ITRAQ-based quantitative proteomic analysis of processed Euphorbia lathyris L. for reducing the intestinal toxicity

Abstract

Background

Euphorbia lathyris L., a Traditional Chinese medicine (TCM), is commonly used for the treatment of hydropsy, ascites, constipation, amenorrhea, and scabies. Semen Euphorbiae Pulveratum, which is another type of Euphorbia lathyris that is commonly used in TCM practice and is obtained by removing the oil from the seed that is called paozhi, has been known to ease diarrhea. Whereas, the mechanisms of reducing intestinal toxicity have not been clearly investigated yet.

Methods

In this study, the isobaric tags for relative and absolute quantitation (iTRAQ) in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic method was applied to investigate the effects of Euphorbia lathyris L. on the protein expression involved in intestinal metabolism, in order to illustrate the potential attenuated mechanism of Euphorbia lathyris L. processing. Differentially expressed proteins (DEPs) in the intestine after treated with Semen Euphorbiae (SE), Semen Euphorbiae Pulveratum (SEP) and Euphorbiae Factor 1 (EFL₁) were identified. The bioinformatics analysis including GO analysis, pathway analysis, and network analysis were done to analyze the key metabolic pathways underlying the attenuation mechanism through protein network in diarrhea. Western blot were performed to validate selected protein and the related pathways.

Results

A number of differentially expressed proteins that may be associated with intestinal inflammation were identified. They mainly constituted by part of the cell. The expression sites of them located within cells and organelles. G protein and Eph/Ephrin signal pathway were controlled jointly by SEP and SE. After processing, the extraction of SEP were mainly reflected in the process of cytoskeleton, glycolysis and gluconeogenesis.

Conclusions

These findings suggest that SE induced an inflammatory response, and activated the Interleukin signaling pathway, such as the Ang/Tie 2 and JAK2/ STAT signaling pathways, which may eventually contribute to injury result from intestinal inflammatory, while SEP could alleviate this injury by down-regulating STAT1 and activating Ang-4 that might reduce the inflammatory response. Our results demonstrated the importance of Ang-4 and STAT1 expression, which are the target proteins in the attenuated of SE after processing based on proteomic investigation. Thus iTRAQ might be a novel candidate method to study scientific connotation of hypothesis that the attenuated of SE after processing expressed lower toxicity from cellular levels.

Background

Euphorbia lathyris L. is an effective but toxic traditional Chinese medicine (TCM) derived from the family of euphorbiaceae. It can expel water retention with drastic purgative effects, namely, breaking up the static blood and eliminating masses and is often used for the treatment of hydropsy, ascites, anuresis and constipation, amenorrhea, scabies [1, 2]. It shows several side effects such as irritation and inflammation intense on the skin, mouth and gastrointestinal tract irritation, carcinogenic, and so on. The gastrointestinal mucosa irritation mainly manifested as serious diarrhea. Traditionally, Semen Euphorbiae Pulveratum (SEP), which is another type of Euphorbia lathyris L., is commonly used in TCM practice and is obtained by removing the oil from the seed which is called paozhi. After processing, the toxicity and the capacity of diarrhea was decreased obviously [3]. Interestingly, considerable research efforts have been devoted to the studies on the effect of SEP and SE on diarrhea. Whereas, the intestine protein changes related to intestinal toxicity and the main mechanisms of reducing toxicity by processing of SE remain poorly understood.

With the improvement of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry [4], considerable research efforts have been devoted to the application of proteomics to find possible involved signals in toxic injure induced by some toxins or to determine the modes of action and mechanisms involved in drug- or chemical-induced toxicity [5, 6]. The isobaric tags for relative and absolute quantitation (iTRAQ) technique is one of the most widely used, innovative and common quantitative proteomics approaches that measure the qualitative and quantitative changes in protein content of a cell or tissue in response to treatment or disease and determine protein-protein and protein-ligand interactions [7]. It can simultaneously analyze 4–8 different specimens, thus increasing throughput while reducing experimental error [8, 9]. iTRAQ labeling coupled with LC-MS/MS is sensitive, automated, and multidimensional and can detect large molecules (> 20 kDa) [10]. ITRAQ is suitable for exploratory studies of the processing mechanisms.

