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The active metabolite of leflunomide, A77 1726, attenuates inflammatory arthritis in mice with spontaneous arthritis via induction of heme oxygenase-1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Leflunomide is a low-molecular-weight compound that is widely used in the treatment of rheumatoid arthritis. Although leflunomide is thought to act through the inhibition of the de novo pyrimidine synthesis, the molecular mechanism of the drug remains largely unknown. We investigated the antiarthritis effects and mechanisms of action of the active metabolite of leflunomide, A77 1726, in interleukin-1 receptor antagonist-knockout (IL-1Ra-KO) mice.

Methods: 14- to 15-week-old male IL-1Ra-KO mice were treated with 10 or 30 mg/kg A77 1726 via intraperitoneal injection three times per week for 6 weeks. The effects of A77 1726 on arthritis severities were assessed by clinical scoring and histological analysis. The serum concentrations of IL-1β, tumor necrosis factor-α (TNF-α), and malondialdehyde were measured by enzyme-linked immunosorbent assay. Histologic analysis of the joints was performed using Safranin O, and immunohistochemical staining. The frequencies of interleukin-17-producing CD4+ T (Th17) cells were analyzed by flow cytometry. Heme oxygenase-1 (HO-1) expression in splenic CD4+ T cells isolated from A77 1726-treated arthritis mice were assessed by western blotting.

Results: A77 1726 treatment induced heme oxygenase-1 (HO-1) in Jurkat cells and primary mouse T cells. Interestingly, A77 1726 inhibited Th17 cell differentiation. In vivo, A77 1726 reduced the clinical arthritis severity of histological inflammation and cartilage destruction. The joints isolated from A77 1726-treated mice showed decreased expression of inducible nitric oxide synthase, nitrotyrosine, TNF-α, and IL-1β. The serum levels of TNF-α, IL-1β, and malondialdehyde were also decreased in A77 1726-treated mice. Whereas the number of Th17 cells in spleens was decreased in A77 1726-treated arthritis mice, a significant increase in the number of Treg cells in spleens was observed. Interestingly, HO-1 expression was significantly higher in splenic CD4+ T cells isolated from A77 1726-treated mice compared with those from vehicle-treated mice, whereas HO-1 expression of splenic non-CD4+ T cells did not differ between groups.

Conclusion: The inhibitory effects of A77 1726 on joint inflammation and oxidative stress in autoimmune arthritis may be associated with HO-1 induction in CD4+ T cells.

No MeSH data available.


Related in: MedlinePlus

In vivo therapeutic effect of A77 1726 on the development of inflammatory arthritis in mice. a Arthritis score and incidence of arthritis in interleukin-1 receptor antagonist-knockout (IL-1Ra-KO) mice following treatment with A77 1726 or vehicle (DMSO). A77 1726 dissolved in DMSO was given intraperitoneally to two different groups (10 or 30 mg/kg; n = 5 mice per group) three times per week for 6 weeks, starting after the first A77 1726 treatment. b Representative histological images of joint tissue sections from IL-1Ra-KO mice. Tissue sections from the joints of each mouse on day 42 after the first A771726 or vehicle treatment were stained with hematoxylin and eosin (H&E; original magnification ×40) and Safranin O (original magnification ×200) to examine the severity of arthritis. c Histological scores of inflammation, bone erosion and cartilage damage in mice treated with A77 1726 or vehicle (n = 5 mice per group) are shown. Values are the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 versus vehicle-treated mice
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Fig2: In vivo therapeutic effect of A77 1726 on the development of inflammatory arthritis in mice. a Arthritis score and incidence of arthritis in interleukin-1 receptor antagonist-knockout (IL-1Ra-KO) mice following treatment with A77 1726 or vehicle (DMSO). A77 1726 dissolved in DMSO was given intraperitoneally to two different groups (10 or 30 mg/kg; n = 5 mice per group) three times per week for 6 weeks, starting after the first A77 1726 treatment. b Representative histological images of joint tissue sections from IL-1Ra-KO mice. Tissue sections from the joints of each mouse on day 42 after the first A771726 or vehicle treatment were stained with hematoxylin and eosin (H&E; original magnification ×40) and Safranin O (original magnification ×200) to examine the severity of arthritis. c Histological scores of inflammation, bone erosion and cartilage damage in mice treated with A77 1726 or vehicle (n = 5 mice per group) are shown. Values are the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 versus vehicle-treated mice

