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In Vitro Nephrotoxicity Induced by Herb-Herb Interaction between Radix Glycyrrhizae and Radix Euphorbiae Pekinensis

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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.
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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) ngerprint 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 signicantly 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 eect 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 ShennongsHerbal Classicthe
extant earliest classic book of Traditional Chinese Medicine
(TCM), which abstractly proposed this concept. Subse-
quently, a book entitled Variorum of Shennongs 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 [13]. 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 scientic 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 ocially
recorded in Chinese pharmacopoeia [6] for the treatment
of oedema, malignant ascites, and urinary retention. In the
clinic, REP exhibits explicit therapeutic eects on relieving
constipation, purging uid, and eliminating phlegm [7, 8].
Modern pharmacological investigations also demonstrated
that its extract exhibited a variety of biological eects, includ-
ing antineoplastic, antiviral, and analgesic activities [911].
RG, which comes from the dried roots of either Glycyrrhiza
uralensis Fisch. or G. glabra L. or G. inate 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 ecacy 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 [1316]. 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 eects on the tissues of
the kidney, heart, and liver of experimental animals than sep-
arate use and leads to signicantly 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
inltration in the renal and hepatic tissues of rats, indicating
some toxic and side eects [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 identication
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).
Dulbeccosmodied Eagles 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 asks, 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-
dihydrouorescein diacetate (DCFH-DA), propidium iodide
(PI), ethylenediaminetetraacetic acid (EDTA), and Annexin
V-uorescein 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 purication 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,
ltered, 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 nal 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=ð1experimental groupsA/control
groupsAÞ× 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 dierent elds 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. Briey,
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 rst 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 buer 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 uorescence 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
uorescence intensity were monitored by HCA coupled with
Hoechst 33342/PI/Rhodamine 123 staining assay. The cells
were rst 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 xed 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 elds per well were taken to
cover the entire well.
2.9. HPLC Analysis. The HPLC ngerprints 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 nal concentration of 0.5 mg
of REP per mL and ltered through a 0.22 μm membrane
lter. 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 ow rate of 1.0 mL/min using acetoni-
trile as mobile phase A and 1.0% acetic acid in puried
water as mobile phase B with gradient elution program
including 28% A (06min), 813% A (621 min), 1320%
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(2130 min), 2035% A (3040 min), 3545% A (40
50 min), 4575% A (5060 min), 7590% A (6065 min),
and 90100% A (6570 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 coecient 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, 0.0020020 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 signicance was evaluated by Studentst-test
for comparison of the mean values. P<0:05 was selected as
the criteria for statistical signicance.
3. Results
3.1. The Cytotoxicity of REP Was Increased when Coused with
RG at the Ratio of 1 : 1, and This Eect 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 signicant dierence 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 signi-
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
signicant protective eect 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 eect of
each RG-REP decoction on MDCK cells was conrmed 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 conrmed the cyto-
toxic eect 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 rmly 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 ow 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 signicant cell
apoptosis. In contrast, treatment with REP or three combined
RG-REP decoctions signicantly 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 signicant 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 7.92102.7 times as many
as the percentage of necrotic cells. These results conrmed
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 uorescence
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 uores-
cence intensity of Rhodamine 123, which is a good indica-
tor for MMP, was not changed signicantly 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
signicant lower uorescence intensity compared with that
of the DMEM control group. In addition, the uorescence
intensity signicantly 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 aect the uorescence
intensity of 2,7-dichlorouorescein (DCF), which is usually
used to reect 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 5.058.68-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 signicantly sup-
pressed in the 3 : 1 RG-REP decoction group compared with
that of the REP decoction group. The DCF uorescence
–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 inuence 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 inuence of the RG-REP decoctions on morphology of MDCK cells (100×). (af) 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 eects 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 aects 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 signicant 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 dierence was not signi-
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
ngerprints 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 identied as DEAX and
DEA, were found to be quantitatively dierent 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 ow-cytometry scatter plot of MDCK cells. (af) 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. (gi) 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 inuence of RG-REP decoctions and compounds on uorescence intensity of Hoechst 33342/Rhodamine 123/PI. (a) Shows
the inuence of each RG-REP decoction on the uorescence intensity of Hoechst 33342; (b) shows the inuence of each RG-REP decoction
on the uorescence intensity of Rhodamine 123; (c) shows the inuence of each RG-REP decoction on the uorescence intensity of PI; (d)
shows the inuence of DEAX and DEA on the uorescence 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. Eect 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 conrmed by the tests of cell
inhibition rate, cellular morphological change, apoptosis,
MMP, and ROS generation.
