Background: Ischemic cerebral infarction is one of cerebrovascular diseases with high incidence, disability rate, and mortality globally, and neuronal cell apoptosis is a crucial cause of brain injury during cerebral infarction. Methods: A middle cerebral artery occlusion (MCAO) model was built in Sprague-Dawley rats to simulate ischemic cerebral infarction. An in vitro model of ischemic cerebral infarction was constructed in BV2 cells with the treatment of oxygen-glucose deprivation (OGD). The role and mechanism of serine/arginine-rich splicing factor 3 (SRSF3) in ischemic cerebral infarction were investigated both in animal and cell models. Results: The expression of SRSF3 was downregulated in MCAO-treated rats. Overexpression of SRSF3 reduced the neurological scores, brain water content, and infarct volume in MCAO-induced rats. Increased apoptosis in neurons accompanied with the abnormal expressions of apoptosis-related proteins in MCAO-induced rats were revised with the upregulation of SRSF3. Also, a diminished cell viability and elevated apoptosis rate were indicated in OGD-induced BV2 cells, which were reversed with the overexpression of SRSF3. Besides, OGD induced an enhancement in the relative protein expression of programmed cell death protein 4 (PDCD4) and a reduction in the relative expression of p-PI3K/PI3K and p-AKT/AKT, which were inverted with the upregulation of SRSF3 in BV2 cells. Overexpression of PDCD4 abolished the role of SRSF3 in cell viability, apoptosis rate, and the level of the PI3K/AKT pathway in OGD-induced BV2 cells. Conclusion: SRSF3 improved ischemic cerebral infarction via PDCD4 in vivo and in vitro, which was closely associated with the PI3K/AKT signaling pathway.

Cerebrovascular disorder is a type of acute neurological disease with high incidence, disability rate, and mortality, which has severely endangered human health [1]. Stroke has been the second leading cause of disability and death worldwide based on the data from the World Health Organization [2]. Ischemic stroke accounts for almost 90% of stroke, which causes poor life quality of patients and a huge public health burden [3]. The blockade of cervical or cerebral blood flow and insufficient perfusion in a short period of time cause the death of the brain cells, owing to hypoxia, which in turn evokes brain dysfunction [5]. Despite the at least partial compensation for brain injury after ischemia from the function of surviving neurons, the repair capacity of brain neurons is finite [6]. Timely blood flow reperfusion can rapidly restore the blood supply for ischemic stroke; however, cerebral ischemia/reperfusion (I/R) injury vastly restricts its development [7]. Although thrombolytic therapy is the most resultful approach for ischemic stroke in a clinic, thrombolysis has a strict treatment time window, and most patients are admitted to the hospital without optimal treatment of thrombolysis [8]. It has been demonstrated that the understanding of mechanisms of ischemic cerebral infarction can contribute to the improvement of prognosis of stroke [9]. Therefore, a better comprehension of the molecular mechanisms and the discovery of potential therapeutic target are of significance for the development of the ischemic stroke therapy.

Serine/arginine-rich splicing factor 3 (SRSF3) belongs to the serine/arginine-rich family, which plays a significant role in the modulation of alternative splicing of pre-mRNA [10], mRNA cytoplasmic transport [12], mRNA stability [13], mRNA localization [14], and translational control [15]. SRSF3 is indispensable for appropriate embryonic development. Embryos with the deficit of SRSF3 fail to form blastocysts and die at the morula stage [16]. The aberrant expression of SRSF3 has been reported in different diseases, such as Alzheimer’s disease [11], heart failure [17], ocular hypertension [18], and cancers [19]. Moreover, the expression of SRSF3 is decreased in the adult mice heart during myocardial infarction [17], and SRSF3 participates in the ameliorative role of miR-486 in fibrotic activity during myocardial infarction [20], indicating that SRSF3 plays an important role in myocardial infarction. However, whether SRSF3 plays a role in ischemic cerebral infarction is unknown.

