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The investigation of anti-inflammatory activity of volatile oil of Angelica sinensis by plasma metabolomics approach
International Immunopharmacology 29 (2015) 269–277
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
The investigation of anti-inflammatory activity of volatile oil of Angelica sinensis by plasma metabolomics approach Wanling Yao, Ling Zhang, Yongli Hua, Peng Ji, Pengling Li, Jinxia Li, Lijia Zhong, Haifu Zhao, Yanming Wei ⁎ Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, PR China
a r t i c l e
i n f o
Article history: Received 1 July 2015 Received in revised form 29 October 2015 Accepted 5 November 2015 Available online 11 November 2015 Keywords: Anti-inflammation Volatile oil of Angelica sinensis (VOAS) Metabolomics
a b s t r a c t Angelica sinensis (AS) is an important medicinal plant, and volatile oil is the main pharmacologically active ingredient. This study was aimed to investigate the anti-inflammatory activity of the volatile oil of A. sinensis (VOAS) and explore its potential anti-inflammatory mechanism by plasma metabolomics approach. Rat acute inflammation was induced by subcutaneous injection of carrageenan in hind paws. Paw edema, histamine (HIS) and 5hydroxytryptamine (5-HT) were detected. Then, we analyzed plasma metabolic profiling of acute inflammation and performed pathway analysis on the metabolite markers reversed after VOAS administration and further integration of metabolic networks. The results showed that VOAS could alleviate the paw edema and decrease plasma HIS and 5-HT levels. Fourteen metabolite markers of acute inflammation were screened, and the levels were all reversed to different degrees after VOAS administration. These metabolite markers mainly related to linoleic acid metabolism, ascorbate and aldarate metabolism, arachidonic acid metabolism, glyoxylate and dicarboxylate metabolism, and glycine, serine and threonine metabolism. In metabolic networks, glycine and arachidonic acid were node molecules. It indicated that VOAS could significantly inhibit systemic inflammatory response triggered by acute local stimulation and it exerted anti-inflammatory activity mainly through regulating the disturbed metabolic networks centered on glycine and arachidonic acid. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Angelica sinensis (AS), commonly known as Danggui in China, is the root of A. sinensis (Oliv.) Diels. Its genuine producing area is in Gansu province, China. It is a popular medicinal plant used in traditional Chinese medicine (TCM) for treatment of menstrual disorders, dysmenorrhea, chronic constipation and other diseases [1–3]. Over 70 compounds have been isolated and identified from AS, including essential oil (such as ligustilide, butylphthalide and senkyunolide A), phthalide dimers, organic acids and their esters (such as ferulic acid), polysaccharides, vitamins, amino acids, and others [4,5]. In previous studies, we studied the hematopoietic function of AS [6] and the hepatoprotective effect of AS polysaccharides [7,8]. Inflammation is a common pathological phenomenon and various inflammatory diseases occupy an extremely important place in human and animal disease spectrum. Excessive or inappropriate inflammatory responses can lead to a range of pathological damage [9]. Increasing traditional Chinese herbs and their active ingredients have become another kind of widely used anti-inflammatory drugs after nonsteroidal and corticosteroid anti-inflammatory drugs. The researches on their anti-inflammatory mechanisms and clinical applications by modern scientific methods have always been the hot spot of the development of new-type anti⁎ Corresponding author. E-mail address: [email protected] (Y. Wei).
http://dx.doi.org/10.1016/j.intimp.2015.11.006 1567-5769/© 2015 Elsevier B.V. All rights reserved.
inflammatory drugs. Volatile oil, an active ingredient of traditional Chinese herb, is abundant in source. It is characterized by smaller molecular weight, rapid absorption and can exert anti-inflammatory action through many ways and many links [10]. Modern pharmacological studies have shown that volatile oils had potent anti-inflammatory activities [11–14]. VOAS is one of the main pharmacologically active ingredients of AS. However, the reports on its anti-inflammatory activity are few at present. Metabolomics, as a novel “-omics” technology in the post gene era, has become an important branch of system biology. The dynamic changes of small molecule (b1 kDa) metabolites represent the physiological and pathological changes of organism [15,16]. Metabolomics has been widely used to observe various metabolic characteristics of control, pathological, and drug-administrated subjects, and further to explore the mechanisms of drug intervention. Moreover, metabolomics is coincident with the integrity and systemic feature of TCM, which usually includes multi-component, multi-pathway, and multi-target treatments [17]. Therefore, metabolomics is a scientific method that can be used to evaluate and explore the mechanisms of medicinal plants used in TCM. In previous work, we explored the intervention of VOAS on local metabolic profiling of carrageenan-induced acute inflammation rats [18]. In order to further research the inhibition effects of VOAS on systemic inflammatory response triggered by local stimulation, the changes of endogenous metabolites in blood were investigated by
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metabolomics approach. This laid the foundation for development of new-type anti-inflammatory drugs. 2. Materials and methods 2.1. Reagents and materials Carrageenan was purchased from Shanghai Zhongqin Chemical Reagent Corporation (Shanghai, China). Aspirin enteric coated tablets were purchased from Bayer healthcare limited company. (Beijing, China). Pyridine was obtained from Kangtai Clinical Reagent Corporation. (Beijing, China). Acetonitrile was obtained from Tedia Corporation (USA). O-methylhydroxylamine hydrochloride, docosane, and Nmethyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) were purchased from Switzerland Fluka. Tween 80 was purchased from Shanghai Genebase Gene-Tech Corporation (Shanghai, China). The ELISA kits for determination of HIS and 5-HT were provided by Nanjing Jiancheng Biological Engineering Institute. (Nanjing, China). 2.2. Preparation of VOAS and phytochemical investigation AS was purchased from Minxian County, Gansu Province, China and authenticated by Prof. Yanming Wei (College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, China). VOAS was prepared according to the Chinese pharmacopeia [19]. AS (200 g) was ground into powder, passed through a 40-mesh sieve, mixed with 1000 mL of distilled water, and soaked for 1 day. Then, the mixture was placed in an extracting device, and subjected to hydro-distillation for 8 h to get volatile oil. Almost 0.6 mL of canary clear oil-like volatile oil was obtained. Anhydrous sodium sulfate was used to remove water from the volatile oil, which was stored in amber laboratory bottle at 4 °C for analysis [20]. Then, more than 40 components were finally identified by GC– MS, and the main components were (Z)-ligustilide (78.61%), (Z)butylidenephthalide (7.99%), (E)-ligustilide (1.66%) and (E)butylidenephthalide (1.29%). Before use, VOAS was dissolved in 2% Tween 80 (diluted by normal saline).
2.3. In vivo experiments protocol Acute toxicity study was previously performed and the lethal dose 50 (LD50) of VOAS was 1.76 mL/kg in rats. Then, 1/5 (0.352 mL/kg), 1/ 10 (0.176 mL/kg) and 1/20 (0.088 mL/kg) of LD50 were selected as the high, middle and low dose, respectively. Sixty male Wistar rats (180–210 g) were supplied by the experimental Animal Center of Lanzhou University, approved no. SCXK (Gan) 2012-0004. The room temperature was regulated at 23 ± 1 °C with 50% ± 5% humidity. A 12 h light/dark cycle was set, and the rats were fed with a standard diet and given free access to water. After acclimatization for 7 days, the rats were randomly divided into six groups with 10 rats in each group: normal control group (Control group); acute inflammation group (Model group); high-dose VOAS group (HD-VOAS group); middle-dose VOAS group (MD-VOAS group); low-dose VOAS group (LD-VOAS); and aspirin positive control group (ASP group). Control and Model groups were gavaged with 2% Tween 80. HD, MD and LD-VOAS groups were gavaged with VOAS (0.352, 0.176, and 0.088 mL/kg/day, respectively). ASP group received aspirin (200 mg/kg/day). Drugs and 2% Tween 80 were orally administrated once daily for 3 consecutive days. Acute inflammation was induced 0.5 h after gavage on the third day. Rats in Model, HD, MD and LD-VOAS groups were subcutaneously injected with 0.1 mL of 1.0% carrageenan in the sub-plantar region of the right hind paws, and corresponding volume of normal saline in Control group [18,21]. The degree of paw edema was calculated by the difference of its volume before and 3 h after carrageenan injection [22]. After that, the rats were anesthetized intraperitoneally with 1% pentobarbital sodium. Blood samples were collected from abdominal aorta. Animal welfare and experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006) and were approved by the Animal Ethics Committee of Gansu Agricultural University. 2.4. Plasma biochemical parameters All the blood samples were collected in heparinized tubes and centrifuged at 3000 rpm (centrifugal radius was 12 cm) for 10 min. Plasma samples were separated and stored at −80 °C for further use. One part of the each plasma sample was used for HIS and 5-HT tests, and the other part was used for GC–MS metabolomics analysis. The plasma samples were thawed at room temperature prior to analysis. HIS and 5-HT tests were performed according to the instructions. 2.5. GC–MS sample preparation
Fig. 1. Effects of different doses of VOAS on carrageenan-induced paw edema in rats. Paw edema was calculated by the difference of its volume before and 3 h after carrageenan injection. Values are expressed as mean ± SD (n = 10). Control indicates normal control group. Model group (acute inflammation group) received 2% Tween 80, ASP group (aspirin positive control group) received aspirin (200 mg/kg/day), and HD, MD and LD-VOAS groups (high, middle and low-dose VOAS groups) received VOAS (0.352, 0.176 and 0.088 ml/kg/day, respectively). For each group, the same lowercase letter (placed next to the numeric value) indicates that there are no significant differences between groups, while different lowercase letters (a, b, c) show significant differences with a P b 0.05. The lowercase letter “a” indicates the maximum value and “a”, “b”, “c”, and “d” decrease in turn.
