Inhibition of DNM1L and mitochondrial fission attenuates inflammatory response in fibroblast‐like synoviocytes of rheumatoid arthritis

Mitochondrial fission and fusion are important for mitochondrial function, and dynamin 1‐like protein (DNM1L) is a key regulator of mitochondrial fission. We investigated the effect of mitochondrial fission on mitochondrial function and inflammation in fibroblast‐like synoviocytes (FLSs) during rheumatoid arthritis (RA). DNM1L expression was determined in synovial tissues (STs) from RA and non‐RA patients. FLSs were isolated from STs and treated with a DNM1L inhibitor (mdivi‐1, mitochondrial division inhibitor 1) or transfected with DNM1L‐specific siRNA. Mitochondrial morphology, DNM1L expression, cell viability, mitochondrial membrane potential, reactive oxygen species (ROS), apoptosis, inflammatory cytokine expression and autophagy were examined. The impact of mdivi‐1 treatment on development and severity of collagen‐induced arthritis (CIA) was determined in mice. Up‐regulated DNM1L expression was associated with reduced mitochondrial length in STs from patients with RA and increased RA severity. Inhibition of DNM1L in FLSs triggered mitochondrial depolarization, mitochondrial elongation, decreased cell viability, production of ROS, IL‐8 and COX‐2, and increased apoptosis. DNM1L deficiency inhibited IL‐1β–mediated AKT/IKK activation, NF‐κBp65 nuclear translocation and LC3B‐related autophagy, but enhanced NFKBIA expression. Treatment of CIA mice with mdivi‐1 decreased disease severity by modulating inflammatory cytokine and ROS production. Our major results are that up‐regulated DNM1L and mitochondrial fission promoted survival, LC3B‐related autophagy and ROS production in FLSs, factors that lead to inflammation by regulating AKT/IKK/NFKBIA/NF‐κB signalling. Thus, inhibition of DNM1L may be a new strategy for treatment of RA.     

Acacetin inhibits expression of matrix metalloproteinases via a MAPK‐dependent mechanism in fibroblast‐like synoviocytes

It is well known that rheumatoid arthritis (RA) is an autoimmune joint disease in which fibroblast‐like synoviocytes (FLSs) play a pivotal role. In this study, we investigated the anti‐arthritic properties of acacetin in FLSs. The expression of matrix metalloproteinase (MMP)‐1, MMP‐3 and MMP‐13 were investigated by quantitative RT‐PCR and western blot at gene and protein levels. At the same time, the phosphorylation of mitogen‐activated protein kinases (MAPK) was investigated. The DNA‐binding activity of NF‐κB was investigated by electrophoretic mobility shift assay. We found that acacetin inhibits p38 and JNK phosphorylation and reduces MMP‐1, MMP‐3 and MMP‐13 expression in interleukin‐1β‐induced FLSs. Our results suggest that acacetin has antiarthritic effects in FLSs. Thus, acacetin should be further studied for the treatment of arthritis.     

Role of protein arginine methyltransferase 5 in inflammation and migration of fibroblast‐like synoviocytes in rheumatoid arthritis

To probe the role of protein arginine methyltransferase 5 (PRMT5) in regulating inflammation, cell proliferation, migration and invasion of fibroblast‐like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA). FLSs were separated from synovial tissues (STs) from patients with RA and osteoarthritis (OA). An inhibitor of PRMT5 (EPZ015666) and short interference RNA (siRNA) against PRMT5 were used to inhibit PRMT5 expression. The standard of protein was measured by Western blot or immunofluorescence. The excretion and genetic expression of inflammatory factors were, respectively, estimated by enzyme‐linked immunosorbent assay (ELISA) and real‐time polymerase chain reaction (PCR). Migration and invasion in vitro were detected by Boyden chamber assay. FLSs proliferation was detected by BrdU incorporation. Increased PRMT5 was discovered in STs and FLSs from patients with RA. In RA FLSs, the level of PRMT5 was up‐regulated by stimulation with IL‐1β and TNF‐α. Inhibition of PRMT5 by EPZ015666 and siRNA‐mediated knockdown reduced IL‐6 and IL‐8 production, and proliferation of RA FLSs. In addition, inhibition of PRMT5 decreased in vitro migration and invasion of RA FLSs. Furthermore, EPZ015666 restrained the phosphorylation of IκB kinaseβ and IκBα, as well as nucleus transsituation of p65 as well as AKT in FLSs. PRMT5 regulated the production of inflammatory factors, cell proliferation, migration and invasion of RA FLS, which was mediated by the NF‐κB and AKT pathways. Our data suggested that targeting PRMT5 to prevent synovial inflammation and destruction might be a promising therapy for RA.     

