| Objective This study aimed to investigate the protective effects of Oxymatrine(OMT)on hypoxic-ischemic brain damage(HIBD)in neonatal rats,and explore the correlation between its protective effects and Wnt/?-Catenin signaling pathway.Methods 1.To observe the protective effect of OMT on neonatal rats with HIBD In vivo: An HIBD model of 7-day-old SD rats was established,and the neonatal rats were randomly divided into three groups: Sham,HI and HI+OMT(120mg/kg)group.The cerebral blood flow was detected by laser speckle imaging,and ELISA was determined the level of S-100 B protein in serum.In vitro: The model of primary hippocampal neuron oxygen glucose deprivation(OGD)injury in neonatal rats was established,cell viability,the level of mitochondrial membrane potential(MMP),and the degree of DNA damage were detected to explore the protective effect of OMT on primary hippocampal neurons.2.To explore the protective effect of OMT on HIBD in neonatal rats and the relationship between Wnt/?-Catenin pathway In vivo: The neonatal rats were divided into Sham,HI,OMT and XAV-939 group,respectively.At 48 h after intraperitoneal injection with OMT or resolvent,cerebral infract volume,histomorphological changes and the amount of TUNEL positive cells were performed to evaluate brain injury.The protein expression of Wnt1,Dvl2,DKK1,Axin1,APC,GSK-3?/p-GSK-3? and ?-Catenin was measured by Western blot.Moreover,gel migration assay was conducted to assess the binding activity of ?-Catenin to Tcf-4.In vitro: After intervention with an inhibitor of XAV-939,the m RNA level of Wnt1 and ?-Catenin was measured by q-PCR.Besides,Western blot was used to detect the protein expression of c-?-Catenin,n-?-Catenin,Tcf-4,Bax,Bcl-2,cleaved-Caspase-3 and cleaved-Caspase-9.Results 1.The neuroprotective effects of OMT on HIBD in neonatal rats.Compared with sham group,the cerebral blood flow in HI group was significantly reduced(P<0.01),and the content of S-100 B protein in serum was markedly increased(P<0.01).In contrast with HI group,OMT(120mg/kg)post-treatment markedly increased the cerebral blood flow(P<0.01),as well as decreased the level of S-100 B protein(P<0.01).Furthermore,the viability of primary hippocampal neurons and the levels of MMP in OGD group were significantly decreased compared with control group(P<0.01).Single cell gel electrophoresis result showed that the degree of DNA damage in OGD group was more obvious than that in control group(P<0.01).However,OMT treatment increased the viability of neurons(P<0.01),MMP levels(P<0.01),and alleviated the DNA damage(P<0.01).2.The underlying mechanism of OMT on HIBD in neonatal rats.The cerebral infarct volume and TUNEL positive cells in HI group were markedly increased than that in sham group(P<0.01),and the histomorphological damage was exacerbation.Compared with sham group,the protein expression of DKK1,Axin1,APC and GSK-3? in HI group were dramatically increased(P<0.01),while the protein levels of Wnt1,Dvl2,p-GSK-3? and ?-Catenin were remarkably decreased(P<0.01).Moreover,the binding activity of ?-Catenin protein with Tcf-4 in HI group was lower than that in sham group.However,OMT post-treatment significantly decreased cerebral infract volume,TUNEL positive cells(P<0.01),and improved the histopathological injury.Besides,the DKK1,Axin1,APC and GSK-3? protein expression in OMT group were reduced(P<0.01),while the expression of Wnt1,Dvl2,p-GSK-3? and ?-Catenin were up-regulated than that in HI group(P<0.01).In addition,OMT increased the binding activity of ?-Catenin protein to Tcf-4.Nevertheless,XAV-939 administration weakened the protective effect of OMT on neonatal rats.In vitro,the m RNA levels of Wnt1 and ?-Catenin,as well as the protein expression of n-?-Catenin,Tcf-4,and Bcl-2 in OGD group were significantly decreased compared with control group(P<0.01).Meanwhile,the protein contents of Bax,cleaved-Caspase-3 and cleaved-Caspase-9 were significantly increased(P<0.01).OMT promoted the m RNA levels of Wnt1 and ?-Catenin,and enhanced the protein expression of n-?-Catenin,Tcf-4,and Bcl-2.Besides,Western blot analyses demonstrated that OMT reduced the expression of Bax,cleaved-Caspase-3 and cleaved-Caspase-9(P<0.01).Whereas,the regulating effect of OMT on primary hippocampal neurons could be partly inhibited by XAV-939.Conclusions We further demonstrated that OMT exerts neuroprotection on neonatal rats subjected to HIBD.The neuroprotective effect of OMT on neonatal rats with HIBD is related to the activation of Wnt/?-Catenin signaling pathway in primary hippocampal neurons. |
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