and neurogenesis were assessed via
   Glut-1/BrdU and DCX/ BrdU dual labeling, respectively. After 14 days of
   ZGP administration, peri-infarct regions exhibited a marked increase in
   Glut-1+/ BrdU+ endothelial cells and vascular density, confirming ZGP’s
   pro-angiogenic effects ([168]Figure 7B-E). Concurrently, ZGP enhanced
   neural progenitor cell proliferation in the subventricular zone (SVZ),
   evidenced by elevated DCX+/BrdU+ cell counts, and facilitated their
   migration toward ischemic lesions ([169]Figure 8A-G). By day 30,
   ZGP-treated mice showed significant sensorimotor functional recovery,
   with error rates in ladder walking reduced and sticker removal time
   shortened compared to stroke controls ([170]Figure 6D). These findings
   provide direct experimental evidence that ZGP concurrently enhances
   vascular and neural regeneration, offering a promising therapeutic
   strategy for ischemic stroke recovery.

   We further elucidated the mechanisms by which ZGP promotes
   neurovascular regeneration and functional recovery. Post-stroke repair
   involves dual strategies: optimizing the regenerative microenvironment
   and enhancing intrinsic cellular repair capacity.[171]22 It is widely
   recognized that boosting VEGF and BDNF levels can enhance the
   regenerative environment following cerebral ischemia, aiding in
   neurovascular growth and axonal restructuring. Furthermore, VEGF
   induction triggers BDNF secretion from endothelial cells, creating a
   feedforward loop that enhances neuronal survival and circuit
   reorganization.[172]23 Western blot analysis of peri-infarct tissues at
   30 days post-stroke revealed that ZGP significantly increased VEGF and
   BDNF levels while elevating the p-mTOR/mTOR ratio compared to stroke
   controls ([173]Figure 11A-D). These results indicate that ZGP
   orchestrates brain repair through coordinated actions: enhancing
   regenerative factor secretion (BDNF/VEGF) to improve microenvironmental
   support, while reactivating mTOR signaling to potentiate intrinsic
   neurovascular growth pathways. This dual mechanism maximizes endogenous
   repair potential, as illustrated in our integrated model ([174]Figure
   12).

Figure 12.

   [175]Figure 12
   [176]Open in a new tab

   ZGP’s mechanisms of action for accelerating ischemic stroke recovery.

   Axonal remodeling is a critical component of post-stroke brain repair.
   The corticospinal tract (CST)—a major projection fiber bundle
   connecting the sensorimotor cortex and spinal cord—plays a pivotal role
   in sensorimotor function recovery through axon regeneration, collateral
   sprouting, and neural network reorganization following cerebral
   ischemia. Diffusion tensor imaging (DTI), a noninvasive MRI technique
   leveraging water molecule’ anisotropic diffusion, provides precise
   quantification of white matter microstructure.[177]39 DTI-derived
   fractional anisotropy (FA) values enable three-dimensional CST
   reconstruction, revealing axonal integrity, myelination status, and
   fiber density. Reduced FA correlates with CST axonal loss and
   functional deficits, making it a validated biomarker for monitoring
   treatment-induced recovery.[178]40 In this study, DTI analysis at 30
   days post-stroke demonstrated that ZGP treatment significantly
   increased ipsilateral CST axon density and integrity, as confirmed by a
   marked elevation in FA values, comparable to the positive control
   cysteamine ([179]Figure 9A-B). Fiber tracking revealed that ZGP
   restored orderly sensorimotor axon projections to the internal capsule,
   counteracting stroke-induced fiber loss and disorganization
   ([180]Figure 10A-B). Crucially, ZGP’s lack of effect on infarct volume
   underscores that its therapeutic efficacy arises from enhancing
   peri-infarct plasticity rather than reducing acute ischemic damage,
   aligning with the core principles of subacute therapeutic strategies.

   While this study elucidates ZGP’s role in promoting neurovascular
   regeneration and corticospinal tract remodeling, certain limitations
   should be acknowledged. First, the impact of ZGP on blood-brain barrier
   (BBB) repair—a critical mediator of neurovascular coupling and
   post-stroke recovery —was not directly assessed. Future studies
   employing BBB-specific markers and functional permeability assays will
   address these gaps. Second, while we identified mTOR signaling as a
   central mechanism, ZGP’s interactions with related
   pathways—particularly insulin-like growth factor-1 (IGF-1)/ osteopontin
   (OPN)/ phosphatase and tensin homolog deleted on chromosome ten (PTEN)
   crosstalk—and its effects on axonal remodeling markers
   (growth-associated protein 43 [GAP43], myelin basic protein [MBP],
   synapsin1 [SYN1]) remain incompletely characterized. Systematic
   interrogation using pathway-specific inhibitors (eg, linsitinib for
   IGF-1 receptor, rapamycin for mTOR) and spatial transcriptomics will
   clarify these mechanisms. These limitations highlight the need for
   further mechanistic exploration to optimize ZGP’s therapeutic potential
   in ischemic stroke.

Conclusion

   Our research results indicate that ZGP can induce neurogenesis and
   angiogenesis, enhance CST remodeling, and facilitate the recovery of
   mice’s neurological function following a focal stroke. The mechanisms
   by which ZGP produces these impacts are connected with the regulation
   of VEGF and BDNF expression, and activating the mTOR pathway. These
   findings provide the first direct evidence that ZGP—a kidney-nourishing
   TCM formula—bridges neurovascular repair by simultaneously upregulating
   trophic factors (VEGF, BDNF) and reactivating mTOR-dependent
   regenerative pathways. Future studies should explore ZGP’s impact on
   blood-brain barrier restoration and validate signaling pathways’
   crosstalk using pathway-specific inhibitors. Clinical translation via
   multimodal neuroimaging (eg, DTI-coupled perfusion MRI) will further
   elucidate its therapeutic potential in stroke patients.

Funding Statement

   This work was supported by National Natural Science Foundation of China
   (Grant No. 82474435,81973794), Natural Science Foundation of Jiangsu
   Province (Grant No. BK20241996), Academic Degree and Postgraduate
   Education Reform Project of Jiangsu Province (Grant No. SJCX24_1012).

Abbreviations

   ZGP, Zuogui Pill; IS, Ischemic stroke; CST, corticospinal tract; TTC,
   2,3,5-triphenyl-2H-tetrazolium chloride; DTI, diffusion tensor imaging;
   BDNF, brain-derived neurotrophic factor; VEGF, vascular endothelial
   growth factor; mTOR, mammalian target of rapamycin; p-mTOR,
   phosphor-mammalian target of rapamycin; BrdU, 5-bromo-20-deoxyuridine;
   DCX, doublecortin; SVZ, subventricular zone; Glut-1, glucose
   transporter type 1; FA, fractional anisotropy; WHO, World Health
   Organization; ROIs, Regions of interests; ANOVA, One-way analysis of
   variance.

Ethics Statement

   The animal study protocol in the study was approved by the Animal
   Ethics Committee of Affiliated Hospital of Nanjing University of
   Chinese Medicine (2021 DW-04-02, February 9, 2021).

Author Contributions

   All authors made a significant contribution to the work reported,
   whether that is in the conception, study design, execution, acquisition
   of data, analysis and interpretation, or in all these areas; took part
   in drafting, revising or critically reviewing the article; gave final
   approval of the version to be published; have agreed on the journal to
   which the article has been submitted; and agree to be accountable for
   all aspects of the work.

Disclosure

   The authors report no conflicts of interest in this work.

References