1,25-(OH)2D3/Vitamin D receptor alleviates systemic lupus erythematosus by downregulating Skp2 and upregulating p27

Abstract

Background: Recent evidence has suggested that the 1,25(OH)₂D₃/Vitamin D receptor (VDR) acts to suppress the immune response associated with systemic lupus erythematosus (SLE), a serious multisystem autoimmune disease. Hence, the aim of the current study was to investigate the mechanism by which 1,25-(OH)₂D₃/VDR influences SLE through regulating the Skp2/p27 signaling pathway.

Methods: Initially, the levels of 1,25(OH)₂D₃, VDR, Skp2, and p27 were measured in collected renal tissues and peripheral blood. Meanwhile, the levels of inflammatory factors, biochemical indicators (BUN, Cr, anti-nRNP IgG, anti-dsDNA IgG) and urinary protein levels were assayed in in VDRinsert and VDR-knockout mice in response to 1,25(OH)₂D₃ supplement. In addition, the distribution of splenic immune cells was observed in these mice.

Results: Among the SLE patients, the levels of 1,25(OH)₂D₃, VDR and p27 were reduced, while the levels of Skp2 were elevated. In addition, the levels of anti-nRNP IgG and anti-dsDNA IgG were increased, suggesting induction of inflammatory responses. Notably, 1,25(OH)₂D₃/VDR mice had lower concentrations of BUN and Cr, urinary protein levels, precipitation intensity of the immune complex and complement, as well as the levels of anti-nRNP IgG and anti-dsDNA IgG in SLE mice. Additionally, 1,25(OH)₂D₃ or VDR reduced the degree of the inflammatory response while acting to regulate the distribution of splenic immune cells.

Conclusion: This study indicated that 1,25-(OH)₂D₃/VDR facilitated the recovery of SLE by downregulating Skp2 and upregulating p27 expression, suggesting the potential of 1,25-(OH)₂D₃/VDR as a promising target for SLE treatment.

Keywords: 1,25(oh)2D3; Inflammatory factors; Renal injury; Skp2; Systemic lupus erythematosus; Vitamin D receptor; p27.

Fig. 1

Incipient SLE patients exhibit reduced Treg cell proportion. The data were analyzed using independent sample t-test. * p < 0.05 vs healthy controls. SLE, systemic lupus erythematosus. Treg, regulatory T cells

Fig. 2

1,25(OH)₂D₃ and VDR are negatively correlated with Skp2 and positively correlated with p27 in SLE patients. a comparison of the 1,25-(OH)₂D₃ content in incipient SLE patients and healthy controls. b mRNA expression of VDR, p27 and Skp2. c correlation between 1,25(OH)₂D₃ and Skp2 expression. d correlation between 1,25(OH)₂D₃ and p27 expression. e correlation of VDR and Skp2 expression. f correlation between VDR and p27 expression. n = 149 for SLE patients and n = 150 for healthy controls; the independent sample t-test was performed to analyze data; * p < 0.05 vs. the healthy controls; VDR, vitamin D receptor; Skp2, S-phase kinase-associated protein 2; SLE, systemic lupus erythematosus

Fig. 3

1,25(OH)₂D₃/VDR alleviates SLE symptoms of SLE mice. a body weight of mice in each group at 0–24 W. b death rate of mice in each group at 0–24 W. n = 20, two-way analysis of variance was performed to analyze data. * p < 0.05 vs. the control group; # p < 0.05 vs. the SLE group; SLE, systemic lupus erythematosus; VDR, vitamin D receptor; W, week

Fig. 4

HE staining mouse renal tissues reflecting the effect of 1,25(OH)₂D₃/VDR on pathological changes (× 400). HE staining of renal tissues in each group; SLE, systemic lupus erythematosus; HE, hematoxylin and eosin; VDR, vitamin D receptor

Fig. 5

PAS staining of mouse renal tissues reflecting the effect of 1,25(OH)₂D₃/VDR on pathological changes of renal tissues of SLE mice (× 400). SLE, systemic lupus erythematosus; PAS, periodic acid Schiff; VDR, vitamin D receptor

