| Background Alzheimer’s disease (AD), as one of progressive neurodegenerative diseases, is the most common form of dementia among older individuals. Two key pathologic features of AD include the extracellular deposition of amyloid-β (Aβ) peptide, which forms oligomers and senile plaques, and the intracellular accumulation of hyperphosphorylated tau protein, which forms neurofibrillary tangles (NFTs). To date, no cure has been developed for AD, and some disease-modifying treatments show side effects and low efficacy. Increasing evidence shows that cyanidin 3-O-glucopyranoside (Cy3G), which is naturally derived from many plants, may provide protection against neurodegenerative diseases including AD; however, its exact mechanisms and the molecular target initiated by Cy3G are still unclear. It is reported that Cy3G can protect against nonalcoholic steatohepatitis, type 2 diabetes mellitus and oxidative stress by activating the PPARy pathway. Therefore, we investigated wether Cy3G could protect against beta-amyloid 25-35 (Aβ25-35)-induced SH-SY5Y cell injury and alleviate cognitive impairment in the APPswe/PS1△E9 (PAP) mouse model of AD through PPARy up-regulation. And we also examined the effect of Cy3G on the intestinal flora of AD mice to provide new idea and means for AD treatment.Methods in vitro study, to investigate the neuroprotective effect of Cy3G against AP25-35-induced injury in SH-SY5Y cells and explore the mechanisms involved, cell viability was assessed by MTT to determine proper concentration of each compound, and Aβ25-35-induced injury model in SH-SY5Y cells was established. Then, cells were divided into five groups:(1) control group:cells were cultured under normal conditions, (2) Aβ25-35 group:cells were treated with 10μM Aβ25-35 for 24h, (3) Cy3G group:cells were treated with 25μM Cy3G alone for 24h. (4) Cy3G+Aβ25-35 group:cells were pretreated with 25μM Cy3G for 24h, and then co-incubated with 10μM AP25-35 for another 24h, (5) GW9662+Cy3G:cells were pretreated with 20μM GW9662 for 3h, and then co-incubated with 25μM Cy3G for 24h, (6) GW9662+Cy3G+Aβ25-35 group:cells were pretreated with 20μM GW9662 for 3h, and then co-incubated with 25μM Cy3G for 24h, the next day injured by 10μM AP25-35 for another 24h. We observed cell as well as organelles morphology or oligomeric/fibrillar forms of Aβ25-35 peptides using transmission electron microscope (TEM). Aβ25-35 binding to the cell surface was determined by the CR assay and scanning electron microscope (SEM). Then the detection of cell apoptosis as well as ROS formation were used by fluorescent probe Hoechst 33258 and DCFH-DA, respectively, and evaluation by flow cytometry. Western blot was used to determine PPARy protein level to verify the direct target molecular of Cy3G.Then, in vivo study, to further verify whether Cy3G could activate PPARy to alleviate cognitive impairment function, seven-month-old PAP mice were randomly divided into model group (PAP), Cy3G treatment group (PAPCy,5mg/kg), GW9662 antagonist group (PAPiCy, lmg/kg) and negative-control group (nPAP). In addition, age-matched and normal wild-type of C57BL/6J mice were selected and divided into vehicle group (WT), Cy3G intervention group (WTCy). Each group containing 10-14 mice, with equal number of male and female mice. After 8-week Cy3G supplementation, behavioral abnormalities or improvements of mice in each group were tested using the open field task, novel object recognition and Morris water maze. In addition, fasting blood glucose level and cerebral glucose metabolism rate of mice in each group were measured by a glucose meter and microPET/CT, respectively. After all animals were sacrificed, the main organs weight and organ coefficients were tested. To detect the liver/ kidney function as well as indicators associated with lipid metabolism, biochemical methods were used. Changes in tissues and brain amyloid deposition were observed by histopathological examination and TEM, respectively. Meanwhile, immunoassay was used to determine the levels of serum insulin and leptin and western blot was used to determine protein levels of Akt, JNK and GSK-3β.Finally, in order to find out the microflora associated with AD development, and to explore the effects of Cy3G on intestinal flora and cytokines related to immune response in PAP mice after 8-week Cy3G supplementation, colonic stools were collected. The high-throughout sequencing technology of 16S ribosomal RNA (rRNA) was used to analyze the structural characteristics of the intestinal microecology, and compare abundance, diversity and structure change of microbial flora in mice among different treatment groups. Changes in pathophysiology and morphological structure of intestinal tract were observed by histopathological examination and TEM. Meanwhile, multi factor detection technology (Luminex xMAP(?)) was used to determine the inflammatory factors in serum, which associated with the immune inflammatory response.Results In an in vitro study, the data indicated that Cy3G-mediated neuroprotection involved the inhibition of Aβ25-35 binding to the cell surface and spontaneous aggregation of Aβ25-35 fibrils at the molecular level. Furthermore, in an in vitro study, Aβ25-35-mediated cytotoxicity, which was caused by inducing apoptotic cell death and ROS formation, was also ameliorated by Cy3G intervention. In addition, upregulation of peroxisome proliferator-activated receptor-γ (PPARγ) protein involved in glucose/lipid metabolism by Cy3G treatment verified that the initiated molecule was Cy3G. GW9662 can inhibited Cy3G activity and reduced the level of PPARy protein.In an in vivo study, Cy3G was shown to alleviate cognitive impairment tested by novel object recognition and Morris water maze without obvious effect on the locomotor activity of APPswe/PS1△E9 mice. Furthermore, Cy3G can improve cerebral glucose uptake (such as hippocampus and frontal lobe) and decrease fasting blood glucose levels of AD mice. In addition, the reduction of brain atrophy, normal lipid metabolism and no obvious liver/kidney toxicity were observed in PAP mice supplemented with Cy3G. In addition, Cy3G also could down-regulate the levels of phospho-JNK protein, otherwise up-regulate the level of phospho-Akt protein and phospho-GSK-3βprotein. However, GW9662 could block the Cy3G biological activity and influence the cognitive function and the glucose/lipid metabolism, but no impact on the expression of Akt, JNK and GSK-3p.In the aspect of intestinal microecology research, the abundance, diversity and structure composition of the microbial community in AD model mice were significantly changed, which may be associated with the changes of AD related pathology and cognitive function. Cy3G supplement can reduce the diversity and abundance of fecal flora in mice of PAPCy, WTCy and PAPiCy groups, especially decrease relative abundance of Cyanobacteria and Firmicutes-to-Bacteroidetes relative abundance ratio, promote the enrichment of Proteobacteria. However, the mice in GW9662 antagonist group have higher relative abundance of Cyanobacteria, lower relative abundance of Lactobacillales or Lactobacillaceae. In addition, Cy3G can effectively improve the abnormal morphology and ultrastructure of ileum mucosal epithelial cells in AD mice, and the PPARy antagonist GW9662 did not counteract Cy3G activity. In the aspect of the immune regulation, Cy3G only can decrease the levels of IL-1β and MCP-1 in the mice of WT group, but has little influence on AD model mice.Conclusion Cy3G intervention can ameliorate amyloid β peptide-induced SH-SY5Y cells injury through PPARy activation. Meanwhile, Cy3G supplement also can partially improve the cognitive dysfunction and cerebral glucose metabolism disorder in as well as reverse intestinal microecology disorder caused by enteric dysbacteriosis and protect intestinal mucosa cells structure in AD model mice. Thus, Cy3G has a good safety profile, which could regulate insulin resistance and inflammatory response and be used for AD prevention and alleviation. In addition, Cy3G also can be used to regulate intestinal flora in AD, which can provided theoretical basis for clinical application. |
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