Effect of LncRNA GAPLINC on the Cell Proliferation of Rheumatoid Arthritis Fibroblast-like Synoviocytes / 中山大学学报(医学科学版)

ObjectiveTo investigate the effect of LncRNA GAPLINC on the cell proliferation of RA-FLSs. MethodsRA-FLSs were cultured from synovial specimens. The expression of LncRNA GAPLINC in RA-FLSs and trauma-FLSs groups was detected by qRCR. GAPLINC suppression was transfected by siRNA and the inhibition efficiency was detected by qRCR. Flow cytometry was adopted to determine the change of cell growth and cell cycle distribution. 【ResμLts】 The expression of LncRNA GAPLINC was significantly higher in RA-FLSs than that of the trauma-FLSs （P<0.05）.Transfection of GAPLINC-siRNA significantly decreased the expression of LncRNA GAPLINC. GAPLINC silence in RA-FLSs revealed significant inhibition in cell proliferation which was showed by the reduced cell number in S phase（P<0.05）. Moreover， flow cytometry assay showed GAPIINC-siRNA treatment group had an accumμLation of cells in the G0/G1 phase and decreased RA-FLSs in the S and G2/M phase（P<0.05）. After GAPLINC knockdown， mRNA and protein levels of Cyclin D1 and PCNA， which were positively correlated with proliferative phenotype， were decreased （P<0.05）， while p21， which was negatively correlated with proliferative phenotype， was up-regμLated （P<0.05）. ConclusionsThe mRNA expression of GAPLINC was higher in RA-FLSs compared with trauma-FLSs ，which was statistically significant（P<0.05）. The silence of LncRNA GAPLINC coμLd significantly inhibit RA-FLSs cell growth and suppress the cell cycle transformation， which suggests that GAPLINC may play a role in the regμLation of proliferation of RA-FLSs， leading to synovial hyperplasia and contributing to RA progression.

Berberine inhibits autophagy and promotes apoptosis of fibroblast-like synovial cells from rheumatoid arthritis patients through the ROS/mTOR signaling pathway / 南方医科大学学报

OBJECTIVE@#To evaluate the regulatory effect of berberine on autophagy and apoptosis balance of fibroblast-like synoviocytes (FLSs) from patients with in rheumatoid arthritis (RA) and explore the mechanism.@*METHODS@#The inhibitory effect of 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L berberine on RA-FLS proliferation was assessed using CCK-8 method. Annexin V/PI and JC-1 immunofluorescence staining was used to analyze the effect of berberine (30 μmol/L) on apoptosis of 25 ng/mL TNF-α- induced RA-FLSs, and Western blotting was performed to detect the changes in the expression levels of autophagy- and apoptosis-related proteins. The cells were further treated with the autophagy inducer RAPA and the autophagy inhibitor chloroquine to observe the changes in autophagic flow by laser confocal detection of mCherry-EGFP-LC3B. RA-FLSs were treated with the reactive oxygen species (ROS) mimic H2O2 or the ROS inhibitor NAC, and the effects of berberine on ROS, mTOR and p-mTOR levels were observed.@*RESULTS@#The results of CCK-8 assay showed that berberine significantly inhibited the proliferation of RA-FLSs in a time- and concentration-dependent manner. Flow cytometry and JC-1 staining showed that berberine (30 μmol/L) significantly increased apoptosis rate (P < 0.01) and reduced the mitochondrial membrane potential of RA-FLSs (P < 0.05). Berberine treatment obviously decreased the ratios of Bcl-2/Bax (P < 0.05) and LC3B-II/I (P < 0.01) and increased the expression of p62 protein in the cells (P < 0.05). Detection of mCherry-EGFP-LC3B autophagy flow revealed obvious autophagy flow block in berberine-treated RA-FLSs. Berberine significantly reduced the level of ROS in TNF-α-induced RA-FLSs and upregulated the expression level of autophagy-related protein p-mTOR (P < 0.01); this effect was regulated by ROS level, and the combined use of RAPA significantly reduced the pro-apoptotic effect of berberine in RA-FLSs (P < 0.01).@*CONCLUSION@#Berberine can inhibit autophagy and promote apoptosis of RA-FLSs by regulating the ROS-mTOR pathway.

Effect of curcumin on the expression of NOD2 receptor and pro-inflammatory cytokines in fibroblast-like synoviocytes (FLSs) of rheumatoid arthritis (RA) patients

