The Effect Of Melatonin On Axonal Hypomyelination Of Periventricular White Matter By Modulating A1/A2 Astrocytic Alteration Via JAK2/STAT3 Pathway In Septic Neonatal Rats

Background:Astrocytic activation play a key role in the pathogenesis of the periventricular white matter(PWM)damage(PWMD)in septic neonate,which would lead to neurological disorders in later life.Activated astrocytes are classified into type A1and A2.Recently,reactive astrocytes were further classified into A1 astrocytes and A2astrocytes.A1 astrocytes are harmful that contribute to the death of neurons and oligodendrocytes,whereas A2 astrocytes upregulate many neurotrophic factors and promote neuronal survival.Melatonin(MEL)has been shown to regulate the activation of astrocytes.MEL is a key neurohormone,as well as its metabolites are endowed with diverse pharmacological properties such as anti-inflammatory,anti-oxidative and anti-apoptotic effects.MEL can improve the long-term neurological dysfunction of septic neonatal rats but with unknown mechanism.Object:This study is designed to investigate the phenotypic alteration of A1/A2 astrocytes in the PWM of septic postnatal rats and explore the role and mechanism of melatonin regulating this process.Methods:Sprague-Dawley rats were randomly assigned to the control group,LPS group and LPS+MEL group.Sepsis model was intraperitoneally injected with LPS(1 mg/kg),and neonatal rats in the LPS+MEL group were administered with MEL(10 mg/kg)7 days after LPS injection.At different time points after injection,rats in each group were divided into five subgroups:1,3,7,14 and 28 days.Double immunofluorescence and enzyme-linked immunosorbent assay(ELISA)were used to inspect the activation of microglia and astrocytes in the PWM.The expression of A1 marker C3 and A2 marker S100A10 were detected by double immunofluorescence staining.The ability of autonomous activity,learning and memory,and the therapeutic effects of melatonin treatment were evaluated by behavioral evaluation.Double immunofluorescence,in situ hybridization and electron microscopy were applied to evaluate the differentiation and maturation of oligodendrocyte precursor cells(OPCs)and axon myelination.In vitro,A1 astrocytes were induced by interleukin 1α(IL-1α),complement component 1q(C1q)and tumor necrosis factorα(TNF-α).Primary astrocytes were divided into six groups:control group,LPS group,LPS+MEL group,LPS+MEL+Luzindole group,LPS+MEL+AG490 group and LPS+MEL+STAT-IN-3 group.Double immunofluorescence and western blot analysis were applied to detect the expression of C3 and S100A10.Western blot(WB)analysis was used to detect the expressions of MT1,and phosphorylation of JAK2and STAT3 in A1 astrocytes after MEL treatment.Real-time q PCR was used to examine the expression of m RNA of the neurotrophic factors LIF and FGF2.Results:1.The number of IBA1~+microglia in the PWM of the postnatal rats was significantly increased at 1 and 3 days after LPS injection compared with control,but returned to normal level at 7 days.While the number of GFAP~+astrocytes was significantly increased from 3 days to 28 days after LPS administration.Compared with the rats in control group,the number of C3~+GFAP~+cells in the PWM of neonatal rats was significantly increased at 7 days after LPS injection,while the number of S100A10~+GFAP~+cells were decreased significantly.2.In open-field test,melatonin significantly increased the duration of movement and distance in the central area of septic rats.In the Morris water maze task,the latency of escaping in the LPS+MEL group was shorter than that in the LPS group,and the number of shuttles was more than that in the LPS group.After MEL treatment,the number of C3~+GFAP~+cells was significantly decreased in the PWM of septic postnatal rats,while the number of S100A10~+GFAP~+cells increased significantly.Ultrastructural observation of electron microscopy showed that the myelin sheath was destroyed and disintegrated in the PWM of septic rats,while the myelin sheath of rats in LPS+MEL group had obviously improved.The expressions of myelin maturation related molecules MAG,MBP and PLP in the PWM of rats in LPS+MEL group were higher than that in the LPS group.3.After co-stimulation of IL-1α,C1q and TNF-α,the expression of C3 in the primary cultured astrocytes were significantly increased,while the expression of S100A10,MT1,P-JAK2 and P-STAT3 decreased significantly.MEL treatment inhibited the high expression of C3 and the low expression of S100A10,MT1,P-JAK2,and P-STAT3 in A1 astrocytes.Furthermore,MEL can reverse the decrease of LIF and FGF2 m RNA levels in astrocytes under the co-stimulation of IL-1α,C1q and TNF-α.Conclusion:MEL may inhibit the activation of A1 astrocytes in the PWM of neonatal rats injected with LPS,and promote the production of A2 astrocytes.MEL may regulate the phenotypic alteration of A1/A2 astrocytes to improve PMWD of septic postnatal rats throughout JAK2/STAT3 pathway.