In our study, we applied iTRAQ approach to processing for Euphorbia lathyris-induced intestinal toxicity and to identify candidate biomarkers for main mechanisms underlying processing of SE. Bioinformatics analysis including GO analysis, pathway analysis, and network analysis were done to find possible differential pathways. Additionally, the investigation suggested that Euphorbiae factor 1(EFL₁), isolated from Euphorbia lathyris, is the main and active diterpenoids which might mediate diarrhea [11]. We also demonstrated EFL₁ group to further compare the DEPs induced by SE and SEP. Finally, western blot analysis was applied further to identify candidate biomarkers, and to confirm and validate significance of the proteomic findings. These results provided a first insight into scientific connotation of hypothesis that the attenuated of SE after processing expressed lower toxicity from cellular levels in mice model and described an efficient method for mechanisms of toxic TCM processing.

Methods

Samples

Experimental animals

KM mice (SPF grade, 18–22 g) were purchased from Sibeifu Co., Ltd. (Beijing, China). All experiments were approved by the Animal Care Committee. Mice were kept at room temperature (23 ± 1 °C) and 55 ± 5% humidity. All experiments were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animal, as adopted by the Committee on Animal Research at Beijing University of Chinese Medicine.

Extracts preparations of semen euphorbiae and semen euphorbiae Pulveratum

Pieces of Euphorbiae Semen (batch number, 1203070692; origin, Jiangxi province, China) were purchased from Anhui Bozhou HuQiao Chinese Herbal Pieces plant. Petroleum ether extract of Semen Euphorbiae, petroleum ether extract of Semen Euphorbiae Pulveratum was provided by Shandong University of Traditional Chinese Medicine. The extraction and isolation methods of Semen Euphorbiae had been published in these articles [12, 13]. Euphorbiae factor 1 was isolated from the petroleum ether extracts of semen Euphorbia by our team [13, 14].

Proteomics extraction procedures

Protein preparation

After 12 h of fasting, KM mice were randomly divided into 4 groups (n = 10 for each group): the group 1 was served as a control, and received only blank 1% sodium carboxymethyl cellulose solution; meanwhile group 2 was the extracts of SE and group 3 was the extraction of SEP, in which the mice were orally administered at the dose of 1.5 ml/20 g and 1.0 ml/20 g, respectively, with the same amount of crude drug. In order to validate the results induced by SE and SEP, group 4 was administered 20 mg/20 g Euphorbiae factor 1(EFL₁) to further verify the protein networks. Mice then received standard diet and water ad libitum. 6 h later, mice were sacrificed, from which the colon were obtained and frozen in liquid nitrogen immediately until they were used for analysis.

Protein isolation

The colon tissue samples were ground into powder in liquid nitrogen, extracted with Lysis buffer (7 M urea (Bio-Rad, 161–0731), 2 M Thiourea (Sigma-Aldrich, T7875), 4% CHAPS (Bio-Rad, 161–0460)) containing complete protease inhibitor Cocktai (Roche, 04693116001). The cell was lysed by sonication at 200 W for 60s and then extracted 30 min at room temperature, centrifuged at 4 °C, 15000 g for 20 min. Before the protein processing, each 5 individual protein samples were mixed equally into 1 specimen. As a result of the strategy, each group contained 2 sample pools, and these sample pools were enrolled to be conducted in subsequent experiments.

Bradford analysis

Total protein concentration of the samples was determined using a Bradford Assay [15]. Standards of BSA were prepared and all samples and standards were analyzed in duplicate. Protein concentrations and standards of BSA were determined at 595 nm on an Multiskan MK3 UV–vis spectrophotometer (Thermo, U.S.) with 10 μL sample reacted with 300 μL Thermo Scientific Pierce Coomassie Plus Bradford Assay (Part No. 23238) 20 min.

Protein reduction, alkylation, and digestion

Filter-aided sample preparation (FASP) method was used to digest protein based on Jacek R Wis’niewski et al. [16]. The 200 μg calculated protein samples were added to centrifuge tube and 25 mM DTT was added and the samples were incubated at 60 °C for 1 h. Samples were incubated for 10 min in the dark after adding 50 mM IAA at room temperature and then centrifuged at 12,000 rpm for 20 min using Ultrafiltration centrifugal tube(NWCO:10 K). 100 μL Dissolution Buffer(iTRAQ ® Kit Dissolution Buffer, AB Sciex, USA, PN:4381664) was added to the filter and centrifuged at 12,000 rpm for 20 min. This step was repeated three times.50 μL trypsin, totally 4 μg, was added and samples were incubated at 37 °C overnight. After trypsin digestion, samples were centrifuged at 12,000 rpm for 20 min, the digested peptides were collected at the bottom of the tube and mixed with 50 μL Dissolution Buffer. Finally 100 μL samples were obtained.

