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TMR Modern Herbal Medicine  2018, Vol. 1 Issue (3): 143-154    DOI: 10.12032/TMRmhm2017A21
Orginal Articles     
Neuroprotective Effect of Bu-Shen-Huo-Xue Extract against High Glucose-induced Apoptosis in PC12 Cells
Zhao Shao-Yang1,2, Dong Xin2, Tu Peng-Fei2, Zeng Ke-Wu2,*(), Wang Xue-Mei1,*()
1Research Studio of Integration of Traditional and Western Medicine, First Hospital, Peking University, Beijing, China
2State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China.
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Objective: To investigate the neuroprotective effect of Bu-Shen-Huo-Xue (BSHX) extract, a polyherbal formula, against High Glucose (HG)-induced neurotoxicity in PC12 cells. Methods: Cell viability assay, Lactate Dehydrogenase (LDH) assay, Reactive Oxygen Species (ROS) detection, Hoechst 33258, Acridine Orange (AO)/Ethidium Bromide (EB) double stain and Mitochondrial Membrane Potential (MMP) assay were performed. In addition, Bax, Bcl-2, caspase-3, cleaved caspase-3, PARP, cleaved PARP, cytochrome c and Mitogen-Activated Protein Kinases (MAPKs) were detected by western blot. Results: BSHX extract increased cell viability and decreased LDH leakage in a concentration-dependent manner in HG-induced PC12 cells. Moreover, BSHX extract decreased the level of intracellular ROS, increased mitochondrial membrane potential, regulated the expressions of Bax and Bcl-2, and inhibited the release of cytochrome c from mitochondria. Furthermore, BSHX extract attenuated the activation of caspase-3 and PARP, and inhibited the phosphorylations of c-Jun N-terminal kinase (JNK) and p38 MAPKs. Conclusion: BSHX extract exhibited significant neuroprotective effect on HG-induced apoptosis in PC12 cells. This effect may be associated with the suppression of ROS generation as well as mitochondria-mediated caspase and JNK/p38 MAPK signaling pathways.

Highlights

High glucose (HG)-induced neurotoxicity is implicated in the pathology of diabetic encephalopathy (DE). In our study, Bu-Shen-Huo-Xue extract (BSHX), a polyherbal formula, exhibits neuroprotective activity on HG-induced PC12 cells and the possible mechanisms may be associated with the suppression of reactive oxygen species (ROS) generation as well as mitochondria-mediated caspase and JNK/p38 MAPK signaling pathways. This study provids a promising agent for the treatment of DE in clinical applications.



Key wordsdiabetic encephalopathy      traditional Chinese medicine      Bu-Shen-Huo-Xue extract      neuroprotective      high glucose     
Received: 05 July 2018      Published: 25 July 2018
Fund:  This work was supported by the National Natural Science Foundation of China (Grant Nos. 81530099, 81573763 and 81773932), the Beijing Municipal Natural Science Foundation (Grant No.7172221), and the National Key Technology R&D Program “New Drug Innovation” of China (Grant No. 2016YFE0116200).
Corresponding Authors: Zeng Ke-Wu,Wang Xue-Mei     E-mail: ZKW@bjmu.edu.cn;wangxuemeibjmu@163.com
Cite this article:

Zhao Shao-Yang, Dong Xin, Tu Peng-Fei, Zeng Ke-Wu, Wang Xue-Mei. Neuroprotective Effect of Bu-Shen-Huo-Xue Extract against High Glucose-induced Apoptosis in PC12 Cells. TMR Modern Herbal Medicine, 2018, 1(3): 143-154. doi: 10.12032/TMRmhm2017A21

URL:

https://www.tmrjournals.com/mhm/EN/10.12032/TMRmhm2017A21     OR     https://www.tmrjournals.com/mhm/EN/Y2018/V1/I3/143

Herbal composition Part used Amounts used (g)
Cuscuta chinensis Lam.
Lycium barbarum L
Rubus chingii Hu.
Schizandra chinensis (Turcz.) Baill.
Plantago asiatica L.
Epimedium brevicornu Maxim.
Hirudo nipponica Whitman
Total mount
Fruit
Fruit
Fruit
Fruit
Fruit
Herb
The whole body
40
40
20
5
10
40
5
160
Table 1 Composition of BSHX
Figure.1 BSHX extract protects PC12 cells from HG-induced injury.
(A) PC12 cells were subjected to HG (75 mM) for 48 h alone, or co-treated with BSHX granule (20, 50, 100 mg/L). (B) PC12 cells were subjected to HG with or without BSHX extract as in (A). All data are presented as mean ± S.D. from independent experiments where n=6. ###P < 0.001 relative to control group; *P < 0.05, ***P < 0.001 relative to HG group.
Figure.2 BSHX extract protects PC12 cells against HG-induced apoptosis via caspase-3/PARP pathway
(A) PC12 cells were subjected to HG (75 mM) with or without BSHX extract for 48 h. Apoptotic nuclei were identified using Hoechst 33258 staining, scale bar = 50μm. (B) The cells were treated as in (A). Apoptotic cells were identified by double staining with AO and EB. The cells which took up both dyes were classified as apoptotic (indicated by arrows in the merged panels). Scale bar = 50μm. (C) PC12 cells were subjected to HG with or without BSHX extract as before, then lysed and immunoblotted for caspase-3, cleaved caspase-3, PARP, cleaved PARP. Data were presented as mean ± S.D. from independent experiments performed in triplicate. ###P < 0.001 relative to control group; *P < 0.05, ***P < 0.001 relative to HG group.
Figure.3 Effect of BSHX extract on ROS production in HG-treated PC12 cells
PC12 cells were subjected to HG (75 mM) with or without BSHX extract for 48 h. Then, PC12 cells were loaded with DCF-DA, and ROS were detected by flow ctytometer. All data are presented as mean ± S.D. from independent experiments performed in triplicate. ###P < 0.001 relative to control group; *P < 0.05, ***P < 0.001 relative to HG group.
Figure.4 BSHX extract protects mitochondrial membrane integrity
(A) The cells were subjected to HG (75 mM) with or without BSHX extract for 48 h. Mitochondrial depolarization was investigated by JC-1 staining assay. The cells with depolarized mitochondria were identified by green fluorescence, and quantified as the percentage of the total cell number. (B) The cells were subjected to HG (75 mM) with or without BSHX extract as before, then mitochondrial and cytosolic fractions were separated and cytochrome c was detected by western blot. (C) The cells were treated as before, then lysed and immunoblotted for Bax and Bcl-2. Data were presented as mean ± S.D. from independent experiments performed in triplicate. ###P < 0.001 relative to control group; *P < 0.05, ***P < 0.001 relative to HG group.
Figure.5 BSHX extract inhibits JNK/p38MAPK pathways in HG-induced PC12 cells
The cells were subjected to HG (75 mM) with or without BSHX extract for 48 h. The protein expression of p-p38/p38, p-JNK/JNK, p-ERK/ERK were analyzed by western blot. Data were presented as mean ± S.D. from independent experiments performed in triplicate. ##P < 0.001 relative to control group; **P < 0.01, ***P < 0.001 relative to HG group.
Figure.6 Mechanism of BSHX extract against HG-induced apoptosis in PC12 cells.
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