As shown in Figure 1(b), DEAX and DEA signicantly
inhibited MDCK cells in a concentration-dependent manner.
Compared with the vehicle control (DMSO) group, the
inhibitory eects were signicantly 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 eects of RG-REP decoctions and compounds on ROS generation. (a) Shows the inuence of decoctions on uorescence
intensity of DCF; (b) shows the inuence of DEAX and DEA on ROS accumulation; (c, d) show the histograms of the uorescence
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 eects 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 ngerprints obtained with decoctions. (a) Shows the ngerprint of 1 : 1 RG-REP decoction with 20
characteristic peaks; (b) shows the ngerprint of RG decoction with 11 peaks out of the above 20 peaks; (c) shows the ngerprint of REP
decoction with 9 peaks out of the above 20 peaks. Peaks 3 and 4 were identied 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 conuence
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 signicantly
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 uorescence
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 uorescence
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. (ac) Show the peaks of DEAX and DEA in HPLC
ngerprints 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 uorescence
intensity signicantly increased in a dose-dependent manner
after adding DEAX or DEA to the medium. The DCF uores-
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 3.065.66 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 ecacy of TCM, great
eorts 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 justies 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 detoxication eect 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 [2628]. Still, others have concluded
that RG had no inuence 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 eect 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 rst 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 inuence of DEAX and DEA on morphology of MDCK cells (100×). (ad) 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 signicant dierence 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 [3234]. This in turn increased/de-
creased the content and bioavailability of one or several sub-
stances and thus resulted in a synergistic/antagonistic eect
[35, 36]. According to this theory, we compared the HPLC
ngerprints 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 eect 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 conrm that DEAX and DEA are denitely 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 signi-
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 conrmed. 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 eects 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 inuences several cellular processes including cell
cycle, cell dierentiation, and apoptosis. However, this eect
is a specic 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 sucient for cell apoptosis increment,
but insucient 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
eect 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 ndings of this
study are available from the corresponding author upon
request.
Conflicts of Interest
The authors declare that there is no conict of interests
regarding the publication of this paper.
AuthorsContributions
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 nal
manuscript.
Acknowledgments
This work was supported by the National Major Scientic
and Technological Special Project for Signicant New Drugs
Development2019ZX09201008-002.
Supplementary Materials
Supplementary Figure 1: the inuence of the RG-REP decoc-
tions on the inhibition rate of MDCK cells. (ac) 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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... Various types of cells, such as immune cells (macrophages, T cells, neutrophils), nucleated cells (red blood cells and platelets), stem cells, have been used as carriers for anticancer drugs. [4][5][6][7][8][9] Therefore, drug delivery combined with cellular immunotherapy may lead to better outcomes in cancer treatment. ...
