Journal Pre-proof Target lipidomics approach to reveal the resolution of inflammation induced by Chinese medicine combination in Liu-Shen-Wan against realgar overexposure to rats Jiaojiao Wang, Lanfang Ding, Jing Zhou, Hongyue Ma, Yuanyuan Wu, Jiajia Wang, Xiang Lv, Shengjin Liu, Hengbin Wang, Yanqing Yan, Niancui Luo, Quan Li, Huiqin Xu, Liuqing Di, Qinan Wu, Jinao Duan PII:
S0378-8741(19)31587-9
DOI:
https://doi.org/10.1016/j.jep.2019.112171
Reference:
JEP 112171
To appear in:
Journal of Ethnopharmacology
Received Date: 20 April 2019 Revised Date:
20 July 2019
Accepted Date: 18 August 2019
Please cite this article as: Wang, J., Ding, L., Zhou, J., Ma, H., Wu, Y., Wang, J., Lv, X., Liu, S., Wang, H., Yan, Y., Luo, N., Li, Q., Xu, H., Di, L., Wu, Q., Duan, J., Target lipidomics approach to reveal the resolution of inflammation induced by Chinese medicine combination in Liu-Shen-Wan against realgar overexposure to rats, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/ j.jep.2019.112171. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
1
Target lipidomics approach to reveal the resolution of inflammation
2
induced by Chinese medicine combination in Liu-Shen-Wan against
3
realgar overexposure to rats
4 5
Jiaojiao Wanga,#, Lanfang Dingc,#, Jing Zhoua,*, Hongyue Maa,*, Yuanyuan Wua, Jiajia Wanga,
6
Xiang Lva, Shengjin Liua, Hengbin Wangd, Yanqing Yand, Niancui Luod, Quan Lid, Huiqin Xua,
7
Liuqing Dib, Qinan Wua, Jinao Duana
8
a. Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources
9
Industrialization, Jiangsu Key Laboratory for High Technology Research of TCM
10
Formulae, and Jiangsu Key Laboratory of efficacy and safety evaluation of TCM,
11
Nanjing University of Chinese Medicine, Nanjing, China.
12
b. School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China; Jiangsu
13
Provincial TCM Engineering Technology Research Center of Highly Efficient Drug
14
Delivery System (DDS), Nanjing, China.
15
c. Nanjing Maternity and Child Health Care Hospital, Nanjing, China.
16
d. Leiyunshang Pharmaceutical Company. Ltd, Suzhou , China.
17
#
Jiaojiao Wang and Lanfang Ding have equal contribution to this paper
18 19
* Corresponding author:
20
Jing Zhou
21
Email:
[email protected] 22
Address: No.138 xianlin avenue, qixia district, nanjing city, jiangsu province
23
Phone Number: +86 13645180005
24 25
Hongyue Ma
26
Email:
[email protected] 27 28
Author Contributions: 1
29
writing—original draft preparation, Jiaojiao Wang and Lanfang Ding;
30
methodology, Jing Zhou and Hongyue Ma;
31
formal analysis, Yuanyuan Wu, Jiajia Wang, Xiang Lv and Shengjin Liu;
32
writing—review and editing, Jing Zhou and Hongyue Ma;
33
project administration, Hengbin Wang, Yanqing Yan, Niancui Luo and Quan Li;
34
funding acquisition, Huiqin Xu, Liuqing Di, Qinan Wu and Jinao Duan.
35 36
[email protected] 37
[email protected] 38
[email protected] 39
[email protected] 40
[email protected] 41
[email protected] 42
lyxhj09@ 163.com
43
[email protected] 44
[email protected] 45
[email protected] 46
[email protected] 2
47
Abstract
48
Ethnopharmacological relevance: Liu-Shen-Wan (LSW) is one of the popular
49
over-the-counter drugs in Asia, which contains realgar (As4S4), used for the treatment
50
of upper respiratory tract inflammation and skin infections. However, the safety and
51
potential risk of this arsenic remain unknown.
52
Aim of the study: The aim of this study was to determine total arsenic in tissue and
53
investigate effects of regular dose and overdose LSW exposure on rat liver.
