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.
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Target lipidomics approach to reveal the resolution of inflammation
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induced by Chinese medicine combination in Liu-Shen-Wan against
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realgar overexposure to rats
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Jiaojiao Wanga,#, Lanfang Dingc,#, Jing Zhoua,*, Hongyue Maa,*, Yuanyuan Wua, Jiajia Wanga,
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Xiang Lva, Shengjin Liua, Hengbin Wangd, Yanqing Yand, Niancui Luod, Quan Lid, Huiqin Xua,
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Liuqing Dib, Qinan Wua, Jinao Duana
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a. Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources
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Industrialization, Jiangsu Key Laboratory for High Technology Research of TCM
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Formulae, and Jiangsu Key Laboratory of efficacy and safety evaluation of TCM,
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Nanjing University of Chinese Medicine, Nanjing, China.
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b. School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China; Jiangsu
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Provincial TCM Engineering Technology Research Center of Highly Efficient Drug
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Delivery System (DDS), Nanjing, China.
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c. Nanjing Maternity and Child Health Care Hospital, Nanjing, China.
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d. Leiyunshang Pharmaceutical Company. Ltd, Suzhou , China.
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#
Jiaojiao Wang and Lanfang Ding have equal contribution to this paper
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* Corresponding author:
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Jing Zhou
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Email:
[email protected] 22
Address: No.138 xianlin avenue, qixia district, nanjing city, jiangsu province
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Phone Number: +86 13645180005
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Hongyue Ma
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Email:
[email protected] 27 28
Author Contributions: 1
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writing—original draft preparation, Jiaojiao Wang and Lanfang Ding;
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methodology, Jing Zhou and Hongyue Ma;
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formal analysis, Yuanyuan Wu, Jiajia Wang, Xiang Lv and Shengjin Liu;
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writing—review and editing, Jing Zhou and Hongyue Ma;
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project administration, Hengbin Wang, Yanqing Yan, Niancui Luo and Quan Li;
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funding acquisition, Huiqin Xu, Liuqing Di, Qinan Wu and Jinao Duan.
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[email protected] 37
[email protected] 38
[email protected] 39
[email protected] 40
[email protected] 41
[email protected] 42
lyxhj09@ 163.com
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[email protected] 44
[email protected] 45
[email protected] 46
[email protected] 2
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Abstract
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Ethnopharmacological relevance: Liu-Shen-Wan (LSW) is one of the popular
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over-the-counter drugs in Asia, which contains realgar (As4S4), used for the treatment
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of upper respiratory tract inflammation and skin infections. However, the safety and
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potential risk of this arsenic remain unknown.
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Aim of the study: The aim of this study was to determine total arsenic in tissue and
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investigate effects of regular dose and overdose LSW exposure on rat liver.
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Materials and methods: We used a target lipidomics approach to quantify
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inflammatory eicosanoids and employed ICP-MS to determine total arsenic in tissue.
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Results: The results showed that oral administration of 8 and 40 mg/kg LSW (1 and 5
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fold human-equivalent dose) induced light changes of liver lipidomic profile in rats,
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which was associated with anti-inflammatory function of LSW. In our recent report,
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we observed that 41 and 134 mg/kg realgar (40 and 132 fold human-equivalent dose)
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stimulated rat liver inflammation through up-regulation of pro-inflammatory
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LOX-derived, CYP-derived HETEs and COX-derived PGs. However, we found that
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LSW in the form of drug combination, containing 41 and 134 mg/kg realger, could
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not stimulate these similar inflammatory responses in rats, although the liver total
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arsenic levels of the realger and LSW groups were same.
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Conclusion: The downregulation of pro-inflammatory showed that the LSW
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containing realger is safer than realger alone administrated to rats. These results
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suggested that Chinese medicines combination could reduce realgar-derived arsenic
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toxicity in rats.
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Key words: Liu Shen Wan; Liver inflammation; Realgar; Reducing toxicity; Chinese
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medicine compatibility.
