The Protective Role of Brain CYP2J in Parkinson’s Disease Models
The Protective Role of Brain CYP2J in Parkinson's Disease Models
Yueran Li 1
Jinhua Wu 1
Xuming Yu 1
Shufang Na 1
Ke Li 0
Zheqiong Yang 1
Xianfei Xie 1
Jing Yang 1
Jiang Yue 1
Lydia W. Tai
0 Demonstration Center for Experimental Basic Medicine Education, School of Basic Medical Sciences, Wuhan University , Wuhan 430071 , China
1 Department of Pharmacology, School of Basic Medical Sciences, Wuhan University , Wuhan 430071 , China
CYP2J proteins are present in the neural cells of human and rodent brain regions. The aim of this study was to investigate the role of brain CYP2J in Parkinson's disease. Rats received right unilateral injection with lipopolysaccharide (LPS) or 6-hydroxydopamine (6-OHDA) in the substantia nigra following transfection with or without the CYP2J3 expression vector. Compared with LPS-treated rats, CYP2J3 transfection significantly decreased apomorphine-induced rotation by 57.3% at day 12 and 47.0% at day 21 after LPS treatment; moreover, CYP2J3 transfection attenuated the accumulation of α-synuclein. Compared with the 6-OHDA group, the number of rotations by rats transfected with CYP2J3 decreased by 59.6% at day 12 and 43.5% at day 21 after 6-OHDA treatment. The loss of dopaminergic neurons and the inhibition of the antioxidative system induced by LPS or 6-OHDA were attenuated following CYP2J3 transfection. The TLR4-MyD88 signaling pathway was involved in the downregulation of brain CYP2J induced by LPS, and CYP2J transfection upregulated the expression of Nrf2 via the inhibition of miR-340 in U251 cells. The data suggest that increased levels of CYP2J in the brain can delay the pathological progression of PD initiated by inflammation or neurotoxins. The alteration of the metabolism of the endogenous substrates (e.g., AA) could affect the risk of neurodegenerative disease.
Parkinson’s disease (PD) is a progressive neurodegenerative
disorder characterized by motor symptoms, including
bradykinesia with resting tremor, rigidity, and gait disturbance. The
major neuropathological findings of PD are the loss of
dopaminergic (DA) neurons and the presence of
α-synucleincontaining aggregates in the substantia nigra (SN). Although
the underlying mechanisms remain unknown, putative
factors in PD pathogenesis include oxidative stress, proteostasis
imbalance, neuroinflammation, environmental toxins, and
Increasing evidence indicates that neuroinflammation is
an important contributor to PD pathogenesis and that
peripheral inflammation contributes to the initiation and/or
progression of the disease by exacerbating and synergizing
with neuroinflammation [
]. Astrocytes play vital roles in
neuroinflammatory processes in PD [
]. They respond to
inflammatory stimulation by producing proinflammatory
cytokines both in vitro and in vivo [
overexpression of mutant α-synuclein causes widespread
astrogliosis, microglial activation, and the degeneration of
DA neuron in mice . Reactive astrogliosis has been
reported in PD animal models and in the affected brain
regions of patients with PD [
]. Astrocytes amplify the
inflammatory signals activated by microglia, and
uncontrolled neuroinflammation ultimately contributes to
]. The injection of lipopolysaccharide (LPS)
into the SN induces the progressive, specific, and irreversible
loss of DA neurons and the accumulation of α-synuclein .