In the present study, the plasma samples were processed according to the method described in ref. [7] with minor modifications. The plasma samples were thawed at room temperature prior to analysis. Firstly, 250 μL of acetonitrile was added into 100 μL of the sample for protein-precipitation. The mixture was ultrasonically extracted for 10 min, followed by centrifugation (4000 rpm) for another 10 min. Then, 300 μL supernatant was transferred to the GC vial and evaporated to dryness under a stream of nitrogen gas. Next, 100 μL of methoxyamine pyridine solution (15 μg/mL) was added to the vial, and methoxymation was performed at 70 °C for 1 h. MSTFA (50 μL, with 1% TMCS) was added, and the silylation was performed at 70 °C for 1 h. After adding 150 μL of n-heptane (with 0.10 mg/mL of docosane as internal standard) and vortex mixing, the mixture was centrifuged for 10 min. The supernatant was transferred to the GC microvial for GC–MS analysis. 2.6. GC–MS analysis GC–MS analysis was carried out using an Agilent 6890N/5973N series GC–MS (Agilent Corporation, USA) equipped with an OV-1701
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Fig. 2. Effects of different doses of VOAS on the levels of HIS (A) and 5-HT (B). The levels of HIS and 5-HT were detected using the corresponding ELISA kits. Values are expressed as mean ± SD (n = 10). Control indicates normal control group. Model group (acute inflammation group) received 2% Tween 80, ASP group (aspirin positive control group) received aspirin (200 mg/kg/day), and HD, MD and LD-VOAS groups (high, middle and low-dose VOAS groups) received VOAS (0.352, 0.176 and 0.088 ml/kg/day, respectively). For each group, the same lowercase letter (placed next to the numeric value) indicates that there are no significant differences between groups, while different lowercase letters (a, b, c) show significant differences with a P b 0.05. The letter “a” indicates the maximum value and “a”, “b”, “c”, and “d” decrease in turn.
capillary column (30 m × 0.15 μm × 15 mm). The initial temperature was maintained at 85 °C for 3 min, and then raised to 280 °C at a rate of 10 °C/min. All samples were injected in split mode at 270 °C. The mass spectrometer was operated in EI mode (positive ion, 70 eV), and the quadrupole was 150 °C. Mass spectra were acquired in full scan mode with repetitive scanning from 60 m/z to 600 m/z for 1 s. The ion source temperature was 230 °C. 2.7. Data processing GC–MS data were exported in comma-separated value (csv) format and then processed by Agilent MSD ChemStation. Raw data were filtered and then subjected to retention time correction and peak alignment [8]. The data matrix was normalized to the sum of all peak areas of each sample, and then the missing value was eliminated via the 80% correction method. Pattern recognition based on principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) was accomplished by using SIMCA-P V11.5 (Umetrics, Sweden) after meancentering and auto scaling. Potential metabolite markers screening was performed according to the value of variable importance in the projection (VIP) (VIP N 1.0) from the PLS-DA model and t-test (P b 0.05). Potential metabolite markers of interest were identified according to the National Institute of Standards and Technology (NIST) mass spectra library. With the completion of PLS-DA, we could try computational systems analysis with the MetaboAnalyst data annotation approach
including a correlation analysis plot of the differential metabolites, and heatmap visualization.
2.8. Statistical analysis Statistically significant differences of paw edema, HIS and 5-HT levels were tested by one-way analysis of variance (ANOVA) followed by the Duncan's multiple range test, and differential metabolites levels were tested by Student's t-test. These were performed on IBM SPSS V.21.0 (SPSS Inc., Chicago, USA) and Microsoft Office Excel 2013. The significant difference was considered at P b 0.05, and extremely significant difference was considered at P b 0.01.
2.9. Construction and analysis of metabolic pathway The construction, interaction, and pathway analysis of potential biomarkers significantly reversed by VOAS was performed with MetaboAnalyst based on database sources, including the Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/) and the Human Metabolome Database (http://www.hmdb.ca/), to identify the affected metabolic pathway analysis and visualization.
3. Results 3.1. Evaluation of the anti-inflammatory activity of VOAS
Fig. 3. Typical GC–MS TIC chromatograms of the plasma samples from Control, Model and MD-VOAS groups.
The inhibition effects of different doses of VOAS on carrageenaninduced paw edema in rats were shown in Fig. 1. The degrees of paw edema in Model group were significantly elevated 3 h after carrageenan injection compared with those in Control group (P b 0.05), indicating that acute inflammation models were successfully established. All drugs significantly attenuated the paw edema of carrageenan-induced acute inflammation rats (P b 0.05). When different doses of VOAS groups were compared with ASP group, there was no significant difference in the paw edema between MD-VOAS group and ASP group (P N 0.05), indicating that the inhibition effect of MD-VOAS on paw edema was close to that of aspirin. The paw edema in HD-VOAS and LD-VOAS groups was significantly higher than that in ASP group (P b 0.05), indicating that their inhibition effects were significantly lower than that of aspirin. The above results indicated that the inhibition effect of MDVOAS on paw edema was the best.
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Fig. 4. Comparison of metabolic profiling between Control and Model groups and screening of the metabolite markers: (A) Scores plot from PCA model classifying Control and Model groups. Modeling diagnostic, R2X = 0.992 and Q2 = 0.944. (B) Scores plot from PLS-DA model classifying Control and Model groups. Modeling diagnostic, R2X = 0.909, R2Y = 0.987, and Q2 = 0.972. (C) Correlation analysis plot of the differential metabolites. (D) Heatmap visualization for the carrageenan-induced acute inflammation rats. The heatmaps were constructed based on the potential biomarkers of importance and implemented in MetaboAnalyst, and they are commonly used for unsupervised clustering. Rows: metabolites; columns: samples; color key indicates metabolite expression value, red: highest and green: lowest.
Table 1 Summary of the potential metabolite markers revealed in this study. No.
Metabolites
R.T. (min)
Chemical class
VIP value
Model
MD-VOAS
Pathways
1 2 3
Arachidonic acid Malic acid
37.29 15.82 24.29
Unsaturated fatty acids Hydroxycarboxylic acids Others
1.873 1.819 1.732
↑## ↓## ↑##
Arachidonic acid metabolism Citrate cycle (TCA cycle) Ascorbate and aldarate metabolism
32.49 13.98 28.51 40.25 11.78 10.89 21.55
Unsaturated fatty acids Common amino acids Saturated fatty acids Lipids Lactic acid Polyamines Aldoses
1.698 1.582 1.522 1.398 1.348 1.300 1.238
↓## ↑## ↓## ↓## ↑## ↑## ↓##
↓⁎⁎ ↑⁎⁎ ↓⁎⁎ ↑⁎⁎ ↓⁎⁎ ↑⁎⁎ ↑⁎⁎ ↓⁎⁎ ↓⁎⁎ ↑⁎⁎
13.32 33.29 10.30 12.03
Tricarboxylic acids Saturated fatty acids Others Amino acids and derivatives
1.168 1.123 1.086 1.044
↓## ↓## ↑## ↓##
4 5 6 7 8 9 10 11 12 13 14
D-Glucuronic acid Linoleic acid Glycine Hexadecanoic acid Cholesterol Lactic acid Cadaverine D-Glucose Citric acid Stearic acid Urea Alanine
↑⁎⁎ ↑⁎⁎ ↓⁎⁎ ↑⁎⁎
Linoleic acid metabolism Glycine, serine and threonine metabolism Fatty acid biosynthesis Steroid biosynthesis Pyruvate metabolism Lysine degradation Starch and sucrose metabolism Glyoxylate and dicarboxylate metabolism Fatty acid biosynthesis Purine metabolism Alanine, aspartate and glutamate metabolism
Note: The up- (or down-) arrows represent the relatively increased (or decreased) levels of the potential metabolite markers in Model group or MD-VOAS group. ## P b 0.01, significant differences compared with Control group. ⁎⁎ P b 0.01, significant differences compared with Model group.
