2型糖尿病脑病小鼠海马中转录因子EB活性与自噬功能的变化

doi:10.3969/j.issn.1674-8115.2023.02.004

摘要/Abstract

摘要：

目的·探讨2型糖尿病脑病小鼠海马中转录因子EB（transcription factor EB，TFEB）及上游蛋白活性和自噬功能的变化。方法·选取20只健康的8周龄雄性C57BL/6J小鼠，随机分为2型糖尿病（type 2 diabetes mellitus，T2DM）组和对照（CON）组，每组10只。T2DM组通过高脂饲料喂养和注射链脲佐菌素（streptozotocin，STZ）造模；CON组以普通饲料喂养，注射等体积柠檬酸钠缓冲液。2组小鼠每周检测随机血糖及体质量，第10周进行Morris水迷宫行为学实验。行为学实验结束后，行经腹腔注射葡萄糖耐量检测（intraperitoneal glucose tolerance test，IPGTT）；之后处死小鼠，留取血液和海马样本。用ELISA试剂盒检测血浆中胰岛素含量；通过Western blotting检测海马组织中淀粉样前体蛋白（amyloid precursor protein，APP）、tau蛋白、磷酸化tau（p-tau）蛋白、自噬溶酶体相关蛋白［包括溶酶体相关膜糖蛋白1（lysosomal-associated membrane protein 1，LAMP1）、微管相关蛋白1A/1B-轻链3（microtubule-related protein 1A/1B-light chain 3，LC3）、P62］、TFEB、磷酸化TFEB（p-TFEB）、哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）、磷酸化mTOR（p-mTOR）与溶酶体钙调通道蛋白MCOLN1（mucolipin TRP cation channel 1，MCOLN1）的表达。采用免疫荧光染色检测小鼠海马组织中β-淀粉样蛋白（β-peptide，Aβ）和p-tau蛋白的沉积。结果·与CON组相比，T2DM组小鼠体质量、随机血糖、血浆中胰岛素水平和IPGTT结果均显著升高（均P<0.05）。水迷宫行为学实验中，T2DM组小鼠逃避潜伏期较CON组显著延长，在目标象限中的停留时间显著缩短，穿越平台次数显著减少（均P<0.05），提示T2DM组小鼠已出现学习记忆功能下降。与CON组相比，T2DM组小鼠的海马组织中Aβ、APP、tau、p-tau蛋白表达水平与p-tau/tau显著升高，自噬相关蛋白LAMP1表达和LC3-Ⅱ/LC3-Ⅰ比值显著下降，P62表达水平显著升高，TFEB和MCOLN1蛋白表达水平显著下降，p-TFEB、mTOR、p-mTOR表达与p-mTOR/mTOR比值显著升高（均P<0.05）。结论·糖尿病脑病小鼠海马组织出现阿尔茨海默病样神经病变，同时海马中TFEB活性降低，自噬功能下调，TFEB上游调节因子MCOLN1表达下降，mTOR活性增强。

关键词: 糖尿病脑病, 自噬, 转录因子EB, 溶酶体钙调通道蛋白, 哺乳动物雷帕霉素靶蛋白

Abstract:

Objective ·To investigate the changes of transcription factor EB (TFEB), the activity of the upstream proteins and autophagy in the hippocampus of mice with type 2 diabetic encephalopathy. Methods ·Twenty healthy 8-week-old male C57BL/6J mice were randomly divided into type 2 diabetes mellitus (T2DM) group and control (CON) group, with 10 mice in each group. The T2DM group was fed with high fat feed and injected with streptozotocin (STZ); the CON group was fed with ordinary diet and injected with equal-volume sodium citrate buffer. The random blood glucose level and the body weight were measured on the mice in both groups weekly. In the 10th week, the Morris water maze behavioral experiments were performed. Then, the intraperitoneal glucose tolerance test (IPGTT) was carried out. The blood and hippocampus samples were collected after the mice were sacrificed. The plasma insulin content was detected by the ELISA kit. The protein levels of amyloid precursor protein (APP), tau protein, phospho-tau (p-tau) protein, autophagy-lysosome-related proteins [including lysosomal-associated membrane protein 1 (LAMP1), microtubule-related protein 1A/1B-light chain 3 (LC3), and P62], TFEB, p-TFEB, mammalian target of rapamycin (mTOR), p-mTOR, and mucolipin TRP cation channel 1 (MCOLN1) in the hippocampus were detected by Western blotting. The deposition of β-peptide (Aβ) and p-tau proteins in the hippocampus was detected by immunofluorescence staining. Results ·Compared with the CON group, the body weight, the random blood glucose level, the plasma insulin level, and the IPGTT results were significantly increased in the T2DM group (P<0.05). In the water maze experiments, the escape latency of the T2DM group in the place navigation increased, and the time spent in the target quadrant and the number of times crossing the platform in the spatial probe decreased (P<0.05), indicating that the ability of learning and memory of mice was damaged in the T2DM group. The levels of Aβ, APP, tau, p-tau, P62, p-TFEB, mTOR and p-mTOR proteins, and the ratios of p-tau/tau and p-mTOR/mTOR significantly increased, while the expression of LAMP1, TFEB and MCOLN1 and the ratio of LC3-Ⅱ/LC3-Ⅰ prominently decreased in the hippocampus of mice in the T2DM group in comparison with the CON group (P<0.05). Conclusion ·Alzheimer's disease-like neuropathy is observed in the hippocampus of diabetic encephalopathy mice, along with decreased TFEB activity, down-regulated autophagy function, decreased expression of MCOLN1, and enhanced mTOR activity in the hippocampus.

