ABSTRACT
Objective To investigate the protective effect of inhibition of hypoxia inducible factor－1 alpha (HIF－1α) and activation of Wnt signaling pathway on brain injury in neonatal rats.Method A total of 312 newborn SD rats were used to establish cerebral white matter damage model (WMD) on day 3.And they were randomly assigned into the model group (WMD group), post－modeling HIF－1αdecomposition inhibition group (PHI group), and post－modeling HIF－1αdecomposition inhibition with activation of Wnt signaling pathway group (PHI+activated Wnt group ) and post－modeling HIF－1αdecomposition inhibition with inhibition of the Wnt signaling pathway group (PHI+inhibition Wnt group).Brain tissues were taken on day 1, 3, 7 and 14 after modeling, respectively.The changes of oligodendrocyte precursor cell (OPC) and oligodendrocyte ( OL ) in brain tissues at different time points were observed by tissue immunofluorescence.The expression of HIF－1αprotein in the brain tissue of each time point was measured by western blot technique.The mRNA level of HIF－1αand Wnt7a in the brain tissue of each time point was detected by RT－qPCR technique.Behaviors of rats were tested by the suspension experiment , the open field experiment and the dark experiment , at 28 d after modeling.Result The numbers of OPC on 1 d and 3 d after modeling and the number of OL on 7 d and 14 d after modeling in PHI +activated Wnt group were significantly higher than the other three groups.The content of HIF－1αin WMD group was the least on 1, 3, 7 and 14 d, but the content of PHI+activated Wnt group was the highest , and the difference was statistically significant (P＜0.05).On 1, 3, 7 and 14 d after modeling, the expression level of HIF－1αand Wnt7a in PHI+activated Wnt group were higher than those in the other three groups , the difference was statistically significant (P＜0.05), and Wnt7a expression was positively correlated with the change of HIF－1α(P＜0.05).There was no significant difference of suspension experiment at 28 d after modeling between groups. Compared with other groups , WMD group had the lowest score on open field experiment ( P＜0.05).The PHI+activation Wnt group had better memory function , and then the PHI group , WMD group.The latent time of dark experiment were significantly shorter in PHI +acitivation Wnt group.There were more mistaken time of dark experiment in WMD group compared with PHI +activation Wnt group.Conclusion Inhibition of HIF－1αdecomposition and activation of Wnt signaling pathway have partially repair effect on brain injury in neonatal rats.
ABSTRACT
ObjectiveTo determine the changes in tumor necrosis factor-α (TNF-α) and interleukin-lβ (IL-1β)expression after bone marrow mesenchymal stem cells (BMSCs) transplantation in a rat pup model of hypoxic-ischemic brain damage (HIBD) and discuss the anti-inflammatory mechanism of BMSCs transplantation in the repair of HIBD.MethodsThree-day-old Sprague-Dawley rat pups were randomly divided into three groups: the transplantation group, in which a model of HIBD was established by ligation of left common carotid artery and hypoxia for two hours followed by the injection of 2μl BMSCs (2×105 cells) into the lateral ventricle; the HIBD model group, in which HIBD model was established and 2μl phosphate saline buffer was injected into the lateral ventricle; and the sham-operation group, in which no intervention was given. Histological changes in the brain were detected by HE staining and the number of cells positive for ectodermal dysplasia-1 (ED-1) staining, which is a specific marker for activated microglia, was detected by immunohistochemistry. The protein and mRNA levels of TNF-α and IL-1β were determined by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. One-way analysis of variance and LSD test were applied for statistical analysis.ResultsHE staining showed that cellular edema and necrocytosis was not observed in cerebral white matter on the 7th post-transplantation day in the transplantation group, but observed in the HIBD model group. In the sham-operation group, cerebral white matter was normal. The number of ED-1 positive cells in the transplantation group (26.3±2.5) was significantly lower than that in the HIBD model group (33.0±4.0), but higher than that in the sham-operation group (2.3±0.6) (LSD test, allP<0.05). The contents of TNF-α and IL-1β both in the transplantation group and the HIBD model group increased and peaked 24 h after transplantation, then gradually decreased, but did not reach normal levels (sham-operation group) on the 7th day. The contents of TNF-α and IL-1β in brain tissue in the HIBD model group [TNF-α: (3.03±0.10), (5.57±0.19), (7.78±0.19), (4.39±0.20), (2.70±019)μg/L; IL-1β:(293.1±7.9), (369.8±17.5), (303.6±23.9), (226.7±21.6), (183.9±33.4) ng/L] were significantly higher than those in the transplantation group [TNF-α: (2.84±0.20), (3.80±0.14), (4.63±0.17), (3.56±0.03), (1.99± 0.17)μg/L; IL-1β: (267.6±14.5), (323.5±26.9), (211.2±24.9), (140.8±7.4), (100.2±8.3) ng/L] at 6, 12, 24, 48 h and 7 days after BMSCs transplantation, respectively. The contents of TNF-α and IL-1β in the sham-operation group [TNF-α:(1.03±0.02), (1.13±0.03), (1.05±0.02), (1.09±0.02), (1.07±0.02)μg/L; IL-1β:(63.6±13.0), (64.0±11.3), (60.8±10.0), (67.9±13.5), (66.2±11.7) ng/L] were significantly lower than those in the transplantation group and HIBD model group (LSD test, allP<0.05). TNF-α and IL-1β mRNA at 24 h after transplantation in the HIBD model group (TNF-α: 2.69±0.43; IL-1β: 3.07±0.38) were significantly higher than those in the transplantation group (TNF-α: 1.61±0.29; IL-1β: 1.08±0.11) and those in the sham-operation group (TNF-α: 0.94±0.16; IL-1β: 1.08±0.11) (LSD test, allP<0.05).ConclusionsBMSCs may play an important role in the recovery of preterm HIBD and the mechanism may involve the inhibition of microglia activation and down-regulation of the expression of inflammatory factors.