Proteomic Analysis of Differentially Expressed Proteins in the Placenta of Anticardiolipin Antibody- (ACA-) Positive Pregnant Mice after Anzi Heji Treatment

Anzi Heji (AZHJ) has been used to treat anticardiolipin antibody- (ACA-) positive pregnant women at risk of spontaneous abortion for many years. The aim of this study was to investigate the protective mechanism of AZHJ in a mouse model of ACA-positive pregnancy at risk of spontaneous abortion using label-free quantitative proteomics. Mice were divided into three groups: normal pregnant mice (control group), ACA-positive pregnant mice administered normal saline (model group), and ACA-positive pregnant mice administered AZHJ (AZHJ group). The model was established by injecting β2-glycoprotein I (GPI) into mice for 18 days. The DEPs and their functions were analyzed by label-free quantitative proteomic and bioinformatic analyses. The levels of IL-6, IL-10, ACA, and TNF-α in the serum and placentas of the mice were measured by enzyme-linked immunosorbent assays (ELISAs). Proteomic data were validated by western blot analysis. The abnormal serum and placental levels of IL-6, ACA, and TNF-α in the model group were reversed by AZHJ. There were 39 upregulated and 10 downregulated DEPs in the AZHJ group relative to the model group. Bioinformatic analysis revealed that the DEPs were mainly involved in nucleic acid binding, signal conduction, and posttranslational modification. The placental levels of T-cell immunoglobulin mucin 3 (Tim-3) and Toll-like receptor 4 (TLR4) expression and AKT phosphorylation in the three groups were consistent with the proteomic findings. Tim-3/AKT signaling is involved in maternal-fetal immune tolerance, while TLR4 is associated with inflammatory responses. Collectively, these results indicate that AZHJ may exert its protective effect in ACA-positive pregnant mice by regulating the maternal-fetal immune tolerance and inflammatory response.


Introduction
Recurrent spontaneous abortion (RSA) refers to three or more consecutive pregnancy losses [1,2]. About 1-3% of healthy women with normal fertility and >15% of women experiencing RSA are positive for anticardiolipin antibody (ACA) [3]. ACA-positive patients have decreased in vitro fertilization, pregnancy, and implantation rates and an increased risk of spontaneous abortion [3,4]. erefore, increasing attention has been paid to the relationship between ACA and RSA.
Extracts from traditional Chinese medicine herbs or other herbs have been used to prevent abortion due to their various abilities to regulate immune responses [5][6][7]. Anzi Heji (AZHJ) is an approved prescription at Jiangsu Province Hospital of Traditional Chinese Medicine, China (approval no. 051226). It has been used to prevent spontaneous abortion (especially in ACA-positive cases) for many years. It has no toxic side effects [7]. e main components of AZHJ are Cuscuta chinensis, sangjisheng, ramie root, Dipsacus asperata, Salvia miltiorrhiza, Atractylodes macrocephala, Scutellaria baicalensis, radix Pseudostellariae, and roasted Glycyrrhizae. Our previous clinical studies revealed that AZHJ can significantly improve the clinical symptoms of ACA-positive pregnant women at risk of spontaneous abortion and improve pregnancy hormone levels [8][9][10].
Fetal preservation was successful in >85% of cases, and the conversion rate of ACA-positive to ACA-negative results was 90% [8][9][10]. However, the mechanism underlying the therapeutic effect of AZHJ remains largely unknown.
In this study, the proteomic changes in ACA-positive pregnant mice in response to AZHJ treatment were investigated using nano-high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) technology.
ere were 39 upregulated and 10 downregulated differentially expressed proteins (DEPs) after AZHJ treatment. is study provides a foundation for future studies investigating the therapeutic mechanisms of AZHJ.

AZHJ Preparation.
AZHJ extraction and quality control were performed as described previously [7].

