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 (EFL[1]) 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. Keywords: Euphorbia lathyris, Proteomics, iTRAQ, Bio-pathway 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 [[39]1, [40]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 [[41]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 [[42]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 [[43]5, [44]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 [[45]7]. It can simultaneously analyze 4–8 different specimens, thus increasing throughput while reducing experimental error [[46]8, [47]9]. iTRAQ labeling coupled with LC-MS/MS is sensitive, automated, and multidimensional and can detect large molecules (> 20 kDa) [[48]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(EFL[1]), isolated from Euphorbia lathyris, is the main and active diterpenoids which might mediate diarrhea [[49]11]. We also demonstrated EFL[1] 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 [[50]12, [51]13]. Euphorbiae factor 1 was isolated from the petroleum ether extracts of semen Euphorbia by our team [[52]13, [53]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(EFL[1]) 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 [[54]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. [[55]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: EFL[1] 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-C[18] 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%ddH[2]O,pH 10;Mobile phaseB:98%ACN,2%ddH[2]O,pH 10);10–13.4 min,5%-8.%B;13.4–31.7 min,8.5%–2 0.5%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 (C[18],3 μm). The samples were loaded at 300 nL/min with mobile phase A: 100% dd H[2]O/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 ([56]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. [[57]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. [58]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. Fig. 1 [59]Open in a new tab 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 [[60]18–[61]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 EFL[1] 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 [62]1). Meanwhile, there were 295 differentially expressed proteins in the colon tissues of mice in union of DEPs of SE and SEP, EFL[1], of which, 70 proteins were down-regulated and 225 proteins up-regulated (Table [63]2). These proteins were subjected to gene-ontology enrichment. Table 1. Related information of differentially expressed protein (DEPs) by iTRAQ analysis after intersection Acc no. (NCBI) Prot names Gene names Control SE SEP EFL[1] Down-regulated proteins [64]Q3TMQ6 Angiogenin-4 Ang4 1 0.5795 0.6082 0.549 [65]Q62010 Oviduct-specific glycoprotein Ovgp1 1 0.4252 0.5825 0.451 [66]Q80ZA0 Intelectin-1b (Intelectin-2) Itln1b 1 0.4847 0.6715 0.498 [67]Q8R1M8 Mucosal pentraxin Mptx1 1 0.5352 0.5652 0.559 V9GXU2 C2 domain-containing protein 3 C2cd3 1 0.5372 0.636 0.463 Up-regulated proteins F6R782 IQ domain-containing protein E Iqce 1 3.496 4.4437 4.691 [68]Q9D1X0 Nucleolar protein 3 (Apoptosis repressor with CARD) Nol3 Arc 1 1.3665 1.5167 1.345 [69]Open in a new tab Acc no Accession number, Prot name Protein name, SE Semen Euphorbiae, SEP Semen Euphorbiae Pulveratum, EFL[1] Euphorbiae Factor 1 Table 2. Summary table showing significantly up-regulated or down-regulated proteins identified by iTRAQ Analysis after combine together Acc no.