小续命汤提取物改善血栓性局灶性脑缺血大鼠脑损伤及利用蛋白质组学探讨其可能治疗靶点

doi:10.5246/jcps.2021.06.036

中国药学(英文版) ›› 2021, Vol. 30 ›› Issue (6): 468-483.DOI: 10.5246/jcps.2021.06.036

小续命汤提取物改善血栓性局灶性脑缺血大鼠脑损伤及利用蛋白质组学探讨其可能治疗靶点

杨滢霖¹^,², 张姗姗¹^,², 刘漫¹^,², 王月华¹^,²^,^*(), 杜冠华¹^,²^,^*()

1. 中国医学科学院药物研究所天然药物活性物质与功能国家重点实验室, 北京 100050
2. 中国医学科学院药物研究所药物靶点研究与新药筛选北京市重点实验室, 北京 100050

收稿日期:2020-11-28 修回日期:2021-02-13 接受日期:2021-03-15 出版日期:2021-06-29 发布日期:2021-06-29
通讯作者: 王月华, 杜冠华
作者简介:
+ Tel.: +86-10-63165313, E-mail: wangyuehua@pku.org.cn
+ Tel.: +86-10-63165184, E-mail: dugh@imm.ac.cn
基金资助:
The National Natural Science Foundation of China (Grant No. 81473383), the Significant New-Drugs Creation of Science and Technology Major Projects (Grant No. 2018ZX09711001-003-019), the Medical and Health Innovation Project of Chinese Academy of Medical Sciences (Grant No. 2016-I2M-3-007), and Innovation Fund for Graduate of Beijing Union Medical College (Grant No. 2018-1007-04).

Xiao-Xu-Ming decoction extract ameliorates brain injury in rats with thrombotic focal ischemic stroke and understanding possible therapeutic targets using proteomics

Yinglin Yang¹^,², Shanshan Zhang¹^,², Man Liu¹^,², Yuehua Wang¹^,²^,^*(), Guanhua Du¹^,²^,^*()

1 State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
2 Beijing Key Laboratory of Drug Target Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China

Received:2020-11-28 Revised:2021-02-13 Accepted:2021-03-15 Online:2021-06-29 Published:2021-06-29
Contact: Yuehua Wang, Guanhua Du

摘要/Abstract

摘要：

缺血性中风严重威胁着人类的健康和生活质量。小续命汤是中风治疗的经典方剂, 前期研究发现小续命汤提取物(XXM)对脑缺血大鼠具有改善脑损伤、减轻神经炎症和神经保护作用。本研究旨在探讨XXM对血栓性局灶性脑缺血的影响及其可能机制。与血栓性局灶性脑缺血大鼠相比, XXM治疗后神经功能和运动能力得到改善, 脑梗死体积明显减少。此外, EB渗漏和紧密连接蛋白表达检测BBB完整性的结果表明, XXM能够维持BBB的完整性, 并改善血栓性脑缺血所致缺血同侧皮质中claudin-1、occludin和ZO-1等紧密连接蛋白的表达。此外, 采用非标记蛋白质组学技术对缺血大脑皮层中的差异蛋白进行了鉴定, 结果表明, 在缺血大脑皮层中检测到132种受XXM调控的差异表达蛋白(DEPs)。生物信息学分析表明, XXM调节蛋白主要参与补体、凝血级联和溶酶体等, 而且Lgals3、Ctsz、Capg、C1qa、S100a4、Grn、Hspb1、Aif1、Anxa1等差异蛋白之间存在相互作用。总之, XXM可改善血栓性局灶性缺血性中风的脑损伤, Lgals3、Ctsz、Capg、C1qa、S100a4、Grn、Hspb1、Aif1、Anxa1可为XXM深入研究提供可能的治疗靶点和研究方向。

关键词: 小续命汤, 血栓性局灶性脑缺血, 血脑屏障, 蛋白质组

Abstract:

Ischemic stroke seriously threatens human health and quality of life. Xiao-Xu-Ming (XXM) decoction has been a classical prescription for stroke therapy. In our previous studies, we have found that XXM exerts neuroprotective effects, improves brain injury, and attenuates neuroinflammation in cerebral ischemia rats. In this study, we investigated the effects and possible mechanism of XXM on thrombotic focal cerebral ischemia. After treatment with XXM, the neurological function and motor abilities were improved, and cerebral infarction volume was significantly decreased compared with rats of thrombotic focal cerebral ischemia. Besides, the results of BBB integrity detected by EB leakage and tight junction (TJ) protein expression showed that XXM could maintain BBB integrity and improve the expressions of TJ proteins, including claudin-1, occluding, and ZO-1, in the ischemic ipsilateral cortex disrupted by thrombotic cerebral ischemia. Furthermore, proteomic techniques were used to identify the differentially expressed proteins (DEPs) in the ischemic cerebral cortex, and the results showed that 132 DEPs regulated by XXM were detected in the ischemic cerebral cortex. Bioinformatic analysis showed that these regulated proteins by XXM were mainly involved in complement and coagulation cascade, and lysosome, etc. Furthermore, there was an interaction among DEPs, including Lgals3, Ctsz, Capg, C1qa, S100a4, Grn, Hspb1, Aif1, and Anxa1, etc. In conclusion, XXM ameliorated brain injury of thrombotic focal ischemic stroke, and Lgals3, Ctsz, Capg, C1qa, S100a4, Grn, Hspb1, Aif1, and Anxa1 could help provide possible therapeutic targets of XXM for ischemic stroke and offer research direction for further research.

