ABSTRACT
Background
Osteoarthritis (OA) is a degenerative joint disease that affects millions worldwide. Synovitis and macrophage polarization are important factors in the development of OA. However, the specific components of synovial fluid (SF) responsible for promoting macrophage polarization remain unclear.
Methods
Semi-quantitative antibody arrays were used to outline the proteome of SF. Differential expression analysis and GO/KEGG were performed on the obtained data. Immunohistochemistry and ELISA were used to investigate the relationship between SF S100A12 levels and synovitis levels in clinalclinical samples. In vitro cell experiments were conducted to investigate the effect of S100A12 on macrophage polarization. Public databases were utilized to predict and construct an S100A12-centered lncRNA-miRNA-mRNA competing endogenous RNA network, which was preliminarily validated using GEO datasets.
Results
The study outlines the protein profile in OA and non-OA SF. The results showed that the S100A12 level was significantly increased in OA SF and inflammatory chondrocytes. The OA synovium had more severe synovitis and higher levels of S100A12 than non-OA synovium. Exogenous S100A12 upregulated the levels of M1 markers and phosphorylated p65 and promoted p65 nuclear translocation, while pretreatment with BAY 11–7082 reversed these changes. It was also discovered that LINC00894 was upregulated in OA and significantly correlated with S100A12, potentially regulating S100A12 expression by acting as a miRNA sponge.
Conclusions
This study demonstrated that S100A12 promotes M1 macrophage polarization through the NF-κB pathway, and found that LINC00894 has the potential to regulate the expression of S100A12 as a therapeutic approach.
1. Background
Osteoarthritis (OA) is a chronic and degenerative joint disease affecting millions worldwide, particularly among older adultsCitation1. It is a debilitating condition that can lead to significant pain, stiffness, and loss of mobility, greatly reducing the quality of life of those affectedCitation2. Osteoarthritis is characterized by cartilage breakdown and osteophyte formation, and low-grade inflammation is also a key factor in its developmentCitation2–4. Despite its high prevalence and impact on public health, there are currently no curative treatments for OA, and the available treatments only provide symptomatic reliefCitation3–6. Therefore, it is essential to investigate further the molecular mechanisms underlying the occurrence and progression of OA in order to develop new and effective therapies to meet the clinical needs of patients.
Synovial fluid (SF) is a viscous, transparent body fluid with a relatively low cellular contentCitation7,Citation8. It is a plasma filtrate filtered out of the vascular synovium and mixed with secretions from surrounding cartilage and synovial tissuesCitation9. Due to its close relationship with various tissues in the joint, SF can provide valuable information on the pathophysiological status of the jointCitation8. Studies have also revealed that SF impacts the development of OA, in addition to its diagnostic potentialCitation10–13. Kulkarni et al.Citation14 demonstrated that SF from OA patients could promote macrophage polarization toward the M1 phenotype, thereby providing a microenvironment conducive to the development of OA. It is widely accepted that M1 polarization of macrophages in OA is an important factor contributing to chronic inflammation and tissue damage, and the accumulation of M1-polarized macrophages is an important characteristic of synovitisCitation15. However, it is unclear which specific components of SF are responsible for these effects.
Proteins are an important component of the biologically active fraction of synovial fluidCitation7,Citation8,Citation16. Antibody microarray is a technology that can detect the levels of a large number of proteins at onceCitation17–19. In this study, the protein profiles of OA and non-OA SF were obtained using antibody microarrays, and it was found that the expression level of S100A12 was significantly increased in OA synovial fluid.
S100A12, also known as calgranulin C, is known for its pro-inflammatory properties and has been implicated in various inflammatory diseases, including Rheumatoid Arthritis (RA), which is a chronic joint disease that shares many similarities with OACitation20–22. Previous studies have shown that S100A12 is upregulated in the synovial fluid of OA patients and that its levels correlate with disease severityCitation23,Citation24. Furthermore, the expression of S100A12 is also higher in cartilage affected by OA compared to unaffected cartilageCitation25. In vitro studies have shown that S100A12 stimulates the expression and release of MMP13 and vascular endothelial growth factor (VEGF) via the RAGE in human osteoarthritic chondrocytesCitation25. These results indicate that S100A12 plays a role in the pathogenesis of the disease.
This study aims to delineate the protein profile of OA SF and investigate the role of S100A12 in regulating macrophage polarization in OA. It was found that S100A12 promotes M1 macrophage polarization via the NF-κB pathway, which is a crucial signaling pathway involved in inflammation. This study suggests that S100A12 May be a potential therapeutic target for the treatment of OA, and further research is warranted to investigate its potential as a target for drug development.
