| Objectives: To explore the neuroprotective effects and the underlying mechanism of baicalin in neonatal rats model surfered from hypoxic ischemic brain damage(HIBD). The present study was divided into two parts:1 The neuroprotective effect of baicalin and its influence on the PI3K/Akt signaling pathway and the expression of GLT-1 in neonatal HIBD ratMethods:(1) Grouping and Model building A total of 162 Seven-day old neonatal Sprague-Dawley(SD)rats were randomly divided into 3 groups(n=54 in each groups):Sham group(Sham), hypoxic ischemic brain damage group(HI) and baicalin treatment group(HI+Ba), each group was further divided into four time points at 2h(n=12),6h(n=12),12h(n=12),24h(n=18). HIBD model was induced as previously Rice described with slight modifications. Sham group were subjected to the separation of left carotid artery but not hypoxia ischemia exposure.Rats in HI+Ba group were given intraperitoneal injection of baicalin immediately after HIBD,while rats in Sham group and HIBD group were given with the same quantity physiological saline.(2) Outcome measures: Observe behavioral changes of rat after HIBD; 2,3,5-triphenyltetrazolium-chloride(TTC)staining method was used to detect cerebral infarction volume at 24 h after HIBD; Pups were decapitated at 2h,6h,12 h,24h after HIBD respectively,Nissle staining was used to speculate the histopathological change;Apoptosis of neuron were detected by Td T-mediated d UTP Nick-End Labeling(TUNEL) method; The expression of phosphorylated Akt(p-Akt) and GLT-1 were measured by immunohistochemistry and Western blot.Results:(1) By observing the neurological behavior and the changes of cerebral histopathology after hypoxic ischemic, it is proved that HIBD model was successfully established.(2) General examination of the brain: the ligated brain hemisphere of the HI group showed obvious pallor,edema and liquefactive necrosis at 24 h after HIBD.While brain edema and necrosis was not serious in the baicalin treatment group(HI+Ba).(3) TTC staining: The infarct ratio in HI group(28.51±2.45)% was markedly higher(P<0.01) than that in baicalin treatment group(13.39±1.69)%. The results indicated that baicalin could reduce the infarct volume of HIBD.(4) Nissl staining:In the Sham group,the cell outline was clear and the structure was compact. Cells were big and have abundant cytoplasm and Nissl body;While in HI group,cells in hippocampus arranged irregularly and edema appeared 2 h after model establishment. Further more, the decreased of cell number in hippocampus was seen in 6h after HIBD. These cells had the characteristics of nerve cell degeneration,such as disappearance of Nissl bodies, nuclear condensation, nuclear fragmentation and nuclear dissolution at 24 h after HIBD. Comparison at the same time point, the histological lesions in baicalin treatment group were much slighter compared to HI group. A large number of blue-stained Nissl bodies could be seen in the HI+Ba group.(5) Apoptosis index(AI): Few TUNEL-positive cells were identified in samples from Sham group pups.As time goes on,TUNEL-positive cells were significantly increased in hippocampus of HI group. AI achieve maximum at 24 h after HIBD.Compared with the HIBD group, the AI decreased at each time point in the groups with administration of baicalin(P<0.05).(6) p-Akt and GLT-1 expression 1)p-Akt expression: Immunohistochemical studies showed that p-Akt positive products were brown granules,mainly localized in cytoplasm.The change of p-Akt protein in the Sham group was not time-dependent. There was no statistically significant differences among the four time points(P>0.05).In HI group, a few weakly Akt positive cells were detected in the ischemic hippocampal CA1 area of ratat 2 h after HIBD.The expression of p-Akt significantly decreased at 2 h after HIBD compared with Sham group and HI+Ba group(P<0.01),but transiently increased and peaked at 6 h, and then decreased again at12 h and 24 h after HIBD.The level of p-Akt expression at12 h and 24 h in HI pups was significantly lower than Sham group and HI+Ba group(P<0.01). In HI+Ba group,p-Akt also decreased at 2 h after HIBD compared with Sham group(P<0.01),but baicalin prevented the reduction of p-Akt at 12 h and 24 h after HIBD. Compared with the HI group, treatment with baicalin persistently increased the level of phospho-Akt at 2h,12 h and 24 h after HIBD(P<0.01).The results of Western blot is consistent with immunohistochemistry results. 2)GLT-1 expression: Immunohistochemical studies showed that GLT-1 positive products were brown granules, mainly localized in cytomembrane.In the Sham group, the IOD/Area of GLT-1 also had no differences among 2h,6h,12 h and 24 h after HIBD(P>0.05).Compared with the Sham group, the expression of GLT-1 decreased at 2h in the HI group(P<0.01),but increased transiently and slightly,peaked at 12 h, and then decreased again at 24 h after HIBD.The level of GLT-1 expression at 24 h in HI group was much lower than Sham group(P<0.05). In HI+Ba group, although GLT-1 also decreased at 2 h after HIBD compared with Sham group(P<0.05), baicalin induced continuously expression of GLT-1 and significantly elevated the expression of GLT-1 at 6h,12 h and 24 h after HIBD compared with HI group and Sham group(P<0.01). The results of Western blot is consistent with immunohistochemistry results.Conclusions:(1) The model of HIBD in neonatal rats were successfully established.