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

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.


Background
Systemic lupus erythematosus (SLE) is a chronic multifactorial autoimmune disease, which involves genetic and environmental factors [1]. SLE is characterized by the production of antinuclear autoantibodies that form immune complexes in different organs, including the skin, joints, kidneys and brain, consequently promoting a chronic inflammatory response [2,3]. A prior study has highlighted estrogen as a contributing factor accounting for the ten times higher incidence of SLE in women, particularly during the reproductive years [4]. Otherwise, the etiological factors of the disease still remain unclear, while endocrine, genetic and environmental factors such as drugs, smoking, ultraviolet radiation, infectious agents, as well as various exposure to chemical agents have been suggested to be potential risk factors [5]. SLE patients often display a wide range of clinical symptoms, ranging from mild joint and skin problems to severe multisystem dysfunction manifesting in renal, neurologic as well as cardiovascular disorders [6]. Therefore, it is necessary to further develop a better understanding of SLE for its early detection and improved treatment.
Vitamin D transmits signals to target cells through the vitamin D receptor (VDR), which is expressed in all immune cells, has been validated to exert a variety of immunomodulatory effects on the prevention of autoimmune diseases [7,8]. Furthermore, 1,25-(OH) 2 D 3 , the active form of vitamin D in vivo has been reported to suppress cell proliferation [9]. Studies have demonstrated that regulatory T cells (Tregs) play a critical role against the progression of SLE symptoms because of their immune-suppressive functions [10,11]. However, Skp2 affects cell cycle control and cell death, and its overexpression could decrease the suppressive effect of Tregs in the progression of autoimmune disorders [12]. Deficiencies of p27, a cell cycle inhibitor, not only reduce the activity and quantity of Tregs, but also induce lupus-like abnormalities [13]. Both Skp2 and p27 play vital roles in mediating the migration, proliferation and invasion of tumor cells as well as balancing immune tolerance in the development of autoimmune diseases [12,14], while 1,25-(OH) 2 D 3 is reported to reduce Skp2 protein expression in metastatic prostate cancer cells [15]. Recent evidence has indicated that the 1,25(OH) 2 D 3 analogs mediate the upregulation of p27 in head and neck squamous carcinoma cells by downregulating Skp2, which leads to p27 stabilization [16]. Mycophenolate mofetil, an inhibitor of guanosine nucleotide synthesis in lymphocytes is useful for the symptomatic treatment of SLE, but there is no effective cure [17], and other drugs, such as corticosteroids, azathioprine, and hydroxychloroquine failing to adequately decrease the risk of organspecific disease [18]. Thus, the central objective of the current study was to investigate the potential role of 1, 25-(OH) 2 D 3 /VDR in the progression of SLE by regulating the Skp2/p27 signaling pathways.

Study population
One hundred and forty-nine patients with incipient SLE were enrolled from March 2015 to April 2016 from the Clinical Medical College, Yangzhou University and Yangzhou First People's Hospital, including 144 females and 5 males with a mean age of 32.72 ± 8.56 years (ranging from 11 to 74 years). All patients conformed to the diagnostic criteria for SLE of the American College of Rheumatology's rheumatoid arthritis electronic clinical quality measures [19] revised in 1997. The exclusion criteria were as follows: 1) patients taking potentially interfering medication such as sex hormones or calcitriol; 2) artificially reducing the interference of light (lightdependent vitamin D formation); and 3) abnormal thyroid function, diabetes, tumors and other autoimmune diseases. In addition, 150 healthy controls were selected. The health controls were selected from people undergoing physical examination at Clinical Medical College, Yangzhou University and Yangzhou First People's Hospital, with all participants well informed regarding the objectives of the experiment. There were no significant differences in gender and age between the incipient SLE patients and healthy controls. Peripheral blood samples and renal tissues from all subjects were collected, with the collected blood samples analyzed using a Japan MEK-7222 automatic blood analyzer within 1 to 4 h. The demographics data of the included subjects is depicted in Table 1.