The intranigral injection of LPS into the striatum and nigra
causes no detectable damage to GABAergic or serotoninergic
neurons, suggesting that DA neurons are selectively
vulnerable to LPS [
In addition to neuroinflammation, oxidative stress is a
critical neurotoxic event in the death of DA neurons [
Actually, the inflammation and oxidative stress are tightly
linked and may be a positive reciprocal feedback loop in
the pathophysiological processes of many chronic diseases
including PD [
]. Nuclear factor erythroid 2-related factor
2 (Nrf2), a key regulator of the antioxidant system, has been
shown to be a therapeutic target for PD. And the genetic
association has been previously reported between functional
polymorphisms of the Nrf2-encoding NFE2L2 gene and the
risk of Parkinson’s disease [
]. The dopamine analog
6-hydroxydopamine (6-OHDA), a typical neurotoxin,
produces high levels of reactive oxygen species and causes
mitochondrial dysfunction . The injection of 6-OHDA into
the striatum causes the progressive loss of nigral DA neurons;
however, no α-synuclein aggregates were observed in this
Cytochrome P450 (CYP) is a superfamily responsible for
the biotransformation of exogenous and endogenous
compounds. The previous study has shown that CYP2J acts as
an arachidonic acid (AA) epoxygenase, and epoxidation of
AA produces four regioisomeric cis-epoxyeicosatrienoic
acids (5,6-, 8,9-, 11,12-, and 14,15-EET) [
]. EETs are
metabolized into dihydroxyeicosatrienoic acids by soluble
epoxide hydrolase [
]. Immunohistochemistry experiments
confirm the presence of CYP2J proteins in the neurons of
human and rodent brain regions [
]. In the present study,
we investigated the protective role of brain CYP2J and
possible mechanisms of action in LPS and 6-OHDA PD models.
2.1. Animals. Male adult Wistar rats weighing 250–300 g,
supplied by the Experimental Animal Center (Hubei, China),
were kept at room temperature (22°C–25°C) on a 12 h
artificial light/dark cycle, with free access to food and water.
Animals were allowed to habituate for 7 days prior to use.
All procedures were approved by the Animal Care
Committee of Wuhan University and complied with the
recommendations of the International Association for the Study of Pain
]. Rat CYP2J3 is the major isoform in the CYP2J
subfamily, which shares a high identity of amino acid sequence with
human CYP2J2 (>70%) [
2.2. Plasmid Construction. The fragment encoding CYP2J2
was cloned into the pcDNA-3.1(+) vector (YouBio Biological
Technology Co., Wuhan, China) after the digestion by
BamHI/EcoRI (Thermo Scientific, Waltham, MA, USA).
The fragment encoding CYP2J3 was cloned into the
pHAGE-CMV-MCS-IZsGreen vector (Stargene SciTech
Development Co., Wuhan, China) after the digestion by
Xhol/HindIII (Thermo Scientific, Waltham, MA, USA).
The fragment encoding primary miR-340 was cloned into
the pcDNA-3.1(−) vector (Invitrogen, Carlsbad, CA, USA)
after the digestion by NotI/BamHI (Thermo Scientific,
Waltham, MA, USA). All final constructs were verified by
DNA sequence analysis.
2.3. Lentiviral Vector (LV) Construction. Recombinant
lentiviruses were produced by transfecting 293T cells with a viral
vector containing the enhanced green fluorescent protein
(eGFP) gene, the CYP2J3 expression vector or a control
vector, and packing and envelope plasmids (psPAX2 and
pMD2.G; Addgene, Cambridge, MA, USA) using
Lipofectamine 2000. The virus-containing medium was harvested
after 48 and 72 h, then concentrated by a two-step
ultracentrifugation procedure after filtration. Titers of the viral
vectors used in this study ranged from 1 to 2 × 109 TU/ml.
2.4. Surgery and Treatment Procedure. The procedure for
implantation of i.c.v. guide cannula was conducted as
previously described [
]. The rats were anesthetized by chloral
hydrate (300 mg/kg, i.p.) and were secured in a stereotaxic
frame (RWD Life Science, Shenzhen, China). The head was
shaved, and a 1 mm hole was drilled using a high-speed drill
(RWD Life Science, Shenzhen, China). A guide cannula
(62003, RWD Life Science, Shenzhen, China) was implanted
at 0.5 mm above the right substantia nigra pars compacta
(SNpc) (Bregma coordinates: AP, 5.3 mm; ML, 2.0 mm; and
DV, 7.8 mm). The insertion cannula for stereotaxic injection
protruded 0.5 mm below the tip of the guide cannula. Guide
cannula was fixed with acrylic dental cement and three
stainless steel screws affixed to the skull. The incision was closed
using 5-0 Dysilk. Animals were administered with
benzylpenicillin (60 mg/kg, s.c.) after the operation and kept warm
until they were awake. Body weights and clinical signs were
monitored closely during postsurgical recovery.