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Background The immunotherapeutic approach utilizing Natural Killer (NK) cells for cancer treatment has garnered significant interest owing to its inherent cytotoxicity, immunomodulatory properties, demonstrated safety in in vivo studies. However, multiple immunosuppressive mechanisms in the tumor microenvironment (TME) suppress the anticancer effect of NK cells in the treatment of solid tumors. Herein, a smart NK cell drug delivery system (DDS) with photo-responsive and TME-responsive properties was designed. Methods The NK cell DDS consists of two parts: the carrier is living NK cell with pH-low (abbreviated as NKpH) insertion peptide on its surface, the cargo is reductive-responsive nanogel (NG) encapsulated siRNA and photosensitizer (abbreviated as SP-NG), the final carrier was abbreviated as SP-NG@ NKpH. Firstly, pHLip helped artificially modified NK cell target and anchor onto cancer and exert the efficacy of cellular immunotherapy. Then, the strategy of combining photoactivation and bioreduction responsiveness achieved the precise release of cargos in cancer cells. Finally, the DDS combined the effect of the immunotherapy of NK cell, the gene therapy of siRNA, and the photodynamic therapy of photosensitizer. Results Under near-infrared laser irradiation, SP-NG@NKpH induced an increase in reactive oxygen species (ROS) within cells, exacerbated cell membrane permeability, and allowed for rapid drug release. Within the tumor microenvironment (TME), NG exhibits highly sensitive reducibility for drug release. The SP-NG released from NK cells can be uptaken by tumor cells. When exposed to near-infrared laser irradiation, SP-NG@NKpH demonstrates significant tumor-targeting specificity and cytotoxicity. Discussion The combined effect of the immunotherapy of NK cell, the gene therapy of siRNA, and the photodynamic therapy of photosensitizer obtained a stronger cancer killing effect in vitro and in vivo. Therefore, this versatile NK cell DDS exhibits a good clinical application prospect.
... Third, herbal interactions are currently not considered in the CIPS software. While there are indeed several studies reporting on interactions between certain drugs [32,33], the data are not sufficient for a full-scale integration. Our team will upgrade CIPS in the future by integrating more data resources as they become available. ...
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Background and objectives: Traditional Chinese Medicine (TCM) is a therapeutic system which has been practiced for thousands of years. Although for much of its history the decoction of medicinal herbs was the most common method of consuming the herbal treatments, TCM prescriptions are now primarily prepared using concentrated Chinese herbal extracts (CCHE) in powder or granular form. However, determining the precise dose of each single Chinese herbal constituent within a prescription creates a challenge in clinical practice due to the potential risk of toxicity. To alleviate this, we invented the Chinese Intelligence Prescription System (CIPS) to calculate the exact dose of each single herb within an individual prescription. Methods: In this study, we applied CIPS in a real-world setting to analyze clinical prescriptions collected and prepared at the TCM Pharmacy of China Medical University Hospital (CMUH). Results: Our investigation revealed that 3% of all prescriptions filled in a 1-month period contained inexact dosages, suggesting that more than 170,000 prescriptions filled in Taiwan in a given month may contain potentially toxic components. We further analyzed the data to determine the excess dosages and outline the possible associated side effects. Conclusions: In conclusion, CIPS offers TCM practitioners the ability to prepare exact Chinese herbal medicine (CHM) prescriptions in order to avoid toxic effects, thereby ensuring patient safety.
... Second, our algorithm does not predict the potential effects of herbal combinations. However, studies have shown that interactions between herbs do exist [9][10][11][12][13][14], thus recombining herbs in different ways may alter the intended function. ...
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Background and Aim The traditional method of taking Chinese Medicine involves creating a decoction by cooking medicinal Chinese herbs. However, this method has become less popular, being replaced by the more convenient method of consuming concentrated Chinese herbal extracts, which creates challenges related to the complexity of stacking multiple formulas. Methods We developed the Chinese Intelligence Prescription System (CIPS) to simplify the prescription process. In this study, we used data from our institutions pharmacy to calculate the number of reductions, average dispensing time, and resulting cost savings. Results The mean number of prescriptions was reduced from 8.19 ± 3.65 to 7.37 ± 3.34 ( p=2.46  ×108p=2.46\;\times10^{-8} p = 2.46 × 10 - 8 ). The reduction in the number of prescriptions directly resulted in decreased dispensing time, reducing it from 1.79 ± 0.25 to 1.63 ± 0.66 min ( p=1.88  ×1014p=1.88\;\times10^{-14} p = 1.88 × 10 - 14 ). The reduced dispensing time totaled 3.75 h per month per pharmacist, equivalent to an annual labor cost savings of 15,488NTDperpharmacist.Inaddition,druglosswasreducedduringtheprescriptionprocess,withameansavingsof15,488 NTD per pharmacist. In addition, drug loss was reduced during the prescription process, with a mean savings of 4,517 NTD per year. The combined savings adds up to a not insignificant 20,005NTDperyearperpharmacist.WhentakingallTCMclinics/hospitalsinTaiwanintoaccount,thetotalannualsavingswouldbe20,005 NTD per year per pharmacist. When taking all TCM clinics/hospitals in Taiwan into account, the total annual savings would be 77 million NTD. Conclusion CIPS assists clinicians and pharmacists to formulate precise prescriptions in a clinical setting to simplify the dispensing process while reducing medical resource waste and labor costs.
