Research Article |
Corresponding author: Anita Mihaylova ( anita.mihaylova@mu-plovdiv.bg ) Academic editor: Magdalena Kondeva-Burdina
© 2024 Nina Doncheva, Anita Mihaylova, Hristina Zlatanova, Mariya Ivanovska, Delian Delev, Ilia Kostadinov.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Doncheva N, Mihaylova A, Zlatanova H, Ivanovska M, Delev D, Kostadinov I (2024) Immunomodulatory properties of cholecalciferol in rats with experimentally induced inflammation. Pharmacia 71: 1-9. https://doi.org/10.3897/pharmacia.71.e122410
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The study aimed to investigate anti-inflammatory and immunomodulatory effects of cholecalciferol (vitamin D3) in rats with complete Freund’s adjuvant-induced arthritis (AIA) and lipopolysaccharide (LPS)-induced inflammation. In the first experiment, rats were treated with cholecalciferol 14 days before or from the day of induction of arthritis. In the second set-up, animals received cholecalciferol for 14 days which was followed by LPS injection. TNF (tumour necrosis factor)-alpha, IL (interleukin)-1β, TGF (transforming growth factor)-β1 levels were measured by enzyme linked immunosorbent assay (ELISA). Cholecalciferol treatment reduced paw oedema and ankle joint diameter in AIA. Significantly lower IL-1β concentrations were found in cholecalciferol-treated arthritic rats. In LPS-challenged rats, cholecalciferol markedly lowered serum TNF-α, whereas an elevation in IL-1β concentrations was observed. Cholecalciferol slightly increased TGF-β1 serum concentration in arthritic rats and non-significantly reduced its level in LPS-challenged animals. Our findings showed that cholecalciferol exerts immunomodulatory properties which probably contribute to its anti-inflammatory effect.
arthritis, cholecalciferol, cytokines, inflammation, lipopolysaccharide
Vitamin D is recognised as a crucial dietary component with diverse physiological functions (
Vitamin D deficiency is a major health problem worldwide. It is associated not only with disorders related to disrupted calcium homeostasis, such as rickets, osteomalacia and increased risk of dental caries (
Vitamin D plays an essential role in the regulation of immune and inflammatory processes. Its deficiency might be associated with chronic inflammation, evidenced by negative correlation between low 25-hydroxyvitamin D levels and serum C-reactive protein (CRP) (
VDR is present in immune cells and vitamin D plays a crucial role in regulating both innate and adaptive immune responses. This vitamin acts by hindering the transformation of monocytes and macrophages into dendritic cells and by halting the generation of inflammatory cytokines. Moreover, vitamin D inhibits the multiplication of Th1, Th17 and B cells (
Inflammatory mediators, such as TNF-α and IL-1β. are central to joint inflammation and bone deformities in rheumatoid arthritis (RA), showing an inverse correlation with vitamin D levels (
The anti-inflammatory properties of vitamin D could be explained with its effect on expression of enzymes responsible for synthesis of pro-inflammatory mediators, influence on intracellular signalling cascades involved in inflammation and interaction with transcription factors essential for regulation of genes for production of pro-inflammatory and anti-inflammatory molecules (
Cholecalciferol (Merck), lipopolysaccharide E. Coli O55 (Sigma Aldrich), complete Freund’s adjuvant (Sigma Aldrich); rat IL-1 beta, TNF-alpha, TGF beta-1 ELISA kits (Diaclone); rat vitamin D3 ELISA kit (MyBioSource).
All experiments were conducted in compliance with the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes. Permission for this study was obtained from the Ethics Committee at the Medical University of Plovdiv, Bulgaria, under protocol № 1/13.02.2020, as well as from the Animal Health and Welfare Directorate of the Bulgarian Food Safety Agency, permit № 249/22.11.2019.
In our study, we used adult male Wistar rats weighing 200 ± 20 grams. They were housed in a controlled laboratory setting with a 12-hour light-dark cycle, maintained at a room temperature of 22 ± 2 °C and air humidity of 55 ± 5%. Tap water and food were available ad libitum. Prior to the experiments, the rats were acclimatised to the laboratory conditions and all experiments were conducted during the daytime.
