Jingpei Guo, Xiaoting Zhang, Junbin Liu, Ke Zhang, Bin Zhou,
1 Center of Interventional Medicine, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, Guangdong Province, 519000, China
2 Center of Cerebrovascular Disease, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, Guangdong Province, 519000, China
3 Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, Guangdong Province 519000, China.
4 Guangdong-Hong Kong-Macao University Joint Laboratory of Interventional medicine, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, Guangdong Province 519000, China.
* Correspondence: KZ, zhangk276@mail.sysu.edu.cn. BZ, zhoub2@mail.sysu.edu.cn.
† These authors contributed equally to this work.
Abstract
The tricho-rhino-phalangeal syndrome-1 gene (TRPS1) is an atypical member of the GATA-type transcription factor family. Prior studies have revealed the effects of TRPS1 in tumors. While prior studies have revealed the role of TRPS1 in tumor biology, its function and underlying mechanisms in ischemic stroke remain largely unexplored.
In this study, a permanent middle cerebral artery occlusion (pMCAO) model was used to simulate ischemic stroke in vivo, while an oxygen-glucose deprivation (OGD) model in PC12 cells was employed to mimic ischemic conditions in vitro. The extent of cerebral injury was assessed by TTC staining and neurological function scoring. Expression levels of TRPS1 were modulated via intracerebroventricular injection of siRNA. To elucidate the functional role of TRPS1 post-stroke, a combination of reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence staining, Western blotting, and flow cytometry was employed.
The results showed that pMCAO significantly increased TRPS1 expression in the ischemic brain. TRPS1 knockdown significantly reduced cerebral infarct volume, improved neurological function, and decreased neuronal apoptosis. Similarly, in vitro OGD conditions led to an upregulation of TRPS1 expression and a concurrent reduction in cell viability. Silencing TRPS1 significantly mitigated OGD-induced neuronal injury, enhancing cell survival and reducing apoptosis. Mechanistically, TRPS1 knockdown activated the JAK2/STAT3 signaling pathway in OGD-treated PC12 cells. Importantly, the neuroprotective effects of TRPS1 silencing were abolished upon inhibition of the JAK2/STAT3 pathway, confirming its critical role in mediating the observed effects. In conclusion, TRPS1 aggravates ischemic neuronal damage by inhibiting the JAK2/STAT3 pathway. Its knockdown confers neuroprotection, suggesting TRPS1 as a potential therapeutic target for ischemic stroke intervention.
- Introduction
Stroke is one of the leading causes of disability and mortality worldwide and is classified into hemorrhagic stroke and ischemic stroke, with over 80% of cases being ischemic strokes[1]. Acute cerebral ischemia leads to severe hypoxia and glucose deprivation, resulting in neuronal apoptosis, inflammation, and blood-brain barrier (BBB) damage[2-4]. The most efficient neuroprotective and neurorestorative treatment for acute ischemic stroke is reperfusion therapy with intravenous recombinant tissue fibrinogen activator or catheter-mediated endovascular thrombectomy[5, 6]. The majority of patients, however, did not receive reperfusion due to the limited treatment time window. As a result, a substantial number of patients miss the most effective treatment period. Therefore, understanding the mechanisms underlying early ischemic brain injury is essential for identifying novel neuroprotective targets and advancing clinical management strategies.
TRPS1 encodes a transcription factor containing a GATA-like zinc finger domain, and initially identified in association with skeletal developmental disorders[7]. Recent studies have demonstrated that TRPS1 is widely expressed across various tissues and regulates key biological processes including cell proliferation, differentiation, and apoptosis[8, 9]. Moreover, studies have shown that TRPS1 is involved in tumor cell proliferation, invasion, and chemoresistance by modulating signaling pathways such as Wnt/β-catenin and TGF-β[10, 11]. However, its function in central nervous system (CNS) disorders remains poorly unexplored. Our preliminary studies were the first to observe a time-dependent upregulation of TRPS1 protein expression in both in vivo and in vitro ischemic models. Notably, TRPS1 expression was positively correlated with the levels of neuronal apoptosis markers such as cleaved caspase-3 and BAX, suggesting that TRPS1 may function as a stress-responsive factor in ischemic brain injury. However, the molecular mechanisms underlying its role remain unclear.
