In silico and in vitro analyses of a novel FoxO1 agonist reducing Aß levels via downregulation of BACE1.

AIMS: FoxO1 is an important target in the treatment of Alzheimer's disease (AD). However, FoxO1-specific agonists and their effects on AD have not yet been reported. This study aimed to identify small molecules that upregulate the activity of FoxO1 to attenuate the symptoms of AD. METHODS: FoxO1 agonists were identified by in silico screening and molecular dynamics simulation. Western blotting and reverse transcription-quantitative polymerase chain reaction assays were used to assess protein and gene expression levels of P21, BIM, and PPARγ downstream of FoxO1 in SH-SY5Y cells, respectively. Western blotting and enzyme-linked immunoassays were performed to explore the effect of FoxO1 agonists on APP metabolism. RESULTS: N-(3-methylisothiazol-5-yl)-2-(2-oxobenzo[d]oxazol-3(2H)-yl) acetamide (compound D) had the highest affinity for FoxO1. Compound D activated FoxO1 and regulated the expression of its downstream target genes, P21, BIM, and PPARγ. In SH-SY5Y cells treated with compound D, BACE1 expression levels were downregulated, and the levels of Aß1-40 and Aß1-42 were also reduced. CONCLUSIONS: We present a novel small-molecule FoxO1 agonist with good anti-AD effects. This study highlights a promising strategy for new drug discovery for AD.

Physalin B reduces Aß secretion through down-regulation of BACE1 expression by activating FoxO1 and inhibiting STAT3 phosphorylation.

Physalin B (PB), one of the major active steroidal constituents of Solanaceae Physalis plants, has a wide variety of biological activities. We found that PB significantly down-regulated ß-amyloid (Aß) secretion in N2a/APPsw cells. However, the underlying mechanisms are not well understood. In the current study, we investigated the changes in key enzymes involved in ß-amyloid precursor protein (APP) metabolism and other APP metabolites by treating N2a/APPsw cells with PB at different concentrations. The results indicated that PB reduced Aß secretion, which was caused by down-regulation of ß-secretase (BACE1) expression, as indicated at both the protein and mRNA levels. Further research revealed that PB regulated BACE1 expression by inducing the activation of forkhead box O1 (FoxO1) and inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3). In addition, the effect of PB on BACE1 expression and Aß secretion was reversed by treatment with FoxO1 siRNA and STAT3 antagonist S3I-201. In conclusion, these data demonstrated that PB can effectively down-regulate the expression of BACE1 to reduce Aßsecretion by activating the expression of FoxO1 and inhibiting the phosphorylation of STAT3.

Alterations of fatty acid composition and metabolism in APP/PS1 transgenic mice.

Accumulating evidence suggests that abnormal fatty acid composition is related to the development of Alzheimer's disease (AD). However, there is no consistency in the fatty acid profile and metabolism associated with AD pathogenesis. This study aims to define the characteristics of fatty acid composition and metabolism in AD. Using 6-month-old APP/PS1 transgenic mice with wild-type mice as a control, we examined the serum lipids, brain fatty acid composition, and the expression levels of various genes involved in liver fatty acid ß-oxidation. The results of our study demonstrate that APP/PS1 mice present decreased serum free fatty acids, altered brain fatty acid profiles, and minimal change in liver fatty acid ß-oxidation. Our results suggest that abnormal fatty acid compositions and contents may play potential roles in AD progression. This study provides further evidence for the metabolic basis of AD pathogenesis.

Effects of chain length of saturated fatty acids on Aß generation in SH-SY5Y cells.

Many studies have shown that saturated fat diet increases the risk of AD. Recently saturated very long chain fatty acids (VLCFAs) have been found be accumulated in AD patients. The variety of saturated fatty acids are found in the diets and human bodies. However, it is not clear which one or more fatty acids are involved in AD pathogenesis. This study investigated the effects of three saturated fatty acids with different carbon chain length (C16:0, C20:0, and C26:0) on amyloid precursor protein (APP) processing and amyloid-ß peptide (Aß) generation. Here, SH-SY5Y cells were treated with vehicle, C16:0, C20:0, and C26:0 (10 µmol/L, 24 h). Compared to the vehicle, C16:0 did not cause any significantly change in APP processing and Aß generation. C20:0 and C26:0 increased Aß levels and the expressions of APP, ß- and γ-secretase and decreased the expression of α-secretase, and C26:0 had the strongest effects among three fatty acids. Moreover, C20:0 and C26:0 significantly increased reactive oxygen species (ROS), and C16:0 had no such effect. These data indicate that saturated fatty acids with different carbon chain length (C16:0, C20:0 and C26:0) have different effects on the process of Aß generation, and fatty acids with longer chain (C20:0 and C26:0) have more potential to promote Aß production and an underlying mechanism of fatty acids action may be related to the elevated oxidative stress. This work supports saturated very long chain fatty acids may play a potential role in the pathogenesis of AD.

