Camostat mesilate inhibits pro-inflammatory cytokine secretion and improves cell viability by regulating MFGE8 and HMGN1 in lipopolysaccharide-stimulated DF-1 chicken embryo fibroblasts

Cell culture

DF-1 chicken embryo fibroblasts (CRL-12203; American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (Thermo Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Thermo Scientific) at 39 °C, and treated with different concentrations of LPS (Sigma-Aldrich, St. Louis, MO, USA) for 6 h.

Reagents

Lipopolysaccharides (from Escherichia coli) (L2630) were purchased from Sigma-Aldrich. CM was obtained from ChemeGen Lnc. (Los Angeles, California, USA). Small interference RNAs (siRNAs) targeting MFGE8 (si-MFGE8#1 and si-MFGE8#2), HMGN1 (si-HMGN1#1 and si-HMGN1#2), and a scrambled siRNA control (si-NC) were synthesized by GenePharma Co., Ltd. (Shanghai, China). Cells were transfected with corresponding siRNAs using Lipofectamine 2000 reagent (Thermo Scientific) according to the instructions of the manufacturer. SiRNA sense sequences were presented in Table S1.

RT-qPCR assay

Total RNA was extracted from DF-1 chicken embryo fibroblasts using Trizol reagent (Thermo Scientific). Next, RNA was reversely transcribed into cDNA first strand using M-MLV Reverse Transcriptase (Thermo Scientific) following the manufacturer’s instructions. The mRNA expression levels of MFGE8, myristoylated alanine-rich protein kinase C substrate (MARCKS), HMGN1, high mobility group nucleosomal binding domain 4 (HMGN2), and GAPDH were measured through the real-time quantitative system using cDNA template, SYBR Green PCR Master Mix (Thermo Scientific) and corresponding quantitative primers. The primer sequences were presented as below: 5′-CGATCTGAACTACATGGTTTAC-3′(forward) and 5′-TTCACTCTGATGCGGTCCAC-3′(reverse) for GAPDH, 5′-GCCACGTCAAAGACTGGAAAC-3′(forward) and 5′-AATGCCTGCACCCCCTTATAC-3′(reverse) for HMGN1, 5′-TGTTAGCACACAGACCGCTT-3′(forward) and 5′-TTCACTCTGATGCGGTCCAC-3′(reverse) for HMGN2, 5′-TTACCACCATTCCAACGG GC-3′(forward) and 5′-GATCCCTTATCGACCCACCC-3′(reverse) for MARCKS, 5′-CAAGGTTTTCCAGGGCAACG-3′(forward) and 5′-GCAACCTGCCGTGTTGAAAT-3′(reverse) for MFGE8.

ELISA assay

DF-1 chicken embryo fibroblasts transfected with or without si-NC, si-MFGE8, or si-HMGN1 were treated with LPS for 6 h and then incubated with CM for an additional 24 h. TNF-α and IL-6 secretion levels were measured using corresponding ELISA kits (MEIMIAN Biotechnology Co., Ltd., Wuhan, China) following the manufacturer’s protocols.

CCK-8 assay

Cell viability was determined through Cell Counting Kit-8 (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocols. Briefly, cells were inoculated into 96-well plates. At the indicated time points after treatment, 10 µl of CCK-8 solution was added into each well (100 µl medium/well). Three hours later, the optical density (OD) values were determined at 450 nm.

Cell apoptosis detection

Cell apoptotic rate was determined by Annexin V-FITC Apoptosis Staining/Detection Kit (Solarbio, Beijing China). Briefly, DF-1 cells were collected and re-suspended in 500 µl of 1 x Binding Buffer. Then, cells were co-incubated with 5 µl of Annexin V-FITC and 5 µl of PI at room temperature in a dark room. Cell apoptotic patterns were analyzed by flow cytometry (Becton Dickinson Co. CA, USA).

TMT-based proteomics analysis

Cell samples (three biological replicates) were lysed using the protein lysis buffer, sonicated, and centrifuged to obtain cell lysates containing proteins. After treated with dithiothreitol (DTT) and iodoacetamide (IAM), proteins were digested into peptide segments with trypsin. Next, the peptide segments post enzymolysis were labeled with TMT and fractionated on an L-3000 high-performance liquid chromatography (HPLC) system. Finally, fractions were analyzed and detected using EASY-nLCTM 1200 Ultra High-Performance Liquid Chromatography (UHPLC, Thermo Scientific) and Q Exactive HF-X Mass Spectrometer (Thermo Scientific). Proteins were identified using Proteome Discoverer 2.2 (Thermo Scientific). Proteins were quantified using Maxquant software. GO and KEGG pathway enrichment analyses were performed using the KOBAS 3.0 website (http://kobas.cbi.pku.edu.cn/kobas3).

