Fig 1: Ovariectomy results in reductions in proteins associated with cholinergic synaptic function in the entorhinal cortex.Lysates were obtained from the medial and lateral entorhinal cortex (MEC and LEC), in groups of animals that received either sham surgery (Sham), ovariectomy (OVX), or ovariectomy and a subdermal implant containing 17-ß estradiol (E2; OVX+E). A. Representative immunoblots of acetylcholinesterase (AChE) and the ß-actin loading control are shown (A1), and the bar graph shows relative expression of AChE protein (A2; n = 6 per group). Note that the reduction in AChE induced by ovariectomy is prevented by administration of E2. Asterisks indicate levels of statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). B. No significant changes were observed in immunoblots (B1) or normalized protein expression (B2) for the vesicular acetylcholine transporter (VAChT; n = 6 per group; vinculin was the loading control). C. Representative immunoblots (C1) and relative protein expression (C2) indicate that the reduction in M1 receptor protein induced by ovariectomy was prevented by administration of E2.
Fig 2: Effects of hAβ1–42 on key mitochondrial elements, synaptic proteins, and cholinergic markers in the entorhinal cortex. (A1) Representative immunoblots of mitochondrial superoxide dismutase 2 (SOD2), mitochondrial cytochrome c (Mito-cyt C), cytosolic cytochrome c (Cyto-cyt C), and β-actin loading control in entorhinal lysates treated with hAβ1–42 and control medium. (A2) Normalized expression of SOD2, Mito-cyt C, and Cyto-cyt C in hAβ1–42-treated entorhinal samples compared to control (n = 6). (B1) Representative immunoblots of postsynaptic density protein (PSD95), presynaptic marker synaptophysin (Synap), and vinculin loading control in entorhinal lysates. (B2) Quantification data showing the normalized expression of both PSD95 and Synap in slices incubated with hAβ1–42 vs. control (n = 6). (C1) Representative immunoblots of cholinergic markers acetylcholinesterase (AChE), vesicular acetylcholine transporter (VAChT), and β-actin (loading control). (C) Bar graphs showing normalized expression of AChE and VAChT in hAβ1–42–treated slices and control (n = 6) (**p < 0.01; ***p < 0.005; ****p < 0.0001).
Fig 3: Electro-acupuncture (EA) treatment regulates cholinergic biomarkers of DG by immunohistochemistry. ChAT and AChE express in the cytoplasm and nerve fibers (brown), and the nuclei are stained with hematoxylin (blue). VAChT expression is granular (brown), without hematoxylin staining of the nuclei. (A) Representative 200 × photomicrographs of ChAT (Top), AChE (Middle), and VAChT (Bottom) expression. (B–D) Statistical results of MOD of ChAT, AChE and VAChT in DG. Red arrows indicate positive protein expression, scale bar = 100 μm. Data are represented as mean ± SD, n = 4/group (*p < 0.05 and **p < 0.01 vs WT group; #p < 0.05 vs 5 × FAD group; ▲p < 0.05 vs 5 × FAD + EA group). ChAT, choline acetyltransferase; AChE, enzyme acetylcholinesterase; VAChT, vesicular acetylcholine transporter; MOD, mean optical density.
Fig 4: Electro-acupuncture (EA) treatment regulates cholinergic biomarkers of MS/VDB by immunohistochemistry. ChAT and AChE express in the cytoplasm and nerve fibers (brown), and the nuclei are stained with hematoxylin (blue). VAChT expression is granular (brown), without hematoxylin staining of the nuclei. (A) Representative 200× photomicrographs of choline acetyltransferase (ChAT) (Top), enzyme acetylcholinesterase (AChE) (Middle), and vesicular acetylcholine transporter (VAChT) (Bottom) expression. (B–D) Statistical results of mean optical density (MOD) of ChAT, AChE and VAChT in MS/VDB. Red arrows indicate positive protein expression, scale bar = 100 µm. Data are represented as mean ± SD, n = 4/group (*p < 0.05 and **p < 0.01 vs WT group; #p < 0.05 vs 5 × FAD group; ?p < 0.05 vs 5 × FAD + EA group). ChAT, choline acetyltransferase; AChE, enzyme acetylcholinesterase; VAChT, vesicular acetylcholine transporter; MOD, mean optical density.
Fig 5: The hippocampal protein levels of acetylcholinesterase but not M1-mAChR are altered in miRNA-132/212-/- mice. Western blot from hippocampal tissue was used to assess the levels of expression of the proteins AChE and mAChR-M1. (A) Demonstrative blots and (B) relative expression of AChE and GAPDH, showing a significant decrease in AChE in miRNA-132/212-/- mice, in comparison to WT mice (* = p < 0.05). (C) Illustrative blots and (D) relative protein expression of mAChR-M1 and GAPDH, showing no differences for mAChR-M1 in miRNA-132/212-/- tissue compared to WT (p > 0.05; t-test). Results are shown as fold change normalized to GAPDH protein levels. Data are shown as mean ± SD, n = 5/group. kDa = kilodalton.
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