Abstract Background Previous studies has shown that nucleotide-binding and oligomerization domain-containing protein 2 (NOD2) is expressed in Fibroblast-like synoviocytes (FLSs) of rheumatoid arthritis (RA) patients which is stimulated by muramyl dipeptide (MDP) present in the joint environment and induces inflammation via the NF-κB pathway. Also, other studies have shown that curcumin inhibits proliferation, migration, invasion, and Inflammation and on the other hand increases the apoptosis of RA FLSs. In this study, we aim to evaluate the effect of curcumin, a natural antiinflammatory micronutrient, on the expression of NOD2 and inflammatory cytokines. Methods Synovial membranes were collected from ten patients diagnosed with RA and ten individuals with traumatic injuries scheduled for knee surgery. The FLSs were isolated and treated with 40 μM curcumin alone or in combination with 20.3 μM MDP for 24 h. mRNA was extracted, and real-time PCR was performed to quantitatively measure gene expression levels of NOD2, p65, IL-6, TNF-α, and IL-1β. Results The study findings indicate that administering MDP alone can significantly increase the mRNA expression levels of IL-6 and IL-1β in the trauma group and TNF-α in the RA group. Conversely, administering curcumin alone or in combination whit MDP can significantly reduce mRNA expression levels of P65 and IL-6 in FLSs of both groups. Moreover, in FLSs of RA patients, a single curcumin treatment leads to a significant reduction in NOD2 gene expression. Conclusion This study provides preliminary in vitro evidence of the potential benefits of curcumin as a nutritional supplement for RA patients. Despite the limitations of the study being an investigation of the FLSs of RA patients, the results demonstrate that curcumin has an anti-inflammatory effect on NOD2 and NF-κB genes. These findings suggest that curcumin could be a promising approach to relieve symptoms of RA.

Morphofunctional analysis of fibroblast-like synoviocytes in human rheumatoid arthritis and mouse collagen-induced arthritis

Abstract Background Fibroblast-like synoviocytes (FLS) play a prominent role in rheumatoid synovitis and degradation of the extracellular matrix through the production of inflammatory cytokines and metalloproteinases (MMPs). Since animal models are frequently used for elucidating the disease mechanism and therapeutic development, it is relevant to study the ultrastructural characteristics and functional responses in human and mouse FLS. The objective of the study was to analyze ultrastructural characteristics, Interleukin-6 (IL-6) and Metalloproteinase-3 (MMP-3) production and the activation of intracellular pathways in Fibroblast like synoviocytes (FLS) cultures obtained from patients with rheumatoid arthritis (RA) and from mice with collagen-induced arthritis (CIA). Methods FLSs were obtained from RA patients (RA-FLSs) (n = 8) and mice with CIA (CIA-FLSs) (n = 4). Morphology was assessed by transmission and scanning electron microscopy. IL-6 and MMP-3 production was measured by ELISA, and activation of intracellular signaling pathways (NF-κB and MAPK: p-ERK1/2, p-P38 and p-JNK) was measured by Western blotting in cultures of RA-FLSs and CIA-FLSs stimulated with tumor necrosis factor-alpha (TNF-α) and IL-1β. Results RA-FLS and CIA-FLS cultures exhibited rich cytoplasm, rough endoplasmic reticula and prominent and well- developed Golgi complexes. Transmission electron microscopy demonstrated the presence of lamellar bodies, which are cytoplasmic structures related to surfactant production, in FLSs from both sources. Increased levels of pinocytosis and numbers of pinocytotic vesicles were observed in RA-FLSs (p < 0.05). Basal production of MMP-3 and IL-6 was present in RA-FLSs and CIA-FLSs. Regarding the production of MMP-3 and IL-6 and the activation of signaling pathways, the present study demonstrated a lower response to IL-1β by CIA-FLSs than by RA-FLSs. Conclusion This study provides a comprehensive understanding of the biology of RA-FLS and CIA-FLS. The differences and similarities in ultrastructural morphology and important inflammatory cytokines shown, contribute to future in vitro studies using RA-FLS and CIA-FLS, in addition, they indicate that the adoption of CIA-FLS for studies should take careful and be well designed, since they do not completely resemble human diseases.

PLCL1 regulates fibroblast-like synoviocytes inflammation via NLRP3 inflammasomes in rheumatoid arthritis

Abstract Background: Phospholipase C-like 1 (PLCL1), a protein that lacks catalytic activity, has similar structures to the PLC family. The aim of this research was to find the function and underlying mechanisms of PLCL1 in fibroblast-like synoviocyte (FLS) of rheumatoid arthritis (RA). Methods: In this study, we first analyzed the expression of PLCL1 in the synovial tissue of RA patients and K/BxN mice by immunohistochemical staining. Then silencing or overexpressing PLCL1 in FLS before stimulating by TNF-α. The levels of IL-6, IL-1β and CXCL8 in FLS and supernatants were detected by Western Blot (WB), Real-Time Quantitative PCR and Enzyme Linked Immunosorbent Assay. We used INF39 to specifically inhibit the activation of NLRP3 inflammasomes, and detected the expression of NLRP3, Cleaved Caspase-1, IL-6 and IL-1β in FLS by WB. Result: When PLCL1 was silenced, the level of IL-6, IL-1β and CXCL8 were down-regulated. When PLCL1 was overexpressed, the level of IL-6, IL-1β and CXCL8 were unregulated. The previous results demonstrated that the mechanism of PLCL1 regulating inflammation in FLS was related to NLRP3 inflammasomes. INF39 could counteract the release of inflammatory cytokines caused by overexpression of PLCL1. Conclusion: Result showed that the function of PLCL1 in RA FLS might be related to the NLRP3 inflammasomes. We finally confirmed our hypothesis with the NLRP3 inhibitor INF39. Our results suggested that PLCL1 might promote the inflammatory response of RA FLS by regulating the NLRP3 inflammasomes.

