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Research Article
In Vitro Nephrotoxicity Induced by Herb-Herb
Interaction between Radix Glycyrrhizae and Radix
Euphorbiae Pekinensis
Meng Chen,
1
Di Geng,
1
Xin Yang,
1
Xiaoxuan Liu,
1
Siqi Liu,
1
Pengmin Ding,
1
Yuesheng Pang,
1
Min Du ,
2
Xiuhua Hu ,
1
and Rufeng Wang
1
1
School of Life Sciences, Beijing University of Chinese Medicine, Yangguang South Street, Fangshan District, Beijing 102488, China
2
Department of Animal Science and School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA
Correspondence should be addressed to Xiuhua Hu; xiuhuahu@126.com and Rufeng Wang; wangrufeng@tsinghua.org.cn
Received 22 October 2019; Revised 17 March 2020; Accepted 27 March 2020; Published 28 April 2020
Academic Editor: Ji C. Bihl
Copyright © 2020 Meng Chen et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Radix Glycyrrhizae (RG)-Radix Euphorbiae Pekinensis (REP) is a representative incompatible herbal pair of Eighteen Incompatible
Medicaments (EIM) and has been disputed in clinical application for a long time. The present study was performed with the
Madin-Darby canine kidney (MDCK) cell line using cell cytotoxicity assay, apoptosis detection, cell cycle measurement, reactive
oxygen species (ROS) determination, and high content analysis (HCA) in combination with high-performance liquid
chromatography (HPLC) fingerprint comparison to clarify whether RG and REP can be concomitantly used from the
perspective of cytotoxicity, investigate the major correlated compounds, and elucidate the underlying mechanisms. The results
showed that the toxicity of REP could be significantly enhanced through its concomitant use with RG in the ratio of 1 : 1, and
this increased toxicity could be weakened with the further increased proportion of RG. 3,3′-di-O-methylellagic acid-4′-O-β-D-
xylopyranoside (DEAX) and 3,3′-di-O-methylellagic acid (DEA) were shown to be mainly responsible for the toxicity induced
by concomitant use of REP and RG. Both RG-REP decoctions and the above two compounds boosted cell apoptosis, cellular
morphological change, ROS accumulation, and mitochondrial membrane potential (MMP) disruption. In conclusion, the
incompatible use of RG and REP is conditionally established because of the bidirectional regulatory effect of RG, and the major
compounds responsible for RG-REP incompatibility are DEAX and DEA, which result in toxicity through activation of
mitochondria-dependent apoptosis induced by increased ROS production. This study provided a basis for understanding the
incompatible use of RG and REP and the EIM theory.
1. Introduction
Drug-drug interaction is a very important aspect in the clinical
practice and is a focus of medical research. In Chinese medi-
cine, an ancient mysterious theory about drug contradiction,
that is, the Eighteen Incompatible Medicaments (EIM), has
always been a disputable issue of medical practitioners. This
theory discourages the concomitant use of eighteen herbal
drug pairs, such as Radix Glycyrrhizae (RG) and Radix
Euphorbiae Pekinensis (REP), Radix Aconiti and Bulbus Fri-
tillariae Cirrhosae, and Radix et Rhizoma Veratri and Radix
Ginseng. Incompatible medicaments in Chinese medicine
stemmed from the famous Shennong’sHerbal Classic—the
extant earliest classic book of Traditional Chinese Medicine
(TCM), which abstractly proposed this concept. Subse-
quently, a book entitled Variorum of Shennong’s Herbal
Classic authored by Hongjing Tao, who was a Chinese phar-
macologist in the Northern and Southern Dynasties, listed
the incompatible herbal drug pairs including those men-
tioned above. Although these incompatible herbal drug pairs
have been avoided as much as possible by TCM practitioners
since ancient times, the experience of concomitant use of
these pairs has been accumulated, and the treatment cases
of the rare and intractable diseases using them have also been
recorded now and then [1–3]. Pharmacologists possess dif-
ferent viewpoints on this concept and have not yet come to
Hindawi
Oxidative Medicine and Cellular Longevity
Volume 2020, Article ID 6894751, 16 pages
https://doi.org/10.1155/2020/6894751
an agreement [4, 5]. Therefore, this theory has been observed
and disputed by Chinese pharmacologists since ancient times,
and it is still an interesting question whether EIM should be
absolutely avoided in the clinical practice and whether there
has been scientific basis to support this occult theory.
REP and RG constitute one of such herbal pairs of EIM.
REP is the dried roots of Euphorbia pekinensis Rupr., which
is a well-known poisonous plant of genus Euphorbia in the
Euphorbiaceae family. This herbal drug has been officially
recorded in Chinese pharmacopoeia [6] for the treatment
of oedema, malignant ascites, and urinary retention. In the
clinic, REP exhibits explicit therapeutic effects on relieving
constipation, purging fluid, and eliminating phlegm [7, 8].
Modern pharmacological investigations also demonstrated
that its extract exhibited a variety of biological effects, includ-
ing antineoplastic, antiviral, and analgesic activities [9–11].
RG, which comes from the dried roots of either Glycyrrhiza
uralensis Fisch. or G. glabra L. or G. inflate Bat., is one of
the most widely used nourishing herbal medicines in TCM.
It is usually used in combination with other herbs and pre-
scribed in approximately 60% of TCM formulas because
most TCM practitioners believe that RG has the capacity to
enhance the efficacy of other ingredients and reduce toxicity
[12]. Meanwhile, RG has been used as a food supplement in
other countries, such as Japan and the UK [13–16]. It is gen-
erally believed that concomitant use of these two herbal drugs
should be forbidden in the clinic. There has been experimen-
tal evidence showing that the concomitant use of REP and
RG results in more severe adverse effects on the tissues of
the kidney, heart, and liver of experimental animals than sep-
arate use and leads to significantly increased levels of alanine
aminotransferase, phosphocreatine kinase, lactate dehydro-
genase, γ-hydroxybutyrate dehydrogenase, urea, and creati-
nine [17]. Furthermore, a study reported that combination
of RG and REP caused vascular congestion and lymphocytic
infiltration in the renal and hepatic tissues of rats, indicating
some toxic and side effects [18]. This reminds us that the
concomitant use of both drugs may be harmful to animals
because of the generation or increment of toxic compounds.