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Fig. 5. VOAS intervention on acute inflammation rats. (A) PLS-DA scores plot derived from plasma levels of 14 metabolite markers in Control group (C, red triangles), Model group (M, green crosses) and MD-VOAS group (MD, blue crosses). (B) Heatmap visualization based on the levels of metabolite markers in Control, Model and MD-VOAS groups. Columns correspond to samples, and variables marked on the right correspond to metabolites. Color key indicates metabolite expression value, red: highest and green: lowest. (C) Bar graphs show the relative peak area ratios of the 14 metabolite markers in Control, Model, and MD-VOAS groups. Data are expressed as mean ± SD. **P b 0.01 versus Model group.
3.2. Plasma biochemical assay The inhibition effects of different doses of VOAS on the levels of plasma HIS and 5-HT were shown in Fig. 2A–B. Compared with Control group, the levels of plasma HIS and 5-HT in Model group were significantly elevated 3 h after carrageenan injection (P b 0.05), indicating that carrageenan-induced local inflammation stimulation triggered systematic inflammatory response. Fig. 2A showed that all drugs significantly decreased the plasma HIS level of carrageenan-induced acute inflammation rats (P b 0.05). When different doses of VOAS groups were compared with ASP group, there was no significant difference in the plasma HIS level between MDVOAS group and ASP group (P N 0.05), indicating that the inhibition effect of MD-VOAS on plasma HIS level was close to that of aspirin. The plasma HIS level in HD-VOAS and LD-VOAS groups was significantly higher than that in ASP group (P b 0.05), indicating that their inhibition effects were significantly lower than that of aspirin. The above results indicated that the inhibition effect of MD-VOAS on plasma HIS level was the best.
Fig. 2B showed that all drugs decreased the plasma 5-HT level of carrageenan-induced acute inflammation rats. There was significant difference between MD-VOAS group and Model group (P b 0.05) while not between HD-VOAS and LD-VOAS groups and Model group (P N 0.05). The above results indicated that the inhibition effect of MD-VOAS on 5-HT was the best. Based on the inhibition effects of different doses of VOAS on paw edema, HIS and 5-HT, middle-dose VOAS was used to research the anti-inflammatory mechanism of VOAS by metabolomics approach. 3.3. GC–MS analysis Prior to the analysis of the experimental samples, the applied GC–MS method was validated including the precision of injection, the withinday stability and the repeatability of sample preparation. The precision of injection was evaluated by analyzing six independently processed replicates; stability was evaluated by analyzing one sample at 0, 1, 2, 3, 6, 9, 12 and 24 h; the repeatability of sample preparation was investigated by preparing six parallel samples using the same preparation
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Fig. 6. Summary of pathway analysis with MetaboAnalyst. A) Linoleic acid metabolism; B) ascorbate and aldarate metabolism; C) arachidonic acid metabolism; D); glyoxylate and dicarboxylate metabolism; and E) glycine, serine and threonine metabolism.
protocol, and then the relative standard deviations (RSD) of retention times and peak intensities were calculated, respectively. The RSDs of retention time for precision, stability and repeatability were estimated to be 0.29–0.48%, 0.29–0.32% and 0.58–1.23%, respectively, and the RSDs of intensity varied within the ranges of 1.37–2.68%, 1.16–2.58% and 1.33–1.85%. The good precision, stability and repeatability indicated that the method could be utilized to the analysis of plasma samples. Typical GC–MS TIC chromatograms of the plasma samples from Control, Model and MD-VOAS groups were shown in Fig. 3. Forty-six endogenous metabolites in the plasma samples were identified by NIST library. These metabolites, mainly including carbohydrates, fatty acids, amino acids, and organic acids, related to glycometabolism, lipid metabolism, and amino acid metabolism. 3.4. Comparison of metabolic profiling between Control and Model groups and screening of the metabolite markers First, the unsupervised PCA analysis on the data of Control and Model groups was performed to visualize general clustering, trends, or outliers among the observations, and also to know whether the two groups could be distinguished based on their metabolic profiling
provided by GC–MS. The PCA scores plot (Fig. 4A) showed that the separation between Control and Model groups was obvious, indicating that the two groups had completely different metabolic profiling. Next, in order to evaluate the systemic changes in the metabolomics of carrageenan-induced acute inflammation rats, a PLS-DA model was established based on the data of Control and Model groups. As shown in the scores plot (Fig. 4B), all samples in the two groups fell well inside the 95% confidence interval, which was represented by an ellipse. The quality parameters of the PLS-DA model between Control and Model groups were: R2X = 0.909, R2Y = 0.987, and Q2 = 0.972. R2Y estimates the goodness of fit of the model that represents the fraction of explained Y-variation, and Q2 estimates the ability of prediction. The cumulative values of R2Y and Q2 are N 0.8, indicating that reliable models are obtained [23]. Therefore, a reliable PLS-DA model was established. Fourteen variables with the VIP N 1.5 and P b 0.05 were selected and considered as potential biomarkers representing the metabolic characteristics of carrageenan-induced acute inflammation rats (Table 1). In order to further investigate the carrageenan-induced acute inflammation metabolic profiling, acquired data were subjected to computational systems analysis with MetaboAnalyst data annotation approach including a correlation analysis plot of the differential metabolites (Fig. 4C), and heatmap visualization (Fig. 4D). Correlation analysis of the 14 differential metabolites are marked on the hierarchical clustering plot which was performed to understand the potential relationships among the differential metabolites, which were shown on the plot in different colors. The heatmap, commonly used for unsupervised clustering, was constructed based on the potential differential metabolites of importance, implemented in MetaboAnalyst, which was consistent with the results in Fig. 4B. From the plots, various differential metabolites could be identified as responsible for the separation between Control and Model groups, and therefore these were viewed as potential biomarkers. 3.5. Metabolomics evaluation of VOAS intervention on acute inflammation rats As shown in Sections 3.1 and 3.2, VOAS has shown obvious inhibition effects on the paw edema and plasma HIS and 5-HT levels of acute inflammation rats. To further reveal the potential antiinflammatory mechanism of VOAS, the metabolomics approach was employed for a second time to determine the metabolic profiling of acute inflammation rats treated with VOAS. A supervised PLS-DA model was established based on the levels of the metabolite markers in Control, Model and MD-VOAS groups. In this model, the first two principal components explained 77.4% and 4.3% of the variance in the data, respectively. According to the scores plot (Fig. 5A), MD-VOAS group was closer to the Control group (the two groups were located in the left side of the figure, which were clearly separated from the
Table 2 Result from pathway analysis with MetaboAnalyst. Total
Expected
Hits
Raw P
−log(P)
Holm P
FDR
Impact
Metabolites
5 9
0.049929 0.089872
1 1
0.04901 0.086602
3.0157 2.4464
1 1
0.70147 0.70147
1 0.4
D-Glucuronic
Arachidonic acid metabolism Glyoxylate and dicarboxylate metabolism Glycine, serine and threonine metabolism Primary bile acid biosynthesis Steroid biosynthesis Citrate cycle (TCA cycle) Starch and sucrose metabolism
36 16 32 46 35 20 23
0.35949 0.15977 0.31954 0.45934 0.3495 0.19971 0.22967
1 1 1 2 1 1 2
0.30643 0.14909 0.27731 0.074614 0.29925 0.18299 0.02079
1.1828 1.9032 1.2826 2.5954 1.2065 1.6983 3.8733
1 1 1 1 1 1 1
1 1 1 0.70147 1 1 0.70147
0.32601 0.2963 0.29197 0.06674 0.05394 0.05356 0.03778
D-Glucose, D-glucuronic
Galactose metabolism Steroid hormone biosynthesis Glutathione metabolism
26 70 26
0.25963 0.699 0.25963
1 1 2
0.23149 0.51349 0.026253
1.4632 0.66653 3.64
1 1 1
1 1 0.70147
0.03644 0.01746 0.00573
Lactic acid Cholesterol Glycine, cadaverine
No.
