Human neuroblastoma cell lines (SH-SY5Y, AW-CCH335; and SK-N-SH, AW-CCH037; Abiowell, Changsha, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (iCell-01000, Icell, Shanghai, China) medium containing 10% fetal bovine serum (AWC0219a, Abiowell) and 1% antibiotics (AWH0529a, Abiowell) in a 5% CO₂ cell incubator at 37°C. In CIRI studies, SH-SY5Y [24–27] and SK-N-SH cells [28–30] have been widely used as in vitro models. SH-SY5Y and SK-N-SH cells were induced by oxygen-glucose deprivation/reperfusion (OGD/R) to construct a CIRI model in vitro. Briefly, to initiate oxygen-glucose deprivation (OGD), SH-SY5Y and SK-N-SH cells were cultured for 4 h [31] or 6 h [29] in a glucose-free DMEM in a three-gas incubator at 37°C, 0.5% O₂, 94.5% N₂, and 5% CO_2, respectively. To simulate reperfusion, the glucose-free DMEM was removed, and the cells were grown in complete DMEM at 37°C for 24 h in 95% air and 5% CO₂. The cells in the control group were cultured under normal conditions and treated in the same way, except that they were not exposed to OGD. After OGD/R, cells were transfected with si-SRSF1 and treated with 100 μM of the NLRP1 activator muramyl dipeptide (MDP) for 24 h [32].

Plasmids overexpression (oe)-C-Myc (HG-HO354870), oe-SRSF1 (HG-HO078166), small interfering (si)-SRSF1 (HG-Si078166), and the negative control (oe-NC and si-NC) were purchased from Changsha Abiowell Biotechnology Co., Ltd. The sequence of siRNA is as follows. si-SRSF1: 5^′-ACTGCCTACATCCGGGTTAAA-3^′. si-NC: 5^′-UUCUCCGAACGUGUCACGUTT-3^′. After the preparation of the plasmid, the Lipofectamine 2000 transfection reagent (11668500, Thermo Fisher, Waltham, MA, USA) (5 μL) was used to transfect the overexpressed/knockdown plasmid (3 μg) into the corresponding group. The transfection efficiency was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB) 48 h after transfection.

The cells were seeded in 24-well plates (3 × 10⁴ per well). 300 μL of CCK8 (C0037, Beyotime, Changsha, China) medium mix was added to the cells. After 4 h of incubation, the optical density values of cells at 450 nm were measured using a microplate reader (MB-530, HEALES, Shenzhen, China).

The cells treated as mentioned above were digested with trypsin without EDTA, followed by centrifugation at 2000 rpm (H1650R, Cence, Changsha, China) for 5 min. The cell pellet was resuspended in 500 μL of binding buffer and then mixed with FAM-YVAD-FMK FITC and propidium iodide (PI) from the FAM-YVAD-FMK FITC cell apoptosis detection kit (ab219935, Abcam, Cambridge, UK). After incubation for 30 min at 37°C, without light, cell apoptosis was detected by flow cytometry (A00-1-1102, Beckman, Brea, CA, USA).

According to the instruction manuals of the lactate dehydrogenase (LDH) assay kit (A020-2-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the LDH level was determined. The optical density at 440 nm was measured using a microplate reader (MB-530, Huisong, Shenzhen, China).

The cells were added to Trizol reagent (15596026, Thermo fisher, MA, USA) for mRNA extraction. cDNA was synthesized with the use of an mRNA reverse transcription kit (CW2569, CWBIO, Beijing, China). The cDNA template was amplified by qRT-PCR using the UltraSYBR Mixture (CW2601, CWBIO). The mRNA expression was assessed by the 2^−ΔΔCt method. β-actin was utilized as an internal control. The primers are listed in Table 1.