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Abbreviations: LSW, Liu-Shen-Wan; ICP-MS, Inductively coupled plasma mass
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spectrometry; APL, Acute promyelocytic leukemia; TCM, Traditional Chinese 3
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medicines; COX, Cyclooxygenases; LOX, Lipoxygenases; CYP, Cytochrome P450;
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HPLC, High Purity Liquid Chromatography; ISs, internal standards; CMC-Na,
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Carboxymethyl Cellulose Sodium; ALT, lanine aminotransferase; ALB, albumin;
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AST, aspartate aminotransferase; TP, total protein; TBIL, total bilirubin; ALP,
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alkaline phosphatase; GLU, glucose; BUN, blood urea nitrogen; CREA, creatinine;
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CHOL, total cholesterol; LDH, lactate dehydrogenase; TG, Triglyceride; CK, creatine
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kinase; K, potassium; Na, sodium; Cl, chlorine; NHJDP, Niu-Huang-Jie-Du-Pian;
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OPLS-DA, orthogonal projections to latent structures discriminant analysis; DHA,
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docosahexaenoic acid; AA, Arachidonic Acid; As2O3, arsenic trioxide; NaAsO2,
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sodium arsenite; Na2HAsO4, arsenate;
85 86
Introduction
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Traditional medicines have gained ever-increasing popularity due to its
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therapeutic effects on many diseases and their natural compounds present as strong
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candidates for the treatment of cancer pain recently. Realgar (As4S4), referred to as
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“Xionghuang”, is one of the traditional mineral medicines in China and as an
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arsenical, and it is known as a poison and paradoxically as a therapeutic agent in small
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doses for the treatment of tonsillitis, herpes zoster, sore throat and convulsions(Liu et
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al., 2013). There are about 440 Chinese medicine preparations in use and about 78
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that contain realgar(Lu et al., 2011). Realgar is the essential component of some
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popular medicinal preparations in Asian and Western countries, used as the oral
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formulation in the treatment of both newly and relapsed/refractory diagnosed acute
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promyelocytic leukemia (APL) currently (Qi et al., 2010). However, chronic exposure
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to realgar also induced potential toxicological risk, produce toxicity to the livers and
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kidneys and cause other arsenic-related diseases because of the highly toxic substance,
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arsenic (Wei et al., 2009). Research showed that the liver was the major target organ
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of arsenic exposure. It has been reported that arsenic trioxide exposure brought about
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the IL-6 mediated inflammatory response, oxidative stress, significantly elevation in 4
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activity of liver enzymes and cellular necrosis in rat livers(Li et al., 2016) recently and
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sometimes led to liver injury . Thus, the arsenic-containing traditional medicines are
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commonly used in clinic treatment and that might bring the risk due to its wide
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exposure.
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It is a traditional concept that Chinese medicine compatibility can attenuate the
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risk of toxic Traditional Chinese medicines. Realgar used to combined with some
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other herbal medicines to reduce toxicity and side effect or improve the efficacy in
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TCMs and Indian Ayurveda medicines. Liu-Shen-Wan (LSW) is one of these
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common medicines, containing realgar (Arsenic sulfide), bezoar (Bos taurus
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domesticus Gmelin), borneol (Dryobalanops aromatica Gaertner. f.), Musk (Moschus
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berezovskii Flerov), toad venom (Bufo bufo gargarizans Cantor)and pearl (Pteria
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martensii (Dunker), for the treatment of upper respiratory inflammation . It has been
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known that the toxicity of realgar was far lower than inorganic arsenite (toxic As(III)
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and As(V)) based on their LD50 in mice (Liu et al., 2008), and we have found that oral
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administration of the pure realgar at maximal dosage could not cause the death of
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mice previously. We assumed that the toxicology risk of realgar was different from its
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preparations in the form of herbal combination, and actions have been taken to
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evaluate whether the compatibility with other Chinese medicines can reduce the
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realgar poisonousness. Several studies showed that the classic toxicological methods of
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investigating arsenic toxicity and its mechanisms in experimental animals are
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insufficient and of limitations, and cannot make early predictions of liver damage
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(Kitchin and Conolly, 2010). So the new research techniques or approaches are
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needed for the prediction of realgar toxicity. Lipidomics had been employed in
