Study on the correlation between Interleukin 4 and febrile seizures

Animal model

A total of 24 specific pathogen-free-grade Sprague-Dawley male rats (7 weeks old and 0.18–0.22 kg; Chongqing Enswell Biotechnology Co., Ltd.) were used in this study. All rats were kept under similar conditions: temperature of 23℃ ± 2℃, humidity of 35–60%, 12 h light/dark cycle, and free access to drinking water and food. After 1 week of acclimatization, the animals were subjected to the experiment. All experimental procedures strictly adhered to the “Regulations on the Management of Experimental Animals” and international ethical guidelines for animal experiments. This study was approved by the Institutional Ethics Committees of the Third Affiliated Hospital of Zunyi Medical University (the First People’s Hospital of Zunyi) (Ethics Review No. [2022]-2-33).

A total of 24 rats were randomly divided into three groups: The normal control (NC) group, the FS model group, and the IL-4 receptor antagonist group (dupilumab). The IL-4 receptor antagonist group was provided with 10 mg/kg dupilumab (SAR-231893, MCE, Shanghai, China) through subcutaneous injection every 3 days, with a total of two doses. The dose of 10 mg/kg was selected based on preliminary dose-response studies [Figure S1a and b], which indicated that this concentration effectively modulated IL-4 receptor activity without causing significant side effects in the rats. The NC group and the FS group were injected with an equal volume of physiological saline. After the subcutaneous injection of dupilumab, the FS model group and the IL-4 receptor antagonist group rats underwent the hot water bath method to establish the FS model.^[13] Specifically, the rats in the FS group and the IL-4 receptor antagonist group were placed in a transparent glass tube (diameter 10 cm and height 50 cm) with small everted holes at the bottom. The tube was vertically placed in a constant-temperature water bath (45℃ ± 0.25℃. Adjusting the depth of the water in the tube with rubber pads at the bottom ensured that the rats stood along the wall of the tube with only their heads exposed. Seizures were induced once in the morning and afternoon for each rat, for a total of 10 inductions over 5 consecutive days. The occurrence of seizures was observed, and the latency, severity, and duration of the seizures were recorded. After the model was established, rats from each group were anesthetized with 1% sodium pentobarbital (0.5 mL/100 g; P3761, Sigma– Aldrich, St. Louis, Missouri, USA) and euthanized by cervical dislocation. Subsequently, peripheral blood, serum, and hippocampal tissue samples were collected for testing.

Racine scoring for FS rats

The severity of seizures in rats was assessed using the Racine scoring method: ^[14] Level 0: No adverse reactions; Level I: Facial clonus and chewing movements; Level II: Muscle spasms primarily consisting of nodding or tail shaking; Level III: Unilateral clonus and spasms; Level IV: Bilateral clonus of forelimbs, accompanied with a standing posture; and Level V: Tonic-clonic seizures, sustained standing, strong body arching, and falling.

Differentially expressed gene (DEG) screening

The dataset Gene Expression Omnibus (GEO) Series accession number 28674 (GSE28674) was obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo/), which contained seven FS samples and 11 normal samples. EdgeR package (Bioconductor, Melbourne, Australia) was used to screen DEGs, with P < 0.05 and |log₂FC| ≥ 1 as the screening conditions. The DAVID database (https://davidbioinformatics.nih.gov/) was used to conduct the gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of upregulated and downregulated DEGs. The K-means clustering algorithm was used to cluster DEGs, and the elbow rule was used to determine the optimal K value. The online database STRING (https://string-db.org/) generated a diagram of protein-protein interaction (PPI) network. The maximal clique centrality (MCC) algorithm was used in the cytoHubba plug-in of Cytoscape 3.8.0 (University of California, California, USA) to screen the top ten genes in the PPI network as key genes.

