图1 PKR沉默Wilson病模型构建及实验流程图

Fig.1 Construction of the PKR-silenced Wilson's disease model and the experimental flow chart.

图2 PKR抑制剂C16对TX小鼠海马PKR磷酸化的抑制作用

Fig.2 Inhibitory effect of C16, a PKR inhibitor, on phosphorylation of PKR in the hippocampus of TX mice. A: Gray-scale band images of phosphorylated PKR and total PKR in the hippocampus of mice. B: Expression levels of phosphorylated PKR and total PKR in the different groups quantified using Western blotting (Mean±SD, n=3). **P<0.01 vs TX group.

图3 PKR抑制背景下"通腑养髓"法对TX小鼠旷场实验中焦虑样行为的影响

Fig.3 Effect of the Modified Gandou Decoction (MGDD) on anxiety-like behaviors of TX mice in open field test. A: Distance traveled in the center zone. B: Time spent in the center zone. C: Distance traveled in the peripheral zone. D: Time spent in the peripheral zone (Mean±SD, n=3). *P<0.05, **P<0.01 vs TX group; #P<0.05 vs C16 group; &&P<0.05 vs TX+MGDD group.

图4 PKR抑制背景下"通腑养髓"法对TX小鼠巴恩斯迷宫空间学习记忆的影响

Fig.4 Effect of MGDD on spatial learning and memory of TX mice. A: Barnes maze test results showing latency to the entry zone of mice in each group. B: Time in the entry zone in each group. C: Representative trajectory of the mice in each group in the test. Data are presented as Mean±SD (n=3-6). *P<0.05, **P<0.01 vs TX group.

图5 PKR抑制背景下"通腑养髓"法对TX小鼠海马神经元凋亡的影响

Fig.5 Effect of the MGDD on the apoptosis of the hippocampus neurons in TX mice. A: Representative immunofluorescence staining images for TUNEL in the hippocampus of mice in each group. Green: TUNEL. Blue: DAPI. Scale bar=200 µm. B: The TUNEL-positive cells of hippocampal neurons in each group of mice (Mean±SD, n=3). **P<0.01 vs TX group; #P<0.05 vs C16 group.

图6 PKR抑制背景下"通腑养髓"法对TX小鼠海马氧化应激损伤的影响

Fig.6 Effect of MGDD on oxidative stress injury in the hippocampus of TX mice. A: Representative immunofluo-rescence staining images for 8-OHdG in the CA1 region of the hippocampus of mice in each group. Red: 8-OHdG. Blue: DAPI. Scale bar=200 µm. B: OHdG-positive cells in the CA1 region of the hippocampus of mice in each group (Mean±SD, n=3). **P<0.01 vs TX group; &P<0.05 vs TX+MGDD group.

图7 PKR抑制背景下"通腑养髓"法对TX小鼠海马突触超微结构的影响

Fig.7 Representative TEM images of synapses in the CA1 region of the hippocampus in each group of mice. The right-side images are enlarged versions of the black boxes on the left images, and the arrows indicate to synaptic vesicles and the pre- and post-synaptic membranes. Scale bars on the left represent 1 µm, and those on the right represent 500 nm.

图8 PKR抑制背景下"通腑养髓"法对TX小鼠海马突触相关蛋白表达的影响

Fig.8 Effect of MGDD on synapse-related protein in the hippocampus of TX mice. A: Gray-scale band images of PSD95, PSD93, Synapsin1, Synaptophysin and α-tubulin in the hippocampus of mice. B: Expression level of synapse-related protein quantified using Western blotting in the different groups (Mean±SD, n=3). **P<0.01 vs TX group; #P<0.05, ##P<0.05 vs C16 group; &P<0.05, &&P<0.05 vs TX+MGDD group.

图9 PKR抑制背景下"通腑养髓"法对TX小鼠海马PKR/eIF2α通路相关基因mRNA表达的影响

Fig.9 Effect of MGDD on PKR/eIF2α‑related mRNA in the hippocampus of TX mice. A-E: Relative expression levels of Pkr, eIF2a, Atf4, Chop and Creb mRNAs (Mean±SD, n=3). *P<0.05, **P<0.01 vs TX group; &P<0.05 vs TX+MGDD group.

图10 PKR抑制背景下"通腑养髓"法对TX小鼠海马PKR/eIF2α/CREB信号通路的关键蛋白表达及磷酸化水平的影响

Fig.10 Effect of MGDD on expression levels of key proteins and phosphorylation in the PKR/eIF2α/CREB signaling pathway in the hippocampus of TX mice. A: Gray-scale band images of PKR, P-PKR, eIF2α, P-eIF2α, CREB, P-CREB and α‑tubulin in the hippocampus of mice. B: Expression levels of phosphorylated protein or total protein. C: Gray-scale band images of ATF4, CHOP and α‑tubulin in the hippocampus of the mice. D: Expression levels of ATF4 and CHOP. Data are presented as Mean±SD (n=3). **P<0.01 vs TX group; ##P<0.05 vs C16 group; &P<0.05, &&P<0.05 vs TX+MGDD group.