Experiment I. A total of 60 rats were randomly divided
into the following groups: control group, LPS group, LPS
treatment following empty vector transfection, and LPS
treatment following LV-CYP2J3 transfection (n = 15). LPS
(from Escherichia coli, serotype O55:B5, Sigma, St. Louis,
MO, USA), dissolved in sterile phosphate-buffed saline
(PBS), was unilaterally injected (10 μg, 2 μl) into the right
SNpc of rats at a rate of 0.2 μl/min by an automatic injector
(CMA 402, CMA Microdialysis AB, Sweden) in the CMA
120 System for Freely Moving Animals (CMA Microdialysis
]. The syringe was left in situ for 5 min. Control
animals were injected with sterile PBS. After injection, the
syringe was left in situ for 5 min. Rats received unilateral
injections of the empty vector or the LV-CYP2J3 vector 3
days before treatment with LPS. One microliter LV was
injected for 5 min at a rate of 0.2 μl/min by an automatic
injector. Using a fluorescent microscope, the expression of
eGFP was used as a visual marker of the injection site and
Experiment II. A total of 40 rats were randomly divided
into the following groups: control group, 6-OHDA group,
6-OHDA treatment following empty vector transfection,
and 6-OHDA treatment following LV-CYP2J3 transfection
(n = 10). 6-OHDA (Sigma, St. Louis, MO, USA) was dissolved
in sterile saline with 0.2% vitamin C. For the 6-OHDA PD
animal model, 6-OHDA (8 μg, 2 μl) was unilaterally injected
into the right SNpc of rats. Control animals were injected with
the vehicle. Rats received unilateral injections of the control
vector or the LV-CYP2J3 vector 3 days before 6-OHDA
treatment. One microliter of LV was injected for 5 min at a rate of
0.2 μl/min by an automatic injector. After injection, the
syringe was left in situ for 5 min.
2.5. Rotational Behavior. The animals were tested for
rotational behavior after treatment with the DA agonist
apomorphine (0.5 mg/kg, s.c.) dissolved in sterile saline 12
and 21 days after LPS or 6-OHDA injections [
]. Rats were
placed in a circular test arena. Rotational activity was
measured for 30 min, beginning 5 min after injection, and
the number of turns was counted [
2.6. Immunohistochemistry. Rats were deeply anesthetized
and perfused with saline followed by cold 4%
paraformaldehyde through the aorta. Brain tissues were quickly removed
and postfixed with 4% paraformaldehyde. The samples were
embedded in paraffin, and a series of 4 μm sections were cut.
Four sections were selected from the SN, and sections from
different groups were matched as closely as possible [
The sections were incubated with a polyclonal rabbit anti-rat
antibody for tyrosine hydroxylase (TH, 1 : 200). After
incubation with biotinylated anti-rabbit IgG (1 : 100), the
antigen-antibody complex was visualized using the
avidin-biotin complex technique (ABC kit, Vector
Laboratories, Burlington, Ont., Canada) followed by a reaction with
3,3′-diaminobenzidine and hydrogen peroxide (DAB kit,
Vector Laboratories, Burlington, Ont., Canada). TH-positive
cells in the lesioned and intact hemispheres were assayed
using the ImageJ software to measure a gray scale value within
the range of 0–200. Background staining was subtracted, and
analysis was performed blinded. The data shown are the
percentage of cells relative to the intact SN side.
2.7. Immunofluorescence. To investigate the effects of CYP2J
on Nrf2 protein levels, brain tissues from both LPS and
6-OHDA PD models were fixed with 4% paraformaldehyde.
The SN was embedded in paraffin and cut into 4 μm. After
deparaffinization and rehydration of the paraffin sections,
antigen retrieval was performed with citrate buffer (0.1 M,
pH 6.0) at 95°C for 10 min. The sections were blocked with
3% BSA for 1 h and incubated with a polyclonal rabbit
antirat CYP2J antibody (1 : 400) and a monoclonal mouse
antirat Nrf2 antibody (1 : 200). The samples were incubated with
the secondary antibodies including a rhodamine-conjugated
AffiniPure Goat anti-rabbit IgG for CYP2J (1 : 100) and a
Cy3-conjugated AffiniPure Donkey anti-mouse IgG for
Nrf2 (1 : 100). Images were analyzed using an Olympus
BX51 fluorescent microscope (Olympus Corporation, Tokyo,
Japan) equipped with an Olympus Micro DP72 camera.
