Chemerin Promotes Mitogen-activated Protein Kinase / Extracellular Regulated Protein Kinases Activation to Induce Inflammatory Factor Production in Rat Synoviocytes

2.1 | Materials

Rat synovial cells (RSC-364) were purchased from Shanghai Zeye Biotechnology Co., Ltd. Other experimental equipment and reagents included: Superclean cell bench (SW-CJ-2D model, Suzhou); ABI 7500 real-time PCR (RT-PCR) instrument (Applied Biosystems Inc, USA); constant temperature carbon dioxide incubator (Thermo Fisher Scientific, USA); Thermo microplate reader (Multiskan Fc); ultrasonic cell disruptor (Z-11D, Japan); Tanon 5200 Multi automatic chemiluminescence/fluorescence image analysis system (Tanon 5200 Multi); fluorescence inverted microscope (Olympus, Japan); 95% Dulbecco's Modified Eagle’s Medium (DMEM) high glucose medium (Solaibao, Beijing); cellular RNA reverse transcription kit (Thermo Fisher Scientific, USA); fluorescent quantitative Polymerase Chain Reaction(PCR) kit (Roche SYBR Green Master, Switzerland); recombinant murine chemerin (RampD, USA); trypsin-ethylene Diamine Tetraacetic Acid (EDTA) digestive solution (Solaibao, USA) Beijing); penicillin/streptomycin; Phosphate Buffered Saline (PBS) solution (Solaibao, Beijing); cell total RNA rapid extraction kit (Solaibao, Beijing); mycoplasma-free fetal bovine serum (Sijiqing, Fig. Zhejiang); High Performance Radio Immunoprecipitation Assay (RIPA) Lysis Solution (Biyuntian), Western and Immunoprecipitation ༈IP༉ Cell Lysis Solution (Biyuntian), Bicinchoninic acid (BCA) Protein Quantification Kit (Syndicate), Antibody electrochemiluminescence (ECL) kit (Syndicate), Immobilon-P 26.5 × 3.75 m Roll polyvinylidene fluoride (PVDF) 0.45 µm (Milipore), PageRuler Prejugated Protein Ladder, 10180 kDa (Thermo), Spectra Multicolor Range High Activity Protein Ladder (Thermo), Horseradish Peroxidase (HRP)-Conjugated Antibody (Anti-rabbit glyceraldehyde 3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase ༈GAPDH༉, Mouse IgG, HRP-linked antibody (Cell Signaling Technology (CST)), Anti-rabbit IgG, HRP-7074 (CST), Anti-ERK1 + ERK2 (abcam), Rabo-p44/42 MAPK (ERK1/2)T mAb (CST), p38 Rabbit mAb (CST), Phospho-p38 Rabbit mAb (CST); Cell Counting Kit-8 (CCK-8) Assay Kit (Dojindo); Rat Matrix Metalloproteinase (MMP)-3 Enzyme-Linked Immunosorbent Assay (ELISA) Kit, Rat MMP-13 ELISA Kit, Rat Tumor Necrosis Factor-α (TNF-α) ELISA Kit, Rat Interleukin (IL)-1β ELISA Kit, and Rat IL-6 ELISA Kit (enzyme-linked Biological, Shanghai).

2.2 | Cell processing

RSC-364 cells were cultured at 37°C in DMEM (10% fetal bovine serum and 1% double antibody) in a 5% carbon dioxide incubator. The cells were subcultured by rinsing twice with sterile PBS followed by digestion in trypsin-EDTA (Solaibao, Beijing), which was evenly applied with observation under an inverted microscope. When 70–80% of the cells began to shrink and round or lift off the surface, 95% DMEM high-glucose culture medium was rapidly added, with gentle agitation using a sterile Papanicolaou dropper. The cells were subcultured at a ratio of 1:2 or 1:3. For cryopreservation, the cells were transferred into centrifuge tubes, the supernatants were removed after centrifugation, and the cell pellets were resuspended in an appropriate amount of cryopreservation solution (containing 20% fetal bovine serum, 10% DMSO, and 70% DMEM medium). The cells were dispensed into 1.5 mL cryopreservation tubes, placed in a programmed cryopreservation box in a − 80°C freezer, and then transferred to liquid nitrogen for long-term preservation. For recovering cryopreserved cells, cryotubes containing the cells were quickly placed in a 37°C water bath for re-warming. After thawing, the tubes were placed in a centrifuge (1500 rpm, 5 min) to remove the supernatant. The cells were then re-suspended in DMEM culture medium and transferred to a culture flask. The rat synoviocytes were evaluated experimentally within two to five passages.