... The quality uniformity and safety of Chinese medicines are still bottlenecks that currently limit the rapid development of the TCM industry [178][179][180]. Currently, there is a lack of toxicity data for many toxic herbal medicines, especially for multi-base source TCM, and there is confusion about the variety of herbs marketed. ...
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As an emerging sequencing technology, single-cell RNA sequencing (scRNA-Seq) has become a powerful tool for describing cell subpopulation classification and cell heterogeneity by achieving high-throughput and multidimensional analysis of individual cells and circumventing the shortcomings of traditional sequencing for detecting the average transcript level of cell populations. It has been applied to life science and medicine research fields such as tracking dynamic cell differentiation, revealing sensitive effector cells, and key molecular events of diseases. This review focuses on the recent technological innovations in scRNA-Seq, highlighting the latest research results with scRNA-Seq as the core technology in frontier research areas such as embryology, histology, oncology, and immunology. In addition, this review outlines the prospects for its innovative application in traditional Chinese medicine (TCM) research and discusses the key issues currently being addressed by scRNA-Seq and its great potential for exploring disease diagnostic targets and uncovering drug therapeutic targets in combination with multiomics technologies.
... Herb-herb interaction commonly occurs during the co-administration of different herbs, which would mimic, magnify, or oppose the effect of drugs (Chen et al. 2020). Therefore, herbherb investigations should be given special attention. ...
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Context Rhynchopylline and pellodendrine are major extractions of commonly used Chinese medicine in gynaecology. The interaction between these two compounds could affect treatment efficiency and even result in toxicity during their co-administration in gynaecological prescription. Objective The pharmacokinetic interaction between rhynchopylline and pellodendrine and the potential mechanism were investigated in this study. Materials and methods Sprague-Dawley rats were randomly divided into four groups to investigate the pharmacokinetic interaction between rhynchopylline (30 mg/kg) and pellodendrine (20 mg/kg) with single dose of these two drugs as the control. The transport of rhynchopylline was evaluated in the Caco-2 cell model. Additionally, the metabolic stability and the activity of corresponding CYP450 enzymes were assessed in rat liver microsomes. Results The pharmacokinetic profile of rhynchopylline was dramatically affected by pellodendrine with the increased area under the pharmacokinetic curve (3080.14 ± 454.54 vs. 1728.08 ± 220.598 μg/L*h), Cmax (395.1 ± 18.58 vs. 249.1 ± 16.20 μg/L), prolonged t1/2 (9.74 ± 2.94 vs. 4.81 ± 0.42 h) and the reduced clearance rate (from 11.39 ± 1.37 to 5.67 ± 1.42 L/h/kg). No significant changes were observed in the pharmacokinetics of pellodendrine. The transport of rhynchopylline was significantly inhibited by pellodendrine with a decreasing efflux ratio (1.43 vs. 1.79). Pellodendrine significantly inhibited the activity of CYP1A2 and CYP2C9 with IC50 values of 22.99 and 16.23 μM, which are critical enzymes responsible for the metabolism of rhynchopylline. Discussion and conclusions The adverse interaction between rhynchopylline and pellodendrine draws attention to the co-administration of these two herbs and provides a reference for further investigations with a broader study population.