2. Methods
2.1. Samples collection
The study was approved by the Institutional Review Board of Shanghai Tongji Hospital. Written informed consent was obtained from each patient prior to their participation. The SF samples were obtained from 10 patients (OA-patients) with primary OA and 10 patients (non-OA) undergoing arthroscopic meniscus repair with no history of arthritic diseases (Supplementary Table S1). Human synovium was obtained from patients undergoing total knee replacement surgery and victims of accidental fractures without evidence of OA.
2.2. Antibody array
To reduce the cost of experiments and biological variation, sample pooling was performed, which is often preferred in microarray experiments to decrease variation between individuals and measure many subjects using relatively few arraysCitation26. SF samples were pooled based on OA or non-OA group and then detected using the RayBio® L-Series Human Antibody Array 1000 (Human L-1000) according to the manufacturer’s instructions. The expression levels of 1000 human proteins can be simultaneously detected with the membrane arrays, including cytokines, chemokines, growth factors, adipokine, proteases, soluble receptors, and other proteins in samples.
The sample pools were dialyzed to remove endogenous biotin before being biotinylated. Biotinylated samples were incubated onto the L-507 and L-493 membranes, and then scanned using ImageQuant LAS4000 Scanner (GE HeaLthcare Corporate, USA). The raw data from the scan was then processed by Raybiotech software for background removal and normalization. Protein intensities were calculated by averaging the readings from the two spots that corresponded to each protein. Differentially expressed proteins (DEPs) are defined as those with foldchange over 1.2 or less than 0.83 (absolute logFC > 0.263).
2.3. Functional annotation
Protein function annotation Gene Ontology (GO) and KEGG pathway are analyzed with the R package “clusterProfiler.” GO analysis includes three subtypes: BP(biological process), MF (molecular function), and CC (cellular component). KEGG systematically analyzes gene functions, linking genomic information with higher-order functional information.
2.4. Analysis based on databases
Public datasets were obtained from the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) database. The datasets of GSE89408, GSE98918, and GSE117999 were selected for analysis. GEO2R was used to perform differential expression analysis. miRDB, miRmap, miRWalk, TargetScan, and LncACTdb databases were used to predict the interactions between mRNA and miRNA as well as miRNA and lncRNA. Several miRNAs with relatively high credibility were obtained by taking the intersection of those predicted in three or more databases. The network was visualized using Cytoscape. The gene microarrays of previously mentioned datasets (GSE98918 and GSE117999) have the ability to detect both mRNA and lncRNA, so the predicted lncRNAs were compared with the detectable range of GPL20844, and 49 common elements were found (). The datasets were processed by R software (version 4.0.3), and the ggplot2 package was used to draw graphs.
2.5. Histomorphometry and immunohistochemistry (IHC)
Synovium samples were fixed with 4% paraformaldehyde for 48 hours and then embedded in paraffin. 4-μm-thick sections were cut for hematoxylin and eosin (H&E) staining. The severity of synovitis was evaluated using a previously reported semi-quantitative scoring system based on three characteristics of synovitis: enlargement of lining cell layer, cellular density of synovial stroma, and leukocytic infiltrateCitation27. For IHC, sections were deparaffinized, antigen retrieval treated, soaked in hydrogen peroxide, and blocked with goat serum. Then, primary (S100A12: Proteintech 16,630–1-AP) and secondary antibodies were used for staining. Finally, the positive signals were visualized using DAB substrate and counterstained with hematoxylin.
2.6. Cell culture and treatment
Healthy human primary chondrocytes were purchased from Procell (Wuhan, China) and used for experiments within the first three passages. SW1353 cells, a human chondrosarcoma cell line, were purchased from Cellcook (Guangzhou, China). Both types of cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) (HyClone, USA). To induce OA state in vitro, the cells were treated with recombinant humanIL-1β (10 ng/ml) for 24 h. THP-1 cell line was obtained from Cellcook and cultured in RPMI-1640 medium containing 10% FBS. All cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. To induce M0 macrophages in vitro, THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA) (100 ng/mL) for 48 h. Then, the differentiated M0 macrophages were stimulated with recombinant human S100A12 protein (ABclonal, RP00009) in the absence or presence of BAY 11–7082 pretreatment (5 μM for 1 h) to verify the effect of S100A12 on macrophage polarization. For secreted proteins detected using WB, 1 µg/ml Brefeldin A (BFA, MedChemExpress) was added for the last 4 hours of cell culture. To investigate the expression and secretion of S100A12 in chondrocytes under an inflammatory environment, human chondrocytes (including human primary chondrocytes and human chondrosarcoma cell line SW1353) were treated with IL-1β.
