ABSTRACT
Background: This study aimed to investigate the expression pattern of astrocyte-derived STC1 in TLE and elucidate the molecular mechanisms by which STC1 regulates neuroinflammation and seizures through the NF-κB signaling pathway. Methods: A TLE model was established by intrahippocampal injection of kainic acid (KA) in mice. STC1 expression levels and cellular localization in the hippocampus of TLE mice were examined. Adeno-associated virus-mediated gene overexpression and shRNA knockdown approaches were employed to investigate the effects of STC1 on neuroinflammatory responses, neuronal survival, and seizure activity. qRT-PCR and immunofluorescence methods were further utilized to evaluate inflammatory cytokine levels and NF-κB signaling pathway activity. Results: STC1 expression was upregulated in hippocampal tissues of TLE mice, with double immunofluorescence showing STC1 predominantly localized in GFAP-positive reactive astrocytes. STC1 overexpression significantly exacerbated KA-induced neuroinflammation, along with enhanced microglial activation. STC1 knockdown attenuated neuroinflammatory responses. Nissl staining and NeuN immunohistochemistry confirmed that STC1 aggravated KA-induced neuronal loss. STC1 overexpression promoted p65 phosphorylation and nuclear translocation, activating the NF-κB signaling pathway. Conclusion: This study reveals the molecular mechanism by which astrocyte-derived STC1 promotes TLE-associated neuroinflammation and neuronal injury through activation of the NF-κB signaling pathway, elucidating the crucial role of the astrocyte-STC1-NF-κB axis in epileptogenesis.
KEYWORDS: Stanniocalcin-1 (STC1), TLE, neuroinflammation, NF-κB, astrocyte activation
Introduction
TLE is a common form of refractory epilepsy characterized by recurrent seizures originating from temporal lobe structures and hippocampal sclerosis, manifested as neuronal loss and glial proliferation [1–3]. Despite advances in antiepileptic drug (AED) therapy, approximately 30% of epilepsy patients respond poorly to existing medications and experience unpredictable seizures [1,4]. Therefore, a comprehensive understanding of TLE pathogenesis is crucial for developing novel therapeutic strategies [5].
Neuroinflammation represents a key component in TLE pathophysiology and has become a research focus in recent years [6,7]. Epileptic seizures can activate microglia and astrocytes, leading to the release of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-18, which further exacerbate neuronal excitability and neuronal damage [8,9]. This inflammatory response forms a vicious cycle that promotes seizure recurrence [10]. Notably, reactive astrocyte proliferation (astrogliosis) plays a critical role in TLE pathogenesis, not only by maintaining the neuronal microenvironment by directly modulating neuroinflammatory processes through the release of various inflammatory factors and cytokines [11]. Studies have revealed that astrocytes in TLE models and patients exhibit characteristic changes in expression profiles associated with multiple signaling pathway activations [12–14]. However, the dynamic regulation of neuroinflammation by astrocytes in TLE pathology and its molecular mechanisms remain incompletely elucidated.
Stanniocalcin-1 (STC1) is a glycoprotein hormone initially discovered in calcium regulation in fish and subsequently detected in various mammalian tissues [15,16]. Recent studies have shown that STC1 is upregulated under stress conditions and participates in multiple pathophysiological processes, including oxidative stress, inflammatory responses, and apoptosis [17,18]. In the central nervous system, STC1 expression is elevated in ischemic brain injury and neurodegenerative diseases, suggesting its potential important role in neuroprotection and neuroinflammation regulation [19,20]. Significantly, recent research has found that STC1 can modulate the NF-κB signaling pathway, a core transcription factor in neuroinflammation [21,22]. In cerebral ischemia-reperfusion injury models, STC1 alleviates neuroinflammation and oxidative stress by regulating NF-κB activity [23]. However, the role of STC1 in TLE pathogenesis and its relationship with astrocyte reactivity and neuroinflammation have not been investigated to date. The NF-κB signaling pathway, as a key regulator of inflammatory responses, plays an important role in seizure generation and neuroinflammation [24,25], but whether STC1 participates in TLE neuroinflammatory processes by modulating the NF-κB signaling pathway remains unclear. Therefore, this study aims to elucidate the role of astrocyte-derived STC1 in TLE pathogenesis and its regulatory relationship with the NF-κB signaling pathway, providing new potential targets for precision treatment of TLE.
Through analysis of transcriptomic data from TLE mouse models, we found that STC1 is upregulated in hippocampal tissue. Preliminary studies indicated that STC1 is primarily expressed in reactive astrocytes and dynamically changes in expression levels in KA-induced TLE models. Based on these findings, we hypothesize that astrocyte-derived STC1 may participate in the TLE neuroinflammatory processes by regulating the NF-κB signaling pathway, thereby affecting seizure activity. This study primarily reveals the mechanism of STC1 in TLE pathophysiology, not only deepening the understanding of TLE pathogenesis providing new potential targets and theoretical foundations for targeted TLE therapy.
Methods
Transcriptomics analysis
The GSE190451 dataset was extracted from the GEO database (GEO: http://www.ncbi.nlm.nih.gov/geo/). GSE190451 contains sequencing results from three normal controls and 3 TLE patients. Genes with absolute log2FC values >1 and p < 0.05 were considered statistically significant and defined as differentially expressed genes (DEGs). Principal Component Analysis was conducted to assess sample clustering and data quality. Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Omicshare analysis platform (https://www.omicshare.com/), an online bioinformatics resource integrating visualization, data mining, and analytical processing. Enrichment analysis was performed using the Homo sapiens database with all expressed genes as the background set.