(NCBI) Pro names Gene names Control SE EFL[1] SEP Up-regulated [70]Q62010 Oviduct-specific glycoprotein Ovgp1 Chit5 Ogp 1 0.4252 0.451 0.5825 [71]A2BDX4 Potassium voltage-gated channel subfamily G member 1 Kcng1 1 0.4347 0.856 0.6645 [72]P97816 Protein S100-G S100 g Calb3 S100d 1 0.4485 0.599 0.653 [73]Q8BV14 Ankyrin repeat domain-containing protein 55 Ankrd55 1 0.4652 0.636 / [74]Q80ZA0 Intelectin-1b (Intelectin-2) Itln1b Itln2 Itlnb 1 0.4847 0.498 0.6715 [75]Q8R1M8 Mucosal pentraxin Mptx1 Mptx 1 0.5352 0.559 0.5652 V9GXU2 C2 domain-containing protein 3 C2cd3 1 0.5372 0.463 0.636 [76]P07146 Anionic trypsin-2 Prss2 Try2 1 0.5465 0.776 0.5967 D6RFD6 Protein RFT1 homolog Rft1 1 0.5687 0.771 4.6342 [77]Q8VCV1 Alpha/beta hydrolase domain-containing protein 17C Abhd17c 1 0.5707 1.271 0.7095 [78]Q3TMQ6 Angiogenin-4 Ang4 1 0.5795 0.549 0.6082 [79]Q08189 Protein-glutamine gamma-glutamyltransferase E Tgm3 Tgase3 1 0.6085 0.728 0.528 [80]Q8CIM3 D-2-hydroxyglutarate dehydrogenase, mitochondrial D2hgdh 1 0.61 0.704 1.0195 [81]Q9D7Z6 Calcium-activated chloride channel regulator 1 Clca1 1 0.649 0.709 0.637 [82]O88273 Gremlin-2 (Protein related to DAN and cerberus) Prdc 1 0.6542 0.906 1.7397 D6RFQ5 p53 and DNA damage-regulated protein 1 Pdrg1 1 0.6585 0.683 0.6567 [83]Q8BYF6 Sodium-coupled monocarboxylate transporter 1 Slc5a8 Smct Smct1 1 0.6667 0.972 0.767 H3BLD0 ATP synthase mitochondrial F1 complex assembly factor 1 Atpaf1 1 0.6687 0.841 0.9637 [84]Q8BXQ3 Leucine-rich repeat and transmembrane domain-containing protein 1 Lrtm1 1 0.6702 0.982 0.573 A0A075B5L8 Protein Igkv4–79 Igkv4–79 1 0.6722 0.688 0.8432 [85]Q3V341 Protein kinase C zeta type Prkcz 1 0.6775 0.606 1.052 [86]O88310 Intelectin-1a Itln1 1 0.6782 0.696 0.7225 [87]Q9D2X6 Colon SVA-like protein Sval1 mcsp mCG_17084 1 0.6782 0.912 0.5127 [88]Q64339 Ubiquitin-like protein ISG15 Isg15 G1p2 Ucrp 1 0.6887 0.922 0.6737 [89]Q810Q5 Normal mucosa of esophagus-specific gene 1 protein Nmes1 1 0.693 0.832 0.5877 [90]P21550 Beta-enolase Eno3 Eno-3 1 0.6965 0.876 0.6672 [91]P56392 Cytochrome c oxidase subunit 7A1, mitochondrial Cox7a1 1 0.7257 0.755 0.655 [92]P30275 Creatine kinase U-type, mitochondrial Ckmt1 1 0.7492 0.851 0.6657 [93]Q6T707 Protein Scd4 (Stearoyl-CoA desaturase-4) Scd4 1 0.768 1.808 1.1152 [94]Q9NYQ2 Hydroxyacid oxidase 2 (HAOX2) Hao2 Hao3 Haox2 1 0.771 0.721 0.658 [95]P09036 Serine protease inhibitor Kazal-type 3 Spink3 1 0.7765 0.987 0.595 [96]P98086 Complement C1q subcomponent subunit A C1qa 1 0.785 0.406 0.8317 F8VPP8 Protein Zc3h7b Zc3h7b 1 0.7887 0.677 0.787 [97]Q5RI75–2 Ras and EF-hand domain-containing protein homolog Rasef 1 0.7892 0.651 0.6965 A2AGQ3 MAP kinase-activating death domain protein Madd 1 0.7932 1.327 1.398 E9QNL5 Sulfotransferase Sult1a1 1 0.796 0.659 0.7287 [98]P00329 Alcohol dehydrogenase 1 Adh1 Adh-1 1 0.7992 1.036 0.5625 [99]Q3UZZ6 Sulfotransferase 1 family member D1 Sult1d1 St1d1 1 0.81 0.632 0.7565 B2RT41 Protein Zfc3h1 Zfc3h1 Ccdc131 1 0.831 0.921 0.6362 [100]P57774 Pro-neuropeptide Y [Cleaved into: Neuropeptide Y Npy 1 0.835 1.436 1.1532 [101]Q3UW68 Calpain-13 (Calcium-activated neutral proteinase 13) Capn13 Gm943 1 0.838 0.987 0.669 [102]P13634 Carbonic anhydrase 1 Ca1 Car1 1 0.8425 0.622 0.818 [103]Q9WUG6 Insulin-like peptide INSL5 (Insulin-like peptide 5) Insl5 Rif Rif2 Zins3 1 0.861 1.429 0.6775 F7BQ76 MPN domain-containing protein (Fragment) Mpnd 1 0.8617 0.603 1.577 [104]P56393 Cytochrome c oxidase subunit 7B, mitochondrial Cox7b 1 0.8755 1.075 0.6255 [105]Q80WK2 Organic solute transporter subunit beta Slc51b Ostb 1 0.881 1.373 1.177 A2A6K0 Troponin I, fast skeletal muscle Tnni2 1 0.886 0.374 0.965 [106]Q7TPR4 Alpha-actinin-1 (Alpha-actinin cytoskeletal isoform) Actn1 1 0.888 0.857 0.6745 G3X940 Histone acetyltransferase Kat6a Myst3 1 0.8887 1.618 1.1427 [107]P01796 Ig heavy chain V-III region A4 0 1 0.8935 1.481 1.0095 G3UVW7 Protein Zfp40 (Zinc finger protein 40) Zfp40 mCG_13522 1 0.9052 1.53 1.0887 [108]Q9EPS2 Peptide YY Pyy 1 0.9135 1.349 0.974 G3XA21 MCG134445, isoform CRA_a (Protein Mroh1) Mroh1 Heatr7a 1 0.922 1.114 1.3435 [109]Q9Z179 SHC SH2 domain-binding protein 1 Shcbp1 Pal 1 0.9295 1.107 1.4725 I6L974 TBC1 domain family member 17 Tbc1d17 1 0.9315 1.155 1.3645 [110]P01631 Ig kappa chain V-II region 26–10 0 1 0.9387 1.75 0.821 [111]P01878 Ig alpha chain C region 0 1 0.942 1.332 0.8282 [112]P57776–2 Elongation factor 1-delta (EF-1-delta) Eef1d 1 0.9477 0.898 0.6252 [113]D3Z6J0 HemK methyltransferase family member 2, isoform CRA_b N6amt1 Hemk2 mCG_130002 1 0.9562 1.524 1.4077 [114]Q9WUH1 Transmembrane protein 115 (Protein PL6 homolog) Tmem115 Pl6 1 0.962 1.161 1.4085 [115]Q8R1U2 Cell growth regulator with EF hand domain protein 1 Cgref1 Cgr11 1 0.9635 0.931 1.4472 A0A087WNJ2 Deleted. 