Pathway name
1 2
Linoleic acid metabolism Ascorbate and aldarate metabolism
3 4 5 6 7 8 9 10 11 12
Linoleic acid acid Arachidonic acid Citric acid Glycine Cholesterol, glycine Cholesterol Citric acid acid
Note: Total is the total number of compounds in the pathway; the Hits is the actually matched number from the user uploaded data; the Raw P is the original P value calculated from the enrichment analysis; the Holm P is the P value adjusted by Holm–Bonferroni method; the FDR P is the P value adjusted using False Discovery Rate; and the Impact is the pathway impact value calculated from pathway topology analysis.
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Fig. 7. Structure of metabolism pathway networks involved in VOAS intervention on acute inflammation.
Model group on the right side), which might suggest that VOAS could reverse the inflammatory reaction process induced by carrageenan. These results showed that pre-treatment of VOAS in rats with acute inflammation induced substantial and characteristic changes in their metabolic profiling. This was quite consistent with the paw edema and plasma HIS and 5-HT results in Sections 3.1 and 3.2. To further evaluate the reversed condition of the potential biomarkers by preadministration of VOAS, t-test was performed. The ratios of relative peak area of the 14 metabolites are shown in Fig. 5C. According to Table 1, Fig. 5C and the heatmap (Fig. 5B) based on the level changes of potential biomarkers in all the three groups, it could be concluded that trends of all the 14 potential biomarkers were reversed by VOAS administration to different degrees. 3.6. Metabolic pathway analysis of VOAS invention on acute inflammation Based on the metabolite markers significantly reversed by VOAS, more detailed analysis of pathways and networks regulated by VOAS were performed using MetaboAnalyst. The potential target metabolic pathway analysis (impact-value ≥ 0.10) revealed that the metabolite markers were important for the invention of VOAS on acute inflammation. Top five metabolic pathways of importance included linoleic acid metabolism, ascorbate and aldarate metabolism, arachidonic acid metabolism, glyoxylate and dicarboxylate metabolism and glycine, serine and threonine metabolism (Fig. 6 and Table 2). The metabolites in the five pathways were mainly linoleic acid, D-glucuronic acid, arachidonic acid, citric acid and glycine. The metabolic networks were structured in Fig. 7 and glycine and arachidonic acid were main nod molecules. 4. Discussion The plasma inflammatory mediators (HIS, 5-HT, NO, etc.) play an important role in evaluating the body's inflammation condition [24]. HIS and 5-HT are both vasoactive amine. HIS can contract vascular endothelium and increase gap between endothelial cells, leading to increase of vascular permeability and exudation of plasma protein and platelet, which cause symptoms of inflammation such as redness, swelling and heat. 5-HT is also known as serotonin. When cell is stimulated by the external environment, 5-HT is released from cell particles into the bloodstream resulting in the increase of vascular permeability, thus leading to inflammation. In the present study, paw edema and the levels of plasma HIS and 5-HT in Model group were significantly higher than
those in Control group, indicating that the acute inflammation model was successfully established. After different doses of VOAS administration, the increased paw edema and inflammatory mediator levels were suppressed, which was compatible with the previous findings [21]. It might be associated with the inhibition of HIS and 5-HT production. From low dose to middle dose, the inhibition effects increased with the increase of dose, and the middle dose peaked. The reason causing this phenomenon might be that low dose didn't arrive at the best blood concentration of inhibiting systemic inflammatory response. But when the dose continued to increase, the inhibition effects declined. This was probably because of negative feedback effect caused by overdose, competitive inhibition between byproducts of former drug metabolism and former drug, or multiple target effect of drugs etc. This doseeffect relationship in our study was similar to the results of antinociceptive effects of Teucrium polium L. total extract and essential oil reported by Mohammad Abdollahi et al. [25], analgesic effects of Eucalyptus essential oils reported by Jeane Silva et al. [26]. In this study, the plasma metabolic characteristics of normal rats and acute inflammation model rats were compared using a GC–MS platform combined with multivariate data analysis. Fourteen differential metabolites were identified, which were reversed by VOAS administration. These metabolites mainly related to linoleic acid metabolism, ascorbate and aldarate metabolism, arachidonic acid metabolism, glyoxylate and dicarboxylate metabolism, and glycine, serine and threonine metabolism. Most of them are directly or indirectly connected with each other (Fig. 7). Glycine, as node molecule, has more connections with other metabolites in different pathways of amino acid metabolism, lipid metabolism and carbohydrate metabolism, whilst arachidonic acid is node molecule in different pathways of lipid metabolism. The results suggested that VOAS exerted anti-inflammatory activity mainly through regulating the metabolic networks centered on glycine and arachidonic acid. In metabolic networks, lipid metabolism is closely related to inflammation [27–29]. Arachidonic acid metabolism is the core of inflammation metabolic network. Arachidonic acid is present in the phospholipid fats in the cell membrane. In response to many inflammatory stimuli, it is released and then oxygenated and further modified to form various eicosanoids, including prostaglandins, thromboxanes and leukotrienes, which act as regulators of inflammation process [30]. In linoleic acid metabolism, δ-6 (D6D) desaturases catalyzes the conversion from linoleic acid to γ-linolenic acid, which is elongated to dihomo-γ-linolenic acid. And dihomo-γ-linolenic acid is in turn
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desaturated to arachidonic acid by δ-5(D5D) desaturases [29]. In the present study, the arachidonic acid level increased and linoleic acid level decreased in Model group compared with those in Control group, which might be ascribed to a large amount of linoleic acid consumption forming arachidonic acid. After VOAS intervention, the level of arachidonic acid decreased and linoleic acid increased, indicating that VOAS reversed the two metabolites. This was similar to the results reported by Yunpeng Qi et al. [21]. Glycine, a key substance in many metabolic reactions, is an inhibitory neurotransmitter in the central nervous system, which can activate a glycine-gated chloride channel (GlyR) expressed in postsynaptic membranes. Activation of the channel allows the influx of chloride, preventing depolarization of the plasma membrane and the potentiation of excitatory signals along the axon [31,32]. Recent studies showed that GlyR was not only distributed in the central nerve cell membrane but also in cell membrane of other tissues such as macrophage, monocytes, neutrophils, T lymphocytes, vascular endothelial cells etc., which participate in inflammation and immune reaction [31,33–35]. Glycine exerts anti-inflammatory activity through inhibiting the activities of these cells. In our study, the level of glycine in Model group increased compared with that in Control group, which was in accordance with previous research results [36–38]. The glycine level increased, probably because enhanced energy metabolism generated pyruvate, and subsequently generated serine and glycine. In the pathway of glycine, serine, and threonine metabolism, hydroxymethyltransferase reversibly converts glycine to serine. After VOAS intervention, the level of glycine decreased, indicating that VOAS could reverse glycine level. In addition, in the ascorbate and aldarate metabolism, D-glucuronic acid can be converted into ascorbate through a sequence of enzymedriven steps [39]. Ascorbate is a powerful water-soluble antioxidant [40] and could also inhibit the activation of myelo-peroxidase/H2O2/ halide system in the leukocytes, thus improving leukocyte movement [41,42]. In our study, the level of D-glucuronic acid in Model group increased compared with that in Control group, indicating that the body was stimulated to release more ascorbate to inhibit inflammatory reaction. Haiyu Liu et al. reported that yeast-induced fever leaded to an intense inflammatory reaction and D-glucuronic acid level increased [39], which was in compatible with our results. After VOAS intervention, the level of D-glucuronic acid decreased, indicating that VOAS reversed D-glucuronic acid level. In glyoxylate and dicarboxylate metabolism, glycine is an important intermediate and glycine and glyoxylate can be mutually conversed by glutamate–glyoxylate aminotransferase [43].
