Study On The Mechanism Of Autophagy Activation Of Phagocytes In Early Immune Disorder After Severe Trauma

Severe trauma kills more than 5 million patients every year,accounting for 10% of the world’s total deaths that year and consuming 14% of the world’s medical resources.It is the leading cause of death for people under 45 years of age,which imposes a huge burden on human health and social development.Although severe trauma accounts for only 3-5% of all injuries,it has many complications,high disability and mortality rate.There are two peaks of death after severe trauma,with fatal organ damage from the initial devastating injury and uncontrolled blood loss being the main causes of the first peak.The second peak of death in patients is usually caused by sepsis or multiple organ failure in the middle to late post-injury period.As medical care continues to improve,more and more patients are surviving early fatal injuries and receiving further treatment in surgical intensive care units,but those who survive these injuries still often end up dying from fatal complications caused by immune disorders.Although researchers all over the world have carried out extensive and in-depth exploration on this problem,the results are not satisfactory at present.Studies have shown that the initial traumatic injury will destroy the macroscopic barrier(skin mucosa,etc.)and microscopic barrier(cell membrane,etc.)of the body,release a variety of dangerous molecules,and lead to the rapid activation of the innate immune system,especially the activation of various phagocytes,such as neutrophils and monocytes,which are an important part of the innate immune system.These cells release a large number of inflammatory mediators,enhance phagocytosis activity and antigen presenting function,bridge the innate immune system and adaptive immune system,further amplify the proinflammatory or anti-inflammatory response after injury,and ultimately induce and promote the occurrence of immune disorders after injury.Phagocytes play an important role in the early immune inflammatory response after trauma.Autophagy is a process that relies on lysosome pathway to degrade cytoplasmic proteins and organelles.It is a self-protection mechanism formed in the long-term evolution of eukaryotic cells.In recent years,many studies have shown that autophagy is related to ontogeny,oxidative damage protection and malignant proliferation of tumor cells.In recent years,as the potential function of autophagy mechanism in immune cells has been gradually clarified,the effect of autophagy on immune function has attracted increasing attention.Studies have shown that autophagy has extensive and profound effects on the immune system.From the basic metabolic function in cells to the differentiation,maturation and functional regulation of various immune cells,Autophagy has gradually been regarded as the core regulatory point controlling immune homeostasis.The efficacy of immune intervention therapy targeting autophagy has also been preliminarily confirmed.Previous studies have shown that autophagy levels of various tissue cells in the body have undergone significant changes after trauma.However,changes in autophagy activation of phagocytes after injury are rarely reported,and whether autophagy of phagocytes is involved in immune disorders after severe trauma is also poorly understood.Therefore,in order to clarify the above problems,the present study observed the characteristics of early immune disorder in patients with severe trauma and the changes in autophagy level of phagocytes,and initially discussed the mechanism of the changes in autophagy of phagocytes in early immune disorder after severe trauma.This study is mainly divided into three parts: the first part observed the clinical features of early post-traumatic immune disorders in patients with severe trauma;the second part analyzed the gene regulatory network of the transcriptome of peripheral white blood cells in patients with severe trauma;the third part discussed the mechanism of autophagy activation of phagocytes in immune disorders after severe trauma.Part I: Analysis of clinical characteristics of early immune disorders in patients with severe traumaObjective: To investigate the clinical features of patients with early immune disorder after severe trauma,as well as the incidence and risk factors of sepsis in this population.Methods: Clinical data of trauma surgery patients admitted to our hospital from July 2019 to December 2021 were retrospectively analyzed to study the occurrence of early immune disorders,the incidence and risk factors of sepsis.Results: A total of 412 trauma patients who met the inclusion criteria were divided into three groups according to the post-injury severity score(ISS),Including 81 patients in the Non-severe trauma group(ISS≤16),203 patients in the severe trauma group(16<ISS≤25)and 128 patients(ISS>25)in the extremely trauma group.The results showed that the higher the severity of trauma in trauma patients,the higher the incidence of immune