Enzyme-linked immunosorbent assay (ELISA) detection

The expression levels of IL-1β, tumor necrosis factor α (TNF-α), and IL-6 in serum were detected using ELISA kits. Rat IL-1β kit (RX302869R, Ruixin Biotech, Quanzhou, China), rat TNF-α ELISA kit (RX302058R, Ruixin Biotech, Quanzhou, China), and rat IL-6 ELISA kit (RX302856R, Ruixin Biotech, Quanzhou, China) were used.

Flow cytometry

Th1/Th2 cell ratio: Heparin anticoagulated blood (1.5 mL) was collected from rats treated in groups, and 3 mL of lymphocyte separation solution (P8630, Solarbio, Beijing, China) was added to a test tube. After diluting the blood with an equal volume of phosphate-buffered saline (PBS) solution, the upper layer of the lymphocyte separation medium was carefully added and centrifuged. After the removal of the test tube, mononuclear cells were carefully absorbed at the liquid-phase junction. Subsequently, 3 mL of PBS solution (PB180327, Pricella, Wuhan, China) was added, mixed well, and centrifuged at ×1500 g for 5 min. The supernatant was then discarded. Each group of cells was resuspended in PBS for washing, transferred to a 1.5 mL Eppendorf (EP) tube, and centrifuged at 1000 rpm for 5 min. The supernatant was then discarded. The cell pellet was carefully resuspended in 100 μL PBS, 5 μL anti-rat CD4 fluorescein isothiocyanate (FITC) (AR00401-50, MULTI SCIENCES, Hangzhou, China) was added, the cells were mixed and incubated at room temperature in the dark for 20 min, and a blank control was set at the same time. After incubation, 1 mL of PBS was added, mixed evenly, and centrifuged. The supernatant was discarded, and this procedure was repeated. After lightly blowing off the centrifuged cells, 100 μL of PBS was added carefully to resuspend the cell pellet. About 5 μL of phycoerythrin (PE) Rat Anti-Mouse Interferon-gamma (554412, BD Biosciences, New Jersey, USA) was added, and the cells were mixed and incubated at room temperature in the dark for 20 min in blank control. After incubation, the suspension was supplemented with 1 mL of PBS, and the previously described procedures were repeated. Subsequently, a fixative solution was added, incubated in the dark, and centrifuged at 500 g for 5 min. A membrane-breaking agent was added and incubated in the dark for 15 min. Followed by incubation, 1 mL of PBS washing solution was supplied. The mixture was centrifuged at ×500 g for 5 min, and the supernatant was discarded. Fluorescently labeled allophycocyanin (APC) Rat Anti-Mouse IL-4 antibody (562045, BD Biosciences, New Jersey, USA) was used, mixed, and cultured, added with 1 mL of washing solution, and centrifuged at ×500 g for 5 min. After we discarded the supernatant, we added 500 mL of PBS or 500 mL of 1% PFA for fixation and analysis on a flow cytometer (CytoFLEX, Beckman, Brea, California, USA).

Cell apoptosis was detected using the Annexin V FITC/propidium iodide (PI) Apoptosis Kit (40302ES50, Yeasen Biotechnology, Shanghai, China). The cell cycle was detected using the Flow Cytometry Cell Cycle Kit (AC12L543, LIFE-iLAB BIO, Shanghai, China). Cell proliferation was detected using the BeyoClick^™ EdU-647 Cell Proliferation Detection Kit (C0081S, Beyotime, China). Rat cells were tested quarterly for mycoplasma contamination using PCR-based methods, and the results were negative.

Quantitative polymerase chain reaction (qPCR) detection

Extracted total RNA was reversely transcribed according to the Goldenstar^™ RT6 complementary deoxyribonucleic acid (cDNA) Synthesis Kit Ver.2 (TSK302M, Qingke, Beijing, China), and fluorescence quantitative analysis was carried out following the guidelines of 2 × T5 Fast qPCR Mix kit (SYBR Green I, TSE002, Qingke, Beijing, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The polymerase chain reaction (PCR) primer sequences are shown in Table 1. Relative expression levels were calculated using the 2^−△△Ct method, which represents the fold difference in target gene expression in the experimental group compared with the control group.

