Objective:To establish contact dermatitis pruritus model and detect itch,anxiety and depression-like behaviors.To observe the activation of the medial prefrontal cortex(mPFC)neurons and provide morphological evi-dence for its involvement in the regulation of itch perception information.Methods:A model of contact dermatitis pruri-tus was constructed with diphenylcyclopyrenone(DCP)applied on the neck and back of the mouse,and the itch per-ception video tracking system was used to detect the scratching behavior.Open field and suspended tail behavioral ex-periments were conducted to explore the anxiety and depression-like behaviors of mice under contact dermatitis.The dis-tribution of c-Fos and p-ERK1/2 positive cells in the mPFC of contact dermatitis model mice was investigated by immu-nofluorescence staining.Results:The contact dermatitis pruritus model was successfully established,and the bouts of scratching in the contact dermatitis model group was significantly higher than that in the control group(P＜0.05).The results of open field test showed that the time and distances were significantly reduced in the central area of the contact dermatitis model group.The results of the tail suspension experiment suggested that the time of immobility increased sig-nificantly in the contact dermatitis model group(P＜0.05).There were more c-Fos positive and p-ERK1/2 positive cells in the mPFC compared with the control group(P＜0.05).Conclusion:Bouts of scratching were increased in the contact dermatitis group and the mice had anxiety and depression-like behaviors.In the contact dermatitis state,mPFC neurons were activated,providing morphological evidence for the involvement of mPFC neurons in contact dermatitis pruritus and accompanying affective disorders such as anxiety and depression.

Objective:To explore the effects of Undaria pinnatifida polysaccharides(UPPs)on depressive-like behavior,neurotransmitter content,oxidative stress,and the hypothalamus-pituitary-adrenal(HPA)axis in rats treated with chronic unpredictable mild stress(CUMS).Methods:A rat model of depression was prepared using the CUMS method,and rats were treated with normal saline(NS)or different doses of UPPs by gavage.The general condition of the rats was observed,and depressive-like behavior was detected by the open field test(OFT),sucrose preference test(SPT),and forced swimming test(FST).The activity or levels of 5-hydroxytryptamine(5-HT),dopamine(DA)and norepinephrine(NE),malondialdehyde(MDA),superoxide disidase(SOD)and catalase(CAT),adrenocorticotrop-ic hormore(ACTH),corticosterone(CORT)in the hippocampus or serum of rats were detected using commercial kits.Western Blot was used to detect the expression level of hippocampal glucocorticoid receptor(GR)protein,and hema-toxylin-eosin(HE)staining was used to observe the tissue structure of hippocampus of rats.Results:The depressive-like behavior of rats in the UPPs medium and high dose groups was significantly improved(P＜0.05).In the UPPs high dose group,the contents of 5-HT,DA,and NE in the hippocampus of rats increased(P＜0.05),while the con-tents of MDA in both serum and hippocampus decreased(P＜0.05),and the activities of SOD and CAT increased(P＜0.05).The contents of ACTH and CORT in serum decreased(P＜0.05).In the UPPs medium dose group,the levels of hippocampal MDA and CAT,as well as serum SOD,ACTH,and CORT were improved(P＜0.05).The expression level of GR protein in the hippocampus increased(P＜0.05),and the pathological changes in the hipp-ocampal dentate gyrus were significantly improved.Conclusion:UPPs can alleviate depressive-like behavior in CUMS rats,and its mechanism may be related to increasing the content of monoamine neurotransmitters in the hippocampus,reducing oxidative stress damage,and HPA axis hyperfunction.