Neuronal apoptosis following cerebral ischemia involves a multitude of complex signaling pathways, with the JAK2/STAT3 pathway playing a pivotal role in mediating neuroprotection. As a classical stress-responsive signaling cascade, JAK2/STAT3 is critically involved in regulating inflammatory responses, oxidative stress, blood-brain barrier integrity, as well as neuronal apoptosis and regeneration[12, 13]. Numerous studies have demonstrated that JAK2/STAT3 activation is strongly associated with reduced neuronal apoptosis following ischemia[13]. Given its diverse functions, identifying upstream regulators of the JAK2/STAT3 pathway is crucial for developing new therapeutic strategies against ischemic brain injury. However, the specific interaction between TRPS1 and the JAK2/STAT3 signaling pathway during ischemic neuronal injury has not been fully defined.
To date, it remains unclear whether TRPS1 contributes to the pathogenesis of stroke through the JAK2/STAT3 signaling axis. Considering the potential role of TRPS1 in regulating apoptosis and its interaction with the STAT3 pathway, this study aims to investigate TRPS1 expression in ischemic brain injury and elucidate its regulatory impact on the JAK2/STAT3 signaling cascade at the molecular level. By clarifying the function of TRPS1 in post-ischemic neuronal injury, this work seeks to provide novel insights into stroke pathogenesis and identify potential neuroprotective targets.
- Method
2.1. Animals
Male Sprague-Dawley (SD) rats (280–300g) bought from the Zhuhai BesTest Bio-Tech Co., Ltd., and Beijing Vital River Laboratory Animal Technology Co., Ltd. All of the rats were raised in a regular light/dark cycle, with plenty of water and food. All animal experiments were approved by the Ethics Committee of the Fifth Hospital of Sun Yat-sen University.
2.2. Permanent Cerebral Ischemia Model
Based on the Zea-Longa method , the animals were anesthetized by intraperitoneal injection of 4% tribromoethanol (1 ml/100 g). The skin was cut about 2 cm along the median line of the neck. The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) are carefully separated. The ECA was ligated, and then artery clips were placed on CCA to stop the blood flow. Next, a small incision was made in the CCA, and a nylon filament with a silicone tip (d = 0.265 mm) was inserted into the ICA slowly through the incision in the CCA about 18 mm, distant from the bifurcation of the CCA to block the middle cerebral artery(MCA) until resistance was felt. A suture was tied to the ECA to hold the filament in place, and then the clip was removed from the CCA. Finally, the incision was closed with a suture, and then the rats were placed under an infrared heat lamp until fully recovered.
2.3. Zea-Longa neurological score
In this study, the Zea-Longa Neurological Score was used to assess the neurological function of rats in the experimental group. Specifically, the Zea-Longa Neurological Score was as follows: (0) No neurological damage; (1) Inability to ex-tend the opposite forepaw completely; (2) Inability to walk straight, circling to the hemiplegic side; (3) Inability to walk straight, collapsing to the hemiplegic side; and (4) Unconscious or could not walk spontaneously. Neurological function scores 1-4 were used as assessment criterion for valid modeling.
2.4. 2, 3, 5-triphenyltetrazolium chloride (TTC) staining
The rats were executed with an overdose of anesthetic, and the brains were isolated and cut into 2-mm-thick coronal sections. The 2-mm-thick sections are incubated in 2% TTC solution at 37°C for 15 minutes in the dark. Subsequently, the sections were fixed with 4% paraformaldehyde for 2 hours and photographed. Infarct volume was expressed as a percentage of total infarct volume/total brain volume by Image J analysis software.
2.5. Stereotactic injection and drug administration
To knock down TRPS1 expression in vivo, we injected interfering fragments of small interfering RNA (siRNA) into the lateral cerebral ventricle with the help of a stereotactic apparatus. Briefly, we fixed the head of rat head on a stereotaxic apparatus, and then extracted 10 μl siRNA with a microsyringe and injected TRPS1 siRNA or disordered siRNA into the right ventricle at a rate of 0.5 μl/min (1.0 mm posterior to the bregma, 1.5 mm lateral, 3.5 mm deep). MCAO was performed 24 h after intracerebroventricular injection of siRNA. AG490 (10 µM) was purchased from MedChemExpress for blocking the JAK/STAT3 signaling pathway. The inhibitor was first dissolved in DMSO and then further diluted with saline to prepare a working solution. AG490 group rats were administered AG490 (10 mg/kg) by intraperitoneal (i.p.) 1 h before ischemia. An equal volume of solvent was administered to Sham or pMCAO rats.
