Saiga antelope horn suppresses febrile seizures in rats by regulating neurotransmitters and the arachidonic acid pathway

Background Saiga antelope horn (SAH) is a traditional Chinese medicine for treating febrile seizure (FS) with precise efficacy, but its mechanism of action and functional substances are still unclear. Given the need for further research on SAH, our group conducted studies to elucidate its mechanisms and active substances. Methods An FS rat pup model was constructed through intraperitoneal injection of LPS and hyperthermia induction. Behavioural indicators of seizures, hippocampal histopathological alterations, serum levels of inflammatory cytokines and hippocampal levels of neurotransmitters were observed and measured to investigate the effects of SAH on FS model rats. Hippocampal metabolomics and network pharmacology analyses were conducted to reveal the differential metabolites, key peptides and pathways involved in the suppression of FS by SAH. Results SAH suppressed FS, decreased the inflammatory response and regulated the Glu-GABA balance. Metabolomic analysis revealed 13 biomarkers of FS, of which SAH improved the levels of 8 differential metabolites. Combined with network pharmacology, a “biomarker-core target-key peptide” network was constructed. The peptides of SAH, such as YGQL and LTGGF, could exert therapeutic effects via the arachidonic acid pathway. Molecular docking and ELISA results indicated that functional peptides of SAH could bind to PTGS2 target, inhibiting the generation of AA and its metabolites in hippocampal samples. Conclusion In summary, the functional peptides contained in SAH are the main material basis for the treatment of FS, potentially acting through neurotransmitter regulation and the arachidonic acid pathway. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13020-024-00949-3.

The following is the Supplementary data to this article: Supplemental Method 1. Nano-LC-MS/MS analysis of SAH

Sample preparation
The proteins of SAH were extract as described with slight modifications.10 mg of animal horn powder was immersed in 2 mL of 50 mM sodium phosphate (pH 7.8) with 2% sodium dodecyl sulfate (SDS) and 20 mM dithiothreitol (DTT), and then incubated overnight at 65℃.To the mixture, 20 μL of 100 mM iodoacetamide (IAA) was added to alkylate Cysteine residue at room temperature for 30 min in the dark.The proteins were precipitated with 5.5 times the volume of 80% acetone overnight, and then followed by 80% acetone wash.The pellet was resuspended in 200 μL of 50 mM Tris (pH = 8) containing 8 M urea.After sonicating in an ice-water bath for 20 min, the supernatant was achieved with centrifugation at 14,000 × g for 5 min.Then the supernatant was diluted with 50 mM Tris solution (pH = 8) to urea concentration below 1 M. Protein digestion was performed by adding trypsin at a 50:1 protein/enzyme ratio at 37°C for 16 h.Subsequently, 10% TFA was added until pH lower than 3 to quench the reaction.The digested peptide samples were desalted using SepPak C18 solid-phase extraction cartridge (Waters, Massachusetts, USA) and then dried in Vacuum Concentrator (Labconco, Kansas City, MO, USA).The peptides were reconstituted in 0.1% formic acid (FA) prior to MS analysis.
The instrument was operated in a data-dependent acquisition under positive mode to automatically switch between full-scan MS and MS/MS.Full-scan MS spectra were acquired with a threshold ion count of 10,000, an isolation width of 2.0 Da, and dynamic exclusion set to 30.0 second.The top 20 most abundant precursor ions were subsequently fragmented in orbitrap by higher energy collision-induced dissociation (HCD) with a target value of 50,000 ions.Source conditions were as follows: electrospray voltage of 2.0 kV and normalized collision energy set at 30.For Full-scan MS and MS/MS, the m/z scan range were set to 350-1800 and 200-2000, respectively.

Protein identification
The raw data were searched via PEAKS Studio Software (8.5 Edition, Bioinformatics Solutions Inc., Waterloo, Canada).The search was performed against the Saiga Keratin database.Search settings included the following: trypsin digestion with missed cleavages allowed to 2, MS1 tolerance of 10 ppm, MS2 tolerance of 0.02 Da, identified peptides filtered to curate a dataset with a false discovery rate (FDR) less than 1% at both the peptide and protein levels, static modification of 57.02 Da on cysteines representing carbamidomethylation from iodoacetamide treatment, dynamic modification of 15.99 Da on methionine representing oxidation, dynamic modification of 42.01 Da on N-terminal of a peptide representing acetylation, and dynamic modification of 0.98 Da on Gln and Asn representing deamidation.

Supplemental table.
Table S1 Gene specific primer pairs used in RT-qPCR.

Table S2
Identification and trends of change for potential biomarkers.