Biomedicine & Pharmacotherapy 134 (2021) 111170

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Huang Lian Jie Du Tang attenuates paraquat-induced mitophagy in human

SH-SY5Y cells: A traditional decoction with a novel therapeutic potential in
treating Parkinson’s disease
I-Jung Lee a, 1, Che-Yi Chao b, 1, Ying-Chen Yang c, Jing-Jy Cheng d, Chuen-Lin Huang e, f,
Chun-Tang Chiou d, Hung-Tse Huang d, Yao-Haur Kuo d, g, Nai-Kuei Huang d, h, i, *
a
Herbal Medicine Department, Yokohama University of Pharmacy, Yokohama, Japan
b
Department of Psychiatry, Cardinal Tien Hospital, New Taipei City 23148, Taiwan, ROC
c
Department of Biotechnology and Animal Science, National Ilan University, Ilan 26047, Taiwan, ROC
d
National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 11267, Taiwan, ROC
e
Medical Research Center, Cardinal Tien Hospital, Hsintien, New Taipei City 23148, Taiwan, ROC
f
Graduate Institute of Physiology & Department of Physiology and Biophysics, National Defense Medical Center, Taipei 11490, Taiwan, ROC
g
Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 40402, Taiwan, ROC
h
The Ph.D. Program for Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan, ROC
i
Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 11031, Taiwan, ROC

A R T I C L E I N F O A B S T R A C T

Keywords: Huang Lian Jie Du Tang (HLJDT) is a traditional Chinese medical decoction for heat-fire clearing and detoxi­
Huang Lian Jie Du Tang cation. Theoretically, the cause of Parkinson’s disease (PD) has been attributed to the dysregulations of internal
Paraquat wind, phlegm, fire, and stasis. Thus, HLJDT has been used to treat PD. However, the molecular mechanism is
Parkinson’s disease
unknown. Besides, paraquat (PQ) as an herbicide has been known to impair midbrain dopaminergic neurons,
Mitochondria
Mitophagy
resemblance to the pathology of PD. Thus, the molecular mechanism of HLJDT in treating PD and PQ-induced in
vitro PD model was investigated in this study. Primarily, the dose-response of PQ (0.1~1 mM)-induced neuro­
toxicity for 24 h was performed in the human neuroblastoma SH-SY5Y cells. The LD50 of PQ is around 0.3 mM
and was applied throughout the following experiments. The neutral red assay was used to estimate cell viability.
Co-transfection of the mitochondrial marker and proapoptotic factor genes were applied to measure the release
of mitochondrial proapoptotic factors during PQ intoxication and HLJDT protection. The fluorescent dyes were
used to detect mitochondrial membrane potential and free radical formation. Western blot and dot-blot analysis
and immunocytochemistry were used to estimate the level of proteins related to apoptosis and mitophagy. PINK1
gene silencing was used to determine the significance of mitophagy during PQ intoxication. In this study, HLJDT
attenuated PQ-induced apoptosis in SH-SY5Y cells. HLJDT reversed PQ-induced decreased mitochondrial
membrane potential and suppressed PQ-induced increased cytosolic and mitochondrial free radical formations
and mitochondrial proapoptotic factor releases. Furthermore, HLJDT mitigated PQ-induced increases in full-
length PINK1, phosphorylations of Parkin and ubiquitin, mitochondrial translocation of phosphorylated Par­
kin, and mitophagy. PINK1 gene silencing attenuated PQ-induced neurotoxicity. Therefore, HLJDT attenuated
PQ-induced cell death by regulating mitophagy.

Abbreviations: BSA, bovine serum albumin; CO3-H2DCFDA, 5-(and 6-)carboxy-2′ ,7′ -dichlorodihydrofluorescein diacetate; CYTO C, cytochrome C; DHE, Dihy­
droethidium; df, degrees of freedom; ER, endoplasmic reticulum; FBS, fetal bovine serum; FCCP, Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; GS, goat
serum; HLJDT, Lian Jie Du Tang; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; PINK1, PTEN-induced kinase 1; PD, Parkinson’s disease; PQ,
Paraquat; ROS, reactive oxygen species; S65, Serine 65; TCM, traditional Chinese medicine; Ub, ubiquitin.
* Corresponding author at: National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 11267, Taiwan, ROC.
E-mail address: andrew@nricm.edu.tw (N.-K. Huang).
1
These two authors devoted equally to this work and are considered to be co-first authors.

