Sodium tanshinone IIA sulfonate improves the ischemic microenvironment by inhibiting apoptosis and promotes the treatment of ischemic brain injury by iPSCs

Sodium tanshinone IIA sulfonate improves the ischemic microenvironment by inhibiting apoptosis and promotes the treatment of ischemic brain injury by iPSCs | 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 Sodium tanshinone IIA sulfonate improves the ischemic microenvironment by inhibiting apoptosis and promotes the treatment of ischemic brain injury by iPSCs Yaoyao Li, Xue Liu, Min Wang, Lin feng Li, Xin yi Wang, Ran Yin, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7950969/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Ischemic stroke is the second leading cause of disability and death worldwide. Sodium tanshinone IIA sulfonate (STS), a well-known Chinese medicine monomer, is commonly used to treat ischemic brain injury because of its anti-apoptotic, anti-oxidative and anti-inflammatory properties. The aim of this study was to clarify the mechanism of STS combined with induced pluripotent stem cells (iPSCs) in treating ischemic brain injury by investigating changes in the ischemic microenvironment in a rat model of cerebral ischemia-reperfusion injury. Methods This study employed magnetic resonance imaging (MRI) to examine functional recovery after a combined therapeutic approach involving STS and iPSCs in vivo. Cerebral ischemia was induced by the middle cerebral artery occlusion approach, and thirty male rats were randomly assigned to five groups: Sham, MCAO, MCAO + STS, MCAO + iPSCs, and MCAO + iPSCs + STS. MRI were conducted on Days 1 and 7, with neurofunctional tests performed every other day. Additionally, 1.0×10⁶GFP-Luc-iPSCs were transplanted on Day 3 and bioluminescence imaging in brains was performed on Days 1 and 5 after transplantation. Proteomic analysis and immunofluorescent analyses were performed on Day 7. Results STS reduced infarct size and enhanced neurological function scores after MCAO. Furthermore, proteomic analysis revealed extensive remodeling of the ischemic microenvironment, with 365 differentially expressed proteins between the Sham and MCAO groups. Proteomic analysis demonstrated that STS primarily improved the ischemic microenvironment by inhibiting apoptosis between the MCAO and STS groups, a finding that was further validated by Western blotting and TUNEL assays. We next assessed iPSCs survival after transplantation. Compared with the iPSCs group, the combined treatment with STS significantly improved iPSCs survival, reduced infarct size and enhanced neurological function scores. Immunofluorescence revealed an increased expression of NeuN and CD31, demonstrating that the combined therapy more effectively promoted neurovascular repair. Moreover, increased GFP co-localization with BDNF, VEGF, and DCX was observed in the combined treatment with STS, indicating STS enhanced iPSCs-mediated paracrine effects and neurogenic potential. Conclusions STS enhances the survival and proliferation of iPSCs by improving the ischemic microenvironment through the suppression of apoptosis and enhances iPSCs-mediated paracrine mechanisms and their potential for differentiation. In summary, STS combined with iPSCs could be a more effective therapeutic approach than using these stem cells individually. Sodium tanshinone IIA sulfonate (STS) induced pluripotent stem cells (iPSCs) middle cerebral artery occlusion (MCAO) magnetic resonance imaging (MRI) ischemic microenvironment apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Stroke is characterized by high morbidity and high mortality, which have caused serious social and economic burdens.[ 1 ] Approximately 87% of strokes are ischemic strokes, in which a blood vessel supplying the brain is obstructed.[ 2 ] Existing therapeutic strategies, including pharmacotherapy, interventional procedures and surgery, have limited efficacy in improving neural function due to a narrow therapeutic window and poor drug penetration across the blood-brain barrier.[ 3 ] Therefore, it is urgent to explore new therapeutic strategies for ischemic stroke. Stem cells hold great promise for the treatment of Central Nervous System diseases.[ 4 ] Several cell types have been proposed to enhance post ischemic brain repair, including induced pluripotent stem cells(iPSCs), mesenchymal stem cells and neural stem cells.[ 5 ] iPSCs can be derived from somatic cells, expanded to large quantities and differentiated into all kinds of cell types, which makes them a promising cell source for therapeutic purposes with fewer ethical concerns.[ 6 ] However, transplantation of iPSCs into ischemic areas had low survival rates due to the harsh microenvironment. Thus, their clinical application is greatly limited.[ 7 ] The brain microenvironment after ischemic stroke includes alterations in ionic homeostasis, overproduction of inflammatory cytokines, and abnormal release of proteases.[ 8 ] When stem cells are transplanted, they are exposed to these harmful factors, more than 80% of implanted cells are sentenced to death 72 hours after injection by the harsh microenvironment.[ 9 ] Traditional Chinese medicine has a long history in the treatment of stroke and has achieved good therapeutic effects[ 10 , 11 ] has been extensively studied for its anti-apoptosis, anti-inflammation, and neurovascular-protective properties.[ 12 ] Tanshinone IIA (Tan IIA) is the effective component isolated from Danshen for treating ischemic stroke.[ 13 ] However, poor water solubility and oral bioavailability limit its biomedical application. Sodium tanshinone IIA sulfonate (STS), a water-soluble derivative of Tan IIA, exhibits high metabolic stability owing to a sulfonic group substitution at the C-16 position.[ 14 ] A clinical trial revealed that STS improved neurologic functional outcomes in acute ischemic stroke patients using traditional thrombolysis therapy[ 15 ] suppresses microglia polarization and neuroinflammation through the regulation of the miR-125b-5p/STAT3 axis to ameliorate neuropathic pain, demonstrating its potential in the treatment of neurological disorders.[ 16 ] Studies have demonstrated that STS can improve the post-ischemic microenvironment through multiple mechanisms, including inhibition of neuronal apoptosis,[ 17 ] suppression of inflammatory responses[ 18 ] and promotion of angiogenesis[ 19 ] modulating these pathological processes, STS reduces secondary injury and promotes endogenous repair. Given the aforementioned points, we hypothesized that the combination of STS and iPSCs transplantation could enhance therapeutic efficacy in ischemic brain injury. To test this hypothesis, we utilized a middle cerebral artery occlusion (MCAO) rat model and conducted proteomic analysis to explore the mechanisms by which STS modulates the ischemic microenvironment. Transfection with lentiviral vectors was applied to iPSCs to trace their survival post-transplantation and immunofluorescence staining was used to evaluate neurovascular repair. Together, these approaches aimed to determine whether STS could potentiate the therapeutic benefits of iPSCs, thereby providing a novel strategy for the treatment of cerebral ischemia. Materials and Methods Stem Cell Culture We would like to thank Professor Jiachuan Wang for donating the human iPSCs cell line. iPSCs were cultured using ncEpic hPSC complete medium (Shownin Biotech, Cat. No. RP01001). After diluting 120 µL of 500 µg/mL vitronectin (VTN) in 9 mL of DMEM/F12, 1.5 mL of the mixture was dispensed into each well of a 6-well plate and left at room temperature for 1 hour before use. Subsequently, the cells were incubated in 2 ml of 0.5 mM EDTA for 7–8 minutes at 37°C. The EDTA was aspirated and replaced with 2 mL of complete medium containing 0.5 µL of 10 mM Blebbistatin (Shownin Biotech, Cat. No. RP01008). The cells were gently pipetted 1–2 times and replated onto VTN-coated 6-well plates at a 1:10 ratio. The plates were gently rocked to distribute the cells evenly. Medium was changed 18–24 hours after seeding and then refreshed daily. Lentiviral transfection of Stem Cells IPSCs were transfected with a lentivirus to express Green Fluorescent Protein and luciferin (GFP-Luc) for in vivo experiments. iPSCs were seeded at a density of 2 × 10⁵ cells per well in 6-well plates. Multiplicity of infection was set at 10. The following day, virus was added to 1 mL of medium per well, followed by a 4-hours incubation. Subsequently, an additional 1 mL of medium was added to bring the total volume to 2 mL. The medium was replaced 24 hours post-infection. Clonal Selection of Stem Cells To obtain iPSCs with a high level of fluorescent marker expression, transfected cells were subjected to clonal selection. GFP-Luc-iPSCs were seeded at a low density of 3 × 10³ cells per well in 6-well plates and cultured for 72 hours. Fluorescence expression was identified using fluorescence microscopy of the GFP-Luc-iPSCs and areas of high expression were marked on the underside of the culture plate. GFP-Luc-iPSCs were treated for 5 minutes with an EDTA solution. The EDTA was then aspirated and replaced with complete medium containing blebbistatin immediately. The marked GFP-Luc-iPSCs were carefully aspirated using a 100 µL pipette and transferred to VTN-coated 24-well plates for continued culture. Animals Healthy male specific-pathogen-free (SPF) grade Sprague–Dawley (SD) rats (220–240 g) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. The rats had ad libitum access to food and water and were housed in a room with a 12:12-hour dark-light cycle. All animal experiments were conducted in compliance with China's animal welfare legislation regarding the care and use of animals and were approved by the Experimental Animal Ethics Committee of Zhejiang Provincial People's Hospital (approval number: 20240927814920). Animal model establishment To induce cerebral ischemia via MCAO, rats were anesthetized using 1.5% sodium pentobarbital (50 mg/kg). A midline incision was performed on the neck to expose the left common carotid artery (CCA), external carotid artery, and internal carotid artery (ICA). The left common and external carotid arteries were isolated and subsequently ligated. A microvascular clip was temporarily applied to the internal carotid artery. A 4–0 nylon suture, equipped with a rounded tip, was inserted through a small incision into the internal carotid artery via the external carotid artery and advanced 18 mm beyond the carotid bifurcation to achieve MCAO. After 90 minutes of ischemia, the filament was removed to permit reperfusion. Sham-operated mice underwent the same surgical procedure, with the exception of filament insertion. Animal Grouping For the first part of the experiment, the rats were divided into three groups as follows: (1) sham group, (2) MCAO group, (3) MCAO + STS group. Rats that did not receive MCAO operation were defined as the sham group and were injected with 1 mL of saline intravenously. The MCAO group mice were injected with 1 mL of saline after MCAO. The MCAO + STS group mice were intraperitoneally injected with 1 mL of STS (20mg/kg).[ 18 ] Three groups received daily injections for 6 consecutive days (once daily). For experiments conducted to compare the survival of stem cells, the rats were randomly divided into two groups: (1) MCAO + iPSCs group, (2) MCAO + iPSCs + STS group. Animals of the above two groups were stereotactically injected with 1×10 6 iPSCs via a brain stereotaxic injector.[ 20 ] Stem Cell Transplantation Procedures On the third day following MCAO, the rats received injections of iPSCs. The rat was placed in a stereotactic instrument (RWD Life Science Co.). A midline skin incision was made on the skull, and subsequently, a small burr hole was drilled through it. A microsyringe was used to deliver 1.0×10⁶ GFP-Luc-iPSCs (20 µL) into the left lateral ventricle over approximately 15 minutes. (anterior–posterior, 0.9 mm to the bregma; mediolateral, 1.5 mm; and dorsoventral, 3.5 mm from dura).[ 21 ] The needle was left in place for an additional 5 min and then removed slowly over 5 min. Bioluminescence imaging We conducted in vivo bioluminescence imaging on the first and fifth days following the injection of GFP-Luc-iPSCs. The D-luciferin potassium salt (Yeasen Biotechnology Shanghai Co., Ltd., Cat. No. 115144-35-9) was dissolved in sterile DPBS to a concentration of 150 mg/kg. The rats were administered 1 ml of D-luciferin potassium salt via tail vein injection before imaging. Imaging began 10 minutes after the administration of 2% isoflurane. Magnetic Resonance Imaging On the first and seventh days after MCAO, all rats underwent MR imaging using a rat-specific MR coil (GE DiscoveryMR 750 3.0T, MODEL: WK602). The MR examination sequences included coronal T2WI, coronal Fast Field Echo (FFE)-T2*WI and axial FFE-T2*WI. These sequences were used to assess changes in the brains of MCAO rats following STS and iPSCs therapies. Neurofunctional Test All rats underwent neurological assessment using the Garcia neurological grading system.