Article pubs.acs.org/jpr
Global and Targeted Metabolomics Evidence of the Protective Effect of Chinese Patent Medicine Jinkui Shenqi Pill on Adrenal Insufficiency after Acute Glucocorticoid Withdrawal in Rats Linjing Zhao,†,‡ Aihua Zhao,§ Tianlu Chen,§ Wenlian Chen,‡ Jiajian Liu,§ Runmin Wei,‡ Jing Su,∥ Xuelan Tang,∥ Keyi Liu,∥ Ran Zhang,∥ Guoxiang Xie,‡ Jun Panee,⊥ Mingfeng Qiu,*,∥ and Wei Jia*,‡,§ †
College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai 201620, China Metabolomics Shared Resource, University of Hawaii Cancer Center, Honolulu, Hawaii 96813, United States § Shanghai Key Laboratory of Diabetes Mellitus and Center for Translational Medicine, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China ∥ School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China ⊥ Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Manoa, Hawaii 96813, United States ‡
S Supporting Information *
ABSTRACT: Glucocorticoids are commonly used in anti-inflammatory and immunomodulatory therapies, but glucocorticoid withdrawal can result in life-threatening risk of adrenal insufficiency. Chinese patented pharmaceutical product Jinkui Shenqi pill (JKSQ) has potent efficacy on clinical adrenal insufficiency resulting from glucocorticoid withdrawal. However, the underlying molecular mechanism remains unclear. We used an animal model to study JKSQ-induced metabolic changes under adrenal insufficiency and healthy conditions. Sprague−Dawley rats were treated with hydrocortisone for 7 days with or without 15 days of JKSQ pretreatment. Sera were collected after 72 h hydrocortisone withdrawal and used for global and free fatty acids (FFAs)-targeted metabolomics analyses using gas chromatography/time-of-flight mass spectrometry and ultraperformance liquid chromatography/quadruple time-of-flight mass spectrometry. Rats without hydrocortisone treatment were used as controls. JKSQ pretreatment normalized the significant changes of 13 serum metabolites in hydrocortisone-withdrawal rats, involving carbohydrates, lipids, and amino acids. The most prominent effect of JKSQ was on the changes of FFAs and some [product FFA]/[precursor FFA] ratios, which represent estimated desaturase and elongase activities. The opposite metabolic responses of JKSQ in adrenal insufficiency rats and normal rats highlighted the “Bian Zheng Lun Zhi” (treatment based on ZHENG differentiation) guideline of TCM and suggested that altered fatty acid metabolism was associated with adrenal insufficiency after glucocorticoid withdrawal and the protective effects of JKSQ. KEYWORDS: glucocorticoid-induced adrenal insufficiency, glucocorticoid withdrawal, Jinkui Shenqi pill, TCM, metabolomics, fatty acid metabolism
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INTRODUCTION Glucocorticoids are steroid hormones that are widely used for the treatment of a variety of diseases due to their antiinflammatory and immunomodulatory properties. However, long-term treatment with supraphysiological doses of glucocorticoids in both human1−5 and adrenal-intact animals6−9 could result in a systemic suppression of the function of the hypothalamus-pituitary-adrenal (HPA) axis. When the drug is abruptly discontinued, recovery of the HPA axis function may be delayed with increased risk of temporary adrenal insufficiency, which is one of the most serious and unpredictable adverse effects in clinical practice.10−12 Currently, the most common treatment for adrenal insufficiency after glucocorticoid withdrawal is chronic glucocorticoid replacement therapy. Despite an optimized glucocorticoid-tapering schedule, patients withdrawing from glucocorticoids still suffer © XXXX American Chemical Society
from fatigue, lack of stamina, loss of energy, reduced muscle strength, weight loss, anorexia, nausea, depression, and anxiety among other symptoms.13−16 In particular, because of the immediate release of orally administered hydrocortisone, glucocorticoid replacement regimens do not fully mimic the cortisol diurnal rhythm regulated by the central biological clock, which leads to increased mortality and impaired quality of life in patients with adrenal insufficiency.17,18 Therefore, new treatments are needed for the management of adrenal insufficiency patients withdrawing from glucocorticoids. In traditional Chinese medicine (TCM), the similar clinic state of adrenal insufficiency after glucocorticoid withdrawal is usually diagnosed as the Kidney-Yang deficiency syndrome Received: May 5, 2016
