Objective : To establish a PTEN gene-downregulated ASD juvenile rat model (VPA-ADV) through combined application of valproic acid (VPA) and PTEN adenovirus (ADV),then to compare the newly constructed animal model with two traditional autistic animal models of VPA and AD,and ultimately to prove the advantages of this model in animal model establishment of acupuncture treatment for ASD. Methods : Wista rats at 12.5 days of gestation were randomly divided into 2 groups.VPA rats were given 600 mg/kg of normal saline (NS) intraperitoneally.Weight,eye opening time and tail deformity were recorded.The newborn mice in NS group were randomly divided into three groups (10 rats in each group):normal (NS) group,virus (ADV) group and virus-negative control (ADV-NC) group;VPA group (20 young rats) was randomly divided into 2 groups (10 rats in each group):valproic acid (VPA) group and valproic acid-binding virus interference (VPA+ADV) group.The body weight,tail length,curved tail,geotaxis text time and skeletal deformity of 5 groups of young rats after birth,the neurobehavioral behavior of 21-day-old rats,and the myelin structure of brain tissue under electron microscope were observed,the expression levels of PTEN,PI3K,AKT,GSK3β and MBP were detected by immunohistochemistry,Western blot and q-PCR. Results: Compared with the NS group,the VPA group had significantly increased malformation rate,slow weight gain,slow tail length growth,and long negative geotaxis reflex time (P<0.05).Compared with the NS group,the weight gain,tail length growth and negative geotaxis reflex time of the three model groups were slower (P<0.05),and the performance of VPA+ADV model was the most significant.There were significant differences in the time of crossing the central grid,the number of crossing the edge grid,and the time of crossing the edge grid in the open field test between the three groups and the NS group (P<0.05).In the selfgrooming experiment,the number of interactions in VPA-ADV model was the least (P<0.05),and the number of digging and self-grooming was the most (P<0.05).In the three-box social experiment,VPA-ADV model had the shortest average number of entries into the social box and the shortest residence time (P<0.05),the time of finding the platform in the water maze experiment was the longest,and the number of times crossing the third quadrant was the least (P<0.05).The structure of the myelin sheath layer in the corpus callosum was observed by transmission electron microscopy.The structure of the NS group was clear and complete.Compared with the NS group,the myelin structure of the ADV-NC group was similar,and the myelin structure of the three model groups was stratified and broken,and there were pathological changes and myelin damage in the ASD.Among them,the myelin sheath of the VPA-ADV model was thickened,stratified,and severe visible disintegration.The results of immunohistochemistry,Western blot and q-PCR showed that the expression of PTEN in VPA+ADV model was down-regulated by about 50%,which was the most obvious.The expression of AKT and MBP increased,and the expression of GSK3β decreased (P<0.05).However,the results of q-PCR showed that the expression of PI3K-mRNA in the three model groups significantly increased (P<0.05),and the change of VPA+ADV model was the most significant. Conclusion The novel PTEN-ASD animal model established by VPA+ADV method is observed to have significant pathological changes that are typical of the manifestation of ASD myelin dysplasia and is determined to have better results than the two traditional autistic animal models.