[ 22 ] The neurological evaluation is a composite of spontaneous activity (abnormal movement), symmetry in the movement of four limbs, forepaw outstretching, climbing, body proprioception, and response to vibrissa touch tests. Neurologic function was graded on a scale of 0–18. The minimum neurological score is 3 and the maximum is 18. The lower the score, the more severe the behavioral deficits. Quantitative proteomic analysis The rats were sacrificed under deep anesthesia, and samples of infarction were taken from the brain. Each group consisted of three biological replicates (Fig. S1 ) . The protein extracts were analyzed using LC-MS/MS. Briefly, the samples were first homogenized by MP FastPrep-24 homogenizer (24×2,6.0M/S, 60s, twice), and then SDT buffer (4%SDS, 100mM Tris-HCl, pH7.6) was added. All samples were digested with trypsin using the filter-aided sample preparation protocol. The resulting peptides were desalted by using C18 cartridge. The solutions containing peptide fragments were dried in a lyophilizer and reconstituted by adding 40 µl 0.1% formic acid. Protein concentration of sample extracts was measured at OD280 nm. Appropriate amounts of iRT standard peptides were mixed into each sample, and each sample was subjected to dataindependent acquisition (DIA) mass spectrometry. The peptides from each sample were analyzed by OrbitrapTM AstralTM mass spectrometer (Thermo Scientific) connected to a Vanquish Neo system liquid chromatography (Thermo Scientific) in the dataindependent acquisition (DIA) mode. DIA data was analyzed with DIA-NN 1.8.1 searching the database from Uniport. A strict cutoff of Log fold-change 2 was used as the qualification criteria. The ratios were sorted by a P-value cutoff of 0.05 to obtain the list of differentially expressed proteins. Immunofluorescent staining Frozen sections of the brain were incubated with DHE in the dark at 37°C for 30 minutes. Then the brain slices were incubated with DAPI at room temperature for 10 minutes. Sections were permeabilized with 0.04% Triton X-100 and blocked with 10% normal goat serum and 0.5% bovine serum albumin in PBS for 1 hour and then treated overnight at 4°C with primary antibodies: neuronal nuclear protein(NeuN)(Proteintech, 26975-1-AP), glial fibrillary acidic protein(GFAP)(Proteintech, 16825-1-AP), ionized calcium-binding adapter molecule 1(Iba-1)(Proteintech, 10904-1-AP), cluster of differentiation 31(CD31)(Proteintech, 28083-1-AP), Brain-derived neurotrophic factor(BDNF)(Proteintech, 25699-1-AP); Vascular endothelial growth factor(VEGF)(Proteintech, 26157-1-AP), and Doublecortin(DCX) (Proteintech, 13925-1-AP); After washing with PBS, the sections were incubated with fluorescence-conjugated secondary antibodies for 1 hour at room temperature. The sections were washed and counterstained with the nuclear dye 4,6-diamino-2-phenylindole. Fluorescence-labeled sections were viewed using a confocal microscope. Histological Examination Brain, heart, liver, and kidney tissues were harvested and fixed in 4% paraformaldehyde for 24–48 hours at 4°C. The tissues were then dehydrated using graded ethanol, cleared in xylene, and embedded in paraffin. Sections (measuring 5 µm) were cut, deparaffinized, and rehydrated. They were stained with Harris hematoxylin, differentiated in acid alcohol, and counterstained with eosin. Following dehydration and clearing, the sections were mounted with neutral resin. The stained sections were imaged under a light microscope to visualize tissue architecture and cellular morphology. TUNEL staining Brain sections (40 µm thickness) were prepared and washed in PBS. Sections were permeabilized with 0.25% Triton X-100 in PBS for 20 min at room temperature. Following permeabilization, sections were treated with 20 µg/mL proteinase K in 10 mM Tris-HCl (pH 7.4–8.0) for 30 min at 37°C. After washing in PBS, sections were incubated with the TUNEL reaction mixture containing TMR red-labeled nucleotides and terminal deoxynucleotidyl transferase (TdT) for 1 h at 37°C in a humidified chamber. Sections were then rinsed in PBS to remove unbound reagents. Nuclei were counterstained with DAPI (2 µg/mL) for 10 min at room temperature to visualize total cell nuclei. Finally, sections were mounted on glass slides using Fluoromount-G and imaged under a fluorescence microscope. Western Blot Analysis The total protein in the peri-infarct hemisphere was extracted, and the protein concentration was determined using the bicinchoninic acid protein assay. The protein samples were then denatured by boiling for 10 min. Then, 20 µg of each protein sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at a constant voltage of 80 V for 100 min. After transfer to polyvinylidene fluoride (PVDF) membrane, the separated protein bands were incubated with individual primary antibodies (Caspase-3, 19677-1-AP, Proteintech; Bax,50599-2-Ig, Proteintech; Bcl2,68103-1-Ig, Proteintech) at 4°C overnight, followed by incubation with the corresponding fluorescent secondary antibody at room temperature for 1 h. The developed bands were observed under Odyssey DLx near-infrared dual-channel laser imaging system. Statistical analysis For statistical analyses, GraphPad Prism 10.0 software (GraphPad software, Inc., La Jolla, CA, USA) was used. All tests were repeated three times or more and the results are shown as mean ± SD. Student's t-test was used to analyze the difference between two groups. For comparisons of multiple groups, one-way analysis of variance followed by Tukey's post hoc test was used. The data are presented as mean ± standard deviation (SD). P < 0.05 was considered statistically significant. Results STS enhances functional recovery following MCAO To evaluate the therapeutic efficacy of STS, rats underwent MCAO surgery on Day 0, followed by daily intraperitoneal injections of STS from Day 1 to Day 6 ( Fig. 1 A ). MRI was utilized to assess brain injury in three groups. On Day 1, compared with the Sham group, rats in both the MCAO group and the MCAO + STS group showed significant brain injury areas. On Day 7, the brain injury area of the MCAO + STS group was significantly reduced compared to that of the untreated MCAO group ( Fig. 1 B ) . Statistical analysis revealed a significant increase in infarct recovery rate in the MCAO + STS group, indicating that STS treatment positively contributes to the recovery of cerebral ischemic injury ( Fig. 1 C ) . MCAO rats treated with STS demonstrated significant improvement in neurological function when compared with the MCAO group on Day 7 ( Fig. 1 D). On Day 7, HE staining revealed intact tissue architecture and clearly defined cell nuclei in the Sham group. In contrast, the MCAO group exhibited extensive tissue damage, characterized by blurred cell contours and widespread neuronal nuclear condensation with deepened staining. Compared to the MCAO group, STS treatment significantly reduced the pathological change of tissue necrosis and nuclear pyknosis, indicating its neuroprotective effect ( Fig. 1 E ) . Quantitative proteomics reveals extensive microenvironmental alterations following MCAO In this work, Astral was applied to obtain the difference in protein expression between the Sham group and the MCAO group in the subacute of ischemic stroke. A total of 4333 proteins were identified and quantified on Day 7. The volcano plots visualized all the identified proteins and highlighted the differentially expressed proteins ( Fig. 2 A ) . The bar chart of 365 differentially expressed proteins is summarized ( Fig. 2 B ) . Compared with the Sham group, a total of 305 proteins were upregulated (red bar), while only 60 proteins were downregulated (blue bar). To gain insight into the potential biological roles of these differentially expressed proteins, Gene Ontology (GO) annotations of proteins were classified into three categories: biological process, cellular component, and molecular function ( Fig. 2 C ) . In the molecular functions, the differentially expressed proteins between the MCAO group and the Sham group on day 7 were mainly distributed in "protein binding (225)", "ion binding (103)", "hydrolase activity (72)", "protein-containing complex binding (71)", "organic cyclic compound binding (69)", "enzyme regulator activity(60)", "catalytic activity, acting on a protein(46)", "heterocyclic compound binding(38)", "transferase activity(37)", "Oxidoreductase activity (23)". The representative cellular component category of these proteins was "organelle (217)", "membrane (181)", "cytoplasm (133)", "extracellular space (98)", "cytosol (69)", "cell junction (69)", "cell projection (66)", "C: protein-containing complex (47)", "C: cell surface (47)", "C: perinuclear region of cytoplasm (40)" categories. Their biological processes were mainly associated with "regulation of biological process (257)", "cellular component organization or biogenesis (144)", "response to chemical (135)", "cellular metabolic process (124)", "anatomical structure development (103)", "organic substance metabolic process (88)", "cellular developmental process (83)", "signal transduction (83)","response to biotic stimulus (78)","establishment of localization (71)". To further corroborate the GO findings and acquire the overall pathway, we performed Gene Set Enrichment Analysis (GSEA) on the rank-ordered whole-proteome lists from Sham and MCAO groups, among which 8 signaling pathways exhibited significant enrichment ( P < 0.001) and have been closely linked to cerebral ischemia (Table. 1) . Among the gene sets analyzed, those related to "Apoptosis",[ 23 ] "Phagosome"[ 24 ] and "Cell adhesion molecules"[ 25 ] have a relatively high gene count (setSize) ( Fig. 2 D ) . This indicates that they play significant roles in the brain microenvironment following MCAO. STS ameliorates the brain microenvironment following MCAO by inhibiting apoptosis Proteomics was used to assess the effects of STS on the brain microenvironment following MCAO. Compared with the MCAO group, STS significantly reshaped the protein expression profile: the heatmap of differentially expressed proteins revealed the top 50 most significantly altered proteins ( Fig. 3 A ) . Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of these differentially expressed proteins indicated significant regulation of the apoptosis pathway ( Fig. 3 B ). Furthermore, GSEA suggested that the apoptosis-related gene set (rno04210) was overall activated, with a normalized enrichment score (NES) of -1.48 and a Pvalue of 0.01 ( Fig. 3 C ) . Western blot analysis further confirmed the upregulation of Bcl2/Bax ( P < 0.01 vs. MCAO group ) and the downregulation of Caspase3 ( P < 0.05 vs. MCAOgroup, Fig. 3 D-E). TUNEL staining showed significantly fewer apoptotic cells following STS treatment, indicating its pronounced anti-apoptotic effect ( Fig. 3 F ) . These findings illustrate that STS ameliorates the brain microenvironment through anti-apoptotic mechanisms, thereby creating a favorable condition for subsequent iPSCs transplantation. Transfection of iPSCs with the GFP-Luc lentiviral vector Immunofluorescence confirmed successful transfection of iPSCs with the GFP-Luc lentiviral vector. GFP positive iPSCs colonies were observed, indicating successful lentiviral transfection of iPSCs ( Fig. 4 A ) . To verify the relationship between luciferase activity and cell number, five tubes containing different numbers of iPSCs transfected with lentivirus were detected. The luminescence intensity increased visibly as the cell number increased, indicating a positive correlation between luciferase activity and cell number ( Fig. 4 B ) . Quantitative analysis further revealed a highly linear relationship between iPSCs number and luciferase luminescence intensity (R² = 0.99, Fig. 4 C). These results demonstrate that transfection of GFP-Luc lentiviral vector enables iPSCs to express GFP and luciferase stably and that the luminescent signal can be used to quantify cell number accurately. STS enhances the functional recovery mediated by iPSCs transplantation after MCAO in rats On the day of transplantation (Day 1), bioluminescence imaging revealed detectable luciferase signals in both the MCAO + iPSCs and MCAO + iPSCs + STS groups, confirming successful cell engraftment. Over time, the luminescence intensity declined in two treatment groups; however, the MCAO + iPSCs + STS group exhibited significantly higher luciferase signals on Day 5 compared with the MCAO + iPSCs group (Fig. 5 A). Quantitative analysis demonstrated that the MCAO + iPSCs + STS group exhibited a smaller reduction in iPSCs number relative to the MCAO + iPSCs group ( P < 0.05 vs. MCAO + iPSCs group, Fig. 5 B). These data indicate that STS promoted the survival of iPSCs in vivo. On Day 7, the combined group exhibited a marked reduction in infarct size, with a higher recovery rate superior to that of MCAO + iPSCs group ( P < 0.05 vs. MCAO + iPSCs group, Fig. 5 C-D). The neurological score in the combined group was higher than that of the MCAO + iPSCs group on Day 7, suggesting that combined treatment is more effective in promoting the recovery of neurological function after MCAO than iPSCs transplantation alone ( Fig. 5 E ) . STS combined with iPSCs enhances neurovascular restoration following MCAO Immunofluorescence staining was used to label neurons, astrocytes, microglia, and endothelial cells in brain tissue using NeuN, GFAP, Iba-1, and CD31, respectively. Compared with the Sham group, the MCAO group exhibited significant reductions in neurons, hypertrophy of astrocytes, activation of microglia, and a decrease in endothelial cell numbers, reflecting neuronal loss, inflammatory reaction, and vascular damage caused by cerebral ischemia. Both the MCAO + STS and the MCAO + iPSCs groups partially restored the balance of neurons and glial cells and increased endothelial cell numbers, indicating that both iPSCs therapy and STS therapy exert protective effects on the neurovascular unit after cerebral ischemia. Notably, compared with MCAO + iPSCs group, MCAO + iPSCs + STS group showed more pronounced neuronal recovery, further reduced glial cell activation, and a more significant increase in endothelial cell numbers on Day 7, suggesting that the combination of STS and iPSCs therapy more effectively promotes neurovascular restoration after cerebral ischemia ( Fig. 6 ) . STS combined with iPSCs enhances the paracrine secretion of BDNF and VEGF To determine whether iPSCs can induce neurogenesis or angiogenesis, immunofluorescence staining of BDNF, VEGF and DCX were performed on brain tissue. BDNF, VEGF, and DCX positive cells were observed evenly distributed in Sham group. In MCAO group, the number of cells positive for BDNF, VEGF, and DCX was significantly reduced, indicating that ischemia resulted in neurotrophic depletion and the exhaustion of endogenous progenitors. The MCAO + iPSCs group exhibited more BDNF, VEGF, and DCX positive cells than those of the MCAO group, indicating that transplanted iPSCs promote tissue repair through paracrine mechanisms. In MCAO + iPSCs + STS group, the number of BDNF, VEGF, and DCX positive cells significantly increased than those of MCAO + iPSCs group, indicating a strong synergistic effect in vivo (Fig. S2 ) . BDNF, VEGF, and DCX expression were then assessed in