Study On Pharmacodynamic Substance Basis And Mechanism Of Xuebijing Injection In Preventing Heart Dysfunction In Septic Mice

Background and Purpose:Background:Sepsis is defined as a systemic organ failure syndrome resulting from immune dysregu Lation caused by microbial infection.Current treatment regimens focus on fluid resuscitation and antibiotic therapy,and it remains a common cause of death in ICU patients worldwide,given the body’s resistance to antibiotics.Cardiac dysfunction is the key to mu Ltiple organ dysfunction syndrome caused by sepsis.However,drμgs for the treatment of sepsis-induced myocardial dysfunction have not been widely studied.Drμgs that improve cardiovascu Lar function and target pro-inflammatory cytokines show potential for the treatment of sepsis and sepsis induced myocardial dysfunction.XBJ（Xuebijing）is a Chinese herbal injection composed of five kinds of Chinese herbal medicines,including safflower,peony,angelica sinensis,ligusticum chuanxiong and salvia miltiorrhiza,which has been approved for the treatment of sepsis and septic shock since 2004 and significantly improved the survival rate of patients with severe pneumonia in a large-scale clinical trial.However,the material basis and mechanism of XBJ in the treatment of heart dysfunction in septic mice are still unclear.Our previous laboratory found that high concentration of XBJ could stimu Late the differentiation of Treg in vitro,moderately inhibit the differentiation of Th17,and improve the survival rate of septic mice（the current low concentration of XBJ clinically used leads to the failure of effective components to play a good role）.We also found that C0127（paeoniflorin,hydroxyl safflower yellow A,protocatechualdehyde,feru Lic acid）,the main component of XBJ,could produce similar prevention and treatment effects to XBJ when used in the prevention and treatment of acute GVHD in mice,showing A certain potential and possibility for the prevention and treatment of GVHD,and could be regarded as the main pharmacological component of XBJ to A certain extent.XBJ injection has the highest content of paeoniflorin and the second content of hydroxysafflor yellow A,and paeoniflorin improves the survival and heart function of rodents treated with lipopolysaccharide.HSYA improves ischemia-induced cardiac hemodynamics by stimu Lating nucleoprotein-mediated angiogenesis.Objective:Based on this,this study aims to further explore the mechanism of Xuebijing in preventing and treating heart dysfunction in sepsis mice and whether its main components C0127 and C0127-2（paeoniflorin,hydroxysafflor yellow A）have similar efficacy with XBJ.So as to improve the safety of clinical medication.Methods:1.References related to XBJ were consu Lted to find the main pharmacodynamic components of XBJ,and the compounds were combined into C0127 and C0127-2according to the concentration ratio of the compounds in Xuebijing.The inflammatory model of H9C2 cells stimu Lated by LPS（1μg/m L）was established in vitro.The expression levels of reactive oxygen species（ROS）and mitochondrial calcium ions in H9C2 cells were detected by high-content imaging system,and the effects of XBJ and C0127S on damaged cardiomyocytes were preliminarily investigated.2.Mouse CLP sepsis model was established,and the mice were randomly divided into 5 groups:sham operation group,CLP group,XBJ group,C0127 group,and C0127-2 group.The efficacy of XBJ,C0127 and C0127-2 were evaluated by the following methods.A.The survival and body temperature of the mice at 7 days were monitored.B.R-T PCR was used to detect the expression level of damage factors in heart tissues and organs of sepsis mice.C.Cardiac function of mice was detected by small animal ultrasound imaging system.D.H&E staining of heart tissue.E.Flow cytometry was used to detect the expression of neutrophils in bone marrow,peripheral blood and heart of mice.3.Transcriptome sequencing（RNA-seq）was performed on the heart tissues of mice in the Sham group,the XBJ group and the C0127s 9m L/kg group respectively,and the RNA-seq results were analyzed using the Proximal Pathway Analysis（IPA）to obtain the potential mechanism of XBJ and C0127-2 in the treatment of septic cardiomyopathy.The results were further verified by real-time reverse transcription-polymerase chain reaction（RT-PCR）,Western blotting（WB）and ELISA.Results:1.XBJ composition of related literatures,found that XBJ efficacy composition mainly includes four categories,corresponding to the highest levels of compounds are selected,according to the four kinds of compounds will determine the content of combination form C0127（paeoniflorin 2mg/m L,hydroxy safflower yellow pigment.A0.5 mg/m L,ferulic acid 0.5 mg/m L,protocatechuic aldehyde 0.5 mg/m L）,then the highest levels of the two compounds in C0127 combination form C0127-2（paeoniflorin,hydroxy safflower yellow pigment A）.In vitro results showed that XBJ and C0127S coud reduce the expression of reactive oxygen species（ROS）and calcium ion(Ca²⁺)overload in H9C2 cells,thereby reducing the inflammatory response of cardiomyocytes.2.In vivo results showed that XBJ and C0127S（C0127 and C0127-2）could prevent and cure septic mice and protect their hearts,mainly in the following aspects:alleviating fever in septic mice and improving the survival rate of septic mice;Decrease the expression of inflammatory factors in the heart tissue of sepsis mice;Reducing the cardiac ejection fraction alleviates the cardiac dysfunction of septic mice;It reduced the edema and the loss of myofibrils in the heart tissue of septic mice and improved the pathological changes of the heart of mice;Reduces the mobilization of neutrophils infiltrate into mouse heart tissue.3.When Fc≥2 and P≤0.05,Sham vs CLP,XBJ vs CLP,C0127 vs CLP,and C0127-2 vs CLP were 952 common differential genes in the four groups.These 952differential genes were analyzed by KEGG and GO.KEGG analysis of RNA-seq showed that,compared with the Sham group,infection and inflammation-related pathways were significantly up-regulated in CLP group,including NF-κB,TNF-αand cytokine receptor signaling pathways,and XBJ and C0127S could down-regulate these inflammation-related signaling pathways.GO analysis showed that the functions of genes related to infection and inflammation were in the top 10,such as the response to LPS,the response to bacteriogenic molecules,and leukocyte migration.These results were confirmed by real-time PCR,Western blot and ELISA.Conclusion:1.In this study,XBJ at a dose of 9 m L/kg inhibited the production of pro-inflammatory cytokines and effectively alleviated sepsis induced cardiac dysfunction.2.The main composition of XBJ,C0127-2（9 m L/kg）,can achieve similar therapeutic effects to XBJ,inhibit the production of pro-inflammatory cytokines,and effectively reduce sepsis induced cardiac dysfunction.It has certain potential and possibility for the prevention and treatment of sepsis and can be regarded as the main pharmacodynamic component of XBJ in the treatment of sepsis patients.