Punicalagin Exerts Protective Effects against Ankylosing Spondylitis by Regulating NF- κ B-TH17/JAK2/STAT3 Signaling and Oxidative Stress

Abstract

Background: Ankylosing spondylitis (AS) is a chronic inflammatory disease characterized by sacroiliitis and spinal rigidity of the axial joints. The role of oxidative stress and increased proinflammatory cytokines is well documented in AS pathogenesis. Punicalagin (2,3-hexahydroxydiphenoyl-gallagyl-D-glucose), an ellagitannin widely present in pomegranates, is found to exhibit potent anti-inflammatory, antiproliferative, and antioxidative effects. The present study was undertaken to investigate the effects of punicalagin in a rodent model of AS.

Methods: BALB/c mice induced spondylitis were sacrificed 24 h after the last injection of proteoglycan extract. Histological scoring was done to assess the degree of the disease. The expression of JAK2/STAT3 proteins and proteins of the nuclear factor-κB (NF-κB) pathway was determined by immunoblotting. Serum levels of inflammatory mediators-TNF-α, IL-1β, IL-6, IL-17A, and IL-23-were assessed. Levels of lipid peroxidation and reactive oxygen species (ROS) were quantified. Antioxidant status as a measure of activities of antioxidant enzymes-catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD)-was determined.

Results: Punicalagin effectively improved antioxidant status and decreased lipid peroxidation, ROS production, and serum levels of inflammatory mediators. NF-κB pathway and JAK2/STAT3 signaling were significantly (p < 0.05) downregulated. Punicalagin effectively regulated the production of cytokines by the Th17 cells and the IL-17A/IL-23 axis.

Conclusion: The observations suggest that punicalagin exerts a protective role in AS via reducing oxidative stress and regulating NF-κB/TH17/JAK2/STAT3 signal. Punicalagin thus could be explored further as a potent candidate compound in the treatment of AS.

Figure 1

Punicalagin reduces peripheral disease progression. The results are presented as the mean ± SD (n = 6). # represents p < 0.05 vs. AS control. A–E represent the mean from different treatment groups that differ at p < 0.05.

Figure 2

Punicalagin reduced IVD damage progression. The results are presented as the mean ± SD (n = 6). # represents p < 0.05 vs. AS control. A–E represent the mean values of experimental groups that differ at p < 0.05.

Figure 3

Punicalagin reduced ROS generation following AS induction (a) and MDA levels (b). The results are presented as the mean ± SD (n = 6). ∗ represents p < 0.05 vs. normal control; # represents p < 0.05 vs. AS control. A–E represent the mean values from the experimental groups that differ at p < 0.05.

Figure 4

Punicalagin enhances the activities of antioxidant enzymes: SOD (a), CAT (b), and GPx (c). The results are presented as the mean ± SD (n = 6). ∗ represents p < 0.05 vs. normal control; # represents p < 0.05 vs. AS control. A–E represent the mean values from different experimental groups that differ at p < 0.05.

Figure 5

Punicalagin regulates NF-κB signaling. Punicalagin downregulated activation of NF-κB (a, b) and inhibitor kinases of NF-κB signaling following AS (a, c). Representative immunoblot. (a) Expression levels of test proteins relative of control expressions set at 100% (the expression of phosphorylated proteins was normalized by respective nonphosphorylated protein expression). The results are presented as the mean ± SD (n = 6). ∗ represents p < 0.05 vs. normal control; # represents p < 0.05 vs. AS control. A–E represent the mean values from different experimental groups that differ at p < 0.05. L1: control; L2: AS control; L3: punicalagin (12.5 mg/kg body weight)+AS; L4: punicalagin (25 mg/kg body weight)+AS; L5: punicalagin (50 mg/kg body weight)+AS; L6: punicalagin (50 mg/kg body weight).

Figure 6

Effects of punicalagin on serum levels of inflammatory mediators. Punicalagin reduced the levels of inflammatory cytokines (a), the activity of Th17 cells (b), and the levels of NO (c). The results are represented as the mean ± SD (n = 6). ∗ represents p < 0.05 vs. normal control; # represents p < 0.05 vs. AS control. A–E represent the mean values from different experimental groups that differ at p < 0.05.

Figure 7

Punicalagin regulated the JAK2/STAT3 signal. Representative immunoblot. (a) Expressions of test proteins relative to control expressions set at 100% (the expression of phosphorylated proteins was normalized by respective nonphosphorylated protein expression). The results are represented as the mean ± SD (n = 6). ∗ represents p < 0.05 vs. control; # represents p < 0.05 vs. AS control. A–E represent the mean values from different experimental groups that differ at p < 0.05. L1: control; L2: AS control; L3: punicalagin (12.5 mg/kg body weight)+AS; L4: punicalagin (25 mg/kg body weight)+AS; L5: punicalagin (50 mg/kg body weight)+AS; L6: punicalagin (50 mg/kg body weight).