4‐Phenylbutyric acid, a potent endoplasmic reticulum stress inhibitor, attenuates the severity of collagen‐induced arthritis in mice via inhibition of proliferation and inflammatory responses of synovial fibroblasts

Figures

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  FIGURE 1. Effect of 4‐phenylbutyric acid (4‐PBA) on the proliferation of interleukin (IL)‐1β‐stimulated rheumatoid arthritis synovial fibroblasts (RASFSs). Proliferation of RASFSs (n = 3–5) in the presence of 1–80 mM of 4‐PBA and absence of IL‐1β for (A) 24 h and (B) 48 h. (C) Cell viability (n = 3–5) in the presence of 1–80 mM of 4‐PBA after 24‐h incubation without IL‐1β stimulation. (D) Proliferation of RASFSs (n = 3) in the presence or absence of 4‐PBA (20 mM) and/or IL‐1β (1 ng/mL). Cell proliferation and viability and cytotoxicity of 4‐PBA were measured by CCK‐8. Results are presented as mean ± SEM. * p < 0.05 versus no 4‐PBA and/or IL‐1β; and # p < 0.05 versus IL‐1β without 4‐PBA

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  FIGURE 2. Effect of 4‐phenylbutyric acid (4‐PBA) on rheumatoid arthritis synovial fibroblasts (RASFSs) by 4‐PBA. (A) Western blot analysis of expression of ER stress markers in RASFSs (n = 3–5). The cells were cultured in the presence or absence of 4‐PBA (20 mM) and/or interleukin (IL)‐1β (1 ng/mL) for 48 h. (B) Expression of 78‐kDa glucose‐regulated protein (GRP78), activating transcription factor 6 (ATF6), AKT, and CHOP of RASFSs was analyzed. Values are mean ± SEM. # p < 0.05 versus no 4‐PBA and/or IL‐1β; and * p < 0.05 versus IL‐1β without 4‐PBA

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  FIGURE 3. Effect of 4‐phenylbutyric acid (4‐PBA) on the matrix metalloproteinases (MMP) production and MAPK activation in interleukin (IL)‐1β‐stimulated rheumatoid arthritis synovial fibroblasts (RASFSs). RASFSs (n = 3–5) were cultured with 4‐PBA (20 mM) for 48 h, then stimulated with IL‐1β (1 ng/mL) for 15 min. Expression of (A) MMPs, TIMPs, and COX‐2 and (B) activation of MAPK/NF‐κB signaling in RASFSs were analyzed by western blotting. Values are mean ± SEM. # p < 0.05 versus no 4‐PBA and/or IL‐1β; and * p < 0.05 versus IL‐1β without 4‐PBA

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  FIGURE 4. Effect of 4‐phenylbutyric acid (4‐PBA) on the expression of ER stress markers in collagen‐induced arthritis (CIA) mice. Western blot analysis was performed to assess the expression of ER stress markers in DBA/1 mice, the CIA mice treated with vehicle, and the CIA mice treated with 4‐PBA (15 mice/group). The protein extract from ankle joints was prepared as described in Material and Methods and used for the analysis of protein expression of 78‐kDa glucose‐regulated protein (GRP78), activating transcription factor 6 (ATF6), AKT, and CHOP. (A) Representative western blotting of the ER stress markers, and (B) they were quantified by densitometric analysis and normalized to control (DBA/1) expression. Results are presented as mean ± SEM of three independent experiments. # p < 0.05 versus DBA/1 without CIA induction; and * p < 0.05 versus the CIA treated with phosphate buffered saline (PBS)

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  FIGURE 5. Effects of 4‐phenylbutyric acid (4‐PBA) on arthritis severity in collagen‐induced arthritis (CIA) mice. (A) Representative front and hind paws of mice of each group, and average paw thickness score and clinical score of CIA mice treated with 4‐PBA (240 mg kg−1 day−1) or phosphate buffered saline (PBS) (vehicle) (15 mice per group). A representative picture of the forepaw and hind paw was taken at the time of euthanasia, 21 days after second immunization. (B) Micro‐CT images and BV/TV of DBA/1 mice (no CIA induction) and CIA mice treated with 4‐PBA (240 mg·kg−1·day−1) or PBS (15 mice/group). Values are presented as mean ± SEM of three independent experiments. BV, bone volume; TV, total volume. # p < 0.05 versus DBA/1 without CIA induction; and * p < 0.05 versus CIA treated with PBS

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  FIGURE 6. Effect of 4‐phenylbutyric acid (4‐PBA) treatment on the expression level of interleukin (IL)‐6, tumor necrosis factor α (TNF‐α), and matrix metalloproteinases (MMPs) in collagen‐induced arthritis (CIA) mice. (A) Concentration of IL‐6 and IL‐1β in the serum and hind joint extracts of DBA/1 mice (no CIA induction) and CIA mice treated with 4‐PBA or phosphate buffered saline (PBS) (15 mice/group), as measured by ELISA. (B) Expression of MMPs, TIMPs, and COX‐2 in hind joint extracts of control mice and mice treated with 4‐PBA or PBS, as shown by western blotting.). Values are presented as mean ± SEM of three independent experiments. # p < 0.05 versus DBA/1 without CIA induction; and * p < 0.05 versus CIA treated with PBS

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  FIGURE 7. Effect of 4‐phenylbutyric acid (4‐PBA) on histopathological changes in the tibiotalar joint of collagen‐induced arthritis (CIA) mice. CIA mice were terminated 3 weeks after the second immunization, and the tibiotalar joints obtained from the mice were stained with (A) H&E, Safranin O, and TRAP. The arrows indicate TRAP‐positive osteoclasts. (B) The average histological score of 4‐PBA or phosphate buffered saline (PBS)‐treated CIA mice (15 mice/group) for synovial inflammation, cartilage damage, and erosion. (C) TRAP‐positive osteoclasts were counted using an automated system for quantitative image analysis. Values are presented as mean ± SEM of three independent experiments. H&E, hematoxylin and eosin; TB, tibia; TL, talus; TRAP, tartrate‐resistant acid phosphatase. * p < 0.05 versus CIA treated with PBS