2.7. Real-time quantitative PCR (RT-qPCR)
In a 6-well culture plate, 5 × 105 cells were seeded in each well for different treatments in preparation for RNA extraction. RNA was isolated from the cells using the FastPure® Cell/Tissue Total RNA Isolation Kit V2 (Vazyme Biotech co.,ltdco., ltd, China) according to the manufacturer’s instructions. Reverse transcription was performed using the PrimeScript RT reagent kit (Takara, Japan). The cDNA was then subjected to qPCR analysis using the qPCR SYBR Green Master Mix (Vazyme, China) on the QuantStudio Dx system (Thermo Fisher, USA). The primer sequences used in this study are listed in Supplementary Table S2. The relative expression levels of target genes were calculated using the 2^−ΔΔCt method, with GAPDH as the internal control. All experiments were performed in triplicate.
2.8. Western blot analysis
5 × 105 cells were seeded into each well of a 6-well culture plate for different treatments in preparation for protein extraction. Total proteins were extracted using RIPA buffer (Epizyme, Shanghai, China) containing protease inhibitor cocktail (Beyotime, Shanghai, China). The protein concentration was measured using BCA protein assay kit (Epizyme, Shanghai, China). Equal amounts of protein were separated on SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% nonfat milk and probed with primary antibodies overnight at 4 °C. The following primary antibodies were used: anti-S100A12 (Proteintech 16,630–1-AP), anti-p65 (santa cruz, sc-8008), anti-phospho-p65 (ABclonal, AP0123), anti-INOS (Proteintech 18,985–1-AP), and anti-β-actin (Beyotime, AF5003). After washing with TBST, the membranes were incubated with the corresponding secondary antibodies for one h at room temperature. The protein bands were detected using the ECL substrate (Beyotime, Shanghai, China) and visualized using the AI600 (GE, USA). Some bands were visualized using the Odyssey CLx Infrared Imaging System (LI-COR, USA).
2.9. Enzyme-linked immunosorbent assay (ELISA)
The levels of S100A12 in synovial fluid and culture supernatants were quantified using a human S100A12 ELISA kit (Abcam, ab282299) according to the manufacturer’s instructions. SF samples were diluted 20 and 2000 times prior to detection.
2.10. Immunofluorescence (IF)
The THP-1 cells were seeded at a density of 1 × 105 cells per well in 6-well culture plates and subjected to different treatments before the IF assay. After treatment, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. The cells were then blocked with 5% goat serum for 1 hour and incubated with primary antibodies at 4 °C overnight. After washing, the cells were incubated with the secondary antibody for 1 hour. The nuclei were counterstained with DAPI. The images were acquired using a fluorescence microscope (Nikon, Japan).
2.11. Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, USA). The results are presented as the mean ± standard deviation (SD). Differences between groups were analyzed using Student’s t-test or one-way ANOVA. Correlations between variables were analyzed using Pearson’s correlation coefficient. A p-value <0.05 was considered statistically significant.
3. Results
3.1. Dysregulated secreted proteins in OA SF and IL-1β-induced chondrocyte S100A12 upregulation
Semi-quantitative antibody arrays were used to outline the proteome of SF (, Supplementary Data 1, 2). The analysis results showed that the levels of 894 proteins changed in OA SF when compared to non-OA SF, with most of them being overexpressed (, , Supplementary Data 3, 4). To determine the functional meaning of the dysregulated secreted proteins in SF, GO and KEGG analysis were performed on DEPs. The results of this study confirmed the important role of SF proteins in intercellular communication (, Supplementary Data 5, 6).
Figure 1. Dysregulated secreted proteins in OA SF and IL-1β-induced chondrocyte S100A12 upregulation. (A) Semi-quantitative antibody arrays were used to outline the proteome of SF. (B) Scatter plot of protein expression levels. (C-D) GO and KEGG analysis on DEPs. (E, G) the expression of S100A12 in mRNA levels increased in chondrocytes under IL-1β stimulation. (F, H) the expression of S100A12 in protein levels increased in chondrocytes under IL-1β stimulation. (I, J) the extracellular S100A12 of chondrocyte supernatant was detected by ELISA under IL-1β stimulation. Data represent the mean ± standard deviation (3 independently repeated experiments); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant (Student t test). BFA, Brefeldin A.