Western blot detection

Total proteins were extracted and separated by electrophoresis and then transferred to a polyvinylidene fluoride membrane (10600023, Amersham, Hessen, Germany). This membrane was sealed with 5% skimmed milk and rinsed 3 times with tris-buffered saline with tween 20 (TBST). After dilution at 1:1000, the primary antibody was added and incubated overnight at 4℃, followed by TBST washing again; the secondary antibody was adjusted to a certain concentration with a seal solution (1:2000) and then incubated at room temperature for 1 h. After three washes with TBST, each time for 10 min, mixed the enhanced chemiluminescence exposure solution (34580, Thermo, Massachusetts, USA) according to the ratio of solution A: B at 1:1, covered the whole film evenly, reacted for 1 min, and placed in the exposure instrument (U.S., Bio-Rad, Universal Hood II) for detection. The primary antibodies were as follows: Jun proto-oncogene (Jun) (A11378, ABclonal, Wuhan, China), Fos proto-oncogene (Fos) (A0236, ABclonal, Wuhan, China), p-Jun proto-oncogene (p-Jun) (AP0048, ABclonal, Wuhan, China), p-Fos proto-oncogene (p-Fos) (AP0038, ABclonal, Wuhan, China), early growth response-1 (Egr1) (A7266, ABclonal, Wuhan, China), IL-4 (A4988, ABclonal, Wuhan, China), Heat shock protein 70 (HSP70) (A23457, ABclonal, Wuhan, China), glial fibrillary acidic protein (GFAP) (A19058, ABclonal, Wuhan, China), cysteine protease-3 (caspase-3) (A11319, ABclonal, Wuhan, China), Bcl-2-associated X protein (Bax) (A19684, ABclonal, Wuhan, China), B-cell lymphoma 2 (Bcl-2) (A19693, ABclonal, Wuhan, China), and GAPDH (A19056, ABclonal, Wuhan, China). The secondary antibody was horseradish peroxidase (HRP)-conjugated Goat anti-rabbit immunoglobulin G (IgG) (H + L) (AS014, ABclonal, Wuhan, China). One-way analysis of variance (ANOVA) was used for group comparisons, and data analysis and graphical representation were facilitated through GraphPad Prism 8.0 software.

Immunofluorescence detection

After dewaxing and rehydration of hippocampal tissue paraffin sections, immunofluorescence staining was performed. Antigen retrieval was carried out for 30 min, and 0.5% Triton X-100 (ST795, Beyotime, Shanghai, China) was applied to the tissue sections for 60 min at room temperature. Subsequently, these samples were treated using goat serum, added with primary antibody (diluted 1:200), and incubated at 4℃ overnight. Furthermore, a secondary antibody (diluted 1:300) was supplied for incubation for 1.5 h. Thereafter, 4’,6-diamidino-2-phenylindole (DAPI) staining (C1005, Beyotime, Shanghai, China) was conducted in the dark for 5 min to stain the nuclei, followed by washing off excess DAPI with PBS. Eventually, samples were sealed by adding an anti-fluorescence quenching reagent. Imaging of the sections was performed using the Mshot inverted microscope (MF53, Guangzhou Ming-Mei Technology Co., Ltd., China). The primary antibodies were as follows: IL-4 (bs-0581R, Bioss antibodies, Beijing, China), Egr1 (bs-1076R, Bioss antibodies, Beijing, China), Jun (bsm-42050M, Bioss antibodies, Beijing, China), p-Jun (bs-3210R, Bioss antibodies, Beijing, China), Fos (bs-10172R, Bioss antibodies, Beijing, China), and p-Fos (bs-3153R, Bioss antibodies, Beijing, China). The secondary antibodies were Cy3 Goat Anti-Mouse IgG (H + L) (AS008, ABclonal, Wuhan, China) and Cy3 Goat Anti-Rabbit IgG (H + L) (AS007, ABclonal, Wuhan, China).