The biological effect of B7-H3 on fibroblast-like synoviocytes in osteoarthritis / 中华风湿病学杂志

Objective:To investigate the effect of B7-H3 gene on the biological function of fibr-oblastlike synoviocytes (FLS) in osteoarthritis (OA).Methods:Synovial tissue of five cases of OA and synovial tissue of 4 normal knee were obtained, and the primary cell lines were isolated and cultured. The expression of B7-H3 in OA synovial tissue and primary OA-FLS were studied by immunohi-stochemistry, real time-poly merase chain reaction (PCR) and FACS. According to sites 996 and 1041 of B7-H3, corresponding siRNA was designed and the expression of B7-H3 in FLS was silenced and down-regulated. The inhibition of B7-H3 and its protein in target cells was determined by Western blot and FACS. The migration and invasion ability of B7-H3 in target cells were analyzed by scratch assay and Transwell assay. CCK8 assay was used to detect cell proliferation ability, and CBA assay was used to detect cytokines and chemokines in cell culture supernatant. GraphPad Prism 8.0 software was used to analyze the experimental data. The normal distribution data was expressed as mean±standard deviation ( Mean± SD). The comparison between data was performed by T test, and P<0.05 was considered statistically significant. Results:The abnormally high expression of B7-H3 in fibro-blast-like synoviocytes of OA was detected. Compared with siNC, si996 and si1041 inhibited the expression of B7-H3 in OA-FLS. In the Transwell migration experiment, the mean cells number of random view in the siNC group, the si996 group, and the si1041 group indicating decreased migration ability of OA-FLS [siNC vs si996 (100.3±3.7) /view vs (48.7±1.2) /view, t=13.24, P<0.001; siNC vs si1041 (100.3±3.7) /view vs (59.7±1.9) /view, t=9.80, P<0.001). In the Transwell invasion experiment, the mean cells number of random view in the siNC group, in the si996 group, and in the si1041 group indicating decreased invasion ability of OA-FLS [siNC vs si996 (127.3±5.6) /view vs (39.7±3.3) /view, t=13.49, P<0.001; siNC vs si1041 (127.3±5.6) /view vs (57.3±1.9) /view, t=11.85, P<0.001]. The secretion of IL-6 [siNC vs si996 (248±21) pg/ml vs (111±12) pg/ml, t=24.08, P=0.002; siNC vs si1041 (248±21) pg/ml vs (46±5) pg/ml, t=13.21, P=0.006], IL-8 [siNC vs si996 (118.1±15.6) pg/ml vs (47.1±5.4) pg/ml, t=6.68, P=0.022; siNC vs si1041 (118.1±15.6) pg/ml vs (10.0±1.3) pg/ml, t=13.08, P=0.006], CXCL8 [siNC vs si996 (178.8±6.4) ng/ml vs (83.2±2.7) ng/ml, t=13.77, P=0.005; siNC vs si1041 (178.8±6.4) ng/ml vs (93.5±2.8) ng/ml, t=12.23, P=0.007] and CCL2 [siNC vs si996 [(184.1±5.1) ng/ml vs (109.4±5.9) ng/ml, t=9.57, P=0.011; siNC vs si1041 (184.1±5.1) ng/ml vs (97.1±1.5) ng/ml, t=16.39, P=0.004] was decreased . Conclusion:B7-H3 may regulate the migration, invasion, cytokine secretion and other biological functions of OA-FLS, providing clues for further study of B7-H3's involvement in the pathogenesis of OA.

Effect of Danggui Niantongtang on Autophagy and Apoptosis of Rheumatoid Arthritis Fibroblast-like Synoviocyte / 中国实验方剂学杂志

ObjectiveTo explore the effect of Danggui Niantongtang （DGNTT） on cell apoptosis and autophagy in rheumatoid arthritis （RA） fibroblast-like synoviocytes （FLS）. MethodRA-FLS were isolated and cultured from the synovial tissue of RA patients. The cells were treated with 10% blank serum （blank control group）， 10% sera containing low， medium and high doses of DGNTT. Wound healing assa and cell invasion test were applied to observe the effect of RA-FLS invasion technique. The apoptosis and autophagy level of RA-FLS cells was detected by Hoechst33342 method and monodansylcadaverine （MDC） staining. The mRNA and protein expression levels of B cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， microtubule-associated protein 1 light chain 3 （LC3）， autophagy key molecular yeast Atg6 homolog 1 （Beclin1） were detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）and Western blot. ResultCompared with the blank control group，each dose of serum could slow down the wound healing and significantly Reduce the number of RA-FLS cells invading the lower chamber（P<0.01），the mRNA and protein expression levels of Bcl-2，LC3，Beclin1 were significantly decreased（P<0.01）， and Bax were significantly increased（P<0.01）. Hoechst33342 results showed that low， medium and high doses DGNTT could promote RA-FLS cell apoptosis. After MDC staining，autophagosome in low， medium and high doses DGNTT decreased significantly（P<0.01）. ConclusionDanggui Niantongtang can effectively inhibit the proliferation of fibroblast-like synoviocytes. Its mechanism may be related to promote apoptosis and inhibit autophagy of fibroblast-like synoviocytes.

