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


 AimChemerin is a chemokine from adipose tissue that specifically binds to the G protein-coupled receptor ChemR23 and has a chemotactic effect on macrophages and dendritic cells. A correlation between chemerin levels in synovial fluid and disease severity has been demonstrated in patients with osteoarthritis. However, the specific mechanism by which chemerin exerts its effects on osteoarthritis remains unclear. In this study, we investigated the mechanism of chemerin-associated synoviocyte inflammation.MethodsCell Counting Kit-8 (CCK-8) assays were used to identify concentrations of chemerin that had an effect on normal rat synoviocytes. The expression changes of mitogen-activated protein kinase kinase (MEK)/extracellular regulated protein kinases (ERK) pathway marker genes, including MEK, ERK, matrix metalloproteinase (MMP)-3 and MMP-13, were detected by fluorescence quantitative polymerase chain reaction (PCR). The phosphorylation of MEK, ERK1/2 and p38 mitogen-activated protein kinases (p38MAPK) by chemerin was analyzed by Western blotting, and the production of inflammatory factors after chemerin treatment was determined by enzyme-linked immunosorbent assay (ELISA). For in vivo assessment of chemerin function, rats were subjected to knee operation to provide a model for arthritis. The knees were then injected with normal saline or recombinant chemerin, and three weeks later, the synovium and knee joint tissue were harvested for HE staining observation and the synovial tissue was harvested for ELISA.ResultsChemerin was demonstrated to enhance the proliferation of synoviocytes in a dose-dependent manner. The stimulatory effect of chemerin on synoviocytes was shown to involve the activation of MEK, ERK1/2 and p38, which was associated with the production of MMP-13, MMP-3, interleukin (IL)-6 and IL-1β by synoviocytes. Inhibition of the ERK1/2 signaling pathway significantly inhibited chemerin-induced MMP-13, MMP-3, IL-6 and IL-1β production. HE staining showed that the degree of synovial hyperplasia and articular cartilage abrasion was more severe in chemerin-treated rats after knee operation. The articular cartilage surface was damaged, and the synovial tissue showed inflammatory cell infiltration. In rats that underwent operation without chemerin treatment, there was a slight inflammatory infiltration and higher levels of inflammatory factors as compared to unoperated rats; however, secretion of the downstream inflammatory factors IL-6, matrix metalloproteinases (MMP-3 and MMP-13) and IL-1β was significantly greater in the drug-treated group (P<0.05).ConclusionChemerin enhances the production of inflammatory factors in synoviocytes by activating the MEK/ERK signaling pathway.


Introduction
Osteoarthritis (OA) is a chronic degenerative disease involving presentation of synovial lesions, which may be indicative of underlying in ammatory responses that precede or elude identi cation by X-ray imaging or Magnetic Resonance Imaging (MRI). 1,2 The pathological process of OA involves in ammatory reaction of the synovium, which may manifest during the initial stages of synovitis as debilitating pain.
Even if patients do not outwardly display clinical symptoms of synovitis, the diseased joints usually have local synovitis, and this in ammatory reaction is most signi cant in the adjacent articular cartilage injury area. 3,4 The original theory was that obese patients are prone to OA because of cartilage destruction caused by biomechanical stress. 5 However, more recent evidence suggests that a large amount of adipose tissue accumulates in obese patients, and that these tissues store energy and endocrine response factors, which can release a large number of cytokines and adipokines to participate in detrimental physiological and pathophysiological processes, such as immune and in ammatory responses, insulin resistance, and tumorigenesis. 6 Consistently, caloric increase in animal models can lead to joint and systemic in ammatory response by promoting the release of adipokines, cytokines and chemokines. 7 These ndings underscore the importance of in ammatory responses in determining the occurrence and outcome of OA.
Chemerin is a recently identi ed chemotactic protein that guides macrophages and dendritic cells that express its receptor (chemokine like receptor 1 (CMKLR1); also known as ChemR23) 8 to in ammatory sites and is involved in both adaptive and innate immunity. 9 Chemerin is expressed in liver cells, white adipose tissue, monocyte-derived macrophages, and immature dendritic cells. 10 In adipose tissues, chemerin is widely distributed and displays endocrine activity, with signi cant correlation in metabolic diseases such as obesity, type 2 diabetes, and cardiovascular disease. [11][12][13] Chemerin levels in synovial uid have been found to correlate with OA severity using the Kellgren-Lawrence grade as a criterion. 14 Furthermore, ChemR23 is expressed in articular synoviocytes, 15 and its expression is associated with the production of a variety of in ammatory mediators that are released in synovial lesions in OA and affect articular cartilage. 3 Evidence suggests that the mitogen-activated protein kinase kinase (MEK)/extracellular regulated protein kinases (ERK) signaling pathway mediates in ammatory responses in OA, and mitogen-activated protein kinases(MAPKs)have been evaluated as potential therapeutic targets. 16,17 However, the mechanisms by which chemerin promotes in ammatory factor production in synoviocytes have not been evaluated. Therefore, in the present study, we investigated the activation of ERK/MEK signaling pathways by chemerin as well as the associated release of in ammatory mediators by joint synoviocytes. Our results provide a new approach for clinical treatment of OA through targeting of chemerin-associated Mitogen-activated protein kinase MAPK signaling pathway.