Dual-luciferase reporter gene assay

The 293T cells (LH-H080, Laibaiha (Shanghai) Biotechnology Co., Ltd., Shanghai, China) were subjected to short tandem repeat (STR) identification to exclude exogenic cell contamination. The STR identification report can be found in the supplementary material. In this study, 293T cells showed negative results on testing quarterly for mycoplasma contamination through PCR-based methods. After reviving and passaging 293T cells, vector construction was performed. Through the predictions from analysis websites, a 2,000 bp fragment of the IL-4 promoter region was synthesized and cloned into the dual-luciferase reporter plasmid pGL4.11-basic, referred to as pGL4.11- wild-type (WT). In addition, mutation sites within the 2,000 bp sequence of the IL-4 promoter were introduced and cloned into the same reporter vector, resulting in the construct promega luciferase (pGL) 4.11-mut. For co-transfection of plasmids, 50 μL of culture medium devoid of antibiotics and serum was added, followed by 1 μg of plasmid DNA (mixed in appropriate groups), and lightly mixed using a pipette. Then, 1.6 μL of nanofusion transfection reagent was added, mixed lightly, and left at room temperature for 5–20 min before adding to the cells in a 12-well plate.

• Verification of Jun binding sites with IL-4, consisting of five groups:

  ① plasmid cloning DNA (pcDNA) 3.1-NC + pGL4.11-NC + Promega Renilla Luciferase-Thymidine Kinase (PRL-TK);

  ② pcDNA3.1-NC + pGL4.11-WT + PRL-TK;

  ③ pcDNA3.1-JUN + pGL4.11-NC + PRL-TK;

  ④ pcDNA3.1-JUN + pGL4.11-WT + PRL-TK;

  ⑤ pcDNA3.1-JUN + pGL4.11-mut + PRL-TK;

• Verification of Fos binding sites with IL-4, consisting of five groups:

  ① pcDNA3.1-NC + pGL4.11-NC + PRL-TK;

  ② pcDNA3.1-NC + pGL4.11-WT + PRL-TK;

  ③ pcDNA3.1-FOS + pGL4.11-NC + PRL-TK;

  ④ pcDNA3.1-FOS + pGL4.11-WT + PRL-TK;

  ⑤ pcDNA3.1-FOS + pGL4.11-mut + PRL-TK;

• Verification of Egr1 binding sites with IL-4, consisting of five groups:

  ① pcDNA3.1-NC + pGL4.11-NC + PRL-TK;

  ② pcDNA3.1-NC + pGL4.11-WT + PRL-TK;

  ③ pcDNA3.1-Egr1 + pGL4.11-NC + PRL-TK;

  ④ pcDNA3.1-Egr1 + pGL4.11-WT + PRL-TK;

  ⑤ pcDNA3.1-Egr1 + pGL4.11-mut + PRL-TK.

Statistical analysis

Group comparisons were executed through one-way ANOVA with the Tukey post hoc test method, and data analysis and graphical representation were facilitated through GraphPad Prism 8.0 software (GraphPad Software, LLC, California, USA). For comparisons between the two groups, the independent samples t-test was used to assess the significance of differences between the means. The measurement data were expressed as mean ± standard deviation. Statistical significance was set at P < 0.05.

Analysis of DEGs

A total of 50 significantly differentially expressed messenger RNAs (mRNAs) were screened from the GSE28674 dataset [Figure 1a], of which 44 were upregulated and six were downregulated [Figure 1b]. These DEGs were mainly enriched in biological processes, such as response to reactive oxygen species, response to mechanical stimuli, and cellular response to chemical stress in GO [Figure 1c]. KEGG enrichment analysis showed that these DEGs were mainly concentrated in signaling pathways such as the mitogen-activated protein kinase (MAPK) signaling pathway, advanced glycation end-products (AGE)-receptor for AGE (RAGE) signaling pathway, TNF signaling pathway, and relaxin signaling pathway [Figure 1d].