However, to our knowledge, there is limited evidence proving
which compound/compounds is/are related to the increased
nephrotoxicity of this herbal pair. In order to obtain the
direct evidence of the underlying toxic mechanism induced
by concomitant use of RG and REP, we carried out the study
to investigate the correlation between the toxicity and com-
pounds in RG-REP decoctions on the basis of chemical com-
position change resulting from a combination process.
2. Materials and Methods
2.1. Materials, Reagents, and Instruments. RG and REP
were purchased from Anguo Chinese crude drug market
in Hebei Province of China, and botanical identification
was done by Prof. Rufeng Wang. Two voucher specimens
(Nos. 201205041 and 201205042 for RG and REP, respec-
tively) have been deposited at the herbarium of School of Life
Sciences, Beijing University of Chinese Medicine, China.
Reference compounds 3,3′-di-O-methylellagic acid-4′-O-
β-D-xylopyranoside (DEAX) and 3,3′-di-O-methylellagic
acid (DEA) were prepared in our laboratory, and their
purities were determined to be >98% by high-performance
liquid chromatography (HPLC).
Dulbecco’smodified Eagle’s medium (DMEM), fetal
bovine serum (FBS), and trypsin were supplied by Gibco Invi-
trogen Corp. (Grand Island, CA, USA). 3-(4,5-dimethyl-2-
thiazolzyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT)
and dimethyl sulfoxide (DMSO) were products purchased
from Sigma Chemical Co. (Deisenhofen, Germany). Cell cul-
ture flasks, 96-well plates, 6-well plates, penicillin, and strep-
tomycin were purchased from Corning Inc. (Cambridge,
MA, USA). Rhodamine 123, Hoechst 33342, 2′,7 ′-dichloro-
dihydrofluorescein diacetate (DCFH-DA), propidium iodide
(PI), ethylenediaminetetraacetic acid (EDTA), and Annexin
V-fluorescein isothiocyanate/PI (Annexin V-FITC/PI) apo-
ptosis detection kit were obtained from Beijing Aoboxing
Biotechnology Co., Ltd. (Beijing, China). A caspase-3/7 assay
kit and cell cycle kit were supplied by EMD Millipore Co.,
Ltd. (Temecula, CA, USA). Acetonitrile of HPLC grade used
for HPLC analysis was produced by Fisher Co. (Pittsburgh,
PA, USA). Deionized water was prepared using a Millipore
water purification system made by Millipore Corporation
(Billerica, MA, USA).
A Bio-Rad Model 550 microplate reader was produced by
Bio-Rad Laboratories (Hercules, CA, USA). A Nikon TE2000
microscope equipped with a high-resolution digital camera
(Nikon DXM 1200F) was manufactured by Nikon Instru-
ments, Inc. (Tokyo, Japan). A CytoFLEX Flow Cytometer
was the product of Beckman Coulter (Brea, CA, USA). A
Muse Cell Analyzer was obtained from Merck Millipore
Corp. (Billerica, MA, USA). An Opera Phenix High Content
Analysis (HCA) confocal microscope equipped with a 20×
Plan Apo objective lens was from PerkinElmer Inc. (Boston,
MA, USA). A Waters 1500 series liquid chromatograph
equipped with a 1525 binary pump, an on-line degasser, a
manual injector, and a 2489 UV/visible detector were the
products of Waters Corp. (Milford, MA, USA).
2.2. Cell Line and Treatment. The Madin-Darby canine kid-
ney (MDCK) cell line was supplied by the Cell Resource Cen-
ter, Peking Union Medical College (CRC/PUMC, China) and
cultured in DMEM containing 10% FBS and 1% penicillin-
streptomycin in an atmosphere of 5% CO
2
and 95% air at
37
°
C and constant humidity. The cells were inoculated in
the 96-well or 6-well plates and cultured overnight prior to
treatment with each RG-REP decoction or each compound.
2.3. Sample Preparation. The crude drug material of RG and
REP was separately pulverized and passed through a 40-mesh
sieve and then used for decoction preparation. Individual
decoctions and combined decoctions which could be further
divided into combination ratios including 1 : 1, 2 : 1, and 3 : 1
(RG : REP, counted by weight of crude drugs) were prepared
according to Table 1. In brief, the powdered material for
each decoction was extracted twice with 10 volumes (v/w)
of distilled water by boiling for 0.5 h each, and the lost water
was made up at any time; then, the extract was combined,
filtered, and concentrated to a solution containing 0.1 g of
REP per mL. The resultant solution was diluted to the
2 Oxidative Medicine and Cellular Longevity
desired concentrations (0.4, 0.7, 1.0, 1.3, 1.6, and 1.9 mg of
REP per mL) using DMEM prior to treating MDCK cells.
For DEA and DEAX, they were accurately weighted and
dissolved in DMSO, respectively. Then, they were diluted
with DMEM to allow the final concentration of DMSO in
the culture medium less than 1.0% (v/v). As such, the maxi-
mum concentrations of DEA and DEAX dissolved in DMEM
were 300 and 500 μM, respectively.
2.4. Cell Cytotoxicity Assay. The inhibition rates of decoctions
or compounds on MDCK cells were detected using MTT
assay. The cells were seeded into 96-well plates at a density
of 3×10
4cells per well and cultured at 37
°
C for 24 h before
being exposed to decoctions or compounds at various con-
centrations. After being treated for 48 or 24 h, the superna-
tant was removed, and the cells were incubated with 100 μL
of MTT solution for 4 h under cell culture conditions. Then,
the liquid part was discarded, and the cells were dissolved in
150 μL of DMSO and shaken for 10 min to dissolve the for-
mazan crystal generated. After that, the absorbance (A)
value of the resultant solution was measured on a micro-
plate reader at the wavelength of 570 nm. The cell inhibi-
tion rates were calculated by the following formula:
cell inhibition rate ð%Þ=ð1−experimental group’sA/control
group’sAÞ× 100%. The concentrations of 50% toxicity
(TC
50
) on MDCK cells were also determined. The MDCK
cellular morphology was observed with a Nikon TE2000
microscope, and three different fields per well were randomly
chosen and photographed.