ObjectiveTo observe the intervention effect of Chaihu and Longgu Mulitang （CLMT） on rat depression model prepared by chronic unpredictable mild stress （CUMS） based on cyclic adenosine monophosphate （cAMP）/protein kinase A （PKA）/cAMP-response element-binding protein （CREB）-brain-derived neurotrophic factor （BDNF） signaling pathway. MethodSixty SD rats were divided into normal group， model group， and CLMT low-， medium- and high-dose groups and fluoxetine group （positive control） according to random number table. They， except the normal group， were treated with CUMS for 49 days to prepare the rat depression model. The CLMT low-， medium- and high-dose groups were given 2.89， 5.78 11.56 g·kg^-1 of CHMD granules， respectively， and the fluoxetine group was given 2.06 mg·kg^-1 of fluoxetine hydrochloride on the 29^th day. The normal group and the model group received equal volume of normal saline for 21 days. The behavioral performance of rats were observed by open field test and forced swim test. The levels of 5-hydroxytryptamine （5-HT）， norepinephrine （NE） and cAMP in rat hippocampus were measured by enzyme-linked immunosorbent assay （ELISA）. The mRNA expressions of PKA， CREB， and BDNF in rat hippocampus were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the protein expressions of PKA and BDNF were detected by Western blot. Immunohistochemistry was used to determine the expression of CREB， and hematoxylin and eosin （HE） staining and Nissl staining were used to observe the morphological changes of hippocampus. ResultCompared with the conditions in the normal group， the immobility time of the model group in the forced swim test was increased （P<0.01） and the total movement distance， residence time in central area， number of entries in central area and movement distance in central area were decreased （P<0.01）. Additionally， the contents of 5-HT， NE and cAMP in hippocampus of the model group as well as the protein expressions of PKA， BDNF and CRE，the mRNA expressions of BDNF and CREB were lower than those in the normal group （P<0.01）. Compared with the model group， the CLMT groups had reduced immobility time （P<0.01）， elevated total movement distance， residence time in central area， number of entries in central area and movement distance in central area （P<0.05， P<0.01）， up-regulated contents of 5-HT， NE and cAMP in hippocampus （P<0.05， P<0.01）， and up-regulated protein expressions of PKA， BDNF and CREB and mRNA expressions of BDNF and CREB （P<0.01）. HE staining and Nissl staining showed that CLMT significantly improved the neuronal structure in rat hippocampus. ConclusionCLMT alleviates the anxiety and depression of rats. These effects may be mediated by regulating monoamine neurotransmitters 5-HT and NE in hippocampus of depressed rats， activating cAMP/PKA/CREB/BDNF signaling pathway， up-regulating the expression of BDNF and protecting hippocampal structure and function .

ObjectiveTo observe the effects of Cuscutae Semen on the learning and memory ability， N6-methyladenosine （m⁶A）-related modification enzymes and total m⁶A level in hippocampus of the offspring of fear-damaged pregnant rats. MethodForty-five pregnant rats were randomly divided into blank group， model group and Cuscutae Semen group. From the 1^st day to the 19^th day of pregnancy， rats in the model group and the Cuscutae Semen group were induced by observing electric shock of other rats. The Cuscutae Semen group was treated with 5 g·kg^-1·d^-1 Cuscutae Semen decoction （ig）， while the other two groups were treated with the same amount of purified water. The offspring were assigned following the grouping method of their maternal generation. The behavioral changes of the offspring were tested by Morris water maze on 21^st day after birth， and the development of hippocampal neurons was observed by transmission electron microscopy. The mRNA and protein expression levels of methyltransferase-like 3 （METTL3）， METTL14， Wilms tumor 1 associated protein （WTAP）， fat mass and obesity-associated protein （FTO） and Alk B homologue 5 （ALKBH5） were detected by Real-time polymerase chain reaction （Real-time PCR）， Western blot and immunohistochemistry （IHC）. The total content of m⁶A in hippocampus was determined by high performance liquid chromatography tandem mass spectrometry （LC-MS/MS）. ResultCompared with the conditions in the blank group， the average latency duration in the model group was prolonged， and the number of entries in the target quadrant， the target quadrant duration and the number of crossing the platform were decreased （P<0.01）. Additionally， the model group had seriously damaged structure of hippocampal CA1 and CA3 neurons， swollen mitochondria， expanded endoplasmic reticulum， and small number of synapses with some having blurred structure， and the expression levels of METTL3， METTL14， FTO， ALKBH5 as well as the total m⁶A level were lower than those in the blank group （P<0.05，P<0.01）. Compared with the model group， the Cuscutae Semen group had shortened average latency duration， increased number of entries in the target quadrant， target quadrant duration and number of crossing the platform （P<0.01）， alleviated damage of hippocampal CA1 and CA3 neurons， fine structure of mitochondrial and endoplasmic reticulum， and clear， intact and dense synapses. And the expression levels of METTL3， METTL14， FTO as well as the total level of m⁶A were up regulated， while the expression level of ALKBH5 was down regulated in the Cuscutae Semen group （P<0.05，P<0.01）. ConclusionCuscutae Semen improved the learning and memory ability of the offspring of the rats affected by fear damaging kidney during pregnancy， protected hippocampal neurons， and up-regulated the expression levels of METTL3， METTL14， FTO and the total m⁶A level in hippocampus.