The microarray expression dataset was downloaded from the GEO database by searching the key words: “RNA,” “peritoneal adhesion,” or “abdominal adhesion” and “Mus musculus” (organism). After screening, the accession number GSE123413 was obtained for analysis. The platform was GPL 21163, Agilent-074809 SurePrint G3 Mouse GE v2 8x60K Microarray. This dataset consisted of the transcriptional profiling of cecum tissues harvested at three time points (i.e. 3 h, 12 h, and 3 d) after the models were prepared by cauterization. The raw data were preprocessed and normalized using the affy package in R (version 3.3.4). Then the differentially expressed genes (DEGs) between sham (SH) and PPA groups at three different time points were screened separately using the limma package. |log₂FC| > 1 and P < 0.05 were considered statistically significant for DEGs. The overlapping DEGs of the three different time points were generated using the online tool Venny (http:// bioinfogp.cnb.csic.es/tools/venny). The heat map of DEGs was obtained with the online tool Morpheus (https://software.broadinstitute.org/morpheus/).

To understand the underlying biological phenomena, gene ontology (GO) terms were used to determine gene annotation. Kyoto Encyclopedia of Genes and Genomes (commonly known as KEGG) enrichment was performed to locate the significant enrichment pathway. Both analyses were implemented on the Database for Annotation, Visualization and Integrated Discovery (commonly known as DAVID; http://david.abcc.ncifcrf.gov/)[7].

To further predict the interaction of peritoneal adhesion-associated protein pairs, the Search Tool for the Retrieval of Interacting Genes (commonly known as STRING)[8] was performed with a confidence score > 0.7 defined as significant. Then PPI integrated networks were mapped by Cytoscape 3.4.0 software[9]. Finally, the plug-in Molecular Complex Detection (commonly known as MCODE) was used to screen the modules of hub genes from the PPI network when node degree > 30 was considered as cut-off criteria.

Twenty male Sprague-Dawley rats (8 wk old, weighing 280 ± 20 g) were purchased from the Qinglongshan Experimental Animal Breeding Farm (Nanjing, China). The rats were randomly divided into two groups: SH (n = 10) and PPA (n = 10). Both groups were housed in a standard condition of 12 h light-dark cycle (light on at 07:00 a.m.) under a controlled temperature of 22 ± 2°C. All animals were provided plenty of food and water and allowed to acclimatize to this condition 3 d before use. All experiments in this study followed the Guidelines of Accommodation and Care for Animals formulated by the Chinese Convention for the protection of vertebrate animals used for experimental and other scientific purposes and were authorized by the Laboratory Animal Management Committee of the Nanjing University of Chinese Medicine.

The cecum cauterization model was established by a previous study[10]. After preoperative fasting for 12 h, the rats were placed under anesthesia with 1.0%–1.5% isoflurane. A 1.5 cm midline incision was made in the abdominal wall after traditional skin preservation and sterilization under aseptic conditions. The cecum was isolated and then cauterized using bipolar forceps to inflict a coagulation function for 1 s. Finally, the cecum was restored into the abdominal cavity, and the abdominal wall was sutured. After 3 d, the rats were sacrificed. Two independent investigators who were blinded to both groups evaluated the adhesion quality on the basis of a five-stage grading score system[11,12] shown in Table 1[13].

After fixation in 10% neutral formalin for 48 h, the cecum tissues were embedded in paraffin, and cut into sections. Masson staining was performed to estimate the collagen deposition. Represented views were visualized under a microscope (DM2500; Leica, Germany).

Total RNA was extracted from the damaged cecum using TRIzol reagent (Invitrogen, United States). The complementary DNA (cDNA) was generated via reverse transcription by using the First Stand cDNA Synthesis Kit (Thermo Fisher Scientific, United States). UltraSYBR One Step RT-qPCR Kit (Cwbio Technology, China) was used according to the manufacturer’s protocol. Based on GAPDH as standardization, the expression levels were conducted by using the 2^−ΔΔCT analysis method. The primer sequences are shown in Table 2.