54
Materials and methods: We used a target lipidomics approach to quantify
55
inflammatory eicosanoids and employed ICP-MS to determine total arsenic in tissue.
56
Results: The results showed that oral administration of 8 and 40 mg/kg LSW (1 and 5
57
fold human-equivalent dose) induced light changes of liver lipidomic profile in rats,
58
which was associated with anti-inflammatory function of LSW. In our recent report,
59
we observed that 41 and 134 mg/kg realgar (40 and 132 fold human-equivalent dose)
60
stimulated rat liver inflammation through up-regulation of pro-inflammatory
61
LOX-derived, CYP-derived HETEs and COX-derived PGs. However, we found that
62
LSW in the form of drug combination, containing 41 and 134 mg/kg realger, could
63
not stimulate these similar inflammatory responses in rats, although the liver total
64
arsenic levels of the realger and LSW groups were same.
65
Conclusion: The downregulation of pro-inflammatory showed that the LSW
66
containing realger is safer than realger alone administrated to rats. These results
67
suggested that Chinese medicines combination could reduce realgar-derived arsenic
68
toxicity in rats.
69 70
Key words: Liu Shen Wan; Liver inflammation; Realgar; Reducing toxicity; Chinese
71
medicine compatibility.
72 73
Abbreviations: LSW, Liu-Shen-Wan; ICP-MS, Inductively coupled plasma mass
74
spectrometry; APL, Acute promyelocytic leukemia; TCM, Traditional Chinese 3
75
medicines; COX, Cyclooxygenases; LOX, Lipoxygenases; CYP, Cytochrome P450;
76
HPLC, High Purity Liquid Chromatography; ISs, internal standards; CMC-Na,
77
Carboxymethyl Cellulose Sodium; ALT, lanine aminotransferase; ALB, albumin;
78
AST, aspartate aminotransferase; TP, total protein; TBIL, total bilirubin; ALP,
79
alkaline phosphatase; GLU, glucose; BUN, blood urea nitrogen; CREA, creatinine;
80
CHOL, total cholesterol; LDH, lactate dehydrogenase; TG, Triglyceride; CK, creatine
81
kinase; K, potassium; Na, sodium; Cl, chlorine; NHJDP, Niu-Huang-Jie-Du-Pian;
82
OPLS-DA, orthogonal projections to latent structures discriminant analysis; DHA,
83
docosahexaenoic acid; AA, Arachidonic Acid; As2O3, arsenic trioxide; NaAsO2,
84
sodium arsenite; Na2HAsO4, arsenate;
85 86
Introduction
87
Traditional medicines have gained ever-increasing popularity due to its
88
therapeutic effects on many diseases and their natural compounds present as strong
89
candidates for the treatment of cancer pain recently. Realgar (As4S4), referred to as
90
“Xionghuang”, is one of the traditional mineral medicines in China and as an
91
arsenical, and it is known as a poison and paradoxically as a therapeutic agent in small
92
doses for the treatment of tonsillitis, herpes zoster, sore throat and convulsions(Liu et
93
al., 2013). There are about 440 Chinese medicine preparations in use and about 78
94
that contain realgar(Lu et al., 2011). Realgar is the essential component of some
95
popular medicinal preparations in Asian and Western countries, used as the oral
96
formulation in the treatment of both newly and relapsed/refractory diagnosed acute
97
promyelocytic leukemia (APL) currently (Qi et al., 2010). However, chronic exposure
98
to realgar also induced potential toxicological risk, produce toxicity to the livers and
99
kidneys and cause other arsenic-related diseases because of the highly toxic substance,
100
arsenic (Wei et al., 2009). Research showed that the liver was the major target organ
101
of arsenic exposure. It has been reported that arsenic trioxide exposure brought about
102
the IL-6 mediated inflammatory response, oxidative stress, significantly elevation in 4
103
activity of liver enzymes and cellular necrosis in rat livers(Li et al., 2016) recently and
104
sometimes led to liver injury . Thus, the arsenic-containing traditional medicines are
105
commonly used in clinic treatment and that might bring the risk due to its wide
106
exposure.