Table 1. The 20 most upregulated proteins in OA SF compared with non-OA SF.
Table 2. The 20 most downregulated proteins in OA SF compared with non-OA SF.
As shown in , the expression of S100A12 in both mRNA and protein levels increased in chondrocytes under IL-1β stimulation. In addition, the results of ELISA show that chondrocytes secrete more extracellular S100A12 in an inflammatory state ().
3.2. S100A12 is involved in the pathology of OA synovitis
In order to investigate the role of S100A12 in the pathology of OA, GEO public datasets were used to investigate the expression changes of S100A12 in various tissues of knee joints under OA state. As shown in , the S100A12 levels in the cartilage, meniscus, and synovium of OA patients are upregulated compared to normal individuals. Through histological assay, it was found that the OA synovium had more severe synovitis and higher levels of S100A12 than non-OA synovium (). Additionally, higher synovitis scores were positively correlated with higher levels of S100A12 in synovial fluid (). These results further support the involvement of S100A12 in the pathology of OA synovitis.
Figure 2. S100A12 is involved in the pathology of OA synovitis. (A) S100A12 levels in different tissues of OA or non-OA joints. (B) HE and S100A12 IHC staining of OA and non-OA synovium (C, D) statistical analysis of synovitis scores and S100A12 IHC in OA and non-OA synovium. Data represent the mean ± standard deviation (3 independently repeated experiments); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant (Student t test). (E) Correlation analysis between synovitis score and S100A12 concentration (Pearson).
3.3. S100A12 promotes M1 macrophage polarization
As shown in , exogenous S100A12 dose-dependently upregulated the expression of INOS (M1 marker). Additionally, S100A12 upregulated the expression of CD80 and CD86, while it did not change the expression of the M2 markers CD163 and CD206 (). Furthermore, treatment with S100A12 significantly upregulated the pro-inflammatory cytokines (including IL-1B, IL-6, and TNF-α) that are regularly released by M1 macrophages ().
Figure 3. S100A12 promotes M1 macrophage polarization. (A) THP-1 cells were induced into M0 macrophages. (B) Western blot analysis of protein levels in THP-1-derived macrophages treated with different concentrations of exogenous S100A12. (C-J) qPCR analysis of mRNA levels in THP-1-derived macrophages treated with exogenous S100A12 (2ug/ml). Data represent the mean ± standard deviation (3 independently repeated experiments); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant (Student t test).
3.4. S100A12 promotes macrophage M1 polarization via the NF-κB signaling pathway
As shown in , the STRING database predicted that S100A12 is associated with multiple molecules in the NF-κB pathway. In addition, the previous proteome analysis also revealed an enrichment in the NF-κB pathway (). The results of WB () and IF () showed that S100A12 treatment upregulated the level of phosphorylated p65 (p-p65) and promoted p65 nuclear translocation, which was consistent with the predicted results. However, pretreatment with NF-κB inhibitor BAY 11–7082 reversed the above changes and downregulated several S100A12-induced M1 markers (). These data suggest that S100A12 promotes macrophage M1 polarization by activating the NF-κB signaling pathway.
Figure 4. S100A12 promotes macrophage M1 polarization via the NF-κB signaling pathway. (A) STRING prediction for S100A12. (B) Western blot analysis of protein levels in THP-1-derived macrophages treated with exogenous S100A12 (2ug/ml). (C, D) Representative immunofluorescence images of THP-1-derived macrophages treated with indicated conditions and quantitative analysis of p65 nuclear translocation. Data represent the mean ± standard deviation (3 independently repeated experiments); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant (Student t test). (E-G) Western blot and quantification analysis of protein levels in THP-1-derived macrophages with or without BAY 11–7082 pretreated. BFA, Brefeldin A. Data represent the mean ± standard deviation (3 independently repeated experiments); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant (ANOVA).
3.5. Construction of the ceRNA network regulating S100A12
To further investigate the upstream mechanism of S100A12, bioinformatics methods were to construct a competing endogenous RNA (ceRNA) network for the regulation of S100A12. Several miRNAs with relatively high credibility were obtained by intersection analysis (, Supplementary Data 7). Then, LncACTdb was used to predict lncRNAs that may bind to these miRNAs, and the lncRNA-miRNA-mRNA network was visualized (). As shown in , differential expression analysis was performed on transcripts (including lncRNA). According to the ceRNA hypothesis, upregulated lncRNAs act as miRNA sponges, thereby increasing the expression of S100A12. Therefore, Venn diagram filtering was performed, and LINC00894, which was upregulated in OA and significantly positively correlated with S100A12, was obtained (, Supplementary Data 8–10).