Fig. 6

1,25(OH)₂D₃/VDR diminishes the precipitation intensity of the immune complex IgG in SLE mice. a the immunofluorescence staining (× 400) of IgG. b the quantitative analysis of fluorescence intensity of IgG in each group. n = 20, one-way analysis of variance was used to analyze data; * p < 0.05 vs. the control group; # p < 0.05 vs. the SLE group; paired t-test was performed to analyze data; $ p < 0.05 vs. 0 W; SLE, systemic lupus erythematosus; VDR, vitamin D receptor; W, week; IgG, immunoglobulin G

Fig. 7

1,25(OH)₂D₃/VDR reduces the precipitation intensity of the immune complex IgA in SLE mice. a the immunofluorescence staining (× 400) of IgA. b the quantitative analysis of fluorescence intensity of IgA in each group. n = 20, one-way analysis of variance was used to analyze data; * p < 0.05 vs. the control group; # p < 0.05 vs. the SLE group; paired t-test was performed to analyze data; $ p < 0.05 vs. 0 W; SLE, systemic lupus erythematosus; VDR, vitamin D receptor; W, week; IgA, immunoglobulin A

Fig. 8

1,25(OH)₂D₃/VDR reduces the precipitation intensity of the immune complex IgM in SLE mice. a the immunofluorescence staining (× 400) of IgM. b the quantitative analysis of fluorescence intensity of IgM in each group. n = 20, one-way analysis of variance was applied to analyze the data; * p < 0.05 vs. the control group; # p < 0.05 vs. the SLE group; paired t-test was performed to analyze data; $ p < 0.05 vs. 0 W; SLE, systemic lupus erythematosus; VDR, vitamin D receptor; W, week; IgM, immunoglobulin M

Fig. 9

1,25(OH)₂D₃/VDR reduces the precipitation intensity of the complement C₃ in SLE mice. a the immunofluorescence staining (× 400) of C₃. b the quantitative analysis of fluorescence intensity of C₃ in each group. n = 20, one-way analysis of variance was used to analyze data; * p < 0.05 vs. the control group; # p < 0.05 vs. the SLE group; paired t-test was performed to analyze data; $ p < 0.05 vs. 0 W; SLE, systemic lupus erythematosus; VDR, vitamin D receptor; W, week

Fig. 10

1,25(OH)₂D₃/VDR reduces the precipitation intensity of the complement C_1q in SLE mice. a the immunofluorescence staining (× 400) of C_1q. b the quantitative analysis of fluorescence intensity of C_1q in each group. n = 20, one-way analysis of variance was used to analyze data; * p < 0.05 vs. the control group; # p < 0.05 vs. the SLE group; paired t-test was performed to analyze data; $ p < 0.05 vs. 0 W; SLE, systemic lupus erythematosus; VDR, vitamin D receptor; W, week

Fig. 11

1,25(OH)₂D₃/VDR regulates the distribution of splenic immune cells (Treg, Th17, Th1, Th2, and CD4-CD8-DN) in SLE mice (%). n = 20, one-way analysis of variance was used to analyze data; * p < 0.05 vs. the control group; # p < 0.05 vs. the SLE group; t-test was conducted; $ p < 0.05 vs. 0 W; SLE, systemic lupus erythematosus; VDR, vitamin D receptor; W, week; Treg, regulatory T; Th17, T helper 17; Th1, T helper 1; Th2, helper 2

Fig. 12

1,25-(OH)₂D₃/VDR downregulates the Skp2 expression and upregulates the p27 expression in SLE mice. a the mRNA expression of Skp2 and p27 determined by RT-qPCR. b the protein expression of Skp2 and p27 measured by western blot analysis. c the statistical analysis of panel B; n = 20, one-way analysis of variance was used to analyze data; * p < 0.05 vs. the control group; # p < 0.05 vs. the SLE group; Skp2, S-phase kinase-associated protein 2; SLE, systemic lupus erythematosus; RT-qPCR, reverse transcription quantitative polymerase chain reaction