Proteomic analysis reveals the potential neuroprotective effects of tetramethylpyrazine dimer in neuro2a/APPswe cells

Article abstract of DOI:10.1039/c9ra03054a

Alzheimer's disease (AD) is a common neurodegenerative disease characterized by pathological processes, including abnormal amyloid deposits and filament tangles, oxidative stress, neuroinflammation, and neurotrophic insufficiency, leading to chronic and prolonged neuronal loss and cognitive deficits. Tetramethylpyrazine (TMP) is one of the main active components of Ligusticum wallichii, a traditional Chinese medicine widely used for brain related disease. Here, we synthesized the TMP derivative tetramethylpyrazine dimer (DTMP), and evaluated the potential mechanisms underlying its potential neuroprotective effects using the murine neuron-like cells (N2a) transfected with the human "Swedish" mutant amyloid precursor protein (N2aAPP). ELISA results indicated that DTMP reduced the levels of Aβ_1-40 and Aβ_1-42 in N2aAPP. Then through proteomic analysis we identified a total of 208 differentially expressed proteins in N2aAPP cells compared to the wild-type N2a cells (N2aWT), including 144 increased and 64 decreased proteins. 449 differentially expressed proteins were revealed in N2aAPP cells on DTMP treatment with 69 increased and 380 decreased proteins. Bioinformatic analysis suggested that these proteins are enriched in mitochondrial function, the electronic transmission chain, ATP binding, oxidative phosphorylation, GTPase function, the transcriptional translation process, amino acid metabolism, nucleotide binding and others. Given the vital role of mitochondria in the pathogenesis of AD, we selected the electron transport chain pathway-related molecules to further validate these findings. Western-blot analysis demonstrated that DTMP significantly increased the levels of complex I (NDUAA), complex II (SDHB), complex III (UCRI), complex IV (COX5A) and complex V (ATP5A) in N2aAPP cells. The modulation of dysregulated proteins implicated in AD pathogenesis implies the pharmacological mechanisms of DTMP and its potential as a novel therapeutic choice in AD.

Full text of DOI:10.1039/c9ra03054a

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Proteomic analysis reveals the potential
neuroprotective effects of tetramethylpyrazine
dimer in neuro2a/APPswe cells†
Cite this: RSC Adv., 2019, 9, 18776
ab
b
d
e
e
f
Xiaoyi Lin, Benhong Xu, Zaijun Zhang, Ying Yang, Gongping Liu, Feiqi Zhu,
b
b
c
ab
b
Xiaohu Ren, Jianjun Liu, Shupeng Li,* Xianfeng Huang* and Xifei Yang*
Alzheimer's disease (AD) is a common neurodegenerative disease characterized by pathological
processes, including abnormal amyloid deposits and filament tangles, oxidative stress,
neuroinflammation, and neurotrophic insufficiency, leading to chronic and prolonged neuronal loss
and cognitive deficits. Tetramethylpyrazine (TMP) is one of the main active components of Ligusticum
wallichii, a traditional Chinese medicine widely used for brain related disease. Here, we synthesized
the TMP derivative tetramethylpyrazine dimer (DTMP), and evaluated the potential mechanisms
underlying its potential neuroprotective effects using the murine neuron-like cells (N2a) transfected
with the human “Swedish” mutant amyloid precursor protein (N2aAPP). ELISA results indicated that
DTMP reduced the levels of Ab1–40 and Ab1–42 in N2aAPP. Then through proteomic analysis we
identified a total of 208 differentially expressed proteins in N2aAPP cells compared to the wild-type
N2a cells (N2aWT), including 144 increased and 64 decreased proteins. 449 differentially expressed
proteins were revealed in N2aAPP cells on DTMP treatment with 69 increased and 380 decreased
proteins. Bioinformatic analysis suggested that these proteins are enriched in mitochondrial function,
the electronic transmission chain, ATP binding, oxidative phosphorylation, GTPase function, the
transcriptional translation process, amino acid metabolism, nucleotide binding and others. Given the
vital role of mitochondria in the pathogenesis of AD, we selected the electron transport chain
pathway-related molecules to further validate these findings. Western-blot analysis demonstrated that
DTMP significantly increased the levels of complex I (NDUAA), complex II (SDHB), complex III (UCRI),
complex IV (COX5A) and complex V (ATP5A) in N2aAPP cells. The modulation of dysregulated proteins
implicated in AD pathogenesis implies the pharmacological mechanisms of DTMP and its potential as
a novel therapeutic choice in AD.