iTRAQ labeling

Each iTRAQ reagent tube (tags-113-121) had 150 μl isopropanol added and vortexed thoroughly, then centrifuged. 50 μl samples (equal to 100 μg digested peptides) were transferred to new tubs and processed according to the manufacturer’s protocol for 8-plex iTRAQ reagent (AB Sciex, PN:4390812) by incubation at RT for 2 h with gentle shaking. The labeled peptide mixtures were then pooled and dried by vacuum centrifugation. Samples were labeled respectively with different isobaric tags as follow: EFL₁ samples labeled 113 and 114, control samples labeled 115 and 116, and extraction of SE samples labeled 117 and 118, extraction of SEP samples labeled 119 and 121. The peptides were labeled with the isobaric tags, incubated at room temperature for 2 h. The labeled peptide mixtures were then pooled and dried by vacuum centrifugation.

iTRAQ-labeled peptide fractionation and proteomic analysis by LC-MS/MS

The iTRAQ-labeled peptide mixtures were re-suspended in buffer A (2% ACN, pH 10) and centrifuged at 14,000 g for 20 min. High pH reversed-phase chromatography was performed to separate the trypsin digestion peptide. The supernatant was loaded onto a 4.6 × 250 mm Durashell-C₁₈ containing 5-μm particles. The peptides were eluted at a flow rate of 0.7 mL/min with a 51-min gradient:0-10 min,5.0% B (Mobile phaseA:2%ACN,98%ddH₂O,pH 10;Mobile phaseB:98%ACN,2%ddH₂O,pH 10);10–13.4 min,5%-8.%B;13.4–31.7 min,8.5%–20.5%B;31.7-41 min,20.5%–31.0%B; 41-46 min,31%–90%B;46-47 min,90.0–95.0%B;47-48 min, 95%–5%B;48-51 min,5%B. The eluted peptides were obtained 40 fractions and finally pooled into 10 fractions through Peak shape.

Then the fraction was re-suspended in 20 μL buffer A (2% ACN, 0.1% FA)and centrifuged at 12,000 rpm for 10 min and 10 μL supernatant was loaded onto a 12 cm × 75 μm EASY-Spray column (C₁₈,3 μm). The samples were loaded at 300 nL/min with mobile phase A: 100% dd H₂O/0.1% Formic acid; mobile phase B: 100% ACN/0.1%FA. The gradient as follows:0-13 min,5–8%B;13-90 min,8030%B;90-100 min,30–50%B;100-105 min,50–95%B;105-115 min,95%B;115-116 min,95–5%B;116-126 min,5%B.

The peptides were subjected to Nano-electrospray ionization followed by mass spectrometry (MS/MS) using a Q-Exactive mass spectrometer (Thermo Scientific) coupled with an online micro flow HPLC system. Key parameter settings for the Thermo Q-Exactive mass spectrometer were as follows:

spray voltage floating (ISVF) 2.3KV, Capillary Temperature:320 °C, Ion source: EASY-Spray source, declustering potential (DP) 100 V.

Full MS:Resolution:70000FWHM;Full Scan AGC target:3e6;Full Scan Max.IT:20 ms;Scan range:300-1800 m/z;

dd-MS2:Resolution:17500 FWHM;AGC target:1e5;Maximum IT:120 ms;Intensity threshold:8.30E + 03;Fragmentation Methods:HCD;NCE:32%;Top N:20.

