The Molecular Machanism Of PP2A/PLD1in Septic Patients With Multiple Trauma

BackgroundPost-traumatic sepsis is the sequence of infection after severe systemic inflammatory response due to severe trauma. It can cause secondary clinical syndromes involving tissue and organ damage. After a few hours of Gram-negative bacterial infection, septic shock can take place, which is characterized mainly by a sharp decline in blood pressure accompanied by fever, diarrhea, and other symptoms of disseminated coagulation. Clinically, sepsis is the most common cause of death in intensive care patients, with the mortality rates ranged from20%to80%. Multiple organ dysfunction and other complications can develop following severe sepsis, resulting in poor prognosis. Post-traumatic sepsis has become a hot topic in recent years, and many scholars have especially focused their laboratory studies on this field.. A PIRO sepsis diagnostic classification system has been proposed for international use. However, because patients differ so profoundly with respect toetiology, background (such as age and underlying disease), and other diverse and complex factors, the molecular mechanisms underlying sepsis are not yet fully understood. Currently, the proposed mechanisms of sepsis involve complex network effects of systemic inflammation, genetic polymorphisms, immune dysfunction, coagulation dysfunction, tissue damage, and infection by many different pathogenic microorganisms and their toxins and other abnormal reactions. Studies have shown that multi-organ pathophysiological changes are closely related to each other.Protein phosphatase-2A (PP2A) is one of the body’s major eukaryotic serine/threonine protein phosphatase. Many different genes encoding the structure’s subunits, which include a variety of different PP2A holoenzymes. These holoenzymes have been found to be controlled by the cell cycle. A variety of biological events are involved in the cell cycle, such as DNA replication, signal transduction, cell differentiation, and malignant transformation which have been studied in biological models such as yeast, fruit flies, and mice. PP2A plays a morphological and developmental role in the signal transduction cascade, where it interacts with other phosphorylases and kinases. It contributes to the downstream regulation of macromolecules that regulate signal transduction. It is also involved in reversible phosphorylation, which can activate protein phosphorylation-dephosphorylation. This suggests that PP2A negatively regulates signal transduction. Some scholars believe that the foreign PP-2A is a critical endogenous regulator of inflammatory cell signaling and endothelial cell function in sepsis-induced obstacles. Molecular target on PP-2A may play an important role in the treatment of sepsis and may have practical pharmacological value as a toxemia target.Phospholipase D (phospholipase D, PLD) is commonly expressed in bacteria, fungi, and mammals. In pathogenic microorganisms, PLD can affect virulence, meiosis, sporulation, and parts of the life cycle. In mammalian cells, PLD functions primarily in membrane transport and in the regulation of mitosis and the actin cytoskeleton. It also plays certain role in cell signal transduction. In the process of pathogenic infection of host cells, pathogens and host cells are both activated, and a PLD cascade takes place. The pathogen can regulate itself using PLD actin filament polymerization and rearrangement of the host cell,causing localized accumulation of actin filaments and phagocytosis of the host cell. In-depth studies of PLD activation of the regulation of infection are important for a thorough understanding of the molecular pathogenic mechanisms that take place in infected host cells. More studies should be performed on the role of PLD1molecular mechanisms of sepsis.The JNK signaling pathway is one of the most important pathways. MAPKs, which are located in the cytoplasm, contain double-phosphorylatted functional areas (composed of three amino acids Thr, pro and Tyr composition) and a cjunN end zone. This activates serine63and serine73which are phosphorylated residues. JNK activation takes place through phosphorylation of the terminal amino acid residues of [implementation]. Once activated, the JNK in the cytoplasm moves to the nucleus. The JNK signaling pathway plays an important role in many physiological and pathological processes involved in the cell cycle, including protein production, apoptosis, and cell stress responses. Physical stress, chemically induced changes in the extracellular environmental changes, and proinflammatory cytokines regulate this pathway. In severe infections, especially those involving sepsis, relevant information can be collected by inhibiting the JNK signaling pathway. This reduces the body’s ability to remove harmful substances, causing aggravated damage.Studies have shown that, in sepsis, molecular mechanisms involving PP2A and PLD1may inhibit the JNK signaling pathway, thereby inhibites the expression of cytokines, worsening infection. However, many of these studies were performed on cell lines and animal models. Until now, no human septic infections have been studied under controlled conditions, and thus the reliability and significance of these studies’ conclusions should be questioned. The molecular mechanisms of action of PP2A and PLD1have not yet been evaluated as well. Purpose:This study was performed on samples of septic blood from patients who had undergone multiple trauma. The samples were examined for the expression and activity of blood protein PP2A and PL. Results showed that PP2A/PLD1is associated with the JNK signaling pathway. Our resultsprovide a new way to further understand the molecular mechanisms of sepsis.Method:1. Twenty-four cases of sepsis attributable to multiple trauma were compared to twenty-two non-septic multiple-trauma patients. Samples were collected at the onset of sepsis and on day1and7. In the former group, fifteen of those cases were evaluated for the expression of c-jun/JNK mRNA and PP2A. Intensive care unit length of stay (LOS, sequential organ failure assessment (SOFA), and multiple organ dysfunction (MOD) scores were assessed for each participant, and correlation analysis was performed. Receiver-operating-characteristic (ROC) curve analysis of PP2A was used to evaluate the diagnosis of sepsis.2. Blood levels of PP2A, PLD1, and JNK protein in septic patients were compared to those of normal controls to determine whether there was any correlation between these levels and septic status.3. A THP-1cell line was subjected to lipopolysaccharide (LPS) stimulation to simulate a septic environment. Western blot analysis and immunoprecipitation (co-immunoprecipitation) technology were used to detect PP2A, while the expression of PLD1and JNK were determined based on differences between JNK activity.4. LPS-stimulated THP-1cells were exposed to siRNA so as to block PP2A expression. Then the JNK signaling pathway, JNK activity, and PLD1expression were detected, to further illustrate the feasibility of these three factors as mechanisms by which PP2A might affect sepsis. Results:1More expressions of PP2A mRNA and protein were observed in septic patients than in either non-septic patients or healthy controls. There was also significantly higher c-jun/JNK mRNA and protein expression when levels of PP2A were low. On day1and7after sepsis,PP2A expression was found to be negatively correlated to LOS, SOFA, and MOD scores. ROC curve analysis showed that the sensitivity of PP2A as a means of distinguishing septic from non-septic patients was87.5%.2Detection of PP2A, PLD1, JNK protein expression in septic patients and healthy controls. In the healthy control group, level of PLD1and JNK were normal, but there was little or no expression of PP2A. In the septic group, there was considerable expression of PP2A but little or no expression of PLD1or JNK. These results indicate that the levels of these three substances may be relevant to sepsis.3In THP-1cell line, co-immunoprecipitation was used to detect interactions among PP2A, PLD1, and JNK, and some relevant interactions were found. The level of JNK activity in the infected cell lines decreased after LPS treatment. This also suggests that these three factors may play a role in the pathogenesis of sepsis.4siRNA was used to block PP2A expression in a LPS-infected THP-1cell line. This caused JNK protein expression and JNK activity to return to normal levels.Conclusion:1. PP2A, which is a negative regulator of the c-Jun N-terminal kinase (JNK) signaling pathway, may be a biomarker of sepsis.2. In sepsis, PP2A, PLD1, and JNK were found to interact with each other.3. The JNK signaling pathway can be inhibited by the protein PP2A. PLD1isinvolved in this process. Among these three factors, the protein PP2A seems to play the leading role.