... In vitro study, casbane diterpenoids from EP are toxic to many cell lines, such as LO-2, 7 IEC-6 8 and MDCK, 9 they can induce cell apoptosis, cellular morphological change, ROS accumulation, and mitochondrial membrane potential (MMP) disruption. [7][8][9] In our previous study, we demonstrated the toxic effects of casbane diterpenoids in vivo, the possible mechanism is due to the disordered expression of aquaporin in intestinal tract caused by diterpenoids from EP, 5 ...
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Gut microbiota disorder will lead to intestinal damage. This study evaluated the influence of total diterpenoids extracted from Euphorbia pekinensis (TDEP) on gut microbiota and intestinal mucosal barrier after long-term administration, and the correlations between gut microbiota and intestinal mucosal barrier were analysed by Spearman correlation analysis. Mice were randomly divided to control group, TDEP groups (4, 8, 16 mg/kg), TDEP (16 mg/kg) + antibiotic group. Two weeks after intragastric administration, inflammatory factors (TNF-α, IL-6, IL-1β) and LPS in serum, short chain fatty acids (SCFAs) in feces were tested by Enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC), respectively. The expression of tight junction (TJ) protein in colon was measured by western blotting. Furthermore, the effects of TDEP on gut microbiota community in mice have been investigated by 16SrDNA high-throughput sequencing. The results showed TDEP significantly increased the levels of inflammatory factors in dose-dependent manners, and decreased the expression of TJ protein and SCFAs, and the composition of gut microbiota of mice in TDEP group was significantly different from that of control group. When antibiotics were added, the diversity of gut microbiota was significantly reduced, and the colon injury was more serious. Finally, through correlation analysis, we have found nine key bacteria (Barnesiella, Muribaculaceae_unclassified, Alloprevotella, Candidatus_Arthromitus, Enterorhabdus, Alistipes, Bilophila, Mucispirillum, Ruminiclostridium) that may be related to colon injury caused by TDEP. Taken together, the disturbance of gut microbiota caused by TDEP may aggravate the colon injury, and its possible mechanism may be related to the decrease of SCFAs in feces, disrupted the expression of TJ protein in colon and increasing the contents of inflammatory factors.
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Euphorbiae pekinensis Radix (EPR) is a traditional Chinese herb commonly used to treat edema, pleural effusion, and ascites. However, counterfeit and adulterated products often appear in the market because of the homonym phenomenon, similar appearance, and artificial forgery of Chinese herbs. This study comprehensively evaluated the quality of EPR using multiple methods. The DNA barcode technique was used to identify EPR, while the UPLC‐Q‐TOF‐MS technique was utilized to analyze the chemical composition of EPR. A total of 15 tannin and phenolic acid components were identified. Furthermore, UPLC fingerprints of EPR and its common counterfeit products were established, and unsupervised and supervised pattern recognition models were developed using these fingerprints. The backpropagation artificial neural network and counter‐propagation artificial neural network models accurately identified counterfeit and adulterated products, with a counterfeit ratio of more than 25%. Finally, the contents of the chemical markers 3,3′‐di‐O‐methyl ellagic acid‐4′‐O‐β‐D‐glucopyranoside, ellagic acid, 3,3′‐di‐O‐methyl ellagic acid‐4′‐O‐β‐ d ‐xylopyranoside, and 3,3′‐di‐O‐methyl ellagic acid were determined to range from 0.05% to 0.11%, 1.95% to 8.52%, 0.27% to 0.86%, and 0.10% to 0.42%, respectively. This proposed strategy offers a general procedure for identifying Chinese herbs and distinguishing between counterfeit and adulterated products.