Bioinformatics analysis

Annotations of identified proteins were done with GO for biological processes, molecular functions and cellular component. The analysis were carried out using the Database for Annotation Visualization and Integrated Discovery. Tagged samples were normalized by comparing median protein ratios for the reference channel. Protein quantitative ratios were calculated from the median of all peptide ratios. The proteins with a relative expression of > 1.32 or < 0.68, and with a P-value < 0.05 selected as statistically significance to ensure up- and downregulation authenticity. The selection parameter was based on the overrepresented GO terms with gene enrichment analysis of p < 0.05. The protein lists were further analyzed by UniProt database (http://www.uniprot.org/uniprot/?query=taxonomy:10090) which gave all canonical pathways, interactions, and network construction with significant enrichment of the input proteins based on data from the UniProt Database, Biocarta, etc. [17]

Western blot analysis

Western blot analysis were performed to confirm the presence of differentially expressed proteins. Colons from mouse were washed with ice-cold saline and triturated under Liquid Nitrogen. 200 mg powder were lysed in 1.5 ml RIPA buffer and incubated on ice for 60 min, sonicated for 60s, followed by centrifugation at 12,000×g for 15 min at 4 °C. The total protein concentration was measured using the BCA protein assay kit (Applygen Technologies Inc. Beijing, China). The supernatant lysates were diluted in 5× SDS sample buffer and boiled for 5–10 min.

Proteins from individual samples were separated on SDS-PAGE gels and transferred electrophoretically onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked for 2 h at room temperature with 3% non-fat dried milk in Tris-buffered saline (TBST, 20 mM Tris-HCl, 137 mM NaCl, and 0.1% Tween 20, pH 7.6). Then, the membranes were incubated overnight at 4 °C in a primary antibody against Anti-STAT1 antibody(Abcam, USA), Rabbit Anti-Angiopoietin 4(Beijing Biosynthesis Biotechnology Co., Ltd.,China), Rabbit and Mouse Anti-β-actin(ZS-Bio. Co., Ltd. Beijing, China). The membranes were then washed with TTBS three times and incubated with horseradish peroxidase-conjugated secondary antibodies (ZS-Bio. Co., Ltd. Beijing, China), Peroxidase-Conjugated Goat anti-Mouse IgG (H + L) (ZB-2305) and Peroxidase-Conjugated Goat anti-Rabbit IgG (H + L) (ZB-2301).Proteins were detected using an enhanced chemiluminescence (ECL) method (Super ECL plus Detection Reagent, Applygen Technologies Inc.P1010). Protein bands were imaged using a ChemiScope 3300 Mini bio-imaging system (Clinx Science Instruments Co., Ltd. (CSI), Shanghai, China). Bands were normalized with β- actin as an internal control. Protein expressions were quantified by chemi analysis and Ang4 and STAT1 were normalized to the beta-actin of each sample. These experiments were each conducted five times.

Results and discussion

Protein profiling

MS raw data files were converted into MGF files using Proteome Discoverer 1.4 (PD 1.4, Thermo), and the MGF data files were searched by using the Mascot search engine (Matrix Science, London, UK; version 2.3.02) to identify proteins. Each confident protein identification involves at least one unique peptide. For protein quantification, it was required that a protein contained at least two unique spectra. The quantitative protein ratios were weighted and normalized by the median ratio in Mascot. As shown in Fig. 1, a total of 393,357 MS/MS spectra which are the secondary mass spectrums were identified by iTRAQ-coupled 2D LC-MS/MS analysis in mice intestine tissues. Among them, 123,136 peptide spectrum-match (PSM) were found. In addition, the LC-MS/MS analysis employed here resulted in identification of 50,007 total peptides with 6727 identified protein groups.

Fig. 1

Basic information statistics of proteome by iTRAQ. MS/MS spectra are the secondary mass spectrums, and PSMs are the secondary mass spectrums after quality control. Protein is identified by Proteome Discoverer 1.4 software

Identification of differentially expressed proteins using iTRAQ labeling and LC-MS/MS