0 1 0.974 0.641 0.7125 E0CYM0 Protein 1700019G17Rik 1700019G17Rik 1 0.9752 1.376 1.0687 D3Z7B5 Protein C330027C09Rik C330027C09Rik 1 0.978 1.336 1.1042 D3Z652 Testis-expressed sequence 35 protein Tex35 1 0.9797 0.993 1.3665 F8VQE9 ANK repeat and PH domain-containing protein 3 Agap3 1 0.9855 1.026 1.6535 [116]O88665 Bromodomain-containing protein 7 Brd7 Bp75 1 0.9895 0.928 1.5765 E9Q933 Transmembrane protein 11, mitochondrial Tmem11 1 0.9942 1.5 1.1595 down-regulated 6NXH9 Keratin, type II cytoskeletal 73 Krt73 Kb36 1 14.265 1.559 1.4102 F6R782 IQ domain-containing protein E Iqce 1 3.496 4.691 4.4437 A0A075B6A3 Protein Igha Igha 1 2.7217 1.208 1.9125 [117]P00687 Alpha-amylase 1 Amy1 1 2.5575 4.341 3.1215 [118]Q8C804 Spindle and centriole-associated protein 1 Spice1 Ccdc52 1 2.3742 1.928 1.8472 [119]O88273 Formin-2 Fmn2 1 2.2107 2.234 3.8712 D3Z1G3 Multiple coagulation factor deficiency protein 2 homolog Mcfd2 1 2.2085 1.694 1.931 A2AHB7 Potassium channel subfamily T member 1 Kcnt1 1 2.181 1.35 5.51 G3UZX8 Probable JmjC domain-containing histone demethylation protein 2C Jmjd1c 1 2.1745 1.124 3.0692 [120]P35991 Tyrosine-protein kinase BTK Btk Bpk 1 2.1057 1.302 1.5725 [121]P70213 Friend virus susceptibility protein 1 Fv1 1 1.847 1.207 1.5947 A0A075B664 Protein Iglv2 Iglv2 1 1.8257 3.016 1.2922 [122]E9Q9F6–2 Cation channel sperm-associated protein subunit delta Catsperd Tmem146 1 1.7907 1.132 0.6605 [123]P57791 CAAX prenyl protease 2 Rce1 Face2 Rce1a 1 1.6772 1.103 1.4677 [124]Q9QZU9 Ubiquitin/ISG15-conjugating enzyme E2 L6 Ube2l6 Ubce8 1 1.648 3.026 2.0062 A2AF82 Activator of 90 kDa heat shock protein ATPase homolog 2 Ahsa2 1 1.6057 1.363 1.5 F8VQM0 Alkaline phosphatase Akp3 1 1.6022 1.282 2.631 [125]P11034 Mast cell protease 1 Mcpt1 1 1.602 1.704 1.5607 [126]Q6ZWN5 40S ribosomal protein S9 Rps9 1 1.5622 1.053 1.3207 [127]Q9DBB8 Trans-1,2-dihydrobenzene-1,2-diol dehydrogenase Dhdh 1 1.5585 1.268 1.6725 [128]Q6NZQ2 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 31 Ddx31 1 1.5305 1.169 1.3397 G5E8C3 G protein-coupled receptor, family C, group 5, member A Gprc5a mCG_22262 1 1.5077 1.168 1.4337 [129]Q91WP6 Serine protease inhibitor A3N Serpina3n Spi2 1 1.502 0.995 1.2225 A2A3U8 LON peptidase N-terminal domain and RING finger protein 3 Lonrf3 1 1.5017 1.274 1.9312 [130]P07759 Serine protease inhibitor A3K Spi2 1 1.4825 0.804 1.2742 [131]Q9DCG2–2 CD302 antigen Cd302 Clec13a 1 1.469 1.116 1.786 [132]P27005 Protein S100-A8 (Calgranulin-A) S100a8 Caga Mrp8 1 1.4637 1.522 1.154 [133]P04227 H-2 class II histocompatibility antigen, A-Q alpha chain H2-Aa 1 1.4617 1.382 0.916 [134]Q8C6B9 Active regulator of SIRT1 Rps19bp1 Aros 1 1.4555 1.094 1.78 [135]P70412 CUB and zona pellucida-like domain-containing protein 1 Cuzd1 Itmap1 1 1.4365 1.325 1.5315 [136]Q9D083–3 Kinetochore protein Spc24 Spc24 Spbc24 1 1.4297 1.978 2.0805 [137]P62984 Ubiquitin-60S ribosomal protein L40 Uba52 Ubcep2 1 1.4247 1.336 1.143 [138]P12804 Fibroleukin Fgl2 Fiblp 1 1.4215 1.407 1.7527 J3QPY0 Protein 1600014C10Rik 1600014C10Rik 1 1.4165 1.485 1.8247 B1AXR3 Perilipin-2 Plin2 1 1.414 0.975 1.3562 [139]Q9ESG9 Membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase Pkmyt1 Myt1 1 1.4137 1.332 1.9545 [140]P07758 Alpha-1-antitrypsin 1–1 (AAT) Serpina1a Dom1 Spi1–1 1 1.4085 0.908 1.1527 [141]Q8C7E9 Cleavage stimulation factor subunit 2 tau variant Cstf2t Kiaa0689 1 1.401 1.082 1.014 F6ZQQ3 26S proteasome non-ATPase regulatory subunit 13 Psmd13 1 1.3935 1.417 2.6332 [142]Q91XL1 Leucine-rich HEV glycoprotein (Protein Lrg1) Lrg1 Lrg lrhg 1 1.3932 0.949 1.327 [143]Q03145 Ephrin type-A