Growth arrest DNA damage-inducible protein 45 β (GADD45B) has been reported to be a regulatory factor for active DNA demethylation and is implicated in the modulation of synaptic plasticity and chronic stress-related psychopathological processes. However, its precise role and mechanism of action in stress susceptibility remain elusive. In this study, we found a significant reduction in GADD45B expression specifically in the ventral, but not the dorsal hippocampal CA1 (dCA1) of stress-susceptible mice. Furthermore, we demonstrated that GADD45B negatively regulates susceptibility to social stress and NMDA receptor-dependent long-term potentiation (LTP) in the ventral hippocampal CA1 (vCA1). Importantly, through pharmacological inhibition using the NMDA receptor antagonist MK801, we provided further evidence supporting the hypothesis that GADD45B potentially modulates susceptibility to social stress by influencing NMDA receptor-mediated LTP. Collectively, these results suggested that modulation of NMDA receptor-mediated synaptic plasticity is a pivotal mechanism underlying the regulation of susceptibility to social stress by GADD45B.
Animals ; Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors* ; CA1 Region, Hippocampal/drug effects* ; Male ; Stress, Psychological/physiopathology* ; Mice ; Neuronal Plasticity/drug effects* ; Long-Term Potentiation/drug effects* ; Mice, Inbred C57BL ; Antigens, Differentiation/metabolism* ; Dizocilpine Maleate/pharmacology* ; Excitatory Amino Acid Antagonists/pharmacology* ; GADD45 Proteins

Objective:To construct the prokaryotic expression vector of Coxsackievirus A16(CA16)viral protein 1(Vp1),express it in Escherichia coli(E.coli)BL21,purify the protein,prepare rabbit polyclonal antibodies,and identify the immunoreactivity of the antibodies.Methods:Bioinformatics online tools were used to predict the amino acid composition,conserved domains,secondary and tertiary structures of the Vp1 protein.The Vp1 gene of CA16 was amplified and cloned into the prokaryotic expression vector pET28a(+).The pET28a-Vp1 was transformed into E.coli BL21,and the expression was induced using isopropyl β-D-thiogalactopyranoside(IPTG).Western blotting method was used to identify the induced expression of Vp1 protein,and the induction time and temperature were optimized to improve the expression efficiency.Two female SPF New Zealand white rabbits were taken,and the purified recombinant protein Vp1 was used to immunize the rabbits to prepare rabbit anti-Vp1 protein polyclonal antibodies.The antibody titer was determined by ELISA method,and the immunoreactivity of the antibodies was identified by Western blotting method.Results:The bioinformatics analysis results showed that the Vp1 gene encoded 297 amino acids,with a relative molecular mass of 33 046.39 and an isoelectric point of 8.32,belonging to a hydrophilic protein;in the secondary structure,α-helix accounted for 15.15％,random coil accounted for 67.68％,and extended strand accounted for 17.17％.Sequencing of the recombinant plasmid pET28a-Vp1 confirmed that the pET28a-Vp1 plasmid was correctly constructed.The Western blotting results showed that the target protein was expressed in the IPTG induction group at a relative molecular mass of 33 000,and the target protein was mainly expressed in inclusion bodies.The optimal induction conditions for protein expression were IPTG concentration of 0.4 mmol·L-1,temperature of 16℃,and induction time of 20 h.The ELISA assay results showed that the titer of the rabbit polyclonal antibody was 1:1 024 000.The Western blotting results showed that the Vp1 rabbit polyclonal antibody could bind to the viral Vp1 protein in the CA16-infected cells.Conclusion:The polyclonal antibody against CA16 Vp1 is successfully prepared,and this antibody has significant binding characteristics to CA16 Vp1,which can be used for the diagnosis of enterovirus infection and the development of treatment methods.

Objective To investigate the role and underlying mechanism of atorvastatin on hyper-glycemia induced hemorrhagic transformation(HT)in a mouse model of cerebral ischemia.Meth-ods A total of 36 SPF-grade male C57BL/6 mice were randomly divided into sham operation group,HT model group and atorvastatin group,with 12 mice in each group.HE staining was used to observe cerebral hemorrhage,immunofluorescent staining was employed to detect the integrity of blood-brain barrier,and Western blotting was applied to measure the protein expression of IgG,ZO-1,occludin,claduin5,MMP-2 and-9 in ischemic penumbra brain tissues.Results Com-pared with sham operation group,the neurological deficit score,mortality rate,HT incidence,HT grading score,IgG fluorescence intensity,and protein levels of IgG,MMP-2 and-9 were signifi-cantly increased,while the protein levels of ZO-1,occludin and claudin5 were obviously decreased in the HT model group(P＜0.01).Atorvastatin treatment resulted in significantly lower neuro-logical deficit score(2.73±1.19 vs 3.91±0.94),mortality rate(16.7％vs 41.6％),HT incidence(58.3％vs 91.6％),HT grading score(1.00±1.04 vs 2.58±1.13),IgG fluorescence intensity(504.30±105.52 a.u vs 859.91±153.28 a.u),and protein levels of IgG(4.55±1.40 vs 12.06± 3.73),MMP-2(1.87±0.41 vs 2.95±0.68)and-9(1.47±0.24 vs 2.12±0.23)(P＜0.05,P＜0.01),and increased protein levels of ZO-1(1.55±0.20 vs 0.53±0.10),occludin(0.92±0.11 vs 0.35±0.07)and claudin5(0.58±0.04 vs 0.30±0.05)(P＜0.01)when compared with the HT model group.Conclusion Atorvastatin can reduce the permeability of blood-brain barrier by in-hibiting the activation of MMP-2 and MMP-9 and up-regulating the protein levels of ZO-1,occlu-din and claudin5,and thus attenuate hyperglycemia-induced HT.