Kainic acid (KA) stereotaxic injection into the right amygdala for TLE mouse model construction
SPF-grade C57BL/6 male wild-type mice, 6–8 weeks old, weighing 22–25 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. [SCXK (Beijing) 2021–0006] 1 week prior to experimentation and housed in an SPF-grade animal facility. The housing environment was quiet and well-ventilated, maintained at a temperature of 22 ± 1°C and humidity of 30–60%, with a 12-hour light/dark cycle. Animals had free access to food and water and were allowed to acclimate for 1 week before selecting those in good condition for the experiment. All mice were fasted for 12 hours before surgery with free access to water. TLE mouse models were established by stereotaxic injection of kainic acid (KA, ab120100; Abcam) into the right amygdala. Male C57BL/6 mice were randomly divided into Sham and KA groups, with 6 mice per group. Mice were anesthetized with intraperitoneal injection of tribromoethanol (40 mg/kg) and surgical procedures were performed after complete loss of consciousness (approximately 1–5 minutes). Anesthesia depth was confirmed by testing the pedal withdrawal reflex and monitoring respiratory patterns. Head fur was shaved, and mice were secured in a stereotaxic apparatus with incisor hooks and ear bars to ensure the head was positioned in the horizontal plane. Body temperature was maintained at 37°C using a feedback-controlled heating pad throughout the procedure. Respiration and heart rate were continuously monitored throughout the procedure to ensure stable vital signs. The scalp was disinfected, and an incision was made along the sagittal line to expose the skull. The periosteum was removed using 3% H₂O₂ to expose the bregma as the coordinate origin. According to the mouse brain stereotaxic atlas, the right amygdala region (AP: −1.2 mm; ML: −2.8 mm; DV: −4.5 mm) was marked. A small burr hole was carefully drilled at the marked location, avoiding damage to brain tissue. The microinjection pump needle was aligned with the drill hole and slowly inserted to the target depth. KA stock solution was prepared by dissolving KA powder in sterile 0.9% NaCl at 4°C and stored at −20°C in aliquots. Working solutions were prepared fresh on the day of injection and kept on ice. For the KA group, 500 nL of KA solution (0.5 μg/μL, dissolved in physiological saline) was injected at a rate of 300 nL/min; for the Sham group, an equal volume of physiological saline was injected at the same location. After injection, the needle was maintained in position for 5 minutes to prevent backflow. After confirming no active bleeding, the scalp was sutured and disinfected with povidone-iodine. Mice were placed on a 37°C constant-temperature heating pad for recovery. Mice were observed continuously post-surgery, and acute epileptic seizure behaviors were recorded within 1 hour after injection, with seizure severity assessed using the Racine scale (0–5 points). This experiment was approved by the Animal Ethics Committee of the Experimental Animal Center.
Manipulation of STC1 expression
Adeno-associated virus (AAV)-mediated gene manipulation was used to achieve STC1 overexpression and silencing. AAV viruses for STC1 overexpression or silencing and their respective control viruses were injected. The injection coordinates were 2.3 mm posterior to bregma, 1.8 mm lateral to the midline, and 2.0 mm in depth. 0.6 μL of AAV was injected at a rate of 0.04 μL/min, and the needle was left in place for 5 minutes before slow withdrawal. The same method was then applied to inject at 1.8 mm right of the midline, followed by suturing and disinfection of the incision and administration of antibiotics. The control group received AAV-Null adenovirus injections at the same locations using the same method.
Pharmacological treatment
BAY 11–7082 (MCE, HY-13453) was first dissolved in DMSO to prepare a stock solution. BAY 11–7082 (5 mg/kg/day) was administered via intraperitoneal injection (i.p.) continuously for 14 days. DMSO stock concentration was 50 mg/mL, and working solutions were prepared daily by diluting the stock solution in sterile phosphate-buffered saline to achieve a final DMSO concentration of ≤ 1%. Control animals received vehicle injections containing equivalent DMSO concentrations. All injections were administered at the same time each day (9:00–10:00 AM) with injection volumes not exceeding 10 mL/kg body weight.
qRT-PCR detection of inflammatory factor
Tissue samples (approximately 20–30 mg) were immediately homogenized in 1 mL TRIzol reagent (Invitrogen 15,596,026) using a TissueLyser II (Qiagen) at 25 Hz for 2 minutes. Total RNA was extracted according to the manufacturer’s protocol and treated with DNase I (Takara, 2270A) to eliminate genomic DNA contamination. RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific), with A260/A280 ratios between 1.8 and 2.2 considered acceptable. cDNA synthesis was performed according to the reverse transcription kit protocol. Each reaction system included 1 μL cDNA and 1 μL each of upstream and downstream primers, using the SYBR GREEN fluorescent quantitative PCR kit for PCR amplification. The PCR program was set as follows: pre-reaction at 95°C for 5 min, denaturation at 95°C for 10 s, annealing at 58°C for 45 s, extension at 72°C for 45 s, with 40 cycles starting from the second step. GAPDH transcription expression level was used as an internal reference, and relative gene expression values were calculated using the 2−ΔΔCt method. All primers were designed using Primer-BLAST (NCBI) and synthesized by Sangon Biotech (Shanghai, China). Primer sequences are shown in Table 1.
Table 1.