2.6. PC12 cell culture culture and Oxygen-glucose deprivation（OGD） model
PC12 cells were cultured (37 °C, 5 % CO2, 95 % air) in 10 cm2 tissue culture dishes with DMEM medium, supplemented with 10 % fetal bovine serum (FBS), 100 μg/mL of streptomycin, and 100 U/mL of penicillin. For OGD treatment, the PC12 cells were washed twice with the phosphate‐buffered saline (PBS, pH 7.4) and then refreshed with the glucose‐free DMEM and then incubated with a gaseous mixture of 5% CO2 / 95% N2 for the indicated time. AG490(a JAK2/STAT3 inhibitor) were added to the medium 1 h prior to OGD treatment.
2.7. Cell transfection
Small interfering RNA for TRPS1 (siRNA-TRPS1) and siRNA negative control (siRNA-NC) were purchased from GenePharma, Shanghai, China. The siRNA was transfected into PC12 cells using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. After 48 h of transfection, the cells were harvested for further analysis. si-TRPS1: sense 5′-GCCUUAAUCCAGAGUUGAATT-3′, antisense 5′-UUCAACUCUGGAUUAAGGCTT-3′.
2.8. TUNEL staining
Neurocyte apoptosis in cortex was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling assay (TUNEL, Beyotime, China). Briefly, the tissue slides were incubated in proteinase K (20 μg/ml) at 37°C for 20 min. After rinsing in PBS three times, the slides were incubated with terminal deoxynucleotidyl transferase enzyme and Cy3-labeled dUTP mixture and kept at 37°C, avoiding light for 1 hour. Tissue apoptosis was then observed and photographed under a fluorescence microscope. In five microscope fields in each section, TUNEL-positive and DAPI-positive cells were counted using Image J. The percentage of TUNEL-positive cells was determined as TUNEL-positive cells/total cells.
2.9. Cell viability assays
Assessment of PC12 cell viability using Cell Counting Kit-8 (CCK-8, Vazyme, Nanjing, China) according to the manufacturer’s instructions. In brief, PC12 cells (100 µL) at 1×104 cells/mL were cultured in 96-well plates at 37 °C for 24 h. then the CCK-8 reagent was added to each well at a ratio of 1:9. After incubation for 2 h at 37 degrees Celsius, the absorbance at 450 nm was detected using a microplate spectrophotometer (Thermo Scientific, USA).
2.10. Flow cytometry
Apoptosis was detected using an Annexin V-FITC Apoptosis Detection Kit I (BD Pharmingen; BD Biosciences). According to the manufacturer’s instructions, the solution was double stained with Annexin V-FITC/propidium iodide (PI), and then apoptotic cells were analyzed by flow cytometry (CytoFLEX, Beckman Coulter, USA).
2.11. Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from brain tissue and PC12 cells using Trizol reagent and Total RNA Kit I (Omega Bio-Tek, USA) according to the manufacturer’s instructions. The cDNA was reverse transcribed using the PrimeScript™ RT Master Mix (Takara Biotechnology, Japan) according to the manufacturer’s instructions. The SYBR green-based RT-qPCR kit (Takara) and real-time PCR detection system (Bio-rad) were used for quantification of messenger RNA (mRNA). Normalized the mRNA expression level of the target gene to the reference gene β-actin, and calculated it using a comparative quantitative method (2 -ΔΔCq). Primer sequences (Huada Gene Research Institute, Shenzhen, China) were as follows:
TRPS1: Forward, 5′-CAAACGGGGAGCAGACTAGG-3’
Reverse, 5′-CCTCGCCTTCACTAGCAACG-3′
β-actin: Forward, 5′-CAAGAAGGTGGTGAAGCAG-3’
Reverse, 5′-AAAGGTGGAAGAATGGGAG-3′
2.12. Western blot analysis
The RIPA protein extraction reagent (Beyotime, Beijing, China) containing the protease inhibitor mixture was used to homogenize brain tissue and cells. Equal amounts of protein samples(50μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (8 % ∼12 %) and transferred to polyvinylidene fluoride membranes. The membrane was then blocked with 5% skimmed milk TBST for 2 hours and incubated with primary antibody at 4 °C overnight. The next day, the membranes were washed and then incubated with secondary antibodies for 1 hour at room temperature. The gray value of each band was quantified using ImageJ software, which was calculated as the gray value of the target protein band divided by the gray value of the internal reference β-actin band. The primary antibodies were as follows: β-actin (1:1000; 60008-1-Ig, Proteintech), TRPS1 (1:1000; 21938-1-AP, Proteintech), Bax (1:1000; 50599-2-Ig, Proteintech), Bcl-2 (1:1000; 68103-1-Ig, Proteintech), Cleaved Caspase-3 (Asp175) (1:1000; 9661, CST), JAK2 (1:1000; 3230, CST), phosphorylated (p)-JAK2 (1:800; 3776, CST), p-STAT3 (1:1000; 9145, CST), STAT3 (1:1000; 9139, CST).
