الفهرس 
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Abstract
Targeting the hepcidin-ferroportin axis is the key mechanism involved in regulating iron homeostasis. This study aims to investigate the intertwined relationship between vitamin D3 status and iron homeostasis in metabolic syndrome induced by a high-fat high-fructose (HFHF) diet in rats.
Vitamin D3 through its anti-inflammatory and anti-oxidant modulatory activities can regulate the hepcidin-ferroportin axis, which in turn restores iron homeostasis. Also, vitamin D3 regulates lipid metabolism and insulin resistance through its receptors (VDR) thus, alleviating the metabolic syndrome.
This study was conducted to investigate the effect of vitamin D3 administration on inflammation and iron dysregulation induced by high fat high fructose diet in rats.
In this study, sixty male albino rats weighing 120±5g were divided into 6 groups, 10 rats for each as follows:
group 1: Negative control group, rats were fed on balanced diet and orally administered distilled water twice per week.
group 2: Positive control group, rats were fed on HFHF diet and orally administered distilled water twice per week.
group 3: Rats were fed on a balanced diet and orally administered 1,25(OH)2D3 (12 μg that equivalent to 480 IU/kg body weight, twice per week).
group 4: Rats were fed on a balanced diet and orally administered 1,25(OH)2D3 (24 μg that equivalent to 960 IU/kg body weight, twice per week).
group 5: Rats were fed on HFHF diet and orally administered 1, 25(OH)2 D3 (12 μg that equivalent to 480 IU/kg body weight, twice per week).
group 6: Rats were fed on HFHF diet and orally administered 1, 25(OH)2 D3 (24 μg that equivalent to 960 IU/kg body weight, twice per week).
All rats had free access to food and water. The body weight of each rat was measured weekly, also food intake was recorded daily, to monitor the body weight changes and to calculate the biological measurements. At the end of experimental period (8weeks) blood samples were collected after 8 hours of fasting from the eyes vein using capillary tube to detect glucose homeostasis, and in order to inhibit glycolysis, samples were collected into tubes containing sodium fluoride. Then after another 4 hours of fasting all rats were sacrificed under sodium barbiturate anesthesia at 200 mg/kg b.wt. Blood samples were collected from the hepatic portal vein and left for 15 minutes at room temperature, then centrifuged at 4000 rpm for 20 minutes for serum separation. Then, serum samples were kept at -20 ºC for further biochemical analysis. Furthermore, liver samples were removed and washed with ice-cold saline solution and then weighed. After that, part of the samples were preserved in 10% formalin solution for microscopic examination, and the other samples were stored at -80 °C for genetic and biochemical analysis. The study follows the guidelines established by the local institutional animal ethics committee of Ain Shams-University (No. ASU-SCI/BIOC/2022/11/2).
At the end of the experimental period, the following parameters were determined:
1. Biological measurements:
- Change in body weight, food intake, feed efficiency ratio (FER), and relative weight of liver.
2. Biochemical parameters:
- Serum adiponectin and resistin levels.
- Nuclear factor erythroid 2-related factor 2 (Nrf2) gene expression in liver tissue, serum heme-oxygenase-1 (HO-1), and malondialdehyde (MDA) concentration.
- Serum interleukin -6 (IL-6), C reactive protein (CRP), and monocyte chemoattractant protein-1 (MCP-1) levels.
- Iron profile (hepatic hepcidin and ferroportin 1 protein gene expression and iron concentration, serum iron, transferrin, total iron binding capacity (TIBC) concentrations).
- Serum lipids profile (total cholesterol (TC), triacylglycerol (TAGs), high-density lipoprotein cholesterol (HDL-C), very low-density lipoprotein cholesterol (VLDL-C), and low-density lipoprotein cholesterol (LDL-C)).
- Determination of serum glucose homeostasis (glucose concentration, insulin level, and the calculation of homeostatic model of assessment for insulin resistance (HOMA-IR)).
- Serum vitamin D3 level and vitamin D receptors (VDR) gene expression in liver tissue.