[Mechanism of MBL inhibiting the LPS-induced DC maturation].

The study was aimed to investigate the mechanism of mannan-binding lectin (MBL) on bacterial lipopolysaccharide (LPS)-induced human peripheral blood monocyte-derived dendritic cell (DC) maturation. The monocytes were prepared from the peripheral blood of healthy adult volunteers. The immature dendritic cells (imDC) were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4. FACS was used to investigate the interaction of MBL with imDC and the impact of MBL on LPS binding to imDC. ELISA and Western blot was used to analyze the interaction of MBL with soluble TLR4 ectodomain protein (sTLR4); Western blot was used to detect LPS-induced NF-κB translocation in imDC. The results showed that MBL could directly bind to imDC in the presence of calcium. sTLR4 protein or LPS could competitively inhibit the binding of MBL to imDC. ELISA and Western blot showed that MBL could evidently bind to sTLR4 protein in a concentration-dependent manner. FACS showed that MBL could competitively inhibit the binding of LPS to imDC by binding to imDC directly. Western blot showed that MBL decreased LPS-induced NF-κB translocation in imDC. It is concluded that MBL may competitively inhibit the binding of LPS to imDC by binding to TLR4 expressed on imDC, resulted in inhibition of LPS-induced DC maturation, suggesting that MBL can regulate DC maturation through ligand-binding. This study provides the good foundation to clarify the mechanism of MBL inhibiting the LPS-induced DC maturation.

[Effects of activation of liver X receptor and peroxisome proliferator-activated receptor alpha on bile acid synthesis in rats].

OBJECTIVE: To explore the effects of the simultaneous activation of liver X receptor (LXR) and peroxisome proliferator-activated receptor alpha (PPARalpha) on bile acid biosynthesis in rats. METHODS: Totally 36 male SD rats were divided into three groups with 12 rats in each group: control group, high cholesterol (HC) group, and high cholesterol + fenofibrate (HC + FENO) group. Total bile acids (serum bile acids plus fecal bile acids) level was assayed. The levels of mRNA for peroxisomal palmitoyl-CoA oxidase (Acox1), LXR, cholesterol 7alpha-hydroxylase (CYP7A1), D-bifunctional protein (DBP), trihydroxycoprostanoyl-CoA oxidase (Acox2), sterol 12alpha-hydroxylase (CYP8B1), and sterol 27-hydroxylase (CYP27A1) in liver were detected by RT-PCR. RESULTS: Total bile acid level was significantly higher in HC + FENO group than in HC group (P < 0.01), and both were significantly higher than that in control group (P < 0.01). Compared with HC group, the mRNA expression of Acox1 and DBP was significantly higher in HC + FENO group (P < 0.01), but no statistical differences was found between HC group and control group. The mRNA levels of LXR and CYP7A1 in HC + FENO group and HC group were not significantly different but were both significantly higher than that in control group (P < 0.01, P < 0.05). No changes were observed in Acox2, CYPSB1, and CYP27A1 mRNA levels among these three groups. CONCLUSION: Simultaneous activation of LXR and PPARalpha can increase of CYP7A1 and DBP mRNA exDression and thus accelerates the biosynthesis of bile acid.

[Relationship between the increase of hepatic D-bifunctional protein activity and bile acid biosynthesis in rats].

OBJECTIVE: To determine the physiological role of D-bifunctional protein (DBP) in bile acid biosynthesis through investigating the effect of increasing activity of DBP on bile acid biosynthesis. METHODS: Twenty male Wistar rats were divided into two groups: diethylhexyl phthalate (DEHP) group (n = 10) and control group (n = 10). Serum triglyceride, total cholesterol, hepatic DBP activity, and fecal bile acids were assayed. The mRNA levels of hepatic peroxisome proliferator-activated receptor alpha (PPARalpha), DBP, and cholesterol 7alpha-hydroxylase (CYP7A1) were detected by RT-PCR. RESULTS: Compared with control group, serum triglyceride level was decreased significantly and PPARalphamRNA level was increased significantly in DEHP group (P < 0.01). Together with a sharp induction of DBP mRNA expression and DBP activity in DEHP group (P < 0.01), the levels of CYP7A1 mRNA and fecal bile acids were significantly increased by 1.9 times and 1.6 times respectively compared to control group (P < 0.01). There was a significantly positive correlation between DBP mRNA level or DBP activity and CYP7A1 mRNA level (r = 0.89, P < 0.01; r = 0.95, P < 0.01). CONCLUSION: The up-regulation of DBP mRNA and activity in liver can result in the increase in CYP7A1 mRNA expression and bile acid biosynthesis, suggesting that DBP may be involved in bile acid biosynthesis together with CYP7A1.