Primer sequences used for qPCR.
| Forward primer | Reverse primer | |
|---|---|---|
| Tnf-α | 5’-TTCTCATTCCTGCTTGTGG-3’ | 5’-CTTGGTGGTTTGCTACGAC-3’ |
| Il-1β | 5’-AAGCCTCGTGCTGTCGGACC-3’ | 5’-TGAGGCCCAAGGCCACAGGT-3’ |
| Il-18 | 5’-GACAGCCTGTGTTCGAGGATATG-3’ | 5’-TGTTCTTACAGGAGAGGGTAGAC-3’ |
| Il-6 | 5’-GGCGGATCGGATGTTGTGAT-3’ | 5’-GGACCCCAGACAATCGGTTG-3’ |
| Gapdh | 5’-AGGTCGGTGTGAACGGATTTG-3’ | 5’-TGTAGACCATGTAGTTGAGGTCA-3’ |
Immunofluorescence staining
At the conclusion of the experiment, animals from each group were first anesthetized, then subjected to cardiac perfusion with 0.9% sodium chloride injection solution (physiological saline) followed by 4% paraformaldehyde tissue fixative (PFA, prepared with 0.1 mol/L PBS solution, pH = 7.2, freshly prepared). Subsequently, brain tissues were harvested. The extracted brain tissues were initially fixed in 4% paraformaldehyde solution for 24 h, followed by embedding and sectioning. Sections were rinsed with PBS buffer. An appropriate amount of PBS solution containing 1.0% Triton X-100 was applied to the specimens and allowed to stand at room temperature for 30 min to permeabilize membranes. Sections were outlined with a PAP pen (Dako) to prevent solution spillage, and 200 μL of permeabilization solution was applied to each section to ensure complete coverage. Following this, a PBS solution containing 3% BSA was applied to the mouse brain sections and left at room temperature for 1 h for blocking. After blocking, the brain tissue sections were immersed in PBST solution and washed 3 times on a decolorization shaker, 5 min each time. After air-drying, 100 μL of PBS solution containing primary antibodies (anti-NeuN antibody, ab177487; anti-GFAP antibody, ab53554; anti-Iba1 antibody, ab178846), 0.5% Triton X-100, and 1% BSA was applied to each mouse brain section and incubated overnight at 4°C. Primary antibody incubation was performed in a humidified chamber to prevent drying. Sections were covered with parafilm and incubated. Following incubation, sections were again immersed in PBST solution and washed 3 times on a decolorization shaker, 5 min each time. After air-drying, approximately 100 μL of PBS solution containing fluorescent secondary antibodies (Alexa488 or Alexa594), 0.5% Triton X-100, and 1% BSA was applied to each brain tissue section. After standing at room temperature for 1 h, sections were washed again 3 times with PBST solution. Finally, once the slides were dry, a mounting medium containing DAPI was applied, and coverslips were carefully placed over the sections. Coverslips were applied carefully to avoid air bubbles, and slides were stored at 4°C in the dark before imaging to allow complete polymerization. To confirm antibody specificity, negative control stainings were performed using identical procedures but omitting the primary antibodies, with only secondary antibodies applied to tissue sections. After the mounting medium solidified, sections were observed using a confocal microscope.
Nissl staining for examination of neuronal morphology
Fixed brain tissues were placed in embedding cassettes, rinsed with running water, and then immersed in 75% alcohol overnight. All dehydration steps were performed at room temperature. Tissue processing was conducted using an automated tissue processor (Leica ASP300S) to ensure consistent timing and reagent quality. On the second day, tissues were dehydrated sequentially in different concentrations of alcohol: 85% → 95% I → 95% II → 100% I → 100% II. Each dehydration step was performed for 2 hours except for the final 100% ethanol step which was extended to 3 hours. Tissues were then cleared in xylene for two changes of 1 hour each. After clearing, tissues were embedded in paraffin. Embedding was performed using a Leica EG1150H embedding station with paraffin wax maintained at 60°C. Following embedding, continuous sections of 4 μm thickness were cut using a microtome. After routine deparaffinization, sections were stained with methyl violet solution for Nissl staining, then differentiated, routinely dehydrated, and mounted. Nissl staining was performed using 0.1% cresyl violet acetate solution (Sigma-Aldrich, C5042) prepared in distilled water and filtered through 0.22 μm filters. Sections were immersed in the staining solution for 3–5 minutes at room temperature with gentle agitation. Staining intensity was monitored under light microscopy to achieve optimal contrast. The morphology of Nissl bodies in brain regions was observed and photographed using an optical microscope, and quantitative analysis was performed.
Western blot
Brain tissue samples were rapidly harvested and immediately homogenized in ice-cold RIPA lysis buffer supplemented with protease inhibitor cocktail (Roche 11,697,498,001) and phosphatase inhibitor cocktail (Roche 04,906,845,001). Samples were homogenized using a tissue homogenizer on ice for 30 seconds with 10-second intervals, then incubated on ice for 30 minutes with occasional vortexing. The lysates were centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatants were collected. Protein concentrations were determined using the BCA protein assay kit (Pierce 23,225) according to the manufacturer’s instructions. Equal amounts of protein (30 μg per lane) were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, IPVH00010) using a wet transfer system at 100 V for 90 minutes at 4°C. Membranes were blocked with 5% nonfat milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature. Incubated overnight at 4°C with primary antibodies: anti-STC1 (1:1000, Abcam, ab124891), anti-GFAP (1:2000, Abcam, ab7260), anti-IBA1 (1:1000, Abcam, ab5076), anti-p-NF-κB p65 (1:1000, CST, #3033), and anti-β-actin (1:3000, CST, #3700) as loading control. Incubated with corresponding HRP-conjugated secondary antibodies (1:5000, Cell Signaling Technology) for 1 hour at room temperature. Visualized using enhanced chemiluminescence (ECL) detection reagent (Pierce 32,106) and imaged using a ChemiDoc imaging system (Bio-Rad). Quantified using ImageJ software and normalized to β-actin expression levels.