Hydrogen sulphide decreases IL‐1β‐induced activation of fibroblast‐like synoviocytes from patients with osteoarthritis

Balneotherapy employing sulphurous thermal water is still applied to patients suffering from diseases of musculoskeletal system like osteoarthritis (OA) but evidence for its clinical effectiveness is scarce. Since the gasotransmitter hydrogen sulphide (H₂S) seems to affect cells involved in degenerative joint diseases, it was the objective of this study to investigate the effects of exogenous H₂S on fibroblast‐like synoviocytes (FLS), which are key players in OA pathogenesis being capable of producing pro‐inflammatory cytokines and matrix degrading enzymes. To address this issue primary FLS derived from OA patients were stimulated with IL‐1β and treated with the H₂S donor NaHS. Cellular responses were analysed by ELISA, quantitative real‐time PCR, phospho‐MAPkinase array and Western blotting. Treatment‐induced effects on cellular structure and synovial architecture were investigated in three‐dimensional extracellular matrix micromasses. NaHS treatment reduced both spontaneous and IL‐1β‐induced secretion of IL‐6, IL‐8 and RANTES in different experimental settings. In addition, NaHS treatment reduced the expression of matrix metallo‐proteinases MMP‐2 and MMP‐14. IL‐1β induced the phosphorylation of several MAPkinases. NaHS treatment partially reduced IL‐1β‐induced activation of several MAPK whereas it increased phosphorylation of pro‐survival factor Akt1/2. When cultured in spherical micromasses, FLS intentionally established a synovial lining layer‐like structure; stimulation with IL‐1β altered the architecture of micromasses leading to hyperplasia of the lining layer which was completely inhibited by concomitant exposure to NaHS. These data suggest that H₂S partially antagonizes IL‐1β stimulation via selective manipulation of the MAPkinase and the PI3K/Akt pathways which may encourage development of novel drugs for treatment of OA.     

H2S transiently blocks IL‐6 expression in rheumatoid arthritic fibroblast‐like synoviocytes and deactivates p44/42 mitogen‐activated protein kinase

Sulfur bath therapy represents the oldest form of treatment for patients with different types of rheumatic disorders. However, scientific reports about the beneficial effects of this form of therapy are controversial, rare and of poor scientific quality. Also, little is known about the role and underlying molecular mechanisms of H₂S. Therefore, this topic encouraged us to investigate the influence of H₂S on fibroblasts isolated from the synovial membrane of RA (rheumatoid arthritis) patients. FLSs (fibroblast‐like synoviocytes) were treated with different concentrations of an exogenous H₂S donor (NaHS). At defined time points, secretion of IL‐6 was quantified by ELISA. Activation/deactivation of MAPKs (mitogen‐activated protein kinases), p38 and p44/42 MAPK (ERK1/2) were confirmed by Western blot experiments. FLSs constitutively express and secrete large quantities of IL‐6 and IL‐8. Data provided prove that, in FLSs, constitutive as well as IL‐1β‐induced expression of IL‐6 is transiently and partially down‐regulated by the short treatment of cells with low concentrations of NaHS. Another key finding is that H₂S deactivates p44/42 MAPK (ERK1/2). Long‐term exposure of FLSs to H₂S provides stimulatory effects, leading to reinforced activation of p38 MAPK and ERK1/2 accompanied by upregulation of IL‐6 expression. Presented data seem of importance for studying (patho‐) physiological functions of H₂S and also for re‐evaluating sulfur spa therapy as one of the oldest forms of therapy for rheumatic disorders.     

miR‐124a inhibits the proliferation and inflammation in rheumatoid arthritis fibroblast‐like synoviocytes via targeting PIK3/NF‐κB pathway

Abnormal hyperplasia of fibroblast‐like synoviocytes (FLS) leads to the progression of rheumatoid arthritis (RA). This study aimed to investigate the role of miR‐124a in the pathogenesis of RA. The viability and cell cycle of FLS in rheumatoid arthritis (RAFLS) were evaluated by Cell Counting Kit 8 and flow cytometry assay. The expression of PIK3CA, Akt, and NF‐κB in RAFLS was examined by real‐time PCR and Western blot analysis. The production of tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 was detected by ELISA. The joint swelling and inflammation in collagen‐induced arthritis (CIA) mice were examined by histological and immunohistochemical analysis. We found that miR‐124a suppressed the viability and proliferation of RAFLS and increased the percentage of cells in the G1 phase. miR‐124a suppressed PIK3CA 3'UTR luciferase reporter activity and decreased the expression of PIK3CA at mRNA and protein levels. Furthermore, miR‐124a inhibited the expression of the key components of the PIK3/Akt/NF‐κB signal pathway and inhibited the expression of pro‐inflammatory factors TNF‐α and IL‐6. Local overexpression of miR‐124a in the joints of CIA mice inhibited inflammation and promoted apoptosis in FLS by decreasing PIK3CA expression. In conclusion, miR‐124a inhibits the proliferation and inflammation in RAFLS via targeting PIK3/NF‐κB pathway. miR‐124a is a promising therapeutic target for RA.     