Identical illumination and camera settings were used within
each data set.
To investigate the effects of CYP2J on α-synuclein
accumulation, the SN from the LPS PD model was co-incubated
with a monoclonal mouse anti-rat α-synuclein antibody
(1 : 400) and a polyclonal rabbit anti-rat TH antibody
(1 : 200). Then, the samples were incubated with the
secondary antibodies which included a rhodamine-conjugated
AffiniPure Goat anti-rabbit IgG for TH (1 : 100) and a
Cy3-conjugated AffiniPure Donkey anti-mouse IgG for
α-synuclein (1 : 100).
To investigate the effects of LPS on the p-CREB levels,
U251 cells were pretreated with CLI-095 (1 μM) or ST 2825
(20 μM) for 1 h before LPS treatment (1 μg/ml, 24 h). The
cells were fixed and permeabilized with 0.5% Triton X-100.
Fixed cells were blocked with 3% fetal calf serum for
30 min. The cells were incubated with monoclonal rabbit
anti-human p-CREB (S133) antibody and goat anti-mouse
alpha tubulin antibody overnight at 4°C. Samples were
incubated with fluorescent-labeled goat anti-rabbit IgG and
fluorescent-labeled goat anti-mouse IgG in PBS for 1 h at
37°C. Then, the cells were washed twice in PBS and incubated
with 4,6-diamidino-2-phenylindole (DAPI) for 5 min.
2.8. Cell Culture and Treatment. Human glioma U251 cells
were grown in Dulbecco’s modified Eagle’s medium
containing 10% fetal bovine serum. To determine whether CYP2J or
AA metabolite plays a role in oxidative stress, U251 cells were
transfected with the CYP2J2 expression vector or treated
with 14,15-EET (1 μM) and
12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA) (1 μM), an inhibitor of soluble
epoxide hydrolase. To investigate the mechanism by which
LPS regulates CYP2J, U251 cells were pretreated with a
specific TLR4 inhibitor (CLI-095, 1 μM) or a specific MyD88
inhibitor (ST 2825, 20 μM) for 1 h before LPS treatment
(1 μg/ml, 24 h).
2.9. Real-Time RT-PCR. Total RNA was isolated using the
TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA
was synthesized using a cDNA Synthesis Kit (Toyobo,
Osaka, Japan) for first-strand synthesis. All real-time
PCR reactions with SYBR Green (Toyobo, Osaka, Japan)
were performed on a CFX connect real-time PCR
detection system (Bio-Rad, Hercules, CA, USA). For miR-340
detection, a small RNA-specific stem-loop RT primer was
used as follows: 5-GTCGTATCCAGTGCGTGTCGTGGA
primers for PCR are listed in Additional file 1 (Table S1).
Human GAPDH was used for the normalization of relative
expression levels, and U6 was used to normalize the relative
expression of miR-340. Gene expression levels were calculated
using the 2−ΔΔCT method relative to internal controls.
2.10. Immunoblotting. The pellets of cells were lysed
using RIPA lysis buffer. For the detection of Nrf2 proteins,
20 μg of total proteins from U251 cells were separated by
SDS-polyacrylamide gel electrophoresis (4% stacking and
10% resolving gels) and then transferred onto PVDF
membranes. These membranes were incubated for 2 h with
a polyclonal rabbit anti-human Nrf2 antibody (1 : 1000).