receptor 2 Epha2 Eck Myk2 1 1.3932 1.186 1.522 [144]Q9QXA1 Cysteine and histidine-rich protein 1 Cyhr1 Chrp 1 1.3902 1.191 1.0515 [145]Q8BHZ4 Zinc finger protein 592 (Zfp-592) Znf592 Kiaa0211 1 1.3865 1.338 1.3052 [146]P07724 Serum albumin Alb Alb-1 Alb1 1 1.3842 0.816 1.2217 V9GX06 Protein Gm11214 Gm11214 1 1.3835 1.098 1.3607 [147]P29699 Alpha-2-HS-glycoprotein (Countertrypin) Ahsg Fetua 1 1.382 0.774 1.1715 [148]P14148 60S ribosomal protein L7 Rpl7 1 1.3705 0.953 1.1725 [149]P42232 Signal transducer and activator of transcription 5B Stat5b 1 1.3705 1.627 1.311 [150]P35980 60S ribosomal protein L18 Rpl18 1 1.3695 0.963 1.176 [151]Q9D1X0 Nucleolar protein 3 (Apoptosis repressor with CARD) Nol3 Arc 1 1.3665 1.345 1.5167 G3X8Z1 Calcium-activated chloride channel regulator 4A mCG_119588 1 1.366 1.008 1.3725 [152]P01741 Ig heavy chain V region (Anti-arsonate antibody) 0 1 1.3647 3.709 1.06 A0A087WQ94 Protein Tns1 Tns1 1 1.3562 1.117 0.9982 A2AAC0 Chymotrypsin-C Ctrc 1 1.354 1.062 1.3185 E9Q8K5 Titin Ttn 1 1.3532 0.744 1.6037 [153]Q3U3Q1–2 Serine/threonine-protein kinase ULK3 Ulk3 1 1.353 1.188 1.574 [154]Q91YU8 Suppressor of SWI4 1 homolog Ppan Ssf1 1 1.3522 1.167 1.1937 [155]Q6LC96 RXR alpha 2 (RXR alpha 3) Rxra RXR alpha 1 1.329 0.984 1.2152 [156]Q3UPV6 Voltage-gated potassium channel subunit beta-2 Kcnab2 1 1.328 1.568 1.183 [157]P62301 40S ribosomal protein S13 Rps13 1 1.3275 1.093 1.1617 [158]P22599 Alpha-1-antitrypsin 1–2 (AAT) (Alpha-1 protease inhibitor 2) Serpina1b Aat2 1 1.326 0.849 1.1235 [159]Q9EP52 Twisted gastrulation protein homolog 1 Twsg1 Tsg 1 1.3242 1.197 0.9917 E9PV04 Protein Gm8994 Gm8994 Gm5576 1 1.3237 1.14 1.2215 [160]P15119 Mast cell protease 2 Mcpt2 1 1.322 1.36 1.0482 [161]Q3ZAR9 Nr2c2 protein (Nuclear receptor subfamily 2 group C member 2) Nr2c2 1 1.3202 1.395 1.158 [162]Q8BSI6 R3H and coiled-coil domain-containing protein 1 R3hcc1 1 1.319 1.279 1.569 [163]Q32M21–2 Gasdermin-A2 Gsdma2 Gsdm2 1 1.3125 1.482 1.233 [164]Q80TL0 Protein phosphatase 1E Ppm1e Camkn 1 1.3082 0.645 1.1805 F6RUC3 Ribonucleoside-diphosphate reductase subunit M2 (Fragment) Rrm2 1 1.3075 1.238 1.4467 [165]A2ALH2 Putative tRNA Ftsj1 1 1.296 1.3 1.5377 [166]Q8BGS0–2 Protein MAK16 homolog (Protein RBM13) Mak16 Rbm13 1 1.2927 1.195 1.334 [167]Q8BHY2 Nucleolar complex protein 4 homolog (NOC4 protein homolog) Noc4l 1 1.2877 1.455 1.5922 [168]Q99J23 GH3 domain-containing protein Ghdc D11lgp1e 1 1.287 1.231 1.3732 [169]O35640 Annexin A8 Anxa8 Anx8 1 1.277 1.55 1.1167 [170]Q60590 Alpha-1-acid glycoprotein 1 Orm1 Agp1 Orm-1 1 1.263 1.116 1.4175 [171]P35461 Lymphocyte antigen 6G (Ly-6G) Ly6g 1 1.2495 0.915 1.331 [172]P42225 Signal transducer and activator of transcription 1 Stat1 1 1.2437 1.533 0.9722 [173]Q8VEJ4 Notchless protein homolog 1 Nle1 1 1.2432 1.251 1.3997 F6S522 Claspin Clspn 1 1.2415 1.134 7.6765 [174]Q8BHN5 RNA-binding protein 45 Rbm45 Drb1 Drbp1 1 1.2387 1.232 1.4235 [175]P31725 Protein S100-A9 S100a9 1 1.2345 1.351 1.028 F8WJ43 Merlin Nf2 1 1.234 1.168 1.441 [176]Q8C3X8 Lipase maturation factor 2 Lmf2 Tmem112b Tmem153 1 1.2307 0.928 1.5145 [177]E9Q8D0 Protein Dnajc21 Dnajc21 1 1.227 1.476 1.1372 [178]Q9QXA1–2 Cysteine and histidine-rich protein 1 Cyhr1 Chrp 1 1.2205 1.374 1.054 [179]Q3UW98 Chloride channel calcium activated 7 Clca4b [180]AI747448 1 1.2187 1.401 1.0597 A0A075B5M8 Protein Igkv12–38 Igkv12–38 1 1.218 1.337 1.2272 [181]Q4QRL3 Coiled-coil domain-containing protein 88B Ccdc88b Ccdc88 1 1.2172 1.485 1.392 [182]Q3TBT3–3 Stimulator of interferon genes protein (mSTING) Tmem173 Eris Mita 1 1.2167 1.441 1.1297 [183]P08905 Lysozyme C-2 (EC 3.2.1.17) (1,4-beta-N-acetylmuramidase C) (Lysozyme C type M) Lyz2 Lyz Lyzs 1 1.2162 1.353 1.0305 [184]Q9DCS1 Transmembrane protein 176A (Gene signature 188) (Kidney-expressed gene 2 protein) Tmem176a Gs188 Keg2 1 1.2157 1.248 1.5587 [185]P84228 Histone H3.2 Hist1h3b 1 1.214 0.511 1.0842 D3Z408 High affinity cGMP-specific 