Through analysis with software, data were processed using the Proteome Discoverer Software 4.0 utilizing the Mascot (Matrix Science,London, U.K.; version 2.3.0) Algorithm. In this algorithm, Parameters set for the searching were iTRAQ eight plex peptide-labeled, trypsin digestion with only two maximum miss cleavage, carboxymate for cysteine residue and oxidation for methionine. The tolerances were specified as ±15 ppm for peptides and ± 20 mmu for MS/MS fragments. The mice protein database was downloaded from UniProt. The false discovery rate (FDR) was controlled at the 1% level. Distributional normality and homogeneity of variance were tested for numerical data. Values were given as mean ± SD. To reduce probability of false peptide identification, only peptides with a fold change cut-off ratio of > 1.32 or < 0.68 and ones with p-values smaller than 0.05 in the analysis (where P-value < 0.05 indicates > 95% confidence of a change in protein concentration irrespective of the magnitude of the change) was selected to designate differentially expressed proteins. The similar experimental design was described in previous study [18,19,20]. Among them, proteins that displayed significantly altered expression levels comparing with the control group were considered as up-regulated or down-regulated differentially expressed proteins (DEPs), respectively. With this filter, we identified 103 DEPs in EFL₁ group, including 82 up-regulated proteins and 21 down-regulated proteins. Besides, regarding to 70 DEPs from SE-treated group compared to control group, 47 proteins were up-regulated, and 23 proteins were down-regulated. Moreover, there were 96 up-regulated proteins and 26 down-regulated proteins, totaling 122 proteins in the SEP-treated groups were identified relative to control. Further analysis indicated that the three test groups shared 7 DEPs in the colon tissues of mice after intersection, of which, 5 proteins were down-regulated and 2 proteins up-regulated (Table 1). Meanwhile, there were 295 differentially expressed proteins in the colon tissues of mice in union of DEPs of SE and SEP, EFL₁, of which, 70 proteins were down-regulated and 225 proteins up-regulated (Table 2). These proteins were subjected to gene-ontology enrichment.

Table 1 Related information of differentially expressed protein (DEPs) by iTRAQ analysis after intersection

Table 2 Summary table showing significantly up-regulated or down-regulated proteins identified by iTRAQ Analysis after combine together

GO ontology analysis

To elucidate the biological significance of these differentially modified proteins, we performed GO analysis and categorized these proteins according to their molecular function and biological process using the GO database. 295 union proteins were selected and separated into 3 categories: biological processes (Fig. 2a), cellular component association (Fig. 2b), and molecular function (Fig. 2c).

Fig. 2

Bioinformatics analysis of the differentially expressed proteins (ratio ≥ 1.32 or ≤ 0.68 fold). a Biological process (b) Cellular component; (c). Molecular function

In the biological process category, the results suggested that most of the DEPs participate in metabolic processes (32.9%), cellular processes (17.10%), biological regulation (12.6%), and response to stimulus (7.70%). In the cellular component analysis, most of the potential biomarkers are concentrated in the cell part (32.80%), organelle (20.90%), extracellular region (19.40%), membrane (11.90%) or macromolecular complex. In the molecular function analysis, the differentially expressed proteins were found to play a role in catalytic activity (34.60%), binding (32.30%), enzymatic activity (9.00%) and structural molecule activity (8.30%),suggesting that their related functions were important in the colon of mice.

On the basis of our findings, it could be concluded that the identified DEPs causing by SE, SEP and EFL₁ were mainly associated with the cellular part. The expression sites of them located within cells and organelles. G protein and Eph/Ephrin signal pathway were controlled jointly by SE and SEP. After processing, the extracts of SEP were mainly reflected in the process of cytoskeleton, glycolysis and gluconeogenesis.

Pathway enrichment analysis and interaction network analysis

MetaCore™ (version 6.18) is an integrated software suite for functional analysis of experimental data. Differential pathways among SE, SEP, EFL ₁ and control were conducted according to the P Value (P < 0.05). All the differential pathways were shown in Tables 3, 4 and 5.

Table 3 Pathway Enrichment analysis of differentially expressed proteins relative to SE compared with control group

Table 4 Pathway Enrichment analysis of differentially expressed proteins relative to SEP compared with control group

Table 5 Pathway Enrichment analysis of differentially expressed proteins relative to EFL₁ compared with control

Comparing with group 1(control), the pathways with higher activity were mainly related to the immune response, and also related to other physiological processes such as development and G protein pathways; the dominant signaling pathways were interleukin signaling pathway, JAK/Stat et al.; the key proteins involved in multiple pathways contain STAT1, SERPINA3, G protein Rap1B and so on. Meanwhile, group 4 (EFL₁) showed that the physiological process with high activity was relatively simple, mainly focused on the immune response and development process. Interleukin signaling pathways, Ang/Tie 2 and NF/kB were found to be the main signaling pathways and the key proteins involved were STAT1 and STAT5; compared with control, group 3 induced cytoskeleton remodeling, glycolysis and gluconeogenesis with higher activities, signaling pathways which contain a variety of major B-Raf pathways, epithelial cells to interstitial cell transition(EMT)-related signaling pathways, cell endocytosis, etc. and PDGF receptors, Ephrin receptors,in which STAT 1 was related to the key proteins.