https://doi.org/10.1016/j.biopha.2020.111170
Received 15 July 2020; Received in revised form 12 December 2020; Accepted 15 December 2020
Available online 28 December 2020
0753-3322/© 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
I.-J. Lee et al.
1. Introduction
As a traditional Chinese medical decoction, Huang Lian Jie Du Tang
(HLJDT) comprises Scutellaria baicalensis Georgi, Gardeniae fructus Ellis,
Phellodendron chinense Schneid, and Coptis chinensis Franch. Its docu­
mentation was firstly addressed in the Wai-Tai-Mi-Yao, an ancient
Chinese medicinal from the Tang Dynasty. The essential effect of this
prescription is used for clearing heat-fire and detoxification. Currently,
it has been investigated for treating cerebrovascular and inflammatory
diseases, ischemia, and neurodegenerative disease, such as Alzheimer’s
disease [1–5]. Besides, according to the traditional Chinese syndrome
differentiation and treatment, the origin of Parkinson’s disease (PD) may
occur due to the dysregulations of internal wind, phlegm, fire, and stasis.
Consequently, HLJDT in treating PD had been prescribed by Chinese
medicine practitioners and substantiated by previous research [6].
However, the molecular mechanism is currently unknown.
PD is an age-associated neurodegenerative disease, with over 6.1
million cases during 2016 globally [7]. However, since there is no an­
tidote for PD medication and only syndrome-alleviating drugs are
available, the need for therapeutic drugs or other complementary and
alternative medicines and the search for novel molecular mechanisms of
PD are urgent. Currently, although both autosomal dominant (such as
SNCA, LRRK2, and VPS35) and recessive inheritance (such as DJ1,
PRKN, and PINK) may result in the familial aggregation of PD [8], only
about 10 percent of these Parkinsonian patients are genetically relevant
[9]. Thus, other risk factors, such as an environment, have also been
identified to play a role in the etiology of PD [10]. Previously, a com­
plete investigation of the toxicological and epidemiologic article has
proposed that paraquat (PQ) as an herbicide has the highest influential
correlation following continual exposure [11].
PQ has been used worldwide for over 60 years. PQ systemic treat­
ment in rodents causes intracellular α-synuclein aggregation [12] and
dopaminergic neuronal loss [13] in the midbrain substantia nigra area,
partly consisting of the pathological features of PD. Furthermore, PQ
also induces neurotoxicity in different cell types [14–16], affording
adequate in vivo and in vitro models for investigating the pathogenic
mechanisms of PD [17,18]. Presently, the pathologic mechanisms of PQ
may involve endoplasmic reticulum and oxidative stresses, dysregulated
autophagic flux, impaired ubiquitin-proteasome function, and mito­
chondrial dysfunction [19,20]. Even so, the discovery of novel patho­
logical mechanisms and neuroprotectants in preventing PQ-induced
neurotoxicity is still required [14,18,21] to provide a further practical
therapeutic approach for PD medication.
On the other hand, mitophagy, which selectively degrades dysfunc­
tional mitochondria by autophagy, has been intensively studied for
mediating neurodegenerative diseases [22]. The process of mitophagy
involves several critical pathways, the most typical being the
PTEN-induced kinase 1 (PINK1)/Parkin-dependent signaling pathway
[23]. In brief, in healthy mitochondria, PINK1 can be transported into
the inner mitochondrial membrane and then digested by proteases.
Nonetheless, in impaired mitochondria with collapsed membrane po­
tential, PINK1 is redirected outside the mitochondria membrane. PINK1
then phosphorylates ubiquitin (Ub) at the site of Serine 65 (S65), which
stimulates the mitochondrial translocation of Parkin [24]. PINK1 also
phosphorylates the recruited Parkin at S65 [25], resulting in the
amplification of mitophagic signal by ubiquitinating mitochondrial
proteins and the subsequent autophagic degradation of dysfunctional
mitochondria [26].
Recently, PINK1/Parkin-mediated mitophagy has been shown to
mediate PQ-mediated apoptosis in human lung epithelial A549 cells
[15]. However, whether PQ also induces PINK1/Parkin-dependent
mitophagy in neuronal cells was unknown. Since PQ could induce
both autophagy (Supplement 1) [27] and apoptosis [14] in SH-SY5Y
cells, the protective mechanism of HLJDT against PQ-induced neuro­
toxicity and the involvement of PINK1/Parkin-mediated mitophagy
during PQ intoxication were investigated.
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Biomedicine & Pharmacotherapy 134 (2021) 111170
2. Materials and methods
2.1. Materials
All reagents were obtained from Sigma-Aldrich/Merck (St. Louis,
MO, USA) except where specified otherwise. Carbonyl cyanide-4-
(trifluoromethoxy)phenylhydrazone (FCCP) was purchased from
Cayman Chemical (Ann Arbor, MI, USA). HyClone Dulbecco’s Modified
Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) was purchased from
GE Healthcare Life Sciences (Issaquah, WA, USA). Dihydroethidium
(DHE), 5-(and 6-)carboxy-2′ ,7′ -dichlorodihydrofluorescein diacetate
(CO3-H2DCFDA) and, MitoTracker® Deep Red FM, fetal bovine serum
(FBS), TMRE, Mito-SOX™, and Lipofectamine® 2000 were purchased
from Thermo Fisher Scientific (Waltham, MA, USA). Anti-TUBULIN
(ab6046) and Parkin (1679) was purchased from Abcam (Cambridge,
MA, USA). Anti-Ub (MAB1510), -ACTIN (MAB1501), and -Ub-S65
(Phospho Serine 65; ABS1513-1) antibodies and Immobilon PVDF
membranes were purchased from Millipore (Millipore, Bedford, MA,
USA). Anti-caspase-3 (9662), -poly(ADP-ribose) polymerase (PARP;
9542), and PINK1 (6946) antibodies were purchased from Cell Signaling
(Beverly, MA, USA). Anti-Parkin-S65 (Phospho Serine 65; CABT-B8840)
antibody was purchased from Creative Diagnostics (Shirley, NY, USA).
Anti-TOM20 (SC-17764) antibody was purchased from Santa Cruz
Biotechnology (Dallas, TX, USA). Secondary antibodies for use in
Western blot analysis were purchased from GE Healthcare (Logan, UT,
USA). Plasmids, pGFP-Cytochrome (Cyto) C (41181), pcDNA3-HtrA2-
GFP (14121), pSmac/Diablo-GFP (40881), PINK1-Myc (13313),
pPINK1-GFP (13316), and pTOM20-Clover (56307) were purchased
from Addgene (Cambridge, MA, USA). pTagRFP-C (FP141) was pur­
chased from Evrogen (Moscow, Russia). GFP and RFP represented the
green and red fluorescence proteins, respectively. pDsRed2-Mito
(expressing as a red fluorescence marker of mitochondria; 632421)
was purchased from Clontech Laboratories (Mountain View, CA, USA).
The siRNAs, ON-TARGETplus™ Control (D-00180-10-05) and human
PINK1 (L-004030-00-0005), and transfection reagent (DharmaFECT™
1) were purchased from Dharmacon™ (Lafayette, CO, USA). The
mitophagy detection kit was purchased from Dojindo Molecular Tech­
nologies (Kumamoto, Japan).
2.2. HLJDT preparation and quality control
HLJDT’s ingredients are derived from the preparation of Coptidis
rhizoma (Huanglian) and Coptis chinensis Franch. of the family Ranun­
culaceae, Scutellariae Radix (Huangqin) and Scutellaria baicalensis Georgi.
of the family Labiatae, Phellodendri chinensis Cortex (Huangbo) and
Phellodendron chinense Schneid. of the family Rutaceae, and Gardeniae
fructus (Zhizi), and Gardenia jasminoides Ellis. of the family Rubiaceae.
These were purchased from the Taipei market and identified by Dr. I-
Jung Lee at the Herbarium of the National Research Institute of Chinese
Medicine, Ministry of Health and Welfare. Samples of these herbs were
deposited in a Traditional Chinese Medicine (TCM) Herbarium with
voucher numbers NHP-1202 for Huanglian, NHP-1203 for Huangqin,
NHP-1155 for Huangbo, and NHP-1286 for Zhizi. HLJDT containing
Huanglian 9 g, Huangqin 6 g, Huangbo 6 g, and Zhizi 9 g in 1200 mL
water was boiled until the volume of water was reduced to 400 mL and
then lyophilized. After reconstitution with deionized water, the HLJDT
was filtered through a 0.2 μm membrane for further experiments.
The HPLC separations were performed on a Shimadzu LC-2040C
series apparatus with a photodiode array detector equipped with a
25 × 0.46 cm i.d. preparative Cosmosil 5C18 AR-II column (Nacalai
Tesque, Kyoto, Japan). A separation column (Cosmosil 5C18-AR-II, five
μm, 250 × 4.6 mm i.d., Japan) was employed to elute at a rate of
1.0 mL/min at room temperature. The mobile phase consisted of water
(A) containing 0.1 % phosphoric acid and acetonitrile (B) using a
gradient program of 5–25 % (B) in 0− 30 min, 25–30 % (B) in
30− 40 min, 30− 40 % (B) in 40− 55 min, and 40–100 % (B) in
I.-J. Lee et al.
55− 65 min. Real-time UV absorption was detected at 365 nm (Supple­
ment 2).
2.3. Cell culture and survival assays
Human neuroblastoma SH-SY5Y cells were cultured in DME/F12
supplemented with 10 % FBS and incubated in a 5 % CO2 incubator at
37 ◦ C. A neutral red uptake assay [28] with a slight modification [29]
was adopted to measure cell viability. Briefly, after seeding
(~3 × 104/cm2) for 2 days, cells were pretreated with or without HLJDT
for 1 h and then added with or without PQ for another 24 h. Cells were
loaded with neutral red (25 μg/mL), incubated at 37 ◦ C for 2 h, washed
once with 200 μL phosphate-buffered saline (PBS), and then added to
100 μL destaining solution (1 % glacial acetic acid, 49 % deionized H2O,
and 50 % ethanol [95 %]). The absorbance (540 nm) of each well was
measured using a micro-enzyme-linked immunosorbent assay reader
(Power Wave X; BioTek, VT, USA). On the other hand, a Cell Meter™
Live Cell TUNEL Apoptosis Assay Kit (Green Fluorescence) (AAT Bio­
quest, Sunnyvale, CA, USA) was used to detect the apoptosis by
following the instruction. In brief, cells growing on an 8-well chamber
slide were stained with Tunnelyte™ for 30 min at the incubator. After
washing once with PBS and adding with reaction buffer, cells were
subjected to confocal analysis. The fluorescently labeled DNA strand
breaks that showed intense green fluorescent staining represented the
apoptotic cells.
2.4. Measurement of mitochondrial membrane potential and reactive
oxygen species (ROS)
After washing with Hank’s Buffered Salt Solution, cells were and
stained with 100 nM TMRE (a marker for measuring mitochondrial
membrane potential) for 15 min or 5 μM Mito-SOX (a marker of mito­
chondrial superoxide) for 10 min, where fluorescence intensity repre­
sents mitochondrial membrane potential and ROS. Cells were then
subjected to image acquisition with fixed exposure times by an inverted
fluorescence microscope (Zeiss Axiovert 200 M; Carl Zeiss, Göttingen,
Germany). At least three frames were acquired and calculated for each
treatment, and 15–30 cells per field were used for quantifications. The
imaging fluorescence intensities were quantified using ImageJ with the
background subtracted.
2.5. Western blot study
A Western blot study was performed as described previously [30]. In
brief, cells were harvested and lysed in an ice-cold lysis buffer (1 mM
dithiothreitol, 20 mM EGTA, 20 mM 4-(2-hydroxyethyl)-1-piper­
azineethanesulfonic acid, 10 % glycerol, 50 mM β-glycerophosphate,
1 mM Na3VO4, 10 mM NaF, 1 mM PMSF, 2 μM aprotinin, 2 μM pep­
statin, 0.5 μM okadaic acid, 100 μM leupeptin, and 1 % Triton X-100).
After sonication, cell debris was discarded by centrifugation at 14,
000 rpm for 10 min and the supernatant used for Western blot study.
Equal amounts of the sample (20 μg/lane) were resolved by poly­
acrylamide gel electrophoresis. Proteins were then electroblotted onto
Immobilon PVDF membranes. Membranes were blocked by using 3 %
bovine serum albumin (BSA) or 5 % skim milk and then incubated with
the first antibodies overnight at 4 ◦ C and the second antibodies for one
hour at room temperature. After washing, membranes were performed
for visualization using an enhanced chemiluminescence system (Perkin
Elmer, Billerica, MA, USA). Membranes were exposed to a Fuji medical
X-ray film (Super RX-N, FUJIFILM Corporation, Tokyo, Japan) to obtain
fluorographic images.
2.6. Measurement of intracellular ROS
Intracellular superoxide and peroxide formations were measured
through staining with DHE and CO3-H2DCFDA, respectively. After
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Biomedicine & Pharmacotherapy 134 (2021) 111170
reacting with superoxide anions, DHE forms a red fluorescent product,
HE. Further, CO3-H2DCFDA is a non-fluorescent and cell-permeable
analog that is converted into CO3-H2DCF fluorescein after intracellular
deacetylation and oxidized into highly fluorescent CO3-DCF. In brief,
after 25 μM CO3-H2DCFDA or 10 μM DHE staining for 15 min, images of
the cells were taken with the fixed exposure times using an inverted
fluorescence microscope (Zeiss Axiovert 200 M). At least three frames
were acquired and calculated for each treatment, and 15–30 cells per
field were used for quantifications. The imaging fluorescent intensities
of the pictures were quantitated using ImageJ (Lakes, NJ, USA) software
with the background subtracted.
2.7. Dot blot analysis
As described in the protocol of Western blot analysis, equal protein
concentrations (10 μg/well) in each group were filtered through a
nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) using the Bio-
Dot Microfiltration Apparatus. During suction, each well was washed
with 200 μl TBST (100 mM Tris-HCl, 150 mM NaCl, and 0.1 % Tween
20; pH 7.4) twice. The transferred blot was blocked in TBST containing 5
% skim milk for 1 h at room temperature and then incubated with the
anti-Ub-S65 (1/5000) in 3 % BSA with 0.02 % NaN3 at 4 ◦ C overnight.
The following method was performed according to the protocol of
Western blot analysis.
2.8. Transient transfection and image detection
The jetPRIME® transfection reagent set (Polyplus-transfection SA,
Illkirch, France) was adopted as a tool to transfer plasmids into cells as
described by the manufacturer’s protocol. After the transfection for
24 h, cells were treated with drugs for another 24 h. Cells were then
fixed and mounted [31], and then observed with a confocal microscope.
2.9. Immunocytochemistry
This protocol was performed as described previously [31]. In brief,
cells were seeded and grown on sterile glass coverslips (3 × 104
cells/cm2). After treatments, cells were fixed by 4 % paraformaldehyde
for 10 min at room temperature. Following fixation, cells were rinsed
three times with PBS. Cells were further permeabilized by using 0.5 %
Triton-100 in PBS for 15 min. A blocking agent composed of 10 % goat
serum (GS) and 0.3 % Triton-100 in PBS was then applied at room
temperature for 90 min. After three washes with PBS, cells were labeled
with the antibodies (1:200), which were dissolved in 1 % BSA and 0.3 %
Triton-100 in PBS and incubated at 4 ◦ C overnight. Cells were washed
three times with PBS at room temperature. Cells with IgG
fluorescein-conjugated secondary antibodies (1:200) were incubated in
1 % BSA/PBS at room temperature for 90 min. After 3 washes with PBS,
cells were mounted on microscope slides with Aqua Poly-Mount (Poly­
sciences, Warrington, PA, USA). Cells were observed with the same gain
by a confocal microscope (LSM 780, Carl Zeiss, Göttingen, Germany).
Protein colocalization was analyzed by ImageJ-Cocol 2.
2.10. Gene silencing
The non-targeting control siRNA and SMART pools of siRNAs that
target human PINK1 were used to silence the expression of PINK1, ac­
cording to standard protocols (Huang et al., 2016).
2.11. Mitophagy detection
The detection of mitophagy was performed according to manufac­
turer protocols. In brief, cells growing on chamber slides (Ibidi GmbH,
Gräfelfing, Germany) were pre-loaded with mitophagy dye (1 μg/mL)
and MitoTracker® Deep Red FM (100 nM) for 30 min and treated with
drugs for another 24 h. Cells were imaged by a confocal microscope
I.-J. Lee et al. Biomedicine & Pharmacotherapy 134 (2021) 111170