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Objective The objective of this study was to decipher chemical interactions between Danshen and Danggui using liquid chromatography–mass spectrometry (LC-MS) and explore the mechanisms of Danshen–Danggui against stroke using network pharmacology and molecular docking. Materials and Methods First, the chemical compounds of Danshen–Danggui were profiled using ultra-high-performance liquid chromatography (HPLC)-quadrupole time-of-flight MS. Accurately characterized compounds in various proportions of Danshen–Danggui were quantified using HPLC combined with triple quadrupole electrospray tandem MS. Network pharmacology was used to uncover the essential mechanisms of action of Danshen–Danggui against stroke. Discovery Studio Software was used for the molecular docking verification of key active chemicals and stroke-related targets. Results A total of 53 compounds were characterized, and 22 accurately identified constituents (10 phenolic acids, 8 phthalides, and 4 tanshinones) were quantified in 15 proportions of Danshen–Danggui. The quantification results showed that Danggui significantly increased the dissolution of most phenolic acids (compounds from Danshen), whereas Danshen promoted the dissolution of most phthalides (compounds from Danggui). Overall, the combination of Danshen and Danggui at a 1:1 ratio resulted in the maximum total dissolution rate. Further network pharmacology and molecular docking results indicated that Danshen–Danggui exerted anti-stroke effects mainly by regulating inflammation-related (tumor necrosis factor, hypoxia-inducible factor, and toll-like receptor) signaling pathways, which ranked among the top three pathways based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Conclusion The chemical compounds in Danshen–Danggui could interact with each other to increase the dissolution of the most active compounds, which could provide a solid basis for uncovering the compatibility mechanisms of Danshen–Danggui and Danshen–Danggui-based formulae.
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Cryptotanshinone and ophiopogonin D are sourced from herbs with similar indications. It is necessary to evaluate their interaction to provide a reference for their clinical prescriptions. The co-administration of cryptotanshinone (30 and 60 mg/kg) and ophiopogonin D was carried out in Sprague-Dawley rats and the pharmacokinetics of cryptotanshinone were analyzed. The Caco-2 cells were employed to evaluate the transport of cryptotanshinone, and the metabolic stability was studied in the rat liver microsomes. Ophiopogonin D significantly increased the Cmax (from 5.56 ± 0.26 to 8.58 ± 0.71 μg/mL and from 15.99 ± 1.81 to 185.12 ± 1.43 μg/mL), half-life (21.72 ± 10.63 vs. 11.47 ± 3.62 h and 12.58 ± 5.97 vs. 8.75 ± 2.71 h) and decreased the clearance rate (0.697 ± 0.36 vs. 1.71 ± 0.15 L/h/kg) and (60 mg/kg and 0.101 ± 0.02 vs. 0.165 ± 0.05 L/h/kg) of cryptotanshinone. In vitro, ophiopogonin D significantly suppressed the transport of cryptotanshinone with the decreasing efflux rate and enhanced the metabolic stability with the reducing intrinsic clearance. The combination of cryptotanshinone and ophiopogonin D induced prolonged exposure and suppressed the transport of cryptotanshinone, which indicated the decreased bioavailability of cryptotanshinone.
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Euphorbia pekinensis (EP) is a commonly used Chinese medicine treating edema with potential hepatorenal toxicity. However, its toxic mechanism and prevention are remained to be explored. Oleanolic acid (OA) is a triterpene acid with potential hepatorenal protective activities. We investigated the protective effect and potential mechanism of OA on EP‐induced hepatorenal toxicity. In this study, rats were given total diterpenes from EP (TDEP, 16 mg/kg) combined with OA (10, 20, 40 mg/kg) by gavage for 4 weeks. The results showed that TDEP administration could lead to a 3–4‐fold increasement in hepatorenal biochemical parameters with histopathological injuries, while OA treatment could ameliorate them in a dose‐dependent manner. At microbial and metabolic levels, intestinal flora and host metabolism were perturbed after TDEP administration. The disturbance of bile acid metabolism was the most significant metabolic pathway, with secondary bile acids increasing while conjugated bile acids decreased. OA treatment can improve the disorder of intestinal flora and metabolic bile acid spectrum. Further correlation analysis screened out that Escherichia‐Shigella, Phascolarctobacterium, Acetatifactor, and Akkermansia were closely related to the bile acid metabolic disorder. In conclusion, oleanolic acid could prevent hepatorenal toxicity induced by EP by regulating bile acids metabolic disorder via intestinal flora improvement.
