Sinomenine inhibits amyloid beta-induced astrocyte activation and protects neurons against indirect toxicity

Abstract

Amyloid beta is a major constituent of the plaques found in the brains of patients suffering from Alzheimer's disease (AD). A growing body of research work suggests that neuroinflammation plays important roles in the development of AD. Thus, considerable efforts are directed towards identification of compounds that can reduce or inhibit neuroinflammation. Here, we show that sinomenine, a compound present in a Chinese medicinal plant, Sinomenium acutum, inhibits oligomeric amyloid beta-induced production of reactive oxygen species (ROS), nitric oxide (NO) and inflammation-related molecules from astrocytic cells. The conditioned medium from oligomeric amyloid beta-treated astrocytic cells induces cell death in the hippocampal neuronal cells. Importantly, sinomenine inhibits this cell death. In addition, this compound has inhibitory effects on the production of ROS, NO and inflammation-related factors from oligomeric amyloid-beta treated human astrocytes. Finally, the conditioned medium from oligomeric amyloid beta-treated human astrocytes induces cell death in the primary culture of human neurons, which is inhibited by sinomenine. Thus, sinomenine inhibits amyloid beta-induced production of toxic factors from astrocytes, and confers protection to hippocampal neuronal cells as well as human neurons against indirect toxicity. The results suggest that this compound could provide beneficial effects in AD and other neurodegenerative conditions by reducing inflammation and neuronal cell death.

Keywords: Alzheimer’s disease; Amyloid beta; Neuroinflammation; Neurotoxicity; Sinomenine.

Fig. 1

Sinomenine inhibits oligomeric amyloid beta-induced production of reactive oxygen species in astrocytic cells. The level of reactive oxygen species (ROS) was examined using the DCFDA reagent. a The oligomeric amyloid beta (ADDL) increases the level of ROS in astrocytic cells. The C8D1A astrocytic cells were treated with lipopolysaccharide (LPS) or ADDL and the levels of ROS were assessed. LPS and ADDL significantly increased the level of ROS in the cells (n = 3). b Sinomenine inhibits ADDL-induced increase in the level of ROS in astrocytic cells. The C8D1A astrocytic cells were treated with ADDL in the absence or presence of sinomenine and the levels of ROS were assessed. ROS production was increased in the ADDL-treated astrocytic cells. However, sinomenine inhibited ADDL-induced increase in ROS production. Sinomenine did not affect the level of ROS in the absence of ADDL treatment (n = 4 in all groups). Asterisk indicates significant difference from control (p < 0.05). # indicates significant difference (p < 0.05) between groups as indicated

Fig. 2

ADDL-induced increase in nitric oxide level in astrocytic cells is inhibited by sinomenine. The C8D1A astrocytic cells were treated with oligomeric amyloid beta (ADDL) in the absence or presence of sinomenine and the levels of nitric oxide (NO) were assessed. ADDL treatment increased NO level in the cells. However, sinomenine inhibited the ADDL-induced increase in the NO level (n = 4). Sinomenine did not affect NO level in the cells in the absence of ADDL treatment. Asterisk indicates significant difference from control (p < 0.05). # indicates significant difference (p < 0.05) between groups as indicated

Fig. 3

Sinomenine inhibits oligomeric amyloid beta-induced production of inflammation-related molecules in the astrocytic cells. The C8D1A astrocytic cells were treated with oligomeric amyloid beta (ADDL) in the absence or presence of sinomenine. The levels of inflammation-related molecules were assessed using cytometric bead array inflammation kit. Significant increase in the inflammation-related molecules was observed in the cells exposed to ADDL. However, sinomenine inhibited ADDL-induced increase in the levels of these molecules (IL-12p70, IL-6, IFN-γ, IL-10, MCP-1, n = 7; TNF, n = 5, in all groups). Sinomenine alone did not affect the levels of these molecules. Asterisks denote significant difference from control (p < 0.05). # indicates significant difference (p < 0.05) between groups as indicated

Fig. 4

Sinomenine protects hippocampal neuronal cells from indirect toxicity. The C8D1A astrocytic cells were treated with oligomeric amyloid beta (ADDL) in the absence or presence of sinomenine. The conditioned medium was used to treat HT22 cells, and cell death was determined by TUNEL assay. The representative images of DAPI- or TUNEL-stained HT22 cells (a) and quantified summary data (b; n = 4) demonstrate that HT22 cells treated with the conditioned medium from ADDL-treated astrocytic cells showed increased number of TUNEL positive cells. However, sinomenine inhibited the ADDL-induced increase in the number of TUNEL positive cells. The HT22 cells treated with the conditioned medium from astrocytic cells that were treated with sinomenine alone, did not show increased number of TUNEL positive cells. Asterisk indicates significant difference from control (p < 0.05). # indicates significant difference (p < 0.05) between groups as indicated. Scale bar, 50 μm

Fig. 5

Sinomenine inhibits oligomeric amyloid beta-induced production of reactive oxygen species and nitric oxide in human astrocytes. The human astrocytes were treated with oligomeric amyloid beta (ADDL) in the absence or presence of sinomenine, and the levels of reactive oxygen species (ROS) and nitric oxide (NO) were assayed. a Effect of sinomenine on ADDL-induced increase in ROS level. The astrocytes treated with ADDL showed higher level of ROS. Sinomenine inhibited ADDL-induced increase in the level of ROS (n = 3). Sinomenine did not affect the level of ROS in the cells that were not treated with ADDL. b Effect of sinomenine on ADDL-induced production of NO. Treatment of astrocytes with ADDL led to a significant increase in the level of NO, whereas sinomenine inhibited ADDL-induced increase in NO level (n = 3). Sinomenine had no significant effect on NO level in the absence of ADDL treatment. Asterisks indicate significant difference from control (p < 0.05). # indicates significant difference (p < 0.05) between groups as indicated

Fig. 6

Sinomenine inhibits oligomeric amyloid beta-induced production of inflammation-related molecules in human astrocytes. The human astrocytes were treated with oligomeric amyloid beta (ADDL) in the absence or presence of sinomenine, and the levels of inflammation-related molecules were assessed. Treatment of astrocytes with ADDL led to an increase in the levels of inflammation-related molecules. Sinomenine inhibited ADDL-induced increase in the levels of these molecules (n = 6). Sinomenine alone did not affect the basal level of these molecules. Asterisks denote significant difference (p < 0.05) and $ indicates p < 0.057 from control. # indicates significant difference (p < 0.05) between groups as indicated

Fig. 7

Sinomenine protects human neurons from indirect toxicity. The human astrocytes were treated with oligomeric amyloid beta (ADDL) in the absence or presence of sinomenine. The human neurons were treated with the conditioned medium from astrocytes and TUNEL assay was used to determine cell death. The representative images of DAPI- or TUNEL-stained neurons are shown in a and quantified summary data (n = 4) are shown in b. The neurons treated with the conditioned medium from ADDL-treated astrocytes showed increased number of TUNEL positive cells. However, ADDL-induced increase in the number of TUNEL positive cells was inhibited by sinomenine. The neurons exposed to the conditioned medium from astrocytes treated with sinomenine alone, did not show increased number of TUNEL positive cells. Asterisk indicates significant difference from control (p < 0.05). # indicates significant difference (p < 0.05) between groups as indicated. Scale bar, 50 μm