Fig. 1. Effect of Huang Lian Jie Du Tang (HLJDT) in attenuating PQ-induced apoptosis in neuronal-like SH-SY5Y cells. (A) SH-SY5Y cells were treated with 0~1 mM
PQ for 24 h, and neutral red survival assays then performed. Cell viability was represented as a percentage of the results from the neutral red assay compared to
controls. Differences between groups in raw data were evaluated through one-way ANOVA and considered significant at p < 0.05. *p < 0.05, compared to the 24 h
control group. (B) Cells pretreated with or without HLJDT at different doses for 1 h were treated with or without 0.3 mM PQ for another 24 h. The neutral red assay
was then performed to measure viability. Differences between groups in raw data were evaluated through two-way ANOVA. *p < 0.05, compared to the group which
was only PQ treated. (C) Cells pretreated with or without 100 μg/mL HLJDT as indicated were treated with or without 0.3 mM PQ over different intervals. Cells were
then gathered and analyzed by Western blotting. The intensities in each group were normalized to the PQ-treated group (100 %). Differences among groups were
evaluated through a Kruskal-Wallis analysis of variance on ranks. *p < 0.05, compared to the control group. These data represented one out of at least three in­
dependent experiments. (D) After pretreating with 100 μg/mL HLJDT for 1 h, cells were treated with or without o.3 mM PQ for 24 h and then subjected to TUNEL
assay. The fluorescence signal was acquired using a confocal microscope, and the apoptotic cells were calculated. Data points represented the mean ± SD of at least
ten frames and 390 cells. Differences among groups were evaluated through one-way ANOVA. *p < 0.05, compared to the control group.

(LSM 780, Carl Zeiss, Göttingen, Germany).
2.12. Statistics
The statistical analysis was performed with SigmaPlot Version 12.5
statistic software package. Data were expressed as mean ± standard
deviation (SD). The differences among groups were performed through
the analysis of the nonparametric Kruskal-Wallis analysis of variance
(ANOVA) on ranks or one-/two-way ANOVA. Post hoc comparison were
calculated using the Student-Newman-Keuls method and the results
were considered significant at p < 0.05.
3. Results
left and lower panels) and CO3-DCF (chi square = 40.0, df = 3,
p < 0.001) (Fig. 2, upper right and lower panels).
3.3. HLJDT reduced PQ-induced mitochondrial dysfunction
HLJDT significantly attenuated PQ-induced decreased and increased
fluorescence intensity of TMRE (chi square = 41.6, df = 3, p < 0.001)
(Fig. 3, upper left and lower panels) and MitoSOX (chi square = 53.7,
df = 3, p < 0.001) (Fig. 3, upper right and lower panels), respectively.
Besides, HLJDT also significantly reduced PQ-induced releases of
mitochondrial proapoptotic factors, CYTO C (chi square = 15.0, df = 3,
p < 0.01) (Fig. 4A), HTRA2 (chi square = 14.9, df = 3, p < 0.001)
(Fig. 4B), and SMAC (chi square = 13.9, df = 3, p < 0.01) (Fig. 4C).