3′,5′-cyclic phosphodiesterase 9A Pde9a 1 1.2137 1.307 1.3392 E9Q4G7 Casein kinase I isoform alpha Csnk1a1 1 1.2105 1.47 1.643 [186]P05533 Lymphocyte antigen 6A-2/6E-1 (Ly-6A.2/Ly-6E.1) (Stem cell antigen 1) (SCA-1) (T-cell-activating protein) (TAP) Ly6a Ly6 1 1.2085 1.378 1.1695 [187]P01844 Ig lambda-2 chain C region Iglc2 1 1.2072 2.441 1.087 G3X8S8 MCG14499 (tRNA-splicing endonuclease subunit Sen15) Tsen15 mCG_14499 1 1.2065 1.143 1.4907 F6QQ13 Selenocysteine insertion sequence-binding protein 2-like (Fragment) Secisbp2l 1 1.2035 1.149 1.3285 [188]P58501 PAX3- and PAX7-binding protein 1 (PAX3/7BP) (GC-rich sequence DNA-binding factor 1) Paxbp1 Gcfc Gcfc1 1 1.2035 1.488 1.2637 [189]Q9JLM9 Growth factor receptor-bound protein 14 (GRB14 adapter protein) Grb14 1 1.1975 0.601 1.1215 [190]P59328–2 WD repeat and HMG-box DNA-binding protein 1 (Acidic nucleoplasmic DNA-binding protein 1) (And-1) Wdhd1 And1 1 1.1922 1.221 1.4022 [191]A2A5Z6–2 E3 ubiquitin-protein ligase SMURF2 (EC 6.3.2.-) (SMAD ubiquitination regulatory factor 2) (SMAD-specific E3 ubiquitin-protein ligase 2) Smurf2 1 1.1902 1.098 1.3955 [192]Q8CIA9 Hippocampus abundant transcript-like protein 1 Hiatl1 1 1.1852 1.098 1.3277 H3BKB9 Protein zwilch homolog (Fragment) Zwilch 1 1.1817 1.114 1.3972 [193]Q5SUA5 Unconventional myosin-Ig Myo1g 1 1.1747 1.196 1.3717 [194]P03991 H-2 class I histocompatibility antigen, K-W28 alpha chain H2-K1 H2-K 1 1.1682 1.554 0.966 [195]Q61542 StAR-related lipid transfer protein 3 (Protein ES 64) (Protein MLN 64) (START domain-containing protein 3) (StARD3) Stard3 Es64 Mln64 1 1.1672 1.663 1.496 [196]A8C756 Thyroid adenoma-associated protein homolog Thada Kiaa1767 1 1.165 1.299 1.382 [197]Q80ZI6 E3 ubiquitin-protein ligase LRSAM1 (EC 6.3.2.-) (Leucine-rich repeat and sterile alpha motif-containing protein 1) (Tsg101-associated ligase) Lrsam1 1 1.1627 1.094 1.59 F6RR81 Protein cordon-bleu (Fragment) Cobl 1 1.1585 1.355 1.1932 [198]Q8R2S8 CD177 antigen (CD antigen CD177) Cd177 1 1.158 1.426 1.0102 A2ALA0 Surfeit locus protein 6 Surf6 1 1.1567 1.218 1.3962 Q5SUW0 Growth factor receptor-bound protein 10 (Fragment) Grb10 1 1.1552 1.019 1.3747 [199]Q9CQS2 H/ACA ribonucleoprotein complex subunit 3 (Nucleolar protein 10) (Nucleolar protein family A member 3) (snoRNP protein NOP10) Nop10 Nola3 1 1.1455 1.37 1.1737 D3YUW8 Pogo transposable element with ZNF domain Pogz 1 1.1365 1.373 1.3605 [200]Q62293 Interferon-gamma-inducible GTPase Ifggb5 protein Tgtp 1 1.1357 1.958 1.0067 [201]Q8BX57–3 PX domain-containing protein kinase-like protein (Modulator of Na,K-ATPase) (MONaKA) Pxk 1 1.1355 0.867 1.3607 E0CYU9 Sjoegren syndrome/scleroderma autoantigen 1 homolog Sssca1 1 1.135 1.705 1.5802 [202]Q9R0X0–3 Mediator of RNA polymerase II transcription subunit 20 (Mediator complex subunit 20) (TRF-proximal protein homolog) Med20 Trfp 1 1.1335 1.088 1.3255 [203]P18527 Ig heavy chain V region 914 0 1 1.133 1.071 0.618 [204]A2A6A1 G patch domain-containing protein 8 Gpatch8 Gpatc8 Kiaa0553 1 1.1295 1.861 1.0447 [205]O35242 Protein FAN (Factor associated with neutral sphingomyelinase activation) (Factor associated with N-SMase activation) Nsmaf Fan 1 1.1275 1.116 1.417 [206]P04184 Thymidine kinase, cytosolic (EC 2.7.1.21) Tk1 Tk-1 1 1.1222 1.288 1.638 [207]Q80VC9–2 Calmodulin-regulated spectrin-associated protein 3 (Protein Nezha) Camsap3 Kiaa1543 1 1.1107 1.308 1.492 [208]S4R2K0 Protein Pdf Pdf 1 1.1082 1.644 1.4732 [209]Q8BZR9 Uncharacterized protein C17orf85 homolog 0 1 1.108 1.132 1.6712 [210]Q8K4Q0–5 Regulatory-associated protein of mTOR (Raptor) (p150 target of rapamycin (TOR)-scaffold protein) Rptor Raptor 1 1.105 1.153 1.4442 [211]Q6P9L6 Kinesin-like protein KIF15 (Kinesin-like protein 2) (Kinesin-like protein 7) Kif15 Klp2 Knsl7 1 1.1012 1.367 1.3967 [212]Q9CR76 Transmembrane protein 186 Tmem186 1 1.0997 0.655 1.0117 [213]Q924Z6–2 Exportin-6 (Exp6) (Ran-binding protein 20) Xpo6 Ranbp20 1 1.0997 1.209 