A network was constructed by protein-protein interaction of the 295 significantly DEPs basing on Analyze Network Algorithm using MetaCore in Fig. 3 (A-D). (Tables 6 and 7).

Fig. 3

Biological networks generated by different groups. a Protein interaction networks of DEPs from four groups after taking the intersection; b, c and d: protein interaction networks of DEPs from four groups after taking union (b: Major Histocompatibility Complex class IInetwork; c: Ubiquitination in Mediating the Cellular Stress Response; d: Interferon-γ-mediated signal transduction and response network); e Explanation of various symbols in the network map. The network of significantly differentially expressed proteins (ratio ≥ 1.32 or ≤ 0.68 fold) was analyzed by MetaCoreTM(version 6.18)software

Table 6 Intersection of differentially expressed protein Networks

Table 7 Union of differentially expressed protein Networks

Obviously, commonly pathways are mainly interleukin-mediated signaling pathways, including IL-7, IL-15, IL-23 and other inflammatory factors both controlled by EFL₁ and SE groups. We supposed that these inflammatory factors activate the interleukin signaling pathway, NF / kB signaling pathway, and then mediate intestinal mucosal barrier injure by up-regulating inflammatory proteins expression which resulting in inflammatory response. While there is no obvious interleukin-mediated inflammatory response in SEP group. Generally speaking, inflammatory response especially interleukin might be closely related to the attenuated mechanism of Semen Euphorbiae.

According to network analysis, four reliable functional networks were found and analyzed. After intersection of four groups, the main protein interaction network was multicellular organism regulation process (only Angiopoietin 4 is the down-regulated differentially expressed protein and NF-κB is a pivotal role which interacts with other proteins in the network most closely, Fig. 3a). DEPs which were taken together mainly participated in the protein interaction networks as shown in Fig. 3b, c and d. MHC II presents endogenous and exogenous antigenic peptides or antigenic polysaccharides (containing 10 differential proteins, the key point is MHC class II in Fig. 3b), stress response (containing 3 up-regulation differential proteins, RelA/P65 and ubiquitin are the central part of network, Fig. 3c), γ- Interferon - mediated signal transduction and response (containing 6 up-regulation,1 down-regulation differential proteins, as shown in Fig. 3d, STAT1 interacted closely with other proteins and play an important role in the networks).

It should be pointed out that Angiopoietin 4 is the only down-regulated differential expressed protein in the interaction network. Subsequently, STAT1 was found to be the key protein shared by the EFL₁, SEP and SE tested groups, compared with the control group. A previous study has implied that the transcription factor NF-κB (nuclear factor kappa B) plays a central role in the regulation of immune and inflammatory responses, as well as in control of cell apoptosis. These proteins participate in the regulation of a wide range of genes involved in immune, inflammatory and apoptosis function [21]. Although the relationship between Angiopoietin 4 and NF-κB has not been reported, according to the network, we could make the hypothesis that SE could increase Angiopoietin 4 and then activate NF-κB to make the body produce immune or inflammatory response. In addition, interferons (IFNs) are important cytokines that play essential roles in antiviral, antibacterial, antitumor and immunomodulatory activities. IFNs primarily signals through the JAK-STAT pathway leading to the activation of signal transducer and activator of STAT and subsequent transcription of target genes [22]. Based on the pathway analysis, IFN-γ could activate STATs through JAK-STAT signal pathway to initiate CIITA (typeIItranscription activator) which as MHC IItrans activator, and then the expression of MHC II were up-regulated to produce immune response and immune regulation so that the mice have diarrhea symptoms after treated with SE group. For these reasons and hypothesis, western blot analysis was then conducted to validate the two differentially expressed proteins- STAT1 and Angiopoietin 4.

Validation of differentially expressed proteins identified by proteomics

Two proteins, STAT1 and Angiopoietin 4 identified DEPs with marked differences in expression determined by iTRAQ based quantitative analysis were selected to be verified by western blot analysis (Figs. 4 and 5). As depicted in Figs. 4 and 5 and Table 8, Angiopoietin 4 protein was significantly down-regulated in SEH, SEPH and EFLH groups as compared with control group (p < 0.05), the expression level of Ang4 in SEH was the lowest; and STAT1 was up-regulated in SEH, SEPH and EFLH groups, which levels were all higher than control group (p < 0.05). Moreover, the groups of low dose of SEL, SEPL and EFLL have no significant differences compared with the control. The results which were found by western blot is consistent with the findings in iTRAQ analysis. Both of Ang-4 and STAT1 expression levels in the mice colon tissue may be dose-dependent with the increase dose of SE and SEP.