Received 24th April 2019
Accepted 7th May 2019
DOI: 10.1039/c9ra03054a
rsc.li/rsc-advances
1
. Introduction
a
College of Pharmaceutical Engineering and Life Sciences, Changzhou University, No.
Alzheimer's disease (AD) is one of the most common neurode-
generative diseases. Approximately 46.8 million people suffer
from AD worldwide and the number is expected to reach 74.7
million by 2030 as the population ages. Characterized path-
2
1, Gehu Middle Road, Wujin District, Changzhou, China 213000. E-mail: huangxf@
1,2
cczu.edu.cn; Fax: +86 13914325607; Tel: +86 13914325607
b
Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease
3,4
Control and Prevention, No. 8, Longyuan Road, Nanshan District, Shenzhen, China
5
18055. E-mail: xifeiyang@gmail.com; Fax: +86 75525508584; Tel: +86 75525601914 ological changes of AD mainly include the formation of b-
c
State Key Laboratory of Oncogenomic, School of Chemical Biology and Biotechnology, amyloid (Ab), neuronal synaptic loss, neurobrillary tangles
Peking University Shenzhen Graduate School, Shenzhen 518055, China. E-mail: lisp@
5,6
(
NFT) and mitochondrial impairment. However, the efficient
therapeutic strategies are limited.
,3,5,6-Tetramethylpyrazine (TMP) is one of the main active
ingredients of Ligusticum wallichii, a traditional Chinese medi-
pkusz.edu.cn; Fax: +86 75526032325; Tel: +86 75526032325
d
Institute of New Drug Research and Guangzhou, Key Laboratory of Innovative
2
Chemical Drug Research in Cardio-Cerebrovascular Diseases, Jinan University
College of Pharmacy, Guangzhou, 510632, China
7
e
Department of Pathophysiology, School of Basic Medicine and the Collaborative cine. TMP has been reported to inhibit platelet aggregation and
2
+
Innovation Center for Brain Science, Key Laboratory of Ministry of Education of thrombolysis, reduce Ca overload, inhibit apoptosis and
China for Neurological Disorders, Tongji Medical College, Huazhong University of
8,9
scavenge free radicals. In the whole brain and focal cerebral
Science and Technology, Wuhan 430030, China
ischemia model, TMP exhibited strong neuroprotective effects
f
Cognitive Impairment Ward of Neurology Department, The 3rd Affiliated Hospital of
10
in both global and focal cerebral ischemic injury models. TMP
can also elevate learning and memory ability, and alleviate
cholinergic dysfunction of D-galactose injury in mice. Some
Shenzhen University, China
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c9ra03054a
11
1
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studies showed TMP can reduce oxidative stress induced Runjie Chemical Reagent Co., Ltd (Nanjing, China); N-bromo
neuronal death, protect against toxicity induced by kainite succinimide were bought from Jiangsu Qiangsheng Functional
12–14
excitability, and benet mitochondrial biogenesis.
Simi- Chemical Co., Ltd (Changshu, China); ascending Sulfur were
larly, diallyl disulde (DADS), a compound in garlic has shown bought from Sinopharm Chemical Reagent Co., Ltd (Shanghai,
15,16
cardioprotective and neuroprotective effects.
It was found China) and dimethyl sulfoxide were purchased from bought from
that the extract containing DADS can prevent APP processing Thermo Fisher Scientic (Waltham, MA, United States). DMSO
17–19
and tau phosphorylation in an AD transgenic mouse model.
Both TMP and DADS showed therapeutic potentials in AD, but
for preparing DTMP (50 mM) and used as a stock solution.
their pharmacological mechanisms are yet to be dened. In this 2.2 Synthesis of DTMP
study, we designed and synthesized a combined molecular of
The lead compound was synthesized as illustrated in Fig. 1.