1.5217 [214]Q8BZT5 Leucine-rich repeat-containing protein 19 Lrrc19 1 1.0952 1.379 1.2207 [215]P11247 Myeloperoxidase (MPO) (EC 1.11.2.2) [Cleaved into: Myeloperoxidase light chain; Myeloperoxidase heavy chain] Mpo 1 1.0945 1.195 1.415 A8DUK4 Beta-globin (Protein Hbb-bs) (Protein Hbb-bt) Hbbt1 Hbb-bs Hbb-bt Hbbt2 1 1.0942 2.14 0.8445 [216]P01630 Ig kappa chain V-II region 7S34.1 0 1 1.094 1.405 1.2252 [217]Q8CGN5 Perilipin-1 (Lipid droplet-associated protein) (Perilipin A) Plin1 Peri Plin 1 1.0895 0.885 1.3687 [218]Q9CQT2 RNA-binding protein 7 (RNA-binding motif protein 7) Rbm7 1 1.0877 1.142 1.333 F7BJK1 Protein Pcdh1 (Fragment) Pcdh1 1 1.0875 0.927 1.8367 [219]Q80TA6–2 Myotubularin-related protein 12 Mtmr12 Kiaa1682 1 1.0835 1.091 1.5237 [220]P54754 Ephrin type-B receptor 3 (EC 2.7.10.1) (Developmental kinase 5) (mDK-5) (Tyrosine-protein kinase receptor SEK-4) Ephb3 Etk2 Mdk5 Sek4 1 1.082 1.341 1.1597 D3Z769 Protein lin-37 homolog (Fragment) Lin37 1 1.0795 1.116 1.5232 A0A075B5X9 Ig heavy chain V region B1–8/186–2 (Fragment) Ighv1–72 1 1.0795 1.415 1.2 F6TLB0 DNA-directed RNA polymerase, mitochondrial (Fragment) Polrmt 1 1.077 1.111 1.3495 A0A087WRI5 Adenylate kinase isoenzyme 6 Ak6 1 1.075 1.346 1.1457 [221]Q8BK35 MCG2065, isoform CRA_c (PreS1 binding protein) (Protein Gltscr2) Gltscr2 mCG_2065 1 1.074 0.953 1.557 [222]Q9CQT0 tRNA(His) guanylyltransferase (EC 2.7.7.79) (tRNA-histidine guanylyltransferase) Thg1l mCG_22296 1 1.0722 1.14 1.4462 A0A075B677 Protein Igkv4–53 Igkv4–53 1 1.0705 1.361 0.986 G3UWZ0 Bromodomain adjacent to zinc finger domain protein 1A Baz1a 1 1.0702 1.564 1.3232 F6R2G3 Mucin-4 (Fragment) Muc4 1 1.0695 1.286 1.3432 [223]Q6GU68 Immunoglobulin superfamily containing leucine-rich repeat protein Islr 1 1.068 1.154 1.3577 E9PWH6 HEAT repeat-containing protein 3 Heatr3 1 1.0605 1.102 1.4625 [224]Q8BLH7 HIRA-interacting protein 3 Hirip3 1 1.0587 1.496 1.4912 [225]Q62264 Thyroid hormone-inducible hepatic protein (Spot 14 protein) (S14) (SPOT14) Thrsp S14 1 1.0582 0.943 1.4015 [226]Q99M73 Keratin, type II cuticular Hb4 (65 kDa type II keratin) (Keratin-84) (K84) (Type II hair keratin Hb4) (Type-II keratin Kb24) Krt84 Krt2–16 Krthb4 1 1.0557 1.17 1.3252 [227]Q9D856 Zinc transporter ZIP5 (Solute carrier family 39 member 5) (Zrt- and Irt-like protein 5) (ZIP-5) Slc39a5 Zip5 1 1.0555 1.484 1.4005 [228]F7BJB9 Protein Morc3 Morc3 1 1.0525 1.375 1.2087 B7ZWM8 Leucine-rich repeat and calponin homology domain-containing protein 3 (Lrch3 protein) Lrch3 1 1.0505 1.128 1.3452 D3Z6K8 Ras-specific guanine nucleotide-releasing factor 2 Rasgrf2 1 1.0482 1.047 1.3875 [229]Q5FWI3 Transmembrane protein 2 Tmem2 Kiaa1412 1 1.0462 1.163 1.4667 G3UZL2 RCC1 and BTB domain-containing protein 1 (Fragment) Rcbtb1 1 1.0417 1.433 1.2967 [230]Q61666–4 Protein HIRA (TUP1-like enhancer of split protein 1) Hira Tuple1 1 1.0405 1.127 1.3862 [231]P53569 CCAAT/enhancer-binding protein zeta (CCAAT-box-binding transcription factor) (CBF) (CCAAT-binding factor) Cebpz Cbf2 Cebpa-rs1 1 1.0367 1.325 1.7395 [232]Q9JJF3 Bifunctional lysine-specific demethylase and histidyl-hydroxylase NO66 (EC 1.14.11.-) (EC 1.14.11.27) (Histone lysine demethylase NO66) No66 Mapjd MNCb-7109 1 1.0337 1.68 1.1642 [233]Q9DAA6 Exosome complex component CSL4 (Exosome component 1) Exosc1 Csl4 1 1.033 1.326 1.2455 A0A087WQR9 NEDD8-conjugating enzyme UBE2F (Fragment) Ube2f 1 1.0292 1.352 1.2977 [234]Q9Z0E6 Interferon-induced guanylate-binding protein 2 (GTP-binding protein 2) (GBP-2) (mGBP-2) (mGBP2) (Guanine nucleotide-binding protein 2) Gbp2 1 1.0292 1.432 0.9467 [235]B7ZMP1–2 Probable Xaa-Pro aminopeptidase 3 (X-Pro aminopeptidase 3) (EC 3.4.11.9) (Aminopeptidase P3) (APP3) Xpnpep3 1 1.0285 0.927 1.4122 D3YWR2 B-cell linker protein Blnk 1 1.0237 1.595 1.1195 H7BX32 Nuclear envelope pore membrane protein POM 121 Pom121 1 1.0165 1.187 1.3717 [236]Q99N87 28S ribosomal protein S5, mitochondrial (MRP-S5) (S5mt) Mrps5 