To assess the anti-inflammatory effects of cholecalciferol in rats with CFA-induced arthritis, the animals were randomly divided into six groups (n = 8):
Group 1: control group: olive oil 0.1 ml/kg bw,
Group 2: positive control group: olive oil 0.1 ml/kg bw + CFA,
Group 3: cholecalciferol 500 IU/kg bw (pretreatment) + CFA,
Group 4: cholecalciferol 1000 IU/kg bw (pretreatment) + CFA,
Group 5: cholecalciferol 500 IU/kg bw + CFA,
Group 6: cholecalciferol 1000 IU/kg bw + CFA.
Groups 1 to 4 were pretreated with the respective substance via oral gavage for 14 days. Groups 5 and 6 received their first dose of cholecalciferol on the day of CFA injection. Experimental arthritis was induced via a single intraplantar injection of 0.1 ml CFA into the right hind paw (day 0). The cholecalciferol treatment continued for 30 days, post arthritis induction, for all animals. At the end of the experiment (day 30), blood samples were taken for serum cytokine measurement.
To evaluate the anti-inflammatory effect of cholecalciferol in rats with LPS-induced systemic inflammation, the animals were randomly divided into four groups (n = 8):
Group 1: control group: olive oil 0.1 ml/kg bw,
Group 2: positive control group: olive oil 0.1 ml/kg bw + LPS,
Group 3: cholecalciferol 500 IU/kg bw + LPS,
Group 4: cholecalciferol 1000 IU/kg bw + LPS.
All animals were pretreated orally, for 2 weeks, with respective substances. On day 15, LPS was intraperitoneally administered at a dose of 1 mg/kg bw to groups 2, 3 and 4. Blood samples for immunological assays were collected four hours, post-administration. In both experiments, the control groups were treated with olive oil, as it served as the diluent for cholecalciferol.
Digital Water Plethysmometer (Ugo Basile, Italy) was employed to assess the CFA-induced inflammatory response, as outlined by
where PV0 is the initial paw volume, PVt is the paw volume at days 1, 15 and 30 following the CFA injection.
Ankle joint diameters were assessed using a digital caliper both before the CFA injection (day 0) and subsequently on days 1, 4, 6, 8, 11, 14, 18, 21, 25, 27 and 30.
Serum levels of IL-1β, TNF-α and TGF beta-1 were measured on days 30 and 15 of the respective experiment using solid-phase ELISA. The assessments were conducted in accordance with the manufacturer’s instructions. Absorbance readings were taken at 450 nm using an ELISA reader and the absorbance values were then converted to concentrations (pg/ml) using a standard curve. The detection limits for IL-1β, TNF-α and TGF beta-1 were 4.4 pg/ml, 15 pg/ml and 48 pg/ml, respectively. Additionally, the detection limit for vitamin D3 was 1.13 pg/ml.
Statistical analysis was performed using IBM SPSS Statistics 19.0. All data are presented as mean ± SEM (standard error of the mean). The data were analysed using repeated measures one-way ANOVA, followed by the Tukey post hoc test. A value of p < 0.05 was accepted for statistically significant.
1.1. Plethysmometer: all experimental groups, as well as the positive control, notably increased paw volume compared to the control rats on day 1 (p < 0.001), day 15 (p < 0.001 for groups 2 and 5; p < 0.05 for groups 3, 4 and 6) and day 30 (p < 0.001 for groups 2, 5 and 6; p < 0.05 for groups 3 and 4). Animals pretreated with cholecalciferol in both doses significantly inhibited paw oedema on the 15th and 30th days compared to the positive control (p < 0.05 and p < 0.001, respectively) (Fig.
1.2. Ankle joint diameters: throughout all testing days, all experimental groups and the positive control exhibited significantly increased joint diameters compared to the control (p < 0.01). Animals pretreated with cholecalciferol at a dose of 500 IU/kg bw notably decreased joint diameter on days 14 and 18 compared to the positive control (p < 0.01 and p < 0.05, respectively). Rats pretreated with the higher dose of vitamin D3 significantly reduced ankle diameter on days 11 (p < 0.01), 14 (p < 0.01), 18 (p < 0.001), 21 (p < 0.05), 25 (p < 0.01), 27 (p < 0.05), and 30 (p < 0.001). Animals that began cholecalciferol (1000 IU/kg bw) treatment on the day of CFA injection showed a significant decrease in joint diameter on days 11 and 14 compared to the positive control (p < 0.01) (Table
Effect of cholecalciferol on ankle joint diameter in rats with CFA-induced arthritis.