Separation of CD4 + T cells
The collected samples were subjected to density gradient centrifugation on a Ficoll-isopaque (Lymphoprep). The residue of the brown-yellow layer of leukocytes was removed from the samples, and the peripheral blood mononuclear cells (PBMCs) were separated. CD25 + cells with removal of CD4 + T cells were used throughout the study to avoid the potential inhibition of CD25 + proliferation by CD4 + cells. We used a CD4 + CD25 + regulatory T cell isolation kit (130-091-301, Miltenyi Biotech, Bergisch Gladbach, Germany) to isolate CD4 + CD25 − T cells from PBMCs by negative selection, based on the manufacturer's instructions. The protein expression of Skp2 and p27 in isolated CD4 + CD25 − T cells was detected by western blot analysis.

Specimen collection
During the 8th, 16th and 24th weeks of treatment, the mice were weighed and anesthetized with 3% pentobarbital sodium (30 mL/kg, Sigma-Aldrich Chemical Company, St. Louis MO, USA) dissolved in normal saline (Wuhan Boster Biological Technology Co., Ltd., Wuhan, Hubei, China) via an intraperitoneal injection. Next, venous blood was collected in heparinized tubes for biochemical assays. The bilateral kidneys of each mouse were removed, with one portion weighed and used for assay of inflammatory factors by reverse transcription quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA) and western blot analysis. The remaining part of the kidney was fixed by immersion with neutral formalin solution, dehydrated using gradient alcohol, cleared by xylene and embedded in paraffin (Thermo Fisher Scientific, San Jose, CA, USA). Next, 5-μm kidney tissue slices were prepared for histopathological observation.

ELISA assay
After dissolving standards, 100 μL portions were added to draw a standard curve on the instructions of the ELISA kit (eBioscience, San Diego, CA, USA). The samples to be tested (100 μL) were added to the wells for a preincubation at 37°C for 90 min. The samples were incubated successively with 100 μL biotinylated antibody for 60 min at 37°C, after which 100 μL enzyme conjugates were added under subdued light for a further 30-min incubation at 37°C, followed by the addition of 100 μL substrate for a further 15-min incubation at 37°C in the dark, whereupon the termination buffer was added. The optical density (OD) value of each tube at 450 nm was determined using a multi-

RT-qPCR
The RNA of renal tissues and CD4 + T cells was extracted using the TRIZOL method (Invitrogen, Inc., Carlsbad, CA, USA). The concentration and purity of the collected RNA were measured using a NanoDrop2000 (Thermo Fisher Scientific Inc., Waltham, MA, USA). According to the gene sequences published in the GenBank database, PCR primers were designed using Primer 5.0 primerdesign software ( Table 2) and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). RT-qPCR was conducted on the ABI PRISM 7500 real-time PCR System (ABI Company, Oyster Bay, NY, USA). The reliability of the PCR results was evaluated using a dissolution curve, with the CT value subsequently obtained (the inflection point of amplification curve). The fold changes between the experiment group and the control group were determined based on the relative quantification 2-△△Ct method [20]. Data are mean values from three independent experiments.

Western blot analysis
The proteins from the renal tissues of mice were extracted and analyzed with a bicinchoninic acid kit (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China). The extracted protein samples were separated using 10% polyacrylamide gel electrophoresis (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China), followed by transferring onto a polyvinylidene fluoride membrane. The membrane was blocked using 5% bovine serum albumin (BSA) for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies to p-Skp2 (