Salvianolic acid C inhibits inflammation and induce apoptosis by regulating nuclear factor-erythroid 2-related factor 2 pathway in rheumatoid arthritis-fibroblast-like synoviocytes cells / 中华风湿病学杂志

Objective:To investigate the effect of salvianolic acid C (SalC) on fibroblast-like synoviocytes and through the role of nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway.Methods:Rheumatoid arthritis-fibroblast-like synoviocytes (RA-FLSs) were exposed to different concentrations of SalC (0.1 μmol/L, 1 μmol/L, 5 μmol/L, 10 μmol/L, 20 μmol/L) for 24-72 h and measured for viability, proliferation, migration and invasion by Cell counting kit 8 (CCK-8) assay, wound-healing and transwell assay. The levels of Tumor Necrosis Factor-α (TNF-α), Interleukin-1 beta (IL-1β) and IL-6 were measured by enzyme linked immunosorbent assay (ELISA). Western blot was used to detect the expression of matrix metallopeptidase (MMP)-9, MMP-13, apoptosis-related proteins and Nrf2 mediated gene. Then we used ML385 to inhibit Nrf2 signaling pathway. RA-FLSs were measured for migration and invasion, and the expression of proteins related to apoptosis, inflammation and Nrf2 pathway.Results:Compared with the control group, SalC inhibited the cell migration significantly (0.1 μmol/L, 0.75±0.05, t=7.65, P<0.001; 5 μmol/L, 0.50±0.05, t=14.25, P<0.001; 10 μmol/L, 0.26±0.05, t=20.67, P<0.001) and invasion (0.1 μmol/L, 0.75±0.11, t=4.93, P<0.001; 5 μmol/L, 0.49±0.06, t=10.32, P<0.001; 10 μmol/L, 0.26±0.07 , t=14.96, P<0.001) of RA-FLs, reduced the levels of MMP-9 (0.1 μmol/L, 0.72±0.10, t=5.60, P<0.001; 5 μmol/L, 0.48±0.08, t=11.03, P<0.001; 10 μmol/L, 0.27±0.06, t=15.94, P<0.001) and MMP-13 (0.1 μmol/L, 0.77±0.06, t=8.66, P<0.001; 5 μmol/L, 0.58±0.06, t=11.03, P<0.001; 10 μmol/L, 0.32±0.13, t=14.22, P<0.001), and promoted apoptosis. SalC reduced the level of pro-inflammatory cytokines significantly ( P<0.001) and activated the expression of Nrf2 signaling pathway proteins Nrf2, CAT, NQO1, SOD1 and GSS ( P<0.001). After ML385 was used to interfere Nrf2, the levels of SalC on Nrf2 pathway proteins, such as Nrf2 (0.68±0.06, t=5.08, P<0.001), CAT (1.44±0.12, t=4.77, P<0.001), NQO1 (0.65±0.12, t=5.04, P<0.001), SOD1 (1.43±0.10, t=6.36, P<0.001) and GSS (1.42±0.10, t=7.60, P<0.001), as well as the levels of TNF-α [(260±22) pg/ml, t=13.75, P<0.001], IL-1β [(701±30) pg/ml, t=12.98, P<0.001], IL-6 [(180±10) pg/ml, t=16.38, P<0.001) were significantly reduced. In addition, ML385 inhibited the inhibition of SalC on cell migration and invasion (0.70±0.09, t=11.24, P<0.001; 0.64±0.04, t=8.03, P<0.001) and induction of apoptosis (24.4±1.8, t=23.02, P<0.001). Conclusion:SalC may inhibit cell activity and inflammatory response, promote apoptosis via the upregulation of Nrf2 signaling pathway. SalC may have therapeutic potential in RA. However, further investigation are needed in animal models and human.

Therapeutic effect of neohesperidin on TNF-α-stimulated human rheumatoid arthritis fibroblast-like synoviocytes / 中国天然药物

During the pathogensis of rheumatoid arthritis (RA), activated RA fibroblast-like synoviocytes (RA-FLSs) combines similar proliferative features as tumor and inflammatory features as osteoarthritis, which eventually leads to joint erosion. Therefore, it is imperative to research and develop new compounds, which can effectively inhibit abnormal activation of RA-FLSs and retard RA progression. Neohesperidin (Neo) is a major active component of flavonoid compounds with anti-inflammation and anti-oxidant properties. In this study, the anti-inflammation, anti-migration, anti-invasion, anti-oxidant and apoptosis-induced effects of Neo on RA-FLSs were explored to investigate the underlying mechanism. The results suggested that Neo decreased the levels of interleukin IL-1β, IL-6, IL-8, TNF-α, MMP-3, MMP-9 and MMP-13 in FLSs. Moreover, Neo blocked the activation of the MAPK signaling pathway. Furthermore, treatment with Neo induced the apoptosis of FLSs, and inhibited the migration of FLSs. It was also found that Neo reduced the accumulation of reactive oxygen species (ROS) induced by TNF-α. Taken together, our results highlighted that Neo may act as a potential and promising therapeutic drug for the management of RA.