1 1.0147 1.41 0.9545 [237]Q8CBC4 Consortin Cnst 1 1.0092 1.33 1.19 A2AER8 Polyglutamine-binding protein 1 Pqbp1 1 1.0077 1.477 0.9262 A8Y5N4 17-beta-hydroxysteroid dehydrogenase 13 Hsd17b13 1 1.006 0.603 0.7325 [238]Q9D8I1 Marginal zone B- and B1-cell-specific protein Mzb1 Pacap 1 1.006 1.348 0.9347 [239]P26618 Platelet-derived growth factor receptor alpha Pdgfra 1 1.0032 1.152 1.4392 [240]P55088–2 Aquaporin-4 (AQP-4) Aqp4 1 1.0005 1.327 0.8442 [241]Open in a new tab Acc no Accession number, Prot name Protein name, SE Semen Euphorbiae, SEP Semen Euphorbiae Pulveratum, EFL[1] Euphorbiae Factor 1 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. [242]2a), cellular component association (Fig. [243]2b), and molecular function (Fig. [244]2c). Fig. 2. Fig. 2 [245]Open in a new tab 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 EFL[1] 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 [246]3, [247]4 and [248]5. Table 3. Pathway Enrichment analysis of differentially expressed proteins relative to SE compared with control group NO Maps pValue 1 Immune response_Oncostatin M signaling via JAK-Stat in mouse cells 0.000195 2 Immune response_Oncostatin M signaling via JAK-Stat in human cells 0.000242 3 Development_Thrombopoetin signaling via JAK-STAT pathway 0.000294 4 Immune response_IL-15 signaling via JAK-STAT cascade 0.000322 5 Development_Transcription factors in segregation of hepatocytic lineage 0.000552 6 Immune response_IL-7 signaling in T lymphocytes 0.000887 7 Immune response_IL-7 signaling in B lymphocytes 0.001136 8 Cell adhesion_Ephrin signaling 0.001244 9 Neurophysiological process_Receptor-mediated axon growth repulsion 0.001244 10 Immune response_IL-5 signaling 0.001300 11 Signal transduction_PTMs in IL-12 signaling pathway 0.001415 12 G-protein signaling_Rap1B regulation pathway 0.013047 13 Protein folding_Membrane trafficking and signal transduction of G-alpha (i) heterotrimeric G-protein 0.022438 14 Immune response_IL-12 signaling pathway 0.027103 15 Development_Glucocorticoid receptor signaling 0.028266 [249]Open in a new tab Table 4. Pathway Enrichment analysis of differentially expressed proteins relative to SEP compared with control group NO Maps pValue 1 Cytoskeleton remodeling_Role of PDGFs in cell migration 0.002188 2 Glycolysis and gluconeogenesis p.3 / Human version 0.002188 3 Glycolysis and gluconeogenesis p.3 0.002188 4 Development_PDGF signaling via STATs and NF-kB 0.003877 5 Normal and pathological TGF-beta-mediated regulation of cell proliferation 0.004119 6 Cell adhesion_Ephrin signaling 0.007559 7 Neurophysiological process_Receptor-mediated axon growth repulsion 0.007559 8 Development_PDGF signaling via MAPK cascades 0.008224 9 Some pathways of EMT in cancer cells 0.009631 10 Aberrant B-Raf signaling in melanoma progression 0.011137 11 Transport_Macropinocytosis regulation by growth factors 0.014439 12 Glycolysis and gluconeogenesis (short map) 0.015773 13 G-protein signaling_Rap1B regulation pathway 0.031748 14 Cell adhesion_Chemokines and adhesion 0.034254 15 Cytoskeleton remodeling_Cytoskeleton remodeling 0.035519 [250]Open in a new tab Table 5. Pathway Enrichment analysis of differentially expressed proteins relative to EFL[1] compared with control NO. Maps pValue 1 Development_Angiopoietin - Tie2 signaling 0.000027 2 Immune response_IL-7 signaling in T lymphocytes 0.000035 3 Immune response_IL-7 signaling in B lymphocytes 0.000051 4 Immune response_Antiviral actions of Interferons 0.000090 5 Immune response_Oncostatin M signaling via JAK-Stat in mouse cells 0.000425 6 Immune response_Oncostatin M signaling via JAK-Stat in human cells 0.000526 7 Development_Thrombopoetin signaling via JAK-STAT pathway 0.000639 8 Immune response_IL-15 signaling via JAK-STAT cascade 0.000699 9 Immune response_IL-23 signaling pathway 0.000827 10 Signal transduction_PTMs in IL-23 signaling pathway 0.001274 11 Development_PDGF signaling via