Programmed cell death protein 4 (PDCD4), located at chromosome 10q24, is a potential downstream target of SRSF3. Kim et al. [14] report that SRSF3 directly interacts with PDCD4 mRNA and regulates translational inhibition via binding to the 5′-untranslated region. The protein expression of PDCD4 is suppressed by SRSF3 through coordinated modulation of alternative splicing, export, and translation [21]. Moreover, inhibition of PDCD4 reduces infarct injury and cortical neuronal apoptosis in cerebral I/R injury [22]. PDCD4 as the target of miR-340-5 p mitigates neuronal injury in oxygen-glucose deprivation (OGD)/reoxygenation [23]. More importantly, PDCD4 is strongly involved in the occurrence of apoptosis [24]. PDCD4 participates in the myocardial I/R injury by inhibiting apoptosis, acting as the target of miR-21 [25], miR-206 [26], miR-200a-3p [27], and miR-499 [28]. Apoptosis is a significant pathogenesis of ischemic brain damage [30]. Thus, based on the abovementioned findings, we speculated that SRSF3 might function on ischemic cerebral infarction by regulating apoptosis via PDCD4.

Therefore, to verify the speculation, a middle cerebral artery occlusion (MCAO) model was built in Sprague-Dawley (SD) rats to simulate ischemic cerebral infarction in vivo. On the other hand, an in vitro model of ischemic cerebral infarction was constructed in BV2 cells with the treatment of OGD. The role of SRSF3 in ischemic cerebral infarction was investigated both in MCAO-induced rats and OGD-induced BV2 cells. Besides, the possible mechanism was explored in vitro.

Animal

Male SD rats (180–220 g) were provided by Junke Biological Co., Ltd. (Nanjing, China), and kept in a laboratory room with 40–60% relative humidity and 12-h/12-h light-dark cycle at 22°C. All the procedures were strictly executed in keeping with the Guide for the Care and Use of Laboratory Animals [31] and the Animal Research Ethics Committee of Heping Hospital Affiliated to Changzhi Medical College.

Construction of the MCAO Model

Based on the previous study [32], rats were introduced with 2% isoflurane (RWD Life Science, Shenzhen, China) for anesthesia, and the right common carotid artery (CCA), external carotid artery, and internal carotid artery were then separated through a ventral midline incision. The distal external carotid artery and proximal CCA were ligated. To obstruct the origin of the right middle cerebral artery, a 4–0 nylon suture (Φ0.26 mm) with the tip embedded in paraffin was interposed from the right CCA to the internal carotid artery and to the circle of Willis. The sham rats were administrated the identical operative programs without the MCAO.

Animal Group and Treatment

To upregulate the expression of SRSF3, the sequence of SRSF3, as well as the scrambled control, was constructed and packed into the adeno-associated virus (AAV) 2 virus (GENECHEM, Shanghai, China). AAV particles (2 μL) were stereotactically delivered into the cortex at 3 sites (AP + 1.2 [site 1], 0.3 [site 2], −0.6 [site 3]; ML + 5.5; DV −3.5 mm from the skull) according to the previous report [32] at the rate of 0.5 μL/min by using an automatic injector. After the injection, the needle was kept for extra 5 min and withdrawn at a short distance for retention for another 2 min. A total of 20 rats were stochastically apportioned into four groups (n = 5), including sham, MCAO, MCAO+scramble, and MCAO+AAV-SRSF3. Rats in MCAO, MCAO+scramble, and MCAO+AAV-SRSF3 groups were first stereotactically injected with phosphate buffer saline (P1020, Solarbio, Beijing, China), AAV particles packed with scrambled control, and AAV particles packed with SRSF3 with a concentration of 4.0 × 1011 genome copy/mL (gc/mL) and then subjected to MCAO. Rats in the sham group were administrated the identical operative programs without the MCAO. After 24 h, rats were used for the assessment of neurological scores, and then rats were sacrificed by the inhalation with excess isoflurane.

Assessment of Neurological Scores

The neurological function was determined with the neurological scores based on the previous description [33]. In brief, 5-point standard scores were used; and a score of 0 indicated no neurological impairment, a score of 1 meant that the left front paw could not be fully extended, a score of 2 indicated left rotation in the front paw, a score of 3 indicated landing on the left front paw, and a score of 4 indicated that rats could not walk spontaneously and felt depressed.

Measurement of Brain Water Content

Rat brains were removed and weighted using an electronic scale (wet weight) and then dried at 105°C overnight. Subsequently, the weight of dried brains was quantified (dry weight). The brain water content was determined by ([wet weight − dry weight]/wet weight) × 100%.

