DAP Inhibits the TNF-α-Induced Inammatory Response by Modulating the MAPK Signaling Pathway in Synovial Cells

Daphnetin(DAP) is extracted from Daphne odora var. marginata and contains coumarin compounds, which have a good anti-inammatory analgesic effect. In this study, we investigated whether daphnetin can reduce the TNF-α-induced inammatory response by inhibiting the MAPK signaling pathway in the synovial cells of CIA rats. A model of synovial cells was constructed using CIA rats induced by TNF-α. The expression of inammatory cytokines in the synovial cells of CIA rats was observed by real-time PCR and ELISA. The expression and nuclear translocation of MAPK signaling pathway proteins were detected by Western blot and immunouorescence assays. The results show that the mRNA and protein levels of IL-6, TGF-β, MMP-3 and MMP-13 were signicantly lower than those in the culture supernatant of the model control group. The synovial cells of CIA rats induced by TNF-α exhibited decreased expression of p-p38, p-ERK1/2 and p-JNK in the nucleus. In conclusion, daphnetin can affect the activation of the MAPK signaling pathway and reduce the expression of inammatory factors by inhibiting the MAPK signaling pathway, which plays a role in anti-rheumatic inammation. by a variety of different extracellular as cytokines, hormones, neurotransmitters, cell adhesion and cell activated serine-threonine protein kinase(8). It was found that the mitogen-activated protein kinase (MAPK) pathway was signicantly activated in the synovial cells of RA patients compared with those of normal subjects. The members of p38, ERK1/2 and JNK were all found in the synovial membranes of RA patients, and these signaling molecules all exist in a phosphorylated form. Studies have shown that the MAPK signal transduction pathway has a regulatory role in the pathogenesis of RA, induction of inammatory cytokine secretion, aggregation of chemotactic inammatory cells, and promotion of T cell activation, differentiation and apoptosis of a variety of cells cells, osteoblasts, Therefore, the MAPK signaling pathway plays a very important role in the and progression of RA. The MAPK pathway is likely to be an important for MMP-3 and MMP-13 are all cytokines regulated by the MAPK signaling pathway. Our study found that daphnetin was able to signicantly downregulate the expression of IL-6, TGF-β, MMP-3 and MMP-13 mRNA in the synovial cells of CIA rats induced by TNF-α. Additionally, we detected the secretion of IL-6, TGF-β, MMP-3 and MMP-13 in the supernatant of the synovial cells of CIA rats with ELISA and found that the results were consistent with the gene expression analysis. This reduction of the inammatory response and tissue cell damage by daphnetin is of great signicance.


Introduction
Rheumatoid arthritis (RA) is a typical systemic autoimmune disease that is characterized by the abnormal hyperplasia of synovial cells, progressive in ltration of in ammatory cells in joint parts, formation of pannus, destruction of bone and cartilage and production of autoantibodies, which lead to joint deformity, loss of function, long disease course, poor prognosis, extensive disability, and serious impacts on the quality of life (1)(2)(3). The pathogenesis of RA has not been fully elucidated, but studies have shown that the abnormal activation of a signal transduction pathway is an important factor that causes abnormal proliferation of synovial cells and excessive secretion of in ammatory factors and plays an important role in the pathogenesis of RA (4)(5)(6)(7). Mitogen-activated protein kinases (MAPK) are a group that is commonly found in most cells and can be stimulated by a variety of different extracellular stimuli, such as cytokines, hormones, neurotransmitters, cell adhesion and cell activated serine-threonine protein kinase(8). It was found that the mitogen-activated protein kinase (MAPK) pathway was signi cantly activated in the synovial cells of RA patients compared with those of normal subjects. The main members of p38, ERK1/2 and JNK were all found in the synovial membranes of RA patients, and these signaling molecules all exist in a phosphorylated form. Studies have shown that the MAPK signal transduction pathway has a regulatory role in the pathogenesis of RA, induction of in ammatory cytokine secretion, aggregation of chemotactic in ammatory cells, and promotion of T cell activation, affecting the proliferation, differentiation and apoptosis of a variety of cells (such as synovial cells, osteoblasts, osteoclasts) (9,10). Therefore, the MAPK signaling pathway plays a very important role in the development and progression of RA. The MAPK pathway is likely to be an important target for drug therapy.
Natural plants contain many anti-in ammatory and antioxidant compounds that can be applied to rheumatoid arthritis and other in ammatory diseases. Daphnetin (7,8-dihydroxycoumarin), extracted from Daphne odora var. marginata (D. marginata), mainly contains coumarin compounds, which are included in our laboratory's long-term study of active ingredients in traditional Chinese medicine, with antiin ammatory, antibacterial, anti-tumor and other pharmacological effects (11)(12)(13). Our previous study found that daphnetin has a signi cant therapeutic effect on CIA rats, signi cantly reducing the symptoms of arthritis in CIA rats by regulating their immune function, improving Foxp3 + Treg levels in cells, balancing the ratio of Th17/Treg cells, and inducing the apoptosis of synovial cells by a Caspasedependent pathway (14,15). This study was based on the results of previous studies; the effect of daphnetin on the synovial membranes of TNF-α-induced CIA rat synovial cells, and the regulation of the MAPK signal transduction pathway was the focus of this study. Tumor necrosis factor-α (TNF-α) is a monocyte or megakaryocyte cell line produced by a variety of e cacies of proin ammatory factors, and there are a variety of ways to increase the in ammatory signal resulting in the over-proliferation and activation of broblast synovial cells. This study aimed to analyze the molecular mechanism of daphnetin against rheumatoid in ammation.