2.5. Cell Apoptosis Detection. The percentages of survival, early
apoptotic, late apoptotic, and necrotic cells were detected
using a caspase-3/7 kit or Annexin V-FITC/PI apoptosis
detection kit. First, MDCK cells at the density of 1:8×10
5cells
per well were cultured in 6-well plates for 24 h and then
treated with each RG-REP decoction for 48 h or each com-
pound for 24 h. After treatment, the cells were trypsinized
with 0.05% trypsin without EDTA and then centrifuged in
PBS three times. Subseque ntly, 5 μL of Muse caspase-3/7
working solution or 5 μL of Annexin V-FITC and 150 μLof
7-AAD working solution or 5 μL of PI solution were succes-
sively added in the dark at room temperature to stain the cells
for 15 min. After that, the stained cells were detected with a
Muse Cell Analyzer or a CytoFLEX Flow Cytometer within
1 h. For each analysis, a total of 10,000 events were recorded.
2.6. Cell Cycle Measurement. The cell cycle distribution of
MDCK cells was analyzed using a cell cycle kit. Briefly,
MDCK cells at the density of 1:8×10
5cells per well were
seeded in 6-well plates for 24 h and then treated with each
RG-REP decoction. About 48 h later, the cells were dissoci-
ated and harvested through centrifugation at 1500 rpm for
5 min. Then, they were permeabilize d in 70% ethanol at
4
°
C for 24 h and washed three times with PBS. The pellets
were lysed in 100 μL of cell cycle reagent in the dark for
30 min and examined using the Muse Cell Analyzer. At least
10,000 events of MDCK cells were typically acquired for
each analysis.
2.7. Intracellular Reactive Oxygen Species (ROS) Production
Measurement. The intracellular ROS production was mea-
sured using DCFH-DA dye as described by the literature
[19]. The MDCK cells at the density of 1:8×10
5cells per well
were first seeded into 6-well plates and incubated overnight.
Next, they were exposed to various concentrations of each
RG-REP decoction for 48 h or each compound for 24 h.
Then, the cells were harvested using 0.05% trypsin without
EDTA and centrifuged at 1500 rpm for 5 min. After that, they
were resuspended in a buffer containing 10 μM DCFH-DA at
37
°
C in the dark for 30 min and washed twice with PBS to
remove unincorporated dye. Finally, the fluorescence inten-
sity of the samples was immediately recorded on the Cyto-
FLEX Flow Cytometer at the excitation and emission
wavelengths of 485 and 530 nm, respectively. A minimum
of 10,000 events were collected on each sample.
2.8. High Content Analysis. The cell appearance and average
fluorescence intensity were monitored by HCA coupled with
Hoechst 33342/PI/Rhodamine 123 staining assay. The cells
were first seeded into 96-well plates at the density of 3×10
4
cells per well. After incubation for 24 h, the medium was
removed, and cells were treated with the medium containing
each RG-REP decoction or each compound. About 48 or
24 h later, they were stained in the dark with PBS containing
5μM Hoechst 33342, 10 μM PI, and 10 μM Rhodamine 123,
under cell culture conditions for 45 min. The stained cells
were washed twice with PBS and maintained in prewarmed
DMEM without phenol red for live cell imaging. Finally,
the MDCK cells were imaged in 96-well plates using an
Opera Phenix HCA confocal microscope equipped with a
20×Plan Apo objective lens (NA 0.45) at three fixed excita-
tion and emission wavelengths for Hoechst 33342 (Ex/Em
350/461 nm), PI (Ex/Em 535/615 nm), and Rhodamine 123
(Ex/Em 507/529 nm). Nine fields per well were taken to
cover the entire well.
2.9. HPLC Analysis. The HPLC fingerprints of each decoction
and content assay of DEAX and DEA were obtained on a
Waters 1500 series chromatograph. Each decoction sample
was appropriately diluted to the final concentration of 0.5 mg
of REP per mL and filtered through a 0.22 μm membrane
filter. Then, each 10.0 μL of the samples was loaded into a
Thermo Hypersil BDS C
18
column (250 mm × 4:6mm,
5μm) at the column temperature of 30
°
C. The chromatog-
raphy was run at a flow rate of 1.0 mL/min using acetoni-
trile as mobile phase A and 1.0% acetic acid in purified
water as mobile phase B with gradient elution program
including 2–8% A (0–6min), 8–13% A (6–21 min), 13–20%
Table 1: The ratios and doses of RG and REP in decoctions.
Decoctions RG (g/L) REP (g/L) Ratio (RG : REP)
RG 4.8 ——
REP —1.6 —
1 : 1 RG-REP 1.6 1.6 1 : 1
2 : 1 RG-REP 3.2 1.6 2 : 1
3 : 1 RG-REP 4.8 1.6 3 : 1
3Oxidative Medicine and Cellular Longevity
A(21–30 min), 20–35% A (30–40 min), 35–45% A (40–
50 min), 45–75% A (50–60 min), 75–90% A (60–65 min),
and 90–100% A (65–70 min). The stock solutions of DEAX
(0.02 μg/mL) and DEA (40 μg/mL) were prepared with meth-
anol and further diluted into a series of working solutions for
the establishment of calibration curves. The calibration
curves for DEAX and DEA were obtained by plotting their
peak area (Y) versus amount (X,inμg). As a result, the regres-
sion equations and coefficient correlations (r) for DEAX and
DEA were Y=2:80374 × 105X+1644:7(r=0:9996, 0.00156–
0.4 μg) and Y=4×10
8X+ 28851 (r=0:9997, 0.00200–20 μg),
respectively.
2.10. Data Analysis. The results presented in this study were
the averages of at least three replicates and were presented as
means ± SD. Statistical analysis was carried out by SPSS 19.0,
and statistical significance was evaluated by Student’st-test
for comparison of the mean values. P<0:05 was selected as
the criteria for statistical significance.
3. Results
3.1. The Cytotoxicity of REP Was Increased when Coused with
RG at the Ratio of 1 : 1, and This Effect Was Alleviated along
with the Increment of RG Dose. The results obtained from
the MTT assay showed that the inhibition rate of the RG
decoction on MDCK cells was decreased even though with-
out significant difference compared with that of the DMEM
control group, suggesting RG was nontoxic to MDCK cells.
In contrast, the cytotoxicity of REP and three combined
decoctions to MDCK cells was more prominent compared
with that of DMEM control group. What is more, a signifi-
cant dose- and time-dependent relationship was established.