Determination of biochemical indicators
The Coomassie brilliant blue method was applied to detect the urine protein content. The day prior to the end of the experiment (at the end of the 24th week), the mice were placed in metal metabolism cages for urine collection, followed by a centrifugation at 6000×g for 20 min and stored at − 80°C. The urine protein was detected in accordance with the instructions of the kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). The BSA standard curve was subsequently plotted, followed by dilution of the supernatant with normal saline at a ratio of 1: 10. After the sample to be assessed was mixed with Coomassie Brilliant Blue dye at a ratio of 1: 10, the OD value at a wavelength of 540 nm was measured in an automatic microplate reader (Labsystems Dargon). After final urine collection, blood was collected retroorbitally and subsequently stored for the assay of blood urea nitrogen (BUN) and serum creatinine (Cr) detection. The continuous monitoring method for urease was carried out in order to determine BUN. The reagents (10 μL) were mixed with 10 μL reagent provided in the kit (Rongsheng Biotech Company Co., Ltd., Shanghai, China) for the prompt colorimetric examination using a spectrophotometer (Thermo Fisher Scientific, San Jose, CA, USA). The absorbance values at 30 s (A1) and 90 s (A2) were then read to calculate BUN content using the formula: BUN (mmol/L) = (sample A2 -sample A1) / (blank A2 -blank A1) × (the concentration of the reagent). The content of serum Cr was detected using the picric acid method. Here, urine samples (100 μL) were mixed with an equal volume of the reagent provided in the kit (Rongsheng Biotech Company Co., Ltd., Shanghai, China) and water-bathed for 30 s at 37°C. The absorbance values at 30 s (A1) and 90 s (A2) at 505 nm were then determined, and the concentration of Cr was calculated as follows: Cr (μmol/ L) = (sample A2 -sample A1)/(blank A2 -blank A1) × (the concentration of the reagent). Data are mean values from three independent experiments.

Hematoxylin and eosin (HE) staining and periodic acid Schiff (PAS) staining
The renal tissues were embedded in paraffin, dewaxed with xylene and hydrated with gradient alcohol (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China). Then, the sections were stained with hematoxylin-eosin and PAS, and images of the section were captured under a microscope (CX31, Olympus Optical Co., Ltd., Tokyo, Japan).

Immunofluorescence
Immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), complement 3 (C3) and complement (C1q) were labeled using fluorescein isothiocyanate (FITC). The renal tissue sections were dewaxed, treated with 3% catalase for 10 min for antigen retrieval, processed with pepsin for 10 min and sealed with fetal bovine serum (Gibco Company, Grand Island, NY, USA) for 1 h at 37°C. Next, the sections were incubated with corresponding fluorescent antibodies against IgG (1: 100), IgA (1:100), IgM (1:80), C3 (1:50) and C1q (1:50) (all of the antibodies and reagents were purchased from DAKO Company, Denmark) at 37°C for 45 min. The sections were then washed and sealed with a buffered glycerol solution (DAKO Company, Denmark), whereupon the precipitation intensity of the immune complex was observed and photographed. The experiment was repeated three times.

Statistical analysis
Data were analyzed using SPSS 21.0 statistical software (IBM Corp. Armonk, NY, USA). Measurement data were expressed as mean ± standard deviation. Data between two groups were compared using t-test when it conforms to the normal distribution, while those among multiple groups were compared by means of one-way analysis of variance. Enumeration data were expressed as either a percentage or rate and examined using a Chi-square test. Pearson correlation analysis was applied to determine the correlation between two variables. p < 0.05 was considered to be indicative of significant difference.

Results
A previous study concluded that similar SLE lesions appeared in VDR-knockout mice. Thus, SLE VDR-insert mice were selected for the purposes of the study to investigate the role of the overexpression of VDR in the alleviation of SLE in mice [21].

Peripheral blood of SLE patients exhibits reduced hemoglobin and platelets
SLE is predominately associated with immunopathogenesis. As such, altered parameters of peripheral blood are associated with the progression and diagnosis of SLE. Compared to healthy controls, the white blood cell count, red blood cell count and the mean platelet volume (MPV) of incipient SLE patients did not significantly differ, while the hemoglobin as well as the platelet count detected were significantly smaller than in controls (p < 0.05; Table 3).
The results suggested there to be a lower level of hemoglobin and fewer platelets among SLE patients.

