The Mechanism Of Notch1 Signaling Regulate Astrogliosis Of Immature Astrocytes After Lipopolysaccharide Stimulated

White mater damage(WMD) in the premature babies is considered as the most common form of injury to the preterm brain. Intrauterine infection/inflammation can cause the damage of the glial cells, which is the most important cause of brain white matter damage in the premature infant. Inflammation of the brain after intrauterine infection/inflammation which affects premature astrocyte proliferation and astrogliosis is a key factor of cerebral white matter damage. Notch1/Hes (Hairy enhallcer of split) signal way may participate in the mechanism of premature astrocyte proliferation and astrogliosis after intrauterine infection/inflammation.Objective:To study the regulation for Notchl signal to the premature astrocyte astrogliosis after infection/inflammation and to the influence for the methylation level of CpG island in STAT3 binding sites of Glail fibrillary acidic protein (GFAP) gene promoter region in astrocytes.To further elucidated the mechanism of premature brain white matter damage in intrauterine infection/inflammation.Method:1.Taking fetal mouse brain tissue for cell culture, immunofluorescence methods were used for evaluation of GFAP+/BrdU+ expression and quantitative real-time PCR (QPCR) were used for evaluation of Proliferating Cell Nuclear Antigen (PNCA) and ki67 expression in astrocytes.Moreover, QPCR was used to analyze Notchl, Notchl intracellulardomains (NICD),as well as Hesl,Hes5 expression in astrocytes after exposed to lipopolysaccharide (LPS).2. On the carrier of Notchl ICD-pEGFP transfection astrocytes increased the expression of Notchl signaling pathways and downstream signals, as well as building Notchl siRNA transfection astrocytes, using RNA interference (RNAi) technology to restrain the expression of Notchl gene. In addition, QPCR was used to analyze Notchl, ICD and Hes1, Hes5 expression in astrocytes after exposed to MW167 intervention. Immunofluorescence methods were used for evaluation of GFAP+/BrdU+ expression and QPCR were used for evaluation of PNCA and Ki67 expression in astrocytes.3. Taking immature astrocytes exposed to LPS, and building different plasmid vector transfection, Overexpression of Notchl signal in astrocytes, we observed the methylation level of CpG island in STAT3 binding sites of GFAP gene promoter region in astrocytes. Results:1. Compared with the control group, the number of GFAP+/BrdU+ cells were significantly increased after exposed with LPS (5ug/ml)(P<0.05). Relative expression of PCNA and Ki67 mRNA were increased(P<0.05). With the increase of time,the expression of Notchl-ICD、Hes1、Hes5 mRNA were raised(P<0.05). Moreover,the expression of Notchl、ICD、Hes1、Hes5 protein were raised(P<0.05).2. After exposed with LPS (5ug/ml), if Notchl was improved,the proportion of the GFAP+/BrdU+ cells were increased and the expression of Notchl-ICD、PNCA、Hes1、 Hes5 mRNA were also increased(P<0.05),as well as Notch1、ICD、PNCA、Ki67、 Hes1、Hes5 protein(P<0.05).CCK8 was increased too(P<0.05).3. After exposed with LPS (5ug/ml), if cut Notchl, the proportion of the GFAP+/BrdU+ cells were reduced and the expression of Notchl-ICD、PNCA、Hes1、 Hes5 mRNA were also reduced(P<0.05),as well as Notchl、ICD、PNCA、Ki67、Hes1、 Hes5 protein(P<0.05).CCK8 was reduced too(P<0.05).4. Compared with the control group, the degree of methylation was decreased obviously in the overexpression of Notchl signal group. Conclusion:1. The LPS stimulation promotes the astrocyte proliferation and astrogliosis.2. The LPS stimulation promotes the expression Notch1 and its downstream signal Hes5 and Hesl.3. In the case of LPS stimulation,Notchl signaling pathway can positively regulate the astrocyte proliferation and astrogliosis.It seem to be working by its downstream signal Hes5 and Hesl.4. The mechanism of Notchl regulating the astrocyte proliferation and astrogliosis after LPS stimulated,may through regulate the demethylation degree of CpG island in STAT3 binding sites of GFAP gene promoter region.