107
It is a traditional concept that Chinese medicine compatibility can attenuate the
108
risk of toxic Traditional Chinese medicines. Realgar used to combined with some
109
other herbal medicines to reduce toxicity and side effect or improve the efficacy in
110
TCMs and Indian Ayurveda medicines. Liu-Shen-Wan (LSW) is one of these
111
common medicines, containing realgar (Arsenic sulfide), bezoar (Bos taurus
112
domesticus Gmelin), borneol (Dryobalanops aromatica Gaertner. f.), Musk (Moschus
113
berezovskii Flerov), toad venom (Bufo bufo gargarizans Cantor)and pearl (Pteria
114
martensii (Dunker), for the treatment of upper respiratory inflammation . It has been
115
known that the toxicity of realgar was far lower than inorganic arsenite (toxic As(III)
116
and As(V)) based on their LD50 in mice (Liu et al., 2008), and we have found that oral
117
administration of the pure realgar at maximal dosage could not cause the death of
118
mice previously. We assumed that the toxicology risk of realgar was different from its
119
preparations in the form of herbal combination, and actions have been taken to
120
evaluate whether the compatibility with other Chinese medicines can reduce the
121
realgar poisonousness. Several studies showed that the classic toxicological methods of
122
investigating arsenic toxicity and its mechanisms in experimental animals are
123
insufficient and of limitations, and cannot make early predictions of liver damage
124
(Kitchin and Conolly, 2010). So the new research techniques or approaches are
125
needed for the prediction of realgar toxicity. Lipidomics had been employed in
126
biological samples for the comprehensive analysis and characterization of the
127
metabolism of lipids at the molecular level and showed great possibilities to identify
128
early related biomarkers in liver injury and other inflammatory diseases recently (Tam
129
et al., 2013; Xie et al., 2016). The content of endogenous metabolites, such us
130
eicosanoids, could precede the development of clinical liver injury and the 5
131
phospholipid markers had higher sensitivity than conventional pharmacological
132
indicators(Rolim et al., 2015). Currently, eicosanoids had been monitored in rat serum
133
after orally administration with a single dose of NaAsO2 and were proved to reflect
134
sensitively acute arsenic toxicity (Chen et al., 2017). Arachidonic acid (AA),an
135
important inflammatory lipid mediator,can regulate liver mitochondria oxidative
136
stress by the cyclooxygenase (COX),lipoxygenase (LOX) and cytochrome P450
137
(CYP450) metabolic pathways. Eicosanoids and its metabolites have certain
138
physiological activities and their epoxidase metabolism, named prostaglandins, are
139
known as inflammatory factors in the body, while lipoxygenase metabolite is regarded
140
as anti-inflammatory ingredients mostly(O'Connell and Watkins, 2010). Thus, it
141
makes sense to monitor some characteristic phospholipid markers to make a more
142
comprehensive understanding of realgar toxicity in the rats.
143
Our recent study showed that realgar at 40 and 132 fold human-equivalent doses
144
significantly induced the liver inflammation in rats through up-regulation of
145
COX-derived mediators (Zhou et al., 2019). However, the safety of Liu-Shen-Wan
146
containing realgar has not been elucidated. In this paper, we use lipidomics combined
147
with the routine testing to evaluate effects of regular dose and overdose LSW on
148
livers in rats. Moreover, we compared toxicity of realgar alone and in combination
149
with other herbal medicines (Liu-Shen-Wan), getting comprehensive understanding of
150
safety and drug risks of LSW.
151 152
Materials and Methods
153
Reagents and samples
154
Liu-Shen-Wan (LSW, batch number Z32020481) and realgar (As4S4, purity:
155
94.5%) were obtained from Leiyunshang Pharmaceutical Company. Ltd (Suzhou,
156
China), which was prepared by mixing Xionghuang, Niu-huang, She-xiang, Chan-shu,
157
Zhen-zhu, etc. The contents of ten representative compounds in LSW analyzed by
158
HPLC method were as follows: gamabufotalin, 0.91 mg/g; arenobufagin, 0.51 mg/g; 6
159
telocinobufagin, 1.51 mg/g; bufotalin, 1.63 mg/g; cinobufotalin, 2.50 mg/g; bufalin,
160
1.87 mg/g; resibufogenin, 1.89 mg/g; cinobufagin, 4.46 mg/g; cholic acid, 25.1 mg/g;
161
bilirubin,
162
CAS-No.67-56-1, batch number AS1922), acetonitrile (HPLC-grade, CAS-No.