2.13. Statistical analysis
The data were analyzed and expressed as mean ± standard deviation (SD) using GraphPad Prism version 9.0 (GraphPad Software, Inc.). Multiple group comparisons were performed using one-way ANOVA and Tukey’s test. P<0.05 was considered to indicate a statistically significant difference.
- Result
3.1. TRPS1 expression is upregulated in ischemic brain tissues of pMCAO rats and OGD-induced PC12 cells
To assess the expression pattern of TRPS1 following cerebral ischemia, a permanent middle cerebral artery occlusion (pMCAO) model was established in Sprague-Dawley rats. RT-qPCR and Western blot analyses revealed that both mRNA and protein levels of TRPS1 were significantly increased in the ischemic brain tissues compared to the sham group, with the highest expression observed at 24 hours post-ischemia (Fig. 1A, B). Consistent with these in vivo findings, in vitro experiments using PC12 cells subjected to oxygen-glucose deprivation (OGD) exhibited a time-dependent increase in TRPS1 expression. Compared to the control group, TRPS1 expression was markedly elevated in OGD-treated cells (Fig. 1C, D). Collectively, these results indicate that TRPS1 is significantly upregulated in response to ischemic injury in both in vivo and in vitro models, supporting its potential involvement in the pathophysiological process of ischemic stroke. Based on these findings, 24-hour post-ischemia time point was selected for subsequent mechanistic analyses.
Fig. 1. TRPS1 expression levels in the in vivo pMCAO model and in the vitro OGD model. (A) TRPS1 mRNA levels in brain tissue of pMCAO rats, detected by qRT-PCR (n=3). (B) TRPS1 protein expression in brain tissues of pMCAO rats, analyzed by Western blot (n = 3). (C) PC12 cells were subjected to OGD to simulate cerebral ischemic injury in vitro, and TRPS1 mRNA levels were measured by qRT-PCR (n = 3). (D) Western blot analysis of TRPS1 protein expression in PC12 cells following OGD treatment (n = 3). *P＜0.05, **P＜0.01, ***P＜0.005 vs. sham group.
3.2. Knockdown of TRPS1 attenuates ischemic brain injury in pMCAO rats
To further evaluate the role of TRPS1 in the progression of ischemic stroke, TRPS1 expression was silenced via intracerebroventricular injection of TRPS1-specific siRNA (TRPS1 siRNA). This intervention significantly reduced TRPS1 mRNA and protein levels in brain tissues, as confirmed by qRT-PCR and Western blot analyses (Fig. 2A, B). As shown in Figure 2C and 2D, compared with the negative control group (pMCAO + siNC), TRPS1 knockdown markedly decreased infarct volume and improved neurological function scores. No significant differences were observed between the pMCAO and pMCAO + siNC groups. Histopathological evaluation using hematoxylin and eosin (HE) staining was conducted to assess ischemia-induced brain injury. As shown in Fig. 2E, neurons in the pMCAO and pMCAO + siNC groups exhibited prominent vacuolization, membrane shrinkage, and nuclear pyknosis, whereas the TRPS1 siRNA group showed a reduced number of apoptotic or damaged neurons, indicating attenuated neuronal injury. Furthermore, TRPS1 knockdown significantly downregulated the expression of cleaved caspase-3 and Bax, while upregulating the anti-apoptotic protein Bcl-2 (Fig. 2F). These findings suggest that silencing TRPS1 mitigates neuronal apoptosis and alleviates cerebral ischemic damage.