Figure 5. Construction of the ceRNA network regulating S100A12. (A) The potential miRnas targeting S100A12 were predicted using miRDB, miRmap, miRwalk, and TargetScan databases. Several common miRnas were obtained through screening with a venn diagram. The horizontal bar represents the number of elements shared in one or more lists. For example, 115 elements exclusively exist in a single list, while 30 elements coexist in 2 lists. (B) LncACTdb was used to predict lncRnas that may bind to these miRnas, and the network was visualized. (C) Venn diagram of predicted lncRnas and lncRnas detectable by GPL20844. (D) Volcano plot for differential analysis. (E) Venn diagram of differentially expressed lncRnas and lncRnas positively correlated with S100A12. (F,G) correlation analysis between LINC00894 expression and S100A12 expression (Pearson).
4. Discussion
In summary, this study identifies S100A12 as a key factor for modulating the M1 macrophage polarization and synovitis, which may further promote the progression of OA (). Mechanistically, S100A12-induced M1 polarization promotes the secretion of inflammatory factors such as IL-1β, which in turn promotes the secretion of more S100A12 by chondrocytes as an exogenous inflammatory factor, forming a vicious cycle. This study suggests that S100A12 May be a potential therapeutic target for the treatment of OA.
Figure 6. The schematic diagram summarizes the content of this study and the role of S100A12 in OA.
The protein profiles of OA and non-OA SF were obtained through high-throughput detection using antibody microarrays. It was found that the S100A12 is expressed at higher levels in the synovial fluid of OA patients, which is consistent with a previous study showing that S100A12 is upregulated in OA SF and that its levels correlate with Kellgren—Lawrence grades and Western Ontario McMaster University Osteoarthritis scoresCitation24.
Results of the study also showed that the expression and secretion of S100A12 in chondrocytes increased in an inflammatory environment, which suggests that chondrocytes under OA state are a source of S100A12 in SF. This finding is supported by the analysis of GEO public datasets, which revealed that the S100A12 mRNA levels in the cartilage and meniscus of OA patients are upregulated compared to normal individuals. These findings agree with a previous study that showed higher expression of S100A12 in human cartilage affected by OA compared to unaffected cartilage, as demonstrated by IHCCitation25.
A previous study has verified that S100A12 is involved in chondrocyte pathogenesisCitation25. However, there is currently no research on the role of S100A12 in the synovial pathology of OA. Through histological assay, we found that the OA synovium had more severe synovitis and higher levels of S100A12 than non-OA synovium. Additionally, higher synovitis scores were positively correlated with higher levels of S100A12 in synovial fluid. This finding suggests that S100A12 is closely related to synovitis, and combined with the results of the increased S100A12 secretion in OA cartilage, it is reasonable to assume that S100A12 promotes the occurrence and development of synovitis in a paracrine manner.
The accumulation of M1-polarized macrophages is an important characteristic of synovitisCitation28. The study showed that exogenous S100A12 upregulated the expression of INOS (M1 marker) and CD80/CD86, while it did not change the expression of the M2 markers CD163 and CD206. The study indicates that S100A12 in synovial fluid exacerbates synovitis by promoting M1 macrophage polarization. To further explore its mechanism, the STRING database was used for prediction, and found that S100A12 May interact with multiple molecules in the NF-κB pathway. The NF-κB signaling pathway can act alone or in combination with other signaling pathways to increase the expression of pro-inflammatory cytokines and chemokines in macrophages, promoting M1 macrophage polarization and playing an essential role in the occurrence and development of synovitisCitation29. This study demonstrated that S100A12 treatment upregulated the level of phosphorylated p65 (p-p65) and promoted p65 nuclear translocation, while pretreatment with NF-κB inhibitor BAY 11–7082 reversed the above changes, which are consistent with the predicted results. In the WB images of , it appears that S100A12 and TNF-α are not visibly downregulated under BAY 11–7082 treatment (although there is a downward trend in the quantified results of three replicates). This could be due to several factors. Firstly, one possible reason could be that the level of TNF-α upregulation under S100A12 stimulation is not as high compared to IL-6 and IL-1β, leading to a false negative appearance under the high sensitivity of the ECL reagent. Another possible reason could be that the partial masking effect of the S100A12-induced TNF-α upregulation through other pathways partially obscured the reversing effect of BAY 11–7082. Moreover, the prominent exogenous S100A12-induced upregulation of S100A12 expression may have resulted in unclear separation between the lanes, leading to the overlapping of ECL luminescent signals.