Animal Experiments.
e animal experiment design and schedule are shown in Figure 1. Eight-week-old BALB/c female (25 ± 2 g) and male (30 ± 2 g) mice were obtained from Cavens Laboratory Animal Co., Ltd (Changzhou, China). e mice were maintained in specific pathogen-free conditions at 25°C and 50-60% humidity with a 12 h/12 h light/dark cycle. e mice were fed standard chow and provided water. e mouse model of ACA-positive pregnancy at risk of spontaneous abortion was established by injecting 400 µg/mL β2-glycoprotein I (GPI) into 20 female BALB/c mice for 18 days, as described previously [11][12][13]. Another 10 female BALB/c mice were injected with normal saline for 18 days and used as the normal controls. e female mice were kept in cages with the male mice (1:1), and the day of vaginal plug appearance was considered day 0 of gestation (E0). e 20 female GPI-treated pregnant BALB/c mice were randomly divided into the AZHJ or model group. e mice in the AZHJ group were intragastrically administered 37.7 mg/g AZHJ for 15 days. e mice in the model group were intragastrically administered 0.1 mL/10 g distilled water for 15 days. e 10 normal controls were also intragastrically administered 0.1 mL/10 g distilled water for 15 days. e pregnant mice were killed on E15. e embryo resorption rate, R, was calculated as R � Re/(Re + F), where Re is the number of resorbed embryos and F is the number of surviving embryos. e mouse experiments were carried out in accordance with guidelines approved by the Ethics Committee of Taicang Hospital of Traditional Chinese Medicine.

Sample Preparation and Protein Digestion.
ree placental samples were obtained from each of the three groups and mixed to create a 5 µl sample for each group.
Radioimmunoprecipitation assay (RIPA) lysis buffer was added to the tissues, which were cut into pieces, mechanically homogenized using a tissue homogenizer three times for 3 s, and kept on ice for 15 min. After centrifugation at 12,000 g for 15 min at 4°C, the supernatant was collected and transferred to an Eppendorf tube. e proteins were redissolved in 500 mM triethylammonium bicarbonate (TEAB). e protein concentration was determined using a bicinchoninic acid (BCA) protein assay. Next, 100 μg protein per group was transferred to a new tube and adjusted to a final volume of 100 μL with 8 M urea. ereafter, 11 μL of 1 M dithiothreitol (DTT) was added and the sample was incubated at 37°C for 1 h and then transferred into a 10K Microcon Centrifugal Filter (Millipore, Billerica, MA, USA). To remove the urea, 100 mM TEAB was added and the samples were centrifuged, and this process was repeated twice. Next, 120 μL of 55 mM iodoacetamide was added and the sample was incubated for 20 min in the dark at room temperature. e proteins were then digested with sequencing-grade modified trypsin (Promega, Madison, WI, USA) and lyophilized.

Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) Analysis.
e peptides were redissolved in 30 μL solvent A (A: 0.1% formic acid in water) and underwent LC-MS/MS analysis using an Orbitrap Fusion coupled to an EASY-nano-LC 1200 system ( ermo Fisher Scientific, Waltham, MA, USA). A 6 μL peptide sample was loaded into a trap column (Acclaim PepMap C18 Column, 75 μm × 2 cm; ermo Fisher Scientific). ey were subsequently separated using an analytical column (Acclaim PepMap C18 Column, 75 μm × 50 cm) with a linear gradient from 5% B (B: 0.1% formic acid in acetonitrile) to 38% B over 120 min. e column flow rate was maintained at 300 nL/ min with a column temperature of 40°C. An electrospray voltage of 2 kV was applied between the spray emitter and the inlet of the mass spectrometer.

Data Analysis.
e MS/MS spectra were processed using PEAKS Studio v8.5 (Bioinformatics Solutions Inc., Waterloo, Canada). PEAKS DB was used to search the UniProt mouse database (v201711, 52194 entries), assuming trypsin as the digestion enzyme and setting the fragment ion mass tolerance to 0.05 Da and the parent ion tolerance to 7 ppm. Carbamidomethylation (C) was specified as the fixed modification, and oxidation (M), deamidation (NQ), and acetylation (protein N-termini) were specified as the variable modifications. e peptides were filtered based on a 1% false discovery rate and a minimum of one unique peptide per protein. Analysis of variance (ANOVA) was used for the peptide and protein abundance calculations. Normalization involved averaging the abundance of all peptides (based on median values). DEPs were defined as proteins with a fold change >1.5 and ≥2 unique peptides with significance >13 (p < 0.05).

