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

High mobility group box 1 (HMGB1), when released extracellularly, plays a pivotal role in the development of spinal cord synapses and exacerbates autoimmune diseases within the central nervous system. In experimental autoimmune encephalomyelitis (EAE), a condition that models multiple sclerosis, the levels of extracellular HMGB1 and interleukin-33 (IL-33) have been found to be inversely correlated. However, the mechanism by which IL-33 deficiency enhances HMGB1 release during EAE remains elusive. Our study elucidates a potential signaling pathway whereby the absence of IL-33 leads to increased binding of P300/CBP-associated factor with HMGB1 in the nuclei of astrocytes, upregulating HMGB1 acetylation and promoting its release from astrocyte nuclei in the spinal cord of EAE mice. Conversely, the addition of IL-33 counteracts the TNF-α-induced increase in HMGB1 and acetylated HMGB1 levels in primary astrocytes. These findings underscore the potential of IL-33-associated signaling pathways as a therapeutic target for EAE treatment.
Animals ; Encephalomyelitis, Autoimmune, Experimental/metabolism* ; Astrocytes/metabolism* ; Interleukin-33/metabolism* ; HMGB1 Protein/metabolism* ; Acetylation ; Mice, Knockout ; Mice, Inbred C57BL ; p300-CBP Transcription Factors/metabolism* ; Mice ; Spinal Cord/metabolism* ; Cells, Cultured ; Female ; Signal Transduction

Objective: To observe the effect of Huangjing Zanyu Capsule (HZC) on sperm mitochondrial membrane potential (MMP) in asthenozoospermia patients. METHODS: We assigned 70 asthenozoospermia patients to a treatment group (n = 39) and a control group (n = 31), the former treated with oral HZC at the dose of 4 capsules tid for 3 months while the latter left untreated. We obtained semen parameters from the patients and detected their sperm mitochondrial membrane potentials (MMP) by JC-1 staining and flow cytometry before and after medication, followed by comparison between the two groups. RESULTS: The total effectiveness rate was 71.05% in the treatment group and natural pregnancy was achieved in 3 cases during the medication. A total of 35 patients in the treatment group and 30 controls completed all the laboratory examinations after a 3-month observation. Compared with the controls, the patients treated with HZC exhibited significant improvement after medication in MMP (variation value: ［1.19 ± 10.36］% vs ［20.28 ± 14.21］%, P <0.01), total sperm motility (variation value: ［3.46 ± 8.67］% vs ［20.68 ± 14.12］%, P <0.01), the percentage of progressively motile sperm (variation value: ［2.26 ± 8.29］% vs ［17.58 ± 12.73］%, P <0.01), and the percentage of morphologically normal sperm (variation value: ［0.23 ± 3.48］% vs ［3.37 ± 3.99］%, P <0.01). MMP was significantly correlated with total sperm motility (r = 0.69, P <0.01), progressive sperm motility (r = 0.75, P <0.01) and normal sperm morphology (r = 0.26, P <0.01). CONCLUSIONS Huangjing Zanyu Capsule can enhance sperm mitochondrial membrane potential and sperm mitochondrial function, thus improving total sperm motility, progressive sperm motility and normal sperm morphology. It is safe and effective for the treatment of asthenospermia.
Asthenozoospermia ; drug therapy ; Capsules ; Case-Control Studies ; Drugs, Chinese Herbal ; administration & dosage ; therapeutic use ; Female ; Flow Cytometry ; Humans ; Male ; Membrane Potential, Mitochondrial ; drug effects ; physiology ; Pregnancy ; Semen ; drug effects ; Sperm Motility ; drug effects ; Spermatozoa ; drug effects ; ultrastructure ; Staining and Labeling

Objective:To prepare GST-Orai2 fusion protein and to prepare polyclonal antibody against Orai2 by immunizing rabbits.To further investigate the function of Orai2,a transmembrane protein,this antibody was used to identify the Orai2 conditional gene knockout mice.Methods:ORF of Orai2 was amplified by PCR and subcloned into pGEX-6p-1 vector.After transforming BL21 (DE3) competent cells,we succeeded in inducing the expression of GST-Orai2 fusion protein using IPTG.Then the GST-Orai2 protein was purified by immobilized Glutathione affinity chromatography and identified by SDS-PAGE.A New England rabbit was immunized with the prepared fusion protein in Freund's adjuvant to prepare specific antibody.Finally,the prepared antibody was identified by Western blot by checking its titer and specificity.Furthermore,we took use of the prepared Orai2 antibody to identify Orai2 conditional gene knockout mice,with comparing to the wildtype ones in the same cage.Results:The purity of purified GST-Orai2 reached to 90% and the concentration was 0.35 mg/ml by BCA kit.We could detect Orai2 protein even in dilution of 1:10,000.Also,the prepared polyclonal antibody agianst Orai2 could detect both overexpressed and endogenous Orai2 protein in mouse-brain,without crossing reaction with Orai1.As well,we found that the Orai2 protein expression was of obvious reduction in Orai2 conditional gene knockout mice,compared with the wildtype ones in the same cage.Conclusion:We successfully obtain the purified GST-Orai2 fusion protein and prepare specific and highly sensitive polyclonal antibody against Orai2.The antibody can be used to detect overexpressed and endogenous Orai2 protein inmouse-brain specifically,and to identify Orai2 conditional gene knockout mice,without any crossing reaction with Orai1.Our work contributes a lot to the future investigation of functions of Orai2.

Background and Purpose:Cyclooxygenase-2(COX-2) plays an important part in tumor genesis,growth,and angiogenesis.Many inhibitors of COX-2 could inhibit proliferation and induce apoptosis of cancer cells. This study investigated the impact of NS398 on anti-proliferation and the induction of apoptosis in human osteosarcoma cell line MG-63.Methods:Cell proliferation is measured by MTT method.Characteristic changes of apoptosis in morphology are observed by fluorescence microscopy、transmission electron microscopy(TEM) and quantitatively by TDT-mediated dUTP-biotin nick end-labeling(TUNEL) assay.The apoptotic rates are calculated by flow cytometry(FCM).Results:The growth inhibition rates of MG-63 cells treated with 1,10,50,100 and 200 ?mol/L NS398 are 14.7%,23.5%,33.6%,52.5% and 81.4%,respectively(P