GFP-labeled iPSCs with or without STS treatment in vitro. In MCAO + iPSCs + STS group, GFP signal was markedly enhanced, indicating increased iPSCs survival. The co-localization signals of BDNF, VEGF, and DCX with GFP were concurrently elevated, indicating enhanced paracrine secretion and an increased potential for differentiation ( Fig. 7 A-C ) . In summary, STS promoted the survival and proliferation of iPSCs in vivo and enhanced iPSCs-mediated neuroprotection and angiogenesis through a paracrine pathway. Safety assessment of STS and/or iPSCs treatment in vivo HE staining demonstrated the histological changes in heart, liver, spleen, and kidney tissues of rats from five groups. In Sham group, the tissues of heart, liver, spleen, and kidneys all exhibited normal histological structures with tightly and orderly arranged cells, showing no apparent pathological changes. The tissues from MCAO, MCAO + STS, MCAO + iPSCs, and MCAO + iPSCs + STS groups also maintained integrity, with normal cellular morphology, and no evident inflammatory cell infiltration or tissue necrosis was observed, indicating that the treatment with STS and iPSCs did not cause significant organ pathological changes in the current study ( Fig. 8 A ) . We also measured the levels of liver function indicators (ALT, AST, ALB), kidney function indicators (CR), and complete blood count indicators (MCV, MPV) in rats from five experimental groups. The results of the serum liver and kidney function tests and the complete blood count showed no statistically significant differences among the five groups ( Fig. 8 B ) . In summary, the application of STS and iPSCs treatments in this study demonstrated good safety in rats, without causing detectable organ damage or dysfunction. Discussion Our results indicate that STS enhances the ischemic microenvironment following MCAO by inhibiting apoptosis, which improves the survival of transplanted iPSCs. Comparison of MCAO + iPSCs group, the surviving iPSCs secrete higher levels of BDNF, VEGF and DCX, thereby promoting neuroprotection and angiogenesis. Here, STS reduced infarct size and enhanced neurological function scores, suggesting that it significantly promotes functional recovery in MCAO rats. Furthermore, proteomic profiling was conducted to elucidate the mechanisms by which STS modulates the ischemic microenvironment. The analysis revealed extensive remodeling of the ischemic microenvironment, with 365 differentially expressed proteins identified. Both KEGG pathway enrichment and GSEA consistently demonstrated that STS primarily acts by inhibiting apoptosis, a finding that was further validated by Western blotting and TUNEL assays. Having established that STS improves the ischemic microenvironment, we next assessed iPSCs survival after transplantation using GFP-Luc-iPSCs. Compared with the iPSCs group, the combined treatment with STS significantly improved iPSCs survival, reduced infarct size and enhanced neurological function scores. Immunofluorescence further demonstrated that the combined therapy more effectively promoted neurovascular repair, as evidenced by increased expression of NeuN and CD31. Moreover, GFP co-localization with BDNF, VEGF and DCX was observed in transplanted iPSCs, indicating enhanced paracrine effects and neurogenic potential. The brain microenvironment experiences extensive and dynamic changes following MCAO, among which neuronal death is a key pathological event. Neuronal death exhibits strict spatiotemporal specificity, with apoptosis being one of the major underlying mechanisms,[ 26 , 27 ] ATP levels plummet to < 5% of baseline within minutes in the ischemic core, necrosis becoming the predominant mode of cell death.[ 28 ] Secondly, ATP is maintained at roughly 20–50% of baseline levels in the penumbra, creating an "energy threshold window" that allows for mitochondrial outer-membrane permeabilization without causing immediate energy collapse.[ 29 ] Within the first 6 hours of ischemia, Bax and Bak proteins translocate from the cytosol to the mitochondrial outer membrane and assemble into oligomeric pore structures.[ 30 ] Then, cytochrome c is released exponentially, reaching a peak between 12–24 hours, where it binds to Apaf-1 to form the apoptosome.[ 31 ] The complex recruits and activates procaspase-9 via Caspase Recruitment Domain (CARD)-CARD interactions, thereby initiating the downstream caspase-3 cascades. In ischemic models, caspase activation can persist for 24 to 72 hours, leading to the cleavage of PARP-1 and ultimately driving the execution phase of apoptosis.[ 32 ] Importantly, glycolytic ATP production sustains the assembly of the apoptosome and the activation of caspases, prolonging neuronal apoptosis for 6–72 hours and establishing a therapeutically accessible "rescue window" in both experimental and clinical settings.[ 33 ] In hippocampal regions distant from the ischemic core, neuronal apoptosis is triggered remotely by spreading depolarization (SD).[ 34 ] SD spreads outward from the infarct border and, upon reaching surrounding tissue, induces sustained depolarization and intracellular Ca 2+ overload, ultimately triggering mitochondria-mediated apoptosis.[ 35 ] Meanwhile, reactive oxygen species and nitric oxide produced during spreading depolarization exacerbate DNA damage and suppress the activity of the anti-apoptotic protein Bcl-2, creating a positive-feedback loop that accelerates apoptosis.[ 34 ] In summary, the disruption of the brain microenvironment induced by MCAO is centered on apoptosis as the critical node, propagating a cascade of damage through the penumbra, and remote hippocampal regions. STS improves the ischemic microenvironment by suppressing apoptosis, thereby significantly promoting the survival of transplanted iPSCs. Previous studies had shown that transplanted iPSCs rapidly undergo apoptosis or necrosis in the brain, with over 90% lost within 72 h.[ 36 , 37 ] The hostile microenvironment markedly impairs transplanted stem-cell survival after MCAO, limiting their therapeutic efficacy. Traditional Chinese medicine exerts neuroprotection against ischemic brain injury by suppressing apoptosis.[ 38 ] The active constituents of Danshen have been reported to exert neuroprotective effects. Among these, STS is a key derivative that has emerged as a promising therapeutic candidate for ischemic stroke.[ 19 , 39 ] Its anti-apoptotic properties are deemed a crucial factor in its neuroprotective functions. Studies have shown that STS inhibits intrinsic apoptotic pathways by upregulating the anti-apoptotic protein Bcl-2, downregulating pro-apoptotic proteins Bax, caspase-3, and caspase-9, stabilizing mitochondrial membrane potential, reducing cytochrome c release, and blocking the caspase cascade.[ 40 , 41 ] Additionally, STS has been shown to inhibit the extrinsic apoptotic pathway by suppressing activation of Fas/FADD/caspase-8 and upregulating c-FLIP and v-FLIP, thereby preventing the formation of the death-inducing signaling complex.[ 40 ] In line with previous studies, the findings of the present study further indicate that STS has a significant therapeutic effect by exerting anti-apoptotic effects. STS markedly decreased the proportion of TUNEL-positive cells in the ischemic brain regions, simultaneously downregulating the expression levels of caspase-3. The results indicate that STS exerts neuroprotective effects by inhibiting the mitochondrial-mediated apoptosis pathway, thereby creating a more favorable microenvironment for the survival and functional integration of transplanted stem cells. In this study, the therapeutic benefits of iPSCs were predominantly mediated through paracrine mechanisms. The principal mechanisms underlying iPSCs-based therapy for ischemic stroke encompass cell replacement via direct neuronal and glial integration and paracrine modulation of the neurovascular[ 42 ] paracrine profile of iPSCs-derived cells is generally categorized into four functional groups, including neurotrophic and anti-apoptotic factors (BDNF and GDNF), pro-angiogenic and vascular remodeling factors (VEGF-A and bFGF), immuno-inflammatory modulators (IL-10 and TGF-β1), and chemotactic and cell-migration factors (SDF-1α and MCP-1).[ 43 ] Research has revealed that conditioned medium from hiPSCs-derived glial progenitor cells promote neuroprotection and angiogenesis by modulating inflammatory cytokines, such as TNF, IL-4, and IL-10, and by upregulating neurotrophic factors, including BDNF, CNTF and GDNF,[ 44 ] hiPSCs-MSC-derived extracellular vesicles activated VEGF/CXCR4 signaling pathways, thereby facilitating neurovascular repair.[ 45 ] BDNF is one of the most critical neurotrophic factors in the central nervous system, which can promote the survival, differentiation and synaptic plasticity of neurons.[ 46 ] VEGF is the central regulator of angiogenesis and vascular permeability, which can facilitate neovascularization and the repair of damaged blood vessels.[ 47 ] In this study, we investigated BDNF and VEGF as representative paracrine mediators, aiming to elucidate the mechanisms through which iPSCs confer neuroprotection in cerebral ischemia. Immunofluorescence results revealed that, compared with MCAO + iPSCs group, MCAO + iPSCs + STS group exhibited markedly stronger BDNF- and VEGF- positive signals that co-localized with GFP-iPSCs. Taken together, the iPSCs-secreted BDNF/VEGF axis synergistically promotes neuronal survival and angiogenesis. Synergistic exogenous interventions substantially augment the therapeutic efficacy of stem cells for ischemic stroke. To promote the survival of stem cells, exogenous intervention primarily relies on two synergistic strategies: Genetic engineering modification of stem cells and improvement of ischemic microenvironment. Genetic engineering mediated by lentivirus or CRISPR/Cas9 enables one-step, high-efficiency integration of anti-apoptotic and pro-reparative genes-such as HO-1, FGF21 and VEGF-into stem cells. Compared with unmodified controls, this strategy elevates the 72-hour survival rate of cells within the ischemic penumbra from < 10% to 40–60% and sustains high-level paracrine output for 4–6 weeks.[ 48 – 50 ] However, the high cost and regulatory hurdles associated with viral vectors limit their large-scale application. Traditional Chinese medicine combined with stem cells exerts therapeutic effects against cerebral ischemia via anti-inflammatory, antioxidant, pro-angiogenic, and neuroregenerative mechanisms. For instance, Human Umbilical Cord-Derived MSCs plus curcumin synergistically activate the AKT/GSK-3β/β-TrCP/Nrf2 axis, up-regulate HO-1 and NQO1 and suppress NF-κB–mediated inflammatory cascades, reducing ROS by 60% within 72 h and increasing stem cell survival from < 10% to 45%.[ 51 ] Another example, Bone Marrow-Derived MSCs co-administered with ginsenoside CK activate GLUT1 and stabilize HIF-1α, persistently inducing VEGF-A and Ang-1 transcription. The result is that the microvascular density in the ischemic penumbra increases by 2.5-fold within 7 days, significantly enhancing angiogenesis.[ 52 ] Tanshinone IIA–loaded nanoparticles co-delivered with NSCs showed a decrease in immune cell and reactive astrocyte activation, resulting in a significant increase in NSC survival. In a porcine MCAO model, this regimen reduces infarct volume by 35% and restores motor function to 75% of baseline within 4 weeks.[ 53 ] In the present study, the combination of STS and iPSCs significantly reduced the infarction size. Immunofluorescence results showed that the expressions of NeuN and CD31 in the STS combined with iPSCs group were significantly increased, while the expressions of GFAP and Iba-1 were significantly decreased. This suggests that the neuronal survival and vascular regeneration in the combined treatment group increased, while glial scars and inflammatory factors decreased. In all, it indicates that the STS promotes the survival and therapeutic effect of iPSCs by improving the cerebral ischemic microenvironment. This study has several limitations. Firstly, iPSCs were transplanted directly into the rat brain without immunosuppression, and we did not specifically assess the impact of xenogeneic rejection on iPSCs survival. Therefore, the extent to which immune rejection may have influenced the therapeutic efficacy remains uncertain. Secondly, this study was performed for only 1 week. We therefore could not form conclusions about longer-term response after iPSCs transplantation, including cell differentiation and tumorigenicity, although we did not find any tumor formation in our study. Therefore, future studies should introduce immunosuppression and extend the observation period. Conclusions In summary, our study demonstrates that STS improves the ischemic microenvironment by reducing apoptosis, thereby further promoting the survival and proliferation of iPSCs. The combination of STS and iPSCs significantly reduces infarct size, improves behavioral scores, and promotes neurogenesis and angiogenesis through the paracrine release of BDNF and VEGF in vivo. STS combined with iPSCs could be a better therapeutic approach than these iPSCs used individually. Abbreviations STS Sodium tanshinone IIA sulfonate iPSCs induced pluripotent stem cells Tan IIA Tanshinone IIA MCAO middle cerebral artery occlusion VTN vitronectin SPF specific-pathogen-free CCA common carotid artery ICA internal carotid artery DIA dataindependent acquisition SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis PVDF polyvinylidene fluoride MRI magnetic resonance imaging GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes GSEA Gene Set Enrichment Analysis GFP green fluorescent protein NeuN neuronal nuclei GFAP Glial fibrillary acidic protein Iba-1 ionized calcium-binding adapter molecule 1 CD31 cluster of differentiation 31 BDNF Brain-derived neurotrophic factor VEGF Vascular endothelial growth factor DCX Doublecortin ALT Alanine Aminotransferase AST Aspartate Aminotransferase CR Creatinine MCV Mean Corpuscular Volume MPV Mean Platelet Volume CARD Caspase Recruitment Domain SD spreading depolarization. Declarations Acknowledgments We thank Home for Researchers (www.home-for-researchers.com) for the online illustration tools used to prepare some of the figures in this work. Author contributions YL, XL, HL, FS and WC conceived and designed research and performed experiments. MW, LL, XW and JZ analyzed data. RY, LZ, XW and QS prepared figures and edited text. YL and XL drafted manuscript, HL, FS and WC edited and revised manuscript. LZ, QS, XW, HL and FS gave final approval of the version to be published. All authors approved to submit this version to this publication. All authors read and approved the final manuscript. Funding This work was partially sponsored by grants from the Traditional Chinese Medicine Science and Technology Project of Zhejiang Provincial Health Commission (no. 2026ZL0189, 2025ZL257), National Natural Science Foundation of China (no. 82001862), Zhejiang Provincial Natural Science Foundation (no. LQ24H180010), and Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (no. 2024KY656, 2024KY737). Availability of data and materials All data generated or analysed during this study are included in this article (and its Additional files). Ethics approval and consent to participate All animal procedures were conducted following the Guidelines for the Ethical Review of Laboratory Animal Welfare (China, 2018) and approved by the Experimental Animal Ethics Committee of Zhejiang Provincial People's Hospital (approval number: 20240927814920). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. 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Table 1 Table. 1 The GSEA results indicated that eight signaling pathways exhibited significant enrichment in the comparison between the Sham and MCAO groups. ID Description setSize NES Pvalue Adjust Pvalue rno04210 Apoptosis 49 1.56 0.004 0.06 rno04145 Phagosome 49 1.70 0.000 0.01 rno04514 Cell adhesion molecules 40 1.56 0.003 0.05 rno04620 Toll-like receptor signaling pathway 27 1.55 0.007 0.07 rno00520 Amino sugar and nucleotide sugar metabolism 24 1.54 0.007 0.07 rno04064 NF-kappa B signaling pathway 24 1.59 0.003 0.05 rno04979 Cholesterol metabolism 22 1.59 0.005 0.06 rno04974 Protein digestion and absorption 21 1.66 0.001 0.04 Supplementary Files renamedeebc3.docx floatimage1.png Graphical abstract Cite Share Download PDF Status: Posted Version 1 posted 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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06:53:23","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":148921,"visible":true,"origin":"","legend":"","description":"","filename":"JTRMD25188310structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7950969/v1/f43adf19376fd06cae26a4bf.xml"},{"id":97112701,"identity":"5d5f42f8-e86b-4081-a3f3-2e2e11da2f55","added_by":"auto","created_at":"2025-12-01 06:53:22","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":162536,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7950969/v1/11ad720193ecdc502182ba21.html"},{"id":97112679,"identity":"b616a095-0e0f-4a10-8f45-a924cf6a522b","added_by":"auto","created_at":"2025-12-01 06:53:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":853129,"visible":true,"origin":"","legend":"\u003cp\u003eSTS enhances functional recovery following MCAO. \u003cstrong\u003eA\u003c/strong\u003e Schematic diagram of the animal experiment for STS treatment of cerebral ischemia. \u003cstrong\u003eB\u003c/strong\u003eMR images before and after treatment in three groups (The yellow dotted line areas represent the infarct region). \u003cstrong\u003eC\u003c/strong\u003e Quantitative analysis of the percentage of infarct recovery area. \u003cstrong\u003eD\u003c/strong\u003e Quantitative analysis of the neurological score before and after treatment in three groups. \u003cstrong\u003eE\u003c/strong\u003e HE staining of the brains in three groups. Scale bar: 200 μm. n=3, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7950969/v1/1e0211d3549c83946a284c5e.png"},{"id":97112653,"identity":"b46c69ca-c455-45e7-9fda-e2663d9af07b","added_by":"auto","created_at":"2025-12-01 06:53:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":508061,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative proteomics reveals extensive microenvironmental alterations following MCAO.\u003cstrong\u003e A\u003c/strong\u003e The volcano plot displays significantly differentially expressed genes between Sham and MCAO groups. \u003cstrong\u003eB\u003c/strong\u003e The bar chart shows the number of up-regulated and down-regulated genes between Sham and MCAO groups. \u003cstrong\u003eC\u003c/strong\u003eGO annotation of differentially expressed proteins between Sham and MCAO groups. The GO annotation covers three aspects: biological process, cellular component, and molecular function. The top ten terms in each category are shown. \u003cstrong\u003eD\u003c/strong\u003e The GSEA plots show the top three significantly enriched signaling pathways between Sham and MCAO groups.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7950969/v1/8bec85d12a614bd5e6bf5679.png"},{"id":97112704,"identity":"dda1c7e3-d739-4a99-a926-713990426d20","added_by":"auto","created_at":"2025-12-01 06:53:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":639094,"visible":true,"origin":"","legend":"\u003cp\u003eSTS ameliorates the brain microenvironment following MCAO by inhibiting apoptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e The heatmap displays the differential gene expression between MCAO and STS groups. The top 50 most significantly differentially expressed genes are shown. \u003cstrong\u003eB\u003c/strong\u003e The dotplot illustrates the significantly enriched signaling pathways in the KEGG pathway analysis between MCAO and STS groups. \u003cstrong\u003eC\u003c/strong\u003e The GSEA plot shows the enrichment analysis results of the apoptosis pathway between MCAO and STS groups. \u003cstrong\u003eD\u003c/strong\u003e Western Blot shows the expression levels of apoptosis-related proteins. \u003cstrong\u003eE\u003c/strong\u003e Quantitative analysis of Western Blot results. \u003cstrong\u003eF\u003c/strong\u003e TUNEL staining images show the expression of apoptosis in three groups. Scale bar: 200 μm. n=3, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7950969/v1/a0cbb89f21d22b0af465c991.png"},{"id":97112722,"identity":"4d27d856-16e5-4590-abed-cc2b7ea3ea75","added_by":"auto","created_at":"2025-12-01 06:53:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":324026,"visible":true,"origin":"","legend":"\u003cp\u003eTransduction of iPSCs with the GFP-Luc lentiviral vector. \u003cstrong\u003eA\u003c/strong\u003eImmunofluorescence images demonstrate the expression of GFP in iPSCs. The left image shows GFP expression (green), the middle image shows the morphology of iPSCs and the right image is a merge of GFP expression and cell morphology. Scale bar: 500μm. \u003cstrong\u003eB\u003c/strong\u003e Luminescent intensity in different numbers of iPSCs. The luminescence intensity increased with increase in the number of iPSCs. \u003cstrong\u003eC\u003c/strong\u003e The correlation between iPSCs numbers and luminescent intensity was confirmed in vitro.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7950969/v1/5c0bd87170be5305efb27635.png"},{"id":97112666,"identity":"0f2c885b-45f3-4d6b-92f1-fa343632861f","added_by":"auto","created_at":"2025-12-01 06:53:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":436789,"visible":true,"origin":"","legend":"\u003cp\u003eSTS enhances functional recovery following iPSCs transplantation in MCAO rats. \u003cstrong\u003eA\u003c/strong\u003e The luminescence intensity in brains of MCAO rats on Day 1 and Day 5 after iPSCs transplantation. \u003cstrong\u003eB\u003c/strong\u003e Quantitative analysis of iPSCs number in two groups. \u003cstrong\u003eC\u003c/strong\u003e MR images before and after iPSCs transplantation in two groups. \u003cstrong\u003eD\u003c/strong\u003e Quantitative analysis of infarct recovery area in two groups. \u003cstrong\u003eE\u003c/strong\u003e Quantitative analysis of the neurological score in two groups. n=3, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7950969/v1/c557afe8da5e4eb353c709e5.png"},{"id":97112668,"identity":"ddd95ef9-a6c4-446c-b9cc-a0479bde6757","added_by":"auto","created_at":"2025-12-01 06:53:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1276412,"visible":true,"origin":"","legend":"\u003cp\u003eSTS combined with iPSCs enhances neurovascular restoration following MCAO. NeuN, GFAP, Iba-1 and CD31 fluorescence staining among five groups on Day 7. Scale bar: 100μm. n=3.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7950969/v1/cf4d80ff38c4f96db6c31b5d.png"},{"id":97112629,"identity":"c5cb02d1-0e90-4bbe-86fc-a8eb6dac556f","added_by":"auto","created_at":"2025-12-01 06:53:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":616600,"visible":true,"origin":"","legend":"\u003cp\u003eSTS combined with iPSCs enhances Paracrine Mechanisms of BDNF and VEGF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-C\u003c/strong\u003e BDNF/DAPI/GFP, VEGF/DAPI/GFP, and DCX/DAPI/GFP fluorescence staining between MCAO+iPSCs and MCAO+iPSCs+STS groups. Scale bar:50μm.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7950969/v1/a90d36a7957bca88420cd21f.png"},{"id":97112683,"identity":"92c6f0b3-2009-4d36-a24c-164736abb439","added_by":"auto","created_at":"2025-12-01 06:53:21","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1316706,"visible":true,"origin":"","legend":"\u003cp\u003eSTS and/or iPSCs treatments are safe in vivo. \u003cstrong\u003eA\u003c/strong\u003e HE staining of heart, liver, spleen, and kidney tissues from rats of five groups. Scale bar:200μm. \u003cstrong\u003eB\u003c/strong\u003e Levels of liver and kidney function indicators and complete blood count in rats from five groups. n=3, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7950969/v1/3f0226b225de1139371ef329.png"},{"id":108181035,"identity":"94b3c314-cb84-40a0-858a-9359c3ea2d28","added_by":"auto","created_at":"2026-04-30 08:56:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6357809,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7950969/v1/0c1e3d98-beb1-4277-9aaf-dc6b18f0bbe5.pdf"},{"id":97112667,"identity":"740316bb-a867-42e6-9046-ab0ae4244178","added_by":"auto","created_at":"2025-12-01 06:53:20","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1970035,"visible":true,"origin":"","legend":"","description":"","filename":"renamedeebc3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7950969/v1/12729f653c08245a1a8afc75.docx"},{"id":97140552,"identity":"427d3bce-a4e5-45e5-a708-bd37fa82d370","added_by":"auto","created_at":"2025-12-01 10:05:14","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":404191,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7950969/v1/14c9526fe99077449f069d3b.png"}],"financialInterests":"","formattedTitle":"Sodium tanshinone IIA sulfonate improves the ischemic microenvironment by inhibiting apoptosis and promotes the treatment of ischemic brain injury by iPSCs","fulltext":[{"header":"Introduction","content":"\u003cp\u003eStroke is characterized by high morbidity and high mortality, which have caused serious social and economic burdens.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Approximately 87% of strokes are ischemic strokes, in which a blood vessel supplying the brain is obstructed.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Existing therapeutic strategies, including pharmacotherapy, interventional procedures and surgery, have limited efficacy in improving neural function due to a narrow therapeutic window and poor drug penetration across the blood-brain barrier.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] Therefore, it is urgent to explore new therapeutic strategies for ischemic stroke.\u003c/p\u003e\u003cp\u003eStem cells hold great promise for the treatment of Central Nervous System diseases.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] Several cell types have been proposed to enhance post ischemic brain repair, including induced pluripotent stem cells(iPSCs), mesenchymal stem cells and neural stem cells.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] iPSCs can be derived from somatic cells, expanded to large quantities and differentiated into all kinds of cell types, which makes them a promising cell source for therapeutic purposes with fewer ethical concerns.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] However, transplantation of iPSCs into ischemic areas had low survival rates due to the harsh microenvironment. Thus, their clinical application is greatly limited.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eThe brain microenvironment after ischemic stroke includes alterations in ionic homeostasis, overproduction of inflammatory cytokines, and abnormal release of proteases.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] When stem cells are transplanted, they are exposed to these harmful factors, more than 80% of implanted cells are sentenced to death 72 hours after injection by the harsh microenvironment.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] Traditional Chinese medicine has a long history in the treatment of stroke and has achieved good therapeutic effects[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] has been extensively studied for its anti-apoptosis, anti-inflammation, and neurovascular-protective properties.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] Tanshinone IIA (Tan IIA) is the effective component isolated from Danshen for treating ischemic stroke.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] However, poor water solubility and oral bioavailability limit its biomedical application. Sodium tanshinone IIA sulfonate (STS), a water-soluble derivative of Tan IIA, exhibits high metabolic stability owing to a sulfonic group substitution at the C-16 position.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] A clinical trial revealed that STS improved neurologic functional outcomes in acute ischemic stroke patients using traditional thrombolysis therapy[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] suppresses microglia polarization and neuroinflammation through the regulation of the miR-125b-5p/STAT3 axis to ameliorate neuropathic pain, demonstrating its potential in the treatment of neurological disorders.