Semaglutide attenuates seizure severity and ameliorates cognitive dysfunction by blocking the NLR family pyrin domain containing 3 inflammasome in pentylenetetrazole‑kindled mice

Abstract

Epilepsy comorbidities and anti‑epileptic drugs (AEDs) are currently the main limitations of epilepsy treatment. Semaglutide is a glucagon like peptide‑1 analogue that has entered the market as a new once‑weekly drug for type II diabetes. The aim of the present study was to investigate the functions of semaglutide in epilepsy and inflammation models, in order to investigate its potential mechanism. In vitro, an inflammation model was established using lipopolysaccharide (LPS) and nigericin stimulation in BV2 cells. In vivo, chronic epilepsy model mice were generated using a pentylenetetrazole (PTZ) kindling method. BV2 cell proliferation was assessed using the Cell Counting Kit‑8. The effects of semaglutide on NLR family pyrin domain containing 3 (NLRP3) inflammasome activation and inflammatory cytokine secretion were determined using western blotting (WB) and ELISA. A lactate dehydrogenase (LDH) assay kit was used to detect the effect of semaglutide on LDH release. Electrocorticography and the modified Racine scale were used to assess seizure severity. Cognitive function was evaluated with behavioral assessment. Morphological changes in the hippocampus were observed with Nissl staining. Double immunofluorescence staining for NeuN and Iba‑1, WB and immunofluorescence analysis of apoptosis‑related proteins were used to evaluate neuronal apoptosis. The NLRP3 inflammasome was assessed by reverse transcription‑quantitative PCR, WB and immunofluorescence staining, and inflammatory cytokine release was evaluated by WB analysis in the hippocampus of C57/BL6J model mouse. Semaglutide attenuated the LPS‑ and nigericin‑induced inflammatory response and LDH release by blocking NLRP3 inflammasome activation in BV2 cells. Moreover, semaglutide decreased seizure severity, alleviated hippocampal neuronal apoptosis, ameliorated cognitive dysfunction, blocked NLRP3 inflammasome activation and decreased inflammatory cytokine secretion in PTZ‑kindled mice. These results indicated that semaglutide reduced seizure severity, exerted neuroprotective effects and ameliorated cognitive dysfunction, possibly via inhibition of NLRP3 inflammasome activation and inflammatory cytokine secretion. Semaglutide may therefore be a novel, promising adjuvant therapeutic for epilepsy and its associated comorbidities.

Keywords: NLR family pyrin domain containing 3 inflammasome; cognitive dysfunction; epilepsy; glucagon like peptide‑1; neuroprotection; semaglutide.

Figure 1

Study experimental protocols. (A) BV2 cell treatment flowchart. (B) Mouse treatment flowchart. CCK8, Cell Counting Kit-8; LPS, lipopolysaccharide; PTZ, pentylenetetrazole.

Figure 2

Effect of semaglutide treatment on BV2 cells mediated by LPS and nigericin. (A) Proliferation of BV2 cells treated with semaglutide at different concentrations (300, 600, 900 and 1,000 nM) was assessed using Cell Counting Kit-8 analysis followed by one-way ANOVA. (B) Representative WB images in different BV2 cell groups. (C-E) Statistical results of WB band intensities (Student's t-test). (F-I) Statistical results of IL-1β, IL-18, IL-6 and TNF-α secretion, as assessed by ELISA (Student's t-test). (J) Semaglutide inhibited LPS- and nigericin-mediated LDH release in BV2 cells (Student's t-test). Data are presented as the means ± SD. ^*P<0.05, ^**P<0.01 and ^***P<0.001. N>3. LPS, lipopolysaccharide; WB, western blotting/blot; LDH, lactate dehydrogenase; NLRP3, NLR family pyrin domain containing 3; ASC, apoptosis-associated speck-like protein.

Figure 3

Semaglutide reduces PTZ-induced seizure severity. (A) Statistical results showed that semaglutide doses of 10 and 25 nM/kg decreased the modified Racine score (two-way ANOVA). (B) Representative electrocorticography results in each group of mice. (C) Both 10 and 25 nM/kg semaglutide decreased the rate of complete kindling. (D) Both the 10 and 25 nM/kg semaglutide increased the latency to generalized seizures (one-way ANOVA). (E) Both the 10 and 25 nmol/kg semaglutide alleviated the duration of generalized seizures (one-way ANOVA). Data are presented as the means ± SD. ^*P<0.05, ^**P<0.01 and ^***P<0.001. N≥17. PTZ, pentylenetetrazole.

