Objective To evaluate the effects of hyperbaric oxygen post-conditioning on the expression of P2X4 receptors in the spinal dorsal horn and hippocampus of rats with neuropathic pain (NP).Methods Seventytwo male Sprague-Dawley rats,aged 8-10 weeks,weighing 300-350 g,were randomly divided into 3 groups (n =24 each) using a random number table:sham operation group (group S),NP group and hyperbaric oxygen postconditioning group (group H).NP was induced by chronic constrictive injury.The rats in group H underwent hyperbaric oxygen treatment once a day for 7 consecutive days starting from 1 day after NP was successfully induced.After the rats were placed in the hyperbaric oxygen chamber,the pressure was increased at a rate of 10 kPa/min until the hyperbaric oxygen was at 2.0 atmosphere absolute,and maintained at this level for 60 min,and then the pressure was decreased at a rate of 10 kPa/min until the normal pressure was reached.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before NP was induced and 1,3,7 and 14 days after NP was induced.After the end of measurement,6 rats were randomly chosen from each group and then sacrificed and the L4-6 segments of the spinal cord and hippocampal tissues were removed for determination of the expression of P2X4 receptors (by immunohistochemistry).Results Compared with group S,the MWT was significantly decreased,TWL was shortened,and P2X4 receptor expression in the spinal dorsal horn and hippocampus was up-regulated in NP and H groups.Compared with group NP,the MWT was significantly increased and TWL was prolonged,and P2X4 receptor expression in the spinal dorsal horn and hippocampus was down-regulated in group H.Conclusion Hyperbaric oxygen post-conditioning mitigates NP by down-regulating the expression of P2X4 receptors in the spinal dorsal horn and hippocampus of rats.

BACKGROUND: Radiation (IR)-induced DNA damage triggers cell cycle arrest and has a suppressive effect on the tumor microenvironment (TME). Wee1, a cell cycle regulator, can eliminate G2/M arrest by phosphorylating cyclin-dependent kinase 1 (CDK1). Meanwhile, programed death-1/programed death ligand-1 (PD-1/PDL-1) blockade is closely related to TME. This study aims to investigate the effects and mechanisms of Wee1 inhibitor AZD1775 and anti-PD-1 antibody (anti-PD-1 Ab) on radiosensitization of hepatoma. METHODS: The anti-tumor activity of AZD1775 and IR was determined by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay on human and mouse hepatoma cells HepG2, Hepa1-6, and H22. The anti-hepatoma mechanism of AZD1775 and IR revealed by flow cytometry and Western blot in vitro . A hepatoma subcutaneous xenograft mice model was constructed on Balb/c mice, which were divided into control group, IR group, AZD1775 group, IR + AZD1775 group, IR + anti-PD-1 Ab group, and the IR + AZD1775 + anti-PD-1 Ab group. Cytotoxic CD8 + T cells in TME were analyzed by flow cytometry. RESULTS: Combining IR with AZD1775 synergistically reduced the viability of hepatoma cells in vitro . AZD1775 exhibited antitumor effects by decreasing CDK1 phosphorylation to reverse the IR-induced G2/M arrest and increasing IR-induced DNA damage. AZD1775 treatment also reduced the proportion of PD-1 + /CD8 + T cells in the spleen of hepatoma subcutaneous xenograft mice. Further studies revealed that AZD1775 and anti-PD-1 Ab could enhance the radiosensitivity of hepatoma by enhancing the levels of interferon γ (IFNγ) + or Ki67 + CD8 T cells and decreasing the levels of CD8 + Tregs cells in the tumor and spleen of the hepatoma mice model, indicating that the improvement of TME was manifested by increasing the cytotoxic factor IFNγ expression, enhancing CD8 + T cells proliferation, and weakening CD8 + T cells depletion. CONCLUSIONS This work suggests that AZD1775 and anti-PD-1 Ab synergistically sensitize hepatoma to radiotherapy by enhancing IR-induced DNA damage and improving cytotoxic CD8 + T cells in TME.
Humans ; Animals ; Mice ; Carcinoma, Hepatocellular/radiotherapy* ; Cell Cycle Proteins/metabolism* ; Protein-Tyrosine Kinases/genetics* ; Apoptosis ; Programmed Cell Death 1 Receptor ; Cell Line, Tumor ; G2 Phase Cell Cycle Checkpoints ; Liver Neoplasms/radiotherapy* ; Tumor Microenvironment ; Pyrazoles ; Pyrimidinones