Fig. 2. Knockdown of TRPS1 attenuates brain tissue damage following cerebral ischemia in rats. (A, B) TRPS1 knockdown efficiency in rat brain tissue was evaluated 24 h after pMCAO by qRT-PCR and Western blot analysis (n = 3). (C) Cerebral infarct volume assessed by TTC staining (n = 3). (D) Neurological deficits were evaluated using the Zea-Longa scoring system (n = 10). (E) Histopathological changes in brain tissue examined by HE staining (n = 3). Scale bar = 100 μm. (F) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 protein levels in brain tissues from sham and pMCAO-treated rats (n = 3). ***P＜0.005 vs. sham group; ###P＜0.005 vs. pMCAO+siNC group.
3.3. TRPS1 knockdown attenuates OGD-induced apoptosis in PC12 cells
Apoptosis has been widely acknowledged as a central mechanism underlying neuronal death following cerebral ischemia. To investigate the functional role of TRPS1 in OGD-induced neuronal injury, an in vitro gene silencing model was established by transfecting PC12 cells with TRPS1-specific siRNA. As shown in Fig. 3A, B, both Western blot and qRT-PCR analyses confirmed that TRPS1 siRNA effectively suppressed the OGD-induced upregulation of TRPS1 at the protein and mRNA levels. PC12 cells were transfected with TRPS1 siRNA prior to OGD exposure. As shown in Fig. 3C, cell viability was significantly reduced in the OGD and OGD + siNC groups compared to the control group, whereas TRPS1 knockdown markedly restored cell viability following OGD insult. TUNEL staining further demonstrated that the proportion of apoptotic cells was significantly increased in the OGD and OGD + siNC groups compared to the normal control, while TRPS1 knockdown significantly reduced the percentage of TUNEL-positive cells (Fig. 3D). Furthermore, Western blot analysis revealed that OGD treatment led to increased apoptosis, as indicated by elevated levels of cleaved caspase-3 and Bax and reduced levels of the anti-apoptotic protein Bcl-2. These findings indicate that TRPS1 knockdown effectively reverses OGD-induced apoptotic signaling, highlighting its protective role against ischemic injury in PC12 cells (Fig. 3E , 3F).
Fig. 3. Knockdown of TRPS1 attenuates apoptosis in OGD/R-treated PC12 cells. (A, B) Following transfection of PC12 cells with TRPS1-specific siRNA, TRPS1 mRNA and protein levels were evaluated by qRT-PCR and Western blot, respectively (n = 3). (C) Cell viability was assessed in each treatment group using the CCK-8 assay. (D) Apoptotic cells were detected by TUNEL staining (n = 3). Scale bar = 50 µm. (E) Protein expression levels of Bax, Bcl-2, and cleaved caspase-3 were analyzed by Western blot (n = 3). (F) Flow cytometry analysis was used to examine apoptosis (n = 3). ***P＜0.005 vs. control group; #P＜0.05, ###P＜0.005 vs. pMCAO+siNC group.
3.4. Knockdown of TRPS1 activates the JAK2/STAT3 signaling pathway in both in vivo and in vitro models of ischemic stroke
The JAK2/STAT3 signaling pathway plays a critical regulatory role in neuronal apoptosis induced by ischemic stroke. Based on this observation, the present study aimed to elucidate whether TRPS1 contributes to the regulation of neuronal apoptosis via modulation of the JAK2/STAT3 pathway. To investigate this, we examined the expression levels of key signaling molecules in the JAK2/STAT3 cascade—namely p-JAK2, JAK2, p-STAT3, and STAT3—using Western blot analysis. As shown in Figure 4, the phosphorylation levels of JAK2 and STAT3 were significantly decreased in both the pMCAO and OGD model groups compared to the sham group. Notably, TRPS1 knockdown led to a marked increase in the levels of p-JAK2 and p-STAT3 relative to the respective model groups. These findings suggest that si-TRPS1 can activate the JAK2/STAT3 signaling pathway.
Fig. 4. si-TRPS1 treatment enhances activation of the JAK2/STAT3 signaling pathway.
(A) Western blot analysis of p-JAK2, JAK2, p-STAT3, STAT3, and β-actin protein levels in brain tissues from different treatment groups (n = 3). (B) Western blot analysis of p-JAK2, JAK2, p-STAT3, STAT3, and β-actin in PC12 cells under indicated conditions (n = 3). **P＜0.01 vs. sham or Control group; ##P＜0.01, ###P＜0.005 vs. pMCAO+siNC or OGD+ siNC group.
