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Hippocampal neuron death is a key factor in vascular dementia (VD) induced by chronic cerebral hypoperfusion (CCH). Dl-3-n-butylphthalide (NBP) is a multiple-effects drug. Therefore, the potential molecular mechanisms underlying CCH and its feasible treatment should be investigated. This study had two main purposes: first, to identify a potential biomarker in a rat model of CCH induced VD using antibody microarrays; and second, to explore the neuroprotective role of NBP at targeting the potential biomarker. Glial cell line-derived neurotrophic factor (GDNF)/GDNF family receptor alpha-1 (GFRα1)/receptor tyrosine kinase (Ret) signaling is altered in the hippocampus of CCH rats; however, NBP treatment improved cognitive function, protected against hippocampal neuron apoptosis via regulation of GDNF/GFRα1/Ret, and activated the phosphorylation AKT (p-AKT) and ERK1/2 (p-ERK1/2) signaling. We also found that 1 h oxygen-glucose deprivation (OGD) followed by 48 h reperfusion (R) in cultured hippocampal neurons led to downregulation of GDNF/GFRα1/Ret. NBP upregulated the signaling and increased neuronal survival. Ret inhibitor (NVP-AST487) inhibits Ret and downstream effectors, including p-AKT and p-ERK1/2. Additionally, both GDNF and GFRα1 expression are markedly inhibited in hippocampal neurons by coincubation with NVP-AST487, particularly under conditions of OGD/R. GDNF/GFRα1/Ret signaling and neuronal viability can be maintained by NBP, which activates p-AKT and p-ERK1/2, increases expression of Bcl-2, and decreases expression of Bax and cleaved caspase-3. The current study showed that GDNF/GFRα1/Ret signaling plays an essential role in the CCH induced VD. NBP was protective against hippocampal neuron apoptosis, and this was associated with regulation of GDNF/GFRα1/Ret and AKT/ERK1/2 signaling pathways, thus reducing cognitive impairment.

Background/AimsChronic cerebral hypoperfusion (CCH) is induced by chronic deficit of brain perfusion, contributes to a persistent or progressive cognitive dysfunction, which is characterized by diverse neuropathological manifestations. There are currently no effective medications available. White matter damage (WMD) and cortical neuron death may be caused by CCH, which are related to cognitive impairment, while the underlying molecular mechanisms remain unclear. In the study, a database of the transcriptome level was built to determine potential biomarkers in cortex of CCH.MethodsCCH was induced in male Sprague-Dawley rats by permanent occlusion of the bilateral common carotid arteries. Rats were randomly divided into three groups: Sham-operated group (n = 24), the 4th and 8th week of CCH groups (total = 56, n = 28 for each group). Cognitive function was evaluated using the Morris water maze task. WMD and neuron damage were detected using diffusion tensor imaging and histological analysis, respectively. Western blotting analysis of various markers was used to examine neuronal death. Whole-transcriptome microarray was performed to assess mRNA, circRNA, and lncRNA expression profiles at 4th and 8th weeks after CCH. Diversified bioinformatic tools were performed to analyze and predict the key biological processes and signaling pathways of differentially expressed RNAs and co-expressed potential target genes. Co-expression networks of mRNA–circRNA–miRNA and lncRNA–mRNA were constructed.ResultsCompared to the sham group, cognitive impairment, disintegration of white matter, blood-brain barrier damage and neuron death were induced by CCH. Neuron death including apoptosis and necroptosis might occur in the cortex of CCH. We constructed the regulatory networks of whole-transcriptomic including differentially expressed mRNAs, circRNAs, and lncRNAs, and related biological functions and pathways involved in neurological disease, cell death and survival, energy and metabolism, et al. Our results also indicated that Cyr61 mRNA may play a role in the CCH-related cortical neuronal death.ConclusionWMD and cortical neuronal death are worthy of attention in the pathogenesis of CCH. Additionally, the present results provide potential evidence at the whole-transcription level for CCH, offering candidate biomarkers and therapeutic targets.