| OBJECTIVESepsis is a systemic inflammatory response syndrome caused by infection and is an organ dysfunction caused by dysregulated host response to infection.The morbidity and mortality of sepsis is extremely high,which is the major severe and leading cause of death in the pediatric intensive care unit(PICU).Sepsis-associated encephalopathy(SAE)is a diffuse brain dysfunction caused by sepsis.As one of the vulnerably damaged organ,brain damage can occur at the early stage of sepsis.IL-6 is a multifunctional inflammatory medium with bidirectional effects of anti-inflammatory,regenerative-and pro-inflammatory,while AMPK,"the cell energy regulator",regulates metabolism and energy balance in cells.Recent studies show that IL-6 can activate AMPK by increasing the concentration of AMP,while AMPK regulates mitochondrial biogenesis and autophagy.Therefore,our research intends to investigate the effects of IL-6 on mitochondrial biogenesis in sepsis-induced astrocytes,and further verify whether the effects rely on AMPK signaling pathway.METHODSPart Ⅰ:The astrocytes isolated from neonatal rat cerebral codex were purified and cultured.The ASs were randomly allocated into five groups:control group,LPS+IFN-γ group,IL-6 group(LPS+IFN-γ+IL-6),siRNA group(LPS+IFN-γ+IL-6+IL-6R siRNA)and NC group(LPS+IFN-γ+IL-6+negative control).All groups were stimulated for 6 hours.Cytokines(TNF-a mRNA,IL-1βmRNA)and reactive oxygen species(ROS)analyses,detection of adenosine triphosphate(ATP),and cell viability,western blots of PGC-1α,NRF-1,TFAM and p-AMPK were performed respectively.Part Ⅱ:The astrocytes were randomly divided into six groups:control group,LPS+IFN-y group,IL-6 group(LPS+IFN-γ+IL-6),C group(LPS+IFN-γ+IL-6+Compound C),siRNA group(LPS+IFN-γ+IL-6+IL-6R siRNA)and siRNA+C group(LPS+IFN-γ+IL-6+IL-6R siRNA+Compound C).All groups were stimulated for 6 hours.Detection of adenosine triphosphate(ATP),mtDNA content and cell viability,western blots of PGC-1α,NRF-1,TFAM and p-AMPK,evaluation of the mitochondrial ultrastructure and volume density were performed respectively.RESULTSPart Ⅰ:1.Incubated with LPS and IFN-y for 6 hours,the relative expression of TNF-αmRNA,IL-1β mRNA and ROS level were significantly increased,compared with the control group.The results suggested that the establishment of septic astrocyte models was successful.2.After treatment with IL-6,the expression of PGC-la,NRF-1 and TFAM as well as the ATP level and cell viability increased significantly.However,the silence of IL-6 receptor led to sharp decline in the above indicators.3.The expression of p-AMPK increased significantly after intervention with IL-6,while the expression of p-AMPK decreased in siRNA group compared with IL-6 group.Part Ⅱ:1.Mitochondrial ultrastructure damage appeared in LPS and IFN-γ stimulated astrocytes,but the expression of NRF-1,TFAM protein were increased,the relative content of mitochondrial DNA and mitochondrial volume density increased respectively,while the level of ATP have not dropped compared with the control group.2.IL-6 treatment significantly reduced the mitochondrial structure damage of astrocytes,and mitochondrial volume density increased significantly.Moreover,the expression of PGC-1α,NRF-1 and TFAM were increased,the relative content of mitochondrial DNA and the level of ATP were increased as well.3.Compared with IL-6 group,blocking the IL-6/AMPK signaling pathway,the mitochondrial structure damage of astrocytes was exacerbated,and mitochondrial volume density decreased greatly.The expression of PGC-1α,NRF-1,TFAM and p-AMPK were decreased,while the relative content of mitochondrial DNA and ATP level dropped sharply.CONCLUSIONS1.IL-6 can enhance mitochondrial biogenesis in septic astrocytes,increase the ATP level and alleviate the mitochondrial structure damage of astrocytes,thereby plays the role of protection and compensation,and eventually improves the viability of astrocytes.2.The above protective effects of IL-6 depend on AMPK signaling pathway. |
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