Mentions: Based on the in vitro results above, we investigated whether treatment with A77 1726 would suppress the development and severity of arthritis in vivo. A77 1726 (10 or 30 mg/kg) was administered for 6 weeks to 14–15-week-old male spontaneous arthritic IL-1Ra-KO mice. At the beginning of A77 1726 treatment, the mice of all the groups showed the signs of joints inflammation. Mean arthritis scores at that time were 2.3, 3.4 and 4.1 in vehicle-, 10 mg/kg-, and 30 mg/kg-treated groups, respectively. At the beginning of treatment, there was no significant difference in arthritis severities between the three groups. Treatment of mice with A77 1726 at 30 mg/kg, but not at 10 mg/kg, significantly ameliorated the arthritis severity compared with that in the wild-type and vehicle (DMSO)-treated IL-1Ra-KO mice (Fig. 2a). The clinical severity of arthritis was similar in the mice treated with 10 mg/kg of A77 1726 and the vehicle-treated mice. However, histological sections of the ankle joints stained with H&E and Safranin O showed that arthritis was less severe in A77 1726-treated mice (at both 10 and 30 mg/kg) compared with the vehicle-treated controls (Fig. 2b). There was a statistically significant reduction in the inflammation, bone erosion and cartilage damage scores of the mice treated with 10 or 30 mg/kg A77 1726 compared with those of vehicle-treated arthritis mice (Fig. 2c).Fig. 2


The active metabolite of leflunomide, A77 1726, attenuates inflammatory arthritis in mice with spontaneous arthritis via induction of heme oxygenase-1
In vivo therapeutic effect of A77 1726 on the development of inflammatory arthritis in mice. a Arthritis score and incidence of arthritis in interleukin-1 receptor antagonist-knockout (IL-1Ra-KO) mice following treatment with A77 1726 or vehicle (DMSO). A77 1726 dissolved in DMSO was given intraperitoneally to two different groups (10 or 30 mg/kg; n = 5 mice per group) three times per week for 6 weeks, starting after the first A77 1726 treatment. b Representative histological images of joint tissue sections from IL-1Ra-KO mice. Tissue sections from the joints of each mouse on day 42 after the first A771726 or vehicle treatment were stained with hematoxylin and eosin (H&E; original magnification ×40) and Safranin O (original magnification ×200) to examine the severity of arthritis. c Histological scores of inflammation, bone erosion and cartilage damage in mice treated with A77 1726 or vehicle (n = 5 mice per group) are shown. Values are the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 versus vehicle-treated mice
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Related In: Results  -  Collection

