Effects of Vitamin D and Simvastatin on Inflammatory and Oxidative Stress Markers of High-Fat Diet-Induced Obese Rats

Research Article Correspondence Author Abdel-Moniem A. Makhlouf Email: rg1118@fayoum.edu.eg Abstract Purpose: This study was aimed to evaluate the antioxidant and antiinflammatory effects of vitamin D and Simvastatin (SIM) on a high-fat diet (HFD) induced-obese rats. Methods: 40 adult male rats were divided into four groups: control group, HFD, HFD + vitamin D, and HFD + SIM for 14 weeks. Vitamin D or SIM supplementation was done for the last 6 weeks. Vitamin D dosage was 500 IU/kg, while SIM dosage was 10 mg/kg. Interleukin-6 (IL-6) concentration and markers of oxidative stress including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), and reduced glutathione(GSH) concentrations in serum were determined using ELISA kits and spectrophotometry methods, respectively. Results: Treatment with vitamin D or SIM could significantly reduce IL-6 and MDA and increases SOD, GPx activities, and GSH levels. Oxidative stress can result not only from increased ROS production but also from dysfunctional antioxidant defenses. Conclusion: From the experimental results, it was observed that SIM and vitamin D could attenuate oxidative stress and inflammation markers associated with obesity.


Introduction
Obesity is a serious problem posing a considerable threat to human health (Ni et al., 2020). Obesity is a multifactorial disease caused by chronic positive energy surplus or secondary to genetic, hypothalamic, and endocrine diseases (Vallgårda et al., 2017). Obesity is characterized by excessive fat deposition into adipose tissue and non-adipose tissues (Kelishadi et al., 2017). Another characteristic feature coupled with obesity is the alteration of the redox state accompanied by metabolic risk factors (Warolin et al., 2014). Obesity and its related metabolic disorders are also strongly linked to oxidative stress. This oxidative status is closely associated with proinflammatory cytokine secretion, which can initiate oxidative stress in a continuous circle (Biswas, 2016). As a result, systemic oxidative stress and inflammation are critical factors in the pathogenesis of obesity-related diseases (Crujeiras et al., 2013).

Diets and drugs
Standard diet and HFD were purchased from El-Gomhorya Company, Cairo, Egypt. HFD was preserved at 4°C until used. SIM (Zocor 10 mg) was purchased from Merck sharp & Dohme Ltd., UK. Vitamin D (Calcitriol 500 IU) was purchased from Sigma Aldrich, USA.

Sample collection
At the end of the experimental period (98 days), the animals were weighed and anesthetized by diethyl ether after overnight fasting. The blood samples were collected through the retro-orbital venous plexus. The blood samples used for serum assessment were collected without using an anticoagulant. First, the blood was allowed to clot for 30 min at 25° C. The samples were then centrifuged at 4,000 rpm for 15 min at 4° C. After that, the supernatants were stored at -80° C for further assessment, except IL-6 samples were stored at -20° C to avoid loss of bioactive rat IL-6.

Glutathione peroxidase and superoxide dismutase assessment
SOD was assayed by a spectrophotometric method based on the inhibition of a superoxideinduced reduced nicotinamide adenine dinucleotide (NADH) oxidation according to Paoletti et al. method Boitard et al., (2014)

Reduced glutathione assessment
GSH was measured using the colorimetric method (Beutler et al., 1963) of Beutler et al. using BioAssay Systems ( USA) kit.

Assessment of interleukin-6
IL-6 activity was measured according to the ELISA method of Engvall and Perlmann (Engvall & Perlmann, 1971). using a ThermoFisher kit (USA).

Statistical analysis
All statistical analyses were performed using SPSS software, version 20. Data were analyzed using one-way analysis of variance (ANOVA) followed by Post hoc-least significant difference (LSD). Data were expressed as the mean ± SD. P < 0.05 was considered statistically significant. All charts were performed using OriginPro for data analysis and graphing software (version 2018).

Change of MDA levels in the studied groups
According to our results ( Fig.2), MDA levels were significantly increased in the HFD group. However, both vitamin D and SIM groups showed a marked depletion compared to the HFD group. Moreover, a significant decrease was detected in the SIM group compared to the vitamin D group.