5. Conclusions In conclusion, VOAS possess evident anti-inflammatory activity. VOAS reversed 14 plasma metabolite markers of acute inflammation. VOAS exerted anti-inflammatory activity mainly through regulating the metabolic networks centered on glycine and arachidonic viewing from plasma metabolomics. Conflict of interest The authors confirm that no conflicts of interest exist. Acknowledgments This study was financially supported by the National Natural Science Foundation of China (No. 31272600). We are grateful to all other staff in the Institute of Traditional Chinese Veterinary Medicine of Gansu Agricultural University for their assistance in the experiments. References [1] I.L. Hook, Danggui to Angelica sinensis root: are potential benefits to European women lost in translation? A review, J. Ethnopharmacol. 152 (2014) 1–13.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
The investigation of anti-inflammatory activity of volatile oil of Angelica sinensis by plasma metabolomics approach Wanling Yao, Ling Zhang, Yongli Hua, Peng Ji, Pengling Li, Jinxia Li, Lijia Zhong, Haifu Zhao, Yanming Wei ⁎ Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, PR China
a r t i c l e
i n f o
Article history: Received 1 July 2015 Received in revised form 29 October 2015 Accepted 5 November 2015 Available online 11 November 2015 Keywords: Anti-inflammation Volatile oil of Angelica sinensis (VOAS) Metabolomics
a b s t r a c t Angelica sinensis (AS) is an important medicinal plant, and volatile oil is the main pharmacologically active ingredient. This study was aimed to investigate the anti-inflammatory activity of the volatile oil of A. sinensis (VOAS) and explore its potential anti-inflammatory mechanism by plasma metabolomics approach. Rat acute inflammation was induced by subcutaneous injection of carrageenan in hind paws. Paw edema, histamine (HIS) and 5hydroxytryptamine (5-HT) were detected. Then, we analyzed plasma metabolic profiling of acute inflammation and performed pathway analysis on the metabolite markers reversed after VOAS administration and further integration of metabolic networks. The results showed that VOAS could alleviate the paw edema and decrease plasma HIS and 5-HT levels. Fourteen metabolite markers of acute inflammation were screened, and the levels were all reversed to different degrees after VOAS administration. These metabolite markers mainly related to linoleic acid metabolism, ascorbate and aldarate metabolism, arachidonic acid metabolism, glyoxylate and dicarboxylate metabolism, and glycine, serine and threonine metabolism. In metabolic networks, glycine and arachidonic acid were node molecules. It indicated that VOAS could significantly inhibit systemic inflammatory response triggered by acute local stimulation and it exerted anti-inflammatory activity mainly through regulating the disturbed metabolic networks centered on glycine and arachidonic acid. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Angelica sinensis (AS), commonly known as Danggui in China, is the root of A. sinensis (Oliv.) Diels. Its genuine producing area is in Gansu province, China. It is a popular medicinal plant used in traditional Chinese medicine (TCM) for treatment of menstrual disorders, dysmenorrhea, chronic constipation and other diseases [1–3]. Over 70 compounds have been isolated and identified from AS, including essential oil (such as ligustilide, butylphthalide and senkyunolide A), phthalide dimers, organic acids and their esters (such as ferulic acid), polysaccharides, vitamins, amino acids, and others [4,5]. In previous studies, we studied the hematopoietic function of AS [6] and the hepatoprotective effect of AS polysaccharides [7,8]. Inflammation is a common pathological phenomenon and various inflammatory diseases occupy an extremely important place in human and animal disease spectrum. Excessive or inappropriate inflammatory responses can lead to a range of pathological damage [9]. Increasing traditional Chinese herbs and their active ingredients have become another kind of widely used anti-inflammatory drugs after nonsteroidal and corticosteroid anti-inflammatory drugs. The researches on their anti-inflammatory mechanisms and clinical applications by modern scientific methods have always been the hot spot of the development of new-type anti⁎ Corresponding author. E-mail address: [email protected] (Y. Wei).
http://dx.doi.org/10.1016/j.intimp.2015.11.006 1567-5769/© 2015 Elsevier B.V. All rights reserved.
inflammatory drugs. Volatile oil, an active ingredient of traditional Chinese herb, is abundant in source. It is characterized by smaller molecular weight, rapid absorption and can exert anti-inflammatory action through many ways and many links [10]. Modern pharmacological studies have shown that volatile oils had potent anti-inflammatory activities [11–14]. VOAS is one of the main pharmacologically active ingredients of AS. However, the reports on its anti-inflammatory activity are few at present. Metabolomics, as a novel “-omics” technology in the post gene era, has become an important branch of system biology. The dynamic changes of small molecule (b1 kDa) metabolites represent the physiological and pathological changes of organism [15,16]. Metabolomics has been widely used to observe various metabolic characteristics of control, pathological, and drug-administrated subjects, and further to explore the mechanisms of drug intervention. Moreover, metabolomics is coincident with the integrity and systemic feature of TCM, which usually includes multi-component, multi-pathway, and multi-target treatments [17]. Therefore, metabolomics is a scientific method that can be used to evaluate and explore the mechanisms of medicinal plants used in TCM. In previous work, we explored the intervention of VOAS on local metabolic profiling of carrageenan-induced acute inflammation rats [18]. In order to further research the inhibition effects of VOAS on systemic inflammatory response triggered by local stimulation, the changes of endogenous metabolites in blood were investigated by
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metabolomics approach. This laid the foundation for development of new-type anti-inflammatory drugs. 2. Materials and methods 2.1. Reagents and materials Carrageenan was purchased from Shanghai Zhongqin Chemical Reagent Corporation (Shanghai, China). Aspirin enteric coated tablets were purchased from Bayer healthcare limited company. (Beijing, China). Pyridine was obtained from Kangtai Clinical Reagent Corporation. (Beijing, China). Acetonitrile was obtained from Tedia Corporation (USA). O-methylhydroxylamine hydrochloride, docosane, and Nmethyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) were purchased from Switzerland Fluka. Tween 80 was purchased from Shanghai Genebase Gene-Tech Corporation (Shanghai, China). The ELISA kits for determination of HIS and 5-HT were provided by Nanjing Jiancheng Biological Engineering Institute. (Nanjing, China). 2.2. Preparation of VOAS and phytochemical investigation AS was purchased from Minxian County, Gansu Province, China and authenticated by Prof. Yanming Wei (College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, China). VOAS was prepared according to the Chinese pharmacopeia [19]. AS (200 g) was ground into powder, passed through a 40-mesh sieve, mixed with 1000 mL of distilled water, and soaked for 1 day. Then, the mixture was placed in an extracting device, and subjected to hydro-distillation for 8 h to get volatile oil. Almost 0.6 mL of canary clear oil-like volatile oil was obtained. Anhydrous sodium sulfate was used to remove water from the volatile oil, which was stored in amber laboratory bottle at 4 °C for analysis [20]. Then, more than 40 components were finally identified by GC– MS, and the main components were (Z)-ligustilide (78.61%), (Z)butylidenephthalide (7.99%), (E)-ligustilide (1.66%) and (E)butylidenephthalide (1.29%). Before use, VOAS was dissolved in 2% Tween 80 (diluted by normal saline).
