| Objectives:I.Investigating the therapeutic effect of scutellarin on HIE in newborn rats.2.Elucidating the relationship between scutellarin and GAP43 in the treatment of hypoxic-ischemic encephalopathy in newborn rats.Methods:1.An OGD model of primary cortical neurons was used to simulate hypoxic-ischemic encephalopathy in newborn rats in vitro and to investigate the effect of scutellarin on neurons.The potential mechanism of scutellarin was elucidated by RNA interference technique.2.The scutellarin-HIE-related targets and potential mechanism was predicted by using network pharmacology and bioinformatics.3.The rat HIE model was used to screen the optimal drug concentration of scutellarin,60 SD rats were randomly divided into 5 groups:sham group,HI+NS group,HI+SCUhigh?HI+SCUmedium and HI+SCUlow,10 rats in each group.The TTC staining was used to evaluate the success of the model,and the Zea-longa score,MWMT,RT,OFT and YMT were used to evaluate the neurobehavior of each animal.The mRNA expression level of GAP43,PTN,JAK2,STAT3 was detected by qRT-PCR.4.After construction of GAP43 knockout rats,the HI model of newborn rats was constructed by classical Vannucci method.40 SD rats were randomly divided into 4 groups:Sham group,GAP43+/++HI+ NS,GAP43+/++HI+SCU,GAP43-/-+HI+SCU,10 rats in each group.The neurological function of each animals was evaluated by neurobehavioral evaluation(Zea-longa score,MWMT,RT,OFT,YMT).The relationship between scutellarin and related signaling pathway molecules was further illuminated by qRT-PCR.Results:Experiment in vitro:1.The OGD model in vitro showed that scutellarin could significantly increase cell viability and cell number of neurons(P<0.05).TUNEL staining showed that scutellarin significantly decreased cell apoptosis(P<0.05).2.qRT-PCR showed a significant increase in GAP43 expression in neurons after treatment with scutellarin(P<0.05).3.The results of GAP43 interference experiment showed that after GAP43 interference,the phenomenon of scutellarin promoting cell proliferation and inhibiting apoptosis was reversed,that is,the apoptosis of OGD+Scu+GAP43-si group increased significantly compared with that of the OGD+Scu+NC group(P<0.05).4.The immunofluorescence staining showed that the number of Tuj positive cells in the OGD+Scu+GAP43-si group decreased significantly compared with the OGD+Scu+NC group(P<0.05).5.qRT-PCR results showed that JAK2?STAT3 and PTN expression in neurons increased significantly after GAP43 interference(P<0.05).Prediction of scutellarin-HIE target:1.Databases such as DRUGBANK?STITCH?SwissTargetPrediction?TCMSP and PUBCHEM indicated that scutellarin has 206 potential targets.2.Databases such as Genecard?Disgenet and OMIM screened 1161 potential targets of HIE.3.GO and KEGG analysis show that scutellain-HIE related targets were involved in a total of 1,721 GO term.4.GeneMania analysis shows that there is interaction between JAK2,STAT3,GAP43 and PTN.Drug concentration screening in vivo:1.TTC staining showed that scutellarin(20 mg/kg)significantly decreased the proportion of cerebral infarction in rats(P<0.05).2.Zea-longa score showed that scutellarin significantly improved the symptoms of neurological deficit in HIE rats(P<0.05).3.The results of MWMT and YMT showed that scutellarin significantly improved the spatial learning and memory ability of HIE rats(P<0.05).4.According to the results of rotarod test,the somatomotor function of HIE rats was improved significantly by scutellarin(P<0.05).5,The results of open field test showed that scutellarin significantly improved anxiety-suppressive behavior in HIE rats exposed to new environment(P<0.05).Experiment of GAP43 Knockout rat in vivo:1.The results of TTC staining showed that the proportion of cerebral infarction and cerebral edema in GAP43-/-rats were significantly aggravated compared with GAP43+/+rats(P<0.05).2.Zea-longa scores showed that the GAP43+/++HI+Scu group returned to normal on the 8th day after operation,while the GAP43+/++HI and GAP43-/-+HI+Scu groups returned to normal on the 9th day.3.The results of MWMT and YMT showed that scutellarin significantly improved spatial learning and memory ability in GAP43+/+rats(P<0.05).However,the spatial learning and memory ability of GAP43-/-rats were not significantly improved.4.The results of rotarod test showed that the longest residence time of rats in GAP43-/-+HI+Scu group decreased significantly compared with GAP43+/++HI+Scu group(P<0.05).5.The results of open field test showed that the number of grooming,the peripheral latentperiod and the central latentperiod of the rats in the GAP43-/-+HI+Scu group were significantly increased compared with GAP43+/++Hl+Scu group(P<0.05).As well as,the peripheral distance,the standing number and the central distance were significantly decreased compared with GAP43+/++HI+Scu group(P<0.05).6.The qRT-PCR results showed that the GAP43 expression in the cortex of GAP43+/++HI+Scu group was significantly increased,and the expression of PTN,JAK2,STAT3 were significantly decreased.Besides,the expression of JAK2,STAT3 and PTN were significantly increased in the GAP43-/-+HI+Scu group.Conclusion:1.Scutellarin significantly improved learning,memory and motor function in HIE rats.2.The effect of scutellarin on learning memory and motor function in HIE rats is related to GAP43.3.The role of scutellarin in improving learning memory and motor function in HIE rats is associated with JAK2/STAT3/PTN/GAP43 signaling pathways. |
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