Pharmacodynamics And Mechanical Study Of Xuebijing Injection In The Treatment Of Sepsis Based On Metabolomics And Bioinformatics

Aims To search for the endogenous metabolic biomarkers related to the occurrence and development of sepsis,and to construct the metabolic regulatory network of the Xuebijing injection,and to explore the "multiple components-multiple targets-multiple pathways" mechanism of the Xuebijing injection in the treatment of sepsis from the perspective of metabolism and protein targets.Methods 1.The chemical components of the Xuebijing injection(XBJ)were analyzed through in vivo and vitro research based on ultra high performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high resolution accurate mass spectrometry(UHPLC-Q-Orbitrap HRMS)technology,which the complex components in vitro and the components migrating to blood from the XBJ were identified.2.The classic rat models of sepsis were established to explore the anti-sepsis effects of the XBJ.The behavior and 72 h survival rate of sepsis rat models were observed.And the expression of inflammatory factors(IL-1?,TNF-?,IL-6)were detected by ELISA method.Then the anti-sepsis effects of the XBJ in sepsis rat models were detected by H&E staining.3.The untargeted metabolomic method was established to search the endogenous biomarkers of sepsis,and the metabolic regulatory network of the XBJ was constructed based on the endogenous biomarkers of sepsis.Finally,the therapeutic mechanism of the XBJ was explored from the perspective of metabolism.4.Based on bioinformatic method,the multiple databases and literatures mining were used to forecast its potential targets of components in the XBJ.And the targets of metabolites,which were associated with sepsis and regulated by the XBJ,were also searched through the same method and compared with the predicted component targets.Then the key target proteins of the XBJ were found,which were used to regulate the metabolic disorder in sepsis.Finally,the "multiple components-multiple targets-multiple pathways" mechanism of the XBJ in the treatment of sepsis was further revealed.Results 1.The method was successfully established to detect the in vitro components of the XBJ based on UHPLC-Q-Orbitrap HRMS technology,and the 162 ingredients of the XBJ were identified,including monoterpene glycosides,phenanthrenequinones,lactonates,flavonoids,phenolic acids and other compounds.According to the different kinds of compounds,the fragmentation patterns in mass spectrum were summarized.Meanwhile,the method of in vivo components detection was constructed,and the 21 components migrating to blood from the XBJ were obtained,including hydroxysafflor yellow A,oxypaeoniflorin,ligustilide,butylidenephthalide,salvianolic acid A,ferulic acid,etc.2.The rat models of sepsis were successfully established by cecal ligation and puncture.The XBJ could improve the behavior and survival rate of sepsis rats.The ELISA results showed that the levels of inflammatory cytokines IL-1?,TNF-? and IL-6 in plasma of the model group were significantly higher than those of the sham group(P<0.05),while the XBJ could significantly inhibit the expression of inflammatory cytokines(P<0.05).The pathological results of H&E staining showed that the liver and lung tissue damage occurred in the model group at 12 h and 24 h,and the XBJ could improve the tissue damage in sepsis rats.3.The untarget metabolomic method associated with the XBJ in the treatment of sepsis was successfully constructed by the UHPLC-Q-Orbitrap HRMS technology.A total of 41 metabolites in plasma related to sepsis were identified,and the 32 metabolites of them could be adjusted by the XBJ.The XBJ play a role in the treatment of sepsis mainly through five major metabolic pathways,including taurine and hypotaurine metabolism,valine,leucine and isoleucine biosynthesis,nicotinate and nicotinamide metabolism,alanine,aspartate and glutamate metabolism,sphingolipid metabolism.Subsequently,Based on the establishing the metabolomic method,the 26 biomarkers related to lung injury caused by sepsis were found and the 15 metabolites of them could be adjusted by the XBJ.It found that the XBJ could improve the lung injury caused by sepsis through regulating D-glutamine and D-glutamate metabolism,alanine,aspartate and glutamate metabolism and sphingolipid metabolism.4.Based on bioinformatic methods,It found that the XBJ could target on 29 multi-proteins to regulate the metabolic disorder in sepsis,such as lipopolysaccharide-binding protein(LBP),adenosine methionine transferase(MAT1A),phospholipase A2(PLA2G1B)and et al.Through these protein targets,the 27 effective substance of the XBJ in the treatment of sepsis were found,including hydroxysafflor yellow A,albiflorin,danshensu,kaempferol and et al.And the "multiple components-multiple targets-multiple pathways" mechanism of the XBJ in the treatment of sepsis was revealed.Conclusions Multiple components in the XBJ could synergistically regulate the metabolic disorder during the occurrence of sepsis,mainly involving amino acid metabolism,lipid metabolism,energy metabolism,etc.The "multiple components-multiple targets-multiple pathways" mechanism of the XBJ in the treatment of sepsis was preliminarily explained from the perspective of metabolism and protein targets,providing scientific basis for clinical rational use of the XBJ and a new idea for the research on the mechanism of traditional Chinese medicine compound preparation.