2.3 | CCK-8 assay

After RSC-364 synovial cells were passaged 3 times, the cells were seeded into two 96-well plates at a concentration of 5 × 10³ cells/well. The plates were incubated in a carbon dioxide atmosphere at 37°C for 24 h, then the plates were divided in half. Half of the plate was treated with MEK inhibitor (10 µM PD98059) for 30 min; then, 100 µL of 0, 0.25, 0.5, or 1.0 µg/mL Reconstitute Chemerin’s medium was added, and the plate was placed back in the incubator. After 24 h, 10 µL of CCK-8 solution was added to each well according to the instructions of the CCK-8 kit (Dojindo); the plated was incubated in the cell culture chamber for another 4 h. The absorbance was measured at 450 nm with a microplate reader. The experiment was repeated three times.

2.4 | Quantitative RT-PCR

The synoviocytes, passaged to the third generation, were seeded in a six-well plate at 4 × 10⁴ cells/well and cultured for 24 h. The cells were divided into two groups: the control group and the chemerin group (0.5 µg/mL recombinant murine chemerin; RampD, USA). After continuous culture for 48 h, total RNA was extracted according to the instructions of the RNA rapid extraction kit (Solaibao, Beijing), and then cDNA was synthesized by reverse transcription (Thermo Fisher Scientific, USA) and quantified using a fluorescence quantitative PCR kit (Roche SYBR Green Master, Switzerland). Quantitative PCR was performed for GAPDH, MMP-3, MMP-13, MEK, and ERK using the primers in Table 1. GAPDH was set as the internal reference. All experimental assays were repeated three times.

2.5 | Western blot analysis

The cells, which were passaged for three generations, were divided into the control and chemerin groups and were seeded in six-well plates at 4 × 10⁴/well. After 24 h of attachment, the cells were stimulated for 10 min and then RIPA lysis buffer (containing protease inhibitors) was added. After incubation on ice, the cells were lysed by sonication and centrifuged, and the supernatant was recovered. The protein concentrations were determined by BCA assay, and equal amounts of protein were mixed with loading buffer in a boiling water bath for 5 min. Electrophoresis was carried out at 80 V initially, and then 120 V until the bromophenol blue had migrated to about 1 cm away from the lower edge of the gel. The membranes were blocked with rocking at room temperature for 1 h and then washed three times with Tris-buffered saline Tween (TBST) for 5 min each. The corresponding antibodies were diluted according to the manufacturer recommendations (HRP-Conjugated GAPDH Antibody, HRP-60004, Fig. Proteintech; Anti-Mouse IgG, HRP-linked Antibody, 7076P2, CST; Anti-rabbit IgG, HRP-linked Antibody, 7074S, CST; Anti-ERK1 + ERK2, ab184699, Abcam; Phospho-p44/42 MAPK (ERK1/2) Rabbit mAb, 4377T, CST; Rabbit mAb, 8690T, CST; Phospho-p38 Rabbit mAb, 4511T, CST). The membranes were incubated with primary antibody at room temperature for 2 h or overnight at 4°C and then washed 3 times with TBST for 5 min each time and incubated with HRP-labeled secondary antibody for 1 h at room temperature. Chromogenic droplets were added to the membranes, and the gray values of the target bands were calculated with Image J software.