Treg cell proportion is reduced in incipient SLE patients
CD4 + and CD25 + cells were isolated from the peripheral blood of incipient SLE patients and healthy controls in order to determine Treg cell proportion. The results obtained indicated that the Treg cell proportion in peripheral blood of incipient SLE patients was reduced when compared with that in peripheral blood of healthy controls (p < 0.05; Fig. 1).
1,25(OH) 2 D 3 and VDR are negatively correlated with Skp2 and positively correlated with p27 in SLE patients The expression of 1,25(OH) 2 D 3 , VDR, Skp2 and p27 in renal tubular cells of incipient SLE patients and healthy controls was determined using RT-qPCR. Compared with healthy controls, the Skp2 mRNA expression in the renal tubular cells of SLE patients was significantly higher, while the mRNA expression of 1,25-(OH) 2 D 3 , VDR and p27 was significantly lower (p < 0.05; Fig. 2a (Fig. 2c-f). These results indicated that 1,25(OH) 2 D 3 /VDR downregulated Skp2 expression and upregulated p27 expression.

1,25(OH) 2 D 3 /VDR alleviates SLE symptoms of SLE mice
The body weight of the mice in the control group progressively increased, and the animals showed no signs of skin ulcers, erosion or urinary system infection. No animals died during the follow-up interval. Compared with the control group, mice in the VDR −/− group displayed symptoms of loose hair, skin ulcers and erosion, urinary systemic infections, and progressive body weight loss (p < 0.05). Aggravated skin ulcerations and lower body weights of SLE mice worsened with time compared with mice at 0 W (p < 0.05). The symptoms of skin ulceration and erosion, infection and weight loss of mice in the SLE + VD 3 group were all less pronounced compared to the mice in the SLE group (p < 0.05). Mice in the SLE + VD 3 + VDRinsert group exhibited distinctly improved symptoms in the hair, skin and the urinary tract, exceeding those of the SLE + VD 3 group. Compared with the SLE group, the weight of the SLE + VD 3 + VDRinsert mice was significantly greater (p < 0.05). There was no significant difference in weight between the SLE + VD 3 + VDRinsert group and the control group ( Fig. 3a-b). Thus, 1,25(OH)2D3/ VDR rescued SLE mice from pathology.

1,25(OH) 2 D 3 /VDR reduces the contents of BUN and Cr and urinary protein levels in SLE mice
The Coomassie brilliant blue method was used to detect the contents of BUN and Cr and urinary protein levels. At 0 W, the contents of BUN and Cr and urinary protein levels of SLE mice (in the SLE, SLE + VD 3 and SLE + VD 3 + VDRinsert groups) were significantly higher than those of control mice (p < 0.05), while the mice in the VDR −/− group showed no significant differences in these markers (p > 0.05). Compared with 0 W, the contents of BUN, and Cr and urinary protein levels of control mice had not changed by W 24 (p > 0.05), while these indexes in mice of the VDR −/− group were significantly increased (p < 0.05). These indexes in the SLE mice had slightly increased by W 24, but the markers in the SLE + VD 3 and SLE + VD 3 + VDRinsert groups had diminished significantly by W 24 (p < 0.05). At 24 W, contents of BUN and Cr and urinary protein levels of mice in the VDR −/− group were significantly elevated relative to baseline (p < 0.05). In comparison with the SLE group, the contents of BUN and Cr and urinary protein levels of mice in the SLE + VD 3 and SLE + VD 3 + VDRinsert groups were significantly reduced at week 24 (p < 0.05) with the contents in the mice in the SLE + VD 3 + VDRinsert group even decreased to the normal level (Table 4). These results indicated that 1,25(OH) 2 D 3 /VDR reduced the elevations of BUN and Cr and urinary protein levels otherwise seen in SLE mice.