2.3. In vivo experiments protocol Acute toxicity study was previously performed and the lethal dose 50 (LD50) of VOAS was 1.76 mL/kg in rats. Then, 1/5 (0.352 mL/kg), 1/ 10 (0.176 mL/kg) and 1/20 (0.088 mL/kg) of LD50 were selected as the high, middle and low dose, respectively. Sixty male Wistar rats (180–210 g) were supplied by the experimental Animal Center of Lanzhou University, approved no. SCXK (Gan) 2012-0004. The room temperature was regulated at 23 ± 1 °C with 50% ± 5% humidity. A 12 h light/dark cycle was set, and the rats were fed with a standard diet and given free access to water. After acclimatization for 7 days, the rats were randomly divided into six groups with 10 rats in each group: normal control group (Control group); acute inflammation group (Model group); high-dose VOAS group (HD-VOAS group); middle-dose VOAS group (MD-VOAS group); low-dose VOAS group (LD-VOAS); and aspirin positive control group (ASP group). Control and Model groups were gavaged with 2% Tween 80. HD, MD and LD-VOAS groups were gavaged with VOAS (0.352, 0.176, and 0.088 mL/kg/day, respectively). ASP group received aspirin (200 mg/kg/day). Drugs and 2% Tween 80 were orally administrated once daily for 3 consecutive days. Acute inflammation was induced 0.5 h after gavage on the third day. Rats in Model, HD, MD and LD-VOAS groups were subcutaneously injected with 0.1 mL of 1.0% carrageenan in the sub-plantar region of the right hind paws, and corresponding volume of normal saline in Control group [18,21]. The degree of paw edema was calculated by the difference of its volume before and 3 h after carrageenan injection [22]. After that, the rats were anesthetized intraperitoneally with 1% pentobarbital sodium. Blood samples were collected from abdominal aorta. Animal welfare and experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006) and were approved by the Animal Ethics Committee of Gansu Agricultural University. 2.4. Plasma biochemical parameters All the blood samples were collected in heparinized tubes and centrifuged at 3000 rpm (centrifugal radius was 12 cm) for 10 min. Plasma samples were separated and stored at −80 °C for further use. One part of the each plasma sample was used for HIS and 5-HT tests, and the other part was used for GC–MS metabolomics analysis. The plasma samples were thawed at room temperature prior to analysis. HIS and 5-HT tests were performed according to the instructions. 2.5. GC–MS sample preparation
Fig. 1. Effects of different doses of VOAS on carrageenan-induced paw edema in rats. Paw edema was calculated by the difference of its volume before and 3 h after carrageenan injection. Values are expressed as mean ± SD (n = 10). Control indicates normal control group. Model group (acute inflammation group) received 2% Tween 80, ASP group (aspirin positive control group) received aspirin (200 mg/kg/day), and HD, MD and LD-VOAS groups (high, middle and low-dose VOAS groups) received VOAS (0.352, 0.176 and 0.088 ml/kg/day, respectively). For each group, the same lowercase letter (placed next to the numeric value) indicates that there are no significant differences between groups, while different lowercase letters (a, b, c) show significant differences with a P b 0.05. The lowercase letter “a” indicates the maximum value and “a”, “b”, “c”, and “d” decrease in turn.
In the present study, the plasma samples were processed according to the method described in ref. [7] with minor modifications. The plasma samples were thawed at room temperature prior to analysis. Firstly, 250 μL of acetonitrile was added into 100 μL of the sample for protein-precipitation. The mixture was ultrasonically extracted for 10 min, followed by centrifugation (4000 rpm) for another 10 min. Then, 300 μL supernatant was transferred to the GC vial and evaporated to dryness under a stream of nitrogen gas. Next, 100 μL of methoxyamine pyridine solution (15 μg/mL) was added to the vial, and methoxymation was performed at 70 °C for 1 h. MSTFA (50 μL, with 1% TMCS) was added, and the silylation was performed at 70 °C for 1 h. After adding 150 μL of n-heptane (with 0.10 mg/mL of docosane as internal standard) and vortex mixing, the mixture was centrifuged for 10 min. The supernatant was transferred to the GC microvial for GC–MS analysis. 2.6. GC–MS analysis GC–MS analysis was carried out using an Agilent 6890N/5973N series GC–MS (Agilent Corporation, USA) equipped with an OV-1701
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Fig. 2. Effects of different doses of VOAS on the levels of HIS (A) and 5-HT (B). The levels of HIS and 5-HT were detected using the corresponding ELISA kits. Values are expressed as mean ± SD (n = 10). Control indicates normal control group. Model group (acute inflammation group) received 2% Tween 80, ASP group (aspirin positive control group) received aspirin (200 mg/kg/day), and HD, MD and LD-VOAS groups (high, middle and low-dose VOAS groups) received VOAS (0.352, 0.176 and 0.088 ml/kg/day, respectively). For each group, the same lowercase letter (placed next to the numeric value) indicates that there are no significant differences between groups, while different lowercase letters (a, b, c) show significant differences with a P b 0.05. The letter “a” indicates the maximum value and “a”, “b”, “c”, and “d” decrease in turn.
capillary column (30 m × 0.15 μm × 15 mm). The initial temperature was maintained at 85 °C for 3 min, and then raised to 280 °C at a rate of 10 °C/min. All samples were injected in split mode at 270 °C. The mass spectrometer was operated in EI mode (positive ion, 70 eV), and the quadrupole was 150 °C. Mass spectra were acquired in full scan mode with repetitive scanning from 60 m/z to 600 m/z for 1 s. The ion source temperature was 230 °C. 2.7. Data processing GC–MS data were exported in comma-separated value (csv) format and then processed by Agilent MSD ChemStation. Raw data were filtered and then subjected to retention time correction and peak alignment [8]. The data matrix was normalized to the sum of all peak areas of each sample, and then the missing value was eliminated via the 80% correction method. Pattern recognition based on principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) was accomplished by using SIMCA-P V11.5 (Umetrics, Sweden) after meancentering and auto scaling. Potential metabolite markers screening was performed according to the value of variable importance in the projection (VIP) (VIP N 1.0) from the PLS-DA model and t-test (P b 0.05). Potential metabolite markers of interest were identified according to the National Institute of Standards and Technology (NIST) mass spectra library. With the completion of PLS-DA, we could try computational systems analysis with the MetaboAnalyst data annotation approach
including a correlation analysis plot of the differential metabolites, and heatmap visualization.
2.8. Statistical analysis Statistically significant differences of paw edema, HIS and 5-HT levels were tested by one-way analysis of variance (ANOVA) followed by the Duncan's multiple range test, and differential metabolites levels were tested by Student's t-test. These were performed on IBM SPSS V.21.0 (SPSS Inc., Chicago, USA) and Microsoft Office Excel 2013. The significant difference was considered at P b 0.05, and extremely significant difference was considered at P b 0.01.
2.9. Construction and analysis of metabolic pathway The construction, interaction, and pathway analysis of potential biomarkers significantly reversed by VOAS was performed with MetaboAnalyst based on database sources, including the Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/) and the Human Metabolome Database (http://www.hmdb.ca/), to identify the affected metabolic pathway analysis and visualization.
3. Results 3.1. Evaluation of the anti-inflammatory activity of VOAS
Fig. 3. Typical GC–MS TIC chromatograms of the plasma samples from Control, Model and MD-VOAS groups.
The inhibition effects of different doses of VOAS on carrageenaninduced paw edema in rats were shown in Fig. 1. The degrees of paw edema in Model group were significantly elevated 3 h after carrageenan injection compared with those in Control group (P b 0.05), indicating that acute inflammation models were successfully established. All drugs significantly attenuated the paw edema of carrageenan-induced acute inflammation rats (P b 0.05). When different doses of VOAS groups were compared with ASP group, there was no significant difference in the paw edema between MD-VOAS group and ASP group (P N 0.05), indicating that the inhibition effect of MD-VOAS on paw edema was close to that of aspirin. The paw edema in HD-VOAS and LD-VOAS groups was significantly higher than that in ASP group (P b 0.05), indicating that their inhibition effects were significantly lower than that of aspirin. The above results indicated that the inhibition effect of MDVOAS on paw edema was the best.
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Fig. 4. Comparison of metabolic profiling between Control and Model groups and screening of the metabolite markers: (A) Scores plot from PCA model classifying Control and Model groups. Modeling diagnostic, R2X = 0.992 and Q2 = 0.944. (B) Scores plot from PLS-DA model classifying Control and Model groups. Modeling diagnostic, R2X = 0.909, R2Y = 0.987, and Q2 = 0.972. (C) Correlation analysis plot of the differential metabolites. (D) Heatmap visualization for the carrageenan-induced acute inflammation rats. The heatmaps were constructed based on the potential biomarkers of importance and implemented in MetaboAnalyst, and they are commonly used for unsupervised clustering. Rows: metabolites; columns: samples; color key indicates metabolite expression value, red: highest and green: lowest.
Table 1 Summary of the potential metabolite markers revealed in this study. No.