The emerging role of fibroblast‐like synoviocytes‐mediated synovitis in osteoarthritis: An update

Osteoarthritis (OA), the most ubiquitous degenerative disease affecting the entire joint, is characterized by cartilage degradation and synovial inflammation. Although the pathogenesis of OA remains poorly understood, synovial inflammation is known to play an important role in OA development. However, studies on OA pathophysiology have focused more on cartilage degeneration and osteophytes, rather than on the inflamed and thickened synovium. Fibroblast‐like synoviocytes (FLS) produce a series of pro‐inflammatory regulators, such as inflammatory cytokines, nitric oxide (NO) and prostaglandin E₂ (PGE₂). These regulators are positively associated with the clinical symptoms of OA, such as inflammatory pain, joint swelling and disease development. A better understanding of the inflammatory immune response in OA‐FLS could provide a novel approach to comprehensive treatment strategies for OA. Here, we have summarized recently published literatures referring to epigenetic modifications, activated signalling pathways and inflammation‐associated factors that are involved in OA‐FLS‐mediated inflammation. In addition, the current related clinical trials and future perspectives were also summarized.     

TAK1基因沉默对TNF-a诱导的滑膜细胞IL-6、IL-8表达的影响

目的：观察转化生长因子β激活激酶1（TAK1）基因沉默对肿瘤坏死因子a （TNF-a）诱导的滑膜细胞中促炎介质白介素-6（IL-6）和白介素-8（IL-8）表达的影响,以探讨TAK1在类风湿关节炎（RA）发病中的作用。方法：采取脂质体转染方法将TAK1特异性的小干扰RNA （siRNA）和阴性对照RNA （scRNA）导入类风湿关节炎的滑膜成纤维细胞株MH7A细胞,然后分别用20 ng/ml TNF-a刺激后,检测细胞内IL-6、IL-8 mRNA的表达和培养上清中IL-6和IL-8分泌情况以及p38、ERK、JNK、p65磷酸化的水平和抑制性蛋白IκBα的变化情况。结果：siRNA-TAK1转染72 h后滑膜细胞中TAK mRNA和蛋白表达的平均抑制率分别为75%和55%。siRNA-TAK1转染下调TNF-a诱导状态下IL-6和IL-8的表达,并能抑制p38、JNK、p65磷酸化和增加IκBα水平。结论：TAK1基因沉默能抑制TNF-a诱导的滑膜细胞IL-6、IL-8表达,可能与其抑制JNK和p38MAPK的活化及NF-κB的活化有关。     

Silencing CDR1as enhances the sensitivity of breast cancer cells to drug resistance by acting as a miR‐7 sponge to down‐regulate REGγ

In our study, we aimed to investigate the role of CDR1as during competitive inhibition of miR‐7 in the regulation of cisplatin chemosensitivity in breast cancer via regulating REGγ. RT‐qPCR was applied to detect the expression of CDR1as and miR‐7 in breast cancer tissues, breast cancer cell lines and corresponding drug‐resistant cell lines. The correlation between CDR1as and miR‐7 and between miR‐7 and REGγ was evaluated. MCF‐7‐R and MDA‐MB‐231‐R cells were selected followed by transfection of a series of mimics, inhibitors or siRNA. The effect of CDR1as on the half maximal inhibitor concentration (IC50), cisplatin sensitivity and cell apoptosis was also analysed. Furthermore, a subcutaneous xenograft nude mouse model was established to further confirm the effect of CDR1as on the chemosensitivity of breast cancer to cisplatin in vivo. Immunohistochemical staining was conducted to test the Ki‐67 expression in nude mice. A positive correlation was found between the drug resistance and CDR1as expression in breast cancer. CDR1as could increase the resistance of breast cancer cells to cisplatin. miR‐7 expression was low, while REGγ was highly expressed in MCF‐7‐R and MDA‐MB‐231‐R cells. CDR1as competitively inhibited miR‐7 and up‐regulated REGγ. Overexpression of miR‐7 could reverse the enhanced sensitivity of silenced CDR1as to drug‐resistant breast cancer cells. Additionally, in vivo experiments demonstrated that CDR1as mediated breast cancer occurrence and its sensitivity to cisplatin. Silencing CDR1as decreased Ki‐67 expression. Silencing CDR1as may inhibit the expression of REGγ by removing the competitive inhibitory effect on miR‐7 and thus enhancing the sensitivity of drug‐resistant breast cancer cells.     