Fig. 4

Relative expression levels of Ang4 and STAT1were normalized to the β-actin which were quantified by densitometric analysis. These experiments were each conducted five times

Fig. 5

Western blotting showing the changes in Ang4 and STAT1 level in mice intestine treated with different doses of SE, SEP and EFL₁ with respect to control-treated mice intestine

Note:Internal reference:β- actin,1.Control, 2.High-dose of SE (SEH, 1.5 ml/20 g), 3.low-dose of SE (SEL, 0.5 ml/20 g), 4. High-dose of SEP (SEPH, 1.0 ml/20 g), 5. Low-dose of SEP (SEPL, 0.33 ml/20 g), 6.High-dose of EFL₁ (EFLH, 20 mg/20 g), 7 Low-dose of EFL₁ (EFLL, 10 mg/20 g)

Table 8 The relative expression of Ang4 and STAT1 in intestinal tissue of mice ($\overline{X}$±S, n = 5)

It is well established that the angiopoietin (Ang) family of growth factors includes Ang1, Ang2, Ang3 and Ang4, all of which bind to the endothelial receptor tyrosine kinase Tie2. Ang3 (mouse) and Ang4 (human) are interspecies orthologs. Tie2 [23] maintains the vascular integrity of mature vessels by enhancing endothelial barrier function and inhibiting apoptosis of endothelial cells. According to the pathway network analysis, as shown in Fig. 3a, we speculated that Semen Euphorbiae might inhibit the expression of Ang-4, which Tie-2 couldn’t be activated, so that the steady state of endothelial cells was broken and the sensitivity of various inflammatory mediators increased, permeability, and thus promoted the occurrence of inflammatory response. The inhibition of Ang 4 by SEP group after processing was weakened comparing to SE group, resulting in lower diarrhea and inflammatory response.

STAT1 has been implicated as a mediator of biological responses to a variety of growth factors and cytokines, based on ligand-dependent tyrosine phosphorylation and activation. Stat1 is a functional transcription factor even in the absence of inducer-mediated activation, participating in the constitutive expression of some genes [24]. JAK2/ STAT pathway signaling is activated by a wide array of cytokines and growth factors leading to the stimulation of cell proliferation, differentiation, and apoptosis [25]. And it is an important way of signal transduction of inflammatory factors.

In addition to being involved in the main JAK2 / STAT signaling pathway, STAT1 could be activated by JAK2 (non-receptor tyrosine) kinase, but also by inflammatory factors such as interleukin-6 (IL-6), tumor necrosis factor (TNF),growth factors such as interferon (IFN) [26], epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and other signal activation.

As the up-regulated proteins induced by each group, STAT1 was induced by SEP group lower than the SE group so that we suspected that STAT 1 was most likely one of target proteins related to intestinal inflammation which might illustrate the attenuated mechanism of Semen Euphorbiae.

Both Ang-4 and STAT1 were surmised to be one of the target proteins inducing by Semen Euphorbiae.

Conclusions

This study used iTRAQ labeling followed by 2D-LC-MS/MS for the quantitative proteomic analysis of intestine samples from KM mice with different groups and control to discover candidate biomarkers for attenuated mechanism of Semen Euphorbiae processing for the first time. These findings suggest that SE induced an inflammatory response, and activated the Interleukin signaling pathway, such as the Ang/Tie 2 and JAK2/ STAT signaling pathways, which may eventually contribute to injury result from intestinal inflammatory, while SEP could ease this injury by reducing STAT1 and activating Ang-4 which could reduce the inflammatory response. Taken together, these results not only provided a novel insight into attenuated mechanism of Semen Euphorbiae, which was marked by a number of DEPs that might be associated with intestinal inflammation, but also the first experimental evidence that the Angiopoietin 4 and STAT1 proteins might be two major candidate biomarkers in the attenuated of SE after processing based on proteomic investigation. Our findings suggest that this screening method has potential valuable in studying mechanism of processing. Future systematic studies will investigate how Semen Euphorbiae regulate the expression of these key proteins and illustrate the problem from a clinical point of view.