Metabolites
R.T. (min)
Chemical class
VIP value
Model
MD-VOAS
Pathways
1 2 3
Arachidonic acid Malic acid
37.29 15.82 24.29
Unsaturated fatty acids Hydroxycarboxylic acids Others
1.873 1.819 1.732
↑## ↓## ↑##
Arachidonic acid metabolism Citrate cycle (TCA cycle) Ascorbate and aldarate metabolism
32.49 13.98 28.51 40.25 11.78 10.89 21.55
Unsaturated fatty acids Common amino acids Saturated fatty acids Lipids Lactic acid Polyamines Aldoses
1.698 1.582 1.522 1.398 1.348 1.300 1.238
↓## ↑## ↓## ↓## ↑## ↑## ↓##
↓⁎⁎ ↑⁎⁎ ↓⁎⁎ ↑⁎⁎ ↓⁎⁎ ↑⁎⁎ ↑⁎⁎ ↓⁎⁎ ↓⁎⁎ ↑⁎⁎
13.32 33.29 10.30 12.03
Tricarboxylic acids Saturated fatty acids Others Amino acids and derivatives
1.168 1.123 1.086 1.044
↓## ↓## ↑## ↓##
4 5 6 7 8 9 10 11 12 13 14
D-Glucuronic acid Linoleic acid Glycine Hexadecanoic acid Cholesterol Lactic acid Cadaverine D-Glucose Citric acid Stearic acid Urea Alanine
↑⁎⁎ ↑⁎⁎ ↓⁎⁎ ↑⁎⁎
Linoleic acid metabolism Glycine, serine and threonine metabolism Fatty acid biosynthesis Steroid biosynthesis Pyruvate metabolism Lysine degradation Starch and sucrose metabolism Glyoxylate and dicarboxylate metabolism Fatty acid biosynthesis Purine metabolism Alanine, aspartate and glutamate metabolism
Note: The up- (or down-) arrows represent the relatively increased (or decreased) levels of the potential metabolite markers in Model group or MD-VOAS group. ## P b 0.01, significant differences compared with Control group. ⁎⁎ P b 0.01, significant differences compared with Model group.
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Fig. 5. VOAS intervention on acute inflammation rats. (A) PLS-DA scores plot derived from plasma levels of 14 metabolite markers in Control group (C, red triangles), Model group (M, green crosses) and MD-VOAS group (MD, blue crosses). (B) Heatmap visualization based on the levels of metabolite markers in Control, Model and MD-VOAS groups. Columns correspond to samples, and variables marked on the right correspond to metabolites. Color key indicates metabolite expression value, red: highest and green: lowest. (C) Bar graphs show the relative peak area ratios of the 14 metabolite markers in Control, Model, and MD-VOAS groups. Data are expressed as mean ± SD. **P b 0.01 versus Model group.
3.2. Plasma biochemical assay The inhibition effects of different doses of VOAS on the levels of plasma HIS and 5-HT were shown in Fig. 2A–B. Compared with Control group, the levels of plasma HIS and 5-HT in Model group were significantly elevated 3 h after carrageenan injection (P b 0.05), indicating that carrageenan-induced local inflammation stimulation triggered systematic inflammatory response. Fig. 2A showed that all drugs significantly decreased the plasma HIS level of carrageenan-induced acute inflammation rats (P b 0.05). When different doses of VOAS groups were compared with ASP group, there was no significant difference in the plasma HIS level between MDVOAS group and ASP group (P N 0.05), indicating that the inhibition effect of MD-VOAS on plasma HIS level was close to that of aspirin. The plasma HIS level in HD-VOAS and LD-VOAS groups was significantly higher than that in ASP group (P b 0.05), indicating that their inhibition effects were significantly lower than that of aspirin. The above results indicated that the inhibition effect of MD-VOAS on plasma HIS level was the best.
Fig. 2B showed that all drugs decreased the plasma 5-HT level of carrageenan-induced acute inflammation rats. There was significant difference between MD-VOAS group and Model group (P b 0.05) while not between HD-VOAS and LD-VOAS groups and Model group (P N 0.05). The above results indicated that the inhibition effect of MD-VOAS on 5-HT was the best. Based on the inhibition effects of different doses of VOAS on paw edema, HIS and 5-HT, middle-dose VOAS was used to research the anti-inflammatory mechanism of VOAS by metabolomics approach. 3.3. GC–MS analysis Prior to the analysis of the experimental samples, the applied GC–MS method was validated including the precision of injection, the withinday stability and the repeatability of sample preparation. The precision of injection was evaluated by analyzing six independently processed replicates; stability was evaluated by analyzing one sample at 0, 1, 2, 3, 6, 9, 12 and 24 h; the repeatability of sample preparation was investigated by preparing six parallel samples using the same preparation
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Fig. 6. Summary of pathway analysis with MetaboAnalyst. A) Linoleic acid metabolism; B) ascorbate and aldarate metabolism; C) arachidonic acid metabolism; D); glyoxylate and dicarboxylate metabolism; and E) glycine, serine and threonine metabolism.
protocol, and then the relative standard deviations (RSD) of retention times and peak intensities were calculated, respectively. The RSDs of retention time for precision, stability and repeatability were estimated to be 0.29–0.48%, 0.29–0.32% and 0.58–1.23%, respectively, and the RSDs of intensity varied within the ranges of 1.37–2.68%, 1.16–2.58% and 1.33–1.85%. The good precision, stability and repeatability indicated that the method could be utilized to the analysis of plasma samples. Typical GC–MS TIC chromatograms of the plasma samples from Control, Model and MD-VOAS groups were shown in Fig. 3. Forty-six endogenous metabolites in the plasma samples were identified by NIST library. These metabolites, mainly including carbohydrates, fatty acids, amino acids, and organic acids, related to glycometabolism, lipid metabolism, and amino acid metabolism. 3.4. Comparison of metabolic profiling between Control and Model groups and screening of the metabolite markers First, the unsupervised PCA analysis on the data of Control and Model groups was performed to visualize general clustering, trends, or outliers among the observations, and also to know whether the two groups could be distinguished based on their metabolic profiling
provided by GC–MS. The PCA scores plot (Fig. 4A) showed that the separation between Control and Model groups was obvious, indicating that the two groups had completely different metabolic profiling. Next, in order to evaluate the systemic changes in the metabolomics of carrageenan-induced acute inflammation rats, a PLS-DA model was established based on the data of Control and Model groups. As shown in the scores plot (Fig. 4B), all samples in the two groups fell well inside the 95% confidence interval, which was represented by an ellipse. The quality parameters of the PLS-DA model between Control and Model groups were: R2X = 0.909, R2Y = 0.987, and Q2 = 0.972. R2Y estimates the goodness of fit of the model that represents the fraction of explained Y-variation, and Q2 estimates the ability of prediction. The cumulative values of R2Y and Q2 are N 0.8, indicating that reliable models are obtained [23]. Therefore, a reliable PLS-DA model was established. Fourteen variables with the VIP N 1.5 and P b 0.05 were selected and considered as potential biomarkers representing the metabolic characteristics of carrageenan-induced acute inflammation rats (Table 1). In order to further investigate the carrageenan-induced acute inflammation metabolic profiling, acquired data were subjected to computational systems analysis with MetaboAnalyst data annotation approach including a correlation analysis plot of the differential metabolites (Fig. 4C), and heatmap visualization (Fig. 4D). Correlation analysis of the 14 differential metabolites are marked on the hierarchical clustering plot which was performed to understand the potential relationships among the differential metabolites, which were shown on the plot in different colors. The heatmap, commonly used for unsupervised clustering, was constructed based on the potential differential metabolites of importance, implemented in MetaboAnalyst, which was consistent with the results in Fig. 4B. From the plots, various differential metabolites could be identified as responsible for the separation between Control and Model groups, and therefore these were viewed as potential biomarkers. 3.5. Metabolomics evaluation of VOAS intervention on acute inflammation rats As shown in Sections 3.1 and 3.2, VOAS has shown obvious inhibition effects on the paw edema and plasma HIS and 5-HT levels of acute inflammation rats. To further reveal the potential antiinflammatory mechanism of VOAS, the metabolomics approach was employed for a second time to determine the metabolic profiling of acute inflammation rats treated with VOAS. A supervised PLS-DA model was established based on the levels of the metabolite markers in Control, Model and MD-VOAS groups. In this model, the first two principal components explained 77.4% and 4.3% of the variance in the data, respectively. According to the scores plot (Fig. 5A), MD-VOAS group was closer to the Control group (the two groups were located in the left side of the figure, which were clearly separated from the
Table 2 Result from pathway analysis with MetaboAnalyst. Total
Expected
Hits
Raw P
−log(P)
Holm P
FDR
Impact
Metabolites
5 9
0.049929 0.089872
1 1
0.04901 0.086602
3.0157 2.4464
1 1
0.70147 0.70147
1 0.4
D-Glucuronic
Arachidonic acid metabolism Glyoxylate and dicarboxylate metabolism Glycine, serine and threonine metabolism Primary bile acid biosynthesis Steroid biosynthesis Citrate cycle (TCA cycle) Starch and sucrose metabolism
36 16 32 46 35 20 23
0.35949 0.15977 0.31954 0.45934 0.3495 0.19971 0.22967
1 1 1 2 1 1 2
0.30643 0.14909 0.27731 0.074614 0.29925 0.18299 0.02079
1.1828 1.9032 1.2826 2.5954 1.2065 1.6983 3.8733
1 1 1 1 1 1 1
1 1 1 0.70147 1 1 0.70147
0.32601 0.2963 0.29197 0.06674 0.05394 0.05356 0.03778
D-Glucose, D-glucuronic
Galactose metabolism Steroid hormone biosynthesis Glutathione metabolism
26 70 26
0.25963 0.699 0.25963
1 1 2
0.23149 0.51349 0.026253
1.4632 0.66653 3.64
1 1 1
1 1 0.70147
0.03644 0.01746 0.00573
Lactic acid Cholesterol Glycine, cadaverine
No.