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biological samples for the comprehensive analysis and characterization of the
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metabolism of lipids at the molecular level and showed great possibilities to identify
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early related biomarkers in liver injury and other inflammatory diseases recently (Tam
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et al., 2013; Xie et al., 2016). The content of endogenous metabolites, such us
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eicosanoids, could precede the development of clinical liver injury and the 5
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phospholipid markers had higher sensitivity than conventional pharmacological
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indicators(Rolim et al., 2015). Currently, eicosanoids had been monitored in rat serum
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after orally administration with a single dose of NaAsO2 and were proved to reflect
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sensitively acute arsenic toxicity (Chen et al., 2017). Arachidonic acid (AA)，an
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important inflammatory lipid mediator，can regulate liver mitochondria oxidative
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stress by the cyclooxygenase (COX)，lipoxygenase (LOX) and cytochrome P450
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(CYP450) metabolic pathways. Eicosanoids and its metabolites have certain
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physiological activities and their epoxidase metabolism, named prostaglandins, are
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known as inflammatory factors in the body, while lipoxygenase metabolite is regarded
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as anti-inflammatory ingredients mostly(O'Connell and Watkins, 2010). Thus, it
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makes sense to monitor some characteristic phospholipid markers to make a more
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comprehensive understanding of realgar toxicity in the rats.
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Our recent study showed that realgar at 40 and 132 fold human-equivalent doses
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significantly induced the liver inflammation in rats through up-regulation of
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COX-derived mediators (Zhou et al., 2019). However, the safety of Liu-Shen-Wan
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containing realgar has not been elucidated. In this paper, we use lipidomics combined
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with the routine testing to evaluate effects of regular dose and overdose LSW on
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livers in rats. Moreover, we compared toxicity of realgar alone and in combination
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with other herbal medicines (Liu-Shen-Wan), getting comprehensive understanding of
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safety and drug risks of LSW.
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Materials and Methods
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Reagents and samples
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Liu-Shen-Wan (LSW, batch number Z32020481) and realgar (As4S4, purity:
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94.5%) were obtained from Leiyunshang Pharmaceutical Company. Ltd (Suzhou,
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China), which was prepared by mixing Xionghuang, Niu-huang, She-xiang, Chan-shu,
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Zhen-zhu, etc. The contents of ten representative compounds in LSW analyzed by
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HPLC method were as follows: gamabufotalin, 0.91 mg/g; arenobufagin, 0.51 mg/g; 6
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telocinobufagin, 1.51 mg/g; bufotalin, 1.63 mg/g; cinobufotalin, 2.50 mg/g; bufalin,
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1.87 mg/g; resibufogenin, 1.89 mg/g; cinobufagin, 4.46 mg/g; cholic acid, 25.1 mg/g;
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bilirubin,
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CAS-No.67-56-1, batch number AS1922), acetonitrile (HPLC-grade, CAS-No.
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75-05-8,
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CAS-No.67-63-0) were purchased from Tedia (Fairfield, Ohio, USA). The
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deuterium-labeled internal standards (ISs) AA-d8 (batch number 20150513) were
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gained from Cayman Chemical (Ann Arbor, MI, USA). Other reagent solutions,
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including formic acid (HPLC-grade, batch number F0507), were obtained from
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Sigma-Aldrich Corp (Louis, MO, USA). The lipid standards (batch number
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15090801), PGF2ɑ was from SantaCruz and the standard substances, 15-HETrE,
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9,10-diHOME, 15-HETE, 13-HODE, 17,18 EpETE, 10-HDoHE, 9,10 EpOME, 19,20
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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
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Fifty healthy male Sprague-Dawley rats weighing 85-95 g used in the study were
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obtained from the Experimental Animal Center of Zhejiang (animal certificate
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number SCXK-zhe-2014-0001). Male animals were housed in standardized
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animal-house at a stable temperature (23±3 °C) and humidity (60±5%) under
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continuous observation. They were allowed free access to a commercial standard diet
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and water ad libitum. Animal experiments were performed in accordance with Guide
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for the Care and Use of Laboratory Animals (National Research Council of USA,
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1996) and related ethical regulations of Nanjing University of Chinese Medicine.