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] Studies have demonstrated that STS can improve the post-ischemic microenvironment through multiple mechanisms, including inhibition of neuronal apoptosis,[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] suppression of inflammatory responses[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and promotion of angiogenesis[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] modulating these pathological processes, STS reduces secondary injury and promotes endogenous repair.\u003c/p\u003e\u003cp\u003eGiven the aforementioned points, we hypothesized that the combination of STS and iPSCs transplantation could enhance therapeutic efficacy in ischemic brain injury. To test this hypothesis, we utilized a middle cerebral artery occlusion (MCAO) rat model and conducted proteomic analysis to explore the mechanisms by which STS modulates the ischemic microenvironment. Transfection with lentiviral vectors was applied to iPSCs to trace their survival post-transplantation and immunofluorescence staining was used to evaluate neurovascular repair. Together, these approaches aimed to determine whether STS could potentiate the therapeutic benefits of iPSCs, thereby providing a novel strategy for the treatment of cerebral ischemia.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStem Cell Culture\u003c/h2\u003e\u003cp\u003eWe would like to thank Professor Jiachuan Wang for donating the human iPSCs cell line. iPSCs were cultured using ncEpic hPSC complete medium (Shownin Biotech, Cat. No. RP01001). After diluting 120 \u0026micro;L of 500 \u0026micro;g/mL vitronectin (VTN) in 9 mL of DMEM/F12, 1.5 mL of the mixture was dispensed into each well of a 6-well plate and left at room temperature for 1 hour before use. Subsequently, the cells were incubated in 2 ml of 0.5 mM EDTA for 7\u0026ndash;8 minutes at 37\u0026deg;C. The EDTA was aspirated and replaced with 2 mL of complete medium containing 0.5 \u0026micro;L of 10 mM Blebbistatin (Shownin Biotech, Cat. No. RP01008). The cells were gently pipetted 1\u0026ndash;2 times and replated onto VTN-coated 6-well plates at a 1:10 ratio. The plates were gently rocked to distribute the cells evenly. Medium was changed 18\u0026ndash;24 hours after seeding and then refreshed daily.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eLentiviral transfection of Stem Cells\u003c/h3\u003e\n\u003cp\u003eIPSCs were transfected with a lentivirus to express Green Fluorescent Protein and luciferin (GFP-Luc) for in vivo experiments. iPSCs were seeded at a density of 2 \u0026times; 10⁵ cells per well in 6-well plates. Multiplicity of infection was set at 10. The following day, virus was added to 1 mL of medium per well, followed by a 4-hours incubation. Subsequently, an additional 1 mL of medium was added to bring the total volume to 2 mL. The medium was replaced 24 hours post-infection.\u003c/p\u003e\n\u003ch3\u003eClonal Selection of Stem Cells\u003c/h3\u003e\n\u003cp\u003eTo obtain iPSCs with a high level of fluorescent marker expression, transfected cells were subjected to clonal selection. GFP-Luc-iPSCs were seeded at a low density of 3 \u0026times; 10\u0026sup3; cells per well in 6-well plates and cultured for 72 hours. Fluorescence expression was identified using fluorescence microscopy of the GFP-Luc-iPSCs and areas of high expression were marked on the underside of the culture plate. GFP-Luc-iPSCs were treated for 5 minutes with an EDTA solution. The EDTA was then aspirated and replaced with complete medium containing blebbistatin immediately. The marked GFP-Luc-iPSCs were carefully aspirated using a 100 \u0026micro;L pipette and transferred to VTN-coated 24-well plates for continued culture.\u003c/p\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eHealthy male specific-pathogen-free (SPF) grade Sprague\u0026ndash;Dawley (SD) rats (220\u0026ndash;240 g) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. The rats had ad libitum access to food and water and were housed in a room with a 12:12-hour dark-light cycle. All animal experiments were conducted in compliance with China's animal welfare legislation regarding the care and use of animals and were approved by the Experimental Animal Ethics Committee of Zhejiang Provincial People's Hospital (approval number: 20240927814920).\u003c/p\u003e\n\u003ch3\u003eAnimal model establishment\u003c/h3\u003e\n\u003cp\u003eTo induce cerebral ischemia via MCAO, rats were anesthetized using 1.5% sodium pentobarbital (50 mg/kg). A midline incision was performed on the neck to expose the left common carotid artery (CCA), external carotid artery, and internal carotid artery (ICA). The left common and external carotid arteries were isolated and subsequently ligated. A microvascular clip was temporarily applied to the internal carotid artery. A 4\u0026ndash;0 nylon suture, equipped with a rounded tip, was inserted through a small incision into the internal carotid artery via the external carotid artery and advanced 18 mm beyond the carotid bifurcation to achieve MCAO. After 90 minutes of ischemia, the filament was removed to permit reperfusion. Sham-operated mice underwent the same surgical procedure, with the exception of filament insertion.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAnimal Grouping\u003c/h2\u003e\u003cp\u003eFor the first part of the experiment, the rats were divided into three groups as follows: (1) sham group, (2) MCAO group, (3) MCAO\u0026thinsp;+\u0026thinsp;STS group. Rats that did not receive MCAO operation were defined as the sham group and were injected with 1 mL of saline intravenously. The MCAO group mice were injected with 1 mL of saline after MCAO. The MCAO\u0026thinsp;+\u0026thinsp;STS group mice were intraperitoneally injected with 1 mL of STS (20mg/kg).[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] Three groups received daily injections for 6 consecutive days (once daily). For experiments conducted to compare the survival of stem cells, the rats were randomly divided into two groups: (1) MCAO\u0026thinsp;+\u0026thinsp;iPSCs group, (2) MCAO\u0026thinsp;+\u0026thinsp;iPSCs\u0026thinsp;+\u0026thinsp;STS group. Animals of the above two groups were stereotactically injected with 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e iPSCs via a brain stereotaxic injector.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStem Cell Transplantation Procedures\u003c/h3\u003e\n\u003cp\u003eOn the third day following MCAO, the rats received injections of iPSCs. The rat was placed in a stereotactic instrument (RWD Life Science Co.). A midline skin incision was made on the skull, and subsequently, a small burr hole was drilled through it. A microsyringe was used to deliver 1.0\u0026times;10⁶ GFP-Luc-iPSCs (20 \u0026micro;L) into the left lateral ventricle over approximately 15 minutes. (anterior\u0026ndash;posterior, 0.9 mm to the bregma; mediolateral, 1.5 mm; and dorsoventral, 3.5 mm from dura).[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] The needle was left in place for an additional 5 min and then removed slowly over 5 min.\u003c/p\u003e\n\u003ch3\u003eBioluminescence imaging\u003c/h3\u003e\n\u003cp\u003eWe conducted in vivo bioluminescence imaging on the first and fifth days following the injection of GFP-Luc-iPSCs. The D-luciferin potassium salt (Yeasen Biotechnology Shanghai Co., Ltd., Cat. No. 115144-35-9) was dissolved in sterile DPBS to a concentration of 150 mg/kg. The rats were administered 1 ml of D-luciferin potassium salt via tail vein injection before imaging. Imaging began 10 minutes after the administration of 2% isoflurane.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMagnetic Resonance Imaging\u003c/h2\u003e\u003cp\u003eOn the first and seventh days after MCAO, all rats underwent MR imaging using a rat-specific MR coil (GE DiscoveryMR 750 3.0T, MODEL: WK602). The MR examination sequences included coronal T2WI, coronal Fast Field Echo (FFE)-T2*WI and axial FFE-T2*WI. These sequences were used to assess changes in the brains of MCAO rats following STS and iPSCs therapies.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eNeurofunctional Test\u003c/h2\u003e\u003cp\u003eAll rats underwent neurological assessment using the Garcia neurological grading system.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] The neurological evaluation is a composite of spontaneous activity (abnormal movement), symmetry in the movement of four limbs, forepaw outstretching, climbing, body proprioception, and response to vibrissa touch tests. Neurologic function was graded on a scale of 0\u0026ndash;18. The minimum neurological score is 3 and the maximum is 18. The lower the score, the more severe the behavioral deficits.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative proteomic analysis\u003c/h2\u003e\u003cp\u003eThe rats were sacrificed under deep anesthesia, and samples of infarction were taken from the brain. Each group consisted of three biological replicates \u003cb\u003e(Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e. The protein extracts were analyzed using LC-MS/MS. Briefly, the samples were first homogenized by MP FastPrep-24 homogenizer (24\u0026times;2,6.0M/S, 60s, twice), and then SDT buffer (4%SDS, 100mM Tris-HCl, pH7.6) was added. All samples were digested with trypsin using the filter-aided sample preparation protocol. The resulting peptides were desalted by using C18 cartridge. The solutions containing peptide fragments were dried in a lyophilizer and reconstituted by adding 40 \u0026micro;l 0.1% formic acid. Protein concentration of sample extracts was measured at OD280 nm. Appropriate amounts of iRT standard peptides were mixed into each sample, and each sample was subjected to dataindependent acquisition (DIA) mass spectrometry. The peptides from each sample were analyzed by OrbitrapTM AstralTM mass spectrometer (Thermo Scientific) connected to a Vanquish Neo system liquid chromatography (Thermo Scientific) in the dataindependent acquisition (DIA) mode. DIA data was analyzed with DIA-NN 1.8.1 searching the database from Uniport. A strict cutoff of Log fold-change\u0026thinsp;\u0026lt;\u0026thinsp;0.5 and fold-change\u0026thinsp;\u0026gt;\u0026thinsp;2 was used as the qualification criteria. The ratios were sorted by a P-value cutoff of 0.05 to obtain the list of differentially expressed proteins.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescent staining\u003c/h2\u003e\u003cp\u003eFrozen sections of the brain were incubated with DHE in the dark at 37\u0026deg;C for 30 minutes. Then the brain slices were incubated with DAPI at room temperature for 10 minutes. Sections were permeabilized with 0.04% Triton X-100 and blocked with 10% normal goat serum and 0.5% bovine serum albumin in PBS for 1 hour and then treated overnight at 4\u0026deg;C with primary antibodies: neuronal nuclear protein(NeuN)(Proteintech, 26975-1-AP), glial fibrillary acidic protein(GFAP)(Proteintech, 16825-1-AP), ionized calcium-binding adapter molecule 1(Iba-1)(Proteintech, 10904-1-AP), cluster of differentiation 31(CD31)(Proteintech, 28083-1-AP), Brain-derived neurotrophic factor(BDNF)(Proteintech, 25699-1-AP); Vascular endothelial growth factor(VEGF)(Proteintech, 26157-1-AP), and Doublecortin(DCX) (Proteintech, 13925-1-AP); After washing with PBS, the sections were incubated with fluorescence-conjugated secondary antibodies for 1 hour at room temperature. The sections were washed and counterstained with the nuclear dye 4,6-diamino-2-phenylindole. Fluorescence-labeled sections were viewed using a confocal microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eHistological Examination\u003c/h2\u003e\u003cp\u003eBrain, heart, liver, and kidney tissues were harvested and fixed in 4% paraformaldehyde for 24\u0026ndash;48 hours at 4\u0026deg;C. The tissues were then dehydrated using graded ethanol, cleared in xylene, and embedded in paraffin. Sections (measuring 5 \u0026micro;m) were cut, deparaffinized, and rehydrated. They were stained with Harris hematoxylin, differentiated in acid alcohol, and counterstained with eosin. Following dehydration and clearing, the sections were mounted with neutral resin. The stained sections were imaged under a light microscope to visualize tissue architecture and cellular morphology.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eTUNEL staining\u003c/h2\u003e\u003cp\u003eBrain sections (40 \u0026micro;m thickness) were prepared and washed in PBS. Sections were permeabilized with 0.25% Triton X-100 in PBS for 20 min at room temperature. Following permeabilization, sections were treated with 20 \u0026micro;g/mL proteinase K in 10 mM Tris-HCl (pH 7.4\u0026ndash;8.0) for 30 min at 37\u0026deg;C. After washing in PBS, sections were incubated with the TUNEL reaction mixture containing TMR red-labeled nucleotides and terminal deoxynucleotidyl transferase (TdT) for 1 h at 37\u0026deg;C in a humidified chamber. Sections were then rinsed in PBS to remove unbound reagents. Nuclei were counterstained with DAPI (2 \u0026micro;g/mL) for 10 min at room temperature to visualize total cell nuclei. Finally, sections were mounted on glass slides using Fluoromount-G and imaged under a fluorescence microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eWestern Blot Analysis\u003c/h2\u003e\u003cp\u003eThe total protein in the peri-infarct hemisphere was extracted, and the protein concentration was determined using the bicinchoninic acid protein assay. The protein samples were then denatured by boiling for 10 min. Then, 20 \u0026micro;g of each protein sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at a constant voltage of 80 V for 100 min. After transfer to polyvinylidene fluoride (PVDF) membrane, the separated protein bands were incubated with individual primary antibodies (Caspase-3, 19677-1-AP, Proteintech; Bax,50599-2-Ig, Proteintech; Bcl2,68103-1-Ig, Proteintech) at 4\u0026deg;C overnight, followed by incubation with the corresponding fluorescent secondary antibody at room temperature for 1 h. The developed bands were observed under Odyssey DLx near-infrared dual-channel laser imaging system.