2,3,5-Triphenyltetrazolium Chloride Staining

Rat brains were rapidly isolated and sectioned into six coronal sections at 2-mm intervals. The slices were hatched with 1% 2,3,5-triphenyltetrazolium chloride staining solution (G3005, Solarbio) for 20 min at 37°C and then pictured. The percentage of infarct volume was quantified with the following formula: the infarct volume/the total contralateral hemispheric volume × 100%, after the infarcted areas were determined using ImageJ software (National Institutes of Health, USA).

Terminal Deoxynucleotidyl Transferase Deoxyuridine Triphosphate Nick End Labeling Assay

The brain tissues were picked and immersed into 4% paraformaldehyde (P1110, Solarbio). Then, the tissues were treated with dehydration and embeddedness. Paraffin-embedded tissues were sectioned into 5-μm slices, and colored with a TUNEL Apoptosis Assay Kit (T2190, Solarbio) for the apoptosis analysis according to the operating manual. The apoptosis rate of ischemic areas was calculated by the selection of ten random nonoverlapping fields (×400). The pictures were imaged using a fluorescence microscopy (Olympus).

Cell Culture

BV2 cells were provided by Procell (CL-0493, Wuhan, China) and grown in DMEM (PM150210, Procell) supplied with 10% fetal bovine serum (FBS, SH30070.01HI, Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (P1400, Solarbio) at 37°C with 5% carbon dioxide (CO2).

Cell Model and Treatment

BV2 cells were cultured with glucose-free DMEM at 37 °C in a hypoxic incubator (including 1% O2, 5% CO2, and 95% N2) for 2 h to induce OGD [32]. The sequences of SRSF3 and PDCD4 were sub-cloned into pcDNA vector plasmids for the overexpression and then transfected into BV2 cells using Lipofectamine 3000 (L3000075, Invitrogen, Carlsbad, CA, USA) according to the previous description [36]. Cells were yielded after 48 h of transfection for the following assays.

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Experiment

According to the previous report [37], BV2 cells with an inoculation density of 5 × 104 cells per well were sowed into 96-well plates and grown at 37°C in 5% CO2. Then, cells were treated with 10 μL MTT solution (M1020, Solarbio) for 4 h; the supernatant was abandoned. Subsequently, 100 μL dimethylsulfoxide (DMSO, D8371, Solarbio) was added to dissolve the crystals. The absorbance at 570 nm was read using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Flow Cytometry

The apoptosis of BV2 cells was examined by the flow cytometry assay following the previous reports [38]. Cells were plated into 24-well plates with a density of 2.5 × 105 cells per well and grown with 5% CO2 at 37°C. Then, cells were yielded and rinsed with phosphate buffer saline. Next, cell apoptosis was determined by the Annexin V-FITC Apoptosis Detection Kit (CA1020, Solarbio) with CellQuest software (BD Biosciences, NJ, USA) on a FACScan flow cytometry.

Western Blot

The Western blot was carried out according to the previous descriptions [40]. In brief, brain tissues and BV2 cells were lysed with a Total Protein Extraction Kit (BC3711, Solarbio) to good harvest the total protein samples. The concentrations of protein were examined with the BCA Protein Assay Kit (PC0020, Solarbio). After isolating with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins samples were shifted onto polyvinylidene fluoride membranes (IPVH00010, EMD Millipore, Billerica, MA, USA). Subsequently, the membranes were sealed with Western blocking buffer (SW3010, Solarbio) and incubated with primary antibodies (anti-SRSF3 [1:1,000, ab198291, Abcam, Cambridge, UK], anti-Bax [1:5,000, ab32503, Abcam], anti-Bcl-2 [1:2,000, ab196495, Abcam], anti-cleaved-caspase 3 [1:1,000, 9661, Cell Signaling Technology, Inc., Danvers, MA, USA], anti-PDCD4 [1:1,000, 9535, Cell Signaling Technology], anti-phosphorylated phosphoinositide 3-kinase [p-PI3K] [1:500, ab182651, Abcam], anti-PI3K [1:1,000, ab191606, Abcam], anti-phosphorylated protein kinase B [p-AKT] [1:1,000, ab38449, Abcam], anti-AKT [1:500, ab8805, Abcam], and anti-β-actin [1:1,000, ab8227, Abcam]) at 4°C overnight. Bounds were treated with goat anti-rabbit IgG H&L (HRP) (ab6721, 1:10,000, Abcam) and then with the ECL Western blotting Detection Kit (Goat IgG) (SW2030, Solarbio). The QUANTITY ONE software (Bio‐Rad, Hercules, CA, USA) was used for the measurement of the band intensity.

