Medermycin Inhibits TNFα-Promoted Inflammatory Reaction in Human Synovial Fibroblasts

Synovial inflammation plays a crucial role in the destruction of joints and the experience of pain in osteoarthritis (OA). Emerging evidence suggests that certain antibiotic agents and their derivatives possess anti-inflammatory properties. Medermycin (MED) has been identified as a potent antibiotic, specifically active against Gram-positive bacteria. In this study, we aimed to investigate the impact of MED on TNFα-induced inflammatory reactions in a synovial cell line, SW-982, as well as primary human synovial fibroblasts (HSF) using RNA sequencing, rtRT-PCR, ELISA, and western blotting. Through the analysis of differentially expressed genes (DEGs), we identified a total of 1478 significantly upregulated genes in SW-982 cells stimulated with TNFα compared to the vehicle control. Among these upregulated genes, MED treatment led to a reduction in 1167 genes, including those encoding proinflammatory cytokines such as IL1B, IL6, and IL8. Pathway analysis revealed the enrichment of DEGs in the TNF and NFκB signaling pathway, further supporting the involvement of MED in modulating inflammatory responses. Subsequent experiments demonstrated that MED inhibited the expression of IL6 and IL8 at both the mRNA and protein levels in both SW982 cells and HSF. Additionally, MED treatment resulted in a reduction in p65 phosphorylation in both cell types, indicating its inhibitory effect on NFκB activation. Interestingly, MED also inhibited Akt phosphorylation in SW982 cells, but not in HSF. Overall, our findings suggest that MED suppresses TNFα-mediated inflammatory cytokine production and p65 phosphorylation. These results highlight the potential therapeutic value of MED in managing inflammatory conditions in OA. Further investigations utilizing articular chondrocytes and animal models of OA may provide valuable insights into the therapeutic potential of MED for this disease.


Introduction
Osteoarthritis (OA), a common degenerative disease, is a primary cause of disability [1].This condition involves alterations in the articular cartilage, synovium, and subchondral bone and is characterized by pain, joint dysfunction, and loss of tissue integrity.Clinically, administration of analgesics and non-steroidal anti-inflammatory drugs (NSAIDs), symptomatic slow acting drugs of OA (SYSADOA) including chondroitin sulfate and glucosamine, surgical intervention using medical devices, and physical therapy are used as therapeutic approaches against OA [2,3].To date, however, no evidence has appeared to indicate that nonsurgical treatment for osteoarthritis, including NSAIDs and physical therapy, is successful in reversing the course of the disease.Therefore, more specific therapeutic approaches need to be researched and developed.
In osteoarthritis, synovial inflammation plays a critical role in joint damage and pain [4,5].The increase in proinflammatory cytokines such as IL1β, IL6, and TNFα during synovial inflammation accelerates the destruction of articular cartilage via matrix degradation enzymes [5,6].The inducible transcription factor NFκB has a key function under inflammatory conditions.Notably, NFκB expression is elevated in the synovium if it affected by radiographic OA [7].Proinflammatory cytokines, including IL6 and IL8, produced by the synovium in osteoarthritis are well-established triggers for the activation of catabolic genes, a process which occurs through mechanisms involving NFκB activation [8,9].Moreover, these cytokines contribute to the experience of pain in OA [10,11].Additionally, NFκB regulates the chemokine ligands that facilitate the recruitment of immune cells during inflammation in the synovium [12,13].These various effects point to the development of NFκB inhibitors as a promising therapeutic approach for the treatment of OA.
Accumulated evidence suggests that some antibiotic agents and their derivatives have anti-inflammatory properties [14][15][16][17].For example, the nematocidal antibiotic jietacin and its derivative exert anti-inflammatory properties via the suppression of NFκB in synovial cells [14].With regard to medermycin (MED), however, a potent antibiotic isolated from Streptomyces sp.K73 with antibiotic activity against Gram-positive bacteria [18], the possibility that this agent has an anti-inflammatory effect has not been examined.
The aim of this study was to determine if MED can reduce inflammatory reactions and reveal the mechanism of action of its effect on human synovial fibroblasts.