| 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 ask. The rat synoviocytes were evaluated experimentally within two to ve passages.

| 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 × 10 3 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.

| Quantitative RT-PCR
The synoviocytes, passaged to the third generation, were seeded in a six-well plate at 4 × 10 4 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 Scienti c, USA) and quanti ed using a uorescence 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.

| 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 × 10 4 /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.

| Enzyme linked immunosorbent assay
After RSC-364 synoviocytes were cultured for 3 passages, the cells were seeded in a six-well plate at 4 × 10 4 /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.

| Animal handling:
Thirty sterile SD rats were divided into three groups. Groups B and C were rst modeled by the modi ed Huths anterior cruciate ligament cutting method 23 . 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.

| HE staining and ELISA
Three weeks after the nal chemerin or saline injection, the rats were injected with an overdose of 1% sodium pentobarbital and sacri ced. 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, para n sections were depara nized with xylene and washed with various levels of ethanol: xylene (I) for 5 minutes → xylene ( ) 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 nal 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).

| 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 signi cant when P < 0.05.

| 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.

| Chemerin promotes the activation of MAPK pathways in synoviocytes
Based on the above ndings 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 signi cantly 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 signi cant 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 ndings are consistent with our mRNA results and indicate that chemerin may regulate cell growth by activating ERK1/2 expression and p38 phosphorylation.

| Chemerin promotes the expression of in ammatory cytokines in a MAPK-dependent manner
Given the role of synovial tissue in ammation in OA progression, 3,4 we evaluated whether chemerin may modulate the production of in ammatory 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 signi cant increase in each of these in ammatory 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 signi cance. These results are consistent with a role for the MEK/ERK pathway in mediating the activation of MMPs and potentially other in ammatory mediators in synoviocytes.

|Chemerin promotes synovial in ammatory hyperplasia and the release of in ammatory factors in rats
To determine whether the in ammatory 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 at shape (Fig. 6). The tissue was relatively loose, with a small amount of capillaries and no obvious in ammatory cell in ltration; the cartilage surface was smooth and at, without damage or bone destruction, and no in ammatory cell in ltration 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 broblasts proliferation was observed, and in ammatory cell in ltration 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 in ltration of in ammatory 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 veri ed that recombinant rat chemerin can also promote synovial tissue to produce corresponding in ammatory mediators in animal experiments. Compared with the control group, each of the assayed in ammatory factors in the chemerin group showed a signi cant increase. In addition, when the control group was compared with the blank group, there was corresponding synovial in ammation, but the degree of lesions and the degree of in ammatory factors were lower than for the drug group (see Fig. 9). These differences are statistically signi cant (P < 0.05), and the results are consistent with the role of synovial cell MMPs and potential other in ammatory mediators in the activation of synovial cells under the action of chemerin.

