The Effect And Mechanism Of 4-octyl Itaconate On Acute Lung Injury Caused By Methicillin-resistant Staphylococcus Aureus

Sepsis,as a serious infectious disease with systemic infection and comorbidity,is the main cause of death in clinical patients caused by infection.Nowadays,sepsis caused by Staphylococcus aureus has surpassed Gram-negative bacteria as the most serious public health problem.The general and improper use of antibiotics makes it more difficult to cure sepsis caused by drug-resistant bacteria.Methicillin-resistant Staphylococcus aureus（MRSA）can cause severe septic lung damage.Its cell wall component,Lipoteichoic acid（LTA）,invades the lung tissue in a persistent manner,stimulating the immune system to act so that neutrophils and macrophages are activated and augmenting the expression of inflammatory factors.At this stage,acute lung injury（ALI）caused by drug-resistant bacteria still lacks effective treatment methods,and new drugs need to be discovered to treat the injury.Itaconate is a metabolite synthesized by an enzyme encoded by immune response gene 1 and participates in the Krebs cycle.It has been proven to be a key regulator of macrophage function.Studies have found that 4-octyl itaconate（4-OI）,a membrane-permeable derivative of itaconate,reduces interleukin 6（IL-6）and inducible nitric oxide synthase（i NOS）in bone marrow macrophages stimulated by toxin and prevents hypoxia-induced cell death and myocardial ischemia-reperfusion injury.The Kelch-like ECH-related protein 1（Keap1）-Nrf2is acknowledged to be one of the most crucial defense systems for modulating oxidative and electrophilic aggression.It is a defense system designed to maintain cell homeostasis in Gram-negative bacteria-induced sepsis.Knockout of Nrf2 eliminated 4-OI-induced neuroprotection against H₂O₂.However,the role of 4-OI in MRSA-induced sepsis and its relationship with Nrf2 have not yet been clarified.In this study,we studied the effect（anti-inflammatory,antioxidant and Nrf2）of 4-OI on MRSA-induced ALI.This study used MRSA-induced ALI in mice with sepsis to determine the role of 4-OI in sepsis induced by MRSA.4-OI treatment significantly increased the survival rate of mice with MRSA sepsis pneumonia,reduced the protein content and total cell number in Bronchoalveolar lavage fluid（BALF）.The results of HE,immunohistochemistry,lung injury score and lung bacterial load test showed that 4-OI could reduce pathological damage,inhibit neutrophil infiltration and reduce the burden of lung bacteria.In addition,4-OI effectively reduced the expression of inflammatory factors in lung tissue,promoted the expression of Nrf2 and its downstream genes,and reduced the content of myeloperoxidase（MPO）and malondialdehyde（MDA）in lung tissue.This project verifies the protective effect of 4-OI on macrophages from LTA-stimulated macrophage damage.4-OI treatment did not affect the viability of RAW264.7 cells.4-OI reduced the expression of the inflammatory protein and the production of ROS caused by LTA in cells,while reducing the expression of inflammatory factors,and increasing the expression of Nrf2 and HO-1 proteins against oxidative stress.By detecting the level of Nrf2 in the cytoplasmic nucleus,an effective nuclear translocation of Nrf2 was found.The results of molecular docking indicated that 4-OI can dock to the binding pocket of Keap1 and can form stable interactions through hydrogen bonds.Both Nrf2 inhibitor（ML385）and Nrf2 knockout eliminated the protective effect of 4-OI on ALI and cell damage in mice caused by MRSA.Taken together,this study reveals:that 4-OI prevents ALI caused by MRSA bacteremia by activating the Nrf2 pathway.