Objective:To analyze the gene variants of a patient affected with Duchenne/Becker muscular dystrophy in a pedigree and further explore the genotype-phenotype correlation for providing basis for family genetic counseling.Methods:The clinical features and family history of family members were collected.Multiplex ligation-dependent probe amplification(MLPA)was utilized to detect copy number variation of target genes.The pathogenic variations were ana-lyzed by whole exome sequencing(WES).The suspected gene variations were verified by Sanger sequencing.For the splice site mutations,mini-gene was constructed and expressed in vitro to detect the number of transcript and cDNA se-quence.Results:The proband of this family is a male,with no obvious involvement of the lower limbs.Laboratory tests showed an elevated level of creatine kinase(CK)in peripheral blood(700-1600 U/L),and electromyography showed myogenic damage.MLPA did not detect pathogenic exon copy number variation in dystrophin(DMD)gene.Genetic testing showed the proband carried a maternal hemizygotic splicing variation of DMD gene(NM_004006.2):c.2622+2T＞C.An in vitro mini-gene splicing assay confirmed that this splicing mutation could affect RNA splicing.According to clinical features and genetic testing results,the proband was speculated first proof of Duchenne/Becker muscular dys-trophy(DMD/BMD)caused by DMD gene mutation.Conclusion:This study identified the pathogenic variation of a proband with DMD/BMD of DMD gene,which enriched the variation spectrum of DMD/BMD in China.It was con-firmed that the splicing variation of the DMD gene c.2622+2T＞C can produce multiple transcripts leading to different functional impairments,and based on the specificity of temporal and spatial expression,it corresponded to the mild clin-ical manifestations of the patient,providing some reference value for the correlation between genotype and phenotype.

Objective:To analyze the effect of depressive state on Alzheimer's disease(AD)mice,3xTg-AD mice were stimulated with chronic social frustration stress(CSDS)and chronic mild unpredictable stress(CUMS),to estab-lish an early AD-induced depression mouse model,and to detect cognitive and behavioral changes,activation of micro-glia in the hippocampus and neuronal loss.Methods:Three-month-old mice were subjected to 8-day stress stimulation alternately,the depressive state of the mice was evaluated by behavior,the evaluation criteria were formulated,and the cognitive behavior was detected and analyzed,and the hippocampal brain tissue sections were stained with immunofluo-rescence to observe the deposition of β-amyloid(Aβ)and the aggregation of microtubule-associated protein(tau),mi-croglia activation and neuronal loss.Results:Depression-related behavioral results showed that the CSDS+CUMS group had depression-related phenotypes.Cognitive-behavioral testing showed that the new object recognition index of the mice in the CSDS+CUMS group was significantly reduced(P＜0.05),and the Morris water maze showed that the spatial memory ability of the CSDS+CUMS group was significantly reduced(P＜0.05),but there was no obvious fear memory loss in the CSDS+CUMS group in the conditioned fear experiment.The results of immunofluorescence staining showed that Aβ deposition appeared in the hippocampus at 4 months of age,the activated microglia increased(P＜0.001),and a certain degree of neuronal loss appeared in the CSDS+CUMS group(P＜0.001);At 8 months of age,the CSDS+CUMS group showed tau protein aggregation early.Conclusion:We established a model of AD-induced de-pression in AD mice,in which 3xTg-AD mice experienced early decline in learning memory and increased AD-related pathological deposition of neurons in the hippocampus,accompanied by microglial activation and neuronal loss.

Objective:To explore the central regulatory mechanisms of exercise to alleviate the behavioral effects of anxiety and depression in post-traumatic stress disorder(PTSD)and to promote hippocampal neurogenesis.Methods:Male C57BL/6J mice were randomly divided into control group(Control),PTSD modeling group(PTSD),PTSD-mod-eling with low-intensity exercise group(PTSD+LE)and PTSD-modeling with high-intensity exercise group(PTSD+HE).A combination of conditioned foot shock(CF)and single-sustained stress(SPS)was used to construct the PTSD compound stress model.The anxiety and depression-like behaviors of mice were assessed using the open field test and the tail suspension test,respectively.Newborn mature neurons and proliferating cells in the DG area of mice hippocam-pus were observed by immunofluorescence double-labeling experiment.The expression of mice hippocampal adiponectin receptor 1(AdipoR1)was detected by Western Blot.Results:The results of the open field test showed that the mice in the PTSD+HE group showed a significantly longer distance and stay in the central area than those in the PTSD group and the PTSD+LE group(P＜0.05);the results of the tail suspension test showed that the immobility time of the mice in the groups with different levels of locomotor training was significantly reduced compared with that of the mice in the PTSD group(P＜0.05),and it was more significant in the PTSD+HE group(P＜0.05).In addition,BrdU+,Br-dU+/NeuN+and MCM2+cell densities in the hippocampal DG region of mice in the PTSD+HE group were significant-ly higher(P＜0.05).And the hippocampal AdipoR1 protein expression of mice in the PTSD+HE group was signifi-cantly upregulated(P＜0.05).Conclusion:Anxiety and depression-like behaviors in PTSD mice are associated with decreased levels of hippocampal neurogenesis.Exercise can improve their anxiety and depression-like behaviors,and the mechanism of the effect may be related to the promotion of AdipoR1 expression and hippocampal neurogenesis.