First, ligustrazine (5.0 g, 36.76 mmol) and benzoyl peroxide
0.84 g, 3.47 mmol) were dissolved in carbon tetrachloride, and
N-bromo succinimide (6.74 g, 3.45 mmol) were added in three
TMP and DADS, named tetramethylpyrazine dimer (DTMP),
and evaluated it neuroprotective effects. The potential mecha-
nism of DTMP neuroprotection was assessed using N2a cells.
Proteomic and bioinformatic strategies were used to identify
differentially expressed proteins in cells with or without DTMP-
treated N2aAPP cells. Our results revealed that DTMP had
a potential therapeutic effect on AD by modulating the dysre-
gulated proteins involved in the pathogenesis of AD, such as
mitochondria-associated molecules.
(
ꢀ
ꢀ
portions at 45 C. Then the temperature was raised to 70 C for
0 hours and then stopped. It was ltered with hot saturated
1
sodium bicarbonate solution, extracted with dichloromethane.
In the second step, sublimed sulfur (122.76 mg, 3.84 mmol) and
sodium sulde (1.815 g, 23.27 mmol) were dissolved in 82.5 ml of
ꢀ
water and reacted at 50 C for 30 minutes, taken out and cooled to
room temperature. First, all of the compound 2 was added, then
dodecyl dimethylammonium bromide (2640 mg, 5.70 mmol),
2. Materials and methods
82.5 ml of chloroform was added and reacted at room temperature.
2
.1 Reagents
4 h later, an appropriate amount of water and dichloromethane
were added for extraction. The organic layer was taken, dried over
anhydrous sodium sulfate, and evaporated, and then puried by
column chromatography to give the desired product of ligustrazine
TMP and di-dodecyl dimethylammonium bromide were bought
from Aladdin Chemical Reagent Co., Ltd (Shanghai, China);
dichloromethane, chloroform, petroleum ether and ethyl acetate
were bought from Shanghai Lingfeng Chemical Reagent Co., Ltd
ꢀ
1
dimer (3.24 g, 52.77%) [Mp: 64.3–65.5 C, H NMR (300 MHz,
CDCl ) d 3.99 (s, 4H), 2.55 (s, 6H), 2.50 (s, 12H)].
3
(Shanghai, China); sodium sulde were bought from Wuxi
Yasheng Chemical Co., Ltd (Wuxi, China); carbon tetrachloride
were bought from Jiangsu Yongfeng Chemical Reagent Co., Ltd
2.3 Cell culture
20
(Danyang, China); benzoyl peroxide were bought from Jiangsu The method was referenced to a previous report.
Fig. 1 Synthesis and chemical structure of DTMP and DTMP reduced the levels of Ab in N2aAPP cell. (A) DTMP synthesis process, (B) levels of
#
Ab1–42 in cell lysates, (C) levels of Ab1–40 in cell lysates, (D) levels of Ab1–42/Ab1–40 in cell lysates. N ¼ 5. p < 0.05 compared to N2aWT, *p < 0.05,
**p < 0.01 compared to N2aAPP. Error bars indicate SEM.
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2.4 ELISA
2.8 Western-blot
Aer 24 hours of DTMP treatment, cells were washed twice with Reference experimental method 2.4 protein sample prepara-
2
1
ꢁ PBS. 200 mL of IP lysis buffer was added to each 25 cm ask tion. Protein samples were combined with loading buffer
ꢀ
for 30 minutes on ice. Cells were scraped from the ask and (Thermo Fisher Scientic) and heated at 96 C for 8 minutes,
collected into a 1.5 mL tube, and centrifuged at 20 000g at 4 C then separated on 10% SDS-PAGE and transferred to PVDF
ꢀ
for 30 min. The resulting supernatant was diluted prior to assay membrane. Aer blocking for 1 h at room temperature, it was
ꢀ
2
0 times. The levels of Ab1–40 and Ab1–42 in each fraction were incubated with primary antibody in 1ꢁ TBST overnight at 4 C.
determined using the ELISA kits (R&D Systems, Minneapolis, The primary antibodies used in our study are anti-b-actin
MN, USA) according to the kit instructions.