The protein levels of Nrf2 were normalized to β-actin
(1 : 1000) to control the loading efficiency. The
immunoblots were visualized using chemiluminescence followed
by exposure to autoradiography films and analyzed using
Gel Documentation & Analysis Systems (Beijing Liuyi
Instrument, Beijing, China). The relative density of each band
was expressed in arbitrary density units after subtracting
2.11. Chromatin Immunoprecipitation (ChIP) Assay. The
ChIP kit (EpiGentek Group Inc., Brooklyn, NY, USA) was
used to determine the binding profile of CYP2J2 genes with
p-CREB protein in cells pretreated with or without CLI-095
(1 μM) or ST 2825 (20 μM) for 1 h before LPS treatment
(1 μg/ml, 24 h). Briefly, the cross-linked chromatin from the
nuclear fraction was sheared into fragments of the proper
length by sonication according to the manufacturer’s
protocol. The chromatin concentration was measured, and
approximately 10% of the chromatin was retained as input
material. The chromatin solution was immunoprecipitated
with rabbit anti-human p-CREB (S133) antibody (1 : 100)
or non-immune mouse IgG as a negative control. The
number of cycles used for PCR amplification were within the
linear range (primers are listed in Table S1), and PCR products
were resolved on a 2% agarose gel. The band intensities were
quantified using an AutoChemi Imaging System (UVP LLC,
Upland, CA, USA).
2.12. Materials. LPS (cat. number L2880) and 6-OHDA
(cat. number H116) were purchased from Sigma (St.
Louis, MO USA), 14,15-EET were purchased from Cayman
Chemical Co. (cat. number 50651, Ann Arbor, MI),
apomorphine (cat. number HY-12723A) and ST 2825 (cat. number
HY-50937) were purchased from MedChem Express
(Princeton, NJ, USA), and CLI-095 was purchased from
InvivoGen (cat. number tlrl-cli95, San Diego, CA, USA).
Primary antibodies to TH (cat. number ab112), Nrf2
(cat. number ab89443), α-synuclein (cat. number ab1903),
and p-CREB (S133) (cat. number ab32096) were purchased
from Abcam (Cambridge, MA, USA). Primary antibodies
to CYP2J were purchased from Merck Millipore (cat. number
ABS1605, CA, USA). Primary antibodies to TH were
purchased from GeneTex Inc. Irvine (cat. number GTX113016,
CA, USA). Primary antibodies to alpha-tubulin were
purchased from Proteintech (cat. number 66031-1-lg, Chicago,
IL, USA). Primary antibodies to β-actin were purchased from
Santa (cat. number sc-8432, Santa Cruz, CA, USA).
Rhodamine-conjugated AffiniPure Goat anti-rabbit IgG was
purchased from Kerui Biotechnology Co. (cat. number
100428, Wuhan, China) and DyLight 405 Donkey
antimouse IgG was purchased from Antgene (cat. number
ANT080, Wuhan, China). FITC-conjugated AffiniPure Goat
anti-rabbit IgG (cat. number AS1110) and CY3-conjugated
AffiniPure Goat anti-mouse IgG (cat. number AS1111) were
purchased from Aspen Biological (Wuhan, China).
Dulbecco’s modified Eagle’s medium (DMEM) was
purchased from HyClone (Rockford, IL, USA); reduced
serum medium Opti-MEM was purchased from Gibco
(Carlsbad, CA, USA). Lipofectamine 2000 was purchased
from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum
(FBS) was purchased from Zhejiang Tianhang Biotechnology
Co. (Zhejiang, China). Other materials were purchased from
standard suppliers or as indicated in the text.
2.13. Statistical Analysis. All data are expressed as arithmetic
mean with a standard error of the mean (SEM). The
normalized mRNA levels of genes from cells and rat brain tissue
were expressed as arbitrary density units. Results from the
ChIP assay were expressed as arbitrary density units and
normalized to input levels. Data were obtained from at least five
separate experiments. One-way ANOVA was used to test for
differences among the groups and was followed by the least
significant difference post hoc test. A value of p < 0 05 was
3.1. LPS Downregulated CYP2J Levels via the TLR4-MyD88
Signaling Pathway in U251 Cells. Our previous study showed
that brain CYP2J2 is the target gene of CREB in astrocytes
]. Compared with controls, mRNA levels of CYP2J2 and
CREB were decreased by 67.4% and 34.8%, respectively, after
LPS treatment for 24 h (Figure 1(a)). The LPS-induced
inhibition of CYP2J2 and CREB mRNA levels was abolished by
CLI-095 (a specific TLR4 inhibitor) and partly attenuated
by ST 2825 (a specific MyD88 inhibitor) compared with the
LPS group. Immunofluorescence data showed that CLI-095
and ST 2825 attenuated LPS-induced decreases in the
p-CREB protein level in cells (Figure 1(b)). ChIP data showed
that p-CREB proteins bound to the CYP2J2 promoter
at −1426 to −1405 bp. Compared with the control, binding
of the p-CREB protein to the CYP2J2 promoter was decreased
by 50% following LPS treatment; however, decreased binding
of the p-CREB protein to the CYP2J2 promoter induced by
LPS was attenuated by CLI-095 and ST 2825 (Figure 1(c)).