Statistical analysis
GraphPad Prism 9 software was used for all statistical analyses. Data normality was assessed using the Shapiro–Wilk test, and homogeneity of variance was evaluated using Bartlett’s test. For comparisons between the two groups, normally distributed data were analyzed using unpaired t-tests, while non-normally distributed data or small sample sizes (n < 6) were analyzed using the Mann-Whitney U test. For multiple group comparisons, normally distributed data were analyzed using one-way ANOVA followed by Tukey’s post hoc test, while non-normally distributed data were analyzed using the Kruskal–Wallis test followed by Dunn’s post hoc test. All data are presented as mean ± SEM. Statistical significance was set at p < 0.05.
Results
STC1 is highly expressed in the hippocampal tissue of TLE mice
To investigate the expression characteristics of STC1 in TLE, we extracted the GSE190451 dataset from the GEO database (GEO: http://www.ncbi.nlm.nih.gov/geo/), which included sequencing analysis results from 3 control and 3 TLE patient tissue samples. Principal component analysis (PCA) revealed differences in gene expression patterns between the TLE model group and Sham control group, with samples from the two groups showing clear separation along the PC1 and PC2 axes (Figure 1A). Hierarchical clustering analysis further confirmed the transcriptomic differences between the two groups and indicated high homogeneity within the TLE group (Figure 1B). Through a differential gene expression analysis, we identified 4427 upregulated genes and 939 downregulated genes (|log2FC| > 1, p < 0.05). Volcano plot analysis highlighted STC1 as one of the most upregulated genes (Figure 1C). To validate the transcriptome analysis results, we quantitatively assessed the expression levels of relevant genes in hippocampal tissue from KA-induced TLE mice using qRT-PCR. The results showed that, compared with the Sham group, the mRNA expression level of the astrocyte marker GFAP was elevated in the TLE model group (p < 0.001) (Figure 1D), and the mRNA expression of the microglial marker IBA1 was also significantly increased (p < 0.01) (Figure 1E). These results indicate glial cell activation in the KA-induced TLE model. Importantly, STC1 mRNA expression was upregulated in the hippocampal tissue of TLE mice compared to the control group (p < 0.001) (Figure 1F), consistent with the transcriptome sequencing results. Western blot analysis confirmed that GFAP, IBA1 and STC1 were high expression in hippocampal tissue 35 days after KA-induced epilepsy surgery in mice (Figure 1G). To further evaluate the pathological changes in the TLE model and the cellular basis of STC1 expression, we performed immunohistochemical analysis. As shown in Figure 1H, the number of NeuN-positive neurons in the hippocampus of TLE mice was reduced (p < 0.01), indicating significant neuronal loss. Meanwhile, the number of GFAP-positive astrocytes increased (p < 0.001) (Figure 1I), and the cells exhibited typical reactive morphological changes, including hypertrophic cell bodies and thickened processes. Similarly, the number of IBA1-positive microglia was significantly increased (Figure 1J), with many displaying an activated phenotype characterized by enlarged cell bodies and shortened processes.
Figure 1.
STC1 is highly expressed in the hippocampal tissue of TLE mice.
A–C. Comprehensive transcriptomic analysis of GSE190451 dataset. Principal component analysis (PCA) (A), hierarchical clustering analysis (B), and volcano plot analysis (C).
D–E. PCR analysis of GFAP (D) and IBA1 (E) expression in hippocampal tissue 35 days after KA–induced epilepsy surgery in mice.
H–J. Immunostaining and quantitative analysis of NeuN (H), GFAP (I), and IBA1 (J) positive cells in brain tissue 35 days after KA–induced epilepsy surgery in mice.
Data are presented as mean ± SEM with individual data points shown. n = 6 represents independent biological replicates (individual animals). Statistical analysis was performed using unpaired t–test. ***p < 0.001, ****p < 0.0001.
Data are presented as mean ± SEM with individual data points shown. n = 6 represents independent biological replicates (individual animals). Statistical analysis was performed using unpaired t–test. ***P < 0.001.
Immunofluorescence analysis revealed STC1 colocalization with GFAP-positive astrocytes in the hippocampus of TLE mice
To determine the expression dynamics of STC1 during TLE progression and its cellular origin, we systematically analyzed the spatiotemporal expression characteristics of STC1 in the KA-induced TLE mouse model. qPCR analysis showed that, compared with the Sham group, STC1 mRNA levels rapidly increased on day 1 after KA injection (7.83 ± 0.91 fold, p < 0.001), remained elevated on day 10 (5.26 ± 0.67 fold, p < 0.01), and were still higher than the control group on day 35 (1.94 ± 0.54 fold, p < 0.01) (Figure 2A). This indicates that STC1 is rapidly upregulated in the early stages of TLE and continues to be expressed during the chronic phase, suggesting that STC1 may be involved in both the acute injury response and chronic epileptogenesis in TLE. To further verify the temporal dynamics of STC1 expression, we used immunofluorescence staining to directly observe changes in STC1 protein expression in hippocampal tissue. The results showed that STC1 protein reached its highest expression level on day 1 after KA injection, decreased by day 10, and further decreased by day 35 but remained above baseline levels (Figure 2(B,C)). This protein expression pattern was generally consistent with the changes in mRNA levels. To identify the cellular source of STC1, we performed multiple-label immunofluorescence co-localization analysis to detect the co-expression of STC1 with different central nervous system cell markers. In the CA3 region of the mouse hippocampus on day 1 after KA injection, STC1 showed a very high co-localization rate with the astrocyte marker GFAP (Figure 2D).
