Oxidation-antioxidation Imbalance Induced By D-serine/NADPH Oxidase Signaling—The Molecular Mechanism Of Hippocampal Neuron Injury In Early Stage Of Vascular Dementia

Objectives To observe neuronal damage of hippocampal CA1 region in early stage of vascular dementia(VD), further focus on D-serine(D-ser) and NADPH oxidase to explore the effects of excitatory amino acids toxicity and oxidative stress on the neuronal damage, as well their mediation after cerebral chronic hypoperfusion. The aim of current study was to reveal the molecular mechanism of VD pathogenesis and discoverd potential new therapeutic targets for VD.Methods 1 Cerebral chronic hypoperfusion in male adult SD rats was induced by bilateral common carotid artery occlusion(BCCAO) with a 7d interval. The animals were randomly divided into six groups: sham, BCCAO(1h, 1d, 3d, 7d, 21d), antisense oligodeoxynucleotide(AS) of serine racemase(SR-AS), missense oligodeoxynucleotides(MS) of SR(SR-MS), special inhibitor of NADPH oxidase gp91ds-tat, and control scrambled group(Scr). SR-AS or MS was continuously administrated using mini-pump by intracerebroventricular(i.c.v.) injection and gp91ds-tat or Scr was subcutaneously treated using mini pump. 2 The markers of oxidative stress damage and anti-oxidants and SR protein expression in hippocampal CA1 region were investigated using immunofluorescence staining and western blot analysis. 3 The activity of NADPH oxidase and D-Ser level in hippocampal CA1 region were mesured utilizing ELISA analysis. 4 The ultrastructions of hippocampal CA1 neurons and vascular were observed using transmission electron microscopy(EM).Results 1 Oxidant/antioxidant imbalance occurs in hippocampal CA1 neurons in the early stage of VD.(1) Compared to sham control, the fluorescence intensity of HEt, an oxygen free radical probe, at 1h, 3d, 7d and 21 d after BCCAO significantly increased, and double immunofluorescent staining or western blot results indicated that levels of 3-NT, marker of ONOO- and 4-HNE, marker of membrane lipid damage were significantly increased in BCCAO 7d, 21 d groups(P<0.05).(2) Protein expressions of Nrf2, an antioxidant transcription factor, and its downstream anti-oxidants SOD2, HO-1 significantly decreased in BCCAO 7d and 21 d groups compared with sham group(P<0.05).(3) Compared with sham group, NADPH oxidase activity markedly enhanced at 1h, 1d, 7d and 21 d after BCCAO(P<0.05). 2 The early stage of VD induced ultrastructural damage of neuron and vascular in hippocampal CA1 region. EM analysis revealed that there are different degrees of neuronal and vascular damage was observed at 1h, 3d and 21 d after BCCAO in hippocampal CA1 region, and the damage was the most severe in BCCAO 21 d group, showing dissolved nuclear membrane, chromatin edge accumulation, dilatation of endoplasmic reticulum, swollen mitochondria, as well as severe perivascular edema. 3 BCCAO increased protein expressons of SR and GFAP in hippocampal CA1 region. Wester blot analysis showed that SR protein expression significantly increased in BCCAO 1h, 3d, 7d and 21 d groups compared with sham group(P<0.05), and the GFAP level in each group of BCCAO(1h, 1d, 3d, 7d and 21d) significantly enhanced compared to sham control(P<0.05). 4 Knockdown of SR protein decreased D-Ser level in hippocampal CA1 region. Continuous central administration of SR-AS significantly reversed the increases in SR protein expression and D-Ser level induced by BCCAO 21d(P<0.05). 5 Either SR-AS or gp91ds-tat prevented hippocampal CA1 neuronal damage through the regulation of oxidation/anti-oxidation balance.(1)Either SR-AS or gp91ds-tat profoundly decreased the activity of NADPH oxidase caused by BCCAO 21d(P<0.05).(2) SR-AS or gp91 dstat not only markedly attenuated protein expression of oxidative stress markers such as 4-HNE, 3-NT, but also upregulated protein levels of anti-oxidant enzymes Nrf2, HO-1, and SOD2, as compared to the BCCAO 21 d group(P<0.05).(3) Either SR knockdown or activity inhibition of NADPH oxidase perfectly prevented early ultrastructural damage of hippocampal CA1 neurons at 21 d after BCCAO.Conclusions 1 Neuronal damage in hippocampal CA1 region were observed in the early stage of VD and the oxidation/antioxidation imbalance contributes to the molecular mechanism. 2 Up-regulation of endogenous D-Ser in the hippocampal CA1 region plays an important role in the NADPH oxidase induction and antioxidant/oxidant imbalance that occurs during early stage BCCAO.