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Figure 1:

Differentially expressed mRNAs between NC group and FS group. (a) Heatmap: The expression levels of differentially expressed genes in each sample [fragments per kilobase per million (FPKM) values] were log-transformed with base 2, and Euclidean distances were calculated. Hierarchical clustering was then performed to obtain the overall clustering results for the samples. In the figure, rows represent genes, and columns represent samples. The differences in gene expression across samples are reflected by changes in the heatmap colors. (b) Volcano map: The X-axis represents the log-transformed fold change of differentially expressed genes in the comparison group. The Y-axis represents the negative log-transformed P-value of the statistical significance of the expression change. Each point represents a specific gene, with red indicating significantly upregulated genes, blue indicating significantly downregulated genes, and gray indicating genes with no significant expression difference. (c) GO enrichment: The X-axis represents GO terms under the BP category, and the Y-axis represents the number of candidate target genes annotated to that term (including its sub-terms). (d) KEGG enrichment: The X-axis represents the number of target genes corresponding to each KEGG pathway, and the Y-axis represents the KEGG pathway names. The color indicates the significance of pathway enrichment; redder colors indicate higher significance. P-value represents statistical significance. mRNA: Messenger RNA, NC: Normal control, FS: Febrile seizures, GO: Gene ontology, BP: Biological process, KEGG: Kyoto encyclopedia of genes and genomes.

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Upstream regulatory genes of IL-4 are Jun, Fos, and Egr1

The K-means clustering algorithm was used for cluster analysis of DEGs, and the elbow rule determined the optimal K value to be 4 [Figure 2a]. DEGs were divided into four clusters, and Jun, Fos, and Egr1 were distributed in the same cluster [Figure 2b]. Among the TOP10 core genes of PPI are Jun, Fos, Egr1, Cyr61, Dusp1, Atf3, Nr4a1, Ctgf, Serpine1, and Btg2 [Figure 2c and d]. Among the TOP10 core genes of PPI, the top three Jun, Fos, and Egr1 were all known upstream transcription factors of IL-4.^[15,16] The analysis of DEGs showed that they were all significantly upregulated in the FS group, suggesting their role as molecular mechanisms associated with increased IL-4 levels.

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Figure 2:

Screening of upstream regulatory genes of IL-4 in the GEO database. (a) Elbow method to determine the optimal K value. (b) Cluster plot; PPI network diagram to screen key genes: (c) Network diagram of all genes. (d) Top 10 gene network diagram, the color ranges from red to yellow, indicating the level of importance, with red representing high importance. IL: Interleukin, PPI: Protein–protein interaction.

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Jun, Fos, and Egr1 can activate the IL-4 promoter

The dual-luciferase reporter gene assay [Figure 3a-c] suggested that the transcription factors Jun, Fos, and Egr1 may activate the IL-4 promoter. This conclusion was based on the observation that their overexpression increased firefly luciferase activity linked to the IL-4 promoter. The activation of the IL-4 promoter by Jun, Fos, and Egr1 was likely achieved through binding to specific DNA sequences within the promoter. This was inferred from the differences in luciferase activity driven by WT and mutant promoters, where the activity of the mutant promoter did not show significant changes, suggesting that the activation by these transcription factors depends on specific DNA binding sites. Furthermore, the experiments confirmed that the overexpression of Jun, Fos, and Egr1 did not cause non-specific alterations in luciferase activity. This was demonstrated by comparing a promoter with a non-specific sequence (NC) with the WT promoter, showing that these transcription factors specifically activated the IL-4 promoter without affecting the non-specific sequence.