STATs and NF-kB 0.001357 12 Immune response_IL-22 signaling pathway 0.001532 13 Development_EPO-induced Jak-STAT pathway 0.001623 14 Development_Growth hormone signaling via STATs and PLC/IP3 0.001623 15 Immune response_IL-9 signaling pathway 0.001717 [251]Open in a new tab 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 (EFL[1]) 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. [252]3 (A-D). (Tables [253]6 and [254]7). Fig. 3. Fig. 3 [255]Open in a new tab 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 Network GO processes Total nodes Seed nodes p-Value zScore gScore Angiopoietin 4, NF-kB, ALDR, TIE2, ATP + PtdIns(4,5)P2 = ADP + PtdIns(3,4,5)P3 response to oxygen-containing compound (70.6%; 1.570e-16), regulation of multicellular organismal process (76.5%; 2.094e-15), response to peptide (47.1%; 1.618e-14), response to stress (82.4%; 2.570e-14), positive regulation of cellular process (88.2%; 3.104e-14) 51 1 0.00245 20.16 22.66 [256]Open in a new tab Table 7. Union of differentially expressed protein Networks Network GO processes p-Value zScore gScore Trypsin II, Chymotrypsin C, Trypsin 3, TATI, RAIG1 antigen processing and presentation of peptide or polysaccharide antigen via MHC class II (27.3%; 6.498e-17) 1.010E-21 48.76 48.76 Ubiquitin, Fetuin-A, UBC, RelA (p65 NF-kB subunit), TRAF2 regulation of response to stress (56.5%; 6.254e-19), positive regulation of NF-kappaB transcription factor activity (28.3%; 2.556e-17) 1.140E-05 14.33 44.33 STAT1, TGTP, Mcpt4 (rodent), Sca-1, Thrombomodulin interferon-gamma-mediated signaling pathway (31.9%; 8.694e-24), response to interferon-gamma (36.2%; 1.175e-23) 2.970E-14 33.37 33.37 [257]Open in a new tab Obviously, commonly pathways are mainly interleukin-mediated signaling pathways, including IL-7, IL-15, IL-23 and other inflammatory factors both controlled by EFL[1] 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. [258]3a). DEPs which were taken together mainly participated in the protein interaction networks as shown in Fig. [259]3b, [260]c and [261]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. [262]3b), stress response (containing 3 up-regulation differential proteins, RelA/P65 and ubiquitin are the central part of network, Fig. [263]3c), γ- Interferon - mediated signal transduction and response (containing 6 up-regulation,1 down-regulation differential proteins, as shown in Fig. [264]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 EFL[1], 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 [[265]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 [[266]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. [267]4 and [268]5). As depicted in Figs. [269]4 and [270]5 and Table [271]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. Fig. 4 [272]Open in a new tab 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. Fig. 5 [273]Open in a new tab Western blotting showing the changes in Ang4 and STAT1 level in mice intestine treated with different doses of SE, SEP and EFL[1] 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 EFL[1] (EFLH, 20 mg/20 g), 7 Low-dose of EFL[1] (EFLL, 10 mg/20 g) Table 8. The relative expression of Ang4 and STAT1 in intestinal tissue of mice ( [MATH: X¯ :MATH] ±S, n = 5) groups Ang4 STAT1 Control 0.865 ± 0.027 0.396 ± 0.019 SEH 0.489 ± 0.084* 0.706 ± 0.167* SEL 0.683 ± 0.218 0.439 ± 0.046 SEPH 0.598 ± 0.142* 0.421 ± 0.076 SEPL 0.803 ± 0.080 0.358 ± 0.086 EFLH 0.582 ± 0.098* 1.326 ± 0.372* EFLL 0.749 ± 0.111 0.731 ± 0.133 [274]Open in a new tab Note: *compared with control (P < 0.05) 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 [[275]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. [276]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 [[277]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 [[278]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) [[279]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. Acknowledgments