Results
MED isolated from Streptomyces tanashiensis 144 MED structure is shown in Figure 1.indicate that nonsurgical treatment for osteoarthritis, including NSAIDs and physical therapy, is successful in reversing the course of the disease.Therefore, more specific therapeutic approaches need to be researched and developed.
In osteoarthritis, synovial inflammation plays a critical role in joint damage and pain [4,5].The increase in proinflammatory cytokines such as IL1β, IL6, and TNFα during synovial inflammation accelerates the destruction of articular cartilage via matrix degradation enzymes [5,6].The inducible transcription factor NFκB has a key function under inflammatory conditions.Notably, NFκB expression is elevated in the synovium if it affected by radiographic OA [7].Proinflammatory cytokines, including IL6 and IL8, produced by the synovium in osteoarthritis are well-established triggers for the activation of catabolic genes, a process which occurs through mechanisms involving NFκB activation [8,9].Moreover, these cytokines contribute to the experience of pain in OA [10,11].Additionally, NFκB regulates the chemokine ligands that facilitate the recruitment of immune cells during inflammation in the synovium [12,13].These various effects point to the development of NFκB inhibitors as a promising therapeutic approach for the treatment of OA.
Accumulated evidence suggests that some antibiotic agents and their derivatives have anti-inflammatory properties [14][15][16][17].For example, the nematocidal antibiotic jietacin and its derivative exert anti-inflammatory properties via the suppression of NFκB in synovial cells [14].With regard to medermycin (MED), however, a potent antibiotic isolated from Streptomyces sp.K73 with antibiotic activity against Gram-positive bacteria [18], the possibility that this agent has an anti-inflammatory effect has not been examined.
The aim of this study was to determine if MED can reduce inflammatory reactions and reveal the mechanism of action of its effect on human synovial fibroblasts.
Consistent with these results using SW982, expression of p65 did not differ between the hrTNFα and vehicle groups (Figure 6A,B), and phosphorylation of p65 was increased in the presence of hrTNFα in HSF (p = 0.004, Figure 6A,C).Phosphorylation of p65 was suppressed in the presence of 250 ng/mL MED (p < 0.001, Figure 6A,B).Akt expression was reduced in the presence of 250 ng/mL MED compared to the vehicle (p = 0.037) and hrTNFα alone (p = 0.003).However, contrary to the results in SW982, p-Akt was increased in presence of 62.5 (p = 0.040), 125 (p = 0.002), and 250 (p = 0.036) ng/mL MED.p < 0.001) in HSF (Figure 4A,C,F).However, levels of IL1β concentration in the samples were found to be below the detection limit of the ELISA, which is set at <2 pg/mL (Figure 4B), which is consistent with a previous study [14].The expression of IL6 and IL8 mRNA, as well as the production of IL6 and IL8, increased following hrTNFα treatment (IL6, p < 0.001; IL6, p = 0.025; IL8, p < 0.001; IL8, p = 0.016; Figure 4D,F).Treatment with 250 ng/mL medermycin reduced the mRNA and protein levels of IL6 and IL8 (IL6, p = 0.004; IL6, p = 0.025; IL8, p = 0.014; IL8, p < 0.002; Figure 4E,F).Pathway analysis suggested that MED regulates NFκB signaling in synovial cells.In contrast, previous studies reported that MED inhibited Akt signaling in a tumor cell line [19,20].As Akt signaling could crosstalk with NF-kb signaling [21][22][23], we subsequently examined the effect of MED on the NFκB and Akt pathways.Expression of p65 did not differ between the hrTNFα and vehicle groups.However, reduced p65 expression was observed in the presence of 125 (p = 0.026) and 250 (p = 0.049) ng/mL MED compared to the hrTNFα groups.hrTNFα induced the phosphorylation of p65 (p < 0.001), which was suppressed in the presence of 250 ng/mL MED (p = 0.040) in SW-982 (Figure 5A,B).Akt and phosphorylated Akt expression did not differ between the hrTNFα and vehicle groups (Figure 5A,D,E).However, Akt expression was reduced in the presence of 125 (p = 0.006) ng/mL MED compared to the hrTNFα groups in SW-982 (Figure 5A,D).The level of p-Akt was also reduced in the presence of 62.5 (p = 0.037), 125 (p = 0.045), and 250 (p = 0.035) ng/mL MED compared to the hrTNFα groups (Figure 5A,E).Consistent with these results using SW982, expression of p65 did not differ between the hrTNFα and vehicle groups (Figure 6A,B), and phosphorylation of p65 was increased in the presence of hrTNFα in HSF (p = 0.004, Figure 6A,C).Phosphorylation of p65 was suppressed in the presence of 250 ng/mL MED (p < 0.001, Figure 6A,B).Akt expression was reduced in the presence of 250 ng/mL MED compared to the vehicle (p = 0.037) and hrTNFα alone (p = 0.003).However, contrary to the results in SW982, p-Akt was increased

