mTOR信号通路在中枢炎症小鼠学习记忆功能障碍中的作用

Role of mTOR signaling pathway in learning and memory dysfunction in mice with neuroinflammation

目的 mTOR信号通路在中枢炎症小鼠学习记忆功能障碍中的作用。方法 C57雄性成年小鼠156只,将其中108只小鼠随机分成空白对照组(Nctrl组,n=36)、溶液对照组(Sham组,n=36)和LPS脑室注射组(LPS组,n=36),LPS组给予脑室注射2 000 ng LPS,Sham组脑室注射等量的人工脑脊液,Nctrl组不做处理。分别在术后第1、2、3天,每组随机抽取6只进行条件恐惧实验,检测海马相关的学习记忆功能,另取6只行颈椎脱臼法处死,取海马组织,用ELISA法测量海马组织IL-1β、TNF-α、Aβ含量,Western blot法测量p-mTOR、p-P70S6K、p-Tau含量;将另48只C57小鼠随机分为对照组(Ctrl组,n=12)、雷帕霉素对照组(Ctrl+RAPA组,n=12)、LPS脑室注射组(LPS组,n=12)和雷帕霉素组(LPS+RAPA组,n=12)。LPS组和LPS+RAPA组脑室注射2 000 ng LPS,Ctrl+RAPA组和LPS+RAPA组在脑室注射前3 d每天腹腔注射0.5 mg/kg雷帕霉素预处理,Ctrl+RAPA组术前3 d每天注射与雷帕霉素等量的DMSO溶剂,Ctrl组不做处理。在术后第1天,每组随机抽取6只进行条件恐惧实验,检测海马相关的学习记忆功能,另外6只行颈椎脱臼法处死,取海马组织,用ELISA法测量海马组织IL-1β、TNF-α、Aβ含量,Western blot法测量p-mTOR、p-P70S6K、p-Tau含量。结果注射后第1,2天,LPS组与Nctrl组比较,小鼠的僵直比降低(D1 43.80%±5.27%vs 66.42%±5.52%;D2 53.35%±4.43%vs66.17%±5.79%)(P均<0.05)、小鼠海马组织的TNF-α浓度升高[D1(26.33±2.27)pg/ml vs(14.68±0.76)pg/ml;D2(21.16±2.18)pg/ml vs(15.11±1.09)pg/ml]、IL-1β[D1(27.36±2.53)pg/ml vs(17.49±1.18)pg/ml;D2(22.58±1.87)pg/ml vs(17.04±1.37)pg/ml](P均<0.05)、小鼠海马组织的p-mTOR(D1 2.54±0.14 vs 1.41±0.14;D2 2.06±0.13 vs 1.39±0.08)、p-P70S6K(D1 0.77±0.08 vs 0.46±0.06;D2 0.68±0.06 vs 0.45±0.05)、p-Tau(D1 1.34±0.13 vs 0.38±0.03;D2 1.21±0.16 vs0.39±0.04)、Aβ蛋白(D1 3.29±0.23 vs 1.96±0.11;D2 2.61±0.21 vs 1.96±0.07)含量均升高(P均<0.05);雷帕霉素处理实验中,LPS+RAPA组与LPS组比较,在LPS注射后的第1天小鼠的僵直比有所提高(52.78%±3.57%vs 42.13%±2.93%,P<0.05),海马组织TNF-α[(18.98±2.07)pg/ml vs(27.17±1.72)pg/ml]、IL-1β[(18.28±1.67)pg/ml vs(28.78±2.57)pg/ml]浓度均有所降低(P均<0.05),小鼠海马组织p-mTOR(1.67±0.11 vs 2.45±0.18)、p-P70S6K(0.65±0.05 vs 0.79±0.06)、p-Tau(0.56±0.07 vs 1.38±0.08)、Aβ蛋白(2.17±0.18 vs 3.27±0.22)含量有均有所降低(P均<0.05)。结论 mTOR信号通路参与了中枢炎症导致的小鼠学习记忆功能障碍。

Objective To investigate the role of mTOR signaling pathway in learning and memory dysfunction in mice with neuroinflammation. Methods One hundred and eight C57 mice were randomly divided into control group（Nctrl group, n=36）, solution control group（Sham group, n=36）, and intraventricular injection of LPS group（LPS group, n=36）. Mice in LPS group received intracerebroventricular injection of 2000 ng LPS, sham group received intracerebroventricular injection of equivalent artificial cerebrospinal fluid, Nctrl group did not receive any treatment. At d1, d2, d3, 6 rats of each group were given contextual fear condition（CFC） test to detect learning and memory function and other 6 rats were sacrificed to collect hippocampal tissue, IL-1β, TNF-α, Aβ were measured by ELISA and p-mTOR, p-P70 S6 K, p-Tau were measured by western blot. Another 48 C57 mice were randomly divided into control group（Ctrl group, n=12）, rapamycin control group（ctrl＋RAPA group, n=12）, intraventricular injection of LPS group（LPS group, n=12）, and rapamycin group（LPS＋RAPA group, n=12）. Mice in LPS group and LPS＋RAPA group received intracerebroventricular injection of 2 000 ng LPS, Ctrl＋RAPA group and LPS＋RAPA group were intraperitoneally injected of rapamycin at 0.5 mg/（kg·day） for 3 days before LPS intracerebroventricular injection. CFC, IL-1 β, TNF-α, p-mTOR, p-P70 S6 K, p-Tau, Aβ were measured with the same methods mentioned above. Results At d1 and d2 after injection, compared with Nctrl group, freezing time ratio in mice of LPS group decreased（d1： 43.80%±5.27% vs 66.42%±5.52%; d2： 53.35%±4.43% vs66.17%±5.79%; P〈0.05 respectively）, while, TNF-α[d1：（26.33±2.27） pg/ml vs（14.68±0.76） pg/ml; d2：（21.16±2.18） pg/ml vs（15.11±1.09） pg/ml] and IL-1β [d1：（27.36±2.53） pg/ml vs（17.49±1.18） pg/ml; d2：（22.58±1.87） pg/ml vs（17.04±1.37） pg/ml]increased in the hippocampus（P〈0.05 respectively）; p-mTOR（d1： 2.54±0.14 vs 1.41±0.14; d2： 2.06±0.13 vs 1.39±0.08）, p-P70 S6 K（d1： 0.77±0.08 vs 0.46±0.06; d2： 0.68±0.06 vs 0.45±0.05）, p-Tau（d1： 1.34±0.13 vs 0.38±0.03; d2： 1.21±0.16 vs0.39±0.04） and Tau protein（d1： 3.29±0.23 vs 1.96±0.11; d2： 2.61±0.21 vs 1.96±0.07） increased in the hippocampus in mice of LPS group（all P〈0.05）. In the mTOR experiment, compared with LPS group, freezing time ratio in mice of LPS＋RAPA group increased（52.78%±3.57% vs 42.13%±2.93%, P〈0.05）, while TNF-α[（18.98±2.07） pg/ml vs（27.17±1.72） pg/ml], IL-1β[（18.28±1.67） pg/ml vs（28.78±2.57） pg/ml] decreased in the hippocampus（P0.05 respectively）, p-mTOR（1.67±0.11 vs 2.45±0.18）, p-P70 S6 K（0.65±0.05 vs 0.79 ±0.06）, p-Tau（0.56±0.07 vs 1.38±0.08） and Tau protein（2.17±0.18 vs 3.27±0.22） decreased in the hippocampus of mice in LPS＋RAPA group（all P〈0.05）. Conclusion mTOR signaling pathway is involved in learning and memory dysfunction of neuroinflammation mice.