(1 : 3000, Santa Cruz, sc-47778), anti-a-tubulin (1 : 3000, Santa
Cruz, sc-73242), anti-NDUAA (1 : 1000, Abcam, ab103026), anti-
SDHB (1 : 1000, Abcam, ab14714), anti-UCRI (1 : 1000, Abcam,
ab131152), anti-COX5A (1 : 3000, Santa Cruz, sc-376907), and
anti-ATP5A (1 : 1000, Abcam, ab14748). The membranes were
washed with TBST and incubated with anti-rabbit or anti-mouse
IgG HRP secondary antibody for 1 hour. Thereaer, the
membrane was washed with TBST three times and visualized
using the chemiluminescent reagent from ECL kit (Thermo
Scientic Pierce ECL, USA) color. Quantitative densitometry
analysis was performed using quantity one soware.
2.5 Sample preparation
Aer 24 hours of 1 mM DTMP treatment, cells were washed twice
with 1ꢁ PBS. 400 mL of urea lysis buffer was added to each 75
2
cm ask for 30 minutes on ice. Collect cells scraped from the
culture ask and sonicated for 2 minutes (6 seconds on and four
seconds off) with Fisher 550 Sonic Dismemberer (Pittsburgh,
Pennsylvania) in 45% of the power until the samples was
transparent. Then, the samples were then cleaved on ice for
ꢀ
30 min and centrifuged at 20 000g for 30 min at 4 C. Protein
concentration was measured using Nanodrop2000 (Thermo
2.9 Statistical analysis
Scientic, NJ) according to the manufacturer's instruction. The
Results are expressed as mean ꢂ SEM. One-way analysis of
variance was used for statistical analysis (GraphPad Prism 7.0,
http://www.graphpad.com/). P < 0.05 were considered statisti-
cally signicant.
1
mg from six individual samples (per group) of the pooled
ꢀ
protein 10 mM dithiothreitol (DTT) at 55 C incubated for
30 min and then incubated at room temperature (dark envi-
ronment) with 25 mM iodoacetamide (IAA) for 1 h. Samples
were diluted to a nal concentration of 1.0 M urea with PBS (pH
8
.0) and digested with trypsin (1 : 100 w/w) (Promega, WI, USA)
3
. Results
ꢀ
for 14 hours at 37 C. Aer digestion, peptides were adjusted to
a pH value of 1–2 with 1% formic acid (FA) and centrifuged at
1
3.1 DTMP synthesis
1
2 000g for 15 min to collect the supernatant, and then reversed Tetramethylpyrazine was used as a raw material and carbon
ꢀ
phase column (Oasis HLB, Waters, USA) desalted and dried in tetrachloride as a solvent. They were heated to 70 C degrees
a vacuum centrifuge and then dissolved in 50 mL triethy- under reux with N-bromo succinimide (NBS) and the catalysis
lammonium bicarbonate buffer (TEAB, 200 mM, pH 8.5).
of benzoyl peroxide (BPO), which carry out a free radical reac-
tion to obtain 2-bromomethyl-3,5,6-trimethylpyrazine. In the
2
second step, sulfur and sodium sulde were rst heated in
water to form polysulde in the aqueous phase. The polysulde
then reacted with dodecyl dimethylammonium bromide
2
.6 TMT labeling
The TMT reagent (dissolved in 40 mL of acetonitrile 0.8 mg TMT)
was added to the peptide solution incubated for 1 h at room
temperature, and then reacted by adding 5% hydroxylamine at
RT for 15 minutes to terminate the reaction. The peptide
samples were labeled as follows: TMT-126, N2aWT; TMT-127,
N2aAPP; TMT-128, DTMP. The labeled from each group were
mixed together, as were the labeled total peptides from each
group, then the mixtures were desalted and dried for fraction-
ation. The TMT-labeled peptide mixtures were individually
fractionated using a high-pH reversed-phase peptide fraction-
ation kit (Thermo Scientic, NJ, USA) and divided into eight
different fractions. Per fraction was dried by using vacuum
centrifuge, then dissolved in 20 mL 0.1% FA for liquid
chromatography-tandem mass spectrometry analysis.
(DDAB), and the product entered the organic phase to undergo
phase transfer catalyzed reaction with compound 2 to obtain
the desired product. The structure of the compound was
1
conrmed by H NMR (Fig. S1†), and its molecular weight was
334.5.