Bioinformatic Analysis. Gene Ontology (GO) and Kyoto
Encyclopedia of Genes and Genomes (KEGG) analyses were used to identify the significantly enriched biological functions and signaling pathways related to the DEPs based on the GO (geneontology.org/) and KEGG (genome.jp/kegg/) databases. To categorize the DEPs based on protein orthologs and paralogs, the Clusters of Orthologous Groups database was used (more specifically, the euKaryotic Orthologous Groups (KOGs) database; ncbi.nlm.nih.gov/COG/). Lastly, a DEP interaction network was constructed using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), as described previously (http://string-db.org/).

Western Blot Analysis.
Total protein was extracted from placental tissues (n � 6 per group) using ice-cold RIPA lysis buffer containing 1% phenylmethanesulfonyl fluoride (PMSF) and a complete protease inhibitor cocktail (Beyotime Institute of Biotechnology, Haimen, China). e protein concentration was determined using a Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Next, 30 µg total protein per group was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. e membranes were blocked with 5% nonfat milk for 1 h at room temperature. ey were then incubated at 4°C overnight with rabbit anti-Tim-3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-TLR4(1:1000; Cell Signaling Technology, Danvers), anti-p-AKT(1:1000; Cell Signaling Technology), anti-AKT (1:1000; Cell Signaling Technology, Danvers), or rabbit anti-β-actin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by adding peroxidase-labeled secondary antibody for 1 h at room temperature. e membranes were then rinsed three times (for 10 min each time) using Tris-buffered saline with Tween 20. Next, the target proteins were detected using enhanced chemiluminescence reagent ( ermo Fisher Scientific) according to the manufacturer's instructions. e relative optical density of the bands of interest was analyzed using ImageJ v1.48 (National Institutes of Health, Bethesda, MD, USA).

Statistical
Analysis. Statistical analysis was performed using SPSS v19.0 (IBM Corp., Armonk, NY, USA). One-way ANOVA was used to analyze differences between multiple groups, and Student's t-test was used to analyze differences between pairs of groups. Results are expressed as the mean ± standard deviation. p < 0.05 was considered to indicate a statistically significant difference.

AZHJ Reversed the Increased Embryo Loss Rate, the Increased ACA and TNF-α Levels, and the Decreased IL-6 Levels in β2-GPI-Treated Mice.
e embryo loss rate of the mice in the model group was significantly higher than in the normal control group (Figure 2(a)). e model group also showed remarkable increases in the levels of ACA (Figure 2(b)) and TNF-α (Figure 2(c)) and decrease in the levels of IL-6 ( Figure 2(d)) in serum and placentas, suggesting that the model had been established successfully. However, AZHJ reversed the β2-GPI-induced increases in embryo loss rate and ACA and TNF-α levels and the decreases in IL-6 levels.
ese results suggest that AZHJ attenuates the β2-GPI-induced responses in ACA-positive pregnant mice at risk of spontaneous abortion.

Identification of DEPs in Placental Tissues.
Nano-HPLC-MS/MS was used to explore the protective mechanisms of AZHJ.

Bioinformatic Analysis of DEPs.
e DEPs (AZHJ vs. model group) were analyzed with regard to GO cellular components (CC), molecular functions (MF), and biological processes (BP) using InterProScan. Regarding the   Evidence-Based Complementary and Alternative Medicine BP terms, the majority of the DEPs were associated with cell cycle process and lipid biosynthetic process ( Figure 3). Regarding the CC terms, the DEPs were most commonly associated with nuclear lumen (Figure 3). Lastly, regarding the MF terms, the DEPs were most commonly associated with nucleic acid binding (Figure 3). e KEGG pathway analysis showed that the DEPs implicated in the protective effects of AZHJ were mainly involved in signaling pathways related to spliceosomes, human papillomavirus infection, the phosphoinositide 3kinase (PI3K)-AKT signaling pathway, and protein processing in the endoplasmic reticulum (Figure 4).
e COG analysis showed that the DEPs were associated with posttranslational modification, protein turnover, chaperones, translation, ribosomal structure, and biogenesis ( Figure 5).
To better understand the protective mechanisms of AZHJ, DEP interaction networks were constructed using STRING. As shown in Figure 6, most of the DEPs in the interaction network exhibited direct or indirect links.