Statistical Analysis

Results were present as mean ± standard deviation. Data analysis was performed by SPSS 20.0 software (IBM, Armonk, NY, USA) with one-way analysis of variance followed by the post hoc Bonferroni test. A significant difference was defined when p < 0.05.

SRSF3 Improved Brain Injury in MCAO-Treated Rats

After the establishment of MCAO model in rats, the relative protein level of SRSF3 was notably reduced in the brain tissues (Fig. 1a). To study the role of SRSF3 in ischemic cerebral infarction, gain-of-function assays were conducted in MCAO-treated rats by upregulating the level of SRSF3 (Fig. 1a). MCAO administration induced a prominent enhancement in the brain water content, neurological scores, and infarct volume in rats, which were all markedly decreased with the overexpression of SRSF3 (Fig. 1b–e). Thus, upregulation of SRSF3 alleviated brain damage in MCAO-treated rats.

Fig. 1.

Overexpression of SRSF3 relieved brain damage in MCAO-treated rats. a Relative protein expression of SRSF3 in the brain tissues was quantified by Western blot. Results were normalized with β-actin. b Brain water content was determined by ([wet weight − dry weight]/wet weight) × 100%. c Neurological function was evaluated with the neurological scores. d, e Infarct volume was assessed by TTC staining. **p < 0.01 versus sham; @p < 0.05 and @@p < 0.01 versus MCAO. TTC, 2,3,5-triphenyltetrazolium chloride.

Fig. 1.

Overexpression of SRSF3 relieved brain damage in MCAO-treated rats. a Relative protein expression of SRSF3 in the brain tissues was quantified by Western blot. Results were normalized with β-actin. b Brain water content was determined by ([wet weight − dry weight]/wet weight) × 100%. c Neurological function was evaluated with the neurological scores. d, e Infarct volume was assessed by TTC staining. **p < 0.01 versus sham; @p < 0.05 and @@p < 0.01 versus MCAO. TTC, 2,3,5-triphenyltetrazolium chloride.

Close modal

SRSF3 Inhibited Brain Neuron Apoptosis in Rats with MCAO

To determine the role of SRSF3 in apoptosis during ischemic cerebral infarction, the brain tissues were subjected to terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling staining. The percent of terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling-positive cells was significantly increased in MCAO-induced rats, accompanied with the enhanced expressions of Bax and cleaved-caspase 3 protein and diminished expression of Bcl-2 protein (Fig. 2a–f). However, upregulation of SRSF3 markedly modified these changes in MCAO-induced rats (Fig. 2a–f). Therefore, overexpression of SRSF3 suppressed brain neuron apoptosis in MCAO-treated rats.

Fig. 2.

Upregulation of SRSF3 repressed brain neuron apoptosis in MCAO-treated rats. a, b Percent of TUNEL-positive cells was quantified with TUNEL staining. Scale bar = 100 μm. c–f Relative protein expression of Bax, Bcl-2, and cleaved-caspase 3 in the brain tissues was examined by Western blot. Results were normalized with β-actin. **p < 0.01 versus sham; @@p < 0.01 versus MCAO. TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling.

Fig. 2.

Upregulation of SRSF3 repressed brain neuron apoptosis in MCAO-treated rats. a, b Percent of TUNEL-positive cells was quantified with TUNEL staining. Scale bar = 100 μm. c–f Relative protein expression of Bax, Bcl-2, and cleaved-caspase 3 in the brain tissues was examined by Western blot. Results were normalized with β-actin. **p < 0.01 versus sham; @@p < 0.01 versus MCAO. TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling.

