Saponins from Anemarrhena asphodeloides Bge. protect neurons from amyloid β-protein fragment 25-35-induced apoptosis / 中国药理学与毒理学杂志

ABSTRACT

AIM To investigate the neuroprotective effects and possible mechanisms of saponins from Anemarrhena asphodeloides Bge. (SAaB) on neuronal damage induced by amyloid β-protein fragments 25-35 (Aβ25-35). METHODS Cultured mouse peritoneal macrophages were stimulated with Aβ25-35 (20 μmol·L-1) for 0.5, 1, 2 and 6 h or preincubated with SAaB (10, 30 and 100 μmol·L-1)for 10 min or mitogen-activated protein kinase (MAPK) specific inhibitors (p38 MAPK inhibitor SB 203580 and MEK specific inhibitor PD98059) for 30 min prior to the addition of Aβ25-35(20 μmol·L-1). After stimulation with Aβ25-35 for the indicated times, total cellular extracts were prepared for Western blotting of extracellular signal-regulated kinase (ERK) and p38 MAPK. After stimulation with Aβ25-35 for 48 h, the supernatants of cultured macrophages were collected for quantification of tumor necrosis factor-α (TNF-α) and nitric oxide (NO) and protein expression of inducible nitric oxide synthase (iNOS) in macrophages was determined by immunocytochemical staining. To determine whether SAaB has protective effect against neuronal apoptosis mediated by Aβ25-35-induced macrophages activation, macrophages were stimulated with Aβ25-35 in the presence or absence of SAaB (10, 30 and 100 μmol·L-1) for 48 h and then the cell-free supernatant of Aβ25-35-stimulated macrophages was transferred to the culture of cerebellar granule neurons for 72 h. Neuronal apoptosis was quantitated by scoring the percentage of cells with apoptotic nuclear morphology after Hoechst 33258 staining. RESULTS Aβ25-35(20 μmol·L-1) significantly induced increase in phospho-ERK1/2 and phospho-p38 MAPK protein expression without affecting total protein levels and in the production of TNF-α and NO in cultured macrophages. Aβ25-35-induced increase of TNF-α production in macrophages involved activation of ERK1/2 signal pathway. Importantly, TNF-α and NO generated by cultured macrophages after Aβ25-35 stimulation may be responsible for the majority of the neuronal apoptosis. SAaB (30 and 100 μmol·L-1) significantly suppressed Aβ25-35-induced increase in phospho-ERK1/2 and phospho-p38 MAPK protein. In addition, SAaB (10, 30 and 100 μmol·L-1) also decreased the level of TNF-α and NO in supernatants of cultured macrophage and inhibited Aβ25-35-induced increase in iNOS protein expression of macrophages. Neuronal apoptosis mediated by Aβ25-35-induced macrophage activation was also significantly attenuated by treatment with SAaB (10, 30 and 100 μmol·L-1). CONCLUSION SAaB protects neurons against the neuronal cell death induced by Aβ25-35. The beneficial effects of SAaB may be related to the reduction of TNF-α and NO from activated macrophage induced by Aβ25-35.