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DOI: 10.1021/acs.jproteome.6b00409 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research (KDS-Yang).19 The Jinkui Shenqi (JKSQ) pill, an ancient traditional Chinese formula first recorded in the Synopsis of Prescriptions of the Golden Chamber, has been applied for treatment of patients with Kidney-Yang deficiency syndrome for thousands of years in China. JKSQ consists of Aconiti Lateralis Rddix Praeparata (Fu Zi), Cinnamomi Ramulus (Gui Zhi), Achyranthis Bidentatae Radix (Niu Xi), Rehmanniae Radix (Di Huang), Corni Fructus (Shan Zhu Yu), Dioscoreae Rhizoma (Shan Yao), Poria (Fu Ling), Aliamria Rhizoma (Ze Xie), Plantaginis Semen (Che Qian Zi), and Moutan Cortex (Mu Dan Pi). Recent research has shown that JKSQ possesses satisfactory therapeutic effects on adrenal insufficiency and related complications resulting from glucocorticoid withdrawal using scores of TCM syndrome and biochemistry analyses.20−23 Nevertheless, the metabolic and molecular mechanisms underlying the protective effect of JKSQ on the stimulation of HPA axis function remain unclear. In this study, a high dose of hydrocortisone was used to induce a pathophysiological condition in rats that mimicked clinical adrenal insufficiency after glucocorticoid withdrawal based on our previous study.24 The rats were either pretreated with JKSQ or with saline before the hydrocortisone administration to investigate the metabolic responses of normal and adrenal insufficiency rats to JKSQ treatment. Metabolites in sera were determined using both untargeted global profiling and free fatty acids (FFAs)-targeted metabolomics techniques using gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) and ultraperformance liquid chromatography/ quadruple time-of-flight mass spectrometry (UPLC/QTOFMS).
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Animals
The animal experiment was performed at the Center for Laboratory Animals, Shanghai Jiao Tong University, and the protocol was approved by the Animal Ethics Committee of the Shanghai Jiao Tong University. Eight-week-old male Sprague− Dawley rats (200 ± 20 g) were purchased from Shanghai Laboratory Animal Company, Ltd. (SLAC, Shanghai, China). All rats were housed individually in a barrier system with regulated temperature (21 ± 1 °C), humidity (60 ± 10%), and 12/12-h light/dark cycle and provided with certified standard rat chow and tap water ad libitum. After two weeks of acclimatization, rats were randomly assigned to four groups (with seven in each group): (1) the normal control group (N), (2) the model group (M), (3) the JKSQ model group (JM), and (4) the JKSQ normal group (JN). Each rat in group JM was pretreated with JKSQ (dissolved in saline) orally at 6 g kg−1 body weight once a day for 15 days and then received an intraperitoneal injection of hydrocortisone in saline (5%) at 50 mg kg−1 of body weight once a day from days 16 to 22. The dosage of JKSQ used in the rats is determined according to an established formula for human−rat drug conversion.25 Rats in group JN were also pretreated with the same dose of JKSQ orally once a day for 15 days and then injected with the same volume of saline intraperitoneally once a day from days 16 to 22. Model group rats were orally administered the same volume of saline daily from days 1 to 15 and then 5% hydrocortisone (50 mg kg−1 body weight) once a day through intraperitoneal injection from days 16 to 22. Normal control rats received saline daily in the same way as mentioned above. Rats were sacrificed by cervical dislocation on day 25, and hydrocortisone or saline were not administered during the last 72 h. Sera and 24 h urine samples were collected at the end of the experiment and stored at −80 °C, pending biochemical, global, and target metabolomics analysis. The 24 h food consumption and body weight of each rat were recorded on days 0, 7, 15, 22 and 25, and the general behavior of the rats was also observed.