disorders(P<0.05).The incidence of early immune disorder was as high as 72.8% in all patients with severe trauma(ISS>16).The incidence of sepsis was 31.5% in severe trauma patients with early immune disorder after injury,peripheral blood TNF-α level,emergency surgical treatment,and prolonged ventilator use were independent risk factors for sepsis in these patients.Conclusion: The higher the severity of trauma,the higher the incidence of post-injury immune disorders in trauma patients,and the incidence of sepsis in severe trauma patients accompanied by early post-injury immune disorders was also significantly increased.Peripheral blood TNF-α level,emergency surgical treatment and long-term use of ventilator are independent risk factors for the high incidence of post-injury sepsis.Timely intervention will help to reduce the incidence of sepsis and improve the prognosis of these patients.Part II: Analysis of gene regulatory network of leukocyte after severe traumaObjective: To analyze the core biological processes and key signaling pathways and genes of peripheral blood leukocytes in patients with severe trauma,and to seek a novel therapeutic target to intervene in immune disorders after severe trauma.Methods: In this study,transcriptome information of peripheral blood immune cells in the mass trauma cohort was analyzed to screen differentially expressed genes.Weighted Gene Co-expression Network Analysis(WGCNA)was adopted to construct co-expression modules and identify key modules.The core biological processes of immune cells after severe trauma were determined by enrichment analysis of the genes contained in the core modules.Results: The light green module is the key module of early immune disorder after severe trauma,and JMY gene was identified as the core gene of this module.The results of gene annotation analysis and Kyoto gene analysis suggest that autophagy activation is the core cell biological process of peripheral blood immune cells in the early stage of severe trauma.Conclusion: The results of enrichment analysis and core gene identification showed that autophagy activation is the core cellular biological process of peripheral blood immune cells in the early stage of severe trauma.Autophagy activation may be closely related to the occurrence of immune disorders after severe trauma,and further studies are needed.Part III: Study on the mechanism of autophagy activation of phagocytes in immune disorders after severe traumaObjective: To observe the changes of autophagy level of immune cells in peripheral blood of patients in the early stage after severe trauma,and to explore the role of autophagy activation of phagocytes in immune disorders after severe trauma for the aim of providing new insights into the pathogenesis and intervention strategies of immune disorders after severe trauma.Methods: Flow cytometry and enzyme linked immunosorbent assay(ELISA)were used to observe the changes of autophagy in peripheral blood immune cells of 48 patients with severe trauma and 21 healthy volunteers.Single cell sequencing was used to analyze the transcriptional profiles of immune cells in peripheral blood of patients with severe trauma and healthy volunteers.After single-cell sequencing data were identified by cell type labeling and cluster labeling,the changes in autophagy levels of immune cells were analyzed through the expression changes of 604 autophagy related genes.Subsequently,the effect of autophagy activation on cell function was investigated by enrichment analysis.Results: Flow cytometry and ELISA analysis indicated significant autophagy activation in monocytes and neutrophils.Single-cell sequencing revealed a significant reduction in adaptive immune cell components and a significant increase in phagocytes(monocytes and neutrophils)in patients with severe trauma compared with healthy controls.Active autophagy was detected in monocytes and neutrophils by monitoring the dynamic transcription characteristics of autophagy in immune cells using autophagy related genes.Further functional gene enrichment analysis showed that exogenous antigen uptake,processing and presentation,MHC Class II protein complex assembly,peptide antigen assembly and lymphocyte-mediated immune function of autophagy activated monocytes were significantly up-regulated during the early stage of severe trauma.Antigen processing and peptide antigen presentation,endogenous antigen presentation,antiviral replication,protein deacetylation,and type I interferon signaling pathways of autophagy activated neutrophiles were significantly up-regulated during the early stage of severe trauma.Conclusion: Autophagy activation of phagocytes such as monocytes and neutrophils play a key role in immune disorders after severe trauma.Autophagy activation may participate in excessive inflammatory response in the early stage of severe trauma by enhancing the multiple immune functions of phagocytes.Early intervention of autophagy activity of phagocytes may be a promising strategy for regulating post-injury immune disorders.