3.1. HLJDT reduced PQ-induced neurotoxicity in human neuroblastoma 3.4. HLJDT reduced PQ-induced the formation of mitophagy and
SH-SY5Y cell elevation of PINK1

PQ significantly and dose-dependently induced cell death as revealed
by the neutral red survival assay (F8,50 = 152.8, p < 0.001) (Fig. 1A).
The LD50 of PQ is 0.256, approximately 0.3 mM, which was adopted for
the following experiments. On the other hand, HLJDT attenuated PQ-
induced cell death significantly and dose-dependently (F1,16 = 121.7,
p < 0.001) (Fig. 1B). HLJDT at 100 μg/mL tended to yield the highest
protection without resulting neurotoxicity and was selected for the
subsequent experiments. HLJDT also reduced PQ-induced increased
cleavage forms of caspase 3 (chi square = 19.7, degrees of freedom
(df) = 9, p < 0.05) and PARP (chi square=22.7, df=9, p < 0.01)
(Fig. 1C). Furthermore, by using the TUNEL assay, HLJDT was
confirmed to attenuate PQ-induced apoptosis (F3,37=22.7, p < 0.001)
(Fig. 1D).
3.2. HLJDT reduced PQ-induced free radical formation
HLJDT significantly attenuated PQ-induced increased fluorescence
intensities of HE (chi square = 43.4, df = 3, p < 0.001) (Fig. 2, upper
HLJDT attenuated PQ-induced mitophagy formation (Fig. 5A).
Further, PQ significantly induced the elevation of PINK1 (chi
square = 20.2, df = 9, p < 0.05) after 8 and 24 h treatments (Fig. 5B).
HLJDT significantly attenuated the PQ-induced elevation of PINK1
(Fig. 5B). Besides, normally, PINK1 colocalized with mitochondria
(Fig. 5C), whereas PQ treatment resulted in the dislocation of PINK1
from mitochondria (F3,116 = 25.9, p < 0.001) (Fig. 5C and D). HLJDT
reversed PQ-induced phenomena (Fig. 5C and D).
3.5. HLJDT reduced the PQ-induced elevation of Ub-S65 and Parkin-S65
phosphorylations
PQ significantly increased the phosphorylations of Ub (Ub-S65) (chi
square = 19.2, df = 9, p < 0.05) (Fig. 6A and B) and Parkin (Parkin-
S65) (chi square = 23.4, df = 9, p < 0.01
) (Fig. 7A and 7B) at S65. Besides, PQ also resulted in increased
colocalizations Ub-S65 and Tom20 (F3,116 = 133.0, p < 0.001) (Fig. 6C)
and Parkin-S65 and Tom20 (F3,116 = 114.1, p < 0.001) (Fig. 7C).

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I.-J. Lee et al. Biomedicine & Pharmacotherapy 134 (2021) 111170

Fig. 2. Effects of Huang Lian Jie Du Tang (HLJDT) on PQ-induced cytosolic reactive oxygen species formation in human neuroblastoma (SH-SY5Y) cells. PQ
(0.3 mM) was administered for 24 h with or without pretreatment with 100 μg/mL HLJDT for 1 h. Cells were then stained with 25 μM CO3-H2DCFDA (upper left
panel) or 10 μM DHE (upper right panel) and subjected to image acquisition and quantitation (lower panel). At least 10 images were randomly acquired for each
group. Fluorescence intensity is represented as a percentage of controls. Differences among groups were evaluated through a Kruskal-Wallis analysis of variance on
ranks. * p < 0.05, compared with controls. The bar represents 50 μm. These data represented one out of three independent experiments that provided similar results.

However, HLJDT attenuated these phenomena (Figs. 6 and 7).
3.6. PINK1 gene silencing reduced PQ-induced neurotoxicity
PINK1 gene silencing resulted in not only the decreased expression of
the PINK1 protein (chi square = 6.7, df = 2, p < 0.05) (Fig. 8A) but also
increased resistance to PQ neurotoxicity (chi square = 9.4, df = 3,
p < 0.05) (Fig. 8B). Alternatively, PINK1 overexpression hampered the
protection of HLJDT in preventing PQ-induced cell death (F8,50 = 152.8,
p < 0.001) (Fig. 8C).
4. Discussion
Since the pathology of PD involves multiple mechanisms [32], it is
unlikely that a single-purpose drug could cope with all of them.
Recently, the use of TCM with multiple ingredients for treating PD has
drawn increasing attention [33–35] and revealed positive findings in
clinical trials [36–38]. In this study, we present another TCM, HLJDT,
and its protective mechanisms for mediating neurotoxicity in an in vitro
model of PD.
4.1. HLJDT prevented PQ-induced neurotoxicity
Accordingly, to screen and study the protective mechanisms of TCM,
an in vitro PD model was applied in a neuronal-like SH-SY5Y human cell
line treated with the herbicide PQ [39]. Thus, in the present study, the
PQ-induced cellular model of PD was reproduced (Fig. 1A) [14], and it
was found that HLJDT exerted protection against PQ-induced apoptosis
(Fig. 1B–D), thus supporting HLJDT as a novel therapy in treating PD.

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I.-J. Lee et al. Biomedicine & Pharmacotherapy 134 (2021) 111170

Fig. 3. Effects of Huang Lian Jie Du Tang (HLJDT) on PQ-induced mitochondrial reactive oxygen species and membrane potential in human neuroblastoma (SH-
SY5Y) cells. PQ (0.3 mM) was administered for another 24 h with or without pretreatment with 100 μg/mL HLJDT for 1 h. Cells were then stained with 100 nM
TMRE (upper left panel) or 5 μM MitoSOX (upper right panel) and subjected to image acquisition and quantitation (lower panel). At least 10 images were randomly
acquired for each group. Fluorescence intensity is represented as a percentage of controls. Differences among groups were evaluated through a Kruskal-Wallis
analysis of variance on ranks. * p < 0.05, compared with controls (n = 3~6). The bar represents 50 μm. These data represented one out of three independent
experiments that provided similar results.

4.2. HLJDT attenuated PQ-induced cytosolic and mitochondrial free
radical formation
Since imbalanced free radical formation may participate in the
pathogenesis of neurodegeneration, such as in PD [40], we thus exam­
ined and found that PQ did induce cytosolic (Fig. 2) and mitochondrial
(Fig. 3, upper right panel) free radical formation, consistent with pre­
vious studies [31,41,42]. Accordingly, although PQ may target mito­
chondria (such as complexes I and III) to generate free radicals [43,44],
there are still other mitochondrial (such as aconitase) and cytosolic
(such as thioredoxin reductase, NADPH oxidase, and nitric oxide syn­
thase) enzymes that may also be responsible for free radical formation
[45]. Since we have already shown that a known antioxidant,
U74389 G, exerts protection against PQ-induced cell death in this sys­
tem [14], the protection of HLJDT may also involve the mediation of an
antioxidative mechanism. Indeed, HLJDT has previously been shown to
contain an antioxidative effect [46]. Recently, HLJDT was shown to
exert anti-inflammatory effects through the regulation of nitric oxide
production and inhibition of cyclooxygenase and lipoxygenase [47,48],
which are responsible for the free radical formation and regarded as the
toxic mechanism of PQ [49,50], further supporting the antioxidative
effects of HLJDT.
4.3. HLJDT attenuated PQ-induced mitochondrial dysfunction
As described above, since PQ is known to interfere with the mito­
chondrial complex activity [43,44] that usually results in decreased
mitochondrial membrane potential and increased free radical formation
[51], we examined and confirmed that PQ induced decreased mito­
chondrial membrane potential (Fig. 3, upper left panel) and increased

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I.-J. Lee et al. Biomedicine & Pharmacotherapy 134 (2021) 111170

Fig. 4. Effect of Huang Lian Jie Du Tang (HLJDT) on PQ-induced mitochondrial release of proapoptotic factors. After co-transfection with (A) pDsRed2-Mito/pGFP-
Cyto C (1:3), (B) pDsRed2-Mito/pGFP-HtrA2 (1:3), and (C) pDsRed2-Mito/pSmac-GFP (1:3) for 24 h, cells were pretreated with 100 μg/mL HLJDT for 1 h and then
received 0.3 mM PQ treatments for another 24 h. Cells were fixed and then subjected to confocal microscopy analysis. The bar represents 5 μm. In each treatment, at
least 15 transfected cells were randomly acquired and the percentage of cells with released mitochondrial proapoptotic factors were counted and calculated. Dif­
ferences among groups were evaluated through a Kruskal-Wallis analysis of variance on ranks. *p < 0.05 compared to the PQ-treated group.