MATERIALS AND METHODS
Reagents and Pharmaceutical Product
Hydrocortisone solution for injection (0.5%) was purchased from Shanghai Xinyi Pharmaceutical Company (Shanghai, China). The targeted fatty acids and the isotopically labeled internal standards (nonadecanoic-d37 acid and tridecanoic-d25 acid) were obtained from Nu-Chek Prep (Elysian, MN, USA), Sigma-Aldrich (St. Louis, MO, USA), and Cambridge Isotopes Laboratories (Andover, MA, USA). L-2-Chlorophenylalanine, BSTFA (1% TMCS), methoxyamine, leucine-enkephalin, and all other chemical standards for metabolites annotation were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol, acetonitrile, chloroform, isopropanol, n-hexane, and pyridine with HPLC grade were purchased from Merck Chemicals (Darmstadt, Germany) and Fisher (Loughborough, UK). Other chemicals were of analytical grade and obtained from China National Pharmaceutical Group Corporation (Shanghai, China). The JKSQ pill (state drug approval document no: Z11020147) was manufactured and supplied by Beijing Tongrentang Science and Technique Development Company, Ltd. (Beijing, China). It is a well-established product with SFDA approved preparation procedures and quality control protocols that are strictly carried out according to the Drug Standards of People’s Republic of China (WS3-B-3892-982011, State Food and Drug Administration). As shown in Table S1, 24 components in JKSQ were identified, mainly involving phenylpropanoids, flavonoid, terpene, phenols, and their glycosides.
Urine 17-Hydroxycorticosteroids Measurement
17-Hydroxycorticosteroid (17-OHCS) was measured in 24 h urine using ELISA kits (Groundwork Biotechnology Diagnosticate Ltd., San Diego, CA, USA) following the manufacturer’s instructions. Global Metabolomics Study by GC/TOFMS
Serum samples were extracted and derivatized according to our previously published procedures.26 An Agilent 6890N gas chromatography (GC) coupled with a Pegasus HT time-offlight mass spectrometry (TOFMS) (Leco Corporation, St. Joseph, MI, USA) was used for sample analysis. Samples were splitlessly injected into a DB-5 MS capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness; (5%-phenyl)-methylpolysiloxane bonded and cross-linked; Agilent J&W Scientific, Folsom, CA, USA) with helium as the carrier gas at a constant flow rate of 1.0 mL min−1. The injection volume was 1 μL. The GC temperature programming for the analysis serum samples was published in our previous reports.26 Temperatures of injection, transfer interface, and ion source were set to 270, 260, and 200 °C, respectively. Electron impact ionization (70 eV) at full scan mode (m/z 30−600) was used. The acquisition rate was 20 spectra s−1. The acquired MS files were exported in NetCDF format by ChromaTOF software (v3.30, Leco Company, CA, USA). The CDF files were extracted using custom scripts (revised Matlab toolbox HDA) in MATLAB 7.1 (The MathWorks, Inc., MA, B
DOI: 10.1021/acs.jproteome.6b00409 J. Proteome Res. XXXX, XXX, XXX−XXX
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Journal of Proteome Research
identification was performed using the accurate mass and retention time in our in-house library containing approximately 100 fatty acids standards.
USA) for data pretreatment procedures such as baseline correction, denoising, smoothing, alignment, time-window splitting, and peak feature extraction (based on a multivariate curve resolution algorithm).27 Three-dimensional data sets including sample information, peak retention time, and peak intensities were obtained. In addition, metabolite identification was processed by comparing the mass fragments with NIST 11 Standard mass spectral databases in ChromaTOF software (v4.34, LECO, USA) with a similarity of more than 70% and then further verified by comparing with the mass fragments and retention time of available reference standards in our in-house library.
Statistical Analysis
Data from the biochemistry determination were expressed as the mean ± SE; Student’s two-tailed, unpaired t test was used to compare the means of the groups. A P value less than 0.05 was considered as statistically significant. Multivariate statistical analysis of orthogonal partial leastsquares projection to latent structure-discriminant analysis (OPLS-DA) was carried out by SIMCA-P 14.0 version (Umetrics, Umeå, Sweden) after mean centering and unit variance scaling. Variable importance in the projection (VIP) values of all the variables from the 7-fold cross-validated OPLSDA model were ranked, and those variables with VIP larger than 1 are considered relevant for group discrimination. All the differentially expressed metabolites between two groups were selected using the Mann−Whitney U test with the critical P value set to 0.05. The fold change (FC) shows the relative intensity ratio of the differential or representative metabolites in normal control or model rats whether or not pretreated with JKSQ. The box plots were generated in SPSS 16.0 (Statistical Package for the Social Sciences, SPSS Ins., IL, USA).
