Plasma Metabolomics And Gender Differences Of Strychnine In Acute Poisoning Rats

Objective:1.The acute poisoning model of strychnine in rats was established to search the biomarkers in plasma of strychnine poisoning by metabonomics techniques and analyze the toxicological mechanism of strychnine.2.The acute poisoning model of strychnine in healthy and castrated rats were established to compare the metabolic differences of strychnine in male and female rats.Metabolomics techniques were used to investigate the differential metabolism of strychnine in male and female rats.Methods:1.Twenty male and twenty female SD rats were randomly divided into experimental group and blank control group.The experimental group was orally administered with 1/4LD₅₀strychnide aqueous solution,while the control group was orally administered with equal dose of water.Blood was collected from orbital vein 30min,120 min and 720 min after administration and plasma was obtained by centrifugation.The changes of plasma metabolites were detected by HPLC-TOF-MS.Fold change analysis,principal component analysis,partial least squares discriminant analysis,orthogonal partial least squares discriminant analysis and other statistical methods were used to analyze the data.Screening the differential metabolites according to the analysis parameters,and looking for meaningful biomarkers.KEGG database was used for bioinformatics analysis to study strychnine toxin-related metabolic pathways and toxicity mechanism.2.6 male and 6 female SD rats were removed from testicles and ovaries as castrated rats group.Another 6 male and 6 female SD rats were selected as healthy rats group.1/4LD₅₀strychnine nitrate solution was orally administered,and blood was collected from orbital veins at 5 min,10 min,15 min,30 min,45 min,1 h,1.5 h,2 h,4 h,6 h,8 h,12 h,24 h,48 h after administration,the blood is then centrifuged to obtain plasma.Plasma concentrations of Strychnine and Strychnine n-oxide were determined by HPLC-MS/MS.Statistical software IBM SPSS Statistics 26 was used to analyze data differences between male and female rats.DAS3.2.8 metabolic kinetics software was used to establish metabolic kinetics model and the metabolic kinetics model parameters of male and female rats were compared.Comparing and analyzing the metabolomics data of healthy male and female rats to analyze the difference of plasma metabolism profile of male and female rats exposed to strychnine,and to analyze the cause and mechanism of the differential metabolism of strychnine in male and female rats.Results:1.24 differential metabolites that may be related to strychnine poisoning were screened from the plasma of female rats.26 differential metabolites which may be related to strychnine poisoning were screened from the plasma of male rats.They are mainly amino acids and their derivatives.A total of 12 possible metabolic pathways related to strychnine metabolism were obtained by KEGG analysis,including Necroptosis,Sphingolipid metabolism,Sphingolipid signaling pathway,Glycine,serine and threonine metabolism,ABC transporters,Phenylalanine metabolism,Arginine and proline metabolism,Linoleic acid metabolism,Biosynthesis of amino acids,Aminoacyl-t RNA biosynthesis and Pyrimidine metabolism.2.The plasma concentration of Strychnine was different in both healthy and castrated rats,but the difference of strychnine N-oxide was not statistically significant.In both healthy and castrated rats,the peak concentration and absorption rate of strychnine in female rats were higher than those in male rats,the metabolic rate was lower than that of male rats.Compared with healthy rats,the absorption rate and metabolism rate of strychnine in castrated rats were significantly slower,and the peak concentration in plasma decreased significantly.Male rats decreased to 38.787%of normal rats and female rats decreased to 38.524%.A total of 22 metabolites that may be related to the differential metabolism of strychnine in male and female rats were screened by metabolomics analysis,and 9 metabolic pathways that may cause the differential metabolism of strychnine in male and female rats were obtained,including ABC transporters,Pyrimidine metabolism,Linoleic acid metabolism,Steroid biosynthesis,Ferroptosis,etc.Conclusion:1.The metabolites associated with acute strychnine poisoning in rat plasma were screened based on HPLC-TOF-MS metabolomics technique.1-Myristoyl-sn-glycero-3-phosphocholine and norethindrone acetate should be considered as biomarkers for strychnine poisoning in female rats,and 1-Myristoyl-sn-glycero-3-phosphocholine and D-Tagatose should be considered as biomarkers for strychnine poisoning in male rats.Sphingolipid metabolism,pyrimidine metabolism and metabolism of amino acids are affected by strychnine,which leads to metabolic disorders and interferes with normal physiological functions of organs and systems.2.The metabolism of strychnine was different in male and female rats,and gender hormones may be involved in the metabolism of strychnine,but it may not be the main factor causing the differential metabolism of strychnine.Metabolomics analysis showed that there were significant differences in plasma metabolic profiles of male and female rats exposed to strychnine.Differential expression of ABC transporters,pyrimidine metabolism,linoleic acid metabolism,steroid biosynthesis,ferroptosis and other metabolic pathways may be responsible for the differential metabolism of strychnine.