mitochondrial free radicals (Fig. 3, upper right panel) [31]. However,
HLJDT pretreatment attenuated PQ-induced increased mitochondrial
free radical formation and decreased membrane potential (Fig. 3),
supporting protection by HLJDT. Accordingly, except for the anti­
oxidative property of HLJDT that may attenuate free radical formation
and the subsequent decreased mitochondrial membrane potential, a
significant component of HLJDT [52] (Supplement 2), berberine, has
been shown to reduce NADPH oxidase activity [53], attenuate mito­
chondrial deficits and redox imbalance [54], and induce mitochondrial
biogenesis [55]. Therefore, the protection of HLJDT in alleviating
PQ-induced decreased mitochondrial membrane potential may be partly
attributed to the effect of berberine. Indeed, the observation that
berberine significantly attenuated PQ-induced cell death (Supplement
3) may partly support the viewpoints mentioned above. However, these
mechanisms still required further investigation.
Alternatively, the increased permeability of the mitochondrial
membrane, which is mediated by different ion channels [56] such as the
mitochondrial apoptosis-induced channel (MAC), has been associated
with the collapse of membrane potential, release of proapoptotic factors,
and subsequent cell death [57,58]. Thus, since HLJDT was able to
attenuate the PQ-induced collapse of membrane potential (Fig. 3, upper
left panel), it also attenuated PQ-induced releases of mitochondrial
proapoptotic factors, including CYTO C (Fig. 4A), HTRA2 (Fig. 4B), and
SMAC (Fig. 4C), further supporting the protection of HLJDT in medi­
ating mitochondrial function.
4.4. HLJDT attenuated PQ-induced mitophagy
Because PQ resulted in mitochondrial dysfunction (Figs. 3 and 4)
[31], which hinders the import and degradation of PINK1 in
mitochondria [25] and induces mitophagy in human lung epithelial-like
cells [15], we examined and found that PQ did induce mitophagy in
human SH-SY5Y cells (Fig. 5A). In parallel, FCCP, a known electron
transport uncoupler and mitophagy inducer [59], also induced neuro­
toxicity and mitophagy in SH-SY5Y cells (Supplement 4), consisting of
the previous study [60]. We thus examined the involvement of the
PINK1/Parkin pathway and found that the level of full-length PINK1 was
increased (Fig. 5B) and dislocated from mitochondria (Fig. 5C and 5D)
after PQ treatment, indicating the activation (autophosphorylation) of
PINK1 [25]. Regarding the idea that activated PINK1 phosphorylates
both Ub and Parkin at Ser65 [60], we have shown that PQ did indeed
result in the phosphorylations of Ub (Fig. 6A) and Parkin (Fig. 7A) at S65
and colocalized with the mitochondrial outer membrane protein
(Fig. 6B, C, and 7 B, C). These data support the participation of the
PINK1/Parkin-dependent signaling pathway in PQ-induced mitophagy.
Thus, HLJDT protection might contribute to protection against
PQ-induced mitochondrial dysfunction and mitophagy.
Recently, mitophagy has been proposed as a drug target in treating
PD [61] or other neurodegenerative diseases [62], suggesting that
mitophagy promotion could be a therapeutic approach. Our finding that
PQ induced mitophagy and cell death tends to contradict this viewpoint.
Although several compounds (such as nicotinamide riboside, urolithin
A, rapamycin, and spermidine) that enhance mitophagy have been
postulated to be beneficial in treating neurodegenerative diseases [63],
we found toxic or no effects from these compounds in this system (data
not shown). Besides, since other Parkinsonian toxins (such as 6-hydrox­
ydopamine, 1-methyl-4-phenylpyridinium, and rotenone) [64] and
FCCP (Supplement 4) all exert neurotoxicity and upregulate mitophagy,
it is unlikely that these Parkinsonian toxins should be used as thera­
peutic drugs for treating PD. Furthermore, the data on dysfunctional

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I.-J. Lee et al. Biomedicine & Pharmacotherapy 134 (2021) 111170

Fig. 5. Effect of Huang Lian Jie Du Tang (HLJDT) on PQ-induced mitophagy and translocation of PINK1. (A) After staining with the dyes for mitophagy and
mitochondria, cells were pretreated with HLJDT (100 μg/mL) for one hour and then treated with or without 0.3 mM PQ for 24 h. Cells were then subjected to
confocal image acquisition. The bar represents 5 μm. (B) Cells pretreated with or without HLJDT (100 μg/mL) for 1 h were treated with or without 0.3 mM PQ over
different intervals. Cells were then gathered and analyzed by Western blot analysis. The intensities in each group were normalized to the control group divided by
that of TUBULIN (100 %). Differences among groups were evaluated through a Kruskal-Wallis analysis of variance on ranks. *p < 0.05, compared to the control
group. (C) After pretreating with HLJDT (100 μg/mL) for 1 h, cells then received 0.3 mM PQ for another 24 h. Cells were fixed and subjected to immunocyto­
chemistry analysis. The bar represents 5 μm. (D) The colocalization maps (scatter plots) of volume pixels were shown. Tom20 emission intensities were plotted on the
x-axis, while PINK1 was plotted on the y-axis. Tom20 and PINK1 colocalization were quantified by measuring Pearsonʾs colocalization coefficients. Data points
represented the mean ± SD of at least 30 images. Differences among groups were evaluated through a Kruskal-Wallis analysis of variance on ranks. *p < 0.05,
compared to the control group. #p < 0.05, compared to the PQ-treated group.

mitochondria or impaired mitophagy in midbrain DAergic neurons are
mainly derived from postmortem PD studies [65,66]. The dynamic
changes of mitochondrial function and mitophagic flux during the pro­
gression or before the onset age of PD are unknown. Therefore, the
concept of enhancing mitophagy for treating neurodegeneration should
be limited and conditionally dependent, and more data are required to
dissect these discrepancies.
On the other hand, since PINK1 is an upstream regulator of the
Parkin function [67], PINK1 knockdown was performed to identify the
significance of the PINK1-dependent pathway during PQ intoxication.
To our surprise, PINK1 knockdown (Fig. 8A) significantly attenuated
PQ-induced neurotoxicity (Fig. 8B), indicating the character of the
PINK1-dependent pathway in regulating PQ-induced mitophagic cell
death. Although the extent of PINK1 knockdown could contribute to its
limited protection (Fig. 8B), other PINK1/Parkin-independent and
receptor-mediated mitophagic pathways [63], such as AMBRA1,
BCL2L13, BINP3, BINP3L, FUNDC1, NBR1, and prohibitin 2, may also
possibly be involved in PQ-induced neurotoxicity. Besides, even though
PINK1 and Parkin deficiency have been suggested to contribute to
neurodegeneration [26], PINK1 depletion rescues the decreased
bone-marrow cellularity and erythroid anemia in ATAD3-depleted mice
[68], and Parkin gene knockout prevents mitophagy- and
autophagy-dependent cell death in insulin-deprived human cortical
neuronal cells [69]. Thus, further studies are required to be done to
reveal the significance of each mitophagic pathway during cellular stress
and death.

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I.-J. Lee et al. Biomedicine & Pharmacotherapy 134 (2021) 111170

Fig. 6. Effect of Huang Lian Jie Du Tang (HLJDT) on the PQ-induced phosphorylation of Ub. (A) Cells pretreated with or without HLJDT (100 μg/mL) for 1 h were
treated with or without 0.3 mM PQ over different intervals. Cells were then harvested and analyzed by dot blot and Western blot analysis. The intensities in each
group were normalized to the control group divided by that of TUBULIN (100 %). Differences among groups were evaluated through a Kruskal-Wallis analysis of
variance on ranks. *p < 0.05, compared to the control group. (B) After pretreating with HLJDT (100 μg/mL) for 1 h, cells then received 0.3 mM PQ for another 24 h.
Cells were fixed and subjected to immunocytochemistry analysis. The bar represents 5 μm. (C) The colocalization maps (scatter plots) of volume pixels are shown. Ub-
S65 emission intensities are plotted on the x-axis and Tom20 on the y-axis. Ub-S65 and Tom20 colocalization were quantified by measuring Pearsonʾs colocalization
coefficients. Data points represented the mean ± SD of at least 30 images. Differences among groups were evaluated through a Kruskal-Wallis analysis of variance on
ranks. *p < 0.05, compared to the control group. #p < 0.05, compared to the PQ-treated group.