163
75-05-8,
164
CAS-No.67-63-0) were purchased from Tedia (Fairfield, Ohio, USA). The
165
deuterium-labeled internal standards (ISs) AA-d8 (batch number 20150513) were
166
gained from Cayman Chemical (Ann Arbor, MI, USA). Other reagent solutions,
167
including formic acid (HPLC-grade, batch number F0507), were obtained from
168
Sigma-Aldrich Corp (Louis, MO, USA). The lipid standards (batch number
169
15090801), PGF2ɑ was from SantaCruz and the standard substances, 15-HETrE,
170
9,10-diHOME, 15-HETE, 13-HODE, 17,18 EpETE, 10-HDoHE, 9,10 EpOME, 19,20
171
EpDPA and arachidonic acid were all from Cayman.
1.58
batch
mg/g
(Supplementary
number
AS1122)
Figure
and
1).
isopropyl
Methanol
alcohol
(HPLC-grade,
(HPLC-grade,
172 173
Animal
174
Fifty healthy male Sprague-Dawley rats weighing 85-95 g used in the study were
175
obtained from the Experimental Animal Center of Zhejiang (animal certificate
176
number SCXK-zhe-2014-0001). Male animals were housed in standardized
177
animal-house at a stable temperature (23±3 °C) and humidity (60±5%) under
178
continuous observation. They were allowed free access to a commercial standard diet
179
and water ad libitum. Animal experiments were performed in accordance with Guide
180
for the Care and Use of Laboratory Animals (National Research Council of USA,
181
1996) and related ethical regulations of Nanjing University of Chinese Medicine.
182 183
Experimental design
184
The preparation LSW samples were dissolved in newly prepared 0.5% CMC-Na
185
(Carboxymethyl Cellulose Sodium) solution to obtain different concentrations of
186
suspending solutions. Rats were divided into six groups randomly. Group 1 (Nor 1) 7
187
received 0.5% CMC-Na as control for one month; group 2 (LL-p) received 8 mg/kg
188
Liu-Shen-Wan for one month; group 3 (LH-p) received 40 mg/kg Liu-Shen-Wan for
189
one month; group 4 (Nor 2) received 0.5% CMC-Na as control for two months; group
190
5 (LL) received 320 mg/kg Liu-Shen-Wan for two months; group 6 (LH) received
191
1000 mg/kg Liu-Shen-Wan for two months. The corresponding drugs were
192
administered via the oral route one time per day. The body weights and food intakes
193
of rats were recorded every day. After 1 or 2 months oral administration, rats were
194
euthanized. Blood examples were collected from abdominal aorta and the isolated
195
serum is stored in a cryogenic refrigerator for biochemical assays. Rat liver tissue
196
were isolated, snap-frozen ( with nitrogen) and stored at -80 °C for later testing.
197 198
Determination of total arsenic
199
The liver samples (0.025g) were spiked into 2 ml concentrated HNO3 and placed
200
in a fume hood standing overnight. After being digested at 150 °C for 1 h, 2 ml H2O2
201
and an additional 1 ml HNO3 was added to the samples, and heated until they had
202
boiled dry. Samples were diluted to a final mass of 4 g with 1% (v/v) HNO3. Then we
203
apply ICP-MS analysis of liver digests to determine the total arsenic under instrument
204
conditions described elsewhere (Nearing et al., 2014) and using indium as an internal
205
standard introduced via a separate sample line. Besides, certified ICP standards were
206
used for calibration.