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Herbal medicines are commonly used as compound formulas in clinical practice to achieve optimal therapeutic effects. However, the combination mechanisms usually lack solid evidence. In this study, we report synergistic interactions through altering pharmacokinetics in Gegen-Qinlian Decoction (GQD), an anti-diabetic Chinese medicine formula. A multi-component pharmacokinetic study of GQD and the single herbs was conducted by simultaneously monitoring 42 major bioactive compounds (markers) in rats plasma using LC/MS/MS within 30 min. GQD could remarkably improve the plasma concentrations of berberine (BER) and other alkaloids in Huang-Lian by at least 30%, and glycyrrhizic acid (GLY) from Gan-Cao played a major role. A Caco-2 cell monolayer test indicated that GLY improved the permeability of BER by inhibiting P-glycoprotein. Although GLY alone did not show observable effects, the co-administration of GLY (ig, 50 or 80 mg/kg) could improve the anti-diabetic effects of berberine (ig, 50 mg/kg) in db/db mice in a dose-dependent manner. The blood glucose decreased by 46.9%, whereas the insulin level increased by 40.8% compared to the control group. This is one of the most systematic studies on the pharmacokinetics of Chinese medicine formulas, and the results demonstrate the significance of pharmacokinetic study in elucidating the combination mechanisms of compound formulas.
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As recorded in Traditional Chinese Medicine (TCM) theory, Gancao (Glycyrrhizae Radix et Rhizoma) could weaken the pharmacological effect or increase the toxicity of Yuanhua (Genkwa Flos). However, the theory has been suspected due to lack of evidence. Here, we investigate whether Gancao could weaken Yuanhua’s diuretic effect, if so, which chemicals and which targets may be involved. Results showed that Yuanhua exerted diuretic effect through down-regulating renal AQP 2, without electrolyte disturbances such as K⁺ loss which has been observed as side-effect of most diuretics. Gancao had no diuretic effect, but could impair Yuanhua’s diuretic effect through up-regulating renal AQP 2. Glycyrrhetinic acid (GRA) in Gancao could up-regulate AQP 2 and counteract the AQP 2 regulation effect of Yuanhuacine (YHC) and Ginkwanin (GKW) in Yuanhua. Network pharmacology method suggested that YHC, GKW and GRA could bind to MEK1/FGFR1 protein and influence ERK-MAPK pathway, which was verified by Western blotting. This study supports TCM theory and reminds that more attention should be paid to the safety and efficacy problems induced by improper combination between herbs. Moreover, we suggested that promising diuretics with less side effects can be developed from Chinese Medicines such as Yuanhua.
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Cisplatin is a classic chemotherapeutic agent widely used to treat different types of cancers including ovarian, head and neck, testicular and uterine cervical carcinomas. However, cisplatin induces acute kidney injury by directly triggering an excessive inflammatory response, oxidative stress, and programmed cell death of renal tubular epithelial cells, all of which lead to high mortality rates in patients. In this study, we examined the protective effect of protocatechuic aldehyde (PA) in vitro in cisplatin-treated tubular epithelial cells and in vivo in cisplatin nephropathy. PA is a monomer of Traditional Chinese Medicine isolated from the root of S. miltiorrhiza (Lamiaceae). Results show that PA prevented cisplatin-induced decline of renal function and histological damage, which was confirmed by attenuation of KIM1 in both mRNA and protein levels. Moreover, PA reduced renal inflammation by suppressing oxidative stress and programmed cell death in response to cisplatin, which was further evidenced by in vitro data. Of note, PA suppressed NAPDH oxidases, including Nox2 and Nox4, in a dosage-dependent manner. Moreover, silencing Nox4, but not Nox2, removed the inhibitory effect of PA on cisplatin-induced renal injury, indicating that Nox4 may play a pivotal role in mediating the protective effect of PA in cisplatin-induced acute kidney injury. Collectively, our data indicate that PA blocks cisplatin-induced acute kidney injury by suppressing Nox-mediated oxidative stress and renal inflammation without compromising anti-tumor activity of cisplatin. These findings suggest that PA and its derivatives may serve as potential protective agents for cancer patients receiving cisplatin treatment.