Close modal

SRSF3 Mitigated OGD-Induced Damage in vitro

Moreover, the role of SRSF3 was also confirmed in an OGD-induced cell model. Results from Figure 3a show that OGD treatment notably declined the cell viability of BV2 cells, which was notably restored with the overexpression of SRSF3. A significant elevation in the apoptosis rate was found in OGD-induced cells, which was notably offset with the upregulation of SRSF3 (Fig. 3b, c). Hence, upregulation of SRSF3 reduced OGD-induced damage in BV2 cells.

Fig. 3.

Upregulation of SRSF3 improved OGD-induced damage in vitro. a Cell viability was measured by MTT assay. b, c Cell apoptosis rate was quantified by flow cytometry assays. *p < 0.05 and **p < 0.01 versus control; @p < 0.05 and @@p < 0.01 versus OGD. MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Fig. 3.

Upregulation of SRSF3 improved OGD-induced damage in vitro. a Cell viability was measured by MTT assay. b, c Cell apoptosis rate was quantified by flow cytometry assays. *p < 0.05 and **p < 0.01 versus control; @p < 0.05 and @@p < 0.01 versus OGD. MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Close modal

SRSF3 Downregulated the Level of PDCD4 and Activated the PI3K/AKT Pathway both in vivo and in vitro

To further resolve the role and mechanism of SRSF3 in ischemic cerebral infarction, the expression of PDCD4, a potential downstream target of SRSF3, was examined by Western blot. The relative protein expression of PDCD4 was notably increased in both MCAO-treated rats and OGD-induced BV2 cells, which was markedly decreased with the overexpression of SRSF3 (Fig. 4, 5). However, OGD treatment notably lowered the relative expression of p-PI3K/PI3K and p-AKT/AKT, which was observably recovered with the upregulation of SRSF3 both in MCAO-treated rats and OGD-induced BV2 cells (Fig. 4, 5). Altogether, SRSF3 reduced the expression of PDCD4 and activated the PI3K/AKT pathway in vivo and in vitro.

Fig. 4.

SRSF3 downregulated the expression of PDCD4 and upregulated the PI3K/AKT signaling pathway in MCAO-treated rats. The relative protein expressions of PDCD4, p-PI3K, PI3K, p-AKT, and AKT in brain tissues were detected by Western blot. Results were normalized with β-actin. **p < 0.01 versus sham; @@p < 0.01 versus MCAO.

Fig. 4.

SRSF3 downregulated the expression of PDCD4 and upregulated the PI3K/AKT signaling pathway in MCAO-treated rats. The relative protein expressions of PDCD4, p-PI3K, PI3K, p-AKT, and AKT in brain tissues were detected by Western blot. Results were normalized with β-actin. **p < 0.01 versus sham; @@p < 0.01 versus MCAO.

Close modal
Fig. 5.

SRSF3 decreased the expression of PDCD4 and activated the PI3K/AKT signaling pathway in OGD-induced BV2 cells. The relative protein expressions of PDCD4, p-PI3K, PI3K, p-AKT, and AKT in BV2 cells were determined by Western blot. Results were normalized with β-actin. **p < 0.01 versus control; @@p < 0.01 versus OGD.

Fig. 5.

SRSF3 decreased the expression of PDCD4 and activated the PI3K/AKT signaling pathway in OGD-induced BV2 cells. The relative protein expressions of PDCD4, p-PI3K, PI3K, p-AKT, and AKT in BV2 cells were determined by Western blot. Results were normalized with β-actin. **p < 0.01 versus control; @@p < 0.01 versus OGD.

Close modal

SRSF3 Relieved OGD-Induced Damage via PDCD4

To address the direct role of PDCD4 in OGD-induced damage, the level of PDCD4 was overexpressed in OGD-induced BV2 cells. The restored cell viability due to the upregulation of SRSF3 in OGD-treated BV2 cells was notably counteracted with the overexpression of PDCD4 (Fig. 6a), while inverse results were found in the cell apoptosis rate (Fig. 6b, c). Besides, upregulation of PDCD4 (Fig. 6d, e) markedly downregulated the SRSF3-induced relative expression of p-PI3K/PI3K and p-AKT/AKT in OGD-induced BV2 cells (Fig. 6d, f, g). Totally, overexpression of SRSF3 alleviated OGD-induced damage via the PI3K/AKT signaling pathway, which is mediated with PDCD4.

Fig. 6.