Discussion
In this study, we found that MED treatment led to a reduction in the upregulation 1167 of 1478 genes which were significantly upregulated in a synovial cell line, SW-98 as well as primary human synovial fibroblasts (HSF) stimulated with TNFα.Pathwa analysis revealed DEGs were enriched in the TNF and NFκB signaling pathways, furth supporting the involvement of MED in modulating inflammatory responses.Furthe MED inhibited the expression of IL6 and IL8 at both the mRNA and protein levels in bo SW982 cells and HSF, and reduced p65 phosphorylation in both cell types, indicating i inhibitory effect on NFκB activation.These findings suggest that MED suppresses TNFα mediated inflammatory cytokine production by targeting p65 phosphorylation and hig light the potential therapeutic value of MED in managing inflammatory conditions in OA Many studies have reported that increased proinflammatory cytokines are associate with OA severity and symptoms [24][25][26][27][28][29].IL1β concentration in SF correlates with radi graphic OA grade [28].Increased IL6 concentration in synovial fluid was observed in en stage OA and its concentration correlated with pain in OA patients [24,26].Additionall serum IL6 concentration is associated with cartilage loss [29].Previous research has esta lished a connection between the level of IL8 in synovial fluid and severity of OA, while n such association has been found with IL8 levels in serum [25].In contrast, Ruan et a demonstrated a certain correlation between serum IL8 levels and the clinical and radi logical assessment of OA severity [25,27].Here, we observed that MED effectively su pressed the production of proinflammatory cytokines IL6 and IL8 in human synovial cel stimulated with TNFα.Further investigations utilizing animal models of OA could she light on the therapeutic potential of this agent for the treatment of OA.
Under normal conditions, NFκB is inactive and sequestered in the cytosol throug its interaction with the inhibitory protein IκB [30,31].Phosphorylation of p65 at specifi sites is crucial for terminating the transcriptional activity of NFκB in the cell nucleus [7 Our findings indicate that MED inhibits p65 phosphorylation in both SW-982 and HS NFκb inhibition partly contributes to the reduction in inflammatory cytokines followin MED treatment.In contrast, previous studies reported an interaction between Akt and th NFκB pathway.Inhibiting Akt phosphorylation has been shown to inhibit the NFκB pat way and subsequently reduce the production of proinflammatory cytokines [22,23

