Vitamin D improves autoimmune diseases by inhibiting Wnt signaling pathway

Abstract

Objective: In this study, we investigated the development of the Wnt signaling pathway in vitamin D (VitD) to improve systemic lupus erythematosus in mice to breakthrough clinical treatment approaches.

Methods: Body weight changes were recorded during rearing. Antinuclear antibodies (ANA), anti-dsDNA, and anti-snRNP were detected in the mouse serum using an enzyme-linked immunosorbent assay. Apoptosis of Th1 and Th2 immune cells in mice was detected using flow cytometry. Reverse transcription polymerase chain reaction was used to detect the expression of T-bet, GATA3, and Wnt3a mRNA in the spleens of each group. Western blot analysis was performed to detect the expression of Wnt1, p-β-catenin, β-catenin, glycogen synthase kinsase3β (GSK-3β), Wnt3a, c-myc, and cyclin D1 protein in mice spleens. β-catenin in mice spleen was visualized using immunohistochemistry.

Results: VitD did not substantial reduce the body weight of MRL/LPR mice, whereas the inhibitor did. VitD notably decreased the concentrations of ANA, anti-double-stranded DNA, and anti-snRNP in the serum of MRL/LPR mice and alleviated apoptosis of Th1 and Th2 cells. VitD markedly increased the expression of T-bet and GATA mRNA in the spleen of MRL/LPR mice and consequently increased the levels of Wnt3a and β-catenin. Western blot analysis revealed that the levels of GSK-3β, p-β-catenin, Wnt1, Wnt3a, c-myc, and cyclin D1 could be reduced by VitD, compared with MRL/LPR. Immunohistochemistry demonstrated that the expression of β-catenin was the most pronounced in the spleen of MRL/LPR mice, and the expression level of β-catenin decreased substantially after VitD intervention.

Conclusions: VitD can further inhibit the nuclear translocation of β-catenin by downregulating the expression of Wnt ligands (Wnt1 and Wnt3a), which reduces the expression of the downstream target gene cyclin D1. Systemic lupus erythematosus in mice was improved by inhibiting the activation of Wnt/β-catenin signal pathway.

Keywords: Wnt/β-catenin signal pathway; autoimmune diseases; vitamin D.

Figure 1

Both vitamin D (VitD) and Wnt pathway inhibitors inhibited autoantibody overexpression in MRL/LPR mice. (A) Changes in body weight after VitD and Wnt pathway inhibitor administration. (B) The concentrations of antinuclear antibodies, antidouble‐stranded DNA, and anti‐SNRPN in the serum were detected using enzyme‐linked immunosorbent assay. Data are presented as the mean ± SD. One‐way analysis of variance was used to compare the data differences among multiple groups, and Tukey test was used for post hoc analysis. *p < .05 indicates a statistical difference between two groups. The sample size for each group was n = 5.

Figure 2

Vitamin D (VitD) and Wnt pathway inhibitors can effectively reverse the proportion of Th1 and Th2 cells and the expression of transcription factors in the spleen of MRL/LPR mice. (A) The levels of of Th1 and Th2 cells in spleen of mice were detected using flow cytometry. (B) Reverse transcription polymerase chain reaction was used to detect the expression of T‐bet, GATA3, Wnt3a, and β‐catenin mRNA in spleen. Data are presented as the mean ± SD. One‐way analysis of variance was used to compare the data differences among multiple groups, and Tukey test was used for post hoc analysis. *p < .05 indicates a statistical difference between two groups. The sample size for each group was n = 5.

Figure 3

Vitamin D (VitD) effectively reduced the expression of Wnt pathway related proteins in MRL/LPR mice. The expression of Wnt1, p‐catenin, GSK‐3β, c‐myc, cyclin D1, and Wnt3a proteins in spleen of MRL/LPR mice was detected using western blot analysis. Data are presented as the mean ± SD. One‐way analysis of variance was used to compare the differences among multiple groups, and Tukey test was used for post hoc analysis. *p < .05 indicates a statistical difference between two groups. The sample size for each group was n = 5.

Figure 4

Vitamin D (VitD) and Wnt pathway inhibitor effectively decreased β‐catenin expression in spleen cells. β‐catenin in mouse spleen was visualized using immunohistochemistry. Data are presented as the mean ± SD. One‐way analysis of variance was used to compare the differences among multiple groups, and Tukey test was used for post hoc analysis. *p < .05 indicates a statistical difference between the two groups. The sample size for each group was n = 5. Scale bar = 50 μm.