Validation of DEPs by Western Blotting.
e levels of Tim-3 and Toll-like receptor 4 (TLR4) expression and AKT phosphorylation were significantly increased in the model group compared to the control group (p < 0.05, Figure 7(a)). However, the levels were significantly reduced by AZHJ. ese findings were consistent with those derived from the proteomic analysis. In addition, serum IL-2 and IFN-c were increased and serum IL-10 was decreased in the model group compared to the control group (p < 0.05, Figures 7(b)-7(d)), and these changes were reversed in the AZHJ group (p < 0.05, Figures 7(b)-7(d)).

Discussion
is study is the first to use a quantitative proteomic analysis to identify the DEPs in placental tissues from ACA-positive pregnant mice after AZHJ treatment. After AZHJ treatment, the serum and placental levels of ACA and TNF-α were decreased, while the serum and placenta levels of IL-6 were increased. ese findings suggest that AZHJ exhibited a  Evidence-Based Complementary and Alternative Medicine significant therapeutic effect in ACA-positive pregnant mice. ACA induces abortion via promoting thrombosis, damaging trophoblasts, and activating inflammation and the complement response by targeting β2-GPI [14,15]. TNF-α plays a critical role in the physiology and pathology of pregnancies by regulating the immune balance at the maternal-fetal interface [16,17]. Excessive TNF-α causes thrombosis and TNF-α can activate immunocompetent cells in the decidual stroma, induce immune-mediated destruction and immune rejection, hinder the maintenance of pregnancy and embryo implantation, disrupt the homeostasis of the maternal uterine environment, and cause spontaneous abortion [16,18,19]. IL-6, which is produced by extravillous trophoblasts and cytotrophoblasts, plays an important role in the normal placental development and pregnancy success by regulating placental cell migration and invasion and trophoblast differentiation and proliferation [20,21]. However, the role of IL-6 in ACA-positive spontaneous abortion is controversial, as Krause et al. [22] showed that the serum IL-6 was increased in ACA-positive pregnant mice, while Karakantza et al. [23] reported the opposite result. Our previous studies showed that serum IL-6 in ACA-positive pregnant mice at risk of spontaneous abortion was significantly increased, but there were no changes in the placental or decidual IL-6 levels [11][12][13]. us, we speculate that IL-6, as a proinflammatory factor, plays a key role in the inflammatory reaction, which may be related to ACA production and ACA-induced intravascular lesions, inflammatory responses, and thrombosis. Our label-free quantitative proteomic analysis identified 49 DEPs in the placentas of ACA-positive pregnant mice after AZHJ treatment. GO and KEGG analyses further revealed that the significantly enriched biological functions and signaling pathways included nucleic acid binding, the PI3K-AKT signaling pathway, and protein processing in the endoplasmic reticulum. ese GO terms and KEGG pathways may shed light on the mechanisms underlying the therapeutic effect of AZHJ. According to the COG analysis, the DEPs were predominantly enriched in posttranslational modification, protein turnover, chaperones, and translation, ribosomal structure, and biogenesis.
1 and 2 cell-mediated immune regulation is a major mechanism of maternal peripheral immune tolerance [16,24]. ere is a significant 2 bias in normal pregnancy,  Evidence-Based Complementary and Alternative Medicine while spontaneous abortion is often associated with a 1 bias [24]. 1 cytokines (including IL-2, IFN-c, and TNF-β) are involved in inflammatory response and immune response [16,24]. 