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Clinically, it is common to find that patients with chronic renal failure（CRF） are prone to cognitive impairment (CI), and the main effect site of cognitive function is the hippocampus of the brain. Circulating endothelial progenitor cells （CEPCs） can migrate to the hippocampus and play a protective role in hippocampal neurons by repairing hippocampal vascular endothelial cells and secreting a variety of neuroprotective effectors.
Therefore, we wanted to investigate the neuroprotective effect of circulating EPCs on hippocampal neurons in chronic renal failure and the underlying mechanism.We would like to know whether circulating EPCs in CRF patients affect the activity of hippocampal neurons through eNOS-BNDF pathway, and whether they affect the expression of SYT-3 and MAP-2, which are important regulators of synaptic transmission.
A co-culture system of mouse hippocampal neurons and human-derived CEPCs was established by Transwel chamber. The co-culture system was divided into 4 groups: ① Ctrl group (purchase of primary mouse hippocampal neuron cells: MN-h resuscitation culture), ② CEPCs-MN-H (normal human peripheral blood EPC co-cultured with mouse hippocampal neurons), ③ CRF-EPC-MN-H (CRF patient's peripheral blood EPC co-cultured with mouse hippocampal neurons), and ④ CRF-EPC-MN-h+L-NAME (with the addition of eNOS inhibitor L-NAME). (1) CCK-8 kit was used to detect the activity of hippocampal neurons in each group. (2) Cell fluorescence was used to detect the expression of synaptotagming-3 (SYT-3) and microtubule-associated protein-2 (MAP-2), which are key synaptic proteins affecting synaptic plasticity of hippocampal neurons. (3) The expressions of nitric oxide synthase (eNOS), brain-derived neurotrophic factor (BDNF), SYT-3 and MAP-2 in hippocampal neurons were detected by Western bolting.
(1) Compared with EPC-MN-h group, the activity of hippocampal neurons in CRF-EPC-MN-h group decreased (p <0.0001). After the intervention with eNOS inhibitor L-NAME, the activity of hippocampal neurons was further decreased (p < 0.01). (2) The results of Western bloting showed that the protein levels of eNOS, BDNF, SYT-3 and MAP-2 were consistent: Compared with Ctrl group, the expressions of eNOS, BDNF, SYT-3 and MAP-2 in EPC-MN-h group were significantly increased (p <0.05), while the expression of CRF-EPC-MN-h group was significantly lower than that of EPC-MN-h group (p <0.05). The expressions of eNOS, BDNF, SYT-3 and MAP-2 in CRF-EPC-MN-h group were further decreased after L-NAME intervention (p <0.05). (3) The expression changes of SYT-3 and MAP-2 were observed in the cell fluorescence, which was consistent with the results of Western bloting.
In the state of chronic renal failure, the number of circulating EPCs are decreased, and its protective effect on hippocampal neurons is diminished. Circulating EPC could directly affect the activity of hippocampal neurons through eNOS-BDNF pathway and regulate the expression of key synaptic proteins SYT-3 and MAP2, which affect the release of neurotransmitters.
Therefore, we propose that circulating EPCs are an important cellular factor pathway that causes hippocampal neuronal damage during kidney injury.