The Mechanism Of MiRNA-155 Involved In Sepsis Associated Encephalopathy (SAE) By Regulating UCP2 Expression In Microglia

Sepsis associated encephalopathy(SAE),a neurological complication that occurs in patients with sepsis,which is explained by various theories.Many studies believe that mitochondrial dysfunction is the main reason for the nervous system disorders or the development of SAE.In recent years,a large number of studies have confirmed that miRNA can be used as an effective biomarker for sepsis and can help distinguish the various stages of sepsis.Multiple miRNAs have different expressions and play different roles in sepsis related organ dysfunction.As one of the earliest miRNAs been extensively studied,miRNA-155(miR-155)is related to many autoimmune-related diseases,tumors and sepsis.Mi R-155 was associated with lipopolysaccharide(LPS)-induced microglial activation and promoted the inflammatory response of microglia.Significant increase of miR-155 level in mitochondria was also observed in the inflammatory response in craniocerebral injury,which may be related to the impairment of mitochondrial function.Mitochondrial decoupling protein 2(UCP2)is highly expressed in the brain and has been shown to have a protective effect on mitochondrial function in septic astrocyte models and may play a protective role in sepsis associated encephalopathy.Whether miR-155 is involved in the occurrence of SAE by affecting the expression of mitochondrial UCP2 in microglia remains unclear.In this study,cell experiments in vitro were conducted to explore whether miR-155 is involved in the occurrence of sepsis associated encephalopathy and to investigate its underlying mechanisms.Objective:To clarify the association between miRNA-155 and sepsis associated encephalopathy(SAE),and to explore the possible pathway of miRNA-155 involved in the regulation of SAE.Methods:step1: LPS(1ug/ml)stimulated mice microglia cells to construct septic encephalopathy cell model.The m RNA expression levels of two inflamatory factor,IL-1β and TNF-α were detected by RT-PCR at 6h,12 h,24h and 48 h,respectively,to determine the successful modeling of sepsis related encephalopathy model,and to determine the optimal modeling time(48h).Step 2: RT-PCR was used to measure the relative expression of miRNA-155 in microglia cells after successful modeling.Step 3: construct miR-155 inhibitor,and determine the expression of miR-155 in the inhibitor group,miR-155 inhibitor NC group and blank control group,and verify the transfection efficiency,so as to prepare for the next knockdown experiment of miR-155.Step 4: Themicroglia cells were grouped into the following groups: a.blank control group: mouse microglia cell routinely cultured for 48h;b.LPS stimulation group : mouse microglia+LPS(1ug/ml)cultured for 48h;c.miR-155 negative knockdown group: inhibitor NC mouse microglia+LPS(1ug/ml)cultured for 48h;d.miR-155 inhibitor group: miR-155-3p inhibitor transfected mouse microglia+LPS(1ug/ml)cultured for 48 h.m RNA expression levels of IL-1β,TNF-α,UCP2 in each group were detected by RT-PCR,and protein expression levels of UCP2 were determined by Western Blot.Results:After the microglia cells were stimulated by LPS(1 μg/m L)for 48 h,the m RNA expression levels of the two inflammatory factors were significantly increased,and the differences were statistically significant,suggesting that the SAE microglia inflammatory model was successful constructed.After the successful modeling,the expression level of miR-155 in microglia cells was detected,and the expression level was significantly increased compared with the blank control group,suggesting that miR-155 may be a miRNA associated with sepsis related encephalopathy.After knockdown of miR-155,the levels of inflammatory factors in the miR-155 negative knockdown group were not significantly changed compared with the LPS-stimulated group,while the m RNA expressions of two inflammatory factors in the miR-155 knockdown group were down-regulated compared with the LPS-stimulated group,with statistically significant differences.It is suggested that knockdown of miR-155 can reduce the inflammatory response of microglia after LPS stimulation.UCP2 m RNA expression and protein expression level of microglia in each group were further determined.UCP2 expression level of LPS stimulation group was significantly lower than that of blank control group.UCP2 m RNA expression in miR-155 knockdown group was significantly higher than that in LPS stimulation group.The protein expression level of UCP2 tended to be the same as its m RNA expression level.These results suggest that miR-155 may inhibit the expression of UCP2,thereby reducing the protective effect of UCP2 on microgliaConclusion:1、LPS stimulation of microglia can induce the increasing of microglia inflammatory cytokines,which can be used as a cellular model for the inflammatory response of microglia in sepsis related encephalopathy.2 、 Mi R-155 may promote mitochondrial damage by inhibiting microglia UCP2 expression and participate in the occurrence of septic encephalopathy.