Effects Of Ginkgolide B On Apoptosis Of Neural Cells In Neonatal Rat Induced By Hypoxia-ischemia And Its Mechanisms

Aim of the studyThe hypoxic ischemic brain damage was established according to the classical Rice.Application of Ginkgolide B,LY294002 inhibitor,U0126 inhibitor for intervention.Then levels of BDNF,P-AKT(ser473),P-ERK1/2,Cleaved caspase-3 and apoptosis of nerve cells in the damaged cortex were performed following HI.To investigate the effects of Ginkgolide B on the expression of BDNF following hypoxia–ischemia in neonatal rats,and the role of PI3K-AKT or MEK-ERK signaling pathway in the anti-apoptosis of Ginkgolide B after brain injury.MethodsFirstly 160 pups at P7 were randomly divided into four groups: Sham-operated group,HI group,GBL(10mg/Kg)group,GBH(20mg/Kg)group.Pups were treated with Ginkgobalide B at 0h,24 h after HI.Each group were randomly sacrificed 10 rats at different time points(6h,12 h,24h,48h)after HI.The levers of Caspase-3 and BDNF gene were detected by polymerase chain reaction(PCR).The results showed that the maximum effect point was 24 h after hypoxia–ischemia,and the effect of high dose Ginkgobalide B was better than that of low dose Ginkgobalide B.Then the other 140 neonatal rats were randomly divided into seven groups:Sham-operated group(Sham,n=20);hypoxia–ischemia group(HI,n=20);hypoxia–ischemia plus 20 mg/kg Ginkgobalide B treatment group(GB,n=20;hypoxia–ischemia plus LY294002(1.8mg/Kg)(LY294002,n=20);hypoxia–ischemia plus LY294002 and Ginkgobalide B treatment group(GB+LY294002,n=20);hypoxia–ischemia plus U0126(0.2mg/Kg)(U0126,n=20);hypoxia–ischemia plus U0126 and Ginkgobalide B treatment group(GB+U0126,n =20).LY294002 or U0126(respectively a PI3 K and MEK inhibitor)were administered to rats with or without Ginkgobalide B at 30 min before HI.The rats in the GB group,GB+LY294002 group and GB+U0126 group were subjected to the Ginkgobalide B treatment immediately after HI.Then all rats were killed at 24 hours after HI.The pathological changes of cerebral white matter were observed by Hematoxylin-eosin(HE).Levels of neuron apoptosis were determined by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling(TUNEL).Levels of BDNF,P-AKT(ser473),P-ERK1/2 and Cleaved caspase-3 in the damaged cortex were performed with Immunohistochemistry and Western blotting at 24 h following HI.All datas were expressed as Mean ± SD.Statistical analysis was performed by using SPSS17.0 to conduct analysis of variance for multiple comparision.Differences have statistical significance to further conduct LSD-t test.? value=0.05 was considered as inspection level.Results1 The results of polymerase chain reaction(PCR)The results showed that the level of Caspase-3 mRNA began to rise at 6h after HI both in the HI group and drugs-treated groups,and reached a peak at 24 h,then declined.Otherwise,drugs-treated groups were lower than HI group.However,the level of BDNFm RNA started decreasing at 6h following hypoxia–ischemia in the HI group and drugs-treated groups,and reached the minimum at 24 h,then elevated.Otherwise,drugs-treated groups were higher than HI group.In addition,the effect of high dose Ginkgobalide B was better than that of low dose Ginkgobalide B.2 The results of hematoxylin-eosin(HE)Ginkgolide B can improve the neuronal edema induced by hypoxia-ischemia in neonatal rats.In the Sham group,the cell morphology was normal,The nuclei was stained dark blue and the cytoplasm was stained pink.At 24 h following hypoxia–ischemia,the brain cells in HI group showed obvious edema,vacuolar change,and sometimes even a balloon.Otherwises,existing vascular endothelial cell swelling,the surrounding space widened.In the GB group,there was a decrease in cell edema,and only a few cells exist vacuolar change.3 The results of terminal deoxynucleotidyl transferase mediated d UTP