The Effect Of Minocycline On VPA Autism Model And Its Mechanism

Research background:Autism（ASD）is a group of highly heterogeneous neurodevelopmental disorders occurring in infants and young children.Clinically,it is characterized by social communication,interaction disorder,limited interest and repetitive behavior.Although autism has a strong genetic component,it is well known that environmental factors such as heavy metals,pesticides,infections and drugs can increase the susceptibility to autism.The study found that exposure to sodium valproate（VPA）during pregnancy increased the risk of autism in children.In addition,animal experiments confirmed that rodents exposed to the drug prenatal showed similar behavioral phenotypic characteristics of ASD patients.The offspring model injected with VPA during pregnancy showed obvious anxiety behavior,reduced time spent in the social interaction area,and abnormal cognitive ability.Sodium valproate（VPA）is widely used as an antiepileptic drug and mood stabilizer.At the same time,it is also a non selective deacetylase（HDAC）inhibitor.VPA can affect the development of nervous system and regulate neurotransmission during pregnancy and early development.Therefore,it is assumed that inhibition of HDAC during the critical window of embryonic brain development contributes to the occurrence of autism.However,due to the complex pathogenesis of autism,the mechanism of autism like behavior in offspring induced by prenatal exposure to VPA is unclear.Compared with transgenic models carrying single autism related gene mutations,VPA model may better represent many idiopathic autism caused by environment \ / epigenetics,so it is the most widely used pharmacological model in preclinical research of autism.Evidence shows that microglia activation can induce neuroinflammation.Abnormal activation of microglia in the brain of ASD patients significantly increases the expression of proinflammatory factors,resulting in abnormal development of nervous system,which may be related to cognitive impairment,social impairment,learning and memory impairment and other symptoms in ASD patients.Minocycline,also known as dimethylaminotetracycline or minocycline,is a semi synthetic second-generation tetracycline antibiotic.It has been mainly used to treat acne,periodontitis,skin diseases and so on for a long time.At present,minocycline has been proved to have anti-inflammatory,immunomodulatory and neuroprotective effects.Minocycline has also become the most effective tetracycline derivative with neuroprotective effects.Minocycline has a certain intervention effect in experimental models of ischemia,traumatic brain injury and neurodegenerative diseases including Parkinson’s disease,Alzheimer’s disease and multiple sclerosis.Minocycline has been proved to have multiple mechanisms of action,which may be a drug affecting multiple targets.Animal evidence shows that the mechanisms of minocycline’s potential therapeutic effects include inhibiting microglia activation and inflammatory development;As an inhibitor of matrix metalloproteinase（MMP）activity,it can inhibit the antigen processing of T lymphocytes;And there are neuroprotective effects in a variety of nervous system diseases,which make it different from other tetracycline drugs.At present,there is no report on the regulatory effect and mechanism of minocycline on offspring autism rat model induced by prenatal exposure to VPA.Based on the above background,this study intends to explore the regulatory role of minocycline in the offspring ASD model induced by prenatal VPA exposure,and the regulatory mechanism of minocycline on ASD like behavior.Methods:The ASD model of offspring of VPA rats was established.The day when there was vaginal plug or sperm was observed on the smear was recorded as 0.5 days of pregnancy（E0.5）by vaginal smear method.The offspring ASD model induced by prenatal VPA exposure was established by intraperitoneal injection of sodium valproate solution at a single dose of 500 mg / kg or 0.9% normal saline at the same dose on 12.5 days of pregnancy（E12.5）,Then the behavioral phenotype of offspring VPA model rats was verified by behavioral pharmacology experiment.At the 21 st day of birth,the offspring were separated from milk and divided into three groups for 7consecutive days.The model group rats were given 20 mg / kg minocycline,40 mg /kg minocycline and equal doses of normal saline by intraperitoneal administration according to their body weight.The effects of minocycline on ASD like behavior after7 days of continuous administration were investigated.On the basis of successful modeling,microglia Iba-1,astrocytes GFAP,inflammatory factors IL-6 and IL-1 in prefrontal（m PFC）and hippocampal（HP）brain were investigated by Western blot β、The expression of IL-10 and the related signal pathways regulating cell proliferation,differentiation and differentiation,and the protein expression changes of MAPK signal pathway Er K,JNK and p38.The effect of minocycline on the morphology and expression of microglia was investigated by immunohistochemical staining（DAB method）.Results:1.Prenatal exposure to VPA did not affect the spontaneous movement of offspring rats in the open field experiment,but prolonged the residence time in the middle area of the open field,increased the number of buried beads in the buried bead experiment,and showed some social obstacles in the three box social experiment,which indicated that the VPA model was successfully constructed,resulting in repeated engraving behavior of offspring rats ASD behaviors such as social disorder,and the administration of 20mg/kg low-dose minocycline alleviated anxiety,improved cognitive ability,and improved the novelty ability of young rats.2.Prenatal exposure to VPA increased the expression of Iba-1 in forebrain and hippocampus and IL-1 in hippocampus β Minocycline inhibited the expression of Iba-1 and IL-1 β Especially in the hippocampus.It shows that prenatal exposure to VPA causes abnormal activation of microglia in the brain of offspring rats,resulting in slight inflammatory reaction,and minocycline can inhibit the activation of microglia.3.Prenatal exposure to VPA significantly increased the expression of Er K in the hippocampus of offspring rats,and increased the expression of p38 and JNK phosphorylated water levels in the forebrain and hippocampus,but there was no significant change in PI3 K / Akt signal pathway related proteins,indicating that VPA model rats may regulate microglia activation by activating MAPK / Er K signal pathway,Minocycline may improve the ASD like behavior of VPA model by reducing this activation.4.Prenatal exposure to VPA induced a significant increase in microglia cell body area in forebrain CG1 and hippocampal CA1 of offspring rats,an increase in the overall area of microglia in hippocampal CA3,CA1 and DG areas,and an increase in the expression of CA1 and DG microglia.Minocycline 20 mg / kg administration could reduce the number and cell body area of microglia in forebrain CG1 and IL areas,as well as hippocampal CA3,CA1 The overall area percentage of DG area and the cell body area percentage of CA1 area were reduced.Minocycline reduced the overall area and cell body area of microglia in hippocampal CA3,CA1 and DG areas of the model group at the dose of 40 mg / kg.These results suggest that minocycline can improve ASD like behavior by inhibiting the abnormal activation of progeny forebrain and hippocampal microglia induced by prenatal exposure to VPA.Conclusion:1.Prenatal VPA exposure induces abnormal activation of HP and m PFC microglia in offspring,promotes the release of inflammatory factors and induces ASD like behavior.2.Prenatal VPA exposure may regulate the abnormal activation of microglia through MAPK pathway.3.Minocycline can reverse the activation of microglia in VPA model by inhibiting MAPK pathway,reduce inflammation and improve ASD like behavior.