Discussion
In this study, we found that MED treatment led to a reduction in the upregulation of 1167 of 1478 genes which were significantly upregulated in a synovial cell line, SW-982, as well as primary human synovial fibroblasts (HSF) stimulated with TNFα.Pathway analysis revealed DEGs were enriched in the TNF and NFκB signaling pathways, further supporting the involvement of MED in modulating inflammatory responses.Further, MED inhibited the expression of IL6 and IL8 at both the mRNA and protein levels in both SW982 cells and HSF, and reduced p65 phosphorylation in both cell types, indicating its inhibitory effect on NFκB activation.These findings suggest that MED suppresses TNFα-mediated inflammatory cytokine production by targeting p65 phosphorylation and highlight the potential therapeutic value of MED in managing inflammatory conditions in OA.
Many studies have reported that increased proinflammatory cytokines are associated with OA severity and symptoms [24][25][26][27][28][29].IL1β concentration in SF correlates with radiographic OA grade [28].Increased IL6 concentration in synovial fluid was observed in end-stage OA and its concentration correlated with pain in OA patients [24,26].Additionally, serum IL6 concentration is associated with cartilage loss [29].Previous research has established a connection between the level of IL8 in synovial fluid and severity of OA, while no such association has been found with IL8 levels in serum [25].In contrast, Ruan et al. demonstrated a certain correlation between serum IL8 levels and the clinical and radiological assessment of OA severity [25,27].Here, we observed that MED effectively suppressed the production of proinflammatory cytokines IL6 and IL8 in human synovial cells stimulated with TNFα.Further investigations utilizing animal models of OA could shed light on the therapeutic potential of this agent for the treatment of OA.
Under normal conditions, NFκB is inactive and sequestered in the cytosol through its interaction with the inhibitory protein IκB [30,31].Phosphorylation of p65 at specific sites is crucial for terminating the transcriptional activity of NFκB in the cell nucleus [7].Our findings indicate that MED inhibits p65 phosphorylation in both SW-982 and HSF.NFκb inhibition partly contributes to the reduction in inflammatory cytokines following MED treatment.In contrast, previous studies reported an interaction between Akt and the NFκB pathway.Inhibiting Akt phosphorylation has been shown to inhibit the NFκB pathway and subsequently reduce the production of proinflammatory cytokines [22,23].
Blockage of the PI3K/Akt pathway with LY294002 has been found to partially decrease the activity of TNFα-activated HSF [23].RNA-Seq analysis revealed that AKT3 gene expression was reduced by MED treatment in SW982 cells.Consistent with RNA-Seq analysis, Akt protein expression levels were reduced by MED treatment in SW982 and its reduction was observed in HSF.In contrast, MED suppressed Akt phosphorylation in SW982, whereas MED increased this phosphorylation in HSF.Our results suggest that MED modulates not only Akt phosphorylation but also AKT transcriptional activity.However, a differential effect on Akt phosphorylation was observed between SW982 and HSF.Therefore, the relationship between the modulation of Akt and the anti-inflammatory effect by MED remains unclear.Additional experiments are needed to identify the key pathway modulated by MED.
In conclusion, MED demonstrated the ability to inhibit the production of proinflammatory cytokines in response to TNFα stimulation.MED also inhibited NFκB phosphorylation.Further research using articular chondrocytes and animal models of osteoarthritis (OA) is needed to explore the therapeutic potential of MED in the treatment of OA.

Purification of Medermycin
Medermycin from Streptomyces tanashiensis 144 was obtained by a previously described method [18].The compound was initially reported as luteomycin [15], but we identified it as medermycin [32] by 1 H and 13 C NMR analyses (Supplementary Figure S1).

Cell Culture
SW-982 cells were obtained from the American Type Culture Collection (Rockville, MD, USA).Primary human synovial fibroblasts (HSFs) derived from OA patients were acquired from Sigma Aldrich (Sigma-Aldrich, St. Louis, MO, USA).Both cell types were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum at a temperature of 37 • C.