Figure 2.
Immunofluorescence analysis revealed STC1 colocalization with GFAP-positive astrocytes in the hippocampus of TLE mice.
A-D. mRNA levels of TNF-α (A), IL-1β (B), IL-6 (C), IL-18 (D) in the hippocampus of mice from different treatment groups.
E-F. mRNA levels of GFAP (E), IBA1 (F) in the hippocampus of mice from different treatment groups.
G-H. Immunostaining analysis of GFAP (G), IBA1 (H) in the hippocampus of mice from different treatment groups.
Data are presented as mean ± SEM with all individual data points displayed. n = 6 represents independent biological replicates (individual animals) per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. **P < 0.01, ***P < 0.001.
STC1 overexpression exacerbates KA-induced neuroinflammation
To investigate the functional role of STC1 in TLE-related neuroinflammation, we constructed an adeno-associated virus (AAV)-mediated STC1 overexpression vector (OE-STC1) and achieved local STC1 overexpression through stereotaxic hippocampal injection. qRT-PCR analysis showed that, compared with the NC+Veh group, the OE-STC1+Veh group exhibited slightly elevated mRNA levels of various pro-inflammatory cytokines, including TNF-α (Figure 3A), IL-1β (Figure 3B), IL-6 (Figure 3C), and IL-18 (Figure 3D). This indicates that STC1 overexpression alone can induce a mild neuroinflammatory response. As expected, all inflammatory factors were upregulated in the NC+KA group, reflecting KA-induced neuroinflammation. Notably, in the OE-STC1+KA group, the expression levels of all pro-inflammatory cytokines were further elevated compared to the NC+KA group. These results clearly demonstrate that STC1 overexpression exacerbated KA-induced neuroinflammatory responses. Further analysis revealed that STC1 overexpression also affected the degree of glial cell activation. qRT-PCR results showed that, compared with the NC+Veh group, the expression levels of the astrocyte marker GFAP (Figure 3E) and the microglial marker IBA1 (Figure 3F) were both elevated in the OE-STC1+Veh group. In the KA-treated groups, the expression levels of GFAP and IBA1 in the OE-STC1+KA group were higher than those in the NC+KA group, indicating that STC1 overexpression enhanced KA-induced glial cell activation. To directly observe the morphological changes of glial cell activation, we performed immunohistochemical staining analysis. The results showed that STC1 overexpression enhanced KA-induced astrocyte reactivity (Figure 3G). Similarly, the IBA1 immunostaining results showed that the number of microglia in the OE-STC1+Veh group slightly increased, but morphological changes were not obvious (Figure 3H). Numerous activated microglia were observed in the NC+KA group, characterized by hypertrophic cell bodies and shortened processes. In the OE-STC1+KA group, the number of activated microglia was greater, and many displayed amoeboid and spherical morphologies, indicating a higher degree of activation (Figure 3H).
Figure 3.
STC1 overexpression exacerbates KA-induced neuroinflammation.
A-D. mRNA levels of TNF-α (A), IL-1β (B), IL-6 (C), IL-18 (D) in the hippocampus of mice from different treatment groups.
E-F. mRNA levels of GFAP (E), IBA1 (F) in the hippocampus of mice from different 33 treatment groups.
G-H. Immunostaining analysis of GFAP (G), IBA1 (H) in the hippocampus of mice from different treatment groups.
Data are presented as mean ± SEM with all individual data points displayed. n=6 represents independent biological replicates (individual animals) per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. **P<0.01, ***P<0.001.
Figure 6.
STC1 overexpression activates the NF-κB signaling pathway in TLE mice.
A. KEGG pathway enrichment analysis reveals that the NF-κB pathway is enriched during TLE model progression. B Representative p-NF-κB immunostaining analysis in hippocampi of mice from different treatment groups with STC1 knockdown. C Western blot analysis of p-NF-κB expression in hippocampi of mice from different treatment groups with STC1 knockdown. D Representative p-NF-κB immunostaining analysis in hippocampi of mice from different treatment groups with STC1 overexpression. E Western blot analysis of p-NF-κB expression in hippocampi of mice from different treatment groups with STC1 overexpression. Data are presented as mean ± SEM with all individual data points displayed. n=6 represents independent biological replicates (individual animals) per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. **P<0.01, ****P<0.0001.