SRSF3 ameliorated OGD-induced damage via PDCD4. a Cell viability was quantified by MTT assay. b, c Cell apoptosis rate was measured by flow cytometry assays. d-g Relative protein expressions of PDCD4, p-PI3K, PI3K, p-AKT, and AKT in BV2 cells were examined by Western blot. Results were normalized with β-actin. **p < 0.01 versus control; #p < 0.05 and ##p < 0.01 versus OGD+pcDNA+vector; @p < 0.05 and @@p < 0.01 versus OGD+pcDNA-SRSF3+vector. MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Fig. 6.

SRSF3 ameliorated OGD-induced damage via PDCD4. a Cell viability was quantified by MTT assay. b, c Cell apoptosis rate was measured by flow cytometry assays. d-g Relative protein expressions of PDCD4, p-PI3K, PI3K, p-AKT, and AKT in BV2 cells were examined by Western blot. Results were normalized with β-actin. **p < 0.01 versus control; #p < 0.05 and ##p < 0.01 versus OGD+pcDNA+vector; @p < 0.05 and @@p < 0.01 versus OGD+pcDNA-SRSF3+vector. MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Close modal

In the present study, the in vivo and in vitro models of ischemic cerebral infarction were constructed in SD rats and BV2 cells with the treatment of MCAO and OGD, respectively. The expression of SRSF3 was downregulated in MCAO-treated rats. Overexpression of SRSF3 improved brain injury and brain neuron apoptosis in rats with MCAO, as well as alleviated OGD-induced damage in BV2 cells. Moreover, SRSF3 downregulated the level of PDCD4 and activated the PI3K/AKT pathway both in vivo and in vitro. Overexpression of PDCD4 abolished the role of SRSF3 in cell viability, apoptosis rate, and the level of the PI3K/AKT pathway in OGD-induced BV2 cells. Collectively, SRSF3 ameliorated ischemic cerebral infarction via PDCD4 in animal and cell models, which was tightly associated with the PI3K/AKT signaling pathway.

Cerebral infarction is a sequence of cascade damage responses that resulted from the injury of neurovascular elements upon ischemia and hypoxia [42]. In the present study, rats were treated with MCAO via the blockade of cervical blood flow to build an in vivo model of ischemic cerebral infarction, in line with plenty of studies [32]. MCAO administration induced a prominent enhancement in the neurological scores, brain water content, and infarct volume in rats, indicating a successful construction of ischemic cerebral infarction in rats. Increased brain water content indicated an occurrence of brain edema. Brain edema is currently classified into cytotoxic (cellular) edema and ionic and vasogenic (extracellular) edema [45]. No increase in BWC occurred in cytotoxic edema since water between the cellular and extracellular compartments has osmotically controlled redistribution. However, in ionic and vasogenic edema, namely, extracellular edema, water flows into the brain from the capillaries across the blood-brain barrier (BBB), which causes an increase in brain water content. Among them, the BBB in ionic edema is intact, while that in vasogenic edema is disrupted [46]. An increase in the brain water content in the present study indicated that brain edema was an extracellular in edema. It is demonstrated that cytotoxic edema occured several minutes after the brain insult, and ionic edema occurred immediately after the onset of cytotoxic edema, and vasogenic edema with protein influx due to BBB disruption manifests within hours after the initial brain insult. Thus, the brain edema due to the increased brain water content that occurred in the present study was vasogenic edema with a disrupted BBB. Also, a downregulated expression of SRSF3 was observed in MCAO-treated rats, along with the previous result [17]. Gain-of-function assays revealed that overexpression of SRSF3 declined these promotions in the neurological scores, brain water content, and infarct volume in MCAO-treated rats, which suggested that SRSF3 alleviated brain damage in MCAO-treated rats. Moreover, upregulation of SRSF3 also reduced the cell viability of BV2 cells treated with OGD, which indicated that SRSF3 decreased OGD-induced damage in BV2 cells. Taken together, SRSF3 improved ischemic injury both in vivo and in vitro.