Statistical analysis

In the proteomics analysis, differences between groups were analyzed using Student’s t-test. P values obtained from Student’s t-test were corrected through false discovery rate (FDR). The P values after FDR correction were termed as Q values. Proteins were regarded as differentially expressed at the fold change ≥1.2 or ≤0.83 and Q value ≤0.05.

In the cell experiments, data analysis was conducted using GraphPad Prism software 7.0 (La Jolla, CA, USA) with the results presenting as mean ± standard deviation. Differences between groups were compared using Student’s t-test. Differences among groups were analyzed through one-way ANOVA (Tukey’s post-hoc test) or two-way ANOVA (Sidak post hoc test). Differences were regarded as statistically significant when the P value was less than 0.05.

CM weakened LPS-induced pro-inflammatory cytokine secretion and cell injury in DF-1 chicken embryo fibroblasts

Firstly, our data revealed that LPS facilitated TNF-α and IL-6 secretions and decreased cell viability in a concentration-dependent manner with the strongest effect at the concentration of 10 µg/ml (Figs. 1A–1C). Hence, 10 µg/ml of LPS was used to construct the cell model. Moreover, our study demonstrated that CM dose-dependently inhibited the secretions of TNF-α and IL-6 in the LPS (10 µg/ml)-exposed DF-1 cell model (Figs. 1D and 1E). Additionally, CM increased cell viability in a time- and concentration-dependent manner in LPS-treated DF-1 chicken embryo fibroblasts (Fig. 1F). These data suggested that CM could protect DF-1 chicken embryo fibroblasts from LPS-induced inflammation and cell injury.

Identification of dysregulated proteins in response to LPS stimulation alone or in combination with CM in DF-1 chicken embryo fibroblasts

Next, dysregulated proteins in DF-1 chicken embryo fibroblasts upon the treatment of LPS alone or in combination with CM were screened out by TMT/mass spectrometry (MS)-based proteomics analysis. Results showed that 273 proteins were differentially expressed (182 up-regulated, 91 down-regulated) in LPS treated DF-1 cell group (DL group) compared to the untreated cell group (D group) (Figs. 2A and 2B). Moreover, 102 proteins were highly expressed and 547 proteins were low expressed in DF-1 chicken embryo fibroblasts co-treated with LPS and CM (DLE group) than that in DF-1 chicken embryo fibroblasts treated with LPS alone (DL group) (Figs. 2A and 2B). In addition, 26 common proteins were identified by Venn analysis among down-regulated proteins in DL vs D group and up-regulated proteins in DLE vs DL group (Fig. 2A). Also, 115 proteins were found to be up-regulated in DL vs D group and down-regulated in DLE vs DL group (Fig. 2B). These proteins with the converse change trends in DLE vs DL and DL vs D groups might be involved in the responses of DF-1 chicken embryo fibroblasts to LPS and CM. The heat map of the proteins with converse change trends in DLE vs DL and DL vs D groups was presented in Fig. S1 and corresponding protein information was displayed in Table S2. Next, GO and KEGG enrichment analysis for these proteins with the converse change trends in DLE vs DL and DL vs D groups were carried out to decrypt the protective mechanisms of CM in LPS-stimulated DF-1 chicken embryo fibroblasts. The top 20 GO enrichment terms based on biological processes, cellular components, and molecular functions were presented in Figs. 2C–2E. GO enrichment analysis showed that these proteins were mainly involved in the regulation of biological processes such as cellular/metabolic/biological processes, cellular component organization or biogenesis, gene expression, protein/nitrogen compound/peptide/amide transport, and apoptotic cell clearness (Fig. 2C, Table S3). KEGG enrichment analysis revealed that 7 KEGG pathways (ribosome, steroid biosynthesis, RNA transport, MAPK signaling pathway, spliceosome, herpes simplex virus 1 infection, and salmonella infection) were significantly enriched by these proteins (Fig. 2F, Table S4). These enrichment analyses suggested that CM could trigger the notable dysregulation of a host of essential genes related to cell development.