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Panax ginseng (GS) and Veratrum nigrum (VN) are representative of incompatible pairs in “eighteen antagonistic medicaments” that have been recorded in the Chinese medicinal literature for over 2,000 years. However, evidence linking interference effects with combination use is scare. Based on the estrogen-like effect of GS described in our previous studies, we undertake a characterization of the interaction on estrogenic activity of GS and VN using in vivo models of immature and ovariectomized (OVX) mice and in vitro studies with MCF-7 cells for further mechanism. VN decreased the estrogenic efficacy of GS on promoting the development of the uterus and vagina in immature mice, and reversing the atrophy of reproductive tissues in OVX mice. VN interfered with the estrogenic efficacy of GS by decreasing the increase of the serum estradiol and the up-regulation of ERα and ERβ expressions by treatment with GS. And VN antagonized the estrogenic efficacy of GS on promoting the viability of MCF-7 cells and up-regulation of protein and gene expressions of ERs. In conclusion, this study provided evidence that GS and VN decreased effects on estrogenic activity, which might be related to regulation of estrogen secretion and ERs.
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The alkaloid components in Strychnos nux-vomical L. uncombined and combined with Glycyrrhiza uralensis Fisch have been investigated by electrospray ionization tandem mass spectrometry ( ESI-MS o) and HPLC. The experimental results demonstrated that the number of strychnine and brucine all declined in combined Strychnos nux-vomical L. with Glycyrrhiza uralensis Fisch, and the concentration level of strychnine fell obviously. The results of ESI-MS were identical to those of HPLC, which provided scientific basis for explanation of detoxicity of Glycyrrhiza uralensis Fisch and the reasonable combination of Strychnos nux-vomical L. .
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Background: Combination of licorice root(LR) and Peking euphorbia root(PER) is one of "eighteen incompatible medicaments" in traditional Chinese medicine, toxicities and side effects of combinations of some "incompatible traditional Chinese medical herbs" have not been found, and even some combinations have been found to improve the therapeutic effects. Objective: To discuss the influence of combination of LR and PER in "eighteen incompatible medicaments" (EIM) on the functions and pathological morphology of the heart, liver and kidney in rats. Design: Randomly controlled experiment. Setting, materials and interventions: The experiment was accomplished in the Animal Center of Xinqiao Hospital, Third Military Medical University of PLA. Twenty Wistar rats, weighing 200 g 300 g, to half males and' half females were divided into four groups as LR, PER, LR + PER and control group with 5 in each. The controls were perfused with normal saline, the dosage, method, time and detection were the same as those in other groups. In LR, PER, and LR + PER groups, gastric perfusion was 0.02 mL/10 g, once a day, 7 days in succession. After 7 days, the blood was collected through cutting head for tests of liver and kidney functions and myocardial zymogram, at the same time, slices of the heart, liver and kidney were made for observation. Main outcome measures: Influence of licorice root and Peking euphorbia root in combination on liver and kidney functions and myocardial zymogram. Results: In LR + PER group, alanine aminotransferase [(1 465.3 ± 140.0) nkat/L] was significantly increased compared with those in control, LR, and PER groups [(625.1 ± 60.0), (723.5 ± 70.0), (1 310.3 ± 131.7) nkat/L] (t = 47.16-54.99, P < 0.01); creatine phosphokinase, lactate dehydrogenase, γ-hydroxybutyrate dehydrogenase level were increased, compared with those in control, LR, and PER groups (t = 55.64-91.85, P < 0.01). There was no significant difference of blood urea nitrogen and creatinine in each group (P > 0.05). Conclusion: Single PER could injure the rat liver functions and this was aggravated by its combination with LR; there were obvious injuries on the rat heart functions and no influence on renal functions in LR + PER group.