These data suggest that the TLR4-MyD88 signaling pathway
is involved in the regulation of CYP2J via CREB following
treatment with LPS.
3.2. CYP2J-Dependent Metabolites Upregulated Antioxidative
Genes via miR-340. Compared with controls, CYP2J2
overexpression significantly increased both the mRNA and
protein levels of Nrf2 in U251 cells (Figures 2(a)–2(c)).
The mRNA levels of genes related to mitochondrial
oxidative defense (SOD1, CAT, and GPX1) in astrocytes were
significantly elevated by CYP2J2 overexpression compared
with controls; however, no changes were observed in the
mRNA levels of genes related to endoplasmic reticulum
stress, including MANF, PERK, IRE1α, and ATF6 (data
not shown). Compared with controls, the level of miR-340
mRNA was significantly decreased following CYP2J2
overexpression (Figure 2(d)). Western blotting and real-time
RT-PCR revealed that miR-340 targeted Nrf2 expression in
astrocytes (Figures 2(e)–2(g)). Following 14,15-EET and/or
AUDA (a soluble epoxide hydrolase inhibitor) treatment of
U251 cells, miR-340 and Keap1 were downregulated;
however, genes related to mitochondrial oxidative defense were
upregulated including Nrf2, SOD1, CAT, and GPX1
3.3. CYP2J Transfection Protected Dopaminergic Neurons in
the LPS PD Animal Model. The accumulation of α-synuclein
was observed after LPS treatment for 12 and 21 days
(Figure 3(a)); however, CYP2J transfection attenuated the
LPS-induced accumulation of α-synuclein. Compared with
the control, the number of DA neurons was decreased by
75.5% after stereotaxic injections of LPS for 21 days
(Figures 3(b) and 3(c)); however, DA neurons remaining in
the SNpc of rats transfected with the CYP2J expression
vector were increased by 2.1-fold compared with the LPS
group. Compared with LPS-treated rats, the number of
LPS (1 휇 g) −
CLI-095 (1 휇 M) −
LPS (1 휇 g) −
CLI-095(1 휇 M) −
+ DMSO +
+ LPS (1 휇 g) −
+ ST 2825 (20 휇 M) −
+ DMSO +
+ LPS (1 휇 g) −
+ ST 2825 (20 휇 M) −
p-CREB (Ser 133)
Control LPS LPS +
LPS (1 휇 g)
CLI-095 (1 휇 M)
ST 2825 (20 휇 M)
IP: anti-lg G
rotations in rats transfected with the CYP2J expression
vector was decreased by 57.3% at day 12 and 47.0% at day
21 after LPS treatment (Figures 3(d) and 3(e)).
3.4. CYP2J Transfection Upregulated Genes Related to
Mitochondrial Oxidative Defense in the LPS PD Animal
Model. The effect of CYP2J expression on Nrf2 protein levels
was investigated by fluorescent immunocytochemistry.
Levels of Nrf2 protein were elevated in cells with high levels
of CYP2J3 after LPS treatment for 12 and 21 days compared
with the LPS group (Figures 4(a) and 4(b)). Compared with
the control, mRNA levels of CREB and CYP2J3 in the SN
were decreased by 31.0% and 45.6%, respectively, following
LPS treatment for 21 days, but miR-340 mRNA levels were
increased by 2.5-fold, and levels of genes related to
mitochondrial oxidative defense were decreased. Compared with
the LPS group, the mRNA levels of Nrf2, SOD1, CAT, and
GPX1 in rat SN transfected with the CYP2J3 expression
vector were increased by 6.0-, 2.6-, 3.2-, and 3.2-fold,
respectively, and miR-340 mRNA levels were decreased by
47.7% (Figures 4(c) and 4(d)).