Apoptosis is a key pathogenesis of ischemic brain damage [30]. An increase in the numbers of apoptotic neurons accompanied with the enhanced expressions of Bax and cleaved-caspase 3 protein and diminished level of Bcl-2 protein was discovered in MCAO-treated rats, which were reversed with the upregulation of SRSF3, indicating that SRSF3 inhibited apoptosis in vivo. Besides, overexpression of SRSF3 reduced an OGD-evoked apoptosis rate in BV2 cells. The inhibitory function of SRSF3 in apoptosis has been revealed in various cancer cells [14]. Thus, SRSF3 suppressed apoptosis in both animal and cell models of ischemic injury. PDCD4, a potential downstream target of SRSF3, participates in the myocardial infarction. For instance, Xu et al. [49] report that miR-145 targeting PDCD4 prevents myocardial infarction in rats. The miR-21/PDCD4 axis is involved in the protective role of serum-derived extracellular vesicles in acute myocardial infarction [50]. Moreover, PDCD4 is strongly involved in the occurrence of apoptosis [24]. LncRNA-GAS5/miR-21/PDCD4 mediates cardiomyocytes apoptosis in myocardial infarction [51]. MiR-532-5p targeting PDCD4 relieves apoptosis in hypoxia-induced cardiomyocyte [52], and miR-208 targeting PDCD4 suppresses apoptosis in mice with acute myocardial infarction [53]. More importantly, PDCD4 is reported to function on cerebral I/R injury both in vivo [22] and in vitro [23]. Here, the level of PDCD4 was increased in MCAO-treated rats and OGD-induced BV2 cells, which was neutralized with the upregulation of SRSF3. Overexpression of PDCD4 abolished the role of SRSF3 in cell viability and the apoptosis rate in OGD-induced BV2 cells in the present study. Totally, these results indicated that SRSF3 relieved OGD-induced damage via PDCD4.

The PI3K/AKT signaling pathway serves a crucial role in promoting the repair and survival of cerebral ischemic neurons [54]. PI3K participates in the differentiation and survival of neurons and glial cells, and the PI3K activation can modulate the level of downstream genes via the activation of AKT in the nervous system [55]. Here, a reduction in the relative expression of p-PI3K/PI3K and p-AKT/AKT was observed in MCAO-treated rats and OGD-induced BV2 cells, which manifested that the PI3K/AKT pathway is downregulated in ischemic injury, in accordance with the previous studies [56‒58]. However, these decreases were inverted with the upregulation of SRSF3 both in vivo and in vitro. SRSF3 has been revealed to be involved in the cell viability and metastasis by the PI3K/AKT/mTOR pathway in cervical cancer [47] and gastric cancer [59]. Thus, the ameliorative role of SRSF3 in ischemic damage might be related to the activation of the PI3K/AKT pathway. Furthermore, overexpression of PDCD4 declined the SRSF3-induced level of the PI3K/AKT pathway in OGD-induced BV2 cells. Taken together, the SRSF3/PDCD4 axis improved ischemic injury, which was tightly associated with the PI3K/AKT pathway.

In conclusion, the level of SRSF3 was downregulated in cerebral ischemic injury. Overexpression of SRSF3 improved ischemic damage in both animal and cell models via the inhibition of apoptosis of neurons. Moreover, the alleviative role of SRSF3 in OGD-induced damage is mediated by PDCD4. Mechanically, SRSF3 activated the PI3K/AKT pathway. Collectively, overexpression of SRSF3 alleviated OGD-induced damage via the PI3K/AKT pathway, which is mediated with PDCD4. However, although the upregulation of SRSF3 is helpful to study the role of SRSF3, downregulation or inhibition or even elimination of SRSF3 may be more convincing. Thus, the loss-of-function assays will be conducted in the future to study the role of the SRSF3 downregulation in MCAO-induced rats. Besides, more preclinical and clinical studies should be conducted in the future. Briefly, the results reveal that SRSF3 can work as a potential target for the treatment of cerebral ischemic injury and even other related cerebrovascular diseases.

This study protocol was reviewed and approved by Heping Hospital Affiliated to Changzhi Medical College, approval number DW2022075.

The authors state that there are no conflicts of interest to disclose.

This study did not receive any financial support.

All the authors contributed to the study conception and design. Material preparation and the experiments were performed by Liangliang Cui. Data collection and analysis were performed by Shuying Zhao. The first draft of the manuscript was written by Hong Liu, and all the authors commented on previous versions of the manuscript. All the authors read and approved the final manuscript.

All data generated or analyzed during this study are included in this published article. The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

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