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Objective To observe the effect of different synovial cell secretions on chondrocytes after LPS-induced inflammation,and to explore the mechanism of two synovial cell secretions causing cartilage damage in the progres-sion of KOA disease.Methods Two kinds of synovial cells were co-cultured at 1∶4 and LPS-induced inflamma-tion.The supernatant and exocrine were extracted,and then the normal and LPS-induced inflammation were extrac-ted.The human cartilage tissue obtained during the operation was isolated and cultured into chondrocytes,which were divided into five groups:the first group was added with FLS secretion,the second group was added with nor-mal FLS secretion,the third group was added with secretion after co-culture of two kinds of synovial cells,the fourth group was added with inflammatory MLS secretion,and the fifth group was added with inflammatory FLS se-cretion.CCK-8 was used to detect the viability of chondrocytes in each group.TNF-α,IL-1β,IL-6 level in the su-pernatant of chondrocytes in each group was detected by ELISA.The protein expression of TLR4,NF-κB,IkK,IκB,ADAMTS5 in chondrocytes of each group was detected by Western blot method.Results CCK-8 showed that the activity of chondrocytes in the three groups of inflammatory secretions decreased compared with the secretions from normal synovial cells(P<0.05);ELISA showed TNF-α,IL-1 β,IL-6 level in the supernatant of group Ⅲ,Ⅳ and V was higher than that of group Ⅰ and Ⅱ(P<0.05),TNF-α,IL-1 β,IL-6 level in group Ⅲ was higher than that in group Ⅳ but lower than that in group Ⅴ(P<0.05).Western blot showed the protein expression of TLR4,NF-κB,IkK,IκB,ADAMTS5 in chondrocytes of group Ⅲ,Ⅳ and Ⅴ was higher than that in group Ⅰ and Ⅱ(P<0.05),the protein expression of TLR4,NF-κB,IkK,IκB,ADAMTS5 in group Ⅲ was higher than that in group Ⅳbut lower than that in group Ⅴ(P<0.05).Conclusion Two kinds of synovial cell-derived secretions after LPS-induced inflammation can regulate cartilage TLRs/NF-κB signal pathway,causing cartilage inflammation.The in-flammatory effect of MLS secretion is stronger than that of FLS secretion,but the inflammatory effect of MLS secre-tion under two co-cultures is weaker than that of MLS secretion alone.
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Rheumatoid arthritis (RA) is an autoimmune disease involving symmetrical small joints, with clinical manifestations such as small joint swelling, morning stiffness, progressive pain, and even joint deformity and loss of function. Due to the complex immune mechanism, the pathogenesis of RA remains unclear. However, studies have shown that the pathogenesis of RA is related to abnormal immune mechanism, increased synovial inflammatory response, abnormal biological behavior of RA fibroblast-like synoviocytes (RA-FLSs), and abnormal degradation of extracellular matrix. Mitogen-activated protein kinase (MAPK) plays a key role in the regulation of cell growth, proliferation, differentiation, and apoptosis. It is involved in the abnormal release and activation of inflammatory mediators in RA, the abnormal proliferation, migration, and invasion of RA-FLSs, synovial angiogenesis, bone erosion, and cartilage destruction. The thousands of years of practical experience show that Chinese medicine can effectively mitigate the clinical symptoms such as joint swelling, morning stiffness, and pain and delay the occurrence of joint deformity in RA patients. Moreover, the Chinese medicine treatment has the advantages of overall regulation, personalized treatment, multiple pathways and targets, high safety, few adverse reactions, and stable quality. Modern studies have confirmed that Chinese medicine can play a role in the prevention and treatment of RA by interfering in the MAPK signaling pathway, reducing pro-inflammatory cytokines, inhibiting the abnormal proliferation, migration, and invasion of RA-FLSs, regulating the apoptosis of RA-FLSs, and protecting extracellular matrix. This article elaborates on the key role of MAPK signaling pathway in the development of RA and reviews the latest research results of Chinese medicine intervention in MAPK signaling pathway for the prevention and treatment RA, aiming to provide a basis for the development of new drugs and the clinical application of Chinese medicine in the prevention and treatment of RA.