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Figure 3:

Regulatory effects of Jun, Fos, and Egr1 on IL-4. (a) Verification of Jun binding sites with IL-4 (n = 3). Compared with pcDNA3.1-NC + pGL4.11-WT + PRL-TK, ^✶✶✶P < 0.001; Compared with pcDNA3.1-JUN + pGL4.11-WT + PRL-TK, ^###P < 0.001. (b) Verification of Fos binding sites with IL-4 (n = 3). Compared with pcDNA3.1-NC + pGL4.11-WT + PRL-TK, ^✶✶✶P < 0.001; Compared with pcDNA3.1-FOS + pGL4.11-WT + PRL-TK, ^###P < 0.001. (c) Verification of Egr1 binding sites with IL-4 (n = 3). Compared with pcDNA3.1-NC + pGL4.11-WT + PRL-TK, ^✶✶✶P < 0.001; Compared with pcDNA3.1-Egr1 + pGL4.11-WT + PRL-TK, ^###P < 0.001. Jun: Jun proto-oncogene, Fos: Fos protooncogene, Egr1: Early growth response-1, IL: Interleukin, NC: Normal control, WT: Wild-type.

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The IL-4 content in the serum was detected by ELISA, indicating that its content in the FS groups was remarkably higher than that in the NC group (P < 0.01, [Figure 4a]). qPCR results implied that the mRNA expression of Jun, Fos, Egr1, and IL-4 in the FS groups significantly increased versus those in the NC group (P < 0.01, [Figure 4b-e]). Western blot of hippocampal tissue revealed considerably higher expression of Jun, Fos, p-Jun, p-Fos, Egr1, and IL-4 in the FS groups versus the NC group (P < 0.05, [Figure 4f-l]). Compared with the NC group, the p-Jun/Jun ratio significantly decreased (P < 0.05), while the p-Fos/Fos ratio significantly increased (P < 0.05) in the FS group [Figure 4m and n].

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Figure 4:

Effect of FS on the expression of IL-4, Jun, Fos, and Egr1. (a) ELISA detection of IL-4 content (n = 3). (b-e) qPCR to detect the expression of IL-4, Jun, Fos, and Egr1 mRNA (n = 3). (f-l) Western blot protein expression of Jun, Fos, p-Jun, p-Fos, Egr1, and IL-4 (n = 3). (m and n) Western blot protein expression of p-Jun/Jun and p-Fos/Fos. Compared with the NC group, ^✶P < 0.05, ^✶✶P < 0.01, and ^✶✶✶P < 0.01. Jun: Jun proto-oncogene, Fos: Fos proto-oncogene, Egr1: Early growth response-1, IL: Interleukin, NC: Normal control, WT: Wild-type, FS: Febrile seizures, ELISA: Enzyme-linked immunosorbent assay, qPCR: Quantitative polymerase chain reaction, mRNA: Messenger RNA.

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Immunohistochemistry analysis of protein expression levels in hippocampal tissue revealed increased expression of Jun, p-Jun, Fos, p-Fos, Egr1, and IL-4 in the FS groups compared with the NC group (P < 0.01, [Figure 5a-h]).

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Figure 5:

Expression levels of Jun, p-Jun, Egr1, Fos, p-Fos, and IL-4 proteins. (a-d) Jun, p-Jun, and Egr1 protein detected by immunohistochemistry in each group (×400, scale 50 μm) (n = 3). (e-h) Fos, p-Fos, and IL-4 proteins detected by immunohistochemistry in each group (×400, scale 50 μm) (n = 3). Compared with NC group ^✶✶P < 0.01, ^✶✶✶P < 0.001. Jun: Jun proto-oncogene, Fos: Fos proto-oncogene, Egr1: Early growth response-1, IL: Interleukin, NC: Normal control.

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Effects of IL-4 receptor blockade on FS

The Racine scores for seizure behavior in rats across different groups [Figure 6a-c] showed that compared with the NC group, the FS group and dupilumab group had a significantly reduced seizure latency (P < 0.01), and a significantly increased seizure duration and Racine scores (P < 0.001). The dupilumab group exhibited significantly reduced seizure latency and significantly increased seizure duration and Racine scores versus the FS group (P < 0.01).

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Figure 6:

Comparison of Seizure Behavior in Rats Across Groups. (a) Seizure latency (n = 12). (b) Seizure duration (n = 12). (c) Racine score (level) (n = 12). ^✶✶P < 0.01, ^✶✶✶P < 0.001 versus the NC group; ^##P < 0.01, ^###P < 0.001 versus the FS group. NC: Normal control group, FS: Febrile seizure.