3.5. CYP2J Transfection Protected Dopaminergic Neurons in
the 6-OHDA PD Animal Model. Compared with the control,
the number of DA neurons decreased by 74.8% after
treatment with 6-OHDA for 21 days; however, in the SNpc of rats
transfected with the CYP2J expression vector, the number
of DA neurons increased by 2.4-fold compared with the
6-OHDA and pHAGE groups (Figures 5(a) and 5(b)).
Compared with 6-OHDA-treated rats, rotations in rats
transfected with the CYP2J3 expression vector were decreased by
59.6% at day 12 and 43.5% at day 21 after 6-OHDA treatment
(Figures 5(c) and 5(d)).
3.6. CYP2J Transfection Upregulated Genes Related to
Mitochondrial Oxidative Defense in the 6-OHDA PD Animal
Model. Compared with the 6-OHDA group, Nrf2 protein
levels were elevated in cells with high levels of CYP2J3 after
14, 15-EET −
6-OHDA treatment for 12 and 21 days indicated by
fluorescent immunocytochemistry (Figures 6(a) and 6(b)).
Compared with the control, CREB and CYP2J3 mRNA levels in
the SN decreased by 16.5% and 55.2%, respectively, following
6-OHDA treatment (Figure 6(c)). However, the
6-OHDAinduced inhibition of genes related to mitochondrial oxidative
defense was attenuated by transfection with CYP2J3.
Compared with the control, mRNA levels of Nrf2, SOD1, CAT,
and GPX1 in rat SN transfected with the CYP2J3 expression
vector were increased by 8.7-, 4.9-, 12.9-, and 6.5-fold,
respectively, and miR-340 mRNA levels were decreased by
48.8% (Figure 6(d)).
This is the first demonstration that brain CYP2J plays a
protective role in PD models induced by inflammation or
oxidative stress. We showed that (i) CYP2J3 transfection
significantly attenuated LPS- or 6-OHDA-induced behavioral
impairment and the loss of DA neurons in the SN; (ii)
the TLR4-MyD88 signaling pathway was involved in the
downregulation of brain CYP2J induced by LPS; and (iii)
both CYP2J transfection and 14,15-EET activated the
Nrf2-antioxidant response element (ARE) pathway via the
inhibition of miR-340. These data indicate that AA
metabolites catalyzed by brain CYPs may affect the mitochondrial
oxidative defense system.
A growing body of basic research shows that the
Nrf2-ARE pathway plays a protective role in both
toxininduced and transgenic mouse models of PD [
oxidative stress, Nrf2 is released from Keap1, translocates
into the nucleus, and binds with the ARE of antioxidant
genes. Our data showed the activation of the Nrf2-ARE
pathway by 14,15-EET treatment in astrocytes, which is
consistent with a previous report that 14,15-EET treatment
significantly increased the accumulation of Nrf2 in lung
epithelial cells [
]. The downstream target genes of Nrf2 related
to mitochondrial oxidative defense were upregulated
following CYP2J transfection, suggesting that Nrf2 can prevent the
damage to mitochondria from oxidant injury. The previous
study showed that the isolated mitochondria from the
myocardium of Nrf2 deficiency mice were more sensitive to
mitochondrial membrane permeability transition and
swelling compared with the samples from the wild-type mice
(Nrf2 protects mitochondrial decay by oxidative stress).
The induction of Nrf2 attenuated microglia-induced
inflammation in the hippocampus of LPS-treated mice as
determined by reduced inducible NO synthase (iNOS) levels and
attenuated the production of proinflammatory cytokines
IL-6 and TNF-α [
]. Nrf2 overexpression inhibited
RAC1dependent activation of nuclear factor-κB (NF-κB),
indicating a cross-talk between Nrf2 and NF-κB pathways [
The activation of the Nrf2-ARE pathway following CYP2J
overexpression or the treatment of EETs may also inhibit
the NF-κB signaling pathway. Recently, Nrf2 was identified
as a regulator of the expression of autophagy genes [
Our data showed the downregulation of Nrf2 and its
downstream genes in both the LPS and 6-OHDA PD models,
suggesting that the Nrf2-ARE pathway is associated with both
neuroinflammation and oxidative stress. Nrf2 may be a
suitable pharmacological target for the treatment of PD .