With the same concentration, the 1 : 1 RG-REP decoction
was more cytotoxic than the REP decoction at all time points
tested. Interestingly, the 3 : 1 RG-REP decoction provided
significant protective effect on the cells compared with the
REP decoction and the 1 : 1 RG-REP decoction. The inhibi-
tion rate of the 2 : 1 RG-REP decoction on the cells was
between those of the 1 : 1 and 3 : 1 RG-REP decoctio ns
(Figure 1(a) and Supplementary Figure 1). Quantitatively,
the TC
50
values of REP, 1 : 1, 2 : 1, and 3 : 1 RG-REP
decoctions on MDCK cells at 12, 24, and 48 h were ranged
from 1.147 to 4.074, from 0.796 to 2.649, and from 0.452 to
1.736 mg/mL, respectively.
3.2. 1 : 1 RG-REP Decoction Boosted Cellular Morphological
Change, Apoptosis, Mitochondrial Membrane Potential
(MMP) Disruption, and ROS Accumulation. The effect of
each RG-REP decoction on MDCK cells was confirmed by
observing cellular morphology under a light inverted micro-
scope. Figure 2 shows the representative images of MDCK
cells in an untreated control group and the groups treated
with each RG-REP decoction at the concentration of
1.0 mg/mL for 48 h. In comparison with that in the DMEM
control group, the cell density in the RG decoction group
increased, while that in the REP decoction group obviously
decreased. The cell morphology of MDCK cells in the REP
group became smaller and round shaped. The lowest cell
density was observed in the 1 : 1 RG-REP decoction group.
Nevertheless, the increasing cell density was observed along
with the proportion increment of RG, i.e., in the 2 : 1 and
3 : 1 RG-REP decoction groups (Figure 2).
The results of HCA coupled with Hoechst 33342/PI/R-
hodamine 123 staining assay further confirmed the cyto-
toxic effect of each RG-REP decoction on morphological
features of MDCK cells. As shown in Figure 3, the majority
of cells in the RG group displayed homogeneous blue nuclei
(Hoechst 33342 positive) and bright green cytoplasm (Rho-
damine 123 positive), and few of them exhibited red nuclei
(PI positive). This demonstrated that most of the cells were
viable (VC) and not in the state of apoptosis or necrosis. In
contrast, in the REP group and the 1 : 1 RG-REP group, an
increased number of deep red nuclei and apoptotic bodies
(AB) were observed, which are the features of late apoptosis
or necrosis.
The morphological change of mitochondria firmly con-
vinced us of the toxicity of RG-REP decoctions. The normal
mitochondria (NM) which exhibited the morphological fea-
tures that were well-structured, interconnected, and rod-
shaped were enriched in the DMEM control group and the
RG group, whereas the mitochondria in the groups of the
REP decoction and the 1 : 1 RG-REP decoction were dot-
like round for MMP disruption. These results implied that
the REP decoction and the 1 : 1 RG-REP decoction might
promote cell apoptosis or necrosis.
The percentages of alive, early apoptotic, late apoptotic,
and dead cells quantitatively measured by flow cytometry
revealed the cell death manner primarily contributed to the
cytotoxicity of RG-REP decoctions. As shown in Figures 4(a)
and 5, treatment with RG did not stimulate significant cell
apoptosis. In contrast, treatment with REP or three combined
RG-REP decoctions significantly increased the percentage of
total apoptotic cells in a dose-dependent manner. Further-
more, the percentage of total apoptotic cells in the REP
decoction group at the concentration of 1.0 mg/mL was
25.8%, whereas it increased to 47.9% in the 1 : 1 RG-REP
decoction group and decreased again to 20.43% when the
ratio of RG-REP increased to 3 : 1. As for the percentages of
necrotic cells in three combined RG-REP decoction groups
which ranged from 0.47 to 2.57%, almost no significant dif-
ference was observed compared to the REP decoction group
which ranged from 0.73 to 1.30%. In general, the percent-
age of total apoptotic cells was 7.92–102.7 times as many
as the percentage of necrotic cells. These results confirmed
that REP decoction and 1 : 1 RG-REP decoction preferably
promoted cell apoptosis, and the latter was stronger than
the former.
The results of HCA through detection of the fluorescence
intensity of Rhodamine 123 also made clear that MMP
disruption was involved in the cell damage induced by
the REP and combined RG-REP decoctions. The fluores-
cence intensity of Rhodamine 123, which is a good indica-
tor for MMP, was not changed significantly by RG at all
experimental concentrations and REP at the low concentra-
tions of 0.5 and 1.0 mg/mL. However, the groups treated with
REP at the high concentration of 2.0 mg/mL and 1 : 1 RG-
REP decoction at all experimental concentrations displayed
4 Oxidative Medicine and Cellular Longevity
significant lower fluorescence intensity compared with that
of the DMEM control group. In addition, the fluorescence
intensity significantly decreased from 37942.7 to 25002.2,
from 44486.35 to 27652.2, and from 48004.7 to 33225.5 com-
pared with that of the REP decoction group after treatment
with 1 : 1 RG-REP decoction at the concen trations of 0.5,
1.0, and 2.0 mg/mL, respectively (Figure 6(c)). These results
indicated that the apoptosis of MDCK cells was ascribed to
the mitochondrial dysfunction.
Since mitochondria are both the primary source and the
target of ROS [20, 21], so the ROS changes were determined.
As shown in Figure 7(a), RG did not affect the fluorescence
intensity of 2′,7′-dichlorofluorescein (DCF), which is usually
used to reflect the amount of ROS, even at the high concen-
tration of 2.0 mg/mL. However, the generation of ROS
increased in a concentration-dependent manner after the
MDCK cells were treated with REP and three combined
decoctions for 48 h. Moreover, ROS induced by the 1 : 1
RG-REP decoction at the concentrations of 0.5, 1.0, and
2.0 mg/mL increased by approximately 5.05–8.68-folds com-
pared with that of the DMEM control group. It was also
increased 135.5, 153.8, and 173.9% compared with that of
the REP decoction group at the same concentrations. In con-
trast, the level of intracellular ROS was significantly sup-
pressed in the 3 : 1 RG-REP decoction group compared with
that of the REP decoction group. The DCF fluorescence
–40
DMEM
RG
REP
0
40
80
120
Inhibition rate on MDCK cells (%)
##
#
#
##
##
##
1: 1 RE-REP
2: 1 RE-REP
3: 1 RE-REP
⁎⁎ ⁎⁎ ⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
12 h
24 h
48 h
(a)
0
DMEM
DMSO
18.25
37.5
75
150
300
500
20
40
60
80
100
Inhibition rate on MDCK cells (%)
Concentration (𝜇M)
⁎⁎
⁎⁎
⁎⁎
⁎⁎ ⁎⁎
⁎⁎
⁎⁎
DEA
DEAX
(b)
Figure 1: The influence of the RG-REP decoctions and the compounds on inhibition rate of MDCK cells. (a) Shows the inhibition rates of
decoctions at the representative concentration of 1.0 mg/mL; (b) shows the inhibition rates of MDCK cells treated with two compounds at
a series of concentrations for 24 h. All data are presented as means ± SD,n=3.∗0:01 < P<0:05 and ∗∗ P<0:01, compared with the
DMEM control group;
#
0:01 < P<0:05 and
##
P<0:01, compared with the REP group.