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The expression analysis of TNF-α, IL-1β, and IL-6 in serum [Figure 7a-c] showed that the expression levels of these three factors were significantly higher in the FS and dupilumab groups compared with those in the NC group (P < 0.001). Compared with the FS group, the dupilumab group showed a significantly higher expression (P < 0.001). The results of qPCR analysis of mRNA expression of HSP70, GFAP, caspase-3, Bax, and Bcl-2 in hippocampal tissue [Figure 7d-h] indicated that the mRNA levels of HSP70, GFAP, caspase-3, and Bax were significantly higher in the FS and dupilumab groups than in the NC group (P < 0.001), whereas the mRNA expression of Bcl-2 was significantly lower in the FS and dupilumab groups than in the NC group (P < 0.001). Compared with the FS group, the dupilumab group showed a significant increase in the expression of HSP70, GFAP, caspase-3, and Bax (P < 0.01) but a significant decrease in the expression of Bcl-2 (P < 0.001). Western blot analysis of protein expression levels in hippocampal tissue [Figure 7i-n] showed that HSP70, GFAP, caspase-3, and Bax were significantly upregulated in the FS and dupilumab groups than in the NC group (P < 0.001). By contrast, Bcl-2 expression was significantly lower in the FS and dupilumab groups than in the NC group (P < 0.001). In addition, compared with the FS group, the dupilumab group exhibited a significant increase in HSP70, GFAP, caspase-3, and Bax expression (P < 0.01) and a significant reduction in Bcl-2 expression (P < 0.001).

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Figure 7:

Effects of IL-4 receptor blocker on FS. (a-c) ELISA detected the expression levels of IL-1β, TNF-α, and IL-6 in serum (n = 3). (d-h) qPCR measured the mRNA expression levels of HSP70, GFAP, caspase-3, Bax, and Bcl-2 in hippocampal tissue (n = 3). (i-n) Western Blot assessed the protein expression levels of HSP70, GFAP, caspase-3, Bcl-2, and Bax in hippocampal tissue (n = 3). ^✶✶✶P < 0.001 versus NC group, ^##P < 0.01, ^###P < 0.001 versus FS group. NC: Normal control group, FS: Febrile seizure, IL-6: Interleukin 6, IL-1β: Interleukin 1β, TNF-α: Tumor necrosis factor α, Jun: Jun proto-oncogene, Fos: Fos proto-oncogene, Egr1: Early growth response-1, IL-4: Interleukin 4, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, HSP70: Heat shock protein 70, caspase-3: Cysteine protease-3, GFAP: Glial fibrillary acidic protein, Bax: Bcl-2-associated X protein, Bcl-2: B-cell lymphoma 2.

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Flow cytometry was performed to analyze the Th1/Th2 cell ratio [Figure 8a and b], cell apoptosis [Figure 8c and d], cell proliferation [Figure 8e and f], and cell cycle [Figure 8g and h] in peripheral blood. The results indicated a significant increase in the Th1/Th2 cell ratio and cell apoptosis (P < 0.05), whereas cell cycle progression and cell proliferation were significantly reduced in the FS and dupilumab groups compared with the NC group (P < 0.001). In the dupilumab group compared with the FS group, the Th1/Th2 cell ratio and cell apoptosis showed a significant increase (P < 0.001), whereas cell cycle progression and cell proliferation exhibited a significant decrease (P < 0.001).

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Figure 8:

Effect of FS on IL-4 content and Th1/Th2 ratio. (a and b) Th1/Th2 ratio (n = 3), (c and d) cell apoptosis (n = 3), (e and f) cell proliferation (n = 3), and (g and h) cell cycle. ^✶P < 0.05, ^✶✶✶P < 0.001 versus NC group; ^###P < 0.001 versus FS group. NC: Normal control group, FS: Febrile seizure, Th1: T helper cell type 1, Th2: T helper cell type 2, IL-4: Interleukin 4.

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