Blockade of intracellular Ca²⁺ suppresses ES-induced p-CREB mediated BDNF transcription and neurite outgrowth.

(A) Relative Ca²⁺ levels in cells treated with Ca²⁺ inhibitors for 30 min and ES for 30 min (mean ± SD; n = 4). *P < 0.05 compared to sham; ^#P < 0.05 compared to ES; F-value = 75.949. (B) p-CREB expression in cells treated with Ca²⁺ inhibitors and ES (mean ± SD; n = 3). Histone expression was used as a loading control. (C) Relative p-CREB levels in cells treated with ES (F-value = 72.756) or ES + Ca²⁺ inhibitors (F-value = 5.649) (mean ± SD; n = 3). *P < 0.001 compared to 0 min ES group; ^#P < 0.05 compared to group with 0 min ES + Ca²⁺ inhibitors. (D) Relative BDNF mRNA levels in cells treated with ES (F-value = 41.627) or ES + Ca²⁺ inhibitors (F-value = 12.051) (mean ± SD; n = 4). β-actin was used as a loading control. *P < 0.05 compared to 0 min ES group; ^#P < 0.05 compared to group with 0 min ES + Ca²⁺ inhibitors. (E) p-CREB expression in cells treated with Ca²⁺ inhibitors, ES, and HF. Histone expression was used as a loading control. (F) Quantification of p-CREB treated with Ca²⁺ inhibitors, ES, and HF (mean ± SD; n = 3). *P < 0.05 compared to Sham group; ^#P < 0.05 compared to ES+; ^&P < 0.05 compared to ES+/Ca²⁺ inhibitors; F-value = 28.325. (G) Relative BDNF protein levels in cells treated with ES (F-value = 22.937) or ES + Ca²⁺ inhibitors (F-value = 10.186) (mean ± SD; n = 4). *P < 0.05 compared to 0 min ES group; ^#P < 0.05 compared to group with 0 min ES + Ca²⁺ inhibitors. (H) Typical images of neurite outgrowth in Sham and cells with treatments. β-tubulin III was used to indicate cell bodies and neuritis. Scale bar (100 μm) applies to i—viii. Quantification of neurite outgrowth in treated cells (mean ± SD; n = 3). Numbers of cell neurites analyzed in each group: (i) 108, (ii) 97, (iii) 112, (iv) 120, (v) 105, (vi) 120, (vii) 117, (viii) 101. *P < 0.001 compared to Sham group; ^#P < 0.05 compared to ES group; ^&P < 0.05 compared to ES+/NIM and CON group; ^$P < 0.05 compared to ES+/THA and DAN group; F-value = 17.125; NIM, nimodipine; CON, omega (ω)-conodoxin-GVIA; THA, thapsigargin; DAN, dantrolene; ES, electrical stimulation; HF, hyperforin.