5Oxidative Medicine and Cellular Longevity
100 𝜇m
(a)
100 𝜇m
(b)
100 𝜇m
(c)
100 𝜇m
(d)
100 𝜇m
(e)
100 𝜇m
(f)
Figure 2: The influence of the RG-REP decoctions on morphology of MDCK cells (100×). (a–f) Show the morphology of MDCK cells under a
light microscope after MDCK cells were treated with DMEM, RG, REP, 1 : 1 RG-REP, 2 : 1 RG-REP, and 3 : 1 RG-REP at 1.0 mg/mL for 48 h.
DMEM
Hoechst 3342PIRhodamine 123Merge
RG REP 1: 1 RG-REP 2: 1 RG-REP 3: 1 RG-REP
Figure 3: The effects of RG-REP decoctions on cell death of MDCK cells (200×). Representative images were obtained using an HCA coupled
with Hoechst 33342/PI/Rhodamine 123 staining assay. VC: viable cell; AB: apoptotic body; NM: normal mitochondrion; MMPD: MMP
disruption.
6 Oxidative Medicine and Cellular Longevity
intensity of the 2 : 1 RG-REP decoction group was betwe en
those of the 1 : 1 and 3 : 1 RG- REP decoction groups. The
above results demonstrated that the accumulation of ROS
played an important role in the apoptosis of MDCK cells
induced by the 1 : 1 RG-REP decoction.
ROS has been reported to play an essential role in cell
cycle progression [22], which led us to investigate whether
each RG-REP decoction treatment affects the cell cycle using
a cell cycle kit. The results showed that the percentage of cells
at the G0/G1 phase in all groups increased in a dose-
dependent manner after being exposed to RG-REP decoc-
tions for 48 h. In the 1 : 1 RG-REP decoction group, it
was 67.68, 72.64, and 82.52% at the concentrations of 0.5,
1.0, and 2.0 mg/mL, respectively, although no significant dif-
ference was observed compared to the REP decoction group.
The cell population at the S phase was subsequently contin-
ued with a decrease although the difference was not signifi-
cant (Figure 8).
3.3. The Contents of DEAX and DEA in 1 : 1 RG-REP
Decoction Were Higher than Those in REP Decoction and
Decreased along with the Increment of RG. The HPLC
0
40
80
120
DMEM RG
0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0
REP 1:1 RG-REP 2:1 RG-REP 3:1 RG-REP
##
##
##
#
#
#
##
##
##
##
##
##
#
##
##
##
#
Concentration (mg/mL)
Cell distribution (%)
⁎⁎ ⁎⁎
⁎⁎
⁎⁎ ⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎
⁎
⁎⁎
⁎⁎
⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎
⁎⁎
⁎⁎
⁎
⁎⁎
⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎
⁎⁎
⁎⁎
Live
Total apoptotic
Necrotic
(a)
0
40
80
120
DMSO DEAX
150 300 500 65 130 260
DEA
Cell distribution (%)
⁎⁎
⁎⁎
⁎⁎
⁎⁎ ⁎⁎
⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
Concentration (𝜇M)
Early apoptotic
Late apoptotic
Necrotic
(b)
Figure 4: Quantitative analysis of the percentages of living, apoptotic, and necrotic cells. (a) Shows the distribution of MDCK cells incubated
with various concentrations of each RG-REP decoction for 48 h; (b) shows the distribution of MDCK cells incubated with various
concentrations of DEAX and DEA for 24 h. Data are presented as means ± SD,n=3.∗0:01 < P<0:05 and ∗∗P<0:01, compared with the
DMEM (or vehicle) control group;
#
0:01 < P<0:05 and
##
P<0:01, compared with the REP group.
7Oxidative Medicine and Cellular Longevity
fingerprints obtained with individual decoctions and com-
bined RG-REP decoctions were compared to identify the
compounds responsible for the cytotoxicity. Two com-
pounds corresponding to peaks 3 and 4 in Figures 9 and
10, which were from REP and identified as DEAX and
DEA, were found to be quantitatively different between the
REP decoction and the three combined decoctions. The con-
tents of these two compounds in the 1 : 1 RG-REP decoction
were higher than those in the REP decoction, and they
decreased along with the increment of RG. That is to say,
the contents of DEAX and DEA were the highest in the
1 : 1 RG-REP decoction and then successively decreased in
the 2 : 1 and 3 : 1 RG-REP decoctions (Figure 11). The con-
tent variation tendency of these two compounds in the
0
1
2
3
4
01234
Dead
1.70%
Apoptotic/dead
6.80%
91.20%
Live
0.30%
Apopt otic
Viabi lit y
Caspase-3/7
(a)
Dead
3.45%
Apoptotic/dead
3.80%
92.05%
Live
0.70%
Apopt otic
0
1
2
3
4
Viabi lit y
01234
Caspase-3/7
(b)
Dead
2.70%
Apoptotic/dead
15.10%
76.90%
Live
5.30%
Apopt otic
0
1
2
3
4
Viabi lit y
01234
Caspase-3/7
(c)
Dead
0.55%
Apoptotic/dead
16.65%
52.05%
Live
30.75%
Apopt otic
0
1
2
3
4
Viabi lit y
01234
Caspase-3/7
(d)
Dead
0.70%
Apoptotic/dead
17.80%
64.90%
Live
16.60%
Apopt otic
0
1
2
3
4
Viabi lit y
01234
Caspase-3/7
(e)
Dead
0.55%
Apoptotic/dead
16.80%
73.80%
Live
8.85%
Apopt otic
0
1
2
3
4
Viabi lit y
01234
Caspase-3/7
(f)
Dead
6.11%
Apoptotic/dead
0.31%
90.44%
Live
3.14%
Apopt otic
103104105
FITC
106
Viabi lit y
102
103
104
105
(g)
Dead
22.62%
Apoptotic/dead
5.62%
66.12%
Live
5.64%
Apopt otic
103104105
FITC
106
Viabi lit y
102
103
104
105
(h)
Dead
7.87%
Apoptotic/dead
3.77%
67.89%
Live
20.47%
Apopt otic
Viabi lit y
103
102
103
104
105
104105
FITC
106
(i)
Figure 5: Representative flow-cytometry scatter plot of MDCK cells. (a–f) Show the percentages of alive (left lower quadrant), early apoptotic
(right lower quadrant), late apoptotic (right upper quadrant), and dead (left upper quadrant) cells after they were treated with DMEM, RG,
REP, 1 :1 RG-REP, 2 :1 RG-REP, and 3 :1 RG-REP at 1.0 mg/mL for 48h. (g–i) show the percentages of the 4 types of cells mentioned above
after being treated with 1.0% DMSO, 130 μM DEA, and 300 μM DEAX for 24 h.