2.6 | Enzyme linked immunosorbent assay

After RSC-364 synoviocytes were cultured for 3 passages, the cells were seeded in a six-well plate at 4 × 10⁴/well and cultured in a 37°C constant temperature incubator for 24 h. They were divided into three groups: the control group, the chemerin group (0.5 µg/mL chemerin) and the inhibitor group. The inhibitor group was pre-treated with MEK inhibitor (10 µM PD98059, CST # 9900S) for 1 h and then recombinant chemerin was added at the same concentration as in the chemerin group. After 48 h, the supernatant was collected to determine the levels of MMP-3, MMP-13, TNF-α, IL-1β and IL-6 secreted by synoviocytes of rats in each group according to ELISA kit (Shanghai) instructions.

2.7 | Animal handling:

Thirty sterile SD rats were divided into three groups. Groups B and C were first modeled by the modified Huths anterior cruciate ligament cutting method²³. After the operation, the rats were intraperitoneally injected with 200,000 units of penicillin for 3 consecutive days and were permitted to roam freely in the cage. One week after modeling, 20 rats were randomly divided into two groups. In group C, each knee joint was injected with about 0.1 ml solution containing recombinant Chemerin, once every three days for three weeks; group B was injected with the same amount of normal saline. In group A, no surgical modeling was performed, and each knee joint was injected with the same amount of normal saline at the same times as the above two groups. The growth environment of the three groups of rats was the same.

2.8 | HE staining and ELISA

Three weeks after the final chemerin or saline injection, the rats were injected with an overdose of 1% sodium pentobarbital and sacrificed. The skin was incised along the middle of the knee joint until the entire knee joint was exposed. An incision was made from the upper edge of the patella to the femur, and then the soft tissue was separated from the tibia with ophthalmic scissors along both sides of the patella. The knee joint cavity was opened, and forceps were used to excise the patella and surrounding tissues. The synovial tissue of the patella, which appeared as a layer of smooth light and pale yellow that continued downwards under the patella, was removed and carefully cut with ophthalmic scissors. HE staining was performed as follows: First, paraffin sections were deparaffinized with xylene and washed with various levels of ethanol: xylene (I) for 5 minutes → xylene (Ⅱ) for 5 minutes → 100% ethanol for 2 minutes → 95% ethanol for 1 minute → 80% ethanol for 1 minute → 75 % Ethanol for 1 minute → distilled water for 2 minutes. Then, the sections were stained with hematoxylin dye solution for 10 minutes and rinsed with autoclaved water. Differentiating solution was applied for 30 seconds, followed by warm water (about 50°C) for 5 minutes, eosin dye solution for 2 minutes, and a final rinse with autoclaved water. Conventional ethanol dehydration was performed step by step (95% ethanol (I) for 1 minute → 95% ethanol (II) for 1 minute → 100% ethanol (I) for 1 minute → 100% ethanol (II) for 1 minute → two Toluene carbolic acid (3:1) for 1 minute → xylene (I) for 1 minute → xylene (II) for 1 minute), and then the sections were sealed with neutral resin, covered with a cover slip, and observed under a microscope.

For ELISA, synovial tissue was excised according to the above method and was added to sterile PBS (1 g:10 ml) and mashed. The suspension was centrifuged at 1000×g for 10 minutes, and the supernatant was recovered. The levels of MMP-3, MMP-13, IL-1β and IL-6 in the synovial tissues were determined according to the instructions of the ELISA kit (enzyme-linked immunosorbent assay, Shanghai).

2.9 | Statistical analysis

SPSS 25.0 software was used for statistical processing, expressed as x ± s. One-way Anova analysis of variance was used for multiple group comparisons. T tests were used for comparisons. The difference was considered statistically significant when P < 0.05.

3.1 | Chemerin regulates the proliferation of synovial cells in a dose-dependent manner

To verify the effect of chemerin on synovial cells, we established a RSC-364 cell culture. As expected, the cells exhibited elongated polygonal shapes (Fig. 1). We performed CCK-8 analysis after 24 h of treatment with different concentrations of recombinant mouse chemerin. The results showed that after 24 h, chemerin promoted proliferation, and the proliferation of chemerin could be prevented by pretreating the cells with the MEK/ERK inhibitor PD98059 (Fig. 2). The difference in the effects of chemerin at 24 h suggests that the effects of chemerin may be time- and dose-dependent. In addition, the results support the use of 0.5 µg/mL chemerin as a dose that affects synovial cells in 24 h in subsequent experiments, and further indicates that the effect of chemerin may be related to the MEK/ERK pathway.