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eFor statistical analyses, GraphPad Prism 10.0 software (GraphPad software, Inc., La Jolla, CA, USA) was used. All tests were repeated three times or more and the results are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Student's t-test was used to analyze the difference between two groups. For comparisons of multiple groups, one-way analysis of variance followed by Tukey's post hoc test was used. The data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eSTS enhances functional recovery following MCAO\u003c/h2\u003e\u003cp\u003eTo evaluate the therapeutic efficacy of STS, rats underwent MCAO surgery on Day 0, followed by daily intraperitoneal injections of STS from Day 1 to Day 6 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e MRI was utilized to assess brain injury in three groups. On Day 1, compared with the Sham group, rats in both the MCAO group and the MCAO\u0026thinsp;+\u0026thinsp;STS group showed significant brain injury areas. On Day 7, the brain injury area of the MCAO\u0026thinsp;+\u0026thinsp;STS group was significantly reduced compared to that of the untreated MCAO group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Statistical analysis revealed a significant increase in infarct recovery rate in the MCAO\u0026thinsp;+\u0026thinsp;STS group, indicating that STS treatment positively contributes to the recovery of cerebral ischemic injury \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. MCAO rats treated with STS demonstrated significant improvement in neurological function when compared with the MCAO group on Day 7 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). On Day 7, HE staining revealed intact tissue architecture and clearly defined cell nuclei in the Sham group. In contrast, the MCAO group exhibited extensive tissue damage, characterized by blurred cell contours and widespread neuronal nuclear condensation with deepened staining. Compared to the MCAO group, STS treatment significantly reduced the pathological change of tissue necrosis and nuclear pyknosis, indicating its neuroprotective effect \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative proteomics reveals extensive microenvironmental alterations following MCAO\u003c/h2\u003e\u003cp\u003eIn this work, Astral was applied to obtain the difference in protein expression between the Sham group and the MCAO group in the subacute of ischemic stroke. A total of 4333 proteins were identified and quantified on Day 7. The volcano plots visualized all the identified proteins and highlighted the differentially expressed proteins \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The bar chart of 365 differentially expressed proteins is summarized \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Compared with the Sham group, a total of 305 proteins were upregulated (red bar), while only 60 proteins were downregulated (blue bar). To gain insight into the potential biological roles of these differentially expressed proteins, Gene Ontology (GO) annotations of proteins were classified into three categories: biological process, cellular component, and molecular function \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. In the molecular functions, the differentially expressed proteins between the MCAO group and the Sham group on day 7 were mainly distributed in \"protein binding (225)\", \"ion binding (103)\", \"hydrolase activity (72)\", \"protein-containing complex binding (71)\", \"organic cyclic compound binding (69)\", \"enzyme regulator activity(60)\", \"catalytic activity, acting on a protein(46)\", \"heterocyclic compound binding(38)\", \"transferase activity(37)\", \"Oxidoreductase activity (23)\". The representative cellular component category of these proteins was \"organelle (217)\", \"membrane (181)\", \"cytoplasm (133)\", \"extracellular space (98)\", \"cytosol (69)\", \"cell junction (69)\", \"cell projection (66)\", \"C: protein-containing complex (47)\", \"C: cell surface (47)\", \"C: perinuclear region of cytoplasm (40)\" categories. Their biological processes were mainly associated with \"regulation of biological process (257)\", \"cellular component organization or biogenesis (144)\", \"response to chemical (135)\", \"cellular metabolic process (124)\", \"anatomical structure development (103)\", \"organic substance metabolic process (88)\", \"cellular developmental process (83)\", \"signal transduction (83)\",\"response to biotic stimulus (78)\",\"establishment of localization (71)\". To further corroborate the GO findings and acquire the overall pathway, we performed Gene Set Enrichment Analysis (GSEA) on the rank-ordered whole-proteome lists from Sham and MCAO groups, among which 8 signaling pathways exhibited significant enrichment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and have been closely linked to cerebral ischemia \u003cb\u003e(Table. 1)\u003c/b\u003e. Among the gene sets analyzed, those related to \"Apoptosis\",[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] \"Phagosome\"[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and \"Cell adhesion molecules\"[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] have a relatively high gene count (setSize) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. This indicates that they play significant roles in the brain microenvironment following MCAO.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eSTS ameliorates the brain microenvironment following MCAO by inhibiting apoptosis\u003c/h2\u003e\u003cp\u003eProteomics was used to assess the effects of STS on the brain microenvironment following MCAO. Compared with the MCAO group, STS significantly reshaped the protein expression profile: the heatmap of differentially expressed proteins revealed the top 50 most significantly altered proteins \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of these differentially expressed proteins indicated significant regulation of the apoptosis pathway \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e Furthermore, GSEA suggested that the apoptosis-related gene set (rno04210) was overall activated, with a normalized enrichment score (NES) of -1.48 and a Pvalue of 0.01 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Western blot analysis further confirmed the upregulation of Bcl2/Bax (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. MCAO group ) and the downregulation of Caspase3 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. MCAOgroup, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). TUNEL staining showed significantly fewer apoptotic cells following STS treatment, indicating its pronounced anti-apoptotic effect \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. These findings illustrate that STS ameliorates the brain microenvironment through anti-apoptotic mechanisms, thereby creating a favorable condition for subsequent iPSCs transplantation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eTransfection of iPSCs with the GFP-Luc lentiviral vector\u003c/h2\u003e\u003cp\u003eImmunofluorescence confirmed successful transfection of iPSCs with the GFP-Luc lentiviral vector. GFP positive iPSCs colonies were observed, indicating successful lentiviral transfection of iPSCs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. To verify the relationship between luciferase activity and cell number, five tubes containing different numbers of iPSCs transfected with lentivirus were detected. The luminescence intensity increased visibly as the cell number increased, indicating a positive correlation between luciferase activity and cell number \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Quantitative analysis further revealed a highly linear relationship between iPSCs number and luciferase luminescence intensity (R\u0026sup2; = 0.99, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results demonstrate that transfection of GFP-Luc lentiviral vector enables iPSCs to express GFP and luciferase stably and that the luminescent signal can be used to quantify cell number accurately.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eSTS enhances the functional recovery mediated by iPSCs transplantation after MCAO in rats\u003c/h2\u003e\u003cp\u003eOn the day of transplantation (Day 1), bioluminescence imaging revealed detectable luciferase signals in both the MCAO\u0026thinsp;+\u0026thinsp;iPSCs and MCAO\u0026thinsp;+\u0026thinsp;iPSCs\u0026thinsp;+\u0026thinsp;STS groups, confirming successful cell engraftment. Over time, the luminescence intensity declined in two treatment groups; however, the MCAO\u0026thinsp;+\u0026thinsp;iPSCs\u0026thinsp;+\u0026thinsp;STS group exhibited significantly higher luciferase signals on Day 5 compared with the MCAO\u0026thinsp;+\u0026thinsp;iPSCs group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Quantitative analysis demonstrated that the MCAO\u0026thinsp;+\u0026thinsp;iPSCs\u0026thinsp;+\u0026thinsp;STS group exhibited a smaller reduction in iPSCs number relative to the MCAO\u0026thinsp;+\u0026thinsp;iPSCs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. MCAO\u0026thinsp;+\u0026thinsp;iPSCs group, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These data indicate that STS promoted the survival of iPSCs in vivo. On Day 7, the combined group exhibited a marked reduction in infarct size, with a higher recovery rate superior to that of MCAO\u0026thinsp;+\u0026thinsp;iPSCs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. MCAO\u0026thinsp;+\u0026thinsp;iPSCs group, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). The neurological score in the combined group was higher than that of the MCAO\u0026thinsp;+\u0026thinsp;iPSCs group on Day 7, suggesting that combined treatment is more effective in promoting the recovery of neurological function after MCAO than iPSCs transplantation alone \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eSTS combined with iPSCs enhances neurovascular restoration following MCAO\u003c/h2\u003e\u003cp\u003eImmunofluorescence staining was used to label neurons, astrocytes, microglia, and endothelial cells in brain tissue using NeuN, GFAP, Iba-1, and CD31, respectively. Compared with the Sham group, the MCAO group exhibited significant reductions in neurons, hypertrophy of astrocytes, activation of microglia, and a decrease in endothelial cell numbers, reflecting neuronal loss, inflammatory reaction, and vascular damage caused by cerebral ischemia. Both the MCAO\u0026thinsp;+\u0026thinsp;STS and the MCAO\u0026thinsp;+\u0026thinsp;iPSCs groups partially restored the balance of neurons and glial cells and increased endothelial cell numbers, indicating that both iPSCs therapy and STS therapy exert protective effects on the neurovascular unit after cerebral ischemia. Notably, compared with MCAO\u0026thinsp;+\u0026thinsp;iPSCs group, MCAO\u0026thinsp;+\u0026thinsp;iPSCs\u0026thinsp;+\u0026thinsp;STS group showed more pronounced neuronal recovery, further reduced glial cell activation, and a more significant increase in endothelial cell numbers on Day 7, suggesting that the combination of STS and iPSCs therapy more effectively promotes neurovascular restoration after cerebral ischemia \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eSTS combined with iPSCs enhances the paracrine secretion of BDNF and VEGF\u003c/h2\u003e\u003cp\u003eTo determine whether iPSCs can induce neurogenesis or angiogenesis, immunofluorescence staining of BDNF, VEGF and DCX were performed on brain tissue. BDNF, VEGF, and DCX positive cells were observed evenly distributed in Sham group. In MCAO group, the number of cells positive for BDNF, VEGF, and DCX was significantly reduced, indicating that ischemia resulted in neurotrophic depletion and the exhaustion of endogenous progenitors. The MCAO\u0026thinsp;+\u0026thinsp;iPSCs group exhibited more BDNF, VEGF, and DCX positive cells than those of the MCAO group, indicating that transplanted iPSCs promote tissue repair through paracrine mechanisms. In MCAO\u0026thinsp;+\u0026thinsp;iPSCs\u0026thinsp;+\u0026thinsp;STS group, the number of BDNF, VEGF, and DCX positive cells significantly increased than those of MCAO\u0026thinsp;+\u0026thinsp;iPSCs group, indicating a strong synergistic effect in vivo \u003cb\u003e(Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e)\u003c/b\u003e. BDNF, VEGF, and DCX expression were then assessed in GFP-labeled iPSCs with or without STS treatment in vitro. In MCAO\u0026thinsp;+\u0026thinsp;iPSCs\u0026thinsp;+\u0026thinsp;STS group, GFP signal was markedly enhanced, indicating increased iPSCs survival. The co-localization signals of BDNF, VEGF, and DCX with GFP were concurrently elevated, indicating enhanced paracrine secretion and an increased potential for differentiation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C\u003cb\u003e)\u003c/b\u003e. In summary, STS promoted the survival and proliferation of iPSCs in vivo and enhanced iPSCs-mediated neuroprotection and angiogenesis through a paracrine pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eSafety assessment of STS and/or iPSCs treatment in vivo\u003c/h2\u003e\u003cp\u003eHE staining demonstrated the histological changes in heart, liver, spleen, and kidney tissues of rats from five groups. In Sham group, the tissues of heart, liver, spleen, and kidneys all exhibited normal histological structures with tightly and orderly arranged cells, showing no apparent pathological changes. The tissues from MCAO, MCAO\u0026thinsp;+\u0026thinsp;STS, MCAO\u0026thinsp;+\u0026thinsp;iPSCs, and MCAO\u0026thinsp;+\u0026thinsp;iPSCs\u0026thinsp;+\u0026thinsp;STS groups also maintained integrity, with normal cellular morphology, and no evident inflammatory cell infiltration or tissue necrosis was observed, indicating that the treatment with STS and iPSCs did not cause significant organ pathological changes in the current study \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. We also measured the levels of liver function indicators (ALT, AST, ALB), kidney function indicators (CR), and complete blood count indicators (MCV, MPV) in rats from five experimental groups. The results of the serum liver and kidney function tests and the complete blood count showed no statistically significant differences among the five groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. In summary, the application of STS and iPSCs treatments in this study demonstrated good safety in rats, without causing detectable organ damage or dysfunction.