STC1 knockdown alleviates KA-induced neuroinflammation
To further confirm the role of STC1 in TLE-related neuroinflammation, we specifically knocked down STC1 expression. qRT-PCR analysis showed that, under non-epileptic conditions, STC1 knockdown did not alter the basal expression levels of pro-inflammatory cytokines in hippocampal tissue. Specifically, compared with the Scr+Veh group, there were no differences in the mRNA levels of TNF-α (Figure 4A), IL-1β (Figure 4B), IL-6 (Figure 4C), and IL-18 (Figure 4D) in the sh-STC1+Veh group. However, in the KA-induced epilepsy model, STC1 knockdown attenuated the neuroinflammatory response. Compared with the Scr+KA group, the mRNA levels of all detected pro-inflammatory cytokines were reduced in the sh-STC1+KA group. These results corroborate the findings from the STC1 overexpression experiments, further confirming that STC1 plays a promoting role in TLE-related neuroinflammation. To assess the effect of STC1 knockdown on glial cell activation, we examined the expression levels of GFAP and IBA1. Under non-epileptic conditions, there were no differences in the mRNA levels of GFAP (Figure 4E) and IBA1 (Figure 4F) between the sh-STC1+Veh group and the Scr+Veh group. However, in the KA-induced epilepsy model, the mRNA levels of GFAP and IBA1 were reduced in the sh-STC1+KA group compared with the Scr+KA group. This indicates that STC1 knockdown can inhibit KA-induced glial cell activation. Immunofluorescence staining results directly demonstrated the effect of STC1 knockdown on glial cell activation. As shown in Figure 4G, under non-epileptic conditions, the GFAP immunostaining intensity in the sh-STC1+Veh group was similar to that in the Scr+Veh group, both displaying normal astrocyte morphology. In the KA-induced epilepsy model, numerous reactive astrocytes were observed in the Scr+KA group, characterized by hypertrophic cell bodies, thickened processes, and significantly enhanced GFAP immunostaining intensity. In contrast, the sh-STC1+KA group showed fewer reactive astrocytes with less severe morphological changes, indicating that STC1 knockdown effectively inhibited KA-induced astrocyte reactivity. Similarly, IBA1 immunostaining results (Figure 4H) showed that in the KA-induced epilepsy model, numerous activated microglia were observed in the Scr+KA group, characterized by enlarged and rounded cell bodies, shortened and reduced processes. The sh-STC1+KA group exhibited fewer activated microglia. This indicates that STC1 knockdown effectively inhibited KA-induced microglial activation.
Figure 4.
STC1 knockdown attenuates KA-induced neuroinflammation.
A-D. mRNA levels of TNF-α (A), IL-1β (B), IL-6 (C), IL-18 (D) in the hippocampus of mice from different treatment groups. E-F. mRNA levels of GFAP (E), IBA1 (F) in the hippocampus of mice from different treatment groups. G-H. Immunostaining analysis of GFAP (G), IBA1 (H) in the hippocampus of mice from different treatment groups. Data are presented as mean ± SEM with all individual data points displayed. n=6 represents independent biological replicates (individual animals) per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. ns: no significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
STC1 exacerbates KA-induced neuronal loss and promotes seizure activity
To evaluate the functional significance of STC1 in TLE pathology, we systematically examined the effects of STC1 expression levels on seizure behavioral manifestations and neuronal survival. Racine scale scoring results showed that, compared with the control group (Scr+KA), the STC1 knockdown group (sh-STC1+KA) exhibited reduced Racine scores (Figure 5A). Concurrently, the seizure duration was significantly shortened in the sh-STC1+KA group. These results indicate that STC1 knockdown can alleviate KA-induced seizures. Conversely, compared with the control group (NC+KA), the STC1 overexpression group (OE-STC1+KA) mice showed increased Racine scores (Figure 5B). Additionally, the seizure duration was prolonged in the OE-STC1+KA group. This suggests that STC1 overexpression aggravates KA-induced seizures, further confirming the promoting role of STC1 in seizure activity. To investigate the effect of STC1 on neuronal survival, we employed Nissl staining to assess neuronal density in the hippocampus of mice from different treatment groups. In the STC1 knockdown experimental groups (Figure 5C), no significant differences in neuronal morphology and density were observed between the Scr+Veh and sh-STC1+Veh groups, indicating that STC1 knockdown alone does not affect the survival of normal neurons. Evident neuronal loss was observed in the Scr+KA group, with neuronal density reduced compared to the control group. In the sh-STC1+KA group, the degree of neuronal loss was attenuated, with increased neuronal density compared to the Scr+KA group. This indicates that STC1 knockdown can mitigate KA-induced neuronal damage. In the STC1 overexpression experimental groups (Figure 5D), compared with the NC+Veh group, the OE-STC1+Veh group showed a slight reduction in neuronal density, suggesting that STC1 overexpression alone may exert mild neurotoxic effects. Neuronal density in the NC+KA group was reduced compared to the NC+Veh group. Neuronal loss was more severe in the OE-STC1+KA group, with further reduced neuronal density compared to the NC+KA group. This indicates that STC1 overexpression exacerbates KA-induced neuronal damage.
Figure 5.
STC1 exacerbates KA-induced neuronal loss.
A. Reduced Racine scale scores in epileptic mice with STC1 knockdown. C. Representative Nissl staining analysis in hippocampi of mice from different treatment groups with STC1 knockdown. D. Representative Nissl staining analysis in hippocampi of mice from different treatment groups with STC1 overexpression. Data are presented as mean ± SEM with all individual data points displayed. n=6 represents independent biological replicates (individual animals) per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. ns: no significant, *P<0.05, **P<0.01, ****P<0.0001.