Recent studies have shown that non-coding RNAs play an important role in regulating inflammation, apoptosis, and extracellular matrix metabolism, and are involved in the development and progression of OACitation30,Citation31. The ceRNA mechanism composed of miRNAs and lncRNAs plays a regulatory role in the expression of mRNACitation32. S100A12 is upregulated in OA and inflammatory states, and exploring the upstream regulatory mechanisms of S100A12 can help better understand the OA mechanism. This present study constructed a ceRNA network centered on S100A12 and identified the potential of LINC00894, which was preliminarily validated through datasets. Previous studies have shown that LINC00894 is closely related to the immune microenvironment and tumor prognosisCitation33,Citation34, and can act as a miRNA sponge to affect gene expression and disease progressionCitation35,Citation36. S100A12 is also closely related to immune cells, which suggests that LINC00894 May be involved in regulating S100A12 and is worthy of further research.
There are a number of recognized limitations in our study. Firstly, the use of THP-1-derived macrophages as a model system may not fully represent the behavior of synovial macrophages in response to S100A12. Further work with available synovial macrophages is needed to ascertain whether these two different populations of macrophages will have the same response to S100A12. Additionally, a limitation of this study is that experimental validation using non-transgenic animals could not be performed due to the lack of the S100A12 gene in rodentsCitation37. Therefore, further in vivo research using transgenic mice is needed. Lastly, the investigation of the ceRNA network in this study is purely predictive and based on correlation analysis, lacking experimental validation of the underlying mechanisms. Further experimental validation is necessary to confirm the predicted interactions within the cerna network.
5. Conclusions
In conclusion, our study suggests that S100A12 plays a role in the pathogenesis of OA by promoting M1 macrophage polarization via the NF-κB pathway. S100A12 May be a potential therapeutic target for the treatment of OA, and further research is warranted to investigate its potential as a target for drug development.
Abbreviations
| OA | = | Osteoarthritis |
| SF | = | Synovial Fluid |
| DEPs | = | Differentially expressed proteins |
| GO | = | Gene Ontology |
| KEGG | = | Kyoto Encyclopedia of Genes and Genomes |
| BFA | = | Brefeldin A |
| IL-1β | = | Interleukin-1 beta |
| IL-6 | = | Interleukin-6 |
| TNF-α | = | Tumor Necrosis Factor-Alpha |
| INOS | = | Inducible Nitric Oxide Synthase |
| IHC | = | Immunohistochemistry |
| IF | = | Immunofluorescence |
| M1 | = | Classically Activated Macrophage |
| M2 | = | Alternatively Activated Macrophage |
| ceRNA | = | Competing Endogenous RNA |
| miRNA | = | Micro RNA |
| lncRNA | = | Long non-coding RNA |
| NF-κB | = | Nuclear Factor Kappa B |
| WB | = | Western Blot |
| ELISA | = | Enzyme-linked Immunosorbent Assay |
| BCA | = | Bicinchoninic Acid |
| SDS-PAGE | = | Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis |
| PVDF | = | Polyvinylidene Fluoride |
| TBST | = | Tris-buffered Saline and Tween 20 |
| DAPI | = | 4,’6-diamidino-2-phenylindole |
| ECL | = | Enhanced Chemiluminescence |
| SD | = | Standard Deviation |
| SEM | = | Standard Error of the Mean |
| ANOVA | = | Analysis of Variance |
| t-test | = | Student’s t-test |
Availability of data and materials
Data generated or analyzed during this study are included in this published article and its supplementary files.
Authors’ contributions
YZ: study design, manuscript writing. ZHL: data collection, review. CC: data analysis. HH: samples collection. ZDL, HCZ, and WBH: data interpretation. WW, BL, and YFY: review. All authors contributed to the article and approved the submitted version.
Consent for publication
Written informed consent was obtained from all participants.
Ethics approval and consent to participate
The study was reviewed and approved by the Institutional Review Board of Shanghai Tongji Hospital (No. SBKT-2023-099).
Supplemental Material
Download Zip (5.9 MB)Acknowledgments
The authors would like to thank all the participating patients.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/03008207.2024.2310852.
Additional information
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References
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