8 Oxidative Medicine and Cellular Longevity
0
12500
25000
37500
50000
#
#
Hoechst 33342
Average flourescence intensity
DMEM
RG
REP
1: 1 RE-REP
2: 1 RE-REP
3: 1 RE-REP
0.5 mg/mL
1.0 mg/mL
2.0 mg/mL
⁎⁎
(a)
0
15000
30000
45000
60000
#
#
Rhodamine 123
Average flourescence intensity
DMEM
RG
REP
1: 1 RE-REP
2: 1 RE-REP
3: 1 RE-REP
0.5 mg/mL
1.0 mg/mL
2.0 mg/mL
⁎⁎
⁎⁎
⁎⁎
⁎
(b)
0
20000
40000
60000
80000
##
##
##
##
PI
Average flourescence intensity
DMEM
RG
REP
1: 1 RE-REP
2: 1 RE-REP
3: 1 RE-REP
0.5 mg/mL
1.0 mg/mL
2.0 mg/mL
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎
⁎
(c)
Hoechst 33342
150 300
DMSO DEAX DEA
Concentration (𝜇M)
500 65 130 260
Rhodamine 123
PI
0
1000
2000
3000
4000
Average flourescence intensity
⁎
⁎⁎⁎
⁎⁎ ⁎⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎
(d)
Figure 6: The influence of RG-REP decoctions and compounds on fluorescence intensity of Hoechst 33342/Rhodamine 123/PI. (a) Shows
the influence of each RG-REP decoction on the fluorescence intensity of Hoechst 33342; (b) shows the influence of each RG-REP decoction
on the fluorescence intensity of Rhodamine 123; (c) shows the influence of each RG-REP decoction on the fluorescence intensity of PI; (d)
shows the influence of DEAX and DEA on the fluorescence intensity of Hoechst 33342, Rhodamine 123, and PI. The results are presented as
means ± SD,n=3.∗0:01 < P<0:05 and ∗∗ P<0:01, compared with the DMEM (or vehicle) control group;
#
0:01 < P<0:05 and
##
P<0:01,
compared with the REP group.
9Oxidative Medicine and Cellular Longevity
experimental groups was consistent with that of the cyto-
toxicity. This implied that the increased cytotoxicity of
the 1 : 1 RG-REP decoction might be attributed to these
two compounds.
3.4. Effect of DEAX and DEA on Cellular Morphology,
Apoptosis, MMP Disruption, and ROS Generation. The piv-
otal role of DEAX and DEA in the increased cytotoxicity of
the 1 : 1 RG-REP decoction was confirmed by the tests of cell
inhibition rate, cellular morphological change, apoptosis,
MMP, and ROS generation.
As shown in Figure 1(b), DEAX and DEA significantly
inhibited MDCK cells in a concentration-dependent manner.
Compared with the vehicle control (DMSO) group, the
inhibitory effects were significantly increased after treatment
with 37.5 μM DEAX or 18.25 μM DEA. Besides, MDCK cells
were more sensitive to DEA than to DEAX at the same con-
centration. DEAX and DEA hereby induced cytotoxicity to
MDCK cells with the TC
50
values of approximately 297.4
and 128.3 μM, respectively.
The micromorphology of MDCK cells after treatment
with 250 μM DEAX and 150 μM DEA for 24 h (Figure 12)
0
1500000
3000000
4500000
6000000
Average flourescence intensity
0.5 mg/mL
1.0 mg/mL
2.0 mg/mL
DMEM
RG
REP
1: 1 RE-REP
2: 1 RE-REP
3: 1 RE-REP
⁎⁎
⁎⁎ ⁎⁎
⁎⁎
⁎⁎ ⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎
⁎⁎
(a)
0
5
10
15
DMSO DEAX
150 300 500 150 300 500
DEA
ROS induction (folds of control)
Concentration (𝜇M)
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
⁎⁎
(b)
0
50
100
150
200
Counts
102103104
FITC
105106
Non-stained
DMSO
DEA 65
𝜇
M
DEA 130
𝜇
M
DEA 260
𝜇
M
(c)
0
50
100
150
200
Counts
103103104
FITC
105106
Non-stained
DMSO
DEA 65
𝜇
M
DEA 130
𝜇
M
DEA 260
𝜇
M
(d)
Figure 7: The effects of RG-REP decoctions and compounds on ROS generation. (a) Shows the influence of decoctions on fluorescence
intensity of DCF; (b) shows the influence of DEAX and DEA on ROS accumulation; (c, d) show the histograms of the fluorescence
intensity of DCF after treatment with DEAX and DEA for 24 h. The results are presented as means ± SD,n=3.∗0:01 < P<0:05 and
∗∗P<0:01, compared with the DMEM (or vehicle) control group.
10 Oxidative Medicine and Cellular Longevity
0%
40%
80%
120%
Cell cycle distribution
Concentration (mg/mL)
G0/G1
S
G2/M
DMEM RG
0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0
REP 1: 1 RE-REP 2: 1 RE-REP 3: 1 RE-REP
Figure 8: The effects of RG-REP decoctions on cell cycle distribution.