Chemicals and reagents
Daphnetin (DAP) standard, purity ≥ 98%, was purchased from Shanghai Source Leaf Biological Technology Co., Ltd. Triptolide (TP) standard, purity ≥ 98%, was purchased from Chengdu Pfeiffer Biotechnology Co., Ltd. Recombinant Rat TNF-α was purchased from PeproTech. NT-CROZ, anti-p38, and anti-JNK were purchased from the United States of America Abcam Corporation. Anti-phospho-p38, antiphospho-p38, anti-phospho-p38, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP were purchased from the United States KPL company. All primers were designed by the BGI gene software and tested by NCBI BLAST. The concentration of TNF-α administered 30 min before grouping was 10 ng/ml according to the literature and preliminary experiments. The concentration of daphnetin used was 20 µg/ml, and the concentration of TP used as the follow-up experimental condition was 10 ng/ml.

Quantitative real-time polymerase chain reaction (RT-qPCR)
After administration, the cells were collected, and the total RNA was extracted using the RNAsimple Total RNA Kit. The ReverTra Ace qPCR RT Kit (TOYOBO, Japan) was used to reverse transcribe the RNA into cDNA, and the SuperReal The PreMix Plus (SYBR Green) kit (Beijing Tiangen Biochemical Technology Co., Ltd.) was quanti ed on a Real-time PCR instrument (ABI7500) (Applied Biosystems, USA) with thermal cycling as follows: 1 cycle at 95°C for 15 min; 40 cycles of 95°C for 10 seconds and 60°C for 32 seconds; and nally, a melting curve analysis was performed. The primers used are as follows (Table 1): Samples were incubated at 37°C for 1 hour. After washing the plates, the biotinylated antibodies were added, and the plates were incubated at 37°C for 1 hour. The plates were washed, the enzyme conjugate was added, and the sample was incubated at 37°C for 30 min. Then, the plate was washed and patted dry, the TBS substrate solution was added, and the samples were incubated at 37°C in light for 10-15 min after the termination solution was added to stop the reaction. The absorbance was measured using a microplate reader at 450 nm, and the results were calculated.

Immuno uorescence
The nuclear translocation of the MAPK pathway proteins p-p38, p-ERK1/2, and p-JNK was observed. After 48 hours of treatment, the culture medium was discarded, and the climbing tablets were washed with PBS 3 times for 3 min each. The samples were incubated in solution of 4% formaldehyde in PBS at room temperature for 15 min, washed with PBS 3 times for 3 min each, and dried. Next, the samples were incubated in 0.5% Triton X-100 at room temperature for 20 min, washed with PBS 3 times for 3 min each, and dried. Samples were then incubated in 10% normal goat serum DPBS solution at room temperature in a closed container for 30 min, dried without washing, and 200 µl of a good anti-working solution per well was added. The samples were placed into a wet box and incubated at 4°C overnight. The next day, the samples were dipped in PBST 3 times for 5 min each, incubated in FITC-labeled goat anti-rabbit Ig G for 2 hours, and dipped in PBST 3 times for 3 min each. DAPI re-infected the nuclei, and the samples were immersed in PBST 4 times for 5 min each and prepared with a 50% glycerol lm. A uorescence microscope was used to observe and collect pictures.

Western blot analysis
After 48 hours of treatment, the culture medium was discarded, and the total protein was extracted with a total protein extraction kit (Shanghai Beibo Biotechnology Co., Ltd.). The protein concentration was measured with the BCA method, and the total protein was separated with 5-12% SDS-polyacrylamide gel electrophoresis. Then the expression of proteins from each condition was detected by immunolabeling with enhanced chemiluminescence to determine p-p38/p38, p-ERK1/2/ERK1/2, and p-JNK/JNK.

