Journal: Acta Pharmaceutica Sinica. B

Article Title: Gestational dexamethasone exposure impacts hippocampal excitatory synaptic transmission and learning and memory function with transgenerational effects

doi: 10.1016/j.apsb.2023.05.013

Figure Lengend Snippet: Prenatal dexamethasone exposure (PDE) induced changes in hippocampal CDK5/NR2B signaling and synaptic damages in multigenerational adult offspring rats. (A1–A3, F1–F3) mRNA expression of hippocampal cyclin-dependent kinase 5 ( Cdk5 ), synapsin I ( Syn1 ) and synaptosome-associated protein 25 ( Snap25 ) ( n = 8–12). (B1–B3) Immunofluorescence co-labeling and quantification for hippocampal CDK5 (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) ( n = 4–5). Scale bar, 600 μm. (C1–C3) Protein levels of hippocampal N -methyl- d -aspartate receptor (NR2B) ( n = 3). (D1–D3, E1–E3) Golgi staining of hippocampal neurons, the neuronal dendrite length, the number of branch points, the density of total dendritic spines and the number of mushroom spines in adult offspring ( n = 3–5). Mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01 vs. respective controls. Using unpaired t test.

Article Snippet: To confirm the role of the glucocorticoid receptor (GR), miR-133a-3p and CDK5, the cells were cultured for 3 days with 500 nmol/L DEX and (or) 2.5 μmol/L RU486 (HY-13683, MedchemExpress Biotechnology Inc., USA, a GR antagonist), 500 nmol/L DEX and (or) 10 μmol/L miR -133a-3p inhibitor (RiboBio Technology, Guangzhou, China), or 500 nmol/L DEX and (or) 25 μmol/L Roscovitine (HY-B0231, Medchem Express Biotechnology Inc., a CDK5 inhibitor), respectively, and harvested for subsequent analysis.

Techniques: Expressing, Immunofluorescence, Labeling, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Gestational dexamethasone exposure impacts hippocampal excitatory synaptic transmission and learning and memory function with transgenerational effects

doi: 10.1016/j.apsb.2023.05.013

Figure Lengend Snippet: Aberrant epigenetic modification of CDK5 was involved in intrauterine origin and multigenerational programming effects of altered hippocampal CDK5/NR2B signaling and synaptic damages in male offspring rats of prenatal dexamethasone exposure (PDE). (A, D, F, K) mRNA expression of hippocampal cyclin-dependent kinase 5 ( Cdk5 ), synapsin I ( Syn1 ), synaptosome-associated protein 25 ( Snap25 ) in H19-7 cell line ( n = 6) or in fetal rats ( n = 8–12). (B, H) Protein levels of hippocampal N -methyl- d -aspartate receptor (NR2B) in H19-7 cell line or fetal rats ( n = 3). (C) Immunofluorescence co-labeling and quantification for NR2B (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) in H19-7 cell line ( n = 3). Scale bar, 50 μm. (E, L, M) Levels of acetyl-histone H3 at lys9 (H3K9ac), H3K14ac, and H3K27ac in the promoter of hippocampal Cdk5 in fetal and adult offspring rats, as well as in H19-7 cell line ( n = 6–8). (G) Immunofluorescence co-labeling and quantification for hippocampal CDK5 (red) and DAPI (blue) in fetal rats ( n = 5). Scale bar, 300 μm. (I, J) Golgi staining of hippocampal neurons, the neuronal dendrite length, the number of branch points, the density of total dendritic spines and the number of mushroom spines in fetal rats ( n = 3–5). Mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01 vs. respective controls. Using unpaired t -test compared means between the control and treated groups. The comparisons among multiple groups were analyzed by one-way ANOVA test, followed by Dunnett- t test.

Article Snippet: To confirm the role of the glucocorticoid receptor (GR), miR-133a-3p and CDK5, the cells were cultured for 3 days with 500 nmol/L DEX and (or) 2.5 μmol/L RU486 (HY-13683, MedchemExpress Biotechnology Inc., USA, a GR antagonist), 500 nmol/L DEX and (or) 10 μmol/L miR -133a-3p inhibitor (RiboBio Technology, Guangzhou, China), or 500 nmol/L DEX and (or) 25 μmol/L Roscovitine (HY-B0231, Medchem Express Biotechnology Inc., a CDK5 inhibitor), respectively, and harvested for subsequent analysis.