Effects of LPS alone or in combination with CM on the expression of MFGE8, MARCKS, HMGN1, and HMGN2 in DF-1 chicken embryo fibroblasts

Among proteins with the inverse alteration trends in DLE vs DL and DL vs D groups, 4 proteins (MFGE8 (Yi, 2016), MARCKS (Green et al., 2011), HMGN1 (Yang et al., 2012), and HMGN2 (Xie et al., 2011)) related to inflammation were screened out for further explorations according to previous documents. Annotation analysis revealed that MFGE8 was involved in the regulation of multiple pathways or biological processes such as protein metabolism, post-translation protein modification, insulin-like growth factor (IGF) transport and uptake by insulin-like growth factor binding proteins (IGFBPs), phagocytosis, and apoptotic cell clearance (Table S5). MARCKS participated in actin/protein kinase C/calmodulin binding, actin filament organization, central nervous system development, and actin crosslink formation (Table S5). HMGN1 could regulate nucleotide-excision repair, chromatin organization, transcription by RNA polymerase II, UV-B/UV-C response, and NAD+ ADP-ribosyltransferase activity (Table S5). HMGN2 was implicated in chromatin organization (Table S5). TMT-MS data revealed that MFGE8 expression was notably increased in DLE vs DL group (1.66-fold) and markedly reduced in DL vs D group (0.81-fold) (Table S2). The fold-change values of MARCKS, HMGN1, and HMGN2 in the DLE vs DL group are 0.61, 0.59, and 0.55, respectively. The fold-change values of MARCKS, HMGN1, and HMGN2 in the DL vs D group are 1.39, 1.54, 1.89, respectively (Table S2). Consistent with the TMT-MS outcomes, RT-qPCR assay also disclosed that MFGE8 expression was markedly reduced, and HMGN1 and HMGN2 expression was notably increased in LPS-stimulated DF-1 chicken embryo fibroblasts (DL group) versus un-stimulated cells (D group) (Figs. 3A, 3C, 3D). However, there was no obvious alteration in MARCKS expression in LPS-stimulated DF-1 chicken embryo fibroblasts compared to untreated cells (Fig. 4B). In line with TMT-MS data, higher MFGE8 expression and lower HMGN1 expression were also validated by RT-qPCR assay in DF-1 chicken embryo fibroblasts with the combined treatment of LPS and CM (DLE group) compared to cells treated with LPS alone (Figs. 3A, 3C). Nevertheless, the addition of CM did not influence MARCKS and HMGN2 expression in LPS-treated DF-1 chicken embryo fibroblasts (Figs. 3B, 3D). Given the consistency of RT-qPCR and proteomics data, HMGN1 and MFGE8 were selected for further investigations.

CM exerted its functions through regulating MFGE8 and HMGN1 expression in LPS-treated DF-1 chicken embryo fibroblasts

To further investigate the functions of MFGE8 and HMGN1, si-NC and siRNAs targeting MFGE8 and HMGN1 were synthesized. Knockdown efficiency analyses revealed that the introduction of si-MFGE8#1 led to the notable down-regulation of MFGE8 mRNA level in DF-1 chicken embryo fibroblasts compared with the si-NC group, while si-MFGE8#2 could not effectively silence MFGE8 in DF-1 chicken embryo fibroblasts (Fig. 4A). Also, a noticeable reduction of HMGN1 mRNA expression level was observed in DF-1 chicken embryo fibroblasts transfected with si-HMGN1#1 or si-HMGN1#2 than that in cells transfected with si-NC (Fig. 4B). Considering the stronger knockdown outcomes of si-MFGE8#1 and si-HMGN1#2 on their respective targets, si-MFGE8#1 and si-HMGN1#2 were selected in the subsequent loss-of-function explorations. Functional analyses revealed that MFGE8 knockdown could weaken the effects of CM on TNF-α and IL-6 secretions, cell viability, and cell apoptosis in LPS-treated DF-1 chicken embryo fibroblasts (Figs. 4C–4F). Moreover, our TMT-MS outcomes showed that CM treatment led to the notable increase of MFGE8 in LPS-treated DF-1 chicken embryo fibroblasts. These data suggested that CM could inhibit TNF-α and IL-6 secretions and improved cell viability by up-regulating MFGE8 in LPS-treated DF-1 chicken embryo fibroblasts. Additionally, HMGN1 knockdown did not have statistically significant influence on TNF-α and IL-6 secretions, cell viability and apoptosis in DF-1 chicken embryo fibroblasts co-treated with LPS and CM (Figs. 4C–4F).