Sarsasapogenin-AA13 ameliorates Aβ-induced cognitive deficits via improving neuroglial capacity on Aβ clearance and antiinflammation

Abstract

Aims: Sarsasapogenin has been reported to improve dementia symptoms somehow, probably through modulating the function of cholinergic system, suppressing neurofibrillary tangles, and inhibiting inflammation. However, the role of sarsasapogenin in response to beta-amyloid (Aβ) remains to be delineated. This study aimed to determine the therapeutic effect of sarsasapogenin-13 (AA13, a sarsasapogenin derivative) on learning and memory impairments in Aβ-injected mice, as well as the role of AA13 in neuroglia-mediated antiinflammation and Aβ clearance.

Methods: Focusing on the role of AA13 in regulating glial responses to Aβ, we conducted behavioral, morphological, and protein expression studies to explore the effects of AA13 on Aβ clearance and inflammatory regulation.

Results: The results indicated that oral administration of AA13 attenuated the memory deficits of intracerebroventricular (i.c.v.) Aβ-injected mice; also, AA13 protected neuroglial cells against Aβ-induced cytotoxicity. The further mechanical studies demonstrated that AA13 reversed the upregulation of proinflammatory M1 markers and increased the expression of antiinflammatory M2 markers in Aβ-treated cells. Furthermore, AA13 facilitated Aβ clearance through promoting Aβ phagocytosis and degradation. AA13 modulated the expression of fatty acid translocase (CD36), insulin-degrading enzyme (IDE), neprilysin (NEP), and endothelin-converting enzyme (ECE) in neuroglia.

Conclusion: The present study indicated that the neuroprotective effect of AA13 might relate to its modulatory effects on microglia activation state, phagocytic ability, and expression of Aβ-degrading enzymes, which makes it a promising therapeutic agent in the early stage of Alzheimer's disease (AD).

Keywords: antiinflammation; beta-amyloid clearance; neuroglia; phagocytosis; sarsasapogenin-AA13.

Figure 1

AA13 attenuated learning and memory impairments in Aβ _1‐42‐injected mice. (A) Chemical structure of sarsasapogenin‐AA13. (B) Experimental protocol diagram. Mice were randomly divided into four groups, namely Sham‐operated (vehicle+vehicle), Aβ _1‐42 model (vehicle+Aβ _1‐42), AA13 low‐dose group (6 mg/kg AA13+Aβ _1‐42), and AA13 high‐dose group (30 mg/kg AA13+Aβ _1‐42). The mice were orally administrated once a day for 7 d followed by Aβ _1‐42 i.c.v. injection surgery; then, morris water maze test was conducted. (C) The typical trajectory of each group in probe trial. (D) Time before reaching the hidden platform during 6 days of training trial. (E) Time spent in target quadrant in probe test. (F) The mean speed during the water maze test. (G) Nissl staining of hippocampal CA1 region of mice. (H) The alteration of Iba1 and GFAP protein expression in the brain. *P<.05 compared with control group. #P<.05 compared with model group . **P<.01 compared with control group. ##P<.01 compared with model group

Figure 2

AA13 protected primary cultured neuroglia against Aβ‐induced cytotoxicity. (A, B) Both astrocytes and microglia were treated by 10 μmol/L Aβ _1‐42 with or without 10 μmol/L AA13 and AA13 alone, respectively, for 24 h. Cell viability was determined using MTT assay. (C, D) The morphological alterations of astrocytes and microglia were visualized by a phase‐contrast microscope. *P<.05, **P<.01, ***P<.001 compared with control group. ##P<.01 compared with Aβ‐treated group

Figure 3

AA13 treatment inhibited the upregulation of M1 markers and increased expression of M2 markers in Aβ‐treated cells. Cells were pretreated with or without 10 μmol/L AA13, then exposed to 10 μmol/L Aβ _1‐42 for 24 h. Western blot was used. Error bar represented integrated optical density values normalized to the internal standard GAPDH. (A, B, E, and F) The M2 markers level, Arg‐1 and Ym_1/2, increased in astrocytes and microglia. (C, D, G, and H) Meanwhile, AA13 restored the Aβ‐induced upregulation of TNF‐α and iNOS protein expression in astrocytes and microglia. *P<.05, **P<.01, ***P<.001 compared with control group. ##P<.01, ###P<.001 compared with Aβ‐treated group

Figure 4

AA13 promoted Aβ _1‐42 clearance in primary cultured neuroglia. Astrocytes and microglia were pretreated with 0, 1, 5, 10 μmol/L AA13 for 24 h followed by the addition of 0.2 μmol/L Aβ _1‐42 for 4 h. (A, B) The extracellular Aβ _1‐42 level of astrocytes and microglia and (C, D) the intracellular Aβ _1‐42 level of astrocytes and microglia were determined using Aβ _1‐42 ELISA kit. (E, F) Combined the extracellular and cytosolic values together to gain the total decline in astrocytes and microglia. *P<.05, **P<.01, ***P<.001 compared with control group

Figure 5

AA13 promoted microglia activation and phagocytosis. Primary microglia were treated with 0, 1, 5, and 10 μmol/L AA13 for 24 h. (A) Western blot was used to semiquantitatively determine the Iba1 immunoreactivity to illustrate the microglial activated state. Error bar represented integrated optical density values normalized to the internal standard GAPDH. (B) Compared with unchallenged cells, the effect of AA13 on microglia was visualized by typical cellular distribution of Iba1 immunoreactivity in green. (C, D) After incubation of 10 μmol/L AA13 for 24 h, fluorescent microbeads in red were added to challenged and unchallenged cells. DAPI‐labeled cell nuclei were shown in blue. (E) AA13 had no significant effect on the expression of MSR1 in microglia. (F) While CD36 level obviously enhanced after AA13 exposure. *P<.05, **P<.01 compared with control group

Figure 6

AA13 affected expression of Aβ‐degrading enzymes in primary cultured glia. Cells were treated with or without AA13 (1, 5, 10 μmol/L) for 36 h. Western blot was used. Error bar represented integrated optical density values normalized to the internal standard GAPDH. (A, B, and C) For astrocytes, AA13 significantly promoted the expression of IDE and neprilysin(NEP) in a dose‐dependent manner, but there was an opposite change in ECE expression. (D, E, and F) As for microglia, the expression of IDE and NEP was slightly inhibited by AA13 exposure, while ECE expression significantly increased in a dose‐dependent manner. *P<.05, **P<.01, ***P<.001 compared with control group