Fig. 7. Effect of Huang Lian Jie Du Tang (HLJDT) on the PQ-induced phosphorylation of Parkin. (A) Cells pretreated with or without HLJDT (100 μg/mL) for 1 h
were treated with or without 0.3 mM PQ over different intervals. Cells were then gathered and analyzed by Western blot analysis. The intensities in each group were
normalized to the control group divided by that of TUBULIN (100 %). Differences among groups were evaluated through a Kruskal-Wallis analysis of variance on
ranks. *p < 0.05, compared to the control group. (B) After pretreating with HLJDT (100 μg/mL) for 1 h, cells then received 0.3 mM PQ for another 24 h. Cells were
fixed and subjected to immunocytochemistry analysis. The bar represents 5 μm. (C) The colocalization maps (scatter plots) of volume pixels are shown. Parkin-S65
emission intensities are plotted on the x-axis and Tom20 on the y-axis. Parkin-S65 and Tom20 colocalization were quantified by measuring Pearsonʾs colocalization
coefficients. s. Data points represented the mean ± SD of at least 30 images. Differences among groups were evaluated through a Kruskal-Wallis analysis of variance
on ranks. *p < 0.05, compared to the control group. #p < 0.05, compared to the PQ-treated group.

5. Conclusion induced neurotoxicity through the mediation of mitochondrial

Currently, the impaired regulation of mitophagy appears as a wide­
spread denominator among various pathological diseases. Therefore,
interferences targeting mitophagy may have therapeutic potential. In
this study, we have found for the first time that HLJDT attenuates PQ-
dysfunction and mitophagy, suggesting that HLJDT may have practical
and therapeutic potential in treating PD or other neurodegenerations
involving similar neurotoxic mechanisms.

9
I.-J. Lee et al. Biomedicine & Pharmacotherapy 134 (2021) 111170

Fig. 8. Effect of PINK1 silencing on PQ-induced cell death. (A) After transfection of non-targeting siRNA (siControl) or siRNA targeting PINK1 (siPINK1) for 48 h,
cells were subjected to Western blot analysis (A) or neutral red survival assay (B). In Western blot analysis, the intensities in each group were normalized to the
control group divided by that of ACTIN (100 %). Differences among groups were evaluated through a Kruskal-Wallis analysis of variance on ranks. *p < 0.05,
compared to the control group. In neutral red survival assay, cells with or without gene silencing were treated with or without 0.3 mM PQ for 24 h. Cell viability was
represented as a percentage of its control group with or without gene silencing. Differences among groups were evaluated through a Kruskal-Wallis analysis of
variance on ranks. *p < 0.05, compared to its control group. #p < 0.05, compared to the PQ-treated group. (C) After transfecting pPINK1-GFP or pPINK1-Myc, cells
were pretreated with HLJDT (100 μg/mL) for 1 h and then treated with or without 0.3 mM PQ for 24 h. Cells were then subjected to Neutral red assay. Cell viability
was represented as a percentage of the results from the neutral red assay compared to controls. Differences between groups in raw data were evaluated through one-
way ANOVA and considered significant at p < 0.05. *p < 0.05, compared to the 24 h control group. #p < 0.05, compared to the PQ-treated group.

Author contributions Appendix A. Supplementary data

I-Jung Lee participated in HLJDT preparation. Hung-Tse Huang and
Yao-Haur Kuo participated in the analysis of the fingerprint of HLJDT.
Che-Yi Chao and Chuen-Lin Huang participated in research design,
experiment conduction, and data analysis. Ying-Chen Yang, Jing-Jy
Cheng, and Chun-Tang Chiou participated in research design, data
interpretation, and manuscript revising. Nai-Kuei Huang wrote the
manuscript and supervised the whole study.
Fundings
The present study was funded by the Ministry of Science and Tech­
nology (MOST 106-2320-B-077-003-MY3 and MOST 109-2320-B-077-
005-MY3), Ministry of Health and Welfare (MM10801-0117), National
Research Institute of Chinese Medicine, Ministry of Health and Welfare
(MOHW108-NRICM-B-325-112104), and Medical Research Center,
Cardinal Tien Hospital (CTH-105A-203, CTH106A-2A19, CTH106A-
2A23, CTH107A-2A10, and CTH108A-2A10).
Declaration of Competing Interest
The authors assert no conflict of interest in this investigation.
Acknowledgments
We thank Mr. B.C. Pruitt, Jr. for the English language editing of this
manuscript.
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.biopha.2020.111170.
References
[1] J. Xu, Y. Murakami, K. Matsumoto, M. Tohda, H. Watanabe, S. Zhang, Q. Yu,
J. Shen, Protective effect of Oren-gedoku-to (Huang-Lian-Jie-Du-Tang) against
impairment of learning and memory induced by transient cerebral ischemia in
mice, J. Ethnopharmacol. 73 (3) (2000) 405–413.
[2] J. Lu, J.S. Wang, L.Y. Kong, Anti-inflammatory effects of Huang-Lian-Jie-Du
decoction, its two fractions and four typical compounds, J. Ethnopharmacol. 134
(3) (2011) 911–918.
[3] M.F. Zeng, L.M. Pan, H.X. Zhu, Q.C. Zhang, L.W. Guo, Comparative
pharmacokinetics of baicalin in plasma after oral administration of Huang-Lian-Jie-
Du-Tang or pure baicalin in MCAO and sham-operated rats, Fitoterapia 81 (6)
(2010) 490–496.
[4] S.S. Durairajan, Y.Y. Huang, P.Y. Yuen, L.L. Chen, K.Y. Kwok, L.F. Liu, J.X. Song, Q.
B. Han, L. Xue, S.K. Chung, J.D. Huang, L. Baum, S. Senapati, M. Li, Effects of
Huanglian-Jie-Du-Tang and its modified formula on the modulation of amyloid-
beta precursor protein processing in Alzheimer’s disease models, PLoS One 9 (3)
(2014), e92954.
[5] Q. Zhang, H. Bian, Y. Li, L. Guo, Y. Tang, H. Zhu, Preconditioning with the
traditional Chinese medicine Huang-Lian-Jie-Du-Tang initiates HIF-1alpha-
dependent neuroprotection against cerebral ischemia in rats, J. Ethnopharmacol.
154 (2) (2014) 443–452.
[6] J. Jin, L.X. Duan, J. Sun, W.J. Zhao, Q.L. Zhou, Protective effect of Huanglian jiedu
decoction on the injury of PC12 cells induced by MPP+, Chin. Pharm. J. 43 (5)
(2008) 344–348.
[7] G.B.D.P.s.D. Collaborators, Global, regional, and national burden of Parkinson’s
disease, 1990–2016: a systematic analysis for the Global Burden of Disease Study
2016, Lancet Neurol. 17 (11) (2018) 939–953.
[8] A. Lunati, S. Lesage, A. Brice, The genetic landscape of Parkinson’s disease, Rev.
Neurol. (Paris) 174 (9) (2018) 628–643.