Stereoselective regulation of MDR1 expression in Caco-2 cells by cetirizine enantiomers

MDR1-encoded P-glycoprotein (P-gp) is a drug efflux transporter mainly expressed in liver, kidney, intestine, brain (at the level of the blood-brain barrier), and placenta. It thus plays important roles in drug absorption, distribution, and excretion. Cetirizine is a second-generation nonsedating antihistamine used to treat allergic disease of respiratory system, skin and eyes. To evaluate P-gp expression and function in Caco-2 cells pretreated with cetirizine enantiomers, we assessed the sensitivity of Caco-2 cells to paclitaxel using the MTT assay and the polarized transport of rhodamine-123 and doxorubicin across Caco-2 monolayers. RT-PCR and flow cytometry were used to assay MDR1 mRNA and P-gp protein respectively. The sensitivity of Caco-2 cells to paclitaxel decreased significantly after cells were pretreated with 100 microM R-cetirizine but increased upon treatment with S-cetirizine. The efflux of rhodamine-123 and doxorubicin was enhanced significantly after Caco-2 monolayers were pretreated with 100 microM R-cetirizine but was reduced by S-cetirizine. The MDR1 mRNA and P-gp levels in Caco-2 cells were increased by 100 microM R-cetirizine and decreased by 100 microM S-cetirizine. These results suggest that R-cetirizine up-regulates MDR1 expression while S-cetirizine down-regulates MDR1 expression.     

FSP1 promotes the biofunctions of adventitial fibroblast through the crosstalk among RAGE,JAK2/STAT3 and Wnt3a/β‐catenin signalling pathways

Emerging evidence indicates that fibroblast‐specific protein 1 (FSP1) provides vital effects in cell biofunctions. However, whether FSP1 influences the adventitial fibroblast (AF) and vascular remodelling remains unclear. Therefore, we investigated the potential role and action mechanism of FSP1‐mediated AF bioactivity. AFs were cultured and stimulated with FSP1 and siRNA‐FSP1 in vitro. Viability assays demonstrated that siRNA‐FSP1 counteracted AFs proliferative, migratory and adherent abilities enhanced with FSP1. Flow cytometry revealed that FSP1 increased AFs number in S phase and decreased cellular apoptosis. Contrarily, siRNA‐FSP1 displayed the contrary results. RT‐PCR, Western blotting and immunocytochemistry showed that FSP1 synchronously up‐regulated the expression of molecules in RAGE, JAK2/STAT3 and Wnt3a/β‐catenin pathways and induced a proinflammatory cytokine profile characterized by high levels of MCP‐1, ICAM‐1 and VCAM‐1. Conversely, FSP1 knockdown reduced the expression of these molecules and cytokines. The increased number of autophagosomes in FSP1‐stimulated group and fewer autophagic corpuscles in siRNA‐FSP1 group was observed by transmission electron microscope (TEM). Autophagy‐related proteins (LC3B, beclin‐1 and Apg7) were higher in FSP1 group than those in other groups. Conversely, the expression of p62 protein was shown an opposite trend of variation. Therefore, these pathways can promote AFs bioactivity, facilitate autophagy and induce the expression of the proinflammatory cytokines. Contrarily, siRNA‐FSP1 intercepts the crosstalk of these pathways, suppresses AF functions, restrains autophagy and attenuates the expression of the inflammatory factors. Our findings indicate that crosstalk among RAGE, STAT3/JAK2 and Wnt3a/β‐catenin signalling pathways may account for the mechanism of AF functions with the stimulation of FSP1.     

Selective reversal of drug resistance in drug-resistant lung adenocarcinoma cells by tumor-specific expression of MDR1 ribozyme gene mediated by retrovirus

According to the fact that CEA gene expressed only in lung adenocarcinoma and not in normal lung cells, a retroviral vector (pCEAMR) was constructed which carried the CEA promoter coupled to MDR1 ribozyme gene. pCEAMR was introduced into drug-resistant lung adenocarcinoma cells GAOK with CEA expression and HeLaK without CEA expression; the expression of pCEAMR and drug resistance in the infected cells were analyzed in vitro and in vivo ; pCEAMR expressed only in CEA-producing GAOK cells and not in non-CEA-producing HeLa cells. The drug resistance to doxorubicin (DOX) decreased 91.5% in the infected GAOK cells and did not change in the infected HeLa cells. In nude mice, DOX could obviously inhibit the growth of the infected GAOK tumors, and had no effect on the growth of the infected HeLa cells. These results indicated that MDR1 ribozyme gene regulated by CEA promoter expressed only in human adenocarcinoma cells and reversed their drug resistance selectively. This gene-drug therapy might serve as an effe