3. Histopathological examination was carried out for hepatic sections for all experimental groups to confirm the biochemical results.
The results of the present study can be summarized as follows:
1) Biological measurements:
The biological data reported that there was a significant increase (P<0.05) in body weight, feed efficiency ratio, and relative liver weight, but in the case of feed intake, there was a significant decrease in the experimental groups fed on HFHF diet (G2, G5 & G6) as compared to -ve control group (P<0.05). On the other hand, Vit.D3 administered groups (G5& G6) showed a significant improvement of these parameters.
2) Biochemical parameters:
2.1. Serum adipokines parameters:
HFHF diet led to a significant decrease in serum adiponectin and a significant increase in resistin as compared with -ve control group. While in Vit.D3 administered groups (G5& G6) there was a significant increase in adiponectin and a significant decrease in resistin as compared to +ve control group.
2.2. Lipid profile:
Consumption of HFHF diet led to a significant elevation in serum TC, TAGs, LDL-C, and VLDL-C levels with a significant reduction in HDL-C in comparison with -ve control group (p<0.05). While administration of Vit.D3 showed a significant improvement in altered lipids profile, with a significant reduction in serum TC, TAGs, LDL-C, and VLDL-C levels and a significant increase of HDL-C as compared to +ve control group(p<0.05).
2.3. Glucose homeostasis:
Results showed a marked significant elevation in serum glucose, insulin, and HOMA-IR values in groups that were fed on HFHF diet as compared to the healthy (–ve control group). While, administration of Vit.D3 in G5 and G6 showed a significant decrement in serum glucose, insulin, and HOMA-IR as compared to +ve control group.
2.4. Oxidative stress markers:
There was a significant decrease in liver Nrf2 gene expression and serum HO-1 in (G2, G5& G6) compared to -ve control group, in contrast, there was a significant increase in serum MDA concentration. Administration of Vit.D3 reversed the elevated levels of oxidative stress markers.
2.5. Serum inflammatory markers:
It was observed that serum IL-6, CRP, and MCP-1 levels were significantly increased in HFHF diet groups compared with -ve control group. On the other hand, there was a significant decrease in serum IL-6, CRP, and MCP-1 levels after Vit.D3 administration.
2.6. Iron profile:
HFHF diet caused significant upregulation (p˂0.05) of the hepcidin gene and downregulation of FPN1 compared to the healthy control group. Compared to the positive control group, the expression of hepcidin was significantly downregulated, and ferroportin expression was significantly upregulated (p˂0.05) in the groups that were orally administered Vit.D3. Furthermore, the results showed a significant decrease in serum iron with a significant increase in liver iron, serum TIBC and transferrin in the HFHF diet groups. While Vit.D3 administration reversed the effect of the HFHF diet on iron indices (p < 0.05).
2.7. Serum vitamin D3 and vitamin D receptor (VDR) gene expression in liver tissue:
There was a significant decrease (p˂0.05) in serum Vit.D3 level as well as a significant downregulation of VDR expression in the positive control group compared with the healthy control group. Whereas Vit.D3 administration significantly restored serum, Vit.D3 level and significantly increased VDR expression compared to the rats fed a HFHF diet.
3) Histopathological examination:
The histopathological examination of liver sections after HFHF diet consumption showed pathological manifestations including fatty change in a diffuse manner all over the hepatocytes in the parenchyma. There was congestion in the portal vein and hyperplasia in the epithelial cells lining the bile ducts with few inflammatory cells infiltration in the portal area. On the other hand, Vit.D3 administration showed marked improvement in hepatocytes thus restoring hepatic architecture.
It was concluded from the results of the current experiment that vitamin D3 regulates iron homeostasis through its anti-inflammatory and antioxidant activities. It also improves lipid levels and reduces insulin resistance. Vitamin D3 reduces inflammation and iron dysregulation induced by a diet high in fat and fructose by targeting the hepcidin-ferroportin 1 axis.