Proteins Expression Profiling of Rat Uteruses with Primary Dysmenorrhea Syndrome


 Purpose The aim of this study was to investigate differentially expressed proteins (DEPs) and their functions in the uteruses of primary dysmenorrhea (PD) rats by using label-free quantitative proteomics analysis.Methods The PD rat model was induced by injecting both estradiol benzoate and oxytocin. Twenty rats were equally divided into two groups: a control group (normal rats), a PD model group (PD rats). Writhing scores and serum levels of Prostaglandin E2 (PGE2) and Prostaglandin F2α (PGF2α) were used to evaluate the success of the rat PD model. The DEPs were identified and analyzed by label-free quantitative proteomics and bioinformatics analyses.Results A total of 276 DEPs were identified, including 119 up-regulated DEPs and 157 down-regulated DEPs. Bioinformatics revealed that the DEPs were mainly associated with ‘protein binding’, ‘metabolism’, ‘signal conduction’ and ‘focal adhesion’. The proteomic findings were verified by western blot analysis, which confirmed that myosin light chain kinase (MLCK), heat shock protein 90 AB1 (HSP90AB1), apolipoprotein A1 (Apoa1), p38 MAP kinase, c-Jun N-terminal kinase (JNK), and extracellular signal-related kinase 1/2 (ERK1/2) were significantly differentially expressed in between the control and PD samples.Conclusions These results provide a deeper understanding the molecular pathogenesis of PD. The DEPs found in the present study may provide new ideas for further study of the mechanism of PD and aid the search for biomarkers for early diagnosis and treatment.


Introduction
Primary dysmenorrhea (PD) refers to recurrent menstrual cramps without identi able pelvic pathology [1,2]. It is estimated that the prevalence of PD ranges from 45-95% [1,2]. The type of syndrome for PD is pain which causes extreme physical and mental suffering to patients [3,4]. Although previous studies focused on the excessive secretion of uterine prostaglandins (PGs) for the understanding etiology of PD [5][6][7], the pathogenesis of PD remains largely unknown. Therefore, identi cation of novel therapeutic targets for PD will be bene cial for a large group of patients.
Label-free quantitative proteomics is a novel tool used for etiological study and biomarker identi cation in various diseases [8,9]. Although the mRNA expression pro le of uterus from PD syndrome rats was reported previously [10], the protein expression pro le of PD remains unknown so far. In this study, label-free quantitative proteomics and bioinformatics analyses were adopted to explore the differentially expressed proteins (DEPs) and their functions in the uteri of PD rats. Our ndings provide a deeper understanding the molecular pathogenesis of PD and aid the search for biomarkers for early diagnosis and treatment.

Animal model establishment
The rat model of PD was established as previously described by us [11]. Then Wistar female rats were used in each group. The animal experiment was approved by Ethics Committee of Taicang Hospital of traditional Chinese Medicine.

Writhing test
The writhing reaction of rats in response to pain was evaluate by writhing scores and calculated as previously described by us [11].
Enzyme-linked immunosorbent assay (ELISA) Serum levels of PGE2, PGF2α, TNF-α and IL-8 were measured using speci c ELISA kits according to the manufacturer's guidelines.

Proteomics analysis
The uteri of three rats per group were collected. Sample preparation, protein digestion, LC-MS/MS analysis, and identi cation of differentially expressed proteins (DEPs) were performed according to our previous study [11]. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were adopted to investigate signi cantly enriched function and signaling pathways of the DEPs. The interaction network of the DEPs was constructed using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING).

Western blotting
Western blotting assays were performed as previously described by us [12]. The following primary antibodies were obtained from Cell Signaling Technology Inc. and used : anti-Apoa1 Statistical analysis SPSS version 19.0 software (IBM Corp., NY) was used for all statistical analyses. Differences between the two groups were analyzed by the Student's t-test. One-way ANOVA was used to make comparisons between multiple groups. Results are expressed as the mean ± standard deviation. Statistical signi cance was assumed at P<0.05.

Results
Induction rat model of PD As shown in Fig.1a, compared with control group, the PD rats exhibited high writhing scores (Fig.1a).
ELISA assay showed that the serum levels of PGE2 and PGF2α were signi cantly higher than those in the control group (Fig.1b). These data suggested that the PD rat model had been established successfully.

Identi cation of DEPs in uterine tissue of PD rats
The protein expression pro les from the uterine tissue of PD rats were investigated by Lab-free based quantitative proteomic method. The proteins expression pattern between the control and PD groups was demonstrated using hierarchical clustering (Fig.2a). Statistically signi cant alterations in proteins between the control and PD groups were identi ed using a volcano plot (Fig.2b). A total of 276 differentially expressed proteins [DEPs, fold change (FC) ≥1.5 and P<0.05] were identi ed in the uterine tissue of PD rats compared with the control group, including 119 up-regulated DEPs and 157 downregulated DEPs (Fig.2c).

Bioinformatics analysis of DEPs
As shown in Fig.3a, GO analysis showed that a total of 4167 biological process (BP) terms were found in the all DEPs, of which 1579 BP terms were signi cantly enriched (P<0.05). A total of 587 terms were found in cell component (CC), of which 276 were signi cantly enriched (P<0.05). A total of 846 terms were found in molecular function (MF), of which 337 were signi cantly enriched (P<0.05). BP analysis showed that the majority of the DEPs were associated with 'response to stimulation', including 'response to oxygen-containing compound', 'response to organic substance', and 'response to endogenous stimulus' (Fig.3b). When the DEPs were analyzed for CC, they were enriched in the cytoplasm and membranebound organelles (Fig.3c). With regards to MF term, the majority of DEPs were associated with binding functions, including protein binding, RNA binding and molecular binding (Fig.3d).
The KEGG pathway analysis showed that the DEPs related to metabolism and focal adhesion signaling pathways (Fig.4a). As shown in Fig.4b, a total of 15 DEPs were involved in the focal adhesion pathway, including 6 up-regulated DEPs and 3 down-regulated DEPs.
The protein-protein interaction (PPI) of the DEPs was analyzed by using STRING. As shown in Fig.5, the DEPs were involved in several signaling pathways or BPs, including metabloic pathways, focal adhesion, spliceosome, biosynthesis of amino acids and platelet activation. Mostof the DEPs in the PPI network exhibited direct or indirect links.

