mycelium polysaccharide-enriched aqueous extract (HE) on an l-glutamic acid (l-Glu)-induced differentiated PC12 (DPC12) cellular apoptosis model and an AlCl3 combined with d-galactose-induced Alzheimers disease mouse model. in the autonomic activity test, improved the endurance time in the rotarod test, and decreased the escape latency time in the water maze test. It also improved the central cholinergic system function in the Alzheimers mice, demonstrated by the fact that it dose-dependently enhanced the acetylcholine (Ach) and choline acetyltransferase (ChAT) concentrations in both the serum and the hypothalamus. Our findings provide experimental evidence that HE may provide neuroprotective candidates for treating or preventing neurodegenerative diseases. that protects DPC12 cells against l-glutamic acid (l-Glu)-induced apoptosis via the mitochondrial apoptotic pathway . water extracts have been reported to show antitumor, antimutagenic, antioxidant, hypolipidemic, and immunomodulatory properties in cell and animal models . Encouragingly, displays neuroprotective properties, such as facilitating nerve growth factor (NGF) expression and secretion and regulating the differentiation and development of cholinergic neurons in in vitro and in vivo experiments . MK-2866 Recently, has been reported to improve visual cognitive memory and mediate spatial short-term memory damage in an A-induced mouse model analyzed MK-2866 via behavioral tests . Taken together, these studies suggest that may have beneficial effects on neurodegenerative diseases. In the present study, the neuroprotection of an polysaccharide-enriched aqueous extract (HE) was investigated in an l-Glu-induced DPC12 cellular apoptosis model and an AlCl3 combined with d-gal-induced AD mouse model. Our data revealed that HE improved cell viability and reversed nuclear apoptotic alternation via the mitochondrial related pathway. The experiments performed in the AD mouse model further confirmed the therapeutic effects of HE on AD, which provided helpful evidence of the benefits of using as a medicine or functional food application in neurodegenerative diseases. 2. Results 2.1. HE-Induced PC12 Cell Differentiation Comparatively, the control cell types were round, triangular, and irregular short spindle, and exhibited well refraction under an inverted microscope. Similarly, regarding NGF, after incubation with 50 and 100 g/mL of HE for 24 and 48 h, respectively, the bodies of the PC12 cells became polygons or polygonal. Combined with the increased HE concentration, the cell axons were similar to neurons, and the differentiation rate was significantly enhanced (Figure 1A). Both NGF and HE strongly enhanced the expressions of -tubulin III in PC12 cells after 24 and 48 h incubation (Figure 1B). Figure 1 (A) HE and NGF induced PC12 cell differentiation determined by inverted microscope. Scale bar: 100 m. (= 6); (B) HE and NGF enhanced the expressions of -tubulin III in PC12 cells. Data are expressed MK-2866 as MK-2866 mean SD (= 6). * … 2.2. HE Improved Cell Viability and MK-2866 Nuclear Apoptotic Alternation In the MTT assay, HE alone had no effect on DPC12 cell proliferation (Figure 2A). Twenty-four-hour exposure to 25 mM of l-Glu resulted in a 46.4% reduction in cell viability (< 0.001; Figure 2B). Meanwhile, 3 h HE preincubation improved cell vitality by 30%, compared with l-Glu-exposed DPC12 cells (< 0.01; Figure 2B). Figure 2 (A) HE showed no effects on cell proliferation in normal DPC12 cells; (B) but strongly enhanced cell viability in l-Glu-induced DPC12 cellular apoptosis model; (C) HE restored l-Glu-induced nucleus morphological apoptotic alterations analyzed via Hoechst ... Under Hoechst 33342 staining, the nontreated cells exhibited homogeneous staining with regular contours and rounded shapes. l-Glu caused asymmetric, bright-blue fluorescence patterns in cells that were significantly prevented by HE pretreatment at doses of 50 and 100 g/mL, indicating HEs ability to successfully inhibit nuclear apoptosis (< 0.001; Figure Rabbit Polyclonal to VRK3. 2C). 2.3. HE Reversed Mitochondrial Dysfunction, Ca2+ Overload and ROS Accumulation JC-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide) molecular probe staining was applied to detect mitochondrial function. Although the control cells exhibited the intense red fluorescence indicative of healthy cells, the 12 h l-Glu-incubated cells exhibited the intense green fluorescence indicative of mitochondrial apoptosis (< 0.001; Figure 3A). Comparatively, HE preincubation strongly enhanced the ratio of red-to-green fluorescence, suggesting its beneficial activity on mitochondria (< 0.001; Figure 3A). Figure 3 (A) The disruption of mitochondrial membrane potential (MMP) caused by 12 h l-Glu exposure was strongly restored by 3 h HE pretreatment analyzed via JC-1 staining (= 6). Scale bar: 100 m; (B) the overaccumulation of reactive oxygen species ... Three-hour HE preincubation followed by another 12 h coexposure to l-Glu strongly reduced high green fluorescence in DCFH-DA (2,7-dichlorofluorescein diacetate) staining, suggesting its inhibition of ROS accumulation (< 0.001; Figure 3B), which was also confirmed using the flow cytometry method (Figure S1). The application of 25 mM of l-Glu resulted in calcium overload in DPC12 cells after a 12 h exposure, as indicated by an extremely high green fluorescence (< 0.001; Figure 3C). HE (50 and 100 g/mL) significantly reduced green fluorescence, revealing its suppressive effect on intracellular calcium overload (< 0.001; Figure 3C). 2.4. The Effects of HE on the Behavior of AD Mice The application of.