207 208
Liver function tests
209
Blood samples were centrifuged at 2000 rpm for 15 min and supernatant were
210
collected as serum. The indicators for liver injury — alanine aminotransferase (ALT),
211
aspartate aminotransferase (AST), total protein (TP), albumin (ALB), total bilirubin
212
(TBIL), alkaline phosphatase (ALP), glucose (GLU), blood urea nitrogen (BUN),
213
creatinine (CREA), total cholesterol (CHOL), lactate dehydrogenase (LDH),
214
triglyceride (TG), creatine kinase (CK), electrolytes potassium (K), sodium (Na) and 8
215
chlorine (Cl) — were assayed using standard commercial kits and automatic
216
biochemical analyzer (HITACHI 7100, Japan).
217 218
Liver histopathology examination
219
Liver of rats was selected and fixed with 10% neutral formalin solution. Then
220
samples were embed with conventional paraffin-embedding protocol and sliced into 4
221
μm thickness tissues. The slides were stained with hematoxylin and eosin and
222
observed under an optical microscope to evaluate the histopathological injury.
223 224
Analysis of lipidomics in livers using UPLC-MS/MS
225
Randomly sample some frozen liver tissue, add 0.9% normal saline to cool liver
226
tissue in a homogenizer and homogenate 5min on the ice. Then separate mediators
227
from livers with the liquid-liquid extraction. 4 ml ethyl acetate and n-hexane
228
(vol/vol=1:1, cooled to -80 °C) were added to the 4.0 ml of 10% liver homogenate,
229
along with the 25ul chloramphenicol methanol solution (1ug/ml). After vortex mixing,
230
the sample was ultra-sonicated at 100 Hz for 20 min in the ice water bath (KQ3200,
231
Kunshan, Jiangsu) centrifuged at 2000 r/min for 5 min and stood at 4 °C for 5 min.
232
Then transfer the supernatants to a fresh tube and the procedures were repeated until
233
the supernatants were completely obtained. Next, combine organic phase in a 10ml
234
centrifuge tube, swirl and volatilize at 37 °C. Centrifuge, dried the supernatants and
235
re-constitute the residue with 500 µl of 90% acetonitrile solution for UPLC-MS/MS
236
analysis.
237
Chromatography was performed on a Synergi reverse-phase C18 column (50×2
238
mm, USA, Phenomenex) using an UPLC system (SHIMADZU LC-20AD XR). The
239
flow rate was 0.30 ml/min and the column was maintained at 35 °C. The mobile phase
240
A was composed of water, acetonitrile and formic acid (water–acetonitrile–formic
241
acid= 70:30:0.02, v/v/v). The mobile phase B was composed of acetonitrile and
242
isopropyl alcohol (acetonitrile–isopropyl alcohol= 50:50, v/v). The column was eluted 9
243
with a linear gradient system: 0–3 min, 0–25% B; 3–11 min, 25–45% B; 11–13 min,
244
45–60% B; 13–18 min, 60–75% B; 18–18.5 min, 90% B; 18.5–20 min, 90% B; 20-21
245
min, 0%; 21-25 min, 0%. The injected sample was 5 µl. The UPLC was directly
246
interfaced with an AB Sciex QTRAP 5500 system (AB SCIEX, Foster City, CA,
247
USA) spectrometer with an ESI source operated in the negative ion mode. Set
248
parameters in the source as follows: ion spray voltage was -4500 V; turbo ion spray
249
temperature was 525 °C; curtain gas was 10 psi; nebulizer gas (GS 1) was 30 psi;
250
heater gas (GS 2) was 30 psi; dwell time was 50 ms. Data were collected in multiple
251
reaction monitoring (MRM) mode by screening parent and daughter ions. The internal
252
standard, AA-d8, was used for the quality control. The data of all eicosanoids content
253
were transferred to SIMCA-P for the principal component analysis (PCA).
254 255
Identification and selection of the clinic literatures
256
To evaluate the adverse effects of realgar-containing preparations and arsenic
257
trioxide in patients, the clinic literatures were collected. The evidence for adverse
258
reactions is consisted of short-term toxicity ( 1
259
month). Relevant literature reporting results of this kind of studies was identified by
260
means of a computerized search of multiple electronic bibliographic databases
261
(PUBMED, CNKI.NET, NCBI, WanFang Date and OvidSP). Using the terms
262
appropriated to each database, the search strategy composed by the adverse reactions
263
of realgar-containing preparations and arsenic, and comparison to results was made.
