Study On The Mechanism Of Human Erythrocytes Hemolysis Caused By Clostridium Perfringens ε Toxin

Clostridium perfringens is a gram-positive rod-shaped anaerobic bacteria,which is widely distributed in nature,human and animal intestines.This bacterium can produce various toxins and has strong pathogenicity,which can cause diseases such as gas gangrene,food poisoning,and necrotizing enteritis.According to the types of toxins produced,Clostridium perfringens can be divided into seven types,A-G type.C.perfringens ε toxin(ETX)is a toxin produced by type B and D Clostridium perfringens ETX can specifically bind to its target cell membrane receptor,forming pores,disrupting the integrity of the cell membrane,leading to cell edema,necrosis,and apoptosis.ETX mainly acts on organs such as the nervous system,heart,and kidneys,causing serious consequences such as acute central nervous system damage and renal failure.In addition,ETX is only second in virulence to botulinum toxin and tetanus toxin,and was once listed as a B-class bioterrorism by the Centers for Disease Control and Prevention in the United States,posing significant biosafety risks.ETX can not only damage internal organs,but also quickly and specifically cause hemolysis of human red blood cells.At present,the hemolytic mechanism of ETX has been partially revealed,including that the hemolytic effect of ETX depends on the P2X7 and P2Y13 receptors on the red blood cell membrane,as well as the presence of MALs.However,the signaling pathway downstream of the P2 receptor is still unclear,therefore,in-depth research on the hemolytic mechanism of ETX is still of great significance.This study first used metabolomics methods to analyze the changes in metabolites in red blood cells induced by ETX.The results showed that metabolic pathways related to alanine,aspartic acid,and glutamate were most affected.Based on literature and metabolomics data,this study further examined the effects of several common amino acids on the hemolysis of ETX.It was found that L-aspartic acid,L-lysine,L-histidine,L-methionine,and L-cysteine can inhibit hemolysis,suggesting that they may be related to intracellular anti oxidative stress.The excessive reactive oxygen species in cells will lead to oxidative stress,and the source of reactive oxygen species is usually organelle.Because red blood cells lack organelle,the source of their reactive oxygen species may be NADPH oxidase(reduced nicotinamide adenine dinucleotide phosphate oxidase).To verify this hypothesis,this experiment tested the effects of an antioxidant containing thiol groups,N-Acetyl-L-cysteine(NAC),and a NADPH oxidase inhibitor,Diphenyleneiodonium chloride(DPI),on the hemolysis of ETX.The results indicate that NADPH oxidase may not participate in hemolysis caused by ETX,but high concentrations of NAC can inhibit hemolysis,so the possibility of reactive oxygen species participating in hemolysis cannot be ruled out.Secondly,we selected three signaling pathways that may be regulated by reactive oxygen species with reference to literature: Ras/Raf,PI3K/Akt,and Mek/Erk signaling pathways.Cross validation of these signaling pathways using different inhibitors showed that all of them may be involved in ETX induced hemolysis.However,immunoblotting experiments were unable to successfully detect activated phosphorylation signaling pathway proteins in red blood cells,and further research is needed to address this issue.This study also screened four drugs with antioxidant properties that can effectively inhibit hemolysis caused by ETX.They are D-mannitol,vitamin C,D-penicillamine,and THPTA,respectively.These drugs may interfere with the mechanism of action of ETX by affecting the redox balance within cells.This provides valuable clues for further studying the detailed mechanism of ETX hemolysis.In summary,this study used metabolomics methods to reveal the correlation between the mechanism of ETX hemolysis and intracellular oxidative stress;Identified potential signaling pathways involved in hemolysis;Four drugs with antioxidant properties have been screened,which can effectively inhibit hemolysis caused by ETX.These findings reveal the mechanism of partial ETX hemolysis and provide new treatment or prevention strategies for injuries or diseases caused by ETX.