Validation of DEPs by western blotting
As shown in Figure 6A, compared with control group, MLCK expression was signi cantly decreased, while Apoa1 and HSP90AB1 expression, as well as the levels of phosphorylated ERK, JNK, and p38, were signi cantly increased in the model group (P < 0.05). However, the total expression levels of ERK, JNK, and p38 remained unchanged. These ndings were consistent with those derived from proteomic analysis.

Discussion
Recently, Label-free quantitative proteomics becomes a popular method in the search for disease-associated proteins [8,9]. To the best of our knowledge, the present study rstly used label-free quantitative proteomics in a rat model of PD to identify the key proteins directly, rather than mRNAs. The in vivo rat model managed with estradiol benzoate and oxytocin is frequently used in PD studies [13][14][15]. The rat model of PD in the present study was successfully established by using this method.
A total of 379 DEPs were identi ed in the present proteomics experiments. GO and KEGG analyses indicated that the DEPs were involved in various BPs and signaling pathways, which may play important roles in the occurrence and development of PD. Protein-protein interaction (PPI) analysis further revealed that the DEPs were involved in several signaling pathways or BPs and performed their functional roles collectively in speci c networks.
The uterus is an organ where lipid distribution plays a critical role for its function. Previous studies showed that increased cholesterol could decrease uterine activity [16]. Cholesterol has been to be enriched in microdomains of the plasma membrane known as rafts and caveolae, which have been implicated in cellular signaling cascades [17]. Apolipoprotein A1 (Apoa1), which is the major protein component of high density lipoprotein (HDL) in plasma, plays an important role in cholesterol transport [18][19][20][21]. In the present study, the expression level of Apoa1 was signi cantly increased in the uterus of PD rats. Based on the above ndings, we speculate that the increased uterine contractions may due to upregulated Apoa1, which led to change of cholesterol content in the uterus of PD.
It is demonstrated that oxytocin signaling and the in ammatory response play critical roles in the occurrence of PD [22][23][24]. MAP kinases, including p38MAP kinase, c-Jun N-terminal kinase (JNK/SAPK), and extracellular signal-related kinase (ERK) were shown to mediate oxytocin signaling and regulate the production of in ammatory cytokines [25][26][27][28]. In the present study, the phosphorylation levels of p38, ERK, and JNK, were all signi cantly up-regulated in the uterus of PD rats, while the total expression of these MAP kinases remained unchanged. It is will be interesting whether these activated MAP kinases play roles in the occurrence of PD by regulating oxytocin signaling and the in ammatory response.
Heat shock protein 90 (HSP90) is a critical molecular chaperone protein that acts on a wide variety of different proteins and cellular processes [29,30]. HSP90 overexpression was demonstrated to be closely related to the occurrence and development of many diseases, such as bronchopulmonary dysplasia, cystic brosis and cancer [31]. HSP90AA1 and HSP90AB1, the two major isoforms of HSP90, has been shown to exert multitude roles in many human diseases due to their interaction with different proteins (its client proteins) [32][33][34][35]. In the present study, the expression level of HSP90AB1 was signi cantly increased in the uterus of PD rats, while the expression level of HSP90AA1 remained unchanged. Previous studies revealed that HSP90 is involved in the regulation of estrogen signaling, which is essential for the progression of PD [36,37]. Therefore, it is reasonable to speculate that up-regulated HSP90AB1 promotes the progression of PD by regulating estrogen signaling.
The increased uterine smooth muscle contraction has been considered as a main cause of PD [38][39][40][41]. Myosin light chain kinase (MLCK) I, a Ca(2+)-calmodulin-activated kinase, regulates smooth muscle contraction by phosphorylation of myosin and is found in many tissues [42,43]. MLCK was shown to be important for regulating uterine smooth muscle contraction [44,45]. In the present study, the expression level of MLCK was signi cantly decreased in the uterus of PD rats, which contrary to we expected.
Whether the decreased MLCK plays a role in uterine smooth muscle contraction of PD remains unknown. Myosin light-chain kinase (MLCK) of smooth muscle consists of an actin-binding domain at the Nterminal, the catalytic domain in the central portion, and the myosin-binding domain at the C-terminal [42,43]. Previous work has suggested that in addition to its kinase activity, MLCK exhibits non-kinase properties within its N-terminus that could in uence cytoskeletal organization of smooth muscle cells [46].
In conclusion, our study rstly analyzed the protein expression pro le of uterus from PD rats by using label-free quantitative proteomics. The identi ed proteins and related signaling pathways might play crucial roles in the development of PD. The identi ed DEPs in the present study may be utilized as candidate biomarkers for PD.

Declarations
Funding This research was funded by the Project of Taicang Science and Technology (Reference number: TC2018JCYL21).

Con icts of interest
The authors declare that they have no con ict of interest.

Availability of data and material
All of the data reported in this article are available from the corresponding author upon reasonable request.  Figure 1 Up-regulation of writhing scores and the serum levels of PGF2α and PGE2 in PD rats. Data represent mean ± SD (n = 10); **p < 0.01 versus normal control group.