STC1 overexpression activates the NF-κB signaling pathway in TLE mice
To explore the molecular mechanism by which STC1 promotes neuroinflammation and seizures, KEGG enrichment analysis was performed on sequencing results from TLE patient tissues. As shown in Figure 6A, transcriptome data analysis identified multiple inflammation-related signaling pathways enriched in the TLE model, among which the NF-κB signaling pathway was significantly enriched. Additionally, the cytokine–cytokine receptor interaction pathways and TNF signaling pathways, which are closely related to the NF-κB pathway, were also notably enriched. These results suggest that the NF-κB signaling pathway may be a key molecular mechanism by which STC1 regulates TLE-related neuroinflammation. To verify the relationship between STC1 and NF-κB pathway activation, we used immunohistochemical methods to detect the expression levels of phosphorylated NF-κB p65 subunit (p-NF-κB p65) in hippocampal tissues from different experimental groups of mice, which is a direct indicator of NF-κB pathway activation. In the STC1 knockdown experiment (Figure 6(B,C)), the p-NF-κB p65 was reduced in the Scr+Veh and sh-STC1+Veh groups. This indicates that under physiological conditions, STC1 knockdown has a weak inhibitory effect on the NF-κB pathway. The Scr+KA group showed enhanced p-NF-κB p65 staining intensity, indicating that KA treatment strongly activated the NF-κB signaling pathway. The p-NF-κB p65 staining intensity in the sh-STC1+KA group was lower than that in the Scr+KA group, indicating that STC1 knockdown can inhibit KA-induced NF-κB pathway activation. In the STC1 overexpression experiment (Figure 6(B,C)), compared with the NC+Veh group, the OE-STC1+Veh group showed enhanced p-NF-κB p65 expression, suggesting that STC1 overexpression alone can activate the NF-κB pathway. The NC+KA group exhibited increased p-NF-κB p65 staining intensity. The p-NF-κB p65 staining intensity in the OE-STC1+KA group was higher than that in the NC+KA group (p < 0.001), indicating that STC1 overexpression enhanced KA-induced NF-κB pathway activation.
Inhibition of the NF-κB pathway attenuates the pro-epileptic effects of STC1
To determine whether STC1 regulates neuroinflammation and seizures through the NF-κB pathway, we administered the specific NF-κB inhibitor BAY 11–7082 to mice in the OE-STC1+KA group. The results showed that, compared with the OE-STC1+KA group, the BAY 11–7082 treatment group exhibited reduced mRNA levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-18) in hippocampal tissue (Figures 7A–7D). More importantly, BAY 11–7082 treatment also alleviated seizures in the OE-STC1+KA group mice, with reduced Racine scores (Figure 7E). The expression of glial cell activation markers (GFAP and IBA1) was also decreased by western blot and IF (Figures 7F-H). This indicates that inhibition of the NF-κB pathway can reverse the enhanced neuroinflammation caused by STC1 overexpression. These results suggest that STC1’s promotion of neuroinflammation and seizures depends, at least partially, on the activation of the NF-κB signaling pathway. Figure 8 illustrates the regulatory effect of STC1 on the NF-κB pathway and its mechanism in exacerbating TLE. This schematic model demonstrates the mechanism by which astrocyte-derived STC1 promotes neuroinflammation and seizures in KA-induced epileptic mice through activation of the NF-κB signaling pathway.
Figure 7.
Inhibition of the NF-κB pathway attenuates the pro-epileptic effects of STC1.
A-D. mRNA levels of TNF-α (A), IL-1β (B), IL-6 (C), IL-18 (D) in the hippocampus of mice from different treatment groups. E. BAY 11-7082 treatment alleviated seizures in OE-STC1+KA mice, with reduced Racine scores. F. Western blot analysis of GFAP and IBA1 in the hippocampus of mice from different treatment groups. G-H. Immunostaining analysis of GFAP (G), IBA1 (H) in the hippocampus of mice from different treatment groups. Data are presented as mean ± SEM with all individual data points displayed. n=6 represents independent biological replicates (individual animals) per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. ns: no significant, **P<0.01, ***P<0.001, ****P<0.0001.
Figure 8.
Schematic diagram illustrating the mechanism by which STC1 promotes epilepsy progression.
This schematic model demonstrates the mechanism by which astrocyte-derived STC1 promotes neuroinflammation and seizures in KA-induced epileptic mice through activation of the NF-κB signaling pathway.
Discussion
This study analyzes the critical role of astrocyte-derived STC1 in the pathogenesis of TLE, revealing that STC1 is upregulated in the hippocampal tissue of TLE mice and primarily localized in reactive astrocytes. Functional investigations demonstrate that STC1 promotes neuroinflammatory responses by activating the NF-κB signaling pathway, exacerbating neuronal damage and seizure activity. These findings not only deepen our understanding of TLE pathological mechanisms provide a new theoretical foundation for developing anti-epileptic strategies targeting the STC1-NF-κB axis.
We observed a significant increase in STC1 expression in the hippocampal tissue of TLE mice, consistent with previous reports of STC1 upregulation under stress conditions [26]. Through co-immunofluorescence, astrocytes were identified as the primary source of STC1 in the TLE model. This finding reveals that astrocytes may participate in neuroinflammatory regulation by secreting STC1 under pathological conditions, complementing our understanding of astrocyte functional diversity. Previous studies have confirmed that astrocytes transform into reactive phenotypes during epileptogenesis, releasing various pro-inflammatory factors and chemokines [27,28]. Our research further elucidates that STC1, as an important factor secreted by astrocytes, plays a key role in astrocyte-mediated neuroinflammatory responses, enriching our knowledge of the functional characteristics of “reactive astrocytes.”
Further investigation revealed that STC1 overexpression aggravated KA-induced neuroinflammatory responses, while STC1 knockdown produced opposite effects. This is similar to observations by Zhu et al. (2024) in Colitis model, where they found STC1 promoted PARP1‐Dependent Cell Death [29]. In the epilepsy model, we found that STC1 promotes neuroinflammatory responses, possibly due to the unique immune microenvironment and astrocyte-neuron interaction mechanisms. Under pathological conditions, STC1 secreted by reactive astrocytes may act on surrounding cells through autocrine and paracrine pathways, amplifying inflammatory cascade reactions. Neuroinflammation and neuronal damage constitute a vicious cycle, mutually promoting each other during epileptogenesis [30]. Pro-inflammatory factors not only directly damage neurons increase neuronal excitability, further exacerbating seizures [31]. Our research suggests that astrocyte-derived STC1 may serve as an important bridge connecting neuroinflammation and neuronal damage, elucidating a new pathway from astrocyte activation to neuronal injury. This finding not only supplements the “second hit” theory of epilepsy pathogenesis [32] provides a new perspective for understanding epileptogenic focus formation and the epileptogenic process.