Day | control | positive control | vit D 500IU/kg + CFA (pretreatment) | vit D 1000 IU/kg + CFA (pretreatment) | vit D 500IU/kg + CFA | vit D 1000 IU/kg + CFA |
---|---|---|---|---|---|---|
1 | 5.18 ± 0.12 | 8.96 ± 0.16* | 9.37 ± 0.11* | 9 ± 0.14* | 9 ± 0.15* | 8.93 ± 0.16* |
4 | 5,12 ± 0.12 | 9.62 ± 0.14* | 9.81 ± 0.19* | 9.81 ± 0.16* | 10.37 ± 0.21* | 8.87 ± 0.17* |
6 | 5.12 ± 0.12 | 8.62 ± 0.17* | 9.18 ± 0.17* | 8.87 ± 0.18* | 8.68 ± 0.16* | 8.25 ± 0.16* |
8 | 5.37 ± 0.15 | 8 ± 0.18* | 8.62 ± 0.18* | 8.18 ± 0.2* | 8 ± 0.14* | 7.81 ± 0.14* |
11 | 5.51 ± 0.1 | 9.1 ± 0.12* | 8.53 ± 0.19* | 8.01 ± 0.2* ^^ | 8.51 ± 0.14* | 7.63 ± 0.14* ^^ |
14 | 5.53 ± 0.11 | 8.71 ± 0.13* | 7.88 ± 0.15* ^^ | 7.93 ± 0.18* ^^ | 8.53 ± 0.15* | 7.87 ± 0.15* ^^ |
18 | 5.51 ± 0.09 | 8.11 ± 0.1* | 7.52 ± 0.13* ^ | 6.87 ± 0.14* ^^^ | 7.93 ± 0.1* | 7.56 ± 0.11* |
21 | 5.6 ± 0.16 | 7.78 ± 0.1* | 7.56 ± 0.13* | 6.87 ± 0.13* ^ | 8.06 ± 0.13* | 7.68 ± 0.12* |
25 | 5.6 ± 0.16 | 7.56 ± 0.14* | 7.25 ± 0.17* | 6.26 ± 0.16* ^^ | 8.12 ± 0.14* | 7.75 ± 0.14* |
27 | 5.62 ± 0.16 | 7.65 ± 0.12* | 7.31 ± 0.18* | 6.16 ± 0.14* ^ | 8.21 ± 0.18* | 7.81 ± 0.13* |
30 | 5.43 ± 0.27 | 7.68 ± 0.1* | 7.56 ± 0.19* | 6.1 ± 0.12* ^^^ | 7.87 ± 0.1* | 7.62 ± 0.12* |
2.1. TNF-α, IL-1β, TGF-β1 and vitamin D3 serum levels in rats with CFA-induced arthritis TNF-α: the positive control and all animals, except those pretreated with 1000 IU/kg cholecalciferol, exhibited significantly higher levels of TNF-α compared to the control group (p < 0.05). Rats pretreated with vitamin D3 notably decreased serum levels of TNF-α compared to the positive control (p < 0.05 and p < 0.01, respectively). Animals receiving a higher dose of vitamin D from the day of AIA induction also significantly decreased TNF-α concentration compared to the positive control (p < 0.05) (Fig.
IL-1β: animals treated solely with CFA exhibited a notable increase in IL-1β serum levels compared to the control group (p < 0.01). All experimental groups showed significantly lower levels of IL-1β compared to the positive control (p < 0.05) (Fig.
TGF-β1: neither control group exhibited a significant difference in serum levels of TGF-β1. Cholecalciferol treatment led to a slight increase in the concentration of this cytokine, but none of the experimental groups reached significance when compared with either of the control animals (Fig.
Vitamin D3: serum levels of cholecalciferol were insignificantly lower in rats with AIA compared to the control group. However, cholecalciferol supplementation resulted in an increase in its serum concentration. Significance was observed in rats pretreated with both doses of vitamin D3 compared to both control groups (p < 0.05 and p < 0.01, respectively). Rats receiving the higher dose of cholecalciferol from the day of arthritis induction exhibited a significant increase in its level compared to the positive control (p < 0.05) (Fig.
The obtained concentrations of cholecalciferol were higher compared with non-treated rats, but remained within the normal range (maximum 181.42 ± 6.8 ng/ml) for related states without reaching toxic levels (
2.2. TNF-α, IL-1β, TGF-β1 and vitamin D3 serum levels in rats with LPS-induced systemic inflammation.
TNF-α: both the positive control (p < 0.01) and the two experimental groups (p < 0.01 and p < 0.05) significantly increased serum levels of TNF-α compared to the control group. Rats treated with the higher dose of cholecalciferol exhibited significantly lower serum levels of TNF-α compared to animals that received only LPS (p < 0.05) (Fig.