RNA Sequencing
The impact of MED on TNFα-stimulated SW-982 cells was analyzed using an RNA sequencing assay.SW-982 cells were plated at a concentration of 2 × 10 5 cells/well in a 6-well plate and incubated for 72 h.The cells were then treated with either DMEM (vehicle) or 10 ng/mL human recombinant TNFα (hrTNFα) along with 250 ng/mL of MED for a duration of 6 h.The concentration of hrTNFα and time point in each experiment was determined based on our previous study [14].The lactate dehydrogenase assay suggested that MED concentrations above 500 ng/mL have cytotoxicity.Therefore, a concentration of up to 250 ng/mL MED was selected for the experiments.Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) as the lysis buffer, followed by purification using the Direct-zol MicroPrep kit from Zymo Research (Orange, CA, USA).The quantity of RNA was measured using a spectrophotometer (Denovix, Wilmington, DE, USA), and its quality was assessed using an Agilent 2100 Bio Analyzer (Agilent, Santa Clara, CA, USA) through microcapillary electrophoresis.The extracted RNA was then used for RNA sequencing analysis.The RNA sequencing procedure was conducted using an MGI DNB-SEQ-G400 sequencer from BGI (Shenzhen, China).Two replicate samples were obtained from the vehicle, hrTNFα, and hrTNFα + MED groups for the purpose of RNA sequencing analysis.

Real Time RT-PCR (rtRT-CR)
We used rtRT-PCR to measure levels of IL1B, IL6, and IL8 expression in the cells to validate the RNA sequencing results and investigate the effects of MED concentrations on expression levels.SW-982 and HSF were treated with either vehicle or 10 ng/mL hrTNFα in the presence of various concentrations of MED (0, 31.3,62.5, 125, and 250 ng/mL) for 6 h (n = 6).First-strand cDNA was synthesized from purified total RNA after the extraction procedure was carried out as described above using the SuperScript ® III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA).Quantitative PCR was carried out using the SYBR green method.Primers used in this study have been described previously [14].A delta-delta CT method was used to determine gene expression (Gene/GAPDH) in the vehicle, and relative expression level was calculated when the average level of gene expression (Gene/GAPDH) was 1.

Enzyme-Linked Immunosorbent Assay (ELISA)
SW-982 cells and HSF were plated at a density of 1 × 10 4 cells/well in 96-well plates.The SW-982 cells were then treated with 10 ng/mL hrTNFα in the presence of different concentrations of MED (0, 31.25,62.5, 125, and 250 ng/mL) for 24 h (n = 6).The culture supernatants were collected, and the concentrations of IL1β, IL6, and IL8 in the supernatants were measured using commercially available ELISA kits (BioLegend, San Diego, CA, USA).

Western Blotting
SW-982 cells and HSF were then treated with 10 ng/mL hrTNFα along with varying concentrations of MED (0, 31.25,62.5, 125, and 250 ng/mL) for 6 h (n = 4).After treatment, total protein was extracted from the samples using sodium dodecyl sulfate (SDS) sample buffer.Cell lysate (3 µg) was separated on SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane.Following blocking with 10% skim milk, the membrane was reacted with anti p65, p-p65, Akt, p-Akt, or GAPDH antibody.Following the primary antibody reaction, the membrane reacted with the HRP-linked secondary antibody.The protein bands on the membrane were visualized using enhanced chemiluminescence (Chemi-Lumi One L; Nacalai Tesque, Kyoto, Japan).The luminescent images were captured using a CCD image and then subjected to luminescent image analysis.Relative expression levels of the target proteins were normalized to the expression of GAPDH, a commonly used housekeeping protein.This analysis was performed using ImageJ software.

Statistical Analysis
The normality of the data distribution was assessed using the Shapiro-Wilk test.Subsequently, differences among the vehicle-, hrTNFα-, and hrTNFα-, and MED-treated cells were compared with either the Kruskal-Wallis test (a non-parametric test) or multiple comparisons test with Bonferroni correction.These statistical analyses were performed using SPSS software version 25.0 from IBM (New York, NY, USA).All statistical tests were two-sided, and a p-value less than 0.05 was considered to indicate statistical significance.