Targeted Metabolomics Study of Serum FFAs by UPLC/Q-TOFMS
In the targeted analysis, FFAs were extracted from serum samples according to published methods with modifications.28 Briefly, 40 μL of serum sample was mixed with 10 μL of isotope-labeled internal standard solution (nonadecanoic-d37 acid in methanol, 5 μg mL−1, or tridecanoic-d25 acid in methanol, 25 μg mL−1) in a microcentrifuge tube. Then, 500 μL of the modified Dole’s mixture (methanol/n-hexane/ phosphoric acid (2M), 40:10:1 (v/v)) was added and vortexed for 2 min. After incubating at room temperature for 20 min, 400 μL of n-hexane and 300 μL of water were added, vortexed, and centrifuged at 12,000 rpm for 10 min. Then, 400 μL of the upper organic layer was transferred into another microcentrifuge tube, and 400 μL of n-hexane was added to the lower layer for further extraction. After vortexing and centrifugation, all of the upper organic phase was combined with the supernatant from the first-round extraction and dried under vacuum. The residue was reconstituted with 80 μL of methanol and subjected to UPLC/Q-TOFMS analysis. An ACQUITY-ultraperformance liquid chromatography (UPLC) system (Waters Corporation, Milford, USA) equipped with a binary solvent delivery system and an autosampler (Waters Corporation, Milford, USA) was used for the separation on a 100 cm × 2.1 mm BEH C18 column with 1.7 μm particles at 40 °C (Waters Corporation, Milford, USA). The optimal mobile phase consisted of water (solvent A) and acetonitrile/isopropyl alcohol (8:2 (v/v)) (solvent B), and the flow rate was set at 400 μL min−1. The injection volume was 5 μL. A gradient elution condition was applied as follows: 70% B over 0−2 min, 70∼75% B over 2−5 min, 75∼80% B over 5−10 min, 80∼90% B over 10−13 min, 90∼100% B over 13−16 min, maintained for an additional 5 min and then returned to 70% B to re-equilibrate over 21−22.5 min. The mass spectrometric data was collected using tandem quadrupole-time-of-flight mass spectrometry (Q-TOFMS) (Manchester, UK). ESI was used as the ionization source, and the analysis was carried out in negative mode. The following parameters were used: capillary voltage, 2500 V; sampling cone, 55 V; extraction cone, 4 V; desolvation temperature, 450 °C; source temperature, 120 °C; desolvation gas flow, 650 L h−1; cone gas flow, 50 L h−1; Lm resolution, 4.7; Hm resolution, 15; scan time, 0.35 s, and inter scan time 0.02s. Leucine-enkephalin was used as the lock mass (m/z 554.2615) at a concentration of 100 ng mL−1 and flow rate of 0.2 mL min−1 with a lockspray frequency of 20 s. UPLC/Q-TOFMS raw data were analyzed using TargetLynx applications manager version 4.1 (Waters Corporation, Milford, MA) to obtain calibration equations and the quantitative concentration of each FFA. Data were manually examined and corrected if errors were found for quality control. Compound
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RESULTS
Hormone Level and General Observation
After 72 h of hydrocortisone withdrawal, the 17-OHCS concentration in the 24 h urine was lower in group M compared with that in groups N (P < 0.01) and JM (P < 0.05), whereas the urine 17-OHCS concentration of group JM was comparable to that of group N, suggesting the beneficial effect of JKSQ in the rapid recovery of HPA axis function following cessation of hydrocortisone treatment (Figure 1). There was no
Figure 1. Concentration of 24 h urinary 17-OHCS in each group (mean ± SE). *P < 0.05 and **P < 0.01 compared to that of the M group. There was no statistically significant difference between groups JM and N.
significant difference in urine 17-OHCS levels between groups JN and N, suggesting that JKSQ treatment did not increase the function of the HPA axis under the normal control condition. In addition, significantly decreased body weight was found on day 22 in the model group compared to that of controls (P < 0.01) and continued to the end of study, which is similar to the weight loss experienced by patients withdrawing from glucocorticoids.29 Although the body weight increased in group C
DOI: 10.1021/acs.jproteome.6b00409 J. Proteome Res. XXXX, XXX, XXX−XXX
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Journal of Proteome Research
aspartic acid, pipecolinic acid, uracil, sorbitol, and others that mainly participate in catecholamine biosynthetic, tricarboxylic acid (TCA) cycle, amino acid, and polyol metabolism. Moreover, different concentrations of four metabolites were found between groups JN and N after 10 days of washout period (from day 16 to day 25). The differential metabolites between the two groups were selected based on the VIP values (VIP > 1.0) by multiple pattern recognition OPLS-DA models (Figure S1), P values (P < 0.05) by the Mann−Whitney U test, and FC of the arithmetic mean values (FC > 1.2 or