3.2 DTMP reduces Ab accumulation
The effect of DTMP on the expression of Ab1–42 and Ab1–40
protein in N2aAPP cells was evaluated by ELISA. N2aAPP
signicantly increased the level of Ab1–42, while DTMP signi-
cantly decreased the level of Ab1–42 as shown in Fig. 1B. N2aAPP
and DTMP did not signicantly reduce the level of Ab1–40 as
shown in Fig. 1C. N2aAPP signicantly increased the relative
expression of Ab1–42/Ab1–40, while DTMP signicantly decreased
the relative expression of Ab1–42/Ab1–40 as shown in Fig. 1D.This
result indicates that DTMP reduces the levels of Ab1–40 and Ab1–
42 in N2aAPP.
2.7 LC-MS/MS analysis and bioinformatics analysis
21,22
The method was referenced to a previous report.
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Fig. 2 Venny analysis of the number of total protein in the two groups and classified aggregated the two proteins together. (A) Venny analyzed
the amount of total protein in the two groups. 118 differentially expressed proteins in N2aAPP cells and N2aAPP cells in the presence of DTMP
and the classification of these differential proteins, (B) mitochondrial function, (C) ATP binding, (D) GTPase function, (E) RNA binding, (F) RNA
binding, (G) nucleotide binding, and others.
Fig. 3 Four bioinformatic analyses of differentially expressed proteins from DTMP-treated and untreated N2aAPP cells. Shows (A) biological
processes, (B) molecular function, (when compared to N2aWT cells). (C) Biological processes, (D) molecular function (when compared to
N2aAPP cells).
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Fig. 4 STRING analysis of protein databases using the difference between N2aAPP in cells and N2aAPP cells are present DTMP protein–protein
interactions. (A) Protein–protein interaction network in 208 differential proteins in N2aAPP cells, (when compared to N2aWT cells), (B) N2aAPP
protein in the presence of DTMP difference of 449 kinds of protein–protein interaction networks, (when compared to N2aAPP cells). Cystoscope
3
.6.0 visualized by using a network to determine the difference in a potential relationship between proteins, protein is removed from the network
is not connected. Interaction represents a gray edge, the node between the red and blue represent the two proteins are increased or decreased.
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Table 1 Bioinformatics analysis was performed on 118 differentially expressed proteins shared by the two groups by using the Gene Set Analysis
Toolkit (Web Gestalt: http://bioinfo.vanderbilt.edu/webgestalt/)
ID
Name
#Gene
FDR
WP730
WP435
WP310
WP567
WP157
WP1770
WP18
WP1248
WP1244
WP578
Glutathione and one carbon metabolism
One carbon metabolism
mRNA processing
Eukaryotic transcription initiation
Glycolysis and gluconeogenesis
One carbon metabolism and related pathways
Heme biosynthesis
Electron transport chain
Estrogen signaling
Leptin insulin overlap
3
2
7
2
2
2
1
2
2
1
1.59Eꢃ01
1.00E+00
1.00E+00
1.00E+00
1.00E+00
1.00E+00
1.00E+00
1.00E+00
1.00E+00
1.00E+00
3
.3 Proteomic proles on DTMP treatment
<0.8. N2aAPP signicantly altered 208 proteins (including
44 increased and 64 decreased), and DTMP signicantly
changed 449 proteins (including 69 increased and 380
decreased). The number of differential proteins in the two
groups were analyzed with Venny in Fig. 2A. (N2aAPP)/
1
Protein changes of N2aWT cells, N2aAPP cells, and N2aAPP
cells treated with 1 mM DTMP were analyzed by TMT marker
proteomics. 3490 proteins were identied with at least one
unique peptide with a false discovery rate (FDR) of less than
(
N2aWT) accounted for 16.7%; (DTMP)/(N2a/APP) accoun-
1
%. The relative protein expression ratios were compared by
ted for 61.4%; these two together accounted for 21.9%. 118
differentially expressed proteins shared by these two and
their classication were shown in Fig. 2B–G, Tables S1 and
S2,† mainly related to mitochondrial function and ATP
calculating the relative abundance of the proteins identied
in each group and then used to determine the increase or
decrease in protein in the two groups [i.e., (N2aAPP)/(N2aWT)
and (DTMP)/(N2aAPP)]. The standard ratios e set as $1.25 or
Fig. 5 Hierarchical clustering of proteins in the mitochondrial electron transport chain in N2aAPP cells and N2aAPP cells in the presence of
DTMP, and signaling in the electron transport chain pathway. (A) Whether there is hierarchical clustering of electron transport chain pathway-
related proteins of DTMP; (B) electron transport chain signaling pathway in N2aAPP cells (compared to N2aWT cells); (C) electron transport chain
signaling pathway in N2aAPP cells in the presence of DTMP (compared to N2aAPP cells). All relevant proteins involved in the ETC pathway are
mapped and used in different ways. Expressing the color series, the decreased protein is blue, the increased protein is red; the unchanged protein
is white.