Discussion
Recent studies have demonstrated that chemerin is an adipokine with chemotactic activity, and that its levels, as well as the levels of other in ammatory factors, are higher in obese individuals. 18 Chemerin promotes the secretion of TNF, IL-1β, IL-6, MMP-1, and MMP-8 by chondrocytes, and these factors play an important role in the development of OA. 19 Furthermore, chemerin has been detected in the synovial uid of OA patients, and its expression is associated with the levels of in ammatory factors. 20 Therefore, we hypothesized that chemerin may regulate in ammatory function in synoviocytes.
Our results provide direct evidence that chemerin promotes the production of in ammatory mediators in synoviocytes. This was demonstrated by an ELISA of a variety of in ammatory cytokines and chemokines, including MMP-3, MMP-13, TNF-α, IL-1β, and IL-6. A previous study suggests that systemic MMP-13 may play a role in knee OA and may be regulated by in ammatory signaling, 21 thus supporting the relevance of our ndings. Furthermore, TNF-α, IL-1β and IL-6 have each been shown to mediate an increase in the expression level of the receptor for chemerin (Chem23), 8 which suggests the possibility of a positive feedback loop that may exacerbate in ammation in OA and other in ammatory conditions. In addition, our results demonstrated that chemerin promotes synovial in ammatory hyperplasia, cartilage damage, and increases in corresponding in ammatory cells and in ammatory factors during in vivo experiments. While the pathogenesis of OA ultimately involves the degeneration of fragments and particles of cartilage, in ammatory responses in the synovium, including in ammatory mediators released by synovial lesions, are known to aggravate articular cartilage destruction, contributing to a vicious cycle that leads to disease progression. 3 Therefore, our results support a model in which chemerin functionally interacts with in ammatory factors and ampli es their effect in mediating cartilage degeneration.
MAPK signaling also is known to play an important role in the pathogenesis of OA. 16,17,22 In this study, we found that chemerin activates MAPK expression in synoviocytes at both the transcriptional and posttranscriptional level. In addition, pretreatment with MAPK pathway-speci c inhibitors attenuated the induction of MMP-3, MMP-13, TNF-α, IL-1β, and IL-6 by chemerin. These results indicate that chemerin may induce or exacerbate the development of OA by regulating the MEK/ERK signaling pathway, thereby affecting the secretion of in ammatory factors by synoviocytes.
In summary, chemerin plays an important role in obesity-induced OA, and our evidence suggests that its function may involve the activation of MAPK signaling in synoviocytes, which leads to the production of in ammatory mediators that ultimately cause cartilage degeneration. Our ndings support the potential of chemerin to serve as a biomarker of disease severity, though additional research will be needed to assess its value in the clinical monitoring and intervention of early OA in obese patients.   Chemerin affects the proliferation of RSC-364 Proliferation was measured by the CCK-8 assay. Cells were pre-treated with the MEK/ERK pathway inhibitor PD98059 where indicated prior to chemerin treatment for 24 h. **** indicates P < 0.01 for the chemerin group compared to the control group; **, *** indicates a statistical difference between the inhibitor group and the chemerin group at P < 0.05 or P < 0.01; ** indicates a statistical difference between the treatment groups at P < 0.05. Results are mean ± SD of 3 independent experiments. Abbreviations: RSC-364, rat synovial cells; CCK-8, cell counting kit-8; MEK, mitogen-activated protein kinase kinase; ERK, extracellular regulated protein kinases; SD, standard   Microscopic observation of synovial tissue and cartilage from the control group of rats. The control group of rats were subjected to knee surgery and then injected with saline. A) synovial tissue microscopic observation; there were more broblast synovial cells than for the blank group; B) cartilage microscopic observation; the cartilage surface was slightly damaged. Microscopic observation of synovial tissue and cartilage from the chemerin group of rats. The chemerin group of rats were subjected to knee surgery and then injected with chemerin. A) synovial tissue microscopic observation; the proliferation of broblasts was obvious, and there was lymphocyte in ltration; B) cartilage microscopic observation; the cartilage surface defect could be seen.

Figure 9
In ammatory factor levels in rats after knee surgery and/or chemerin injection. N represents the blank group (injection of saline only), C represents the medication group (surgery + drugs), and N0 represents the control group (surgery + saline). Levels of in ammatory factors were determined by ELISA. *, ** indicates comparison with group N: P <0.05; *** indicates comparison with group N: P <0.01. The result is the mean ± + standard deviation of 3 independent experiments.