nick end labeling(TUNEL)Ginkgolide B can reduce the apoptosis induced by hypoxia-ischemia in neonatal rats.Compared with the sham-operated group,the TUNEL-positive cells significantly increased(p<0.01).Treatment with Ginkgobalide B obviously decreased cell apoptosis(P<0.01,20 mg/kg Ginkgobalide B vs.HI group).4 The results of immunohistochemicalGinkgolide B can downregulate the positive cells of Cleaved caspase-3(CC3),upregulate that of BDNF and P-AKT(ser473),and reduce the level of apoptosis induced by hypoxia-ischemia.This effect can be inhibited by LY294002 inhibitors.However,the positive cells of P-ERK1/2 in each group have no obvious change.The main results are as follows:Compared with the sham operated group,the positive cells of of BDNF and P-AKT(ser473)significantly decreased in HI group,but that of CC3 significantly increased(p<0.05).Compared with the HI group,treatment with Ginkgobalide B(20mg/kg)obviously increased the expression of BDNF and P-AKT(ser473),and decreased the positive cells of CC3(P<0.05).However,the positive cells of P-ERK1/2 have no significant change among these groups(P>0.05).Compared with Ginkgobalide B(20mg/kg)group,the positive cells of of BDNF and P-AKT(ser473)significantly decreased both in the LY294002 group and GB / LY294002 group,and that of CC3 significantly increased(P<0.05).However,there were no significant differences between the LY294002 group and GB/LY294002 group in the expression of BDNF,P-AKT(ser473)and CC3(P>0.05).Compared with the U0126 group,the positive cells of BDNF significantly increased in the GB group and GB/U0126 group,and that of CC3 significantly decreased(P<0.05).However,there were no significant differences between the GB group and GB/U0126 group in the expression of BDNF and CC3.In the present of U0126 inhibitors,that of P-ERK1/2 significantly reduced.5 The results of Western blottingGinkgolide B can decrease the protein levels of Cleaved caspase-3(CC3),upregulate the expression of BDNF and P-AKT(ser473).This effect can be inhibited by LY294002 inhibitors.However,the expression of P-ERK1/2 in each group has no obvious change.The main results are as follows :In HI group,the expression of BDNF and P-AKT(ser473)significantly decreased,and that of CC3 significantly increased compared with the sham operated group(P<0.05).In GB group,the expression of BDNF and P-AKT(ser473)obviously increased and that of CC3 decreased compared with the HI group(P<0.05).However,the expression of P-ERK1/2 has no significant change among these groups.Compared with Ginkgobalide B(20mg/kg)group,the protein levels of BDNF and P-AKT(ser473)significantly decreased in the LY294002 group and GB / LY294002 group,and that of CC3 significantly increased(P<0.05).However,there were no significant differences between the LY294002 group and GB/LY294002 group in the expression of BDNF,P-AKT(ser473)and CC3(P>0.05).Compared with the U0126 group,the levels of BDNF and P-AKT(ser473)significantly increased,and that of CC3 significantly decreased in the GB group and GB/U0126 group(P<0.05).Otherwise,P-ERK1/2 almost has no expression both in U0126 group and GB/U0126 group.Conclusion1 Treatment with Ginkgobiloba B can inhibit that the upregulation of Caspase-3m RNA and downregulation of BDNFm RNA in neonatal rats induced by hypoxia–ischemia,And the maximum difference was 24 hours after hypoxiaischemia,the effect of high dose Ginkgolide B was better than that of low dose Ginkgolide B;2 Treatment with Ginkgobiloba B can decrease the expression of CC3 and apoptosis of neurons after hypoxia–ischemia in neonatal rats;3 Ginkgolide B can play a neuroprotective role through promoting the secretion of BDNF in the brain of neonatal rats following HI;4 The neuroprotective effect of Ginkgobalide B appears to be mediated via the PI3K-AKT signaling pathway but not MEK-ERK Pathway.