CLCF1/NF-?B signaling pathway regulates macrophage efferocytosis to ameliorate neurological damage and cognitive dysfunction following CO poisoning

Severe carbon monoxide (CO) poisoning can induce structural damage to the nervous system, leading to long-term cognitive dysfunction in patients. The proper termination of the inflammatory response caused by neuronal cell damage is a prerequisite for tissue repair. Macrophages can clear cell corpses/fragments caused by brain injury through efferocytosis and produce cytokines to coordinate immune responses, thereby promoting neuronal repair and regeneration. However, in the microenvironment of the nervous system affected by CO poisoning, the function of macrophages is inhibited. Our study found that CLCF1 can regulate the secretion of cytokines such as TNF-a, IL-1ß, and IL-10 through the NF-?B signaling pathway, thereby affecting neuronal cell repair and regeneration. Overall design: In a series of experiments, we first conducted knockdown or overexpression experiments on the CLCF1 gene, selecting two effective targets (Target a-126 and Target b-266) from three shRNAs designed for CLCF1. CLCF1 cDNA sequence was inserted into a lentiviral vector, transformed into DH5a E. coli, and plasmids were extracted using the EndoFree Plasmid Maxi Kit. Subsequently, a mixture of psPAX2, pMD2.G, and the transfer vector in OPTIMEM was transfected into 293T cells using PEI MAX. After 48 hours, lentiviruses were harvested by centrifuging at 800g for 10 minutes and then used to treat BMDM for 48 hours to assess the efficiency of knockdown or overexpression. Next, an acute CO poisoning model in rats was prepared using the method described by Li et al., where rats inhaled 1000 ppm of CO gas for 40 minutes followed by 3000 ppm for 20 minutes. The successful model was indicated by confusion and high HbCO concentration (>60%). Post-modelling, blood was drawn from the femoral artery and HbCO concentration was measured using a blood gas analyzer. In the lipid injection experiment in rats, chloroquine phosphate liposomes and PBS liposomes (control) were equilibrated to 25°C, and 0.5 mL of liposomes were drawn into a 3 mL syringe, adjusting the dosage according to body weight, and injected through the tail vein or retro-orbital sinus after anesthesia with isoflurane. Finally, in macrophage reconstitution, two days post chloroquine phosphate liposome treatment, rats were reconstituted with BMDM. Cells were detached using trypsin-EDTA, washed with PBS, resuspended, and then slowly injected into the rats through the tail vein or retro-orbital sinus.