1,25(OH) 2 D 3 /VDR reduces the precipitation intensity of the immune complex (IgG, IgA, IgM) and complement (C 3 , C 1q ) in SLE mice
Immunopathological changes and the precipitation intensity of the immune complex of mice in each group were observed via immunofluorescence staining. At 0 W, compared with the control and VDR −/− groups, the intensity of immune proteins and complement depositions of SLE mice displayed an obvious "full house" phenomenon, characterized by positive staining for IgG, IgA, IgM, C 3 , and C 1q . At week 24, the deposition intensity of the immune complex (IgG, IgA, IgM) and complement (C 3 , C 1q ) of mice in the control group had not changed significantly (p > 0.05). The deposition intensity of these immunological markers of mice in the VDR −/− group was   The results obtained indicated that at 0 W, compared with mice in the control and VDR −/− groups, the IL-4, IL-10, IL-17 and INF-γ levels of SLE mice (in the SLE, SLE + VD 3 , and SLE + VD 3 + VDRinsert groups) were significantly increased (p < 0.05). At the 24th week, the levels of these factors in mice in the control group had not changed significantly (p > 0.05). Compared with the control group, the levels of these factors in mice in the VDR −/− group were significantly elevated (p < 0.05). Compared with the SLE group, the levels of these factors in mice in the SLE + VD 3 and SLE + VD 3 + VDRinsert groups were significantly lower (p < 0.05). By the 24th week, there was no significant difference identified in the IL-4, IL-10, IL-17 and INF-γ levels between the SLE + VD 3 + VDRinsert and control groups (Table 5).

1,25(OH) 2 D 3 /VDR decreases the levels of anti-nRNP IgG and anti-dsDNA IgG in SLE mice
ELISA was applied in order to detect the levels of anti-nRNP IgG and anti-dsDNA IgG in SLE mice. Based on the ELISA results, before treatment (0 W), the levels of anti-nRNP IgG and anti-dsDNA IgG in SLE mice (in the SLE, SLE + VD 3 , SLE + VD 3 + VDRinsert groups) were profoundly upregulated compared with the levels in the control and VDR −/− groups (p < 0.05). With 24 W treatment, the levels of anti-nRNP IgG and anti-dsDNA IgG in the control group displayed no significant difference relative to baseline (p > 0.05), while the levels of anti-nRNP IgG and anti-dsDNA IgG in the VDR −/− group were elevated when compared with those in the control group (p < 0.05). Compared with the SLE group, the levels of anti-nRNP IgG and anti-dsDNA IgG were significantly downregulated in the SLE + VD 3 and SLE + VD 3 + VDRinsert groups after treatment (p < 0.05), while the SLE + VD 3 + VDRinsert group displayed no significant difference in the antibody levels versus control mice at 24 W (Table 6). These findings demonstrated that 1, 25(OH) 2 D 3 /VDR decreased the levels of anti-nRNP IgG and anti-dsDNA IgG in SLE mice.

1,25(OH) 2 D 3 /VDR regulates splenic immune cells in SLE mice
Flow cytometry analysis showed that the numbers of T helper cells (Th17, Th1, Th2) and CD4 − CD8 − DN cells were significantly increased in the SLE mice (in the SLE, SLE + VD 3 , SLE + VD 3 + VDRinsert groups) when compared with numbers in the control and VDR −/− groups, while the number of Tregs was reduced (p < 0.05). The  Notes: one-way analysis of variance was performed to analyze data; * p < 0.05 compared with the control group; # p < 0.05 compared with the SLE group; the paired t-test was performed to analyze data; $ p < 0.05 compared with the 0 W; SLE systemic lupus erythematosus, VDR vitamin D receptor; BUN blood urea nitrogen, Cr creatinine numbers of Treg, Th17, Th1, Th2, and CD4 − CD8 − DN cells exhibited no significant difference in the control group at the 24 W (all p > 0.05), while the VDR −/− group displayed increased numbers of Th17, Th1, Th2, and CD4 − CD8 − DN cells compared with the control group (p < 0.05). In comparison to the SLE group, the numbers of Th17, Th1, Th2, and CD4 − CD8 − DN cells were significantly decreased in the SLE + VD 3 and SLE + VD 3 + VDRinsert groups after treatment, while the number of Tregs was enhanced (p < 0.05), with no significant difference detected in relation to the numbers of Treg, Th17, Th1, Th2, and CD4 − CD8 − DN cells in the SLE + VD 3 + VDRinsert group relative to control mice at 24 W (Fig. 11). These results reveal that 1,25(OH) 2 D 3 /VDR mediates the number of splenic immune cells in SLE mice.