2 cells produce anti-inflammatory IL-4, IL-5, IL-6, and IL-10, which contribute to the success of pregnancy [16,24]. IL-6 contributes to normal embryo implantation in early pregnancy [16,24]. As an immunosuppressive factor, IL-10 plays an important role in maintaining immune tolerance [16,24]. In addition, IL-10 can downregulate 1 cytokines in macrophages and inhibit NF-κB, thus playing an immunomodulatory role [16,24]. Tim-3 is a Tim family receptor protein that is a type I transmembrane protein [25]. It is expressed in a variety of cells, including 1, 17, NK, and NKT cells, Tregs, dendritic cells, monocytes, macrophages, and decidual stromal cells [26]. It plays an important regulatory role in  Evidence-Based Complementary and Alternative Medicine the occurrence and development of female reproductive system diseases and is closely related to the occurrence of spontaneous abortion [26]. Blocking Tim-3 in vitro decreases IL-4 and IL-10 production by CD 8 + T cells, increases IFN-c production, disrupts the 1/ 2 balance, and affects the maternal-fetal tolerance [27]. Galectin-9 (Gal-9), which is related to the activation of T cells, is a ligand of Tim-3 [26]. High Tim-3 expression in the peripheral blood and decidual tissues of patients with RSA has been reported to be related to the occurrence and development of RSA [28,29]. Other studies reported that soluble Tim-3 and Gal-9 are increased in the serum of patients with unexplained RSA, which indicated that the soluble costimulatory molecule Tim-3 may regulate the differentiation of 1 and 2 cells in patients with unexplained RSA, with the 1/ 2 balance being biased toward 1 [30][31][32]. e combination of Tim-3 and Gal-9 may weaken the Tim-3/ Gal-9 signaling pathway and transmit inhibitory signals, thus participating in the occurrence and development of unexplained RSA. In the present study, the placental level of Tim-3 protein was significantly increased in the ACApositive pregnant mice and significantly decreased after AZHJ treatment. e differences in the serum levels of 1/ 2-type cytokines (such as IL-2, IFN-c, and IL-10) between the model and AZHJ groups might be at least partly attributable to the altered Tim-3 expression. e PI3K-AKT signaling pathway plays an important role in the occurrence and development of RSA (including unexplained RSA) [33,34]. Inhibition of this pathway promotes trophoblast apoptosis and suppresses trophoblast proliferation and migration [33,34]. is pathway also controls the differentiation of Treg and 17 cells [35]. We found that the phosphorylation level of AKT in the placentas of ACA-positive pregnant mice was increased significantly, while it was decreased by AZHJ. PI3K-AKT signaling is an important downstream target of Tim-3 [36]. Furthermore, Tim-3/Gal-9 signaling in peripheral NK cells promotes maternal-fetal tolerance by regulating the PI3K-AKT signaling pathway [28]. us, we speculated that the reduced phosphorylation level of AKT may be due to the downregulation of Tim-3 expression by AZHJ treatment.
TLRs play important roles in reproductive processes such as ovulation, spermatogenesis, sperm capacitation, fertilization, and pregnancy [37]. A recent study showed that decreased TLR4 expression in response to ligand treatment of spermatozoa is associated with unexplained RSA [38]. However, a study by Li et al. [39] revealed that increased TLR4 expression in decidual and chorionic tissues was closely related to the occurrence of RSA. In the present study, increased TLR4 expression was observed in the placentas of ACA-positive pregnant mice. To the best of our knowledge, the present study is the first to evaluate TLR4 expression in ACA-positive pregnant mice at risk of spontaneous abortion. Our results revealed that TLR4 expression was significantly decreased by AZHJ, suggesting that it may be a critical downstream target of AZHJ. Tim-3 is involved in regulating TLR4 signaling, and whether TLR4 was regulated by Tim-3 after AZHJ treatment needs to be investigated in the future.

Conclusion
is study provides evidence that AZHJ exerts a protective effect against spontaneous abortion in ACA-positive pregnant mice. Our data suggest that the mechanisms underlying these effects include inhibiting Tim-3/AKT signaling and downregulating TLR4. Our data provide guidance and a useful foundation for investigating new treatments for ACApositive spontaneous abortion.

Data Availability
All of the data reported in this article are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.