Screening and analysis for autophagy / 中南大学学报(医学版)

OBJECTIVES@#Long non-coding RNA (lncRNA) has become a key epigenetic regulator that regulates gene expression and affects a variety of biological processes. LncRNA plays an important role in the occurrence and development of rheumatoid arthritis (RA). The study on lncRNA in peripheral blood cells of RA patients has been reported. However, there is no study on autophagy regulation by lncRNA in RA patients. This study aims to provide a new direction for the diagnosis and treatment of RA via screening the changes of lncRNAs in RA fibroblast-like synoviocytes (RA-FLSs) before and after autophagy and finding the key lncRNAs targeting RA-FLSs autophagy.@*METHODS@#Synovial tissues of 6 RA patients after knee and hip joint surgery were obtained, and RA-FLSs were cultured to the 5th generation for further experiments (tissue culture method). After treatment with mTOR inhibitor PP242, the expression of LC3-II was detected by Western blotting. Total RNAs of 3 cases of RA-FLSs before and after treatment with mTOR inhibitor PP242 were extracted by TRIzol and screened by Agilent Human ceRNA Microarray 2019 (4×180 K, design ID: 086188) chip. The lncRNAs with significantly changed expression levels were selected (difference multiple≥2.0, @*RESULTS@#RA-FLSs were successfully isolated and cultured from the synovial tissues of the patient's knee or hip joint. After 6 RA-FLSs were treated with PP242, the expression level of autophagy marker protein LC3-II was increased (@*CONCLUSIONS@#Differentially expressed lncRNAs in RA-FLSs have been identified with microarray analysis. In RA, differential expression of lncRNAs is involved in the autophagy of RA-FLSs. The underlying mechanisms based on bioinformatics analysis include regulating the secretion of cytokines, such as IL-6, TGF-β, TNF-α and IL-17, participating in the immune cell differentiation, such as Th17, Th1, Th2 cells and osteoclasts, as well as regulating the autophagy pathway, MAPK, FoxO, and other signaling pathways. It has been verified that the expression of ENST0000584721.1 is up-regulated and ENST0000615939.1 is down-regulated after autophagy of RA FLSs, which provides a good experimental basis for further study on the mechanism of lncRNA in RA-FLSs autophagy.

Effect of Xinfeng Capsules-containing serum on TNF-α-induced apoptosis and inflammation of fibroblast-like synoviocytes in rheumatoid arthritis / 中国中药杂志

The aim of this paper was to observe the effect of Xinfeng Capsules(XFC)-containing serum on the apoptosis and inflammation of fibroblast-like synoviocytes(FLS) in rheumatoid arthritis(RA) induced by tumor necrosis factor-α(TNF-α), so as to investigate the mechanism of XFC in the treatment of RA. RA-FLS immortalized cell line was established, and XFC drug-containing serum was prepared. CCK-8, ELISA, RT-qPCR, immunofluorescence and TUNEL were used to observe the effect of XFC-containing serum on RA-FLS apoptosis and inflammatory indexes. CCK-8 results showed that the optimal concentration and time of TNF-α on RA-FLS were 10 ng·mL~(-1) and 48 h, respectively; and the optimal concentration and time of XFC on RA-FLS were 6.48 mg·g~(-1) and 72 h, respectively. The results of ELISA showed that compared with RA-FLS group, the expressions of TNF-α, IL-1β, IL-6, IL-8 in TNF-α+RA-FLS group were significantly increased, while the expressions of IL-4 and IL-10 were significantly decreased(P<0.01); after intervention with XFC-containing serum, the expressions of TNF-α, IL-1β, IL-6, IL-8 were significantly decreased, whereas the expressions of IL-4 and IL-10 were significantly increased(P<0.01). The results of RT-qPCR showed that compared with RA-FLS group, the mRNA expressions of Fas, FasL, caspase-3, caspase-8, Bax, Bcl-X1 in TNF-α+RA-FLS group were significantly decreased, while the mRNA expression of Bcl-2 was significantly increased(P<0.001); after intervention with XFC-containing serum, the mRNA expressions of Fas, FasL, caspase-3, caspase-8, Bax, Bcl-X1 were significantly increased, whereas the mRNA expression of Bcl-2 was significantly decreased(P<0.01). The results of immunofluorescence showed that compared with RA-FLS group, the protein expressions of caspase-3 and Bax in TNF-α+RA-FLS group was significantly lower than those in RA-FLS group(P<0.05); after intervention with XFC-containing serum, the protein expressions of caspase-3 and Bax were significantly increased, whereas the protein expression of Bcl-2 was significantly decreased(P<0.05). TUNEL results showed that compared with RA-FLS group, the apoptosis of TNF-α+RA-FLS group was decreased(P<0.05); after intervention with XFC-containing serum, the apoptosis was significantly increased(P<0.05). One of the mechanisms of XFC in the treatment of RA is to promote the apoptosis of RA-FLS and inhibit its inflammatory reaction.