1,25-(OH) 2 D 3 /VDR downregulates the Skp2 expression and upregulates the p27 expression
RT-qPCR and western blot analysis that before treatment (0 W), the mRNA expression of Skp2 in SLE mice (in the SLE, SLE + VD 3 and SLE + VD 3 + VDRinsert groups) was significantly increased, while the  The mRNA expression of Skp2 in the VDR −/− group was notably elevated while that of p27 was markedly decreased (p < 0.05), with an opposite trend identified in the SLE + VD 3 and SLE + VD 3 + VDRinsert groups (p < 0.05). Compared with the control group, the mRNA expression of Skp2 in the VDR −/− group displayed notable increases, while that of p27 was significantly decreased (p < 0.05). Compared with the SLE group, the mRNA expression of Skp2 in the SLE + VD 3 and SLE + VD 3 + VDRinsert groups was significantly decreased, while that of p27 was significantly increased, with the changes in the SLE + VD 3 + VDRinsert group being more significant (p < 0.05; Fig. 12a). Compared with the control group, the relative protein expression of p-Skp2 in the VDR −/− group was significantly increased after 24 W of treatment, while that of p27 was significantly decreased (p < 0.05). Compared with the SLE group, the relative protein expression of Skp2 in the SLE + VD 3 and SLE + VD 3 + VDRinsert groups was significantly decreased, while that of p27 was significantly increased (p < 0.05; Fig. 12b, c). The results demonstrated that 1,25-(OH) 2 D 3 /VDR downregulated the expression of Skp2 in addition to upregulated expression of p27.

