Investigation On The Molecular Mechanism Of Artesunate’s Protective Effect On The Sepsis Induced Immunosuppression Mice

Objective:Sepsis(3.0)is a life-threatening organ dysfunction syndrome caused by host reaction disorders due to infection or infectious factors.It is a common complication of clinical trauma/burns and infection,and a leading cause of death in critically ill patients.Increasing evidences suggest that sepsis development involves two distinct stages,an initial cytokine storm(hyper-inflammation)and subsequent immunosuppression(hypo-inflammation),which exist concomitantly and dominate in different stages.Although the timeline of immunological alterations in sepsis is not well understood,the uncontrolled cytokine storm is responsible for deaths that occur within the first few days,whereas immunosuppression predominantly causes mortality during the later stages.Immunosuppression caused by repeated infection is the main cause of late sepsis death,in which the innate and acquired immune system play an important role.For a long time,studies on sepsis have focused on cytokine storms and their associated multiple organ dysfunction syndrome(MODS).However,no effective drugs emerged or some were withdrawn from the market due to excessive adverse reactions.In recent years,clinical reports have shown that although patients survived the early cytokine storm stage,many died from the later infection and MODS.Therefore,the sepsis immunosuppression stage become the new focus of the sepsis research.Based on the pathophysiology of sepsis,we consider that the ideal treatment for sepsis are supposed to be an immunomodulator that is not restricted by the "time window" of treatment,which plays an anti-inflammatory role in the cytokine storm stage,while it reverses the immunosuppression stage as an immune enhancer.Our research group has been committed to the study of the pathogenesis and therapeutic measures of sepsis and the pharmacological action and mechanism of artesunate(AS)for a long time.In addition,our previous studies found that AS has a good anti-inflammatory effect.AS protects various mouse models of sepsis and significantly reduces serum levels of the inflammatory cytokines,such as TNF-α and IL-6 and et al.Furthermore,AS showed a significant therapeutic effect on LPS-tolerant mice challenged with PA.Combined with other reports,we speculate that artesunate might be a bidirectional immunomodulator.To investigate the effect of AS on sepsis-induced immunosuppression mice,the cecal ligation and puncture(CLP)immunosuppression mice and second hit immunosuppression mice were employed.The level of pro-inflammation cytokines in blood,spleen and lungs were measured in immunosuppression mice.The results showed AS increased the low level of pro-inflammation cytokines in immunosuppression mice.Besides,the seven-day survival rate,bacterial clearance and the level of pro-inflammation cytokines in blood,spleen and lungs were measured in the second hit mice.The results showed AS increased the above three indicators in the second hit mice.As is known,monocyte/macrophage system is the first line of defense against pathogenic microorganisms in the body,and play a crucial role in the identification of pathogenic microorganisms,clearance of pathogenic microorganisms,and antigen presentation.The body’s ability to clear bacteria will be impaired when the function of monocyte/macrophage system is inhibited.In the immunosuppression stage of sepsis,the function of monocyte/macrophage system reduced.Thus,restoring its function to normal level is of great significance for improving the survival rate of patients with sepsis.To investigate the effect of AS in LPS tolerance cell model,the bacterial clearance and the level of pro-inflammtion cytokines were measured in LPS tolerance monocyte/macrophage.In addition,the effect of autophagy activators and autophagy inhibitors on LPS tolerance cells treated by AS was investigated,and the effects of AS on autophagy activities in LPS tolerance cells were also detected.The results showed that AS increased the bacterial clearance and the level of pro-inflammation cytokines in LPS tolerance cells,in which autophagy might involve in.To explore the potential signal molecules or targets involved in the role of AS,the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)was to predict the candidate of interaction molecules of AS.Vitamin D receptor(VDR)was screened out by Quantitative real-time PCR(q PCR)and Western Blot(WB).The effect of knocking-down and overexpressing Vdr were investigated in LPS tolerance cells treated by AS.As well as,the interaction between AS an VDR were measured in vitro.The results showed VDR was closely related to the formation of LPS tolerance and the role of AS.VDR is a member of the steroid hormone/thyroid hormone receptor superfamily.As a nuclear transcription factor,VDR has more than 1,000 target genes,which are involved in calcium and phosphorus metabolism,immunity,inflammation and other genes.Therefore,VDR mediated effects are relatively extensive and complex.Atg16l1,a key gene in autophagy formation,is a target gene of VDR,and VDR regulate the autophagy process by regulating the transcription of Atg16l1.Cathelicidins,a direct target gene of VDR,is closely related to bacterial clearance and play a role in bacterial clearance by regulating autophagy.It has been reported that VDR interacts with the nuclear transcription factor NF-κB p65 to mediate its regulation of the downstream inflammatory factors.Nuclear transcription factor NF-κB is widely involved in inflammatory response and immune regulation,which is the hub of complex biological signal transduction network.It transcribes and regulates inflammatory cytokines such as TNF-α and IL-6.Therefore,this study further studied the regulation of VDR on its downstream target gene and its interaction with NF-κB p65.Firstly,WB,immunofluorescence,Chromatin Immunoprecipitation(Ch IP)and enzyme-linked immunosorbent assay(ELISA)were used to investigate the nuclear translocation of VDR,the transcriptional regulation of Atg16l1 and the regulation of m CRAMP.Then,the interaction between VDR and NF-κB p65 was examined by WB,Immunoprecipitation(Co-IP),and ELISA.The results showed that AS inhibited the transcriptional regulation of VDR to its target gene Atg16l1 negatively and inhibited the regulation of VDR on m CRAMP.In addition,AS inhibited the interaction between VDR and NF-κB p65 to promote nuclear translocation of NF-κB p65 and its regulation of downstream inflammation-related signaling pathways.This study was to investigate the therapeutic effect of AS on sepsis induced immunosuppression and its mechanism.The results show that AS exerts its“pro-inflammatory” effect in the immunosuppression stage in contrast to its“anti-inflammatory” effect in the cytokines storm stage.Our present results showed a schematic of the mechanism of AS’s effect through two ways: AS interacts with VDR,to preventing its nuclear translocation of VDR,which decreases its negative regulation of autophagy related target genes,such as Atg16l1,and increases autophagy.Additionally,AS interaction with VDR prevents the VDR-NF-κB p65 interaction,promoting the nuclear translocation of NF-κB p65,leading to increased transcription of its target genes,such as pro-inflammatory cytokine.These two activities increase bacterial clearance and pro-inflammatory cytokine releases from macrophages.The preliminary results of our group showed that AS exerted an anti-inflammatory effect on the cytokine storm stage of sepsis.Therefore,we considered that an immunoregulator might be an ideal drug to treat sepsis caused by bacterial or virus without limitation of treatment time.Based on our previous and current results,AS might be a bidirectional immunoregulator for both the hyper-and hypo-inflammatory stages of sepsis.