The Effects And Mechanisms Of Kirenol On Perinatal White Matter Injury In Mice

White Matter Injury(WMI)is the main cause of cerebral palsy,and it is also the most common type of brain injury in preterm infants.At present,the treatment of brain injury in premature infants is very limited.Therefore,it is of great significance for the family and society of premature infants to explore new treatment methods to improve the damage of nervous system development caused by brain injury in premature infants.Neuroinflammation caused by perinatal infection is an important factor leading to preterm delivery and impaired white matter development.White matter is mainly composed of myelinated axons and glial cells,which plays an important role in signal transduction.Oligodendrocytes are the basic cells that form myelin.Oligodendrocyte precursor cells are highly sensitive to adverse stimuli during the perinatal period.Perinatal infection can have a negative effect on the development of oligodendrocytes by activating microglia and releasing inflammatory factors,resulting in the maturation stagnation of oligodendrocytes,and finally the disturbance of myelin formation in developing white matter,resulting in perinatal WMI,cognitive,motor defects,affective disorders and other neurological sequelae.Kirenol(Kir)is a diterpene derivative,which exists in the plant of Compositae.It has been reported that it has a variety of biological activities,including neuroprotective,anticancer,heart protection,anti-photoaging,anti-arthritis and so on.However,at present,there is no literature to report the therapeutic effect of Kirenol on perinatal white matter injury.Under the above background,we speculate that Kirenol can reduce white matter damage and neurological dysfunction caused by perinatal infection by targeting microglia,and carry out the following studies.Ⅰ The protective effects and mechanisms of Kirenol in perinatal WMIThe perinatal WMI model was established to determine the therapeutic effect of Kirenol on the abnormal development of white matter myelin caused by perinatal infection.Kunming mouse pups were selected as experimental subjects,and the perinatal WMI model was established by intraperitoneal injection of LPS at 2 to 5 days after birth to detect the behavior of mice after treatment behavior and molecular changes related to oligodendrocytes were evaluated to evaluate the lineage development and neurological function recovery of oligodendrocytes.The results are as follows:(1)First,the neural behavior of mice was detected,and the test was mainly divided into two stages,namely short-term(P7)and long-term(P28).The results showed that compared with the control group,LPS group showed obvious anxiety-like phenomenon,which could improve the anxiety of mice after treatment with Kirenol.(2)Myelin formation was evaluated by LFB staining,MBP,CNPase immunofluorescence staining,qPCR and Western Blotting.The results showed that the expression of MBP,CNPase was increased and the decrease of myelin formation was improved after treatment with Kirenol.(3)The expression of oligodendrocyte precursor cell marker PDGFR-α was detected by immunofluorescence staining.The results showed an abnormal increase in oligodendrocytes after treatment with Kirenol compared with the LPS group.(4)The expression of Olig2,Sox10,the key transcription factor affecting the differentiation/maturation of oligodendrocyte lineage,was detected by immunofluorescence and qPCR assay.The results showed that the expression of Olig2,Sox10 decreased in LPS group and increased in LPS+Kir group.Ⅱ The effects and mechanisms of kirenol on microglia after perinatal WMIThe first part of the results showed that Kirenol can alleviate the neurobehavioral disorders and myelination disorders caused by perinatal WMI by promoting the expression of sox10 and Olig2,the key transcription factors of differentiation/maturation of oligodendrocyte lineages.In addition,studies have shown that microglia also play an important role in perinatal white matter damage,and after microglia are activated,they produce various forms of biological effects through the cytokines they release,and are involved in regulating oligodendrocyte differentiation/maturation and myelin formation.Next,we conducted an indepth study on the role and mechanism of Kirenol in perinatal WMI microglia,and achieved the following results:(1)In vivo experiments,immunofluorescence detected the number of Iba-1+ microglia,and qPCR detected the expression levels of inflammatory factors IL-1β,TNF-α and IL-6.The results showed that Kirenol relieved the activation of microglia and the release of inflammatory factors in areas of white matter injury.(2)In vitro,MTT results showed that Kirenol had no significant effect on the viability of microglia at the concentration of 25μmol/L.Detection of M1 and M2 marker expression in microglia by qPCR experiments.The results showed that the surface markers IL-1β,TNF-α,IL-6,CD32 and CD86 of microglial M1 in the LPS+Kir group were significantly downregulated compared with the LPS group,and the expression of TGF-β surface markers of microglia type M2 was increased.(3)Through the Western Blotting experiment,the phosphorylation levels of the inflammation-related signaling pathways AKT and ERK were detected.The results showed that Kirenol downregulated the level of AKT phosphorylation after lippolysaccharide stimulation,which had no effect on the ERK signaling pathway.We further demonstrated by using the AKT activator SC79 for a revertative experiment that Kirenol may inhibit microglia activation and reduce inflammatory factor release through the AKT signaling pathway.(4)Western Blotting results showed that H3K4me3 expression decreased and H3K27me3 expression increased in LPS group,while H3K9me3 expression decreased in H3K9me3,but had no significant effect on H3K27me3 expression.qPCR results showed that Kirenol increased the expression of H3K9me3 demethyltransferase KDM4D and decreased the expression of methyltransferase KMT1C,KMT1E.This study demonstrates that Kirenol can promote the transcriptional maturation of OPCs after injury by regulating the expression of oligodendrocyte lineage differentiation/maturation of key transcription factors Olig2 and Sox10,as well as inhibiting microglia activation,alleviating inflammatory factor release and improving myelination disorders in perinatal WMI mice,and alleviating motor coordination ability and mood disorders in mice.This study suggests that Kirenol has a good effect in the treatment of brain injury associated with inflammation of the central nervous system,and has great application value in clinical treatment.