IL-1β: animals injected solely with LPS, as well as the two experimental groups receiving cholecalciferol, exhibited a notable elevation in serum levels of IL-1β compared to the control rats (p < 0.001) (Fig.
TGF-β1: rats from the positive control group showed a significant increase in serum levels of TGF-β1 compared to the control group (p < 0.05). However, animals from the two groups treated with vitamin D3 at doses of 500 and 1000 IU/kg bw exhibited a decrease in serum concentration of TGF-β1, although this change did not reach statistical significance (Fig.
Vitamin D3: administration of LPS led to a slight reduction in cholecalciferol serum levels compared with the control group. However, both experimental groups exhibited a significant increase in vitamin D serum levels compared with the positive control (p < 0.05 and p < 0.01, respectively) (Fig.
Beyond its role in maintaining calcium-phosphorus homeostasis, vitamin D plays a pivotal role in both innate and adaptive immunity. Deficiency in this vitamin has been linked to an increased risk of autoimmune and inflammatory disorders. The principal finding of our current study is that cholecalciferol suppresses inflammation and demonstrates immunomodulatory effects in two distinct experimental models: adjuvant-induced arthritis (an autoimmune-mediated chronic inflammatory process) and LPS-induced acute systemic inflammation.
Vitamin D deficiency has been observed in patients with various autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, type 1 diabetes mellitus, multiple sclerosis and thyroiditis (
Treatment with vitamin D following CFA induction of arthritis has a protective effect, with a significant decrease in inflammatory markers (e.g. TNF-α, IL-6, rheumatoid factor), beneficial effects on serum lipids and an increase in red blood cells, haemoglobin, haematocrit and platelets (
Vitamin D could ameliorate inflammation in AIA through several mechanisms. A recent review discussed the role of small molecules, such as prostaglandins, leukotrienes, nitric oxide, reactive oxygen species and lipoxins in the pathogenesis of inflammation observed in rheumatoid arthritis (
TNF-α plays a crucial role in inflammation’s pathogenesis. It causes vasodilation by stimulating inducible nitric oxide (NO) synthetase, increases vascular permeability and expression of cell adhesion molecules and stimulates production of reactive oxygen species. TNF-α is primarily released by macrophages, but other cells can also produce this pro-inflammatory cytokine, such as neutrophils, NK-cells, B-lymphocytes etc. (
Bacterial LPS is one of the primary triggers of TNF-α synthesis. LPS is a component of the outer membrane of Gram-negative bacteria. It stimulates the innate immune response and causes a robust inflammatory reaction (
IL-1β is an important molecule implicated in the development of autoinflammatory and autoimmune diseases. It has also been shown to play a role in the pathogenesis of ischemic injury in stroke, type 2 diabetes due to its cytotoxic effects on pancreatic beta cells, osteoarthritis, gout, myeloma and heart failure following myocardial infarction. Blockade of IL-1β signal pathways results in an improvement of the condition in the above-mentioned diseases (
IL-1β is mainly produced by monocytes and macrophages (
TGF-β is produced by a variety of cells, including non-immune cells and has pleiotropic effects in the body. It is important for the development and maintenance of immune tolerance, but may also promote inflammation and enhance autoimmune reactions (
LPS markedly increased TGF-β1 serum levels. Our results agree with early in vivo studies about the stimulatory effect of LPS on the production of this cytokine from murine macrophages (
Cholecalciferol demonstrates significant immunomodulatory properties, which likely contribute to its anti-inflammatory effects. In both arthritic and LPS-challenged rats, vitamin D3 significantly reduced TNF-α serum levels. However, the impact on IL-1β and TGF-β1 concentrations varied depending on the inflammatory model. Therefore, vitamin D supplementation may serve as a beneficial adjunct in the treatment of disorders associated with heightened TNF-α signalling. Cholecalciferol could exhibit a preventative effect in such diseases, as evidenced by the superior outcomes observed in animals pretreated with this vitamin.
The authors of this manuscript have declared that no conflict of interests exists.
Conceptualisation, ND, AM and IK; methodology, ND, AM, IK and MI; investigation, ND, AM, IK and MI; analysis, HZ and DD; writing, original draft preparation, AM, IK and HZ; writing, review and editing, IK and HZ; supervision, IK and DD.
This study was funded by Medical University of Plovdiv, Bulgaria, grant № DPDP-20/2019.