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Experimental design
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The preparation LSW samples were dissolved in newly prepared 0.5% CMC-Na
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(Carboxymethyl Cellulose Sodium) solution to obtain different concentrations of
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suspending solutions. Rats were divided into six groups randomly. Group 1 (Nor 1) 7
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received 0.5% CMC-Na as control for one month; group 2 (LL-p) received 8 mg/kg
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Liu-Shen-Wan for one month; group 3 (LH-p) received 40 mg/kg Liu-Shen-Wan for
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one month; group 4 (Nor 2) received 0.5% CMC-Na as control for two months; group
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5 (LL) received 320 mg/kg Liu-Shen-Wan for two months; group 6 (LH) received
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1000 mg/kg Liu-Shen-Wan for two months. The corresponding drugs were
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administered via the oral route one time per day. The body weights and food intakes
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of rats were recorded every day. After 1 or 2 months oral administration, rats were
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euthanized. Blood examples were collected from abdominal aorta and the isolated
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serum is stored in a cryogenic refrigerator for biochemical assays. Rat liver tissue
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were isolated, snap-frozen ( with nitrogen) and stored at -80 °C for later testing.
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Determination of total arsenic
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The liver samples (0.025g) were spiked into 2 ml concentrated HNO3 and placed
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in a fume hood standing overnight. After being digested at 150 °C for 1 h, 2 ml H2O2
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and an additional 1 ml HNO3 was added to the samples, and heated until they had
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boiled dry. Samples were diluted to a final mass of 4 g with 1% (v/v) HNO3. Then we
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apply ICP-MS analysis of liver digests to determine the total arsenic under instrument
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conditions described elsewhere (Nearing et al., 2014) and using indium as an internal
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standard introduced via a separate sample line. Besides, certified ICP standards were
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used for calibration.
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Liver function tests
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Blood samples were centrifuged at 2000 rpm for 15 min and supernatant were
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collected as serum. The indicators for liver injury — alanine aminotransferase (ALT),
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aspartate aminotransferase (AST), total protein (TP), albumin (ALB), total bilirubin
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(TBIL), alkaline phosphatase (ALP), glucose (GLU), blood urea nitrogen (BUN),
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creatinine (CREA), total cholesterol (CHOL), lactate dehydrogenase (LDH),
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triglyceride (TG), creatine kinase (CK), electrolytes potassium (K), sodium (Na) and 8
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chlorine (Cl) — were assayed using standard commercial kits and automatic
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biochemical analyzer (HITACHI 7100, Japan).
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Liver histopathology examination
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Liver of rats was selected and fixed with 10% neutral formalin solution. Then
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samples were embed with conventional paraffin-embedding protocol and sliced into 4
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μm thickness tissues. The slides were stained with hematoxylin and eosin and
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observed under an optical microscope to evaluate the histopathological injury.
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Analysis of lipidomics in livers using UPLC-MS/MS
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Randomly sample some frozen liver tissue, add 0.9% normal saline to cool liver
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tissue in a homogenizer and homogenate 5min on the ice. Then separate mediators
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from livers with the liquid-liquid extraction. 4 ml ethyl acetate and n-hexane
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(vol/vol=1:1, cooled to -80 °C) were added to the 4.0 ml of 10% liver homogenate,
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along with the 25ul chloramphenicol methanol solution (1ug/ml). After vortex mixing,
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the sample was ultra-sonicated at 100 Hz for 20 min in the ice water bath (KQ3200,
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Kunshan, Jiangsu) centrifuged at 2000 r/min for 5 min and stood at 4 °C for 5 min.
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Then transfer the supernatants to a fresh tube and the procedures were repeated until
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the supernatants were completely obtained. Next, combine organic phase in a 10ml
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centrifuge tube, swirl and volatilize at 37 °C. Centrifuge, dried the supernatants and
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re-constitute the residue with 500 µl of 90% acetonitrile solution for UPLC-MS/MS
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analysis.