264
Besides, two reviewers independently screened the eligibility of the articles first on
265
the title and the abstract, and then on the full text.
266 267
Statistics
268
The results were expressed as mean ± SD and statistical analysis was performed
269
using two-tailed unpaired Student’s t-test. A P value of less than 0.05 was considered
10
270
statistically significant and P less than 0.01 was deemed outstanding statistically
271
significant.
272 273
RESULTS
274
Comparison of adverse clinical reactions of two realgar-containing preparations
275
Table 1 has summarized the common adverse clinical outcomes of two
276
realgar-containing preparations (Liu-Shen-Wan, LSW, and Niu-Huang-Jie-Du-Pian,
277
NHJDP) and the arsenic trioxide injection. For the short-term toxicity ( 1 month), LSW induced liver inflammation
281
and other clinical symptoms in about 2.6% case numbers. However, the NHJDP or
282
arsenic trioxide injection seemed to be more toxic on liver than LSW with liver
283
damage induction rate of 11.5% and 7.9%, respectively. Besides, the NHJDP caused
284
the chronic arsenism (13.5%, 7/52). In summary, the LSW had lower adverse clinical
285
reactions on livers than realgar and another realgar-containing preparations (NHJDP).
286 287
Total arsenic content in rat livers
288
In this experimental study, we firstly determined total arsenic content in rat livers.
289
The oral administration of 8 and 40 mg/kg LSW (1 and 5 fold human equivalent doses)
290
for one month significantly increased the total arsenic contents of livers from the
291
0.83±0.11 µg/g in the normal control to 2.24±0.55 µg/g and 3.56±0.66 µg/g,
292
respectively (Figure 1). LSW overexposure (320 and 1000 mg/kg LSW, 40 and 132
293
fold human equivalent doses) to rats for two months could elevate the total arsenic
294
contents in livers from 1.03±0.21 µg/g in the normal control to 31.73±10.24 µg/g and
295
32.6±4.67 µg, respectively (Figure 1). Here, we noticed that the oral administration of
296
41 and 134 mg/kg realgar (equivalent to 320 and 1000 mg/kg LSW) for two months
297
had the similar total arsenic (30.28±14.73 µg/g and 31.15±5.64 µg/g (our data 11
298
published (Zhou et al., 2019)) as the LSW. This illustrated that total arsenic intakes of
299
rats exposed to the realgar alone and its TCM preparation were the same, and
300
LSW in the form of herbal combination did not reduce the total arsenic content in
301
realgar-treated rats.
302 303
Effects of LSW exposure on the biochemical markers
304
As shown in figure 2, compared with the normal control groups, the weight and
305
the amount of food intake in rats orally administrated of LSW had no marked change
306
(P > 0.05). That indicated LSW had no significant effects on the growth and
307
development of rats.
308
Then, we evaluated the function of rat liver and biochemical markers treated with
309
LSW. It showed that serum enzyme activity did not have significant changes,
310
including ALT, AST, TBIL, ALP, ALB, LDH, GLU, TP et al. (Table 2). There was
311
slight fluctuation in individual index, but no statistical significant differences between
312
treated and normal groups were observed (P > 0.05) generally. Meanwhile, the results
313
of liver histopathology examination showed the same evidence, presenting no cell
314
necrosis, interstitial vasodilation, congestion, abnormalities and histological changes
315
in rats after exposure to LSW (figure 3). These evidences illustrated that oral
316
administration of regular dose and overdose LSW for one or two months did not cause
317
liver injuries in rats.
318 319
Intervention of herbal combination in LSW on lipid markers
320
To investigate the effects of different doses LSW, the supervised method,
321
orthogonal projections to latent structures discriminant analysis (OPLS-DA), was
322
used to separate predictive variables responsible for the inter-group differences (figure
323
4A). The fold changes (FC) in content which are more than 1.5 were selected as
324
biomarkers and the significant markers (P