Key words: diabetic encephalopathy, autophagy, transcription factor EB (TFEB), mucolipin TRP cation channel 1 (MCOLN1), mammalian target of rapamycin (mTOR)

中图分类号:

R587.2

陈奕馨, 程丽珍, 林祎嘉, 苗雅. 2型糖尿病脑病小鼠海马中转录因子EB活性与自噬功能的变化[J]. 上海交通大学学报（医学版）, 2023, 43(2): 162-170.

CHEN Yixin, CHENG Lizhen, LIN Yijia, MIAO Ya. Change of transcription factor EB activity and autophagy in hippocampus of type 2 diabetic encephalopathy mice[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(2): 162-170.

图/表 6

图1 2组小鼠糖代谢水平的比较 (n=10)Note： A. Weight. B. RBG. C. Plasma insulin. D. Results of IPGTT. ①P=0.000, compared with the CON group.

Fig 1 Comparison of glucose metabolic levels between the two groups of mice (n=10)

图2 2组小鼠学习记忆功能的比较 (n=10)Note： A. Typical swimming traces of the two groups of mice in the place navigation and the spatial probe. B. Comparison of the escape latency between the two groups in the place navigation. C. Comparison of the number of times crossing the platform between the two groups in the spatial probe. D. Comparison of the time spent in the target quadrant between the two groups in the spatial probe. ①P=0.006, ②P=0.002, compared with the CON group.

Fig 2 Comparison of learning and memory function between the two groups of mice (n=10)

图3 2组小鼠海马APP、p-tau、tau和Aβ蛋白水平的比较 (n=10)Note： A. The Western blotting images of APP, p-tau and tau proteins in the hippocampi of mice in the two groups. B. Statistical histogram of the protein bands in the Western blotting analysis. C. The immunofluorescence images of Aβ protein in the hippocampi of mice in the two groups. D. Statistical histogram of the fluorescence intensity of Aβ. E. The immunofluorescence images of p-tau protein in the hippocampi of mice in the two groups. F. Statistical histogram of the fluorescence intensity of p-tau. ①P=0.004, ②P=0.000, ③P=0.032, ④P=0.003, compared with the CON group.

Fig 3 Comparison of APP, p-tau, tau and Aβ proteins in the hippocampi of mice between the two groups (n=10)

图4 2组小鼠海马中LC3-Ⅰ/Ⅱ、P62和LAMP1蛋白水平的比较 (n=10)Note： A. The Western blot images of LC3-Ⅰ/Ⅱ, P62 and LAMP1 proteins in the hippocampi of mice in the two groups. B. Statistical histogram of the protein bands in the Western blotting analysis. ①P=0.032, ②P=0.016, ③P=0.040, compared with the CON group.

Fig 4 Comparison of LC3-Ⅰ/Ⅱ, P62 and LAMP1 proteins in the hippocampi of mice between the two groups (n=10)

图5 2组小鼠海马中p-TFEB和TFEB蛋白水平的比较 (n=10)Note： A. The Western blotting images of p-TFEB and TFEB proteins in the hippocampi of mice in the two groups. B. Statistical histogram of the protein bands in the Western blotting analysis. ①P=0.001, ②P=0.028, ③P=0.018, compared with the CON group.

Fig 5 Comparison of p-TFEB and TFEB proteins in the hippocampi of mice between the two groups (n=10)

图6 2组小鼠海马中p-mTOR、mTOR和MCOLN1蛋白水平的比较 (n=10)Note： A. The Western blotting images of p-mTOR and mTOR proteins in the hippocampi of mice in the two groups. B. Statistical histogram of the p-mTOR and mTOR protein bands in the Western blotting analysis. C. The Western blotting images of MCOLN1 protein in the hippocampi of mice in the two groups. D. Statistical histogram of the MCOLN1 protein bands in the Western blotting analysis. ①P=0.014, ②P=0.044, ③P=0.048, ④P=0.005, compared with the CON group.

Fig 6 Comparison of p-mTOR, mTOR, and MCOLN1 proteins in the hippocampi of mice between the two groups (n=10)

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