ABSTRACT
Objective:To explore the effects and mechanism of PR-957 on hypoxic-ischemic brain damage(HIBD) of newborn rats.Methods:A total of 54 rats aged seven days were recruited and randomly divided into sham operation group, HIBD group and PR-957 intraperitoneal group.HIBD model was established according to modified Rice method.PR-957 group was given intraperitoneal injection PR-957(20 mg/kg) after hypoxic-ischemic.Model group was intraperitoneally injected with equal volume of DMSO.Ligation or hypoxic treatment were not given in sham operation group.HE staining was applied to observe inflammation in cortex.Immunohistochemical analysis was adopted to observe the expression of interleukin(IL)-17 and IL-10 in brain tissue.Western blot was carried out to test the protein level of low molecular poly peptide 7 (LMP7), forkhead box P3(FOXP3), and retinoic acid-recepter-related orphan receptor gamma t(RORγt). The proportion of T helper cells 17(Th17)/Treg was detected by flow cytometry.Results:HE staining displayed that sham operation group brain structure was basically normal, HIBD group revealed significant inflammation in the left cerebral cortex, while some pathological improvement was observed in PR-957 group.Immunohistochemistry: IL-10 positive cells in left cortex of HIBD group [(12.11±3.73)%] were lower than sham operation group[(29.12±3.95)%] and PR-957 group[(22.61±6.59)%], and the differences were statistically significant (all P<0.05). IL-17 positive cells in the left cortex of HIBD group [(35.55±4.85)%] were higher than sham operation group [(8.48±2.58)%] and PR-957 group [(19.16±4.31)%], and the differences were statistically significant (all P<0.05). Western blot: the expression of LMP7 and RORγt in HIBD group (1.01±0.12, 0.71±0.10) were higher than those in sham operation group (0.50±0.10, 0.34±0.07) and PR-957 group (0.65±0.13, 0.54±0.07), and the differences were statistically significant ( P<0.05). The expression of FOXP3 in HIBD group (0.44±0.10) was lower than sham operation group (0.93±0.07) and PR-957 group (0.68±0.09), and the differences were statistically significant (all P<0.05). The flow cytometry of peripheral blood: the Th17/Treg ratio of HIBD group (0.66±0.24) was higher than sham operation group (0.20±0.09) and PR-957 group (0.45±0.18), and the differences were statistically significant (all P<0.05). Conclusion:PR-957 can regulate the immune balance of Th17/Treg cells and reduce the inflammatory in the brain tissues of HIBD newborn rats.
ABSTRACT
Objective:To study the effect of melatonin (MEL) on the pyroptosis of hippocampus in neonatal rats with hypoxic-ischemic brain damage (HIBD), and the related mechanism.Methods:The animal model of HIBD was established by the modified Rice method.According to the random number table, a total of 105 Sprague-Dawley (SD) rats aged 7 days were divided into 7 groups (15 rats in each group): sham operation (Sham) group, model (HIBD) group, MEL treatment group (5, 10 and 20 mg/kg), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway inhibitor (LY294002) treatment group and MEL+ LY294002 group.The hippocampus neuronal morphology and the changes of nissl bodies were observed through HE staining and nissl staining.The mRNA expression levels of Nod-like receptor family 3 (NLRP3), apoptosis-associated speck-like protein containing a card (ASC), Caspase-1, gasdermin D (GSDMD), interleukin-1β (IL-1β) and interleukin-18 (IL-18) in the left hippocampus of rats were detected by real-time fluorescence quantitative PCR.The protein expression level of the above indexes and the level of phosphorylated Akt (p-Akt) were measured by Western blot.Results:Compared with the Sham group, the number of cell layers in hippocampal CA1 region in the HIBD group decreased, the cell arrangement was irregular, and there were less nissl bodies.Besides, the mRNA expression levels of NLRP3 (1.98±0.08 vs.0.86±0.13), ASC (1.40±0.12 vs.0.81±0.07), Caspase-1 (1.46±0.10 vs.0.75±0.09), GSDMD (1.35±0.10 vs.0.81±0.10), IL-18 (1.23±0.08 vs.0.23±0.04), IL-1β (1.83±0.09 vs.0.57±0.08) and p-Akt (1.12±0.12 vs.0.54±0.07) in the HIBD group were significant higher than those in the Sham group (all P<0.05). Compared with the HIBD group, there were more cell layers in hippocampal CA1 region of the MEL group (10 mg/kg), the arrangement of cells was more regular, and the number of nissl bodies increased.The mRNA expression levels of NLRP3 (1.04±0.10), ASC (0.91±0.06), Caspase-1 (0.63±0.06), GSDMD (1.01±0.09), IL-18 (0.65±0.05) and IL-1β (0.63±0.10) in the MEL group were statistically significantly lower than those in the HIBD group (all P<0.05). Compared with the MEL group (10 mg/kg), the arrangement of cells in hippocampal CA1 region of the MEL+ LY294002 group was relatively disordered, the nissl bodies declined, the p-Akt protein level (0.87±0.09 vs.1.99±0.27) decreased significantly, and the Caspase-1(p20) protein level (0.85±0.09 vs.0.58±0.09) increased significantly (all P<0.05). Conclusions:MEL may inhibit the hippocampal pyroptosis in neonatal rats with HIBD by activating the Akt signaling pathway, thereby protecting the brain.