α-倒捻子素对肌萎缩侧索硬化的作用及其机制

doi:10.3969/j.issn.1674-8115.2022.09.013

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (9): 1265-1274.doi: 10.3969/j.issn.1674-8115.2022.09.013

• 论著 · 基础研究 • 上一篇

α-倒捻子素对肌萎缩侧索硬化的作用及其机制

王大元¹(), 徐见容¹, 江淦¹, 宋清香¹, 陈钧², 宋华华¹, 谷晓¹, 高小玲¹()

^1.上海交通大学医学院药理学与化学生物学系，上海 200025
^2.复旦大学药学院，上海 201203

收稿日期:2022-05-29 接受日期:2022-08-24 出版日期:2022-09-17 发布日期:2022-09-17
通讯作者: 高小玲 E-mail:wangdayuan@sjtu.edu.cn;shellygao1@sjtu.edu.cn
作者简介:王大元（1994—），男，博士生；电子信箱：wangdayuan@sjtu.edu.cn。
基金资助:
国家自然科学基金(81722043);上海市高水平地方高校创新团队(SHSMU-ZDCX20211201)

Effect of α-mangostin on amyotrophic lateral sclerosis and its mechanism

WANG Dayuan¹(), XU Jianrong¹, JIANG Gan¹, SONG Qingxiang¹, CHEN Jun², SONG Huahua¹, GU Xiao¹, GAO Xiaoling¹()

^1.Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
^2.Fudan University School of Pharmacy, Shanghai 201203, China

Received:2022-05-29 Accepted:2022-08-24 Online:2022-09-17 Published:2022-09-17
Contact: GAO Xiaoling E-mail:wangdayuan@sjtu.edu.cn;shellygao1@sjtu.edu.cn
Supported by:
National Natural Science Foundation of China(81722043);Innovative Research Team of High-Level Local Universities in Shanghai(SHSMU-ZDCX20211201)

摘要/Abstract

摘要：

目的·探究α-倒捻子素（α-mangostin，AM）对超氧化物歧化酶1（superoxide dismutase 1，SOD1）突变型肌萎缩侧索硬化（amyotrophic lateral sclerosis，ALS）的干预作用及可能的机制。方法·通过试剂盒检测小胶质细胞ATP水平变化，探究AM对SOD1聚集体处理的小胶质细胞（SOD1-ALS小胶质细胞模型）能量平衡的调控作用。通过Western blotting检测AMP活化蛋白激酶（AMP-activated protein kinase，AMPK）及其代谢调节通路中关键分子肝脏激酶B1和PPARγ协同激活因子-1α的激活水平，探究AM对疾病状态小胶质细胞AMPK通路的调控作用。通过化合物C抑制AMPK激活后检测ATP变化，探究AMPK在AM调控疾病状态小胶质细胞能量平衡中的作用。抑制AMPK激活的同时通过ELISA检测SOD1-ALS小胶质细胞对SOD1的摄取率和降解率，探究AMPK介导的能量调控在AM调节SOD1-ALS小胶质细胞清除SOD1中的作用。通过Western blotting和免疫荧光检测探究AM对疾病状态小胶质细胞自噬的作用及自噬在AM调节小胶质细胞降解SOD1中的作用。通过制备纳米颗粒包载的AM（NPAM）实现AM的体内给药。通过尾静脉注射的方式对SOD1*G93A ALS模型小鼠连续给予NPAM 30 d，1次/d。通过免疫组织化学染色检测NPAM给药后ALS小鼠腰段脊髓SOD1蛋白的聚集情况，通过尼氏染色和NeuN免疫组织化学染色检测神经元的损伤和丢失程度，通过转棒实验和后肢握紧实验评价ALS小鼠的运动能力，通过记录体质量和生存期评价ALS小鼠的疾病进程。结果·AM恢复了疾病状态小胶质细胞的ATP水平，增强了其对SOD1聚集体的摄取和通过自噬的降解；抑制AMPK通路后，AM的这些作用消失。NPAM减少了SOD1*G93A ALS模型小鼠腰段脊髓SOD1聚集体，减少了腰段脊髓神经元的损伤和丢失，还缓解了ALS小鼠的运动损伤和体质量丢失。结论·AM通过AMPK介导的能量平衡恢复作用增强了SOD1-ALS小胶质细胞模型对SOD1聚集体的清除，并显著改善了ALS模型小鼠的神经病理变化及疾病症状。

关键词: 肌萎缩侧索硬化, α-倒捻子素, 小胶质细胞, 超氧化物歧化酶1, 能量平衡, AMP活化蛋白激酶

Abstract:

Objective ·To study the effect of α-mangostin (AM) on the superoxide dismutase 1 (SOD1) mutant amyotrophic lateral sclerosis (ALS) and the possible mechanism. Methods ·The ATP level in the microglia was measured by kits to investigate the effect of AM on the energy balance in the SOD1 aggregates-treated microglia (SOD1-ALS model microglia). Western blotting was used to detect the activation levels of AMP-activated protein kinase (AMPK) and the key molecules in its metabolic regulation pathway including liver kinase B1 and PPARγ coactivator 1α, in order to explore the regulatory role of AM on AMPK pathway in the disease-associated microglia. The ATP level was also detected after AMPK activation being inhibited by compound C to explore the role of AMPK in the regulation of energy balance by AM in the SOD1-ALS microglia. The microglial uptake and degradation of SOD1 aggregates was detected by using ELISA with or without inhibition of AMPK activation, to explore the role of AMPK-mediated energy regulation in AM regulating SOD1 clearance in the disease-associated microglia. Western blotting and immunofluorescence were used to study the effect of AM on microglial autophagy and the role of autophagy in AM regulating the degradation of SOD1 in the microglia. AM-encapsulated nanoparticles (NPAMs) were prepared to realize the in vivo treatment of AM. NPAMs were injected into the SOD1*G93A ALS model mice through the caudal vein for 30 d, once a day. SOD1 aggregates were detected by immunohistochemistry in the lumbar spinal cords of the ALS mice. Neuronal damage and loss were measured by using Nissl staining and anti-NeuN immunohistochemistry. Motor ability of the ALS mice was evaluated by rotarod test and hindlimb clasping test. Disease progress of the ALS mice was evaluated by recording body mass and survival time. Results ·AM increased the ATP level in the diseased-associated microglia and enhanced microglial phagocytosis and autophagy-mediated degradation of SOD1 aggregates. While inhibiting AMPK pathway abolished these effects of AM, NPAM reduced SOD1 aggregates in the lumbar spinal cord of the SOD1*G93A ALS model mice, and reduced the neuron damage and loss. NPAM also alleviated the body mass loss and the damaged motor ability of the ALS mice. Conclusion ·AM enhances microglial clearance of SOD1 aggregates through AMPK-mediated recovery of energy balance, and thus alleviates the neuropathological changes and symptoms in ALS mice.

Key words: amyotrophic lateral sclerosis (ALS), α-mangostin (AM), microglia, superoxide dismutase 1 (SOD1), energy balance, AMP activated protein kinase (AMPK)

中图分类号:

R966

王大元, 徐见容, 江淦, 宋清香, 陈钧, 宋华华, 谷晓, 高小玲. α-倒捻子素对肌萎缩侧索硬化的作用及其机制[J]. 上海交通大学学报（医学版）, 2022, 42(9): 1265-1274.

WANG Dayuan, XU Jianrong, JIANG Gan, SONG Qingxiang, CHEN Jun, SONG Huahua, GU Xiao, GAO Xiaoling. Effect of α-mangostin on amyotrophic lateral sclerosis and its mechanism[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(9): 1265-1274.

图/表 3

图1 AM、NPAM通过AMPK通路恢复SOD1-ALS小胶质细胞ATP产能Note： A. ATP levels in the microglia. B. Western blotting analysis of AMPK, p-AMPK and GAPDH expression in the microglia (n=3). C. Western blotting analysis of LKB1, p-LKB1 and GAPDH expression in the microglia (n=3). D. Western blotting analysis of PGC-1α and GAPDH expression in the microglia (n=3). ①P=0.000, ②P=0.001, compared with the Blank group. C—compound C; PFF was a positive control.

Fig 1 AM and NPAM restoring ATP production through AMPK pathway in the SOD1-ALS microglia

图2 AM、NPAM增强小胶质细胞对SOD1聚集体的摄取和通过自噬的降解Note： A. Measurement of SOD1 uptake rate and degradation rate by using ELISA (n=3). B. Western blotting analysis of LC3-Ⅰ, LC3-Ⅱ and GAPDH expression in the microglia (n=3). C. Representative images of SOD1 and LC3B immunofluorescence. Scale bar=25 μm in the merged image; scale bar=5 μm in the magnified image. ①P=0.000, compared with the SOD1 group (A) or the Blank group (B).

Fig 2 AM and NPAM enhancing microglial phagocytosis and autophagy-mediated degradation of SOD1 aggregates

图3 NPAM减少了SOD1*G93A ALS模型小鼠腰段脊髓SOD1聚集体并缓解了疾病症状Note： A. Representative images of SOD1 staining in the lumbar spinal cords by immunohistochemistry. Scale bar=200 μm. B. Quantification of SOD1 integrated density by using ImageJ software (n=3). C. Representative images of Nissl staining in the ventral horns of lumbar spinal cords. The red arrows indicate normal neurons. Scale bar=100 μm. D. Representative images of NeuN staining in the lumbar spinal cords by immunohistochemistry. Scale bar=200 μm. E. Quantification of NeuN positive cells by using ImageJ software (n=3). F. Rotarod test (n=6). G. Hindlimb clasping test (n=6). H. Records of the body mass (n=6). I. Survival time (n=6). ①P=0.000, ②P=0.002, ③P=0.042, compared with SOD1*G93A+NPAM group (B and E) or SOD1*G93A+Saline group (F?H).

Fig 3 NPAM decreasing SOD1 aggregates and ameliorating the neuropathology in SOD1*G93A ALS mice

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α-倒捻子素对肌萎缩侧索硬化的作用及其机制

Effect of α-mangostin on amyotrophic lateral sclerosis and its mechanism

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