Tissue proteins were extracted by the RIPA Protein Extraction Kit (Beyotime Biotechnology, China) according to routine protocols. The extracted proteins were added to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes. Then the membranes were incubated with primary antibody at 4°C overnight against Toll-like receptor 4 (TLR4, 1:200), myeloid differentiation primary response protein 88 (MyD88, 1:200), nuclear factor kappa B (NF-κB, 1:200), and β-actin (1:2000) (all from Santa Cruz Biotechnology, United States). After incubation with secondary antibody for 1 h at 37°C, protein expression was viewed by the Chemiluminescence Imaging system (Bio-Rad, United States).

Statistical analyses were conducted using SPSS 19.0 software. All data are presented as the mean ± SD. Statistical comparisons within the two groups were made by the unpaired Student’s t-test. P values less than 0.05 were considered statistically significant.

The GSE123413 expression profile dataset consisted of the expression data matrix of 56743 gene probes. The raw data were processed and normalized with R software, as presented in Figure 1A. We identified the DEGs of three time points and found 457 overlapping genes. Of these genes, 446 expressed genes were altered, among which 183 were upregulated and 263 were downregulated. The expression levels of dysregulated genes are shown in Figure 1B.

Open in New Tab   Full Size Figure   Download Figure

Figure 1 Box plots of data normalization and hierarchical cluster heatmap. A: Box plots of data normalization. The blue box plot represents the data before normalization, whereas the red box plot represents the normalized data; B: Heatmap of the obtained DEGs.

Analyses of the above-obtained 446 genes were performed using DAVID for biological process annotation and KEGG pathway enrichment. GO term assignment analysis suggested that the most enriched GOs were enriched in inflammatory response (GO: 0006954), neutrophil chemotaxis (GO: 0030593), cytokine activity (GO: 0005125), cellular response to interleukin-1 (IL-1) (GO: 0071347), immune response (GO: 0006955), and response to lipopolysaccharide (GO: 0032496), as presented in Figure 2A. According to pathway enrichment analysis, the enriched altered genes were involved in PPA-related pathways such as the cytokine-cytokine receptor interaction and tumor necrosis factor (TNF) signaling pathway (Figure 2B).

Open in New Tab   Full Size Figure   Download Figure

Figure 2 GO annotation and pathway enrichment of DEGs in adhesion formation. A: GO analysis of DEGs (top 20); B: KEGG enrichment of DEGs (top 20). GO: Gene ontology; DEGs: Differentially expressed genes; KEGG: Kyoto Encyclopedia of Genes and Genomes.

The 743 pairs involving 376 proteins were mapped in the PPI network through the STRING website. The genes with relatively high degrees were considered hub genes related to peritoneal adhesion such as TNF (degree = 43), IL-6 (degree = 42), C-X-C motif chemokine ligand 2 (CXCL2; degree = 39), CXCL1 (degree = 38), and IL-1β (degree = 34). These genes may play pivotal roles in PPA progression. To further depict the complex relationship, the obtained interaction pairs were constructed using Cytoscape software (Figure 3A). After analysis using the plug-in MCODE, the top two significant modules were identified and enriched in the inflammatory response by DAVID. Module 1 involved 18 genes and 153 connections, and module 2 implicated 30 genes and 176 connections that were markedly enriched in the TLR signaling pathway, as shown in Figure 3B and Table 3.

Open in New Tab   Full Size Figure   Download Figure

Figure 3 Visualization of the PPI network of identified DEGs. A: Network of the adhesion formation-associated genes; B: Two significant modules selected from the PPI network. Red: Greater degree; Yellow: Lesser degree. DEG: Differentially expressed genes; PPI: Protein–protein interaction.

The incisions of all rats were primarily healed, without infection or other complications. The adhesion grades and scores among groups are shown in Table 4. In the SH group, the adhesion grade ranged from 0 to 1, which indicated the absence of adhesion or minor adhesion. However, the adhesion grade ranged from 2 to 4, and the score was approximately 3.4, which suggested that dense connective tissues were present in the PPA groups. The magnitude of peritoneal adhesions is presented in Figure 4.

Open in New Tab   Full Size Figure   Download Figure

Figure 4 Representative images of PPA in each group. A: PPA group; B: Sham group.

