Effect of nigranoic acid on Ca2+ influx and its downstream signal mechanism in NGF-differentiated PC12 cells

Ethnopharmacological relevance: Schisandra chinensis has a long history of use as a famous traditional Chinese medicine. The plants of genus Schisandra, especially Schisandra neglecta, Schisandra rubriflora, and Schisandra sphaerandra are used in the same way as Schisandra chinensis in the folk medicine to treat insomnia, fatigue, increasing intelligence, and tranquilizing. Many studies showed that lignans were the major active components of Schisandra genus, whereas the bioactivity of abundant triterpenoids in Schisandra genus, such as nigranoic acid (SBB1, 3,4-secocycloartene triterpenoid), has not been examined yet in neuropathology. Materials and methods: After treating with SBB1, intracellular Ca2+ concentration was analyzed by Ca2+ fluorescent indicator (Fluo-4 AM) in NGF-differentiated PC12 cells. Intracellular nitric oxide (NO) level was analyzed using NO fluorescent indicator (DAF-FM). The expression of extracellular signal regulated kinase 1 and 2 (ERK1/2) was analyzed by western blotting, and the temporal mRNA for BDNF and c-fos was analyzed using reverse transcription quantitative PCR. Result: We found that SBB1 induced Ca2+ influx in a time- and concentration-dependent manner, which was significantly attenuated in Ca2+ free media. SBB1 promoted the intracellular NO production which depended on increasing cytoplasmic Ca2+ level. Moreover, SBB1 stimulated activation of ERK1/2 through Ca2+-CaMKII pathway. In addition, we found that SBB1 increased the expression of BDNF and c-fos mRNA. Conclusion: These results suggest that SBB1 is able to promote NO production and stimulate phosphorylation of ERK1/2 through Ca2+ influx, further impact expression of BDNF and c-fos, which provides evidence for the effects of SBB1 that may be benefit to enhance mental and intellectual functions. (C) 2014 Elsevier Ireland Ltd. All rights reserved.