Effect of swertiamarin, gentiopicrin and sweroside on cell apoptosis and expression of Bcl-2 in rheumatoid arthritis fibroblast-like synoviocytes / 中国中药杂志

The aim of this paper was to discuss the effect of swertiamarin, gentiopicrin and sweroside on rheumatoid arthritis fibroblast-like synoviocytes(RA-FLSs) and B-cell lymphoma-2(Bcl-2) and their mechanisms. ZINC database and RCSB PDB database were retrieved for 3 D chemical structures of swertiamarin, gentiopicrin and sweroside and 3 D target protein structures. AutoDock Mgltools 1.5.6, AutoDockVina 1.1.2 and pyMOL 2.2.0 were applied for molecular docking to analyze the relationship between Bcl-2(1 GJH) target protein and important ingredients. The cell apoptosis of RA-FLSs was tested by Annexin V-FITC. The Bcl-2 protein expression of RA-FLSs treated with different ingredients was tested by Western blot. The Bcl-2 mRNA expression of RA-FLSs treated with different ingredients was tested by RT-PCR. Swertiamarin, gentiopicrin and sweroside were docked well with Bcl-2(1 GJH). The binding energy of swertiamarin was-6.9 kcal·mol~(-1), the binding energy of gentiopicrin was-6.7 kcal·mol~(-1) and the binding energy of sweroside was-6.4 kcal·mol~(-1). Compared with the blank group, the Bcl-2 protein expression of each group were reduced, while that of the gentiopicrin group was the highest(P<0.01). Compared with the blank group, the Bcl-2 mRNA expression of each groups were reduced. Gentiopicrin can reduce the Bcl-2 protein expression and the Bcl-2 mRNA expression, so as to promote the RA-FLSs apoptosis.

Effect and Mechanism of Yuxuebi Tablet Against Collagen-Induced Arthritis of Rats / 中国实验方剂学杂志

Objective:To explore the intervention effect of Yuxuebi tablet （YXB） on collagen-induced arthritis （CIA） in rats and its anti-inflammatory mechanism. Method:Following CIA modeling， the rats in the drug administration groups were separately treated with intragastric administration of YXB （0.1， 0.2， and 0.4 g·kg^-1） and methotrexate （MTX， 0.4 mg·kg^-1）， once a day. The incidence of CIA， mechanical pain threshold （MPT） and cold pain threshold （CPT） were evaluated once every three days. After continuous administration for 30 days， the peripheral blood of rats was collected for the determination of platelet （PLT） count and fibrinogen （FIB） content. The hematoxylin-eosin （HE） staining was conducted to analyze the pathological changes in joint tissues. The protein expression levels of interleukin （IL）-1<italic>β</italic>， IL-8， nuclear transcription factor-<italic>κ</italic>B （NF-<italic>κ</italic>B） p65， phosphorylated NF-<italic>κ</italic>B （p-NF-<italic>κ</italic>B） p65， Ras， and Raf-1 in joint tissues of CIA rats were detected by immunohistochemistry （IHC） and Western blot. The rheumatoid arthritis fibroblast-like synoviocytes （RA-FLS） were induced by tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>， 10 μg·L^-1） <italic>in vitro</italic> and then subjected to transwell migration/invasion assay， followed by the detection of protein expression levels of Ras， Raf-1， and p-NF-<italic>κ</italic>B p65 in RA-FLS by Western blot. Result:Compared with the control group， the model group exhibited an increased incidence of CIA， significantly decreased MPT （<italic>P</italic><0.05，<italic>P</italic><0.01）， elevated CPT （<italic>P</italic><0.01） and PLT and FIB in the peripheral blood， worsened histopathological score of joints， enhanced RA-FLS migration and invasion， and up-regulated inflammatory factors （<italic>P</italic><0.01）. The comparison with the model group revealed that YXB at different doses obviously reduced the incidence of CIA， increased MPT， down-regulated CPT and PLT and FIB in the peripheral blood （<italic>P</italic><0.05，<italic>P</italic><0.01）， ameliorated the pathological changes like synovial hyperplasia and bone and cartilage destruction （<italic>P</italic><0.05，<italic>P</italic><0.01）， and inhibited RA-FLS migration and invasion. Besides， the low-， medium-， and high-dose YXB reversed the IL-1<italic>β</italic>， IL-8， Ras， Raf-1， and p-NF-<italic>κ</italic>B p65 expression in joint tissues of CIA rats to different extents， as well as the protein expression of Ras， Raf-1 and p-NF-<italic>κ</italic>B p65 in RA-FLS （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:YXB reduces the incidence of CIA， ameliorates the clinical symptoms of RA and the pathological changes in joint tissues， and inhibits the formation of synovium， which may be attributed to its inhibition against Ras/Raf-1/NF-<italic>κ</italic>B signaling pathway.

IL-22 increases the production of sFRP3 by FLS in inflammatory joint diseases

Rheumatoid arthritis (RA), psoriatic arthritis (PsA), and ankylosing spondylitis (AS) are inflammatory diseases with different bone remodeling patterns. Fibroblast-like synoviocytes (FLS) are cells involved in the transition from an acute and reparable phase to a chronic and persistent stage in these diseases. The distinction of joint phenotypes involves inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-17, and IL-22 directly or through key signaling pathways such as Wnt. To evaluate the role of FLS as the source of Wnt antagonists (sFRP3/FRZB and Dkk1) in the synovia, levels of TNF- α, IL-17, IL-22, Dkk1, and sFRP3 were measured by ELISA directly in the synovial fluid of patients with RA, PsA, or AS. Dkk1 and sFRP3 were also measured in the FLS culture supernatants after different inflammatory stimulus. sFRP3 and Dkk1 are constitutively expressed by FLS. IL-22 and sFRP3 were positively correlated (r=0.76; P<0.01) in synovial fluid. The stimulation of FLS with IL-22, but not TNF-alpha and IL-17, increased the production of sFRP3. No stimulus altered the basal expression of Dkk1. These results showed, for the first time, the ability of IL-22 to increase the expression of sFRP3/FRZB by human FLS in both in vitro and ex vivo models. This finding linked IL-22 to local inhibition of Wnt signaling and possibly to blockade of osteogenesis. Furthermore, FLS presented as a source of this inhibitor in synovial fluid, assigning to this cell a bone injury mechanism.