Discussion
Accumulating evidence has elucidated a close correlation between 1,25-(OH) 2 D 3 /VDR and SLE [22,23]. Moreover, Skp2 and p27 have been shown to function in autoimmune disease development [12,13]. In the present study, our aim was to investigate the effects of 1, 25-(OH) 2 D 3 /VDR treatment in SLE that are obtained by regulation the Skp2/p27 signaling pathway, with the hope of developing a novel therapeutic target for the treatment of SLE. Our results demonstrated that 1,25-(OH) 2 D 3 /VDR promotes the recovery of SLE in mice by downregulating Skp2 and upregulating p27 expression. First, we found that the hemoglobin and platelet count in patients with incipient SLE were significantly decreased compared with those in healthy controls. A previous study has revealed that patients with SLE exhibit markedly decreased platelet counts [24]. Importantly, the present study found that the expression of Skp2 in the incipient SLE patients was notably increased, while the expression of 1,25-(OH) 2 D 3 , VDR and p27 was markedly decreased, highlighting the relationship between SLE and 1,25-(OH) 2 D 3 /VDR. Moreover, the variant of VDR may negatively influence the clinical presentations in the process of SLE [7]. Furthermore, VDR led to the reduced production of many interleukins and IFN-γ, and dysregulation of T cells contributed to an excessive production of antibodies acting against DNA and self-proteins [25]. Furthermore, 1, 25-(OH) 2 D 3 regulates the expression of various apoptosis  factors which directly induce apoptosis via caspase activation, demonstrating its ability to interfere with the immunostimulatory effects of SLE [26]. A key observation of our study was that 1,25(OH) 2 D 3 /VDR regulated splenic immune cells in SLE mice. Tregs, including induced Tregs and naturally arising Foxp3 + nTregs, together play vital roles in immune escape and immunotherapy failure in cancer patients [27]. Therefore, maintaining the appropriate quantity of Tregs is vitally important in immunological fitness. However, Skp2 is a critical functional and molecular switch for Tregs, and excessive expression of Skp2 in Foxp3 + nTregs weakens the expression of Foxp3 and subsequently attenuates its suppressive function [12]. 1,25-(OH) 2 D 3 /VDR was found to downregulated the expression of Skp2 and upregulated the expression of p27 in SLE patients. Iglesias et al. concluded that p27, as a cell cycle inhibitor, pays a crucial role in regulating Tregs in addition to demonstrating an ability to enhance the activity and differentiation of Tregs as a positive regulator of the signaling pathway in CD4+ cells [13]. The Pearson correlation analysis highlighted a correlation between Skp2 and 1,25-(OH) 2 D 3 /VDR, suggesting them to be negatively related, while a positive association between p27 and 1,25-(OH) 2 D 3 /VDR was detected. Yang et al., revealed diminished Skp2 protein levels in cells in response to 1,25-(OH) 2 D 3 after 24 and 48 h of treatment [15]. In addition, 1,25(OH) 2 D 3 analogs have been shown to act via two cell type-dependent mechanisms to elevate the expression of p27 and decrease the expression of Skp2 [16]. The deteriorated symptoms of mice without VDRinsert also further underlined the vital significance of VDR in SLE, highlighting the potential of 1,25-(OH) 2 D 3 /VDR as a promising treatment for SLE.
1,25(OH) 2 D 3 /VDR downregulates the levels of IL-4, IL-10, IL-17 and INF-γ in SLE mice. It has been indicated that the cytokine production in SLE is promoted Fig. 12 1,25-(OH) 2 D 3 /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 by the alterations in Th1 and Th2, which may exacerbate the condition of patients suffering from the condition [28]. Cytokines formed by Th17 cells, such as IL-17, play crucial roles in the process of inflammation which can result in tissue damage, with upregulated levels of IL-17 observed in patients with a variety of autoimmune diseases, including SLE [22]. The activity of these cytokines can trigger a chain of inflammatory responses, leading to autoantibody production by B cells in SLE [29]. Hence, when compared with the control and VDR −/− mice, the inflammatory factors IL-4, IL-10, IL-17 and INF-γ were all significantly elevated. However, other evidence has suggested that vitamin D can inhibit the expansion of T cells and modulate the expression of cytokines with a Th2 bias [28], which in the present context resulted in a decrease in the inflammatory cytokines in the SLE + VD 3 and SLE + VD 3 + VDRinsert groups. Other observations revealed continually increased expression of inflammatory factors in the VDR −/− group, which ultimately emphasized the efficacy of VDR in SLE.

Conclusion
Taken together, the key evidence obtained during the current study suggests that 1,25(OH) 2 D 3 /VDR acts to stimulate the recovery from SLE symptoms by downregulating Skp2 and upregulating the p27, highlighting a promising novel therapeutic target for future SLE treatment.
Additional file 1: Figure S1. Construction of targeting vector for VDR ablation. A schematic representation of the VDR gene is displayed on the basis of the structure of the human gene and characterization of the sequences from exon 3 to exon 9 of the mouse gene. The exons are numbered and showed by solid boxes. A partial restriction map is demonstrated for the following enzymes: R, EcoRI; X, XbaI; S, SacI. The XbaI site indicated by the asterisk was derived from the phage arm. A 5kb XbaI fragment 59 to exon 3 and a 3.5-kb XbaI fragment 39 to exon 3 of the mouse VDR gene were applied as the targeting sequences. The SacI-XbaI fragment applied as a probe for identifying homologous recombinants is showed as well.