NF-κB is a key transcription factor regulating inflammatory gene expression and plays a central role in epileptogenesis [33,34]. We found that STC1 overexpression increases p65 phosphorylation levels, and subsequently activates downstream pro-inflammatory gene expression. This is consistent with observations in cerebral ischemia models [35]. In the context of epilepsy, STC1 demonstrates a role in promoting NF-κB activation. This conclusion requires further molecular mechanism studies for validation. Our research reveals a novel astrocyte-STC1-NF-κB-neuroinflammation axis in TLE pathogenesis, providing a new framework for understanding the immuno-neurological mechanisms of epilepsy.
Interventions targeting STC1 may alleviate neuroinflammatory responses, protect neurons, and consequently inhibit seizures. Compared to direct NF-κB inhibition, targeting upstream STC1 may provide more specific and safer therapeutic strategies, as NF-κB also plays an important role in maintaining normal physiological functions. Additionally, since STC1 is primarily upregulated under pathological conditions, interventions targeting STC1 may have higher specificity and fewer side effects. Considering that approximately 30% of epilepsy patients respond poorly to existing drug treatments, developing novel therapeutic strategies targeting STC1 has significant clinical translational value.
Several limitations should be acknowledged in this study. The analysis of human TLE samples relied on the GSE190451 dataset, which included only three TLE patients and three controls. While this dataset provided valuable initial evidence for STC1 upregulation in human TLE and served as a rationale for the mechanistic investigations, the limited sample size constrains the generalizability of the translational conclusions. Future studies should validate these findings in larger, independent human cohorts and clinical samples to strengthen the clinical relevance of the results. Second, this study tested only one level of STC1 overexpression and knockdown without conducting dose–response analyses. This limitation makes it unclear whether the observed effects reflect physiological ranges or experimental extremes. Future dose-dependent studies would be valuable to determine the optimal levels of STC1 modulation and to better understand the physiological relevance of STC1 in epileptogenesis. Third, the present investigation focused exclusively on the NF-κB signaling pathway. While this choice was based on the observed inflammatory phenotype and the established role of NF-κB in neuroinflammation and epileptogenesis, STC1 is known to regulate other important mechanisms including oxidative stress and apoptosis. Future studies should examine whether STC1 influences epileptogenesis through ROS generation, mitochondrial dysfunction, or apoptotic pathways, which could provide a more comprehensive understanding of STC1’s role in epilepsy and identify additional therapeutic targets. While immunofluorescence analysis demonstrated STC1 expression in GFAP-positive astrocytes, the study did not systematically examine STC1 expression in other major brain cell types, including neurons and microglia. A comprehensive cellular localization analysis using multiple cell-type-specific markers (such as NeuN for neurons and Iba1 for microglia) would be valuable to fully characterize the cellular distribution of STC1 in TLE and to determine whether STC1 contributes to neuronal dysfunction or microglial activation in addition to astrocytic responses. In addtion, this study primarily employed a KA-induced TLE model without verifying consistency in other epilepsy models. Different epilepsy models may exhibit different pathological characteristics and molecular mechanisms, making it essential to validate the role of STC1 in multiple epilepsy models.
Conclusion
This study reveals a novel mechanism by which astrocyte-derived STC1 promotes TLE-associated neuroinflammation and neuronal injury through the activation of the NF-κB signaling pathway, providing a new perspective for understanding the pathophysiological processes of epilepsy and indicating a new direction for developing anti-epileptic therapeutic strategies targeting neuroinflammation. These findings are not only significant for epilepsy research provide new insights into the role of astrocytes in neurological diseases.
Abbreviation list
- cDNA
Complementary DNA
- GFAP
Glial fibrillary acidic protein
- IL-1β
Interleukin-1 beta
- IL-6
Interleukin-6
- KA
Kainic acid
- NeuN
Neuronal nuclei
- NF-κB
Nuclear factor kappa B
- PBS
Phosphate-buffered saline
- PFA
Paraformaldehyde
- shRNA
Short hairpin RNA
- STC1
Stanniocalcin-1
- TLE
Temporal lobe epilepsy
- TNF-α
Tumor necrosis factor alpha
- SPF
Specific pathogen-free
- ROS
Reactive oxygen species
- PCA
Principal component analysis
- KEGG
Kyoto Encyclopedia of Genes and Genomes
- GO
Gene Ontology
- DEG
Differentially expressed gene
- DMSO
Dimethyl sulfoxide
Supplementary Material
Funding Statement
The authors would like to thank Natural Science Foundation of Heilongjiang Province (LH2023H074), Projects of Health Commission of Heilongjiang Province (20230202040115), Heilongjiang Provincial Traditional Chinese Medicine Research Project (ZHY2024–047).
Disclosure statement
The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this study.
Authors’ contributions
H.C. devised the concepts and directions for this study. K.L and Q.S. collected the literature and analyzed the data. Y.G. and W.Z. conducted the literature analysis and wrote the initial draft of the paper, with contributions from all the authors. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
The experiments met the ethical requirements and were approved by the ethics committee of Heilongjiang University Of Chinese Medicine (YSY-DWLL-2023337).
Data availability statement
Data included in article/supp. material/referenced in article.
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/15384101.2025.2566293
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Data Availability Statement
Data included in article/supp. material/referenced in article.