Licochalcone A Inhibits Proliferation and Induces Apoptosis of MH7A Cells by Regulating MAPK Signaling Pathway / 中国实验方剂学杂志

Objective:To study the effects of licochalcone A （LCA） on the proliferation and apoptosis of rheumatoid arthritis fibroblast-like synoviocytes （MH7A） as well as the related inflammatory factors， also to reveal the relevance between mitogen activated protein kinase （MAPK） signaling pathway and LCA regulation of MH7A cell proliferation and apoptosis. Method:MH7A cells were cultured and divided into blank group， LCA groups （10，20，40 μmol·L-1）. The proliferation of MH7A cells was detected by methylthiazolyldiphenyl-tetrazolium bromide（MTT）and immunofluorescence staining. The cell cycle of MH7A cells was determined by flow cytometry after PI staining and apoptosis was detected by flow cytometry after Annexin V/PI staining. The effect of LCA on interleukin-1β（IL-1β） mRNA was detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）. Western blot was used to detect the effect of LCA on the key proteins of MAPK signaling pathway， meanwhile， PD98059， a specific ERK inhibitor， was used to observe the expression levels of p-ERK and IL-1β. Result:Compared with blank group， LCA could inhibit the proliferation of MH7A cells in a dose-dependent manner， and the number of living cells decreased significantly（P<0.01）， while the number of early apoptotic cells increased significantly（P<0.01）. Compared with the tumor necrosis factor-α （TNF-α，10 μg·L-1）group， LCA could reverse the expression of IL-1β mRNA induced by TNF-α（P<0.01）. and compared with the blank group， LCA also promoted the phosphorylation of ERK， JNK and p38 in a dose-dependent manner（P<0.01）. After ERK inhibitor PD98059 inhibited ERK phosphorylation， the inhibitory effect of LCA 10， 20 μmol·L-1 on IL-1β disappeared. Conclusion:LCA can inhibit the proliferation and induce apoptosis of MH7A cells， which may be related to the phosphorylation of MAPK pathway related proteins， and then inhibit the expression of inflammatory factors.

decoction induces apoptosis by activating Fas/caspase-8 pathway in rheumatoid arthritis fibroblast-like synoviocytes / 南方医科大学学报

OBJECTIVE@#To explore the effect of decoction (DGNTD) on cell apoptosis and TNF receptor super family 6 (Fas)/caspase-8 pathway in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS).@*METHODS@#FLS isolated from the synovial tissue of RA patients were cultured and identified using immunofluorescence staining. The cells were treated with 10% blank serum (blank control group), 10% sera containing low, moderate or high doses of DGNTD, or 20 μmol/mL KR-33493 (a Fas inhibitor) combined with 10% serum containing high-dose DGNTD. MTT assay was used to detect the proliferation of the cells after the treatments. Apoptosis of the cells was detected at 48 h in each group using Hoechst 33342 staining and flow cytometry with annexin V-FITC/PI staining. The mRNA and protein expressions of Fas, FADD, caspase-8 and caspase-3 in the cells at 48 h were detected using qPCR and Western blotting.@*RESULTS@#Immunofluorescence staining identified the cultured cells as FLS. Treatment with DGNTD-containing sera significantly inhibited the proliferation of FLS, and the inhibitory effects were enhanced as the dose and intervention time increased ( < 0.05). Hoechst 33342 staining and flow cytometry showed that the sera containing different doses of DGNTD significantly promoted apoptosis of FLS ( < 0.05). The expression levels of Fas, FADD, caspase-8, and caspase-3 at both mRNA and protein levels were significantly increased in the cells after treatment with different doses of DGNTD-containing sera ( < 0.05). The application of KR-33493 obviously reversed the effects of DGNTD on the FLS ( < 0.05).@*CONCLUSIONS@#DGNTD can induce apoptosis of the FLS by activating Fas/caspase-8 signaling pathway.

Resveratrol mediates apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis by down-regulating mnsod / 中国药理学通报

Aim: To investigate the role of manganese superoxide dismutase (MS0D) protein expression in the proliferation and apoptosis of fibroblast-like synoviocyte (RA-FLS) in rheumatoid arthritis mediated by resveratrol(Res). Methods: After RA-FLSs was treated with Res, the expression of MnSOD protein was detected by CCK-8 method, and the expression of Mn- SOD protein was detected by, Western blot. After lentivirus infection with RA-FLSs, 8 mg · L-1 purine mycin was used to screen the infected cells, and three stable cell lines were established; MnSOD overexpression, MnSOD interference and MnSOD control. The mitochondria reactive oxygen species (mtROS) levels of each cell line treated with 5 μmol · L-1 hydrogen peroxide (H2O2) and 200 mol · L-1 Res were detected by laser confocal microscopy, and cell apoptosis was detected by flow cytometry. Results Res could inhibit the proliferation and the expression of MnSOD in RA- FLSs. The lentivirus-mediated MnSOD regulation could significantly increase or decrease the expression of Mn- SOD in RA-FLSs. MnSOD over expression could decrease the level of mtROS and apoptosis of the RA- FLSs treated with 5 μmol · L-1 H2O2 and 200 μmol · L-1 Res. While the MnSOD RNAi showed the opposite results. Conclusions: Res may up-regulate mtROS levels by inhibiting the expression of MnSOD, thus promoting the apoptosis of RA-FLSs.