AU
0.000
0.010
0.020
0.030
0.040
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 50.0045.00 60.0055.00
5
678
910 11
12
13 14
15
16 1
217
3
18
19
420
Min
(a)
AU
0.000
0.002
0.004
0.006
0.008
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 50.0045.00 60.0055.00
9
10
11
12 15
1
2
17
18
19
20
Min
(b)
AU
0.000
0.010
0.020
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 50.0045.00 60.0055.00
5
67
813
14
16
3
4
Min
(c)
Figure 9: Comparison of HPLC fingerprints obtained with decoctions. (a) Shows the fingerprint of 1 : 1 RG-REP decoction with 20
characteristic peaks; (b) shows the fingerprint of RG decoction with 11 peaks out of the above 20 peaks; (c) shows the fingerprint of REP
decoction with 9 peaks out of the above 20 peaks. Peaks 3 and 4 were identified as DEAX and DEA, respectively.
11Oxidative Medicine and Cellular Longevity
was consistent with that of the cytotoxicity (Figure 1(b)). The
cells in the DMSO control group grew into a confluence
monolayer and exhibited a normal spindle shape. However,
the cells in the DEAX and DEA groups displayed obvious
damages, such as cytoplasm shrinkage, membrane blebbing,
and cell-cell separation increment. The damages in these
two groups were similar to those induced by cisplatin, a
nephrotoxic positive drug, at the concentration of 60 μM.
The cellular morphology observed by HCA also showed that
an increased number of cells with apoptotic bodies (AB) and
disrupted MMP were observed after treatment with DEAX or
DEA, while the number of viable cells (VC) was significantly
less than that in the DMSO control group (Figure 13).
The exact percentage of apoptotic cells was deter-
mined by Annexin V-FITC/PI labelling assay. As shown in
Figures 4(b) and 5, the percentage of the apoptotic cells in
the DMSO control group was 3:43 ± 0:07%, whereas it chan-
ged to 2:88 ± 0:33,24:89 ± 0:76, and 60:22 ± 0:64%, respec-
tively, after treatment with 150, 300, and 500 μM DEAX for
24 h. These data corresponded with 0.35-, 2.99-, and 5.16-
folds of those of necrotic cells. As for DEA, exposing MDCK
cells to 130 and 260 μM for 24 h obviously increased the per-
centages of apoptotic and necrotic cells compared with the
DMSO control group. What is more, the percentages of
necrotic cells after treatment with 65, 130, and 260 μM
DEA for 24 h corresponded with 4.32-, 2.10-, and 3.77-folds
of those of apoptotic cells, respectively.
As shown in Figure 6(d), the Rhodamine 123 fluorescence
intensity of cells treated with 150, 300, and 500 μM DEAX
corresponded with 80.29, 77.39, and 71.71% of those of cells
in the DMSO control group. Similarly, the fluorescence
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 50.0045.00 60.0055.00
0.000
0.030
0.020
0.010
0.040
AU
DEAX
DEA
Min
(a)
0.000
0.005
0.010
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 50.0045.00 60.0055.00
DEAX DEA
AU
Min
(b)
0.000
0.010
0.020
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 50.0045.00 60.0055.00
DEAX DEA
AU
Min
(c)
Figure 10: Quantitative comparison of DEAX and DEA in RG-REP decoctions by HPLC. (a–c) Show the peaks of DEAX and DEA in HPLC
fingerprints obtained with an equal quantity of 1 : 1, 2 : 1, and 3 : 1 RG-REP decoctions, respectively.
0
5
10
15
20
Contents of DEAX and DEA (10–3 𝜇g)
DEAX
DEA
REP
1: 1 RE-REP
2: 1 RE-REP
3: 1 RE-REP
⁎⁎
⁎⁎
⁎
⁎
Figure 11: The contents of DEAX and DEA in RG-REP decoctions.
The results are presented as means ± SD,n=3.∗0:01 < P<0:05 and
∗∗P<0:01, compared with the REP group.
12 Oxidative Medicine and Cellular Longevity
intensity of cells treated with 65, 130, and 260 μM DEA cor-
responded with 89.8, 71.6, and 68.4% of that of cells in the
DMSO control group, respectively. These results implied that
DEAX and DEA inducing cytotoxicity was partially due to
the MMP disruption in MDCK cells.
As shown in Figures 7(b)–7(d), the DCF fluorescence
intensity significantly increased in a dose-dependent manner
after adding DEAX or DEA to the medium. The DCF fluores-
cence intensity of the cells exposed to DEAX was approxi-
mately 9.15-12.1 times of that of the cells in the DMSO
control group. Similarly, the amount of ROS produced in
the DEA treatment group was 3.06–5.66 times of that of the
DMSO control group. These data indicated that oxidative
stress was an important pathway leading to MDCK cell apo-
ptosis induced by DEAX or DEA.
4. Discussion
Out of concerns for the safety and efficacy of TCM, great
efforts have been made to reveal the mechanisms behind
the herbal drug-drug interaction [8, 23]. In this study, the
cytotoxicity of both individual decoctions and three com-
bined decoctions of RG and REP was systematically com-
pared and the toxic components were traced, so as to
elucidate the mechanism of RG-REP incompatibility and
the possible toxic substances. Our results demonstrated that
RG-REP incompatibility was conditionally established due
to the RG dose-dependent REP toxicity change which was
evidenced by MTT test, morphological observation, and cas-
pase-3/7 assay. The toxicity of REP could be bidirectionally
regulated by RG, and the combination ratio of both drugs
was the determinant for incompatibility/compatibility. On
the one hand, RG can unusually increase the toxicity of
REP by inhibiting cell growth, deteriorating cell health status,
and promoting cell apoptosis when it is used in nearly equal
quantity with the latter and thereby justifies the incompati-
bility. On the other hand, the toxicity of REP can be attenu-
ated by the further increased quantity of RG which supports
the usual detoxification effect of RG and thus underlies the
compatibility. Previous studies have not come to an agree-
ment on the toxicity change resulted by concomitant use
of RG and REP. Some researchers have proven that the
toxicity of REP could be strengthened by RG [18, 24,
25], while others obtained the contrary conclusion based
on their experiments [26–28]. Still, others have concluded
that RG had no influence on the toxicity of REP [29, 30].
Although this issue remains unsettled, our research pro-
vided substantial evidence that RG plays a two-way regula-
tory effect on the toxicity of the RG-REP herb pair. The
similar bidirectional regulation of RG was also observed
when it was coused with another EIM associated herb, Flos
Genkwa [31].