Objective:Combined retrograde tracing with optogenetic methods,we are investigating the functional role of dorsal raphe nucleus(DRN)GABAergic neurons projecting to lateral habenula(LHb)terminals in anxiety-like behaviors.Methods:The specific retrograde tracing virus AAVretro-Ef1α-DIO-mCherry was injected into the LHb of Vgat-Cre mice.After the viral expression,Multi-brain slides scanning microscope imaging scan and observe the up-stream distribution of GABAergic neural projection in LHb.By retrograde tracing,opto-activated virus AAV2/9-Ef1a-DIO-ChR2-mCherry(ChR2 group)and control virus AAV2/9-Ef1a-DIO-mCherry(mCherry group)were injected into the DRN of Vgat-cre mice respectively.Three weeks after virus expression,DRNGABA neurons and the DRNCABA-LHb neural terminals were activated by optogenetic to observe their role in anxiety-like behaviors.Results:According to the results of retrograde tracing,the midbrain DRN is one of the major GABAergic neural projection brain areas in the LHb nucleus.Optogenetic stimulation of DRNGABA neurons,compared with the mCherry group,the ChR2 group showed sig-nificantly longer total moving distance,central area moving time and distance in the open field test(OFT);In the ele-vated plus maze(EPM),the open arm moving time and distance was significantly increased.When DRN GABA-LHb neural terminals were stimulated,compared with the mCherry group,the ChR2 group showed a significantly longer cen-tral zone moving time,distance and total moving distance in the OFT.During the elevated plus maze(EPM),the open arm moving time was significantly increased.Conclusion:The specific activation of the DRNGABA neuron as well as the DRNGABA-LHb neural projections showed that both significantly improved anxiety-like behaviors in mice.This provides new ideas and evidence for the treatment of anxiety,depression and other psychiatric disorders.

Objective:Biological markers of the ventrolateral orbitofrontal cortex(vlOFC)involved in pain regula-tion were screened.Methods:Chronic inflammatory pain was induced in male C57BL/6J mice by injection of complete Freund's adjuvant(CFA)into the left posterior plantar.Paw withdrawal threshold(PWT)and paw withdrawal latency(PWL)were detected to evalue hyperalgesia.Transcriptome sequencing was performed on fresh tissue from vlOFC of mice after behavioral tests.The differentially expressed genes(DEGs)were screened by bioinformatics method,and their biological functions and pathways were enriched.Results:Compared with the PBS group,the left hindpaw me-chanical pain threshold and the paw withdrawal latency caused by heat pain were significantly reduced in the CFA group(P＜0.001).The DEGs of vlOFC in the two groups were 497,of which 143 were up-regulated and 354 were down-reg-ulated.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGGs)analysis showed that:In chro-nic inflammatory pain model mice,DEGs of vlOFC were mainly manifested in biological processes such as organic cation transport,neurotransmitter transport,and regulation of cytoplasmic calcium ion concentration.It is related to G protein-coupled receptors(GPCRs),neuropeptides and ammonium transport.DEGs mainly focuses on neuroactive ligand-receptor interactions,cytokine-cytokine receptor interactions,and cAMP signaling pathways.Reactome functional en-richment analysis showed that the pathway with the highest number of DEGs enriched and the lowest P value-adjusted was GPCRs ligand binding.Conclusion:Ion transport,neurotransmitter transport and binding,and GPCRs-related ac-tivities in vlOFC are involved in the regulation of chronic inflammatory pain.