After extracting proteins with RIPA (P0013B, Beyotime), the total protein concentration in cells was determined by the BCA method. The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (FFN08, Beyotime). Membranes were blocked with 5% (w/v) skim milk powder for 90 min and incubated with primary antibodies overnight at 4°C. Subsequently, the protein bands were incubated with secondary antibody HRP goat anti-mouse IgG (SA00001-1, 1:5000, Proteintech, Rosemont, IL, USA) or HRP goat anti-rabbit IgG (SA00001-1, 1:5000, Proteintech) at room temperature for 90 min. β-actin was used as an internal control. Information on the antibodies used is listed in Table 2. ECL chemiluminescence solution (32209, Thermo Fisher) was used to incubate the membrane for 1 min, and the gel imaging system (ChemiScope6100, CLINX, Shanghai, China.) was used for imaging. The exposed image strips were analyzed with Quantity One professional grayscale analysis software (v4.6.8, BIO-RAD).

The cells were collected 48 h after transfection and lysed in RIPA Lysis Buffer supplemented with protease/phosphatase inhibitors (P1050, Beyotime) to prepare cell lysates. The extracted cell proteins were randomly divided into Input, IgG, and IP groups. In the IgG and IP group centrifuge tube, cell proteins were respectively incubated overnight at 4°C with normal rabbit IgG antibody (B900610, Proteintech) or SRSF1 antibody (12929-2-AP, Rabbit, 1:8000, Proteintech). 20 μL of protein A/G magnetic beads (PR40025, Proteintech) were added to each tube to capture the antibody-protein complexes overnight at 4°C. SRSF1 (12929-2-AP, Rabbit, 1:8000, Proteintech) and NLRP1 (A16212, Rabbit, 1:1000, Proteintech) were analyzed by WB.

After the cells were fixed in 4% paraformaldehyde for 30 min, 0.3% Triton X-100 was used to permeable for 30 min. 5% BSA (SW3015, Solarbio, Beijing, China) was sealed at 37°C for 90 min. Sections were incubated overnight with primary antibodies Caspase1 (AWA44822, 1:100, Abiowell) at 4°C. Sections were incubated with secondary antibody, including CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (1:200, SA00013-2, Proteintech, USA) at 37°C for 60 min. The sections were preserved in buffered glycerin and observed under a fluorescence microscope (BA210T, Motic, Singapore).

All data were expressed as the mean ± standard deviation. Statistical comparisons of the results were conducted using GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA). An unpaired two-tailed Student’s t-test was employed to analyze the data between the two groups. One-way analysis of variance with Tukey’s multiple comparisons test was used for multi-group comparisons. When p < 0.05, it was considered to be statistically significant.

Firstly, pyroptosis in neuroblastoma cells induced by OGD/R was investigated. OGD/R-induced neuroblastoma cell viability decreased (Fig. 1A). The OGD/R model promoted neuroblastoma pyroptosis (Fig. 1B). NLRP1, Caspase1, ASC, interleukin-1beta (IL-1β), and IL-18 expression were elevated in neuroblastoma cells subjected to OGD/R (Fig. 1C). LDH level was elevated in neuroblastoma cells subjected to OGD/R (Fig. 1D). C-Myc and SRSF1 expression were decreased in neuroblastoma cells subjected to OGD/R (Fig. 1E). Our findings suggested that neuroblastoma cells induced by OGD/R exhibited pyroptosis.

Previous studies have shown that c-Myc is involved in pyroptosis [33]. Here, c-Myc effects on neuroblastoma pyroptosis induced by OGD/R were further explored. Oe-c-Myc increased c-Myc expression in neuroblastoma cells subjected to OGD/R (Fig. 2A). Oe-c-Myc increased cell viability in neuroblastoma subjected to OGD/R (Fig. 2B). Oe-c-Myc inhibited neuroblastoma pyroptosis subjected to OGD/R (Fig. 2C). Oe-c-Myc inhibited NLRP1, Caspase1, ASC, IL-1β, and IL-18 expression in neuroblastoma cells subjected to OGD/R (Fig. 2D). Oe-c-Myc reduced LDH levels in neuroblastoma cells subjected to OGD/R (Fig. 2E). In addition, immunofluorescence results show that oe-c-Myc inhibited Caspase1 expression in neuroblastoma cells subjected to OGD/R (Fig. 2F). Our findings suggested that c-Myc inhibited neuroblastoma pyroptosis subjected to OGD/R.