(2) Neurons in hippocampus were subjected to a serious injury and apoptosis in neonatal rat HIBD model.(3) The expression of p-Akt and GLT-1 in hippocampus tissue of neonatal rat after HIBD was significantly decreased at the early stage post HIBD,and increased transiently and slightly, but decreased again at 24 h after HIBD.(4) Baicalin can improve the pathological changes,reduce infarct volume and inhibit apoptosis in hippocampus of HIBD rat,which possibly results from activatingPI3K/Akt signaling pathway and increasing the levels of GLT-1.2 Blocking PI3K/Akt signal pathway to further explore the mechanism underlying the neuroprotective effect of baicalin.Methods:(1) Grouping:A total of 72 Seven-day old newborn SD rats were randomly divided into 4 groups(n=18 in each groups):Sham group(Sham),hypoxic ischemic brain damage group(HI),HIBD+baicalin+DMSO group(HI+Ba+DM)and HIBD+ baicalin+LY294002 group(HI+Ba+LY). In HI+Ba+DM group: 5μl 2% DMSO was injected intracerebroventricularly 30 min before HIBD, In HI+ Ba+LY group: 5μl inhibitor LY294002(dissolved in DMSO) was injected intracerebroventricularly 30 min before HIBD. Rats in HI+Ba+DM group and HI+ Ba+LY group were given intraperitoneal injection of baicalin immediately after HIBD.Sham group and HI pups received an equal volume physiological saline.(2) Outcome measures: All rats were sacrificed and the brain tissue samples were obtained at 24 h after HIBD. Infarction volume of injured brain was assessed by TTC staining(n=6); Nissle staining was used to speculate the histopathological change; Apoptosis of neurons were detected by TUNEL method(n=6); Western blot was applied to analyse the expression of p-Akt and GLT-1(n=6).Results:(1) TTC staining:No infarction was observed in Sham group. HI group showed a large area of infarction at 24 h after HIBD.Baicalin treatment group with vehicle DMSO(HI+Ba+DM) Significantly reduce cerebral infarct volumes compared with HI group(P<0.01). In HI+Ba+LY group,the use of PI3K/Akt inhibitor LY294002 weakened the effect of baicalin to reduce the infarct volumes in rat.The infarct ratio in HI+Ba+LY group was markedly higher(P<0.01)than that in HI+Ba+DM group, but lower than HI group(P<0.01).(2)Nissl staining:Cellular morphology was normal in the sham group. In HI group,the cell outline was fuzzy,the cell arranged sparsely and the Nissl bodies cannot be seen as well. The cell number in hippocampus of HI group was reducedobviously compared with sham group.The structure of neuron in HI+Ba+DM was similar with that of sham operation group rats and better than that of rats in HI group.A large number of Nissl bodies can be observed in HI+Ba+DM group.Baicalin ameliorate the pathological changes of damaged brain.but the ameliorating injury effect of baicalin was abolished by pretreatment with PI3K/Akt inhibitor LY294002.Such changes in HI+ Ba+LY group include: disappearance of Nissl bodies, nuclear condensation, nuclear fragmentation and nuclear dissolution.These histological injury was similar to the changes in HI group.(3) Apoptosis index(AI) :Almost no TUNEL-positive cells were observed in brains subjected to sham surgery. In HI group, numerous neurons were strongly positive for TUNEL staining. In the samples of baicalin treatment group, the number of apoptotic cells was significantly decreased compared with HI group(P<0.01).Pretreatment with inhibitor LY294002 weakened the effect of baicalin to reduce the AI in rat.The AI was markedly increased at 24 h after HIBD in HI+Ba+LY group as compared with the HI+Ba+DM group(P<0.01).However,the AI in the HI+Ba+LY group was still decreased compared with the HI group(P<0.01).(4) p-Akt and GLT-1 expression The expression of p-Akt and GLT-1 in HI group both decreased significantly compared with sham group at 24 h after HIBD(P<0.01),which were markedly up-regulated by baicalin post-treatment compared with sham group and HI group(P<0.01).LY294002 can completely abolished the elevation of Akt phosphorylation induced by baicalein in rats(P<0.01).The expression of p-Akt data showed that there was no statistical differences between HI group and HI+Ba+LY group(P>0.05). However, LY294002 only partially blocked the elevation of GLT-1 induced by baicalein in rat(P<0.01).The expression of GLT-1 in HI+Ba+LY group was higher than HI group(P<0.01).Conclusions:(1) PI3K/Akt inhibitor LY294002 blocked the increase of p-Akt and GLT-1 evoked by baicalein and abolished the associated neuroprotective effect.(2) Baicalin protects brains against hypoxic-ischemic injury by up-regulating GLT-1 via the PI3K/Akt signaling pathway. |
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