Techniques: Modification, Expressing, Immunofluorescence, Labeling, Staining, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Gestational dexamethasone exposure impacts hippocampal excitatory synaptic transmission and learning and memory function with transgenerational effects

doi: 10.1016/j.apsb.2023.05.013

Figure Lengend Snippet: Intervention of CDK5 could reverse the changes of hippocampal NR2B phosphorylation and learning and memory impairment in male offspring rats of prenatal dexamethasone exposure (PDE). (A–C) The H19-7 cell line was treated with 500 nmol/L DEX and (or) 25 μmol/L roscovitine. (A) Immunofluorescence co-labeling and quantification for N -methyl- d -aspartate receptor (NR2B) (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) ( n = 3–4). Scale bar, 50 μm. (B) Protein levels of NR2B ( n = 3). (C) mRNA expression of Synapsin I ( Syn1 ) and Synaptosome-associated protein 25 ( Snap25 ) ( n = 6). (D) Diagram summarizing the experimental procedure. 3 weeks after the appropriate adeno-associated virus (AAV) injection, rats were undergone novel object recognition (NOR) experiments and Morris water maze (MWM) tests. (E) mRNA expression of hippocampal cyclin-dependent kinase 5 ( Cdk5 ) 3 weeks after AAV injection ( n = 8). (F) The discrimination ratio in NOR experiment ( n = 8–10). (G) (left) The escape latencies and movement distances to find the hidden platform during the five consecutive days of the training phase. (middle) Representative swim patterns of rats for spatial orientation during the hidden platform trials. The escape latencies and movement distances to find the hidden platform during the hidden platform trials. (right) Representative swim patterns of rats for spatial orientation during the probe trials. The number of swim crossings over the previous platform location and time spent in the previous platform location during the probe trials ( n = 8–9). (H) Protein levels of hippocampal NR2B ( n = 3). (I) mRNA expression of hippocampal Syn1 and Snap25 ( n = 8). Mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01 vs. Control; # P < 0.05, ## P < 0.01 vs. DEX or PDE. Using one-way ANOVA.

Article Snippet: To confirm the role of the glucocorticoid receptor (GR), miR-133a-3p and CDK5, the cells were cultured for 3 days with 500 nmol/L DEX and (or) 2.5 μmol/L RU486 (HY-13683, MedchemExpress Biotechnology Inc., USA, a GR antagonist), 500 nmol/L DEX and (or) 10 μmol/L miR -133a-3p inhibitor (RiboBio Technology, Guangzhou, China), or 500 nmol/L DEX and (or) 25 μmol/L Roscovitine (HY-B0231, Medchem Express Biotechnology Inc., a CDK5 inhibitor), respectively, and harvested for subsequent analysis.

Techniques: Phospho-proteomics, Immunofluorescence, Labeling, Expressing, Virus, Injection, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Gestational dexamethasone exposure impacts hippocampal excitatory synaptic transmission and learning and memory function with transgenerational effects

doi: 10.1016/j.apsb.2023.05.013

Figure Lengend Snippet: The high expression and transmission of oocyte miR-133a-3p might mediate the epigenetic regulation of hippocampal Cdk5 in multi-generation of male offspring rats of prenatal dexamethasone exposure (PDE). (A, B) Heat map and volcano map showing the top 20 differentially expressed miRNAs in the hippocampus of PDE male fetal rats on gestational day 20 (GD20). (C) Sequence conservation of miR-133a-3p in rat, human, mouse, and zebrafish. (D) Signaling enrichment map of miR-133a-3p targeted genes. (E) Relative expression of miR-133a-3p in the hippocampus, long bones, cartilage, liver, adrenal gland, kidney, and placenta in male fetal rats ( n = 4–6). (F, G) Relative expression of miR-133a-3p and mRNA expression of Sirt1 in the hippocampus of male fetal and adult offspring rats of F1, F2, and F3 generations, as well as in H19-7 cell line ( n = 6–8). (H) Protein levels of SIRT1 in the hippocampus of male fetal and adult offspring rats of F1, F2, and F3 generations, as well as in H19-7 cell line ( n = 3). (I) (left) The complementary sequences of miR-133a-3p were discovered in 3′UTR of Sirt1 mRNA using Target Scan; (right) Relative luciferase activity was analyzed after wild-type or mutant 3′UTR reporter plasmids were co-transfected with different plasmids in H19-7 cell line ( n = 6). (J–L) Relative expression of miR-133a-3p , mRNA expression of Sirt1 and Cdk5 , and the levels of H3K9ac and H3K27ac in the promoter of Cdk5 in the H19-7 cell line treated with 500 nmol/L DEX and (or) 10 μmol/L miR -133a-3p inhibitor ( n = 6). (M) Expression of miR-133a-3p in oocytes in adult offspring rats of F1 and F2 generations ( n = 6–8). Mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01 vs. Control; # P < 0.05, ## P < 0.01 vs. DEX. Using unpaired t test compared means between the control and treated groups. Multiple groups comparison was analyzed by one-way ANOVA test, followed by Dunnett- t -test.