Someone may wonder why both incompatibility and
compatibility simultaneously occurred when RG and REP
were used together, i.e., 1 :1 combination increased the toxic-
ity while 2 : 1 and 3 : 1 combinations decreased toxicity. As
usual, changes of components and toxicity induced by
drug-drug interaction should be considered. To address this
issue, we first compared the toxicity of the mixtures of indi-
vidually decocted RG and REP at the ratios of 1 : 1, 2 : 1,
100 𝜇m
(a)
100 𝜇m
(b)
100 𝜇m
(c)
100 𝜇m
(d)
Figure 12: The influence of DEAX and DEA on morphology of MDCK cells (100×). (a–d) Show the morphology of MDCK cells under a light
microscope after they were treated with 1.0% DMSO, 60 μM cisplatin, 130 μM DEA, and 300 μM DEAX for 24 h, respectively.
13Oxidative Medicine and Cellular Longevity
and 3 : 1, respectively. The results (data were not shown)
showed that the inhibition rates of the above three mixtures
on MDCK cells had no significant difference compared
with that of the REP decoction. This indicated that the
key compound interaction from RG-REP may occur during
the decoction process. Many researchers have found that
when RG was decocted together with other herbs, natural
compounds interacted with each other and led to their
increased/decreased solubility and even generation of new
substances accordingly [32–34]. This in turn increased/de-
creased the content and bioavailability of one or several sub-
stances and thus resulted in a synergistic/antagonistic effect
[35, 36]. According to this theory, we compared the HPLC
fingerprints of individual decoctions and combined decoc-
tions to evaluate the composition change during the decoc-
tion preparation for the purpose of tracing toxic substances
responsible for RG-REP incompatibility. As a result, the con-
tents of DEA and DEAX in each decoction changed consis-
tently with their toxic effect on MDCK cells. Therefore, RG
should have an impact on the dissolution of both com-
pounds, which may contribute to the toxicity change during
RG-REP concomitant use.
To confirm that DEAX and DEA are definitely responsi-
ble for the toxicity change ascribed to RG-REP concomitant
use, we performed validation experiments with these two
compounds. The results showed that the compounds signifi-
cantly decreased cell viability in a concentration-dependent
manner and induced the cellular morphological changes
described above. All of these were in agreement with the tox-
icity change of RG-REP decoctions, and thus, the roles of both
compounds were confirmed. Literature [37] also reported
that DEA could inhibit the proliferation of HepG2 cells,
which provided a strong support for our conclusion.
The next question is how the toxicity was induced by the
1 : 1 RG-REP decoction, DEAX, and DEA. To this end, the
percentages of living, apoptotic, and necrotic cells after treat-
ment with the decoctions or compounds were analyzed. The
results evidenced that apoptosis was the primary cell death
manner induced by these substances. It has been reported
that ROS is an important response to cellular injury and
DMEM
Hoechst 33342PIRhodamine 123Merge
Positive drug DEA DEAX
Figure 13: The effects of the compounds on cell death of MDCK cells (200×). Representative images were obtained using an HCA coupled
with Hoechst 33342/PI/Rhodamine 123 staining assay. VC: viable cell; AB: apoptotic body; NM: normal mitochondrion; MMPD: MMP
disruption.
14 Oxidative Medicine and Cellular Longevity
apoptotic cell death [22]. Excessive ROS impaired the integ-
rity of the mitochondrial membrane, which subsequently
caused MMP disruption, and eventually led to apoptosis
through the mitochondrial apoptotic pathway [38]. Our
results showed that the 1 : 1 RG-REP decoction, DEAX, and
DEA resulted in remarkably decreased MMP (Figure 6),
which was associated with the dysfunctional mitochondria.
This tendency was positively correlated with cytotoxicity
and ROS generation (Figure 7). What is more, ROS alterna-
tively influences several cellular processes including cell
cycle, cell differentiation, and apoptosis. However, this effect
is a specific procedure depending on the amount and dura-
tion of ROS exposure [39]. In this study, we observed the
invisible increment of the G0/G1 phase in the 1 : 1 RG-REP
decoction group compared with the REP decoction group
(Figure 8). It demonstrated that the cell cycle arrest might
not play a key role in the apoptosis induced by RG-REP
concomitant use. Thus, the ROS accumulation thereby
induced was only sufficient for cell apoptosis increment,
but insufficient for G0/G1 cell cycle arrest. Furthermore,
ROS-mediated mitochondrial dysfunction could be associ-
ated with the increased cell apoptosis and eventually
resulted in toxicity to MDCK cells. Previous studies also
indicated that mitochondrial dysfunction caused by ROS
overproduction is the main reason for nephrotoxicity
induced by some drugs [40, 41].
5. Conclusions
The incompatibility of RG-REP concomitant use is condi-
tionally established because of the bidirectional regulatory
effect of RG. The major compounds responsible for RG-
REP incompatibility are DEAX and DEA, which results in
toxicity through activation of mitochondria-dependent apo-
ptosis induced by the increased ROS production.
Data Availability
The experimental data used to support the findings of this
study are available from the corresponding author upon
request.
Conflicts of Interest
The authors declare that there is no conflict of interests
regarding the publication of this paper.
Authors’Contributions
RFW, MC, and MD conceived and designed the experiments.
MC, DG, and XY performed the experiments. XXL, YSP, and
XHH analyzed the data. MC and RFW drafted the manu-
script. MC, RFW, PMD, SQL, and XHH performed the revi-
sion and checking. All authors read and approved the final
manuscript.
Acknowledgments
This work was supported by the National Major Scientific
and Technological Special Project for “Significant New Drugs
Development”2019ZX09201008-002.
Supplementary Materials
Supplementary Figure 1: the influence of the RG-REP decoc-
tions on the inhibition rate of MDCK cells. (a–c) show the
inhibition rates of RG, REP, 0.5 : 1 RG-REP, 0.75 : 1 RG-
REP, 1 : 1 RG-REP, 2 : 1 RG-REP, and 3 : 1 RG-REP on
MDCK cells at a series of concentrations at 12, 24, and
48 h, respectively. All data are presented as means ± SD,
n=3.∗0:01 < P<0:05 and ∗∗P<0:01, compared with the
REP group. (Supplementary Materials)
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