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Fig2: In vivo therapeutic effect of A77 1726 on the development of inflammatory arthritis in mice. a Arthritis score and incidence of arthritis in interleukin-1 receptor antagonist-knockout (IL-1Ra-KO) mice following treatment with A77 1726 or vehicle (DMSO). A77 1726 dissolved in DMSO was given intraperitoneally to two different groups (10 or 30 mg/kg; n = 5 mice per group) three times per week for 6 weeks, starting after the first A77 1726 treatment. b Representative histological images of joint tissue sections from IL-1Ra-KO mice. Tissue sections from the joints of each mouse on day 42 after the first A771726 or vehicle treatment were stained with hematoxylin and eosin (H&E; original magnification ×40) and Safranin O (original magnification ×200) to examine the severity of arthritis. c Histological scores of inflammation, bone erosion and cartilage damage in mice treated with A77 1726 or vehicle (n = 5 mice per group) are shown. Values are the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 versus vehicle-treated mice
Mentions: Based on the in vitro results above, we investigated whether treatment with A77 1726 would suppress the development and severity of arthritis in vivo. A77 1726 (10 or 30 mg/kg) was administered for 6 weeks to 14–15-week-old male spontaneous arthritic IL-1Ra-KO mice. At the beginning of A77 1726 treatment, the mice of all the groups showed the signs of joints inflammation. Mean arthritis scores at that time were 2.3, 3.4 and 4.1 in vehicle-, 10 mg/kg-, and 30 mg/kg-treated groups, respectively. At the beginning of treatment, there was no significant difference in arthritis severities between the three groups. Treatment of mice with A77 1726 at 30 mg/kg, but not at 10 mg/kg, significantly ameliorated the arthritis severity compared with that in the wild-type and vehicle (DMSO)-treated IL-1Ra-KO mice (Fig. 2a). The clinical severity of arthritis was similar in the mice treated with 10 mg/kg of A77 1726 and the vehicle-treated mice. However, histological sections of the ankle joints stained with H&E and Safranin O showed that arthritis was less severe in A77 1726-treated mice (at both 10 and 30 mg/kg) compared with the vehicle-treated controls (Fig. 2b). There was a statistically significant reduction in the inflammation, bone erosion and cartilage damage scores of the mice treated with 10 or 30 mg/kg A77 1726 compared with those of vehicle-treated arthritis mice (Fig. 2c).Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Background: Leflunomide is a low-molecular-weight compound that is widely used in the treatment of rheumatoid arthritis. Although leflunomide is thought to act through the inhibition of the de novo pyrimidine synthesis, the molecular mechanism of the drug remains largely unknown. We investigated the antiarthritis effects and mechanisms of action of the active metabolite of leflunomide, A77 1726, in interleukin-1 receptor antagonist-knockout (IL-1Ra-KO) mice.

Methods: 14- to 15-week-old male IL-1Ra-KO mice were treated with 10 or 30&nbsp;mg/kg A77 1726 via intraperitoneal injection three times per week for 6&nbsp;weeks. The effects of A77 1726 on arthritis severities were assessed by clinical scoring and histological analysis. The serum concentrations of IL-1&beta;, tumor necrosis factor-&alpha; (TNF-&alpha;), and malondialdehyde were measured by enzyme-linked immunosorbent assay. Histologic analysis of the joints was performed using Safranin O, and immunohistochemical staining. The frequencies of interleukin-17-producing CD4+ T (Th17) cells were analyzed by flow cytometry. Heme oxygenase-1 (HO-1) expression in splenic CD4+ T cells isolated from A77 1726-treated arthritis mice were assessed by western blotting.

Results: A77 1726 treatment induced heme oxygenase-1 (HO-1) in Jurkat cells and primary mouse T cells. Interestingly, A77 1726 inhibited Th17 cell differentiation. In vivo, A77 1726 reduced the clinical arthritis severity of histological inflammation and cartilage destruction. The joints isolated from A77 1726-treated mice showed decreased expression of inducible nitric oxide synthase, nitrotyrosine, TNF-&alpha;, and IL-1&beta;. The serum levels of TNF-&alpha;, IL-1&beta;, and malondialdehyde were also decreased in A77 1726-treated mice. Whereas the number of Th17 cells in spleens was decreased in A77 1726-treated arthritis mice, a significant increase in the number of Treg cells in spleens was observed. Interestingly, HO-1 expression was significantly higher in splenic CD4+ T cells isolated from A77 1726-treated mice compared with those from vehicle-treated mice, whereas HO-1 expression of splenic non-CD4+ T cells did not differ between groups.

Conclusion: The inhibitory effects of A77 1726 on joint inflammation and oxidative stress in autoimmune arthritis may be associated with HO-1 induction in CD4+ T cells.

No MeSH data available.


Related in: MedlinePlus