Abstract
Depressive disorder is a common psychiatric disease which ranks among the leading cause of disability worldwide. The antidepressants presently used had low cure rate and caused a variety of side-effects. The screening of antidepressant drugs is usually used classic behavioural tests and neuroprotective strategy. Longistyline C, a natural stilbene isolated from the leaves of Cajanuscajan (L.) Millsp, was firstly investigated the antidepressant effect using animal behavioural tests, and studied the neuroprotection and its possible signaling pathways on glutamate-induced injury in PC12 cells. The results of animal test demonstrated that longistyline C had the antidepressant activity, which the effect is similar to the positive control. In current study, we investigated the effect of longistyline C on glutamate-induced injury in PC12 cells and explored its possible signaling pathways. The results demonstrated that pretreatment with longistyline C at the concentrations of 2–8 μmol/L for 24 h had a significant reduction of the cytotoxicity induced by glutamate (15 mmol/L) in PC12 cells using MTT, lactate dehydrogenase (LDH) release assay and Annexin V—PI double staining. Subsequently, we found that pretreatment with longistyline C (8 μmol/L) could drastically down-regulate the over-expression of NMDAR/NR2B and Ca2+/calmodulin-dependent protein kinase II (CaMKII), up-regulate the expressions of p-ERK and p-CREB and alleviate ER stress. In conclusison, longistyline C is most possibly through regulating NMDAR/NR2B-ERK1/2 related pathway and restoring endoplasmic reticulum function to exert neuroprotective effect against glutamate-induced injury in PC12 cells.
Highlights
MethodsDulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY), penicillin (100 IU/ml Sigma, St. Louis, MO), streptomycin (10 μg/ml; Sigma), 10% heat-inactivated fetal calf serum (Gibco, Grand Island, NY)
Depressive disorder (MD) is a common psychiatric disease which ranks among the leading cause of disability worldwide [1]
Longistyline C was isolated from the leaves of pigeonpea by our laboratory [18] and the purity is over 95% which was detected by high performance liquid chromatography (HPLC) method
Summary
Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY), penicillin (100 IU/ml Sigma, St. Louis, MO), streptomycin (10 μg/ml; Sigma), 10% heat-inactivated fetal calf serum (Gibco, Grand Island, NY). PC12 cells were purchased from the Cell Bank of Peking Union Medical College (Beijing, China). Methyl thiazolyl tetrazolium (MTT), dimethylsulfoxide (DMSO) and glutamate were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA), Fura-2/AM (Beyotime Institute of Biotechnology, Jingsu, China). Antibodies used for western blot analysis, primary antibodies: 1:1000 Anti-GADPH, 1:200 Anti-ERK1/2, 1:200 Anti-βActin, 1:100 Anti-GRP78, 1:200 Anti-p-ERk1/2,1:250 Anti-Caspase-12, 1:100 Anti-GADD153, 1:200 Anti-XBP-1, 1:500 Anti-Caspase-9, 1:1000 Anti-NMDAR/NR2B, 1:1000 Anti-CaMKII, 1:1000 Anti-CREB and 1:1000 Anti-p-CREB (Cell signaling Technology, USA); secondary antibodies: 1:1000 Anti-mouse-HRP and 1:1000 Anti-rabbit-HRP (Santa Cruz biotechnology, USA); Nonfat milk powder and bovine serum albumin (Becton, Dickinson and Company, USA).
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