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

Figures

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  Figure 1. Enhanced mitochondrial fission in STs of RA patients correlates with disease severity. A, Representative TEM images of mitochondrial morphology in STs and FLS. Scale bars: 1 µm. B, qRT‐PCR analysis of DNM1L in STs. C, IHC analysis of DNM1L expression in STs. Scale bars: 50 µm. D, Western blot analysis of DNM1L in STs. E, Correlation of the ratio of DNM1L‐to‐MFN1 with the level of serum anti‐CCP, DAS28 and ESR in RA patients. Data are the mean ± SD of each group. N = 10 RA patients and n = 3 non‐RA patients. *P < .05 vs the control

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  Figure 2. DNM1L deficiency alters mitochondria morphology and mitochondrial membrane potential in FLSs. (A, B) FLSs from RA patients were transfected with control (siCtrl) or DNM1L‐specific siRNA (siDNM1L) for 72 h, and the relative levels of DNM1L expression were determined by Western blot and qRT‐PCR. C, Effect of mdivi‐1 concentration on DNM1L GTPase activity. D, Representative confocal microscopy images of mitochondrial morphology in different groups of FLSs, with staining by MitoTracker Green and quantitation of these data. Scale bars: 20 µm. E, Representative confocal microscopy images of the effect of DNM1L deficiency on mitochondrial membrane depolarization (JC‐1 fluorescence) and quantitation of these data. The ratio of red to green signal was determined for quantitative analysis. Scale bars: 100 µm. Data are representative images or expressed as the mean ± SD of each group from three separate experiments. *P < .05; **P < .01; ***P < .001

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  Figure 3. DNM1L deficiency in FLSs reduces their viability and production of pro‐inflammatory cytokines, and increases apoptosis. A, Cell viability was determined using the CCK‐8 assay. (B, C) Western blot and qRT‐PCR analyses of COX‐2 and IL‐8 expression in FLSs (mdivi‐1 concentration = 50 µmol/L). D, Representative flow cytometry data of apoptotic FLSs after staining with FITC‐Annexin V and PI, and quantitation of these data. Data are representative flow cytometry charts, images or expressed as mean ± SD of each group from three separate experiments. *P < .05; **P < .01; ***P < .001; ***P < .0001

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  Figure 4. DNM1L deficiency reduces ROS production and autophagy in FLS. A, Representative DHE and DAPI staining, and quantitation of these data. AOD (average optical density) indicates the absolute absorption of DHE signal. Scale bars: 100 µm. B, Effect of mdivi‐1 on IL‐1β– and IL‐1β/H2O2–mediated AKT activation in FLSs. FLSs were treated with mdivi‐1 in the presence of IL‐1β for 10 min, and with or without, 100 µmol/L H2O2 for 12 h. C, Western blot analysis of LC3B‐I and LC3B‐II in the different groups of FLSs. Data are representative images or expressed as the mean ± SD of each group of cells from three separate experiments. *P < .05; **P < .01; ***P < .001

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  Figure 5. DNM1L deficiency attenuates the IL‐1β–induced NF‐κB activation in FLSs. A, The relative levels of AKT, IKK and NFKBIA expression and AKT and IKK phosphorylation were determined by Western blot. B, The nuclear translocation of NF‐κBp65 in the different groups of FLSs was determined by immunofluorescent microscopy. Scale bars: 50 µm. Data are representative images or expressed as the mean ± SD of each group of cells from three separate experiments. *P < .05; **P < .01; ***P < .001; ***P < .0001

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  Figure 6. Treatment with mdivi‐1 ameliorates the severity of CIA in mice. After established CIA, the mice were randomized and treated with DMSO or 0.2 mg mdivi‐1 every other day for 2 wk. (A‐C) Their CIA clinical scores, paw thickness and affected paws were measured. D, Representative images of paws in the different groups of mice. E, The relative levels of inflammatory molecules in the joints of different groups of mice were determined by qRT‐PCR. F, Histological examination of the hind paws after H&E or toluidine blue staining. Scale bar: 100 µm. G, Histological examination of the liver and kidney tissues after H&E staining. Scale bar: 100 µm. H, DHE staining of ROS contents in the crystal synovial membranes of mice. Scale bar: 200 µm. Data are representative images or expressed as the mean ± SD of each group of mice (n = 7). *P < .05; **P < .01