Statistical analysis
All results are presented as the mean ± S.E.M. Parametric data were evaluated by one-way ANOVA with SPSS statistical software. P-values less than 0.05 were considered statistically signi cant in all cases. Because of the activation of the MAPK signaling pathway in the synovial cells of rheumatoid arthritis patients, the main proteins in this pathway, p38, ERK, and JNK, can be found in the synoviocytes of rheumatoid arthritis patients, and these proteins all exist in their phosphorylated form. When p38, ERK1/2, and JNK were phosphorylated and activate in the nucleus, they regulate important pathophysiological processes of cells. Therefore, we used cell immuno uorescence to detect the effect of daphnetin on TNFα-induced CIA rat synoviocytes (P > 0.05), and the expression levels of p-ERK1/2 and p-JNK were decreased. The effect of p-JNK was especially notable (Fig. 3a, b, c).
3.4 Effects of daphnetin on the expression of p38, ERK1/2, and JNK and their phosphorylation ratios in the synovial cells of CIA rats induced by TNF-α p38, ERK1/2, JNK are the three most important families in the study of the MAPK signaling pathway. P38, ERK1/2, and JNK phosphorylation and activation regulate the vital life process of cells; the phosphorylation level of proteins in a pathway and the regulation of that pathway are closely related. To investigate the effect of daphnetin on the MAPK signaling pathway in the synovial cells of CIA rats, we detected the expression of the MAPK signaling pathway factors p-p38/p38 and p-ERK1 in the synovial cells of CIA rats induced by TNF-α with Western blot analysis (P < 0.05). The expression of p-p38, p-ERK1/2 and p-JNK were signi cantly decreased as determined by the Western blot results. The results showed that the expression levels of p-p38, p-ERK1/2 as well as the phosphorylation ratios p-JNK/JNK, p-p38/p38, p-ERK1/2/ERK1/2 were also signi cantly reduced (Fig. 4).

Effects of Daphnetin on MAPK Signaling Pathway in Synoviocytes of CIA Rats Induced by TNF -α On the expression of MAPK related cytokines
IL-6 is a multicellular cytokine that is a key cytokine in RA. The pathogenic effects of IL-6 on RA mainly promote the differentiation of T cells and B cells associated with in ammation. The acute phase of this response promotes protein synthesis induces osteoclast differentiation and the loss of joint tissue, and IL-1 is synergistically induced by the production of MMP, resulting in increased bone and cartilage damage (20). Studies have shown that RA patients with signi cantly increased serum levels of TGF-β. TGF-β is a growth factor with multiple effects that can induce the secretion of the extracellular matrix and is involved in the expression of in ammatory factors and the differentiation of a variety of immune cells.
The activation and proliferation of macrophages and broblasts have an important impact promoting angiogenesis, pannus formation, and synovial brosis (21). The expression of MMP-3 and MMP-13 in the synovial uid and synovium of patients with active RA was signi cantly increased compared to controls. MMP-3, also known as matrix lysin 1, is secreted by synovial cells, chondrocytes, etc., can make collagen types II, III, IV, IX, and XI and multiple proteoglycan matrices, degrades laminin, and is positively correlated with RA. MMP-13, also known as collagenase-3, is mainly secreted by chondrocytes and has the highest degradation e ciency for type II collagen, which is the most abundant collagen in the cartilage matrix. Therefore, MMP-13 is also the limiting enzyme in the speed of matrix collagen degradation. IL-6, TGF-β, MMP-3 and MMP-13 are all cytokines regulated by the MAPK signaling pathway.
Our study found that daphnetin was able to signi cantly downregulate the expression of IL-6, TGF-β, MMP-3 and MMP-13 mRNA in the synovial cells of CIA rats induced by TNF-α. Additionally, we detected the secretion of IL-6, TGF-β, MMP-3 and MMP-13 in the supernatant of the synovial cells of CIA rats with ELISA and found that the results were consistent with the gene expression analysis. This reduction of the in ammatory response and tissue cell damage by daphnetin is of great signi cance.
Effects of daphnetin on MAPK pathway protein expression, phosphorylation and nuclear translocation in CIA rat synoviocytes induced by TNF-α The MAPK signaling pathway undergoes stepwise activation after various stimuli. The phosphorylation of the pathway protein is transferred to the nucleus to be activated, and the activated protein regulates the transcription, translation and synthesis of the downstream proteins and participates in the important life processes in the cell. We investigated the effects of daphnetin on the protein expression and nuclear translocation of the MAPK signaling pathway molecules p-p38, p-ERK1/2, p-JNK in the synovial cells of CIA rats induced by TNF-α with an immuno uorescence assay.
The results showed that the p38, ERK1/2, JNK proteins were signi cantly activated in the model group and the C group. Additionally, phosphorylated p38, ERK1/2 and JNK were translocated into the nucleus, and their expression levels in the nucleus were stronger than those in the cytoplasm. The expression of p-p38, p-ERK1/2 and p-JNK was signi cantly decreased in the nucleus compared with that in the cytoplasm and compared with the expression of p38, ERK1/2 and JNK, respectively. The p-ERK1/2-FITC uorescence intensity was signi cantly lower in the nucleus than in the cytoplasm. The expression and phosphorylation of MAPK pathway proteins were detected by Western blot analysis. We found that the p-p38, p-ERK1/2, and p-JNK protein expression levels in the model group were signi cantly increased, while daphnetin signi cantly reduced the expression of p38, ERK1/2, and JNK in the synovial cells of CIA rats induced by TNF-α. The phosphorylation levels of ERK1/2 and JNK were lower in the DAP group than in the C and TP groups, and there was no signi cant difference in p38 phosphorylation level between the C and TP groups.
p-p38, p-ERK1/2, p-JNK immuno uorescence and Western blot results con rmed that daphnetin could inhibit the phosphorylation and activation of the MAPK signal transduction pathway, which affected the regulation of various biological activities of cells by MAPK and was closely related to the pharmacological effects of daphnetin.