A new method for isolation and culture of fibroblast-like synovial cells from adjuvant arthritis rabbits / 第二军医大学学报

[Abstract] Objective To establish a new, simple method for isolation and culture of fibroblast-like synoviocytes (FLSs) from adjuvant arthritis (AA) rabbits using suspended explant culture method. Methods Sixteen healthy New Zealand white rabbits, weighing 2.0-2.5 kg, were randomly divided into two groups: the normal control group (n＝8) and the model group (n＝8). Rabbit models of AA were prepared by injection with complete Freund’s adjuvant at multiple sites and time points. The joint synovial layers were obtained. The FLSs of rabbits with AA were cultured by suspended explant culture method and explant culture method. The growth and morphological characteristics of cells were observed, and the cell viability was measured by CCK-8 assay. The expression of vimentin was detected by immunocytochemistry. Results The tarsometatarsal joints and toes of rabbits in the model group were swollen more obviously than those in the normal control group. After the toes were dissected, obvious microvascular proliferation and inflammation were observed in the model group. The results of H-E staining showed that synoviocytes in the normal control group were arranged in a regular manner like beads, while those in the model group were arranged in a disordered manner, suggesting that the AA model was successfully prepared. The operating durations required for suspended explant culture method and explant culture method were about 25 min and 1 h, respectively. The morphology and viability were similar for the cells obtained by the two methods. The positive rates of vimentin in the suspended explant culture method and explant culture method group were 98.01% and 98.35%, respectively. Conclusion Isolation and culture of FLSs by suspended explant culture method has been successfully established, and the method is simple and needs a short time.

Effect of Gancao Fuzitang on Synovial Fibroblast-like Cell Proliferation in Adjuvant Arthritis Mice Model / 中国实验方剂学杂志

Objective:To investigate the effect of Gancao Fuzitang on the proliferation of fibroblast-like synoviocytes (FLS) in synovial tissue of mice with adjuvant-induced arthritis (AA). Method:The 48 male Balb/c mice were randomly divided into 4 groups:normal control group, model group(arthritis induced through injection with adjuvant), Gancao Fuzitang group and triptolide group. The normal control group and the model control group were orally given physiological saline. After the successful modeling, the mice of the other two groups were treated with Gancao Fuzitang or triptolide orally for 18 days. Then the animals were euthanized, the paw edema of the animals was measured, and arthritic ankles were observed by histological examination. Tumor necrosis factor (TNF)-α was examined by immunoassays. The proliferation of synovial cells of foot joints was detected by immunohistochemical expressions of Vimentin. The expressions of Cyclin D1, proliferating cell nuclear antigen (PCNA), p53 and p21 were detected by Western blot. Result:Compared with the normal control group, the mice of synovial hyperplasia and bone erosion in model group were obvious. The joint swelling degree, TNF-α level and proliferation of FLS were increased (P1,PCNA were up-regulated(PPα, cell cycles (Cyclin D1 and PCNA), and increased protein expressions of p53 and p21 (PConclusion:Gancao Fuzitang has a therapeutic effect on AA mice, and the mechanism might be associated with its anti-inflammatory effect and the effects in regulating Cyclin D1, PCNA, p53 and p21 expressions and inhibiting FLS proliferation.

Construction and screening of fibroblast-like synoviocytes in human rheumatoid arthritis stably overexpressing lncRNA HOTAIR / 医学研究生学报

Objective　The HOTAIR gene is closely related to pannus formation in rheumatoid arthritis (RA). This study aimed to construct and screen fibroblast-like synoviocytes in human RA (HFLS-RA) stably overexpressing lncRNA HOTAIR, and to pave the way for further study of the role of lncRNA HOTAIR in the pathogenesis of RA.　Methods　LncRNA HOTAIR was cloned and linked to the PMT406 vector digested by BamHI-HF-HF and XhoI. The constructed plasmids were sequenced, identified and then transfected into 293T cells to pack lentivirus. The HFLS-RA cells were infected with the recombinant and empty vector lentiviruses, and purinomycin was employed to screen the lncRNA HOTAIR-overexpressed and control cell lines. The total RNA was extracted from the blank, negatively transfected and overexpressed cells by Trizol, and the cDNA obtained by reverse transcription was amplified by qPCR, followed by determination of the expression of lncRNA HOTAIR.　Results　The relative expression of lncRNA HOTAIR was significantly higher in the overexpression group than in the blank control and negative transfection groups (30.329 ± 3.860 vs 1.001 ± 0.048 and 0.892 ± 0.247, P 0.05).　Conclusion　The HFLS-RA cell line stably overexpressing lncRNA HOTAIR was successfully constructed, which has provided some experimental evidence for further investigation of the role of lncRNA HOTAIR in the pathogenesis of RA.