Objective:To investigate the effect of panax notoginseng saponins(PNS)on the expression of tumor necrosis factor-α(TNF-α)in lipopolysaccharide(LPS)-induced activated BV2 microglia through p38 mitogen-activa-ted protein kinase(p38 MAPK)pathway.Methods:BV2 microglia were divided into control group,LPS activated group and LPS+panax notoginseng saponins intervention group(LPS+PNS).The CCK-8 method was used to detect the viability of BV2 microglia and determine the optimal drug intervention concentration.Western Blot and immunofluo-rescence were used to detect the expression of p38 MAPK and TNF-α and the phosphorylation level of p38 MAPK(p-p38 MAPK)in BV2 microglia.Results:Compared with the blank control group,there was no significant difference in the cell viability of BV2 microglia,and finally 100 mg/L was selected as the drug intervention concentration.Western Blot and immunofluorescence results indicated that after LPS activation,the expression of TNF-α and the phosphoryla-tion level of p38 MAPK in BV2 microglia were significantly increased(P＜0.05).After PNS intervention,compared with LPS-activated group,the expression of TNF-α and the phosphorylation level of p38 MAPK were significantly decreased(P＜0.05).After treatment with p38 MAPK pathway inhibitor(SB203580),there was no significant differ-ence in the expression levels of p-p38 MAPK and TNF-α in PNS combined with SB203580 group(LPS+PNS+I)com-pared with LPS+PNS group(P＞0.05).In addition,the changes of p38 MAPK in each group were not statistically sig-nificant(P＞0.05).Conclusion:PNS may inhibit the expression of inflammatory factor TNF-α secreted by activated BV2 microglia through p38 MAPK pathway.

Objective:To observe the effects of electroacupuncture(EA)intervention on norepinephrine(NE)andα2A adrenergic receptors(α2A-R)in the dorsal root ganglion(DRG)of mice with spared nerve injury(SNI).Methods:Male C57BL/6 mice were randomly divided into sham surgery group(Sham),model group(SNI),negative control EA group(SNI+NC-EA),and EA group(SNI+EA).Mechanical and thermal stimuli were used to measure the paw withdrawal mechanical threshold(PWMT)and paw withdrawal thermal latency(PWTL).Immunofluorescence staining was used to detect the sprouting of sympathetic nerve fibers and the co-localization of α2A-R with large-diameter sensory neurons in mouse DRG.Enzyme-linked immunosorbent assay(ELISA)kits were used to measure NE levels in mouse serum and DRG,and Western Blot was used to detect tyrosine hydroxylase(TH)and α2A-R expression levels in DRG.Results:After SNI,the PWMT and PWTL were significantly decreased,and after electroacupuncture treatment,PWMT and PWTL were reversed and increased.Immune fluorescence staining showed that sympathetic ganglion sprouting increased in DRG after SNI,and significantly decreased after electroacupuncture;After SNI,NE,α2A-R,and TH in DRG all significantly increased,and their expression decreased after electroacupuncture intervention,but NE in the serum did not change significantly.Conclusion:In the SNI model,electroacupuncture may regulate the sympathetic-sensory coupling by inhibiting the release of NE and the expression of α2A-R in DRG,thereby producing analgesic effects.

Objective:To observe the role of microRNA-92a(miRNA-92a or miR-92a)in the differentiation of CD4+T cells in the central nervous system of mice with experimental autoimmune encephalomyelitis(EAE);to study the process of miR-92a regulating cell differentiation through the glycolytic pathway,and to investigate the molecular mechanism by which miR-92a affects the pathological process of EAE targeting mTOR as a downstream key molecule.Methods:After EAE models were successfully constructed on C57BL/6J or miR-92a-/-mice,spinal cord CD4+T cells were isolated and cultured in vitro,the proportions of Th1,Th2,Th17 and Treg cells were measured by flow cytometry,the level of glycolysis was measured using Seahorse,and the level of related gene changes was measured using RT-qPCR;naive CD4+T cells cultured in vitro were induced to differentiate into Th1 or Treg cells,on the basis of which miR-92a levels were regulated and combined with glycolytic agonists or inhibitors to detect cell differentiation;mTOR and p-Akt expression changes in CD4+T cells of C57BL/6J or miR-92a-/-mice were detected using Western Blot,and glycolysis levels in CD4+T cell and cell differentiation were measured after overexpression of mTOR with plasmid transfection.Results:miR-92a could lead to the destruction of CD4+T cell differentiation balance,the ratio of Th1 and Th17 cells was increased,and that of Th2 and Treg cells decreased after EAE;miR-92a regulated CD4+T cell differentiation by promoting glycolysis;Treg cell differentiation was promoted after inhibiting glycolysis;miR-92a could increase the level of glycolysis by activating the mTOR signaling pathway,thus affecting cell differentiation.Conclusion:miR-92a promotes T cell glycolysis and disrupts the balance of CD4+T cell differentiation by activating mTOR signaling pathway,which is involved in the pathological process of MS and EAE.