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur results indicate that STS enhances the ischemic microenvironment following MCAO by inhibiting apoptosis, which improves the survival of transplanted iPSCs. Comparison of MCAO\u0026thinsp;+\u0026thinsp;iPSCs group, the surviving iPSCs secrete higher levels of BDNF, VEGF and DCX, thereby promoting neuroprotection and angiogenesis. Here, STS reduced infarct size and enhanced neurological function scores, suggesting that it significantly promotes functional recovery in MCAO rats. Furthermore, proteomic profiling was conducted to elucidate the mechanisms by which STS modulates the ischemic microenvironment. The analysis revealed extensive remodeling of the ischemic microenvironment, with 365 differentially expressed proteins identified. Both KEGG pathway enrichment and GSEA consistently demonstrated that STS primarily acts by inhibiting apoptosis, a finding that was further validated by Western blotting and TUNEL assays. Having established that STS improves the ischemic microenvironment, we next assessed iPSCs survival after transplantation using GFP-Luc-iPSCs. Compared with the iPSCs group, the combined treatment with STS significantly improved iPSCs survival, reduced infarct size and enhanced neurological function scores. Immunofluorescence further demonstrated that the combined therapy more effectively promoted neurovascular repair, as evidenced by increased expression of NeuN and CD31. Moreover, GFP co-localization with BDNF, VEGF and DCX was observed in transplanted iPSCs, indicating enhanced paracrine effects and neurogenic potential.\u003c/p\u003e\u003cp\u003eThe brain microenvironment experiences extensive and dynamic changes following MCAO, among which neuronal death is a key pathological event. Neuronal death exhibits strict spatiotemporal specificity, with apoptosis being one of the major underlying mechanisms,[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] ATP levels plummet to \u0026lt;\u0026thinsp;5% of baseline within minutes in the ischemic core, necrosis becoming the predominant mode of cell death.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] Secondly, ATP is maintained at roughly 20\u0026ndash;50% of baseline levels in the penumbra, creating an \"energy threshold window\" that allows for mitochondrial outer-membrane permeabilization without causing immediate energy collapse.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] Within the first 6 hours of ischemia, Bax and Bak proteins translocate from the cytosol to the mitochondrial outer membrane and assemble into oligomeric pore structures.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] Then, cytochrome c is released exponentially, reaching a peak between 12\u0026ndash;24 hours, where it binds to Apaf-1 to form the apoptosome.[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] The complex recruits and activates procaspase-9 via Caspase Recruitment Domain (CARD)-CARD interactions, thereby initiating the downstream caspase-3 cascades. In ischemic models, caspase activation can persist for 24 to 72 hours, leading to the cleavage of PARP-1 and ultimately driving the execution phase of apoptosis.[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] Importantly, glycolytic ATP production sustains the assembly of the apoptosome and the activation of caspases, prolonging neuronal apoptosis for 6\u0026ndash;72 hours and establishing a therapeutically accessible \"rescue window\" in both experimental and clinical settings.[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] In hippocampal regions distant from the ischemic core, neuronal apoptosis is triggered remotely by spreading depolarization (SD).[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] SD spreads outward from the infarct border and, upon reaching surrounding tissue, induces sustained depolarization and intracellular Ca\u003csup\u003e2+\u003c/sup\u003e overload, ultimately triggering mitochondria-mediated apoptosis.[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] Meanwhile, reactive oxygen species and nitric oxide produced during spreading depolarization exacerbate DNA damage and suppress the activity of the anti-apoptotic protein Bcl-2, creating a positive-feedback loop that accelerates apoptosis.[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] In summary, the disruption of the brain microenvironment induced by MCAO is centered on apoptosis as the critical node, propagating a cascade of damage through the penumbra, and remote hippocampal regions.\u003c/p\u003e\u003cp\u003eSTS improves the ischemic microenvironment by suppressing apoptosis, thereby significantly promoting the survival of transplanted iPSCs. Previous studies had shown that transplanted iPSCs rapidly undergo apoptosis or necrosis in the brain, with over 90% lost within 72 h.[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] The hostile microenvironment markedly impairs transplanted stem-cell survival after MCAO, limiting their therapeutic efficacy. Traditional Chinese medicine exerts neuroprotection against ischemic brain injury by suppressing apoptosis.[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] The active constituents of Danshen have been reported to exert neuroprotective effects. Among these, STS is a key derivative that has emerged as a promising therapeutic candidate for ischemic stroke.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] Its anti-apoptotic properties are deemed a crucial factor in its neuroprotective functions. Studies have shown that STS inhibits intrinsic apoptotic pathways by upregulating the anti-apoptotic protein Bcl-2, downregulating pro-apoptotic proteins Bax, caspase-3, and caspase-9, stabilizing mitochondrial membrane potential, reducing cytochrome c release, and blocking the caspase cascade.[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] Additionally, STS has been shown to inhibit the extrinsic apoptotic pathway by suppressing activation of Fas/FADD/caspase-8 and upregulating c-FLIP and v-FLIP, thereby preventing the formation of the death-inducing signaling complex.[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] In line with previous studies, the findings of the present study further indicate that STS has a significant therapeutic effect by exerting anti-apoptotic effects. STS markedly decreased the proportion of TUNEL-positive cells in the ischemic brain regions, simultaneously downregulating the expression levels of caspase-3. The results indicate that STS exerts neuroprotective effects by inhibiting the mitochondrial-mediated apoptosis pathway, thereby creating a more favorable microenvironment for the survival and functional integration of transplanted stem cells.\u003c/p\u003e\u003cp\u003eIn this study, the therapeutic benefits of iPSCs were predominantly mediated through paracrine mechanisms. The principal mechanisms underlying iPSCs-based therapy for ischemic stroke encompass cell replacement via direct neuronal and glial integration and paracrine modulation of the neurovascular[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] paracrine profile of iPSCs-derived cells is generally categorized into four functional groups, including neurotrophic and anti-apoptotic factors (BDNF and GDNF), pro-angiogenic and vascular remodeling factors (VEGF-A and bFGF), immuno-inflammatory modulators (IL-10 and TGF-β1), and chemotactic and cell-migration factors (SDF-1α and MCP-1).[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] Research has revealed that conditioned medium from hiPSCs-derived glial progenitor cells promote neuroprotection and angiogenesis by modulating inflammatory cytokines, such as TNF, IL-4, and IL-10, and by upregulating neurotrophic factors, including BDNF, CNTF and GDNF,[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] hiPSCs-MSC-derived extracellular vesicles activated VEGF/CXCR4 signaling pathways, thereby facilitating neurovascular repair.[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] BDNF is one of the most critical neurotrophic factors in the central nervous system, which can promote the survival, differentiation and synaptic plasticity of neurons.[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] VEGF is the central regulator of angiogenesis and vascular permeability, which can facilitate neovascularization and the repair of damaged blood vessels.[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] In this study, we investigated BDNF and VEGF as representative paracrine mediators, aiming to elucidate the mechanisms through which iPSCs confer neuroprotection in cerebral ischemia. Immunofluorescence results revealed that, compared with MCAO\u0026thinsp;+\u0026thinsp;iPSCs group, MCAO\u0026thinsp;+\u0026thinsp;iPSCs\u0026thinsp;+\u0026thinsp;STS group exhibited markedly stronger BDNF- and VEGF- positive signals that co-localized with GFP-iPSCs. Taken together, the iPSCs-secreted BDNF/VEGF axis synergistically promotes neuronal survival and angiogenesis.\u003c/p\u003e\u003cp\u003eSynergistic exogenous interventions substantially augment the therapeutic efficacy of stem cells for ischemic stroke. To promote the survival of stem cells, exogenous intervention primarily relies on two synergistic strategies: Genetic engineering modification of stem cells and improvement of ischemic microenvironment. Genetic engineering mediated by lentivirus or CRISPR/Cas9 enables one-step, high-efficiency integration of anti-apoptotic and pro-reparative genes-such as HO-1, FGF21 and VEGF-into stem cells. Compared with unmodified controls, this strategy elevates the 72-hour survival rate of cells within the ischemic penumbra from \u0026lt;\u0026thinsp;10% to 40\u0026ndash;60% and sustains high-level paracrine output for 4\u0026ndash;6 weeks.[\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] However, the high cost and regulatory hurdles associated with viral vectors limit their large-scale application. Traditional Chinese medicine combined with stem cells exerts therapeutic effects against cerebral ischemia via anti-inflammatory, antioxidant, pro-angiogenic, and neuroregenerative mechanisms. For instance, Human Umbilical Cord-Derived MSCs plus curcumin synergistically activate the AKT/GSK-3β/β-TrCP/Nrf2 axis, up-regulate HO-1 and NQO1 and suppress NF-κB\u0026ndash;mediated inflammatory cascades, reducing ROS by 60% within 72 h and increasing stem cell survival from \u0026lt;\u0026thinsp;10% to 45%.[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] Another example, Bone Marrow-Derived MSCs co-administered with ginsenoside CK activate GLUT1 and stabilize HIF-1α, persistently inducing VEGF-A and Ang-1 transcription. The result is that the microvascular density in the ischemic penumbra increases by 2.5-fold within 7 days, significantly enhancing angiogenesis.[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] Tanshinone IIA\u0026ndash;loaded nanoparticles co-delivered with NSCs showed a decrease in immune cell and reactive astrocyte activation, resulting in a significant increase in NSC survival. In a porcine MCAO model, this regimen reduces infarct volume by 35% and restores motor function to 75% of baseline within 4 weeks.[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] In the present study, the combination of STS and iPSCs significantly reduced the infarction size. Immunofluorescence results showed that the expressions of NeuN and CD31 in the STS combined with iPSCs group were significantly increased, while the expressions of GFAP and Iba-1 were significantly decreased. This suggests that the neuronal survival and vascular regeneration in the combined treatment group increased, while glial scars and inflammatory factors decreased. In all, it indicates that the STS promotes the survival and therapeutic effect of iPSCs by improving the cerebral ischemic microenvironment.\u003c/p\u003e\u003cp\u003eThis study has several limitations. Firstly, iPSCs were transplanted directly into the rat brain without immunosuppression, and we did not specifically assess the impact of xenogeneic rejection on iPSCs survival. Therefore, the extent to which immune rejection may have influenced the therapeutic efficacy remains uncertain. Secondly, this study was performed for only 1 week. We therefore could not form conclusions about longer-term response after iPSCs transplantation, including cell differentiation and tumorigenicity, although we did not find any tumor formation in our study. Therefore, future studies should introduce immunosuppression and extend the observation period.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, our study demonstrates that STS improves the ischemic microenvironment by reducing apoptosis, thereby further promoting the survival and proliferation of iPSCs. The combination of STS and iPSCs significantly reduces infarct size, improves behavioral scores, and promotes neurogenesis and angiogenesis through the paracrine release of BDNF and VEGF in vivo. STS combined with iPSCs could be a better therapeutic approach than these iPSCs used individually.