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Fig. 6 The relevant proteins on the electron transport chain were verified by Western blot analysis. b-Actin and a-tubulin were used as a loading
#
##
###
control. N ¼ 3. p < 0.05, p < 0.01,
p < 0.001 compared to N2aWT cells, *p < 0.05, **p < 0.01 compared to N2aAPP. Error bars indicate SEM.
binding, GTPase function related, RNA binding, DNA
binding, nucleotide binding, and others.
The electron transport chain pathway-associated proteins
were hierarchically clustered from the 3490 proteins (Fig. 5).
Using Gene Ontology (GO) annotation, we analyzed the The majority of proteins involved in ETC were changed, and
biological characteristics (biological processes and molecular these proteins (NDUFB1, NDUFA5, NDUFA10, COX5A, ATP5A
functions) of differentially expressed proteins. Gene ontology and SLC25A4) were up-regulated in DTMP-treated N2aAPP cells.
enrichment analysis identied the top ten enrichment of bio-
logical processes and molecular functions of 208 differential 3.4 Conrmation of differential protein expression by
proteins in N2aAPP cells (Fig. 3A, B, Tables S3 and S4†), bio- Western-blot analysis
logical processes include macromolecular complex subunit
organization, response to metal ion, macromolecular complex
assembly, and ossication; molecular functions include: chro-
matin binding, RNA binding, purine nucleotide binding, and
nucleotide binding. While DTMP-treated N2aAPP cells were
enriched in the top ten classes of biological processes and
molecular functions of 449 differentially expressed proteins
To conrm proteomic data, we veried the expression of
differential proteins by Western blot analysis. Five related
proteins complex I (NDUAA), complex II (SDHB), complex III
(UCRI), complex IV (COX5A) and complex V (ATP5A) in the
electron transport chain were selected for validation. The
expression levels of NDUAA, SDHB, UCRI and COX5A were
decreased in N2a/APP cells, while the treatment of DTMP
increased the levels of NDUAA and COX5A in N2aAPP cells
(Fig. 3C, D, Tables S5 and S6†), biological processes include cell
cycle, cell division, protein localization, cell cycle process and
cell cycle phase; molecular function include: RNA binding,
nucleotide binding, purine nucleotide binding, and chromatin
binding. To illustrate protein–protein interactions from
N2aAPP and DTMP differential proteins, we used the STRING
database to construct a protein network. The network was
visualized by using Cystoscope 3.6.0 to determine the potential
relationship as shown in Fig. 4. Many interacting proteins are
involved in AD processes, such as mitochondrial function and
electron transport chain, RNA binding, DNA binding, ATP
binding and GTPase function, which are highly compatible with
the pathogenesis of AD. These characteristics were further re-
(
Fig. 6). Overall, the Western blot results (NDUAA, UCRI and
COX5A) are consistent with the proteomic data.