Pathway name
1 2
Linoleic acid metabolism Ascorbate and aldarate metabolism
3 4 5 6 7 8 9 10 11 12
Linoleic acid acid Arachidonic acid Citric acid Glycine Cholesterol, glycine Cholesterol Citric acid acid
Note: Total is the total number of compounds in the pathway; the Hits is the actually matched number from the user uploaded data; the Raw P is the original P value calculated from the enrichment analysis; the Holm P is the P value adjusted by Holm–Bonferroni method; the FDR P is the P value adjusted using False Discovery Rate; and the Impact is the pathway impact value calculated from pathway topology analysis.
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Fig. 7. Structure of metabolism pathway networks involved in VOAS intervention on acute inflammation.
Model group on the right side), which might suggest that VOAS could reverse the inflammatory reaction process induced by carrageenan. These results showed that pre-treatment of VOAS in rats with acute inflammation induced substantial and characteristic changes in their metabolic profiling. This was quite consistent with the paw edema and plasma HIS and 5-HT results in Sections 3.1 and 3.2. To further evaluate the reversed condition of the potential biomarkers by preadministration of VOAS, t-test was performed. The ratios of relative peak area of the 14 metabolites are shown in Fig. 5C. According to Table 1, Fig. 5C and the heatmap (Fig. 5B) based on the level changes of potential biomarkers in all the three groups, it could be concluded that trends of all the 14 potential biomarkers were reversed by VOAS administration to different degrees. 3.6. Metabolic pathway analysis of VOAS invention on acute inflammation Based on the metabolite markers significantly reversed by VOAS, more detailed analysis of pathways and networks regulated by VOAS were performed using MetaboAnalyst. The potential target metabolic pathway analysis (impact-value ≥ 0.10) revealed that the metabolite markers were important for the invention of VOAS on acute inflammation. Top five metabolic pathways of importance included linoleic acid metabolism, ascorbate and aldarate metabolism, arachidonic acid metabolism, glyoxylate and dicarboxylate metabolism and glycine, serine and threonine metabolism (Fig. 6 and Table 2). The metabolites in the five pathways were mainly linoleic acid, D-glucuronic acid, arachidonic acid, citric acid and glycine. The metabolic networks were structured in Fig. 7 and glycine and arachidonic acid were main nod molecules. 4. Discussion The plasma inflammatory mediators (HIS, 5-HT, NO, etc.) play an important role in evaluating the body's inflammation condition [24]. HIS and 5-HT are both vasoactive amine. HIS can contract vascular endothelium and increase gap between endothelial cells, leading to increase of vascular permeability and exudation of plasma protein and platelet, which cause symptoms of inflammation such as redness, swelling and heat. 5-HT is also known as serotonin. When cell is stimulated by the external environment, 5-HT is released from cell particles into the bloodstream resulting in the increase of vascular permeability, thus leading to inflammation. In the present study, paw edema and the levels of plasma HIS and 5-HT in Model group were significantly higher than
those in Control group, indicating that the acute inflammation model was successfully established. After different doses of VOAS administration, the increased paw edema and inflammatory mediator levels were suppressed, which was compatible with the previous findings [21]. It might be associated with the inhibition of HIS and 5-HT production. From low dose to middle dose, the inhibition effects increased with the increase of dose, and the middle dose peaked. The reason causing this phenomenon might be that low dose didn't arrive at the best blood concentration of inhibiting systemic inflammatory response. But when the dose continued to increase, the inhibition effects declined. This was probably because of negative feedback effect caused by overdose, competitive inhibition between byproducts of former drug metabolism and former drug, or multiple target effect of drugs etc. This doseeffect relationship in our study was similar to the results of antinociceptive effects of Teucrium polium L. total extract and essential oil reported by Mohammad Abdollahi et al. [25], analgesic effects of Eucalyptus essential oils reported by Jeane Silva et al. [26]. In this study, the plasma metabolic characteristics of normal rats and acute inflammation model rats were compared using a GC–MS platform combined with multivariate data analysis. Fourteen differential metabolites were identified, which were reversed by VOAS administration. These metabolites mainly related to linoleic acid metabolism, ascorbate and aldarate metabolism, arachidonic acid metabolism, glyoxylate and dicarboxylate metabolism, and glycine, serine and threonine metabolism. Most of them are directly or indirectly connected with each other (Fig. 7). Glycine, as node molecule, has more connections with other metabolites in different pathways of amino acid metabolism, lipid metabolism and carbohydrate metabolism, whilst arachidonic acid is node molecule in different pathways of lipid metabolism. The results suggested that VOAS exerted anti-inflammatory activity mainly through regulating the metabolic networks centered on glycine and arachidonic acid. In metabolic networks, lipid metabolism is closely related to inflammation [27–29]. Arachidonic acid metabolism is the core of inflammation metabolic network. Arachidonic acid is present in the phospholipid fats in the cell membrane. In response to many inflammatory stimuli, it is released and then oxygenated and further modified to form various eicosanoids, including prostaglandins, thromboxanes and leukotrienes, which act as regulators of inflammation process [30]. In linoleic acid metabolism, δ-6 (D6D) desaturases catalyzes the conversion from linoleic acid to γ-linolenic acid, which is elongated to dihomo-γ-linolenic acid. And dihomo-γ-linolenic acid is in turn
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desaturated to arachidonic acid by δ-5(D5D) desaturases [29]. In the present study, the arachidonic acid level increased and linoleic acid level decreased in Model group compared with those in Control group, which might be ascribed to a large amount of linoleic acid consumption forming arachidonic acid. After VOAS intervention, the level of arachidonic acid decreased and linoleic acid increased, indicating that VOAS reversed the two metabolites. This was similar to the results reported by Yunpeng Qi et al. [21]. Glycine, a key substance in many metabolic reactions, is an inhibitory neurotransmitter in the central nervous system, which can activate a glycine-gated chloride channel (GlyR) expressed in postsynaptic membranes. Activation of the channel allows the influx of chloride, preventing depolarization of the plasma membrane and the potentiation of excitatory signals along the axon [31,32]. Recent studies showed that GlyR was not only distributed in the central nerve cell membrane but also in cell membrane of other tissues such as macrophage, monocytes, neutrophils, T lymphocytes, vascular endothelial cells etc., which participate in inflammation and immune reaction [31,33–35]. Glycine exerts anti-inflammatory activity through inhibiting the activities of these cells. In our study, the level of glycine in Model group increased compared with that in Control group, which was in accordance with previous research results [36–38]. The glycine level increased, probably because enhanced energy metabolism generated pyruvate, and subsequently generated serine and glycine. In the pathway of glycine, serine, and threonine metabolism, hydroxymethyltransferase reversibly converts glycine to serine. After VOAS intervention, the level of glycine decreased, indicating that VOAS could reverse glycine level. In addition, in the ascorbate and aldarate metabolism, D-glucuronic acid can be converted into ascorbate through a sequence of enzymedriven steps [39]. Ascorbate is a powerful water-soluble antioxidant [40] and could also inhibit the activation of myelo-peroxidase/H2O2/ halide system in the leukocytes, thus improving leukocyte movement [41,42]. In our study, the level of D-glucuronic acid in Model group increased compared with that in Control group, indicating that the body was stimulated to release more ascorbate to inhibit inflammatory reaction. Haiyu Liu et al. reported that yeast-induced fever leaded to an intense inflammatory reaction and D-glucuronic acid level increased [39], which was in compatible with our results. After VOAS intervention, the level of D-glucuronic acid decreased, indicating that VOAS reversed D-glucuronic acid level. In glyoxylate and dicarboxylate metabolism, glycine is an important intermediate and glycine and glyoxylate can be mutually conversed by glutamate–glyoxylate aminotransferase [43].
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