Figure 2 .
Figure 2. RNA sequencing analyses of vehicle, hrTNFα, and hrTNFα + medermycin (MED) groups.(A) Venn diagram analysis of DEGs (differentially expressed genes) showing the overlapping region between the hrTNFα/vehicle dataset and the hrTNFα + Medermycin (MED)/hrTNFα dataset.This indicates common genes that are differentially expressed in both conditions.(B) Bubble diagram representing the enrichment analysis of 20 KEGG pathways associated with the DEGs.Each bubble represents a specific pathway, and the size of the bubble corresponds to the number of genes involved in that pathway.Two independent experiments were carried out for each analysis.

Figure 2 .
Figure 2. RNA sequencing analyses of vehicle, hrTNFα, and hrTNFα + medermycin (MED) groups.(A) Venn diagram analysis of DEGs (differentially expressed genes) showing the overlapping region between the hrTNFα/vehicle dataset and the hrTNFα + Medermycin (MED)/hrTNFα dataset.This indicates common genes that are differentially expressed in both conditions.(B) Bubble diagram representing the enrichment analysis of 20 KEGG pathways associated with the DEGs.Each bubble represents a specific pathway, and the size of the bubble corresponds to the number of genes involved in that pathway.Two independent experiments were carried out for each analysis.

Figure 4 .
Figure 4. Impact of medermycin on proinflammatory cytokine expression and production in primary human synovial fibroblasts (HSFs).IL1B mRNA by rtRT-PCR (A) and IL1β protein concentration by ELISA (B).IL6 mRNA by rtRT-PCR (C) and IL6 protein concentration by ELISA (D).IL8 mRNA by rtRT-PCR (E) and IL8 protein concentration by ELISA (F).HSF treated with DMEM (vehicle), hrTNFα, or hrTNFα + medermycin.a p < 0.05 compared to vehicle, b p < 0.05 compared to hrTNFα.Three independent experiments were carried out for each analysis.

Figure 4 .
Figure 4. Impact of medermycin on proinflammatory cytokine expression and production in primary human synovial fibroblasts (HSFs).IL1B mRNA by rtRT-PCR (A) and IL1β protein concentration by ELISA (B).IL6 mRNA by rtRT-PCR (C) and IL6 protein concentration by ELISA (D).IL8 mRNA by rtRT-PCR (E) and IL8 protein concentration by ELISA (F).HSF treated with DMEM (vehicle), hrTNFα, or hrTNFα + medermycin.a p < 0.05 compared to vehicle, b p < 0.05 compared to hrTNFα.Three independent experiments were carried out for each analysis.

Figure 6 .
Figure 6.Impact of medermycin on the NFκB and Akt pathway in human primary synovial fibroblasts (HSFs).Western blotting was performed to analyze the protein expression levels of p65, phosphorylated p65 (p-p65), Akt, phosphorylated Akt (p-Akt), and GAPDH (A) (n = 4).Densitometry analysis of the western blot protein bands for p65 (B), p-p65 (C), Akt (D), and p-Akt (E) was conducted, with normalization to the expression of GAPDH.HSF were treated with different treatments: DMEM (vehicle), hrTNFα alone, or hrTNFα in combination with medermycin (at concentrations of 31.3, 62.5, 125, and 250 ng/mL).a p < 0.05 compared with vehicle, b p < 0.05 compared to hrTNFα groups.Three independent experiments were carried out for each analysis.

Author Contributions:
Conceptualization, G.I., M.T. and K.U.; validation, Y.I. and M.M.; formal analysis, S.I. and M.M.; investigation, S.I., M.I. (Makoto Itakura), K.M. and G.I.; resources, Y.I., G.I., T.H. and M.I. (Masato Iwatsuki); data curation, S.I., M.I. (Makoto Itakura), K.M. and M.M.; writing-original draft, S.I. and K.U.; writing-review and editing, M.T. and K.U.; visualization, K.U.; supervision, M.T.; project administration, T.H., M.I. (Masato Iwatsuki) and K.U.; funding acquisition, G.I. and K.U.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by JSPS KAKENHI Grant Number 23K08617.Additionally, the investigation received support from the Research Support Project for Life Sciences Research and Drug Discovery, specifically the Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS), funded by the Agency for Medical Research and Development Grant Number