10
I.-J. Lee et al.
[9] B. Thomas, M.F. Beal, Parkinson’s disease, Hum. Mol. Genet. 16 (Spec No. 2)
(2007) R183–94.
[10] N. Ball, W.P. Teo, S. Chandra, J. Chapman, Parkinson’s disease and the
environment, Front. Neurol. 10 (2019) 218.
[11] C.M. Tanner, F. Kamel, G.W. Ross, J.A. Hoppin, S.M. Goldman, M. Korell,
C. Marras, G.S. Bhudhikanok, M. Kasten, A.R. Chade, K. Comyns, M.B. Richards,
C. Meng, B. Priestley, H.H. Fernandez, F. Cambi, D.M. Umbach, A. Blair, D.
P. Sandler, J.W. Langston, Rotenone, paraquat, and Parkinson’s disease, Environ.
Health Perspect. 119 (6) (2011) 866–872.
[12] A.B. Manning-Bog, A.L. McCormack, J. Li, V.N. Uversky, A.L. Fink, D.A. Di Monte,
The herbicide paraquat causes up-regulation and aggregation of alpha-synuclein in
mice: paraquat and alpha-synuclein, J. Biol. Chem. 277 (3) (2002) 1641–1644.
[13] A.I. Brooks, C.A. Chadwick, H.A. Gelbard, D.A. Cory-Slechta, H.J. Federoff,
Paraquat elicited neurobehavioral syndrome caused by dopaminergic neuron loss,
Brain Res. 823 (1-2) (1999) 1–10.
[14] J.M. Yang, H.M. Huang, J.J. Cheng, C.L. Huang, Y.C. Lee, C.T. Chiou, H.T. Huang,
N.K. Huang, Y.C. Yang, LGK974, a PORCUPINE inhibitor, mitigates cytotoxicity in
an in vitro model of Parkinson’s disease by interfering with the WNT/beta-
CATENIN pathway, Toxicology 410 (2018) 65–72.
[15] D.Z. Sun, C.Q. Song, Y.M. Xu, R. Wang, W. Liu, Z. Liu, X.S. Dong, Involvement of
PINK1/Parkin-mediated mitophagy in paraquat-induced apoptosis in human lung
epithelial-like A549 cells, Toxicol. In Vitro 53 (2018) 148–159.
[16] C.R. Reczek, K. Birsoy, H. Kong, I. Martinez-Reyes, T. Wang, P. Gao, D.M. Sabatini,
N.S. Chandel, A CRISPR screen identifies a pathway required for paraquat-induced
cell death, Nat. Chem. Biol. 13 (12) (2017) 1274–1279.
[17] K. Tieu, A guide to neurotoxic animal models of Parkinson’s disease, Cold Spring
Harb. Perspect. Med. 1 (1) (2011), a009316.
[18] S.J. Chinta, G. Woods, M. Demaria, A. Rane, Y. Zou, A. McQuade, S. Rajagopalan,
C. Limbad, D.T. Madden, J. Campisi, J.K. Andersen, Cellular senescence is induced
by the environmental neurotoxin paraquat and contributes to neuropathology
linked to Parkinson’s disease, Cell Rep. 22 (4) (2018) 930–940.
[19] X.F. Zhang, M. Thompson, Y.H. Xu, Multifactorial theory applied to the
neurotoxicity of paraquat and paraquat-induced mechanisms of developing
Parkinson’s disease, Lab. Invest. 96 (5) (2016) 496–507.
[20] E. Janda, C. Isidoro, C. Carresi, V. Mollace, Defective autophagy in Parkinson’s
disease: role of oxidative stress, Mol. Neurobiol. 46 (3) (2012) 639–661.
[21] D.C. Berwick, K. Harvey, The importance of Wnt signalling for neurodegeneration
in Parkinson’s disease, Biochem. Soc. Trans. 40 (5) (2012) 1123–1128.
[22] M. Martinez-Vicente, Neuronal mitophagy in neurodegenerative diseases, Front.
Mol. Neurosci. 10 (2017) 64.
[23] R.J. Youle, D.P. Narendra, Mechanisms of mitophagy, Nat. Rev. Mol. Cell Biol. 12
(1) (2011) 9–14.
[24] F. Koyano, K. Okatsu, H. Kosako, Y. Tamura, E. Go, M. Kimura, Y. Kimura,
H. Tsuchiya, H. Yoshihara, T. Hirokawa, T. Endo, E.A. Fon, J.F. Trempe, Y. Saeki,
K. Tanaka, N. Matsuda, Ubiquitin is phosphorylated by PINK1 to activate parkin,
Nature 510 (7503) (2014) 162–166.
[25] C. Kondapalli, A. Kazlauskaite, N. Zhang, H.I. Woodroof, D.G. Campbell,
R. Gourlay, L. Burchell, H. Walden, T.J. Macartney, M. Deak, A. Knebel, D.
R. Alessi, M.M. Muqit, PINK1 is activated by mitochondrial membrane potential
depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine
65, Open Biol. 2 (5) (2012), 120080.
[26] K. Palikaras, E. Lionaki, N. Tavernarakis, Mechanisms of mitophagy in cellular
homeostasis, physiology and pathology, Nat. Cell Biol. 20 (9) (2018) 1013–1022.
[27] R.A. Gonzalez-Polo, M. Niso-Santano, M.A. Ortiz-Ortiz, A. Gomez-Martin, J.
M. Moran, L. Garcia-Rubio, J. Francisco-Morcillo, C. Zaragoza, G. Soler, J.
M. Fuentes, Inhibition of paraquat-induced autophagy accelerates the apoptotic
cell death in neuroblastoma SH-SY5Y cells, Toxicol. Sci. 97 (2) (2007) 448–458.
[28] G. Repetto, A. del Peso, J.L. Zurita, Neutral red uptake assay for the estimation of
cell viability/cytotoxicity, Nat. Protoc. 3 (7) (2008) 1125–1131.
[29] C.L. Huang, Y.C. Lee, Y.C. Yang, T.Y. Kuo, N.K. Huang, Minocycline prevents
paraquat-induced cell death through attenuating endoplasmic reticulum stress and
mitochondrial dysfunction, Toxicol. Lett. 209 (3) (2012) 203–210.
[30] N.K. Huang, Y.W. Lin, C.L. Huang, R.O. Messing, Y. Chern, Activation of protein
kinase A and atypical protein kinase C by A(2A) adenosine receptors antagonizes
apoptosis due to serum deprivation in PC12 cells, J. Biol. Chem. 276 (17) (2001)
13838–13846.
[31] C.L. Huang, C.C. Chao, Y.C. Lee, M.K. Lu, J.J. Cheng, Y.C. Yang, V.C. Wang, W.
C. Chang, N.K. Huang, Paraquat induces cell death through impairing
mitochondrial membrane permeability, Mol. Neurobiol. 53 (4) (2016) 2169–2188.
[32] P. Maiti, J. Manna, G.L. Dunbar, Current understanding of the molecular
mechanisms in Parkinson’s disease: targets for potential treatments, Transl.
Neurodegener. 6 (2017) 28.
[33] X. Li, Y. Zhang, Y. Wang, J. Xu, P. Xin, Y. Meng, Q. Wang, H. Kuang, The
mechanisms of traditional chinese medicine underlying the prevention and
treatment of Parkinson’s disease, Front. Pharmacol. 8 (2017) 634.
[34] C. Tang, Y. Ye, Y. Feng, R.J. Quinn, TCM, brain function and drug space, Nat. Prod.
Rep. 33 (1) (2016) 6–25.
[35] C. Wang, X. Yang, G.D. Mellick, Y. Feng, Meeting the challenge: using cytological
profiling to discover chemical probes from traditional chinese medicines against
Parkinson’s disease, ACS Chem. Neurosci. 7 (12) (2016) 1628–1634.
[36] W.F. Kum, S.S. Durairajan, Z.X. Bian, S.C. Man, Y.C. Lam, L.X. Xie, J.H. Lu,
Y. Wang, X.Z. Huang, M. Li, Treatment of idiopathic Parkinson’s disease with
traditional chinese herbal medicine: a randomized placebo-controlled pilot clinical
study, Evid. Complement. Alternat. Med. 2011 (2011), 724353.
[37] K.K. Chua, A. Wong, K.W. Chan, Y.K. Lau, Z.X. Bian, J.H. Lu, L.F. Liu, L.L. Chen, K.
H. Chan, K.P. Tse, A. Chan, J.X. Song, J. Wu, L.X. Zhu, V. Mok, M. Li, A randomized
11
Biomedicine & Pharmacotherapy 134 (2021) 111170
controlled trial of chinese medicine on nonmotor symptoms in Parkinson’s disease,
Parkinsons Dis. 2017 (2017), 1902708.
[38] Q. Ye, X.L. Yuan, C.X. Yuan, H.Z. Zhang, X.M. Yang, Zishenpingchan granules for
