The Expression And Impact Of Uncoupling Protein 2 In Astrocytes Under Sepsis Conditions

OBJECTIVESepsis is an organ dysfunction caused by imbalance of body response to infection.It has a high morbidity and mortality,and is the main cause of death in pediatric intensive care unit(PICU).Sepsis-associated encephalopathy(SAE)is a diffuse brain dysfunction caused by sepsis,which can occur in the early stage of sepsis.The etiology of SAE infection often lies outside the central nervous system.Uncoupling protein 2(UCP2)is a member of the mitochondrial uncoupling protein family.It is widely distributed in brain,heart,lung and other organs and tissues,and participates in a number of pathophysiological processes.Studies have shown that UCP2 may have neuroprotective effects by reducing cell inflammation and inhibiting apoptosis in stroke and acute brain injury models.Our previous studies have confirmed that UCP2 can regulate mitochondrial membrane potential(MMP)and ATP synthesis by decoupling in septic cardiomyocyte model.Therefore,this study was designed to investigate the expression of UCP2 in astrocyte under sepsis conditions and its effect on mitochondrial function.METHODSPart 1:The astrocytes which were isolated from neonatal rat cerebral codex were purified and cultured.And then randomly allocated into four groups:Control group,LPS+IFN-?+6 hours group,LPS+IFN-?+12 hours group and LPS+IFN-?+24 hours group.Cell samples were collected after treated.IL-1? mRNA and TNF-a mRNA expression were detected by RT-PCR;reactive oxygen species(ROS)analyses,detection of adenosine triphosphate(ATP),and western blots of UCP2 were performed respectively.Part 2:ASs were randomly allocated into four groups:shRNA-NC group;shRNA-NC+LPS+IFN-y group;shRNA-UCP2 group and shRNA-UCP2+LPS+IFN-? group.After transfecting with adenovirus shRNA-UCP2 for 48 hours,the cells were treated with LPS(150 ng/mL)+ IFN-?(200 U/mL)for 24 hours.The levels of inflammatory factors,ATP,reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)were measured.The mitochondrial ultrastructure of each group was observed by transmission electron microscopy.RESULTS1.LPS with IFN-y co-stimulated ASs,increase the expression of IL-10 mRNA and TNF-? mRNA in AS at 6 hours,12 hours and 24 hours,suggesting that the sepsis model was successfully built.2.The up-regulation of UCP2 expression in astrocytes under sepsis conditions was confirmed western blotting assay.3.After silencing UCP2 expression by adenovirus shRNA,the inflammatory factor levels,ATP and ROS levels were significantly increased in the AS under sepsis conditions;mitochondrial membrane potential was significantly decreased,and mitochondrial ultrastructural damage was significantly aggravated.CONCLUSIONSUCP2 may played a protective role in astrocytes under sepsis conditions.Its specific role may be related to UCP2 stabilizing mitochondrial membrane potential,regulating ATP synthesis and reducing Mitochondrial structural damage.