Fig. 5
LIPUS increases spine density and irisin secretion via upregulating the Fndc5 level in hippocampal neurons. Primary hippocampal neurons were isolated from C57/BL6 wild-type E17 embryos. A Schematic diagram of LIPUS in vitro. LIPUS was daily applied for a stimulation time of 5 min with a ISPTA of 110 mW/cm2 at a period of 2 days. B Primary hippocampal neurons at DIV 7 were treated with LIPUS and then harvested 2 days later. Cell viability of hippocampal neurons were assessed using CCK-8 Kit. Fndc5 expression of hippocampal neurons were assessed by qPCR. C, D Primary hippocampal neurons at DIV 5 were transduced with lentivirus carrying three specified shRNA hairpin against Fndc5 (shFndc5-1, shFndc5-2 and shFndc5-3) or luciferase (shLuc), then harvested 4 days later. Fndc5 expression was assessed by qPCR (C), Cell viability was assessed using CCK-8 Kit (D). E–H Primary hippocampal neurons at DIV 7 were injured by 6 h OGD, and then harvested 2 days later. Dendritic spines of primary hippocampal neurons were labeled using Phalloidin in Immunohistochemistry (E). Spine density of primary hippocampal neurons were analyzed between neurons with no treatment (control), neurons with OGD injure (OGD), OGD-injured neurons with LIPUS treatment (OGD + LIPUS), OGD-injured neurons with LIPUS treatment and shFndc5-1 infection (OGD + LIPUS + shFndc5-1), OGD-injured neurons with LIPUS treatment and shLuC infection (OGD + LIPUS + shLuc) (F). Fndc5 mRNA expression of hippocampal neurons with different interventions were analyzed in histogram (G). Irisin level in supernatants of hippocampal neurons with different interventions were detected using EILSA (H). Culture supernatants of hippocampal neurons with different interventions were further used as neuron-derived conditioned medium (NCM). (n = 6 per group, 6 independent experiments from 6 different neurons preps. All data are expressed as mean ± SEM, **P < 0.01, ***P < 0.001, ns means no statistical significance)