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AIM:To observe how total flavonoids of Pterocarya hupehensis Skan(PHSTF)affects the migra-tion and invasion of human rheumatoid fibroblast-like synoviocyte line MH7A.METHODS:The MH7A cells were divided into control group(without any treatment),low-,medium-and high-dose(6.25,12.5 and 25 mg/L,respectively)PHSTF groups,phosphatidylinositol 3-kinase(PI3K)inhibitor 740Y-P(10 μmol/L)group,and 740Y-P(10 μmol/L)+high-dose(25 mg/L)PHSTF group.The viability of the MH7A cells was determined by CCK-8 assay,while the migration and inva-sion were assessed by scratch and Transwell assays.The protein levels of matrix metalloproteinase-2(MMP-2),MMP-9,PI3K,p-PI3K,AKT and p-AKT were detected by Western blot.RESULTS:Compared with control group,the cell via-bility in PHSTF treatment groups was reduced(P<0.05),the cell wound healing area was significantly decreased(P<0.01),migratory and invasive cells in the lower chamber were significantly reduced(P<0.01),and the protein expres-sion of MMP-2 and MMP-9 and the ratios of p-PI3K/PI3K and pAKT/AKT were decreased(P<0.01).Compared with high-dose PHSTF group,the addition of PI3K/AKT pathway agonist 740Y-P significantly increased the migration and invasion ability of MH7A cells(P<0.01)and elevated the protein expression of MMP-2 and MMP-9 and the ratios of p-PI3K/PI3K and pAKT/AKT(P<0.01)under the treatment with PHSTF.CONCLUSION:Total flavonoids of Pterocarya hupehensis Skan could inhibit the migration and invasion of MH7A cells by regulating the PI3K/AKT signaling pathway.
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Objective:To explore the effect of low intensity pulsed ultrasound(LIPUS)on inhibiting the abnormal cell phenotype of fibroblast-like synoviocytes in rheumatoid arthritis(RA-FLS)and possible mechanism. Method:Synoviocytes were isolated by using enzyme digestion,and the morphology of cells was observed un-der microscope.At the same time,the expression of Vimentin protein was detected by immunofluorescence method to identify RA-FLS.Cells cultured in vitro were divided into four groups:control group,LIPUS group,tumor necrosis factor(TNF-α)group and TNF-α+LIPUS group or three groups:control group,interleu-kin-6(IL-6)group and IL-6+LIPUS group.The effects of LIPUS on RA-FLS cell viability and proliferation were detected by CCK8 and EDU assay respectively,and the effects of LIPUS on RA-FLS migration were ob-served by scratch test and Transwell migration assay.RT-qPCR was used to detect the gene expression of im-portant cytokines,chemokines and matrix metalloproteinases(MMPs)in RA-FLS.ELISA was used to further detect the effect of LIPUS on the expression of IL-6,a key effector of RA-FLS,and the effects of LIPUS on mitogen-activated protein kinase(MAPK)signaling pathway in RA-FLS were detected by Western Blot. Result:Purified RA-FLS were obtained.Firstly,LIPUS could suppress the cell activity(P<0.00l)and prolifer-ation(P=0.007)induced by TNF-α in RA-FLS cultured in vitro.However,the migration and the transcription levels of MMPs related to migration(MMP2 and MMP9)were not significantly different between groups(P>0.05).LIPUS could inhibit the high expression of IL-6 and interleukin-8(IL-8)at the mRNA level in the in-flammatory environment induced by TNF-α(P<0.001),but there was no significant difference in the suppres-sion of interleukin-1β(IL-1β),MMP1 and MMP13(P>0.05).In addition,compared with untreated group,LI-PUS could inhibit the secretion of IL-6 in RA-FLS induced by TNF-α(P<0.001),and also inhibited the pro-liferation of RA-FLS induced by IL-6(P-0.003).Finally,LIPUS could inhibit the phosphorylation of p38 MAPK and c-Jun N-terminal kinase(JNK)in MAPK signaling pathway(P=0.033),but the effect on the phosphorylation of extracellular signal-regulated kinas 1/2(ERK1/2)was not significantly(P>0.05). Conclusion:LIPUS could reduce the abnormal proliferation of RA-FLS in inflammatory state without affecting its migration,which might be related to the inhibition of p38/JNK-IL-6 signaling pathway.
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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.