3.5. TRPS1 exerts its protective effects against OGD-induced injury via the JAK2/STAT3 signaling pathway
To further determine whether the protective effects of TRPS1 against OGD-induced cellular injury are mediated through the JAK2/STAT3 signaling pathway, PC12 cells transfected with either si-TRPS1 or si-NC were subjected to OGD treatment in the presence or absence of the JAK2 inhibitor AG490. TUNEL staining and flow cytometry analysis revealed that TRPS1 knockdown significantly reduced apoptosis under OGD + siNC conditions compared with the si-NC group (Fig. 5A, 5B). However, co-treatment with AG490 markedly reversed the anti-apoptotic effects observed in the si-TRPS1 group, as evidenced by an increased apoptotic rate. Western blot analysis further confirmed these observations. The addition of AG490 partially abrogated the protective effects of TRPS1 knockdown, as indicated by decreased expression of the anti-apoptotic protein Bcl-2, along with increased levels of the pro-apoptotic proteins Bax and cleaved caspase-3 (Fig. 5C). These findings suggest that the anti-apoptotic role of TRPS1 in OGD-induced neuronal injury is largely dependent on the activation of the JAK2/STAT3 signaling pathway.
Fig. 5. TRPS1 attenuates OGD-induced apoptosis via the JAK2/STAT3 signaling pathway in PC12 cells.
(A) TUNEL staining was used to assess apoptotic cell death in PC12 cells transfected with si-NC or si-TRPS1 and exposed to OGD, with or without the JAK2 inhibitor AG490 (n = 3). Scale bar = 50 μm. (B) Flow cytometry analysis of apoptosis across different treatment groups (n = 3). (C) Western blot analysis of apoptosis-related proteins, including Bcl-2, Bax, and cleaved caspase-3, in PC12 cells subjected to OGD following siRNA transfection and AG490 treatment (n = 3). *P＜0.05 **P＜0.01, ***P＜0.005 vs. OGD+siNC group; ###P＜0.005 vs. OGD+ siTRPS1 group.
3.6. JAK2/STAT3 Signaling Mediates the Neuroprotective Effects of TRPS1 Knockdown in Cerebral Ischemic Injury
Previous studies have shown that TRPS1 knockdown activates the JAK2/STAT3 signaling pathway in the pMCAO rat model. To further elucidate the underlying regulatory mechanism, the present study employed the JAK2-specific inhibitor AG490 for pharmacological intervention. As illustrated in Fig. 6A, compared with the pMCAO + siNC group, TRPS1 knockdown significantly decreased the expression of pro-apoptotic markers cleaved caspase-3 and Bax, while increasing the expression of the anti-apoptotic protein Bcl-2. However, treatment with AG490 effectively reversed the neuroprotective effects conferred by TRPS1 knockdown. These findings suggest that TRPS1 exerts its protective role against ischemic brain injury primarily through the modulation of key nodes within the JAK2/STAT3 signaling pathway, ultimately influencing the expression of apoptosis-related proteins.
Fig. 6. TRPS1 knockdown attenuates ischemia-induced neuronal apoptosis via activation of the JAK2/STAT3 signaling pathway.
(A) Western blot analysis of apoptosis-related proteins (cleaved caspase-3, Bax, and Bcl-2) in ischemic brain tissues from different treatment groups (n = 3). **P＜0.01 vs. pMCAO+siNC group. ##P＜0.01 vs. pMCAO+ siTRPS1 group.
- Discussion
Ischemic stroke is a highly prevalent neurological disorder with substantial morbidity and long-term disability[14]. Its core pathological feature is neuronal injury and death resulting from cerebral ischemia and hypoxia. Although significant progress has been made in acute reperfusion therapies, secondary injuries such as inflammation, oxidative stress, and apoptosis remain critical contributors to poor prognosis[15, 16]. Consequently, there is a pressing need to identify novel molecular targets that can modulate neuronal injury, thereby offering new therapeutic avenues to improve clinical outcomes in patients with ischemic stroke[17].