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSTS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSodium tanshinone IIA sulfonate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eiPSCs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003einduced pluripotent stem cells\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTan IIA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTanshinone IIA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMCAO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emiddle cerebral artery occlusion\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eVTN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003evitronectin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSPF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003especific-pathogen-free\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCCA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecommon carotid artery\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eICA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003einternal carotid artery\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDIA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003edataindependent acquisition\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSDS-PAGE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003esodium dodecyl sulfate polyacrylamide gel electrophoresis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePVDF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003epolyvinylidene fluoride\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMRI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emagnetic resonance imaging\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGene Ontology\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGSEA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGene Set Enrichment Analysis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGFP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003egreen fluorescent protein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNeuN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eneuronal nuclei\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGFAP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlial fibrillary acidic protein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIba-1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eionized calcium-binding adapter molecule 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCD31\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecluster of differentiation 31\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBDNF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBrain-derived neurotrophic factor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eVEGF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eVascular endothelial growth factor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDCX\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDoublecortin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eALT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAlanine Aminotransferase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAST\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAspartate Aminotransferase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCreatinine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMCV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMean Corpuscular Volume\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMPV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMean Platelet Volume\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCARD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCaspase Recruitment Domain\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003espreading depolarization.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Home for Researchers (www.home-for-researchers.com) for the online illustration tools used to prepare some of the figures in this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYL, XL, HL, FS and WC conceived and designed research and performed experiments. MW, LL, XW and JZ analyzed data. RY, LZ, XW and QS prepared figures and edited text. YL and XL drafted manuscript, HL, FS and WC edited and revised manuscript. LZ, QS, XW, HL and FS gave final approval of the version to be published. All authors approved to submit this version to this publication.\u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially sponsored by grants from the Traditional Chinese Medicine Science and Technology Project of Zhejiang Provincial Health Commission (no. 2026ZL0189, 2025ZL257), National Natural Science Foundation of China (no. 82001862), Zhejiang Provincial Natural Science Foundation (no. LQ24H180010), and Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (no. 2024KY656, 2024KY737).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article (and its Additional files).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were conducted following the Guidelines for the Ethical Review of Laboratory Animal Welfare (China, 2018) and approved by the Experimental Animal Ethics Committee of Zhejiang Provincial People\u0026apos;s Hospital (approval number: 20240927814920).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u0026nbsp;\u003c/sup\u003eEmergency and Critical Care Center, Department of Emergency Medicine, Zhejiang Provincial People\u0026apos;s Hospital (Affiliated People\u0026apos;s Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, 310014, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u0026nbsp;\u003c/sup\u003eCancer Center, Department of Nuclear Medicine, Zhejiang Provincial People\u0026apos;s Hospital (Affiliated People\u0026apos;s Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, 310014, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u0026nbsp;\u003c/sup\u003eCenter for Rehabilitation Medicine, Department of Radiology, Zhejiang Provincial People\u0026apos;s Hospital (Affiliated People\u0026apos;s Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, 310014, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHilkens NA, Casolla B, Leung TW, de Leeuw FE. 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VEGF overexpression in transplanted NSCs promote recovery of neurological function in rats with cerebral ischemia by modulating the Wnt signal transduction pathway. Neurosci Lett. 2024;824:137668.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Y, Huang J, Wang J, Xia S, Ran H, Gao L, et al. Human umbilical cord-derived mesenchymal stem cell transplantation supplemented with curcumin improves the outcomes of ischemic stroke via AKT/GSK-3beta/beta-TrCP/Nrf2 axis. J Neuroinflammation. 2023;20:49.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen X, Qian W, Zhang Y, Zhao P, Lin X, Yang S, et al. Ginsenoside CK cooperates with bone mesenchymal stem cells to enhance angiogenesis post-stroke via GLUT1 and HIF-1alpha/VEGF pathway. Phytother Res. 2024;38:4321\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaiser EE, Waters ES, Yang X, Fagan MM, Scheulin KM, Sneed SE, et al. Tanshinone IIA-Loaded Nanoparticle and Neural Stem Cell Therapy Enhances Recovery in a Pig Ischemic Stroke Model. Stem Cells Transl Med. 2022;11:1061\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003e\u003cstrong\u003eTable.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GSEA results indicated that eight signaling pathways exhibited significant enrichment in the comparison between the Sham and MCAO groups.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"540\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.4712%;\"\u003e\n \u003cp\u003eID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.0334%;\"\u003e\n \u003cp\u003eDescription\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003esetSize\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003eNES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003ePvalue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003eAdjust Pvalue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.4712%;\"\u003e\n \u003cp\u003erno04210\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.0334%;\"\u003e\n \u003cp\u003eApoptosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e1.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.004\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.06\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.4712%;\"\u003e\n \u003cp\u003erno04145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.0334%;\"\u003e\n \u003cp\u003ePhagosome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e1.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.000\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.01\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.4712%;\"\u003e\n \u003cp\u003erno04514\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.0334%;\"\u003e\n \u003cp\u003eCell adhesion molecules\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e1.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.003\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.05\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.4712%;\"\u003e\n \u003cp\u003erno04620\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.0334%;\"\u003e\n \u003cp\u003eToll-like receptor signaling pathway\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e1.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.007\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.07\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.4712%;\"\u003e\n \u003cp\u003erno00520\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.0334%;\"\u003e\n \u003cp\u003eAmino sugar and nucleotide sugar metabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e1.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.007\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.07\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.4712%;\"\u003e\n \u003cp\u003erno04064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.0334%;\"\u003e\n \u003cp\u003eNF-kappa B signaling pathway\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e1.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.003\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.05\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.4712%;\"\u003e\n \u003cp\u003erno04979\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.0334%;\"\u003e\n \u003cp\u003eCholesterol metabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e1.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.005\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.06\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.4712%;\"\u003e\n \u003cp\u003erno04974\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.0334%;\"\u003e\n \u003cp\u003eProtein digestion and absorption\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e1.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.001\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.8738%;\"\u003e\n \u003cp\u003e0.04\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Sodium tanshinone IIA sulfonate (STS), induced pluripotent stem cells (iPSCs), middle cerebral artery occlusion (MCAO), magnetic resonance imaging (MRI), ischemic microenvironment, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-7950969/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7950969/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIschemic stroke is the second leading cause of disability and death worldwide. Sodium tanshinone IIA sulfonate (STS), a well-known Chinese medicine monomer, is commonly used to treat ischemic brain injury because of its anti-apoptotic, anti-oxidative and anti-inflammatory properties. The aim of this study was to clarify the mechanism of STS combined with induced pluripotent stem cells (iPSCs) in treating ischemic brain injury by investigating changes in the ischemic microenvironment in a rat model of cerebral ischemia-reperfusion injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study employed magnetic resonance imaging (MRI) to examine functional recovery after a combined therapeutic approach involving STS and iPSCs in vivo. Cerebral ischemia was induced by the middle cerebral artery occlusion approach, and thirty male rats were randomly assigned to five groups: Sham, MCAO, MCAO + STS, MCAO + iPSCs, and MCAO + iPSCs + STS. MRI were conducted on Days 1 and 7, with neurofunctional tests performed every other day. Additionally, 1.0×10⁶GFP-Luc-iPSCs were transplanted on Day 3 and bioluminescence imaging in brains was performed on Days 1 and 5 after transplantation. Proteomic analysis and immunofluorescent analyses were performed on Day 7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSTS reduced infarct size and enhanced neurological function scores after MCAO. Furthermore, proteomic analysis revealed extensive remodeling of the ischemic microenvironment, with 365 differentially expressed proteins between the Sham and MCAO groups. Proteomic analysis demonstrated that STS primarily improved the ischemic microenvironment by inhibiting apoptosis between the MCAO and STS groups, a finding that was further validated by Western blotting and TUNEL assays. We next assessed iPSCs survival after transplantation. Compared with the iPSCs group, the combined treatment with STS significantly improved iPSCs survival, reduced infarct size and enhanced neurological function scores. Immunofluorescence revealed an increased expression of NeuN and CD31, demonstrating that the combined therapy more effectively promoted neurovascular repair. Moreover, increased GFP co-localization with BDNF, VEGF, and DCX was observed in the combined treatment with STS, indicating STS enhanced iPSCs-mediated paracrine effects and neurogenic potential.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSTS enhances the survival and proliferation of iPSCs by improving the ischemic microenvironment through the suppression of apoptosis and enhances iPSCs-mediated paracrine mechanisms and their potential for differentiation. In summary, STS combined with iPSCs could be a more effective therapeutic approach than using these stem cells individually.\u003c/p\u003e","manuscriptTitle":"Sodium tanshinone IIA sulfonate improves the ischemic microenvironment by inhibiting apoptosis and promotes the treatment of ischemic brain injury by iPSCs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 06:53:08","doi":"10.21203/rs.3.rs-7950969/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6f5de3a8-314c-4467-8c1f-de87886fc10b","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T15:31:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-01 06:53:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7950969","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7950969","identity":"rs-7950969","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}