Cardiovascular EETs have been reported to inhibit
reactive oxygen species, the inflammatory reaction,
vascular smooth muscle migration, and enhancement of the
fibrinolytic pathway [
]. Our data suggest that EETs may
play a protective role in PD. A previous study showed that
sEH deficiency or inhibition attenuated MPTP-induced
dopamine neuron loss and behavioral deficits [
]. Our data
showed that 14,15-EET decreased miR-340 levels and that
the LPS-induced elevation of miR-340 levels was attenuated
by CYP2J overexpression. The overexpression of miR-340
decreased Nrf2 mRNA levels in neural cells, which is
consistent with a previous study showing that miR-340 directly
targets Nrf2 mRNA in hepatocytes [
The TLR4-MyD88 signaling pathway is involved in
decreasing CYP2J in astrocytes via CREB following LPS
treatment. Previous studies have shown that α-synuclein
secreted from neurons induced a TLR4-dependent
inflammatory response in primary astrocyte cultures [
accumulation of α-synuclein in astrocytes following the
overexpression of mutant SNCA (encoding α-synuclein) under an
astrocyte-specific promoter resulted in the degeneration of
DA neurons in the SNpc and the ventral tegmental area
(VTA), as well as the onset of a movement disorder [
Data from this study indicate that activation of the TLR4
signaling pathway results in the downregulation of antioxidative
systems in astrocytes via inhibition of brain CYP2J.
Alterations of the CYP2J-dependent metabolism of endogenous
substrates may attenuate the LPS- or 6-OHDA-induced loss
of DA neurons and accumulation of α-synuclein in the SN.
In conclusion, our data demonstrated that the elevated
CYP2J levels strengthen the antioxidative defense system
by altering miRNA profiles within the brain. This suggests
that the alteration of the metabolism of the endogenous
6-OHDA + pHAGE
6-OHDA + pHAGE-CYP2J3
substrates (e.g., AA) could affect the risk of
neurodegenerative disease induced by neuroinflammation or neurotoxins.
The CYP2J-mediated metabolites may activate the
Nrf2ARE pathway and delay PD progression by ameliorating
AA: Arachidonic acid
ANOVA: Analysis of variance
AUDA: 12-(3-Adamantan-1-yl-ureido)dodecanoic acid
CYP2J2: Cytochrome P450 2J2
EETs: cis-Epoxyeicosatrienoic acids
LV: Lentiviral vector
PBS: Phosphate-buffed saline
PD: Parkinson’s disease
SN: Substantia nigra
SNpc: Substantia nigra pars compacta
Toll-like receptor 4
Ventral tegmental area.
The data used to support the findings of this study are
available from the corresponding author upon request.
Declaration of Transparency and Scientific Rigour. This
declaration acknowledges that this paper adheres to the principles
for transparent reporting and scientific rigour of preclinical
research recommended by funding agencies, publishers, and
other organizations engaged with supporting research.
Conflicts of Interest
The authors declare no conflict of interest.
Yueran Li, Jinhua Wu, Shufang Na, and Xuming Yu
performed the experiments. Yueran Li, Jinhua Wu, Ke Li, and
Jiang Yue analyzed the data. Zheqiong Yang, Xianfei Xie, Jing
Yang, and Jiang Yue designed the study. Jiang Yue wrote the
article. All the authors gave final approval for the article to be
published. Yueran Li and Jinhua Wu contributed equally to
this work. Yueran Li and Jinhua Wu are co-first author.
The authors thank the members of the Animal Care
Committee of Wuhan University and the staff at the animal
facility for being helpful and taking good care of our animals.
This study was supported by the National Natural Science
Foundation of China (nos. 81173122, 30973582, and
30960334), the Fundamental Research Funds for the Central
Universities (2042014kf0281), and the Innovation Seed Fund
of Wuhan University School of Medicine.
Primers used in this study are listed in
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