Subject(s)
Humans , Synoviocytes , Berberine/metabolism , Reactive Oxygen Species/metabolism , Tumor Necrosis Factor-alpha/metabolism , Hydrogen Peroxide/metabolism , Sincalide/metabolism , Cell Proliferation , Arthritis, Rheumatoid/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Apoptosis , Fibroblasts , Autophagy , Cells, CulturedABSTRACT
Aim To study the effect of baicalin on the activation of NLRP3 inflammasomes in human fibroblast like synoviocytes of rheumatoid arthritis ( HFLS-RA) and its mechanism. Methods To confirm that baicalin alleviated the activation of NLRP3 inflammasome in HFLS-RA, immunofluorescence was used to observe the expression of NLRP3 before and after baicalin treatment. Western blot was used to detect the protein expression of p-PI3K, p-Akt, NF-κB p65, NL-RP3, ASC and caspase-1 after baicalin treatment for 48 h, and ELISA was employed to detect the contents of IL-1 and IL-18 in the supernatents. In order to explore the mechanism of baicalin alleviating the activation of NLRP3 inflammasome, double luciferin and Westen blot analysis were applied to verify the corresponding relationship between let-7i-3p and PIK3CA. RT-qPCR was utilized to determine the expression of let-7i-3p and PI3K before and after baicalin intervention. let-7i-3p interference was used to verify whether baicalin mitigated the activation of enhanced NLRP3 inflammasomes. Results Baicalin (50, 100 mg · L
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Aim To investigate the regulatory effects of 3-bromopyruvate (3-BrPA) on apoptosis and autophagy of fibroblast-like synoviocytes (FLS) in rats based on AMPK/mTOR signaling pathway and the underlying mechanism. Methods FLS of rats in vitro were cultured and induced by tumor necrosis factor-α (TNF-α) to construct a model of rheumatoid arthritis (R A). MTT assay was used to explore the optimal concentration of TNF-α and 3 -BrPA for induction and treatment of FLS. The effects of 3-BrPA on the migration and invasion of FLS were detected by Wound healing assay and Transwell assay. The apoptosis of FLS was tested by flow cytometry and mitochondrial membrane potential assay kit (JC-1). Moreover, FLS autophagic flux was detected by mCherry-EGFP-LC3B-overexpressed plasmids, and the expression of apoptosis/autophagy-related proteins as well as AMPK/mTOR pathway-related proteins were detected by Western blot. Results 3-BrPA (15 μmol • L) significantly inhibited the proliferation, migration, and invasion of FLS stimulated by TNF-a (25 μg • L
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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<sup>-1</sup>) and methotrexate (MTX, 0.4 mg·kg<sup>-1</sup>), 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<sup>-1</sup>) <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.
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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.
Subject(s)
Humans , Apoptosis , Arthritis, Rheumatoid/genetics , Cell Proliferation , Cells, Cultured , Fibroblasts , Iridoid Glucosides , Molecular Docking Simulation , Proto-Oncogene Proteins c-bcl-2/genetics , Pyrones , SynoviocytesABSTRACT
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.
Subject(s)
Humans , Apoptosis , Arthritis, Rheumatoid/genetics , Capsules , Cells, Cultured , Drugs, Chinese Herbal , Fibroblasts , Inflammation , Synovial Membrane , Synoviocytes , Tumor Necrosis Factor-alpha/geneticsABSTRACT
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.
Subject(s)
Humans , Arthritis, Rheumatoid/genetics , Autophagy/genetics , Cell Proliferation , Cells, Cultured , Fibroblasts , RNA, Long Noncoding/genetics , Reproducibility of Results , SynoviocytesABSTRACT
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.
Subject(s)
Humans , Arthritis, Rheumatoid/drug therapy , Cell Movement , Cell Proliferation , Cells, Cultured , Fibroblasts , Hesperidin/analogs & derivatives , Synoviocytes , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Metabolomics studies have shown metabolic disorders in patients with rheumatoid arthritis (R A) . However, the metabolic characteristics of individual cell types associated with RA have sparsely been elucidated. Fibroblast-like synoviocytes (FLS) are the main cell types at the synovial tissue of joint, which can lead to joint destruction through invading articular cartilage. In the microenvironment of inflammatory tissues, the metabolism of glucose, lipids, amino acids, glutamine and other key nutrients in RA-FLS changes, which are involved in the abnormal activation of FLS and disease activities. The research progress on key molecules of RA-FLS energy metabolism abnormalityin recent years were summarized in this paper, providing a basis for elucidating the pathological mechanism of RA and discovering new drug targets.