知母皂苷对Aβ_(25-35)引起小脑颗粒细胞损伤的保护作用及其机制探讨

Influence of anemarsaponin on Aβ_(25-35)-induced damage to granular cell in cerebellum

目的观察知母皂苷对淀粉样β蛋白25-35(Aβ25-35)激活小鼠巨噬细胞引起小脑颗粒细胞损伤的保护作用及探讨其作用机制。方法小鼠小脑颗粒细胞与腹腔巨噬细胞联合培养及腹腔巨噬细胞单独培养,损伤组加入Aβ25-35(20μmol/L),保护组同时加入不同质量浓度知母皂苷(10、30、100μmol/L)。联合培养的细胞通过免疫组化染色观察微管相关蛋白-2(MAP-2)的表达和测定乳酸脱氢酶(LDH)。单独培养的巨噬细胞用ELISA法和Griess法分别测定培养液中白细胞介素-1β(IL-1β)和一氧化氮(NO)。Western blot检测巨噬细胞磷酸化p38MAPK表达水平。结果知母皂苷组可使MAP-2表达阳性的小脑颗粒细胞数目较Aβ25-35组明显增加,LDH漏出量减少;巨噬细胞培养液中IL-1β和NO生成量减少以及巨噬细胞磷酸化p38MAPK表达水平明显降低。SB203580也能抑制Aβ25-35引起的IL-1β和NO生成量增加。结论 知母皂苷通过抑制Aβ25-35激活巨噬细胞p38MAPK通路,使IL-1β和NO表达水平降低,从而对小脑颗粒细胞起到保护作用。

AIM To investigate the effects of anemarsaponin on granular cell in cerebellum mediated by amyloid β-protein-induced macrophage activation in vitro.METHODS The mouse peritoneal macrophages were preincubated with anemarsaponin（10,30 and 100 μmol/L）,then stimulated with Aβ25-35（20 μmol/L）.Total cellular extracts were prepared for Western blot analysis of phospho-p38MAPK.The supernatants of macrophages were collected and analyzed for interleukin-1β（IL-1β） and nitric oxide（NO） generation.The cells of neuron-macrophage co-cultures were exposed to Aβ25-35（20 μmol/L） in the absence or presence of various concentrations of anemarsaponin（10,30 and 100 μmol/L）.The expressions of Microtubule-associated protein-2（MAP-2） were detected by immunocytochemical technique.The supernatants of co-cultured cells were measured the leakage of LDH.RESULTS Incubation of cultured macrophages with Aβ25-35 elicited the increase in the production of IL-1β and NO.Aβ25-35 also significantly induced the increase in phospho-p38MAPK protein expression.Anemarsaponin decreased these Aβ25-35-induced changes in a concentration dependent manner.Aβ25-35 increased LDH efflux and decreased the expression of MAP-2 of neuronal cells in neuron-macrophage co-cultures cells significantly.As to the anemarsaponin groups,however,the Aβ25-35-induced damages to cerebella granule neurons were inhibited in a dose dependent manner.CONCLUSION Anemarsaponin could significantly inhibit the over-release of IL-1β and NO induced by Aβ25-35 in cultured macrophages,which takes part in the down regulation of p38MAPK signal transduction pathway.