Article Snippet: To confirm the role of the glucocorticoid receptor (GR), miR-133a-3p and CDK5, the cells were cultured for 3 days with 500 nmol/L DEX and (or) 2.5 μmol/L RU486 (HY-13683, MedchemExpress Biotechnology Inc., USA, a GR antagonist), 500 nmol/L DEX and (or) 10 μmol/L miR -133a-3p inhibitor (RiboBio Technology, Guangzhou, China), or 500 nmol/L DEX and (or) 25 μmol/L Roscovitine (HY-B0231, Medchem Express Biotechnology Inc., a CDK5 inhibitor), respectively, and harvested for subsequent analysis.

Techniques: Expressing, Transmission Assay, Sequencing, Luciferase, Activity Assay, Mutagenesis, Transfection, Control, Comparison

Journal: Acta Pharmaceutica Sinica. B

Article Title: Gestational dexamethasone exposure impacts hippocampal excitatory synaptic transmission and learning and memory function with transgenerational effects

doi: 10.1016/j.apsb.2023.05.013

Figure Lengend Snippet: miR -133a-3p was confirmed to mediate dexamethasone-induced changes in brain SIRT1/CDK5/NR2B signaling axis by using zebrafish embryos. (A) (left) Schematic diagram of the microinjection of miR-133a Morpholinos (miR-133a MO) into zebrafish embryos at zygote stage. After microinjection, the zebrafish embryos were treated with 50 μmol/L dexamethasone (DEX) or not; (right) Relative expression of miR-133a-3p in zebrafish embryos at 120-h post fertilization (hpf) ( n = 6). (B) Probability of survival and hatching rate of zebrafish embryos at 24, 48, 72, 96, and 120 hpf ( n = 12). (C, E) mRNA expression of silent information regulator 1 ( sirt1 ), cyclin-dependent kinase 5 ( cdk5 ), synapsin I ( syn1 ) and synaptosome-associated protein 25 ( snap25 ) in zebrafish at 120 hpf ( n = 6). (D) Protein levels of Sirt1, Cdk5 and N -methyl- d -aspartate receptor (Nr2b) in zebrafish at 120 hpf ( n = 3). (F) The spontaneous tail contractions (STC) of zebrafish embryos at 24 hpf ( n = 10). Mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01 vs. Control; # P < 0.05, ## P < 0.01 vs. DEX. Using one-way ANOVA.

Article Snippet: To confirm the role of the glucocorticoid receptor (GR), miR-133a-3p and CDK5, the cells were cultured for 3 days with 500 nmol/L DEX and (or) 2.5 μmol/L RU486 (HY-13683, MedchemExpress Biotechnology Inc., USA, a GR antagonist), 500 nmol/L DEX and (or) 10 μmol/L miR -133a-3p inhibitor (RiboBio Technology, Guangzhou, China), or 500 nmol/L DEX and (or) 25 μmol/L Roscovitine (HY-B0231, Medchem Express Biotechnology Inc., a CDK5 inhibitor), respectively, and harvested for subsequent analysis.

Techniques: Microinjection, Expressing, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Gestational dexamethasone exposure impacts hippocampal excitatory synaptic transmission and learning and memory function with transgenerational effects

doi: 10.1016/j.apsb.2023.05.013

Figure Lengend Snippet: The level or expression of CDK5 in peripheral blood might serve as a potential early-warning marker for learning and memory impairment in offspring of prenatal dexamethasone exposure (PDE). (A–C) Serum CDK5 levels and mRNA expression of CDK5 in peripheral blood mononuclear cell (PBMC) ( n = 9–14), and the correlation analysis of CDK5 in serum or PBMC and in hippocampus of male offspring rats ( n = 7–12). (D–H) The correlation analysis of CDK5 in serum or PBMC and novel object recognition (NOR) experiments or Morris water maze (MWM) tests of male adult offspring rats ( n = 11–12). (I) Maternal age, corrected age, and birth weight of clinical infants ( n = 18–21). (J) (left) Serum CDK5 levels and mRNA expression of CDK5 in PBMC of clinical male infants. (right) The correlation analysis of CDK5 in serum and in PBMC of clinical male infants ( n = 18–21). Mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01 vs. Control. Using unpaired t test compared means between the control and treated groups. Pearson correlation analysis was used to analyze the correlation between two indicators.

Article Snippet: To confirm the role of the glucocorticoid receptor (GR), miR-133a-3p and CDK5, the cells were cultured for 3 days with 500 nmol/L DEX and (or) 2.5 μmol/L RU486 (HY-13683, MedchemExpress Biotechnology Inc., USA, a GR antagonist), 500 nmol/L DEX and (or) 10 μmol/L miR -133a-3p inhibitor (RiboBio Technology, Guangzhou, China), or 500 nmol/L DEX and (or) 25 μmol/L Roscovitine (HY-B0231, Medchem Express Biotechnology Inc., a CDK5 inhibitor), respectively, and harvested for subsequent analysis.

Techniques: Expressing, Marker, Control