Microscopically, collagen fibers appeared blue and muscle fibers were red in color. The model group displayed a deep and extensive range of blue, which indicated increased collagen fiber accumulation. However, the SH group displayed a light and narrow range of blue color, which suggested decreased collagen fiber accumulation (Figure 5).

Open in New Tab   Full Size Figure   Download Figure

Figure 5 Representative images of Masson staining of adhesive tissues (200×). A: Masson staining in the PPA group; B: Masson staining in the SH group. PPA: Postoperative peritoneal adhesion; SH: Sham.

To verify the bioinformatics analysis, the expression levels of the hub genes (TNF, IL-1β, IL6, CXCL1, and CXCL2) of the cecum tissues were quantified by quantitative PCR (qPCR) between the adhesion models and SH controls. The results indicated that the mRNA expression levels significantly increased (P < 0.05, Figure 6) in PPA groups compared with the SH groups.

Open in New Tab   Full Size Figure   Download Figure

Figure 6 Representative mRNA expression level by qPCR. Expression levels of the mRNAs (TNF-α, IL1β, IL6, CXCL1, and CXCL2) were performed using qPCR, results are expressed as mean ± SD. PPA: Postoperative peritoneal adhesion; SH: Sham.

In our study, functional enrichment of modules analysis suggested that the significant hub genes were markedly enriched in the TLR signaling pathway. Increasing evidence[14-16] suggests that TLR4, MyD88, and NF-κB play essential roles in the inflammatory pathway such as Toll-like signaling. On the basis of this evidence, we measured the expression levels of the abovementioned proteins by western blotting. The protein expression levels of TLR4, MyD88, and NF-κB in the PPA groups were significantly upregulated compared with those in the SH group (Figure 7).

Open in New Tab   Full Size Figure   Download Figure

Figure 7 Relative protein levels by western blot analysis. The expression levels of TLR4, MyD88, and NF-κB were obtained using western blot analysis. The results are expressed as mean ± SD. PPA: Postoperative peritoneal adhesion; SH: Sham.

Postoperative peritoneal adhesion (PPA), characterized with abdominal pain, female infertility, and even bowel obstruction after surgery, has always been a major concern. The occurrence and formation of adhesion are from complex biological processes. However, the molecular mechanisms of the basis of microarray data profile, followed by PPA formation, are largely unknown.

The occurrence and development of PPA is a complex biological process, during which many genes and pathways are involved in the pathogenesis of adhesion formation. As such, we developed microarray analysis combined with experimental methods to understand the underlying mechanisms of PPA at the transcriptomic and molecular levels.

The aim of this study was to uncover the molecular mechanisms of PPA formation after surgery using bioinformatics analysis, and to validate the results using rodent adhesion models.

The gene expression profile was retrieved from the Gene Expression Omnibus database for our analysis. A panel of key altered genes and related pathways involved in adhesion formation were identified using bioinformatics analysis methods. And the microarray results were verified by performing quantitative PCR and western blotting in vivo preliminarily.

In total, 446 expressed genes were altered in peritoneal adhesion. We found that several hub genes (e.g., TNF, IL-1β, IL-6, CXCL1, CXCL2) were marked as significant biomarkers associated with PPA. Functional analysis suggested that these genes were enriched in the Toll-like receptor signaling pathway. According to the Kyoto Encyclopedia of Genes and Genomes pathway and published studies, TLR4, MyD88, and NF-κB played essential roles in Toll-like signaling transduction. Here, we gained a regulatory evidence chain of TLR4/MyD88/NF-κB/inflammatory cytokines/peritoneal adhesion involved in the pathogenesis of PPA. The results of the microarray analysis were consistent with the animal experiments.

Our findings provide initial evidence about the regulatory evidence chain of TLR4/MyD88/NF-κB/inflammatory cytokines/peritoneal adhesion in the pathogenesis of PPA. Future studies are required to validate the results.

These findings may extend our understanding of the molecular mechanisms of PPA. Further functional and gene knockout studies are warranted to elucidate the exact effects on the transcriptional expression of genes regulated by NF-κB axis activation.