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Chromatography was performed on a Synergi reverse-phase C18 column (50×2
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mm, USA, Phenomenex) using an UPLC system (SHIMADZU LC-20AD XR). The
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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
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isopropyl alcohol (acetonitrile–isopropyl alcohol= 50:50, v/v). The column was eluted 9
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with a linear gradient system: 0–3 min, 0–25% B; 3–11 min, 25–45% B; 11–13 min,
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45–60% B; 13–18 min, 60–75% B; 18–18.5 min, 90% B; 18.5–20 min, 90% B; 20-21
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min, 0%; 21-25 min, 0%. The injected sample was 5 µl. The UPLC was directly
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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
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temperature was 525 °C; curtain gas was 10 psi; nebulizer gas (GS 1) was 30 psi;
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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
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standard, AA-d8, was used for the quality control. The data of all eicosanoids content
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were transferred to SIMCA-P for the principal component analysis (PCA).
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Identification and selection of the clinic literatures
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To evaluate the adverse effects of realgar-containing preparations and arsenic
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trioxide in patients, the clinic literatures were collected. The evidence for adverse
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reactions is consisted of short-term toxicity ( 1
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month). Relevant literature reporting results of this kind of studies was identified by
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means of a computerized search of multiple electronic bibliographic databases
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(PUBMED, CNKI.NET, NCBI, WanFang Date and OvidSP). Using the terms
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appropriated to each database, the search strategy composed by the adverse reactions
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of realgar-containing preparations and arsenic, and comparison to results was made.
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Besides, two reviewers independently screened the eligibility of the articles first on
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the title and the abstract, and then on the full text.
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Statistics
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The results were expressed as mean ± SD and statistical analysis was performed
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using two-tailed unpaired Student’s t-test. A P value of less than 0.05 was considered
10
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statistically significant and P less than 0.01 was deemed outstanding statistically
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significant.
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RESULTS
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Comparison of adverse clinical reactions of two realgar-containing preparations
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Table 1 has summarized the common adverse clinical outcomes of two
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realgar-containing preparations (Liu-Shen-Wan, LSW, and Niu-Huang-Jie-Du-Pian,
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NHJDP) and the arsenic trioxide injection. For the short-term toxicity ( 1 month), LSW induced liver inflammation
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and other clinical symptoms in about 2.6% case numbers. However, the NHJDP or
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arsenic trioxide injection seemed to be more toxic on liver than LSW with liver
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damage induction rate of 11.5% and 7.9%, respectively. Besides, the NHJDP caused
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the chronic arsenism (13.5%, 7/52). In summary, the LSW had lower adverse clinical
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reactions on livers than realgar and another realgar-containing preparations (NHJDP).
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Total arsenic content in rat livers
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In this experimental study, we firstly determined total arsenic content in rat livers.
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The oral administration of 8 and 40 mg/kg LSW (1 and 5 fold human equivalent doses)
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for one month significantly increased the total arsenic contents of livers from the
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0.83±0.11 µg/g in the normal control to 2.24±0.55 µg/g and 3.56±0.66 µg/g,
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respectively (Figure 1). LSW overexposure (320 and 1000 mg/kg LSW, 40 and 132
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fold human equivalent doses) to rats for two months could elevate the total arsenic
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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
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41 and 134 mg/kg realgar (equivalent to 320 and 1000 mg/kg LSW) for two months
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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
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rats exposed to the realgar alone and its TCM preparation were the same, and
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LSW in the form of herbal combination did not reduce the total arsenic content in
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realgar-treated rats.
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Effects of LSW exposure on the biochemical markers
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As shown in figure 2, compared with the normal control groups, the weight and
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the amount of food intake in rats orally administrated of LSW had no marked change
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(P > 0.05). That indicated LSW had no significant effects on the growth and
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development of rats.
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Then, we evaluated the function of rat liver and biochemical markers treated with
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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
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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