As a transcription factor, TRPS1 has also been found in other studies to be involved in the regulation of cell proliferation and apoptosis. Notably, in tumor cells, its suppressive function enhances cellular sensitivity to chemotherapy, further supporting its broad role in apoptosis regulation[18]. Several studies have highlighted the association of TRPS1 with apoptosis. For instance, TRPS1 promotes apoptosis in ATDC5 chondrocytes by inhibiting the expression of the anti-apoptotic protein BCL2[19]. Moreover, TRPS1 has been identified as a repressor of STAT3 expression and regulates chondrocyte proliferation and apoptosis by modulating cyclin D1 and BCL2 levels. Knockout of TRPS1 leads to upregulation of BCL2 and decreased apoptosis of growth plate chondrocytes[20]. These findings suggest that TRPS1 is closely involved in the regulation of cell apoptosis. In this context, our study reveals that TRPS1 is significantly upregulated following ischemic brain injury, indicating a pivotal role in the pathophysiology of cerebral ischemia. Upon siRNA-mediated silencing of TRPS1, we demonstrated that it exerts a neuroprotective effect under ischemic conditions.
The “double-edged sword” effect of the JAK2/STAT3 signaling pathway in cerebral ischemia has been widely reported, as this pathway is involved in multiple pathological processes following stroke, including neuronal apoptosis, inflammatory responses, and glial activation[13]. Therefore, the regulation of this pathway by TRPS1 may play a pivotal role not only in promoting neuronal survival but also in modulating the inflammatory microenvironment of brain tissue, thereby further enhancing its neuroprotective effects. Consistent with our findings, many studies have suggested that activation of the JAK2/STAT3 pathway can alleviate ischemic brain injury. For instance, activation of the epidermal growth factor receptor (EGFR) has been shown to mitigate cerebral ischemia/reperfusion injury in rats via stimulation of the JAK2/STAT3 signaling pathway[21]. Similarly, leptin exerts neuroprotective effects in pMCAO rat models, which are associated with increased phosphorylation of STAT3 in different cell populations within the injured brain tissue[22]. Additionally, adiponectin (APN) has been reported to protect HT22 neuronal cells from OGD-induced mitochondrial oxidative stress and apoptosis through JAK2/STAT3 pathway activation[23].
This study investigated the neuroprotective role of TRPS1 in ischemic brain injury and its underlying molecular mechanisms. By employing both pMCAO rat model and OGD cellular model, we found that the upregulation of TRPS1 under ischemic stress conditions holds significant pathophysiological relevance. Notably, knockdown of TRPS1 significantly reduced infarct volume, improved neurological scores, and decreased neuronal apoptosis, effects that were associated with the activation of the JAK2/STAT3 signaling pathway. Furthermore, TRPS1 suppression led to enhanced phosphorylation of JAK2 and STAT3, suggesting a potential negative regulatory role of TRPS1 on this pathway, ultimately attenuating neuronal injury. These findings expand our understanding of the molecular regulatory network in ischemic brain injury and provide a strong rationale for developing therapeutic strategies targeting apoptosis-related pathways[24].
Despite these advancements, limitations remain: 1) The direct molecular mechanism by which TRPS1 regulates JAK2/STAT3 phosphorylation requires clarification through phosphoproteomics to unravel interaction networks[25]; 2) The correlation between TRPS1 expression profiles in clinical samples and stroke prognosis warrants validation[26]; 3) Potential off-target effects of long-term TRPS1 inhibition need systematic evaluation[27]. Future research should focus on developing TRPS1-specific small-molecule modulators and employing conditional knockout models to investigate its spatiotemporal regulatory mechanisms.
- Conclusion
This study reveals that TRPS1 is significantly upregulated in ischemic stroke and plays a neuroprotective role by reducing neuronal apoptosis. Specifically, TRPS1 knockdown alleviates neuronal injury in both OGD and pMCAO models by activating the JAK2/STAT3 signaling pathway, reducing the expression of pro-apoptotic proteins, and enhancing the levels of anti-apoptotic factors. These findings not only expand our understanding of the molecular mechanisms underlying ischemic stroke but also highlight TRPS1 as a novel and promising therapeutic target for its treatment.
Author’s contributions
JG, KZ, and BZ conceived and designed the experiments. JG, XZ, and JL performed the experiments. JG and XZ analyzed the data and prepared the Figures. JG, KZ and BZ drafted the manuscript. XZ and KZ revised the manuscript. All authors read and approved the final manuscript.
Funding
This study was supported by the National Natural Science Foundation of China (No. 82102164).
Beijing Science and Technology Innovation Medical Development Foundation (KC2021-JX-0186-4).
Declaration of Competing Interest
The authors declare that they have no competing interests.
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