the treatment of Parkinson’s disease: a randomized, double-blind, placebo-
controlled clinical trial, Neural Regen. Res. 13 (7) (2018) 1269–1275.
[39] H. Xicoy, B. Wieringa, G.J. Martens, The SH-SY5Y cell line in Parkinson’s disease
research: a systematic review, Mol. Neurodegener. 12 (1) (2017) 10.
[40] L. Puspita, S.Y. Chung, J.W. Shim, Oxidative stress and cellular pathologies in
Parkinson’s disease, Mol. Brain 10 (1) (2017) 53.
[41] W. Yang, E. Tiffany-Castiglioni, The bipyridyl herbicide paraquat produces
oxidative stress-mediated toxicity in human neuroblastoma SH-SY5Y cells:
relevance to the dopaminergic pathogenesis, J. Toxicol. Environ. Health A 68 (22)
(2005) 1939–1961.
[42] F. Wang, R. Franco, M. Skotak, G. Hu, N. Chandra, Mechanical stretch exacerbates
the cell death in SH-SY5Y cells exposed to paraquat: mitochondrial dysfunction and
oxidative stress, Neurotoxicology 41 (2014) 54–63.
[43] H.M. Cocheme, M.P. Murphy, Complex I is the major site of mitochondrial
superoxide production by paraquat, J. Biol. Chem. 283 (4) (2008) 1786–1798.
[44] P.R. Castello, D.A. Drechsel, M. Patel, Mitochondria are a major source of
paraquat-induced reactive oxygen species production in the brain, J. Biol. Chem.
282 (19) (2007) 14186–14193.
[45] R. Franco, S. Li, H. Rodriguez-Rocha, M. Burns, M.I. Panayiotidis, Molecular
mechanisms of pesticide-induced neurotoxicity: relevance to Parkinson’s disease,
Chem. Biol. Interact. 188 (2) (2010) 289–300.
[46] S. Fushitani, K. Minakuchi, K. Tsuchiya, M. Takasugi, K. Murakami, [Studies on
attenuation of post-ischemic brain injury by kampo medicines-inhibitory effects of
free radical production. II], Yakugaku Zasshi 115 (8) (1995) 611–617.
[47] H. Zeng, X. Liu, S. Dou, W. Xu, N. Li, X. Liu, W. Zhang, Z. Hu, R. Liu, Huang-Lian-
Jie-Du-Tang exerts anti-inflammatory effects in rats through inhibition of nitric
oxide production and eicosanoid biosynthesis via the lipoxygenase pathway,
J. Pharm. Pharmacol. 61 (12) (2009) 1699–1707.
[48] L. Li, H. Zeng, L. Shan, X. Yuan, Y. Li, R. Liu, W. Zhang, The different inhibitory
effects of Huang-Lian-Jie-Du-Tang on cyclooxygenase 2 and 5-lipoxygenase,
J. Ethnopharmacol. 143 (2) (2012) 732–739.
[49] W. Yang, E. Tiffany-Castiglioni, M.Y. Lee, I.H. Son, Paraquat induces
cyclooxygenase-2 (COX-2) implicated toxicity in human neuroblastoma SH-SY5Y
cells, Toxicol. Lett. 199 (3) (2010) 239–246.
[50] B.J. Day, M. Patel, L. Calavetta, L.Y. Chang, J.S. Stamler, A mechanism of paraquat
toxicity involving nitric oxide synthase, Proc. Natl. Acad. Sci. U S A 96 (22) (1999)
12760–12765.
[51] S. Marchi, C. Giorgi, J.M. Suski, C. Agnoletto, A. Bononi, M. Bonora, E. De Marchi,
S. Missiroli, S. Patergnani, F. Poletti, A. Rimessi, J. Duszynski, M.R. Wieckowski,
P. Pinton, Mitochondria-ros crosstalk in the control of cell death and aging,
J. Signal Transduct. 2012 (2012), 329635.
[52] Q. Yi, X.E. He, K.F. Luo, G.S. Zhang, Y.H. Liu, Q. Xue, N. Hou, W.L. Chen, J.D. Luo,
Protection of long-term treatment with huang-lian-jie-du-tang on vascular
endothelium in rats with type 2 diabetes mellitus, Curr. Ther. Res. Clin. Exp. 73 (6)
(2012) 174–185.
[53] M. Paul, M. Hemshekhar, K. Kemparaju, K.S. Girish, Berberine mitigates high
glucose-potentiated platelet aggregation and apoptosis by modulating aldose
reductase and NADPH oxidase activity, Free Radic. Biol. Med. 130 (2019)
196–205.
[54] V.G. Yerra, A.K. Kalvala, B. Sherkhane, A. Areti, A. Kumar, Adenosine
monophosphate-activated protein kinase modulation by berberine attenuates
mitochondrial deficits and redox imbalance in experimental diabetic neuropathy,
Neuropharmacology 131 (2018) 256–270.
[55] A.P. Gomes, F.V. Duarte, P. Nunes, B.P. Hubbard, J.S. Teodoro, A.T. Varela, J.
G. Jones, D.A. Sinclair, C.M. Palmeira, A.P. Rolo, Berberine protects against high
fat diet-induced dysfunction in muscle mitochondria by inducing SIRT1-dependent
mitochondrial biogenesis, Biochim. Biophys. Acta 1822 (2) (2012) 185–195.
[56] B. O’Rourke, Mitochondrial ion channels, Annu. Rev. Physiol. 69 (2007) 19–49.
[57] G. Kroemer, L. Galluzzi, C. Brenner, Mitochondrial membrane permeabilization in
cell death, Physiol. Rev. 87 (1) (2007) 99–163.
[58] A.P. Halestrap, What is the mitochondrial permeability transition pore? J. Mol.
Cell. Cardiol. 46 (6) (2009) 821–831.
[59] N.D. Georgakopoulos, G. Wells, M. Campanella, The pharmacological regulation of
cellular mitophagy, Nat. Chem. Biol. 13 (2) (2017) 136–146.
[60] E.F. Fang, T.B. Waltz, H. Kassahun, Q. Lu, J.S. Kerr, M. Morevati, E.M. Fivenson, B.
N. Wollman, K. Marosi, M.A. Wilson, W.B. Iser, D.M. Eckley, Y. Zhang,
E. Lehrmann, I.G. Goldberg, M. Scheibye-Knudsen, M.P. Mattson, H. Nilsen, V.
A. Bohr, K.G. Becker, Tomatidine enhances lifespan and healthspan in C. elegans
through mitophagy induction via the SKN-1/Nrf2 pathway, Sci. Rep. 7 (2017)
46208.
[61] J. Liu, W. Liu, R. Li, H. Yang, Mitophagy in Parkinson’s disease: from pathogenesis
to treatment, Cells 8 (7) (2019).
[62] K. Kingwell, Turning up mitophagy in Alzheimer disease, Nat. Rev. Drug Discov.
(2019).
[63] G. Lou, K. Palikaras, S. Lautrup, M. Scheibye-Knudsen, N. Tavernarakis, E.F. Fang,
Mitophagy and neuroprotection, Trends Mol. Med. 26 (1) (2020) 8–20.
[64] R.K. Dagda, T. Das Banerjee, E. Janda, How Parkinsonian toxins dysregulate the
autophagy machinery, Int. J. Mol. Sci. 14 (11) (2013) 22163–22189.
[65] F. Gao, J. Yang, D. Wang, C. Li, Y. Fu, H. Wang, W. He, J. Zhang, Mitophagy in
parkinson’s disease: pathogenic and therapeutic implications, Front. Neurol. 8
(2017) 527.
[66] C. Chen, D.M. Turnbull, A.K. Reeve, Mitochondrial dysfunction in Parkinson’s
disease-cause or consequence? Biology (Basel) 8 (2) (2019).
I.-J. Lee et al.
[67] S.M. Jin, R.J. Youle, PINK1- and Parkin-mediated mitophagy at a glance, J. Cell.
Sci. 125 (Pt 4) (2012) 795–799.
[68] G. Jin, C. Xu, X. Zhang, J. Long, A.H. Rezaeian, C. Liu, M.E. Furth, S. Kridel,
B. Pasche, X.W. Bian, H.K. Lin, Atad3a suppresses Pink1-dependent mitophagy to
12
Biomedicine & Pharmacotherapy 134 (2021) 111170
maintain homeostasis of hematopoietic progenitor cells, Nat. Immunol. 19 (1)
(2018) 29–40.
[69] H. Park, K.M. Chung, H.K. An, J.E. Gim, J. Hong, H. Woo, B. Cho, C. Moon, S.
W. Yu, Parkin promotes mitophagic cell death in adult hippocampal neural stem
cells following insulin withdrawal, Front. Mol. Neurosci. 12 (2019) 46.