Conclusions
Daphnetin can signi cantly inhibit the phosphorylation and activation of the MAPK signaling pathway in the synovial cells of CIA rats induced by TNF-α and decrease the expression level of in ammatory cytokines regulated by the MAPK signaling pathway. It is suggested that daphnetin can regulate the MAPK signaling pathway, inhibiting the activation of this pathway and thereby affecting the important biological processes of cells to inhibit rheumatic in ammation.
Our study was only to explore the effect of daphnetin on the MAPK signaling pathway in the synovial cells of CIA rats induced by TNF-α. We also continue to study the speci c sites and mechanisms of daphnetin. In addition, we are further exploring whether rhenium can exert anti-rheumatic in ammatory effects through other RA-related signal transduction pathways. Figure 1 The relative expression of IL-6, TGF-β, MMP-3 and MMP-13 mRNA in the synovial cells of CIA rats. The expression of IL-6, TGF-β, MMP-3 and MMP-13 mRNA was signi cantly increased after TNF-α stimulation (P <0.05). The expression of IL-6, TGF-β, MMP-3 and MMP-13 mRNA in the DAP group was signi cantly lower than that in the model group (P <0.05). There was no signi cant difference between the two groups (P> 0.05). All experiments were performed in triplicate and repeated three times. The data are presented as the means ± SEM and were analyzed by one-way ANOVA (n=9/group). •P < 0.05 compared with the model group. Immuno uorescence of p-p38, p-ERK1/2, and p-JNK in the synovial cells of CIA rats. The p-p38, p-ERK1/2 and p-JNK proteins were labeled with FITC and green uorescence. The uorescence intensities of p-p38, p-ERK1/2 and p-JNK in the DAP group was signi cantly higher than those in the model group.

Declarations
The uorescence intensity of p-p38, p-ERK1/2 and p-JNK and the expression of it in the nucleus was signi cantly reduced. p-ERK1/2 nuclear translocation was signi cantly inhibited, as shown by the signi cantly lower p-ERK1/2-FITC uorescence in the nucleus than in the cytoplasm(n=3/group). Western blot results and relative protein expression. The expression of the MAPK signaling pathway molecules p38, ERK1/2, JNK and their phosphorylation ratios in the synovial cells of CIA rats induced by TNF-α were detected by Western blotting. The expression of p-p38, p-ERK1/2 and p-JNK was signi cantly increased after induction with TNF-α, and the expression of p-p38, p-ERK1/2 and p-JNK protein was signi cantly decreased with daphnetin treatment. The expression of the p-p38/p38, p-ERK1/2/ERK1/2, p-JNK/JNK protein ratios in the DAP group was also signi cantly lower than that in the model group (P <0.05). The ratio of p-p38/p38 protein expression was signi cantly lower in the DAP group than in the C group and TP group (P> 0.05). All experiments were performed in triplicate and repeated three times. The data are presented as the means ± SEM and were analyzed by one-way ANOVA (n=9/group). •P < 0.05 compared with the model group.