Influences of IL-6R Antibody on PMMA Bone Cement-mediated Expression of OPG and RANKL in Synovial Fibroblasts

Effect of interleukin-6 receptor （IL-6R） antibody on polymethyl methacrylate （PMMA） bone cement-mediated expression of osteoprotegerin （OPG） and ：receptor activator of nuclear fac- tor-kappaB ligand （RANKL） in synovial fibroblasts was investigated. Synovial tissue obtained from to- tal knee arthroplasty was digested and cultured. Inverted microscope was employed to observe the synovial cells and immunocytochemistry （SABC method） staining was used to identify synovial fibro- blasts. This experiment was divided into three groups according to different culture media： PMMA group （75μg/mL PMMA bone cement particles）, IL-6R antibody group （10 ng/mL IL-6R antibody＋75 μg/mL PMMA bone cement particles）, and control group （no IL-6R antibody or PMMA bone cement particles）. Influence of IL-6R antibody and PMMA on proliferation of synovial fibroblasts was meas- ured by cell counting kit-8 （CCK-8）. ELISA method was used to measure OPG and RANKL levels in culture solution. Fluorescence quantitative real-time PCR （FQ-PCR） was used to detect the expression of OPG and RANKL mRNA. After three consecutive passages, more than 95% of the primary synovial cells became long spindle fibroblast-like cells. SABC staining results showed that the fibroblast-like cells were negative for anti-CD68 antibody and positive for anti-vimentin antibody, with brown madder stained. CCK-8 test demonstrated that the absorbance （A） value at 450 nm was significantly lower in IL-6R antibody group than in PMMA group and control group （P〈0.01）, but there was no statistically significant difference in A value at 450 nm between the control group and PMMA group （P〉0.05）. Re- suits of ELISA indicated that the expression of OPG was significantly higher in IL-6R antibody group than in PMMA group and control group （P〈0.01）. The expression of RANKL was inhibited （P〈0.05）, and the ratio of OPG/RANKL was significantly increased in IL-6R antibody group as compared with PMMA group and control group. There was no significant difference in the expression of OPG between control group and PMMA group （P〉0.05）, but the expression of RANKL was higher in PMMA group than in control group （P〈0.05）, and there was a significant difference in the ratio of OPG/RANKL be- tween them （P〈0.05）. Results of FQ-PCR revealed the expression of RANKL mRNA was significantly inhibited （P〈0.01） and the expression of OPG mRNA was significantly increased （P〈0.01） in IL-6R an- tibody group as compared with PMMA group and control group. The expression of RANKL mRNA was higher in PMMA group than in control group （P〈0.05）, but the expression of OPG mRNA had no sig- nificant difference between them （P〉0.05）. IL-6R antibody could significantly increase the expression of OPC~ but inhibit the expression of RANKL, which might provide a theoretical basis of molecular bi- ology for the prevention and treatment of aseptic loosening of prosthesis.

Extracellular sulfatase-2 is overexpressed in rheumatoid arthritis and mediates the TNF-α-induced inflammatory activation of synovial fibroblasts

Extracellular sulfatase-2(Sulf-2)influences receptor-ligand binding and subsequent signaling by chemokines and growth factors,yet Sulf-2 remains unexplored in inflammatory cytokine signaling in the context of rheumatoid arthritis(RA).In the present study,we characterized Sulf-2 expression in RA and investigated its potential role in TNF-α-induced synovial inflammation using primary human RA synovial fibroblasts(RASFs).Sulf-2 expression was significantly higher in serum and synovial tissues from patients with RA and in synovium and serum from hTNFtg mice.RNA sequencing analysis of TNF-α-stimulated RASFs showed that Sulf-2 siRNA modulated~2500 genes compared to scrambled siRNA.Ingenuity Pathway Analysis of RNA sequencing data identified Sulf-2 as a primary target in fibroblasts and macrophages in RA.Western blot,ELISA,and qRT‒PCR analyses confirmed that Sulf-2 knockdown reduced the TNF-α-induced expression of ICAM1,VCAM1,CAD11,PDPN,CCL5,CX3CL1,CXCL10,and CXCL11.Signaling studies identified the protein kinase C-delta(PKCδ)and c-Jun N-terminal kinase(JNK)pathways as key in the TNF-α-mediated induction of proteins related to cellular adhesion and invasion.Knockdown of Sulf-2 abrogated TNF-α-induced RASF proliferation.Sulf-2 knockdown with siRNA and inhibition by OKN-007 suppressed the TNF-α-induced phosphorylation of PKCδand JNK,thereby suppressing the nuclear translocation and DNA binding activity of the transcription factors AP-1 and NF-κBp65 in human RASFs.Interestingly,Sulf-2 expression positively correlated with the expression of TNF receptor 1,and coimmunoprecipitation assays demonstrated the binding of these two proteins,suggesting they exhibit crosstalk in TNF-αsignaling.This study identified a novel role of Sulf-2 in TNF-αsignaling and the activation of RA synoviocytes,providing the rationale for evaluating the therapeutic targeting of Sulf-2 in preclinical models of RA.

TXNDC5 synergizes with HSC70 to exacerbate the inflammatory phenotype of synovial fibroblasts in rheumatoid arthritis through NF-κB signaling

The upregulated expression of thioredoxin domain-containing protein 5(TXNDC5)is associated with rheumatoid arthritis in patients and model mice.However,the underlying mechanism by which TXNDC5 influences the pathological activation of rheumatoid arthritis synovial fibroblasts(RASFs)remains unknown.In this study,we show that TXNDC5 expression in RASFs and their cytokine production are significantly upregulated in response to LPS,TNF-αand IL-6,but suppressed by transfection with TXNDC5-siRNA.TXNDC5 is further validated as the direct target of NF-κB signaling.Mechanistically,TXNDC5 directly interacts with heat shock cognate 70 protein(HSC70)to sequester it in the cytoplasm,and HSC70 silencing exerts the same effects as TXNDC5 on the biological activity of RASFs(for example,decreased cell viability,invasion and cytokine production).Furthermore,HSC70 activates NF-κB signaling by destabilizing IκBβprotein in the absence of LPS or facilitating its nuclear translocation in the presence of LPS.Importantly,TXNDC5 can also regulate the activity of NF-κB signaling in a HSC70-IκBβ-dependent manner.Taken together,by linking HSC70 and NF-κB signaling,TXNDC5 plays a pro-inflammatory role in RASFs,highlighting a potential approach to treat RA by blocking the TXNDC5/HSC70 interaction.

Biological regulation on synovial fibroblast and the treatment of rheumatoid arthritis by nobiletin-loaded tetrahedral framework nucleic acids cargo tank

The hyperplasia and destruction of synovial tissue have an important impact on the development of rheumatoid arthritis(RA), the abnormal proliferation and migration of synovial fibroblast in synovial tissue is similar to tumor cells. Targeting anomalous synovial fibroblast and designing a high bioavailability nano drug delivery system can reduce the dosage for the treatment of rheumatoid arthritis and it is of great significance to reduce toxic and side effects and improve curative effect. In this experiment, the nobiletin-loaded tetrahedral framework nucleic acids cargo tank was established, carrying antiinflammatory small molecule monomer drug nobiletin with minimal bioavailability. Both in vitro cell experiments and in vivo animal studies proved the nano cargo tank enhance the role of nobiletin in reducing the invasiveness of pathological synovial fibroblast and promote their apoptosis, effectively alleviate the disease development of rheumatoid arthritis.