4
. Discussion
In this study, our methods of proteomics research DTMP neu-
roprotective effect on neuroblastoma N2a cells in AD-related
injuries, using human Swedish mutant amyloid precursor
gene transfected mouse neural element N2a cells as an AD
model. The results of ELISA showed that DTMP has neuro-
protective effect on N2aAPP cells. Proteomic analysis of proteins
expressed by N2aAPP 208 cells and proteins differentially DTMP

ected by using the web-based Gene Set Analysis Toolkit (Web
4
49 differentially. Bioinformatics analysis indicated that these
Gestalt: http://bioinfo.vanderbilt.edu/webgestalt/) for 118
differentially expressed proteins shared by the two groups
shown in Table 1. In view of the important role of mitochondria
in the pathogenesis of AD, we selected electron transport chain
pathway-related molecules to further demonstrate the potential
neuroprotective effects of DTMP.
proteins are mainly involved in mitochondrial function, elec-
tron transport chain, ATP binding, oxidative phosphorylation,
GTPase function, and nucleotide binding, suggesting that
multiple molecular processes may be involved in the neuro-
protective effects of DTMP on neuroblastoma N2a cells.
Mitochondria are an organelle in eukaryotic cells, in addi-
tion to being responsible for energy supply, as a regulator of cell
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2
3
death and a major feature of neurodegeneration. Due to
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a role in the pathogenesis of AD. In our research, many
proteins in the electron transport chain have changed. Next, we
focus on these differentially expressed proteins that are closely
related to mitochondrial function and electron transport chain.
It has been reported that dysfunction of the mitochondrial
electron transport chain will lead to ATP synthesis, free radical
production and oxidative damage leading to neuronal
a
multifactorial
disorder
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COX17 and complex V in ATP5I, ATP5H and ATP5J. The results
of the experiment indicated that these proteins have been
down-regulated, supporting previous studies that mitochon-
29,30
drial disorders will lead to neurodegenerative diseases.
Also
of interest, seven differentially expressed proteins in DTMP-
treated N2aAPP cells are involved in the mitochondrial elec-
tron transport chain, including: complex I in NDUFAB1,
NDUFA10; complex III in UCRB; complex IV in COX17 and
28
complex V in ATP5O, ATP5H and ATP5F1. The results showed
that proteins from these increases have taken place, so DTMP
may have a role in the treatment of neurodegenerative diseases.
5. Conclusion
2014, 2014, 107501.
1
0 Y. Chang, G. Hsiao, S. H. Chen, Y. C. Chen, J. H. Lin,
K. H. Lin, et al., Tetramethylpyrazine suppresses HIF-
In summary, these data indicate that DTMP may be used as
a potential drug candidate to ameliorate the symptoms of AD by
modulating the mitochondrial electron transport chain
involved in the expression of the relevant protein.
1alpha, TNF-alpha, and activated caspase-3 expression in
middle cerebral artery occlusion-induced brain ischemia in
rats, Acta Pharmacol. Sin., 2007, 28(3), 327–333.
1
1
1 C. Zhang, S. Z. Wang, P. P. Zuo, X. Cui and J. Cai, Protective
effect of tetramethylpyrazine on learning and memory
function in D-galactose-lesioned mice, Chin. Med. Sci. J.,
Conflicts of interest
The authors declare that they have no conict of interest to
disclose.
2
004, 19(3), 180–184.
2 S. Y. Li, Y. H. Jia, W. G. Sun, Y. Tang, G. S. An, J. H. Ni, et al.,
Stabilization of mitochondrial function by
Acknowledgements
tetramethylpyrazine protects against kainate-induced
oxidative lesions in the rat hippocampus, Free Radical Biol.
Med., 2010, 48(4), 597–608.
3 H. T. Liu, Y. G. Du, J. L. He, W. J. Chen, W. M. Li, Z. Yang,
et al., Tetramethylpyrazine inhibits production of nitric
oxide and inducible nitric oxide synthase in
lipopolysaccharide-induced N9 microglial cells through
blockade of MAPK and PI3K/Akt signaling pathways, and
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This work was supported by National Natural Science Founda-
tion of China (81673134, 81401570), Shenzhen Special Fund
Project on Strategic Emerging Industry Development
1
(
JCYJ20160428143433768,
JCYJ20170818111012390,
JCYJ20150529153646078,
JCYJ20150529164656093,
JCYJ20160422143433757, JCYJ20150529112551484 and
JCYJ20170810163329510) and Sanming Project of Medicine
in Shenzhen (SZSM201611090).
1
4 Z. Tan, Neural protection by naturopathic compounds-an
example of tetramethylpyrazine from retina to brain, J.
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