Key words: Xiao-Xu-Ming decoction, Thrombotic focal ischemic stroke, Blood-brain barrier, Proteomics

Supporting:

杨滢霖, 张姗姗, 刘漫, 王月华, 杜冠华. 小续命汤提取物改善血栓性局灶性脑缺血大鼠脑损伤及利用蛋白质组学探讨其可能治疗靶点[J]. 中国药学(英文版), 2021, 30(6): 468-483.

Yinglin Yang, Shanshan Zhang, Man Liu, Yuehua Wang, Guanhua Du. Xiao-Xu-Ming decoction extract ameliorates brain injury in rats with thrombotic focal ischemic stroke and understanding possible therapeutic targets using proteomics[J]. Journal of Chinese Pharmaceutical Sciences, 2021, 30(6): 468-483.

图/表 8

Table 1. Primer list for RT-PCR.

Figure 1. Effect of XXM on neurobehavioral function in rats with thrombotic focal cerebral ischemia. (A) mNSS test (n = 25); (B) Longa test (n = 15); (C) Coat-hanger tests (n = 15); (D) Rotarod tests (n = 15). Data were presented as mean ± SD. ##P < 0.01 vs. sham group; **P < 0.01 vs. IS group.

Figure 2. Effect of XXM on cerebral infarction volume in rats with thrombotic focal cerebral ischemia. (A) Representative images of cerebral infarction by TTC-stained coronal sections of the brain; (B) Infarct volume calculated from section thickness and infarct area as a result of TTC staining (n = 8); (C) Representative images of cerebral infarction by MRI detection; (D) The infarct volume calculated from section thickness and infarct area as a result of MRI scanning (n = 4). Values are mean ± SD; ##P < 0.01 vs. Sham group; **P < 0.01 vs. IS group.

Figure 3. Effect of XXM on BBB integrity and tight junction (TJ) protein expression in rats with thrombotic focal cerebral ischemia. (A) The coronal sections of brain in EB leakage test; (B) Statistical analysis of EB content (n = 8); (C) The expression of claudin-1, occluding and ZO-1. (D) The relative expression of claudin-1 (n = 6); (E) The relative expression of occludin (n = 6); (F) The relative expression of ZO-1 (n = 6). Data were presented as mean ± SD. #P < 0.05, ##P < 0.01 vs. sham group; *P < 0.05, **P < 0.01 vs. IS group.

Figure 4. Clustering heatmap of the significant proteins by proteomics in ischemic cortex tissues of rats with thrombotic focal cerebral ischemia.

Figure 5. GO analysis of the DEPs in the ischemic cortex of rats with thrombotic focal cerebral ischemia. The cut-off of differential expression of mRNA and protein was set at 1.5-fold change and P < 0.05. The above x-axis shows the value of –Log (P-value), the below x-axis shows the number of increased/decreased genes, and the right y-axis shows the functional categories of the increased or decreased genes.

Figure 6. The KEGG analysis of DEPs in the ischemic cortex of rats with thrombotic focal cerebral ischemia. (A) KEGG analysis of DEPs; (B) Interactions of the DEPs through network analysis using the String database. Network nodes represent proteins (each node represents all the proteins produced by a single, protein-coding gene locus). Edges represent protein-protein associations. Colored nodes: query proteins and first shell of interactions. White nodes: the second shell of interactions. Empty nodes: proteins of unknown three-dimensional structure. Filled nodes: some three-dimensional structure is known or predicted.

Figure 7. mRNA expression by qRT-PCR analysis in the ischemic cortex of rats with thrombotic focal cerebral ischemia. Data were presented as mean ± SD (n = 3). ##P < 0.01 vs. sham group; **P < 0.01 vs. IS group.

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小续命汤提取物改善血栓性局灶性脑缺血大鼠脑损伤及利用蛋白质组学探讨其可能治疗靶点

Xiao-Xu-Ming decoction extract ameliorates brain injury in rats with thrombotic focal ischemic stroke and understanding possible therapeutic targets using proteomics

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