Co-IP experiment results showed that there is an interaction between the c-Myc protein and the SRSF1 protein [19]. Here, c-Myc was positively correlated with SRSF1 (Fig. 3A). Oe-c-Myc promoted c-Myc and SRSF1 levels in neuroblastoma cells subjected to OGD/R, which were reversed by si-SRSF1 (Fig. 3B,C). Oe-c-Myc increased cell viability in neuroblastoma subjected to OGD/R, which was reversed by si-SRSF1 (Fig. 3D). Oe-c-Myc inhibited pyroptosis in neuroblastoma cells subjected to OGD/R. Si-SRSF1 reversed the inhibitory effect of oe-c-Myc on the pyroptosis of neuroblastoma cells subjected to OGD/R. Si-SRSF1 promoted pyroptosis in neuroblastoma cells subjected to OGD/R (Fig. 3E). Oe-c-Myc inhibited NLRP1, Caspase1, ASC, IL-1β, and IL-18 expression in neuroblastoma cells subjected to OGD/R. Si-SRSF1 reversed this effect and promoted NLRP1, Caspase1, ASC, IL-1β, and IL-18 levels in neuroblastoma cells subjected to OGD/R (Fig. 3F). Oe-c-Myc reduced LDH levels in neuroblastoma cells subjected to OGD/R. Si-SRSF1 reversed this effect and increased LDH levels in neuroblastoma cells subjected to OGD/R (Fig. 3G). Moreover, immunofluorescence results showed that oe-c-Myc reduced Caspase1 expression in neuroblastoma cells subjected to OGD/R. Si-SRSF1 prevented this phenomenon (Fig. 3H). Our findings suggested that c-Myc inhibited neuroblastoma pyroptosis subjected to OGD/R via the SRSF1/NLRP1 axis.

SRSF1 effects on OGD/R-induced neuroblastoma pyroptosis were further investigated. Oe-SRSF1 increased SRSF1 expression in neuroblastoma cells subjected to OGD/R (Fig. 4A). Oe-SRSF1 increases neuroblastoma cell viability subjected to OGD/R (Fig. 4B). Oe-SRSF1 inhibited neuroblastoma pyroptosis subjected to OGD/R (Fig. 4C). Oe-SRSF1 inhibited NLRP1, Caspase1, ASC, IL-1β, and IL-18 expression in neuroblastoma cells subjected to OGD/R (Fig. 4D). Oe-SRSF1 reduced LDH levels in neuroblastoma cells subjected to OGD/R (Fig. 4E). Immunofluorescence results showed that oe-SRSF1 reduced Caspase1 expression in neuroblastoma cells subjected to OGD/R (Fig. 4F). Co-IP results showed that SRSF1 interacted with NLRP1 (Fig. 4G). SRSF1 was negatively correlated with NLRP1 (Fig. 4H). Our findings suggested that SRSF1 reduced neuroblastoma pyroptosis subjected to OGD/R.

The mechanism of SRSF1 on OGD/R-induced neuroblastoma pyroptosis was further explored. Oe-SRSF1 promoted SRSF1 expression in OGD/R-induced neuroblastoma cells and inhibited NLRP1 expression. Compared with the oe-SRSF1 group, there was no significant difference in SRSF1 expression in the oe-SRSF1+MDP group, while promoting NLRP1 expression (Fig. 5A). Oe-SRSF1 increased OGD/R-induced neuroblastoma cell viability. These effects were interrupted by MDP (Fig. 5B). Oe-SRSF1 inhibited OGD/R-induced neuroblastoma pyroptosis. MDP reversed this effect (Fig. 5C). Oe-SRSF1 inhibited Caspase1, ASC, IL-1β, and IL-18 expression in neuroblastoma cells subjected to OGD/R, and this effect was reversed by MDP (Fig. 5D). Oe-SRSF1 reduced LDH levels in neuroblastoma cells subjected to OGD/R. MDP reversed the inhibition of oe-SRSF1 on LDH level in OGD/R-induced neuroblastoma cells (Fig. 5E). Immunofluorescence results showed that oe-SRSF1 decreased Caspase1 expression in neuroblastoma cells subjected to OGD/R, which was interrupted by MDP (Fig. 5F). Our findings suggested that SRSF1 reduced OGD/R-induced neuroblastoma pyroptosis by inhibiting NLRP1 activation.