Figure 4

Assessment of behavioral parameters. (A) Statistical results showed that the DI of each group in the novel object recognition test. Both 10 and 25 nM/kg semaglutide increased the DI in comparison to PTZ group (one-way ANOVA). (B and C) Statistical results showed that both the 10 and 25 nM/kg semaglutide increased the active avoidance time in the active avoidance test in comparison with PTZ group (two-way repeated ANOVA). (D and E) Statistical results showed that both the 10 and 25 nM/kg doses of semaglutide reduced the shock time and increased the latency to enter the dark cage in the passive avoidance test in comparison with PTZ group (one-way ANOVA). (F-J) Statistical results for each group of mice in the Morris water maze test. (F) Statistical results showed that both doses of semaglutide (10 and 25 nM/kg) reduced the escape latency in comparison with PTZ group (two-way repeated ANOVA test). (G and H) Representative tracks and statistical results showed that both the 10 and 25 nM/kg doses of semaglutide reduced the escape latency on the first and fifth days of training in comparison with PTZ group (two-way ANOVA). (I and J) Statistical results showed that the time spent in the target district and the number of crossed times were increased after treatment with semaglutide at doses of 10 and 25 nM/kg in comparison with PTZ group (one-way ANOVA). Data are presented as the means ± SD. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001. N≥8. DI, discriminant index; PTZ, pentylenetetrazole.

Figure 5

Results of Nissl staining and double immunofluorescence staining for NeuN and Iba-1 in the mouse hippocampus. (A) Representative images and (B and C) quantitative data for the number of Nissl-positive hippocampal neurons. Both 10 and 25 nM/kg semaglutide increased the numbers of Nissl-positive hippocampal neurons in the CA1 and CA3 areas (one-way ANOVA). (D) Representative immunofluorescence images and (E and F) densitometric analysis of NeuN and Iba-1 showing that both doses of semaglutide (enhanced the fluorescence intensity of Neun and decreased the fluorescence intensity of Iba-1 in the CA1 and CA3 areas (two-way ANOVA). Data are presented as the means ± SD. ^*P<0.05, ^**P<0.01 and ^***P<0.001. N≥3. PTZ, pentylenetetrazole.

Figure 6

Immunofluorescence and WB analyses of apoptosis-related proteins in mouse hippocampal tissue. (A) Representative immunofluorescence images and (B) densitometric analysis of active caspase-3; both 10 and 25 nM/kg semaglutide decreased the fluorescence intensity of active caspase-3 in the CA1 and CA3 regions (one-way ANOVA). (C) Representative immunofluorescence images and (D) densitometric analysis of Bax showing that both doses of semaglutide decreased the fluorescence intensity of Bax in the CA1 and CA3 regions (one-way ANOVA). (E) Representative immunofluorescence images and (F) densitometric analysis of Bcl-2 showing that both 10 and 25 nM/kg semaglutide increased the fluorescence intensity of Bcl-2 in the CA1 and CA3 regions (one-way ANOVA). (G) Representative WB images of active caspase-3, Bax and Bcl-2 in the different groups. (H and I) Statistical results of the immunoblot analysis showing that 10 and 25 nM/kg semaglutide reduced the band intensity of active caspase-3 (one-way ANOVA) and increased the Bcl-2/Bax ratio (two-way ANOVA). Data are presented as the means ± SD. ^*P<0.05, ^**P<0.01 and ^***P<0.001. N≥3. WB, western blot/blotting; PTZ, pentylenetetrazole.

Figure 7

Semaglutide affects NLRP3 inflammasome activation. Reverse transcription-quantitative PCR analysis results showing that both the 10 and 25 nM/kg semaglutide reduced the mRNA levels of (A) NLRP3, (B) ASC and (C) caspase-1 p20 (one-way ANOVA). (D) Representative images and (E-G) statistical results of immunoblot analysis showing that both 10 and 25 nM/kg semaglutide reduced the band intensities of the NLRP3 inflammasome (one-way ANOVA). (H-M) Representative immunofluorescence images and densitometric analysis showing that both doses of semaglutide (10 and 25 nM/kg) lowered the fluorescence intensities in the CA1 and CA3 regions (one-way ANOVA). Data are presented as the means ± SD. ^*P<0.05, ^**P<0.01 and ^***P<0.001. N≥3. NLRP3, NLR family pyrin domain containing 3; ASC, apoptosis-associated speck-like protein; PTZ, pentylenetetrazole.

Figure 8

(A) Representative images and (B-E) statistical results of immunoblot analysis showing that both 10 and 25 nM/kg semaglutide reduced the band intensities of IL-1β, IL-18, IL-6 and TNF-α in hippocampal tissues (one-way ANOVA). Data are presented as the means ± SD. ^*P<0.05, ^**P<0.01, ^***P<0.001. N≥3. PTZ, pentylenetetrazole.

Figure 9

Hypothetical mechanism of semaglutide action in PTZ-kindled mice. PTZ, pentylenetetrazole; NLRP3, NLR family pyrin domain containing 3; ASC, apoptosis-associated speck-like protein; GLP-1R, glucagon like peptide-1.