3.2 | Chemerin promotes the activation of MAPK pathways in synoviocytes

Based on the above findings and given the important role of MAPK pathways in the pathogenesis of OA,^16,17 we sought to determine whether chemerin may regulate the expression of MAPKs. After 48 h, the gene expression of MEK and ERK in synoviocytes that were cultured in the presence of 0.5 µg/mL recombinant mouse chemerin were significantly increased compared to the levels in the control group. Further, the expression of MMP-3 and MMP-13 genes also increased (Fig. 3).

MAPK activity is often also regulated by phosphorylation. Therefore, we also evaluated the total and phosphorylated levels of MAPK proteins after 10 min of exposure of the synoviocytes to chemerin. Western blot analysis demonstrated a significant increase in expression of total ERK1/2, pERK1/2, MEK, pMEK and p-p38 after chemerin treatment, though levels of total p38 were unaltered (Fig. 4). These findings are consistent with our mRNA results and indicate that chemerin may regulate cell growth by activating ERK1/2 expression and p38 phosphorylation.

3.3 | Chemerin promotes the expression of inflammatory cytokines in a MAPK-dependent manner

Given the role of synovial tissue inflammation in OA progression,^3,4 we evaluated whether chemerin may modulate the production of inflammatory mediators in synoviocytes, and whether MAPKs may be involved in this process. Cells were stimulated with chemerin for 48 h in the presence of absence of the MEK/ERK pathway inhibitor PD98059, and ELISA assays were performed to evaluate the concentrations of MMP-3, MMP-13, TNF-α, IL-1β, and IL-6 in culture supernatants. The results demonstrate that chemerin induced a significant increase in each of these inflammatory modulators, and that the increase in MMP-3 and MMP-13 was abolished in synoviocytes pretreated with PD98059 (Fig. 5). Levels of TNF-α, IL-1β, and IL-6 also appeared to be reduced by PD98059, and the decrease did reach statistical significance. These results are consistent with a role for the MEK/ERK pathway in mediating the activation of MMPs and potentially other inflammatory mediators in synoviocytes.

3.4 |Chemerin promotes synovial inflammatory hyperplasia and the release of inflammatory factors in rats

To determine whether the inflammatory effects of chemerin are recapitulated in an in vivo model, we divided rats into three groups. One group (the “blank group”) did not receive surgery and received injections of saline. The second group (the “control” group) received knee surgery to induce arthritis followed by saline injections, and the third group (the “chemerin” group) received knee surgery followed by chemerin injections. Observation of HE-stained synovial tissue under a microscope showed that the tissue from the blank group contained 1–2 layers of synovial cells arranged regularly and with a mostly flat shape (Fig. 6). The tissue was relatively loose, with a small amount of capillaries and no obvious inflammatory cell infiltration; the cartilage surface was smooth and flat, without damage or bone destruction, and no inflammatory cell infiltration could be seen under the microscope. For the control group (Fig. 7), the synovial tissue was more hyperplasic than that of the blank group. A small amount of synovial fibroblasts proliferation was observed, and inflammatory cell infiltration could be seen in the intercellular space. There was slight cartilage destruction, but no obvious bone destruction. Finally, in the chemerin group (Fig. 8), the synovial tissue showed obvious signs of proliferation, and the infiltration of inflammatory cells (mostly lymphocytes) was more than that of the control group. There was also obvious vascular proliferation, and the continuity of the articular cartilage surface was interrupted, with obvious cartilage defects.

ELISA results verified that recombinant rat chemerin can also promote synovial tissue to produce corresponding inflammatory mediators in animal experiments. Compared with the control group, each of the assayed inflammatory factors in the chemerin group showed a significant increase. In addition, when the control group was compared with the blank group, there was corresponding synovial inflammation, but the degree of lesions and the degree of inflammatory factors were lower than for the drug group (see Fig. 9). These differences are statistically significant (P < 0.05), and the results are consistent with the role of synovial cell MMPs and potential other inflammatory mediators in the activation of synovial cells under the action of chemerin.