Change of SOD and GPx activities in the studied groups
A notable decrease in SOD activities in the HFD group compared to the control group. Conversely, a significant rise was detected in vitamin D and SIM groups versus the HFD group; also, an increase was detected in the SIM group compared to the HFD group and (Fig.2). The current study revealed a significant upwards in GPx in the HFD group compared to the control group. In contrast, a marked decline was detected in the vitamin D and SIM groups compared to the HFD group. However, a non-significant decrease was detected between the two groups (Fig.2).

Change of GSH levels in the studied groups
Results from the current study showed a significant decrease in the HFD group compared to the control group in serum GSH levels. A further marked decline in GSH levels was detected in both vitamin D and SIM groups compared to the HFD group. However, there was a significant rise in the SIM group compared to the vitamin D group (Fig.2). A significant rise was detected in IL-6 levels in the HFD group compared to the control group. A remarkable drop was detected in both vitamin D and SIM groups compared to the HFD group. However, the SIM group showed a significant decrease compared to the vitamin D group (Fig.2). Obesity is a real pathology as it represents a risk factor for various metabolic diseases (De Lorenzo et al., 2019). Regardless of whether obesity is a condition or a disease, it arises from multiple etiologic determinants, either acquired or inherited (d'Autume et al., 2012). These determinants favor positive energy balance and weight gain (Hall et al., 2009;Popkin & Hawkes, 2016). Feeding (HFD) to rats was proved to be a valuable model of the putative effects of dietary fat in humans (López et al., 2003). Therefore, rat models are valuable tools for inducing obesity as they will readily gain weight when fed high-fat diets (Von Diemen et al., 2006).

Change of IL-6 levels in the studied groups
Oxidative stress is an imbalance between tissue free radicals, reactive oxygen species (ROS), and antioxidants (Fernández-Sánchez et al., 2011). Lipid peroxidation is the first step of cellular membrane damage (Repetto et al., 2012). It constitutes a complex chain reaction of free radicals that leads to degradation of polyunsaturated fatty acids (PUFAs) in the cell membrane (Halliwell & Gutteridge, (1984) and in turn, profound alternations of the cell membrane structure and function (van Ginkel & Sevanian, 1994). MDA is one of the end-products of the lipid peroxidation process. Thus, increased MDA level is a crucial indicator of lipid peroxidation and oxidative status (Ayala et al., 2014).
The results of the current study were supported by Mahmoudi et al., which demonstrated that the elevation of serum MDA levels is an indicator of lipid damage due to obesity development (Mahmoudi et al., 2019). Also, reduction of MDA levels after vitamin D supplementation was matched by Mostafa et al., which elucidated vitamin D's significant role in ameliorating lipid peroxidation levels and, in turn, inhibiting HFD-induced oxidative stress in obese males Wistar rats (Mostafa et al., 2016). Furthermore, MDA levels were found to be markedly decreased, reaching almost the control level in the SIM group. These results agreed that Eweda et al. proved that SIM could prevent lipid peroxidation and ameliorate oxidative stress induced by the HFD (Eweda et al., 2018) Furthermore, oxidative stress can result not only from increased ROS production but also from dysfunctional antioxidant defenses. SOD is the first defense against ROS toxicity (Fang et al., 2013) as it catalyzes superoxide radicals dismutation to the less toxic hydrogen peroxide (H2O2) (McCord, 1987). Also, GPx, a selenoenzyme, plays a significant role in H2O2 and hydroperoxide reduction to non-toxic products (Freeman, & Crapo, 1982). It also has a predominant role in the redox cycling of the reduced glutathione (GSH) to the oxidized glutathione (GSSG), necessary to maintain cells' thiol content to maintain the cell's redox state (Agrawal et al., 2014). Therefore, the decrease in SOD and GPx activities could be ascribed to the exhaustion of these enzymes in fighting free radicals generated during the development of obesity (Das et al., 2013;Noeman et al., 2011).
On the other hand, the antioxidant effect of vitamin D due to its ability to decrease superoxide anion formation; hence these enzymes will not be exhausted in encountering the radicals after vitamin D supplementation. Thus the concentration of SOD and GPx were upregulated (Polidoro et al., 2013). Furthermore, Liang et al. and Pan et al. proved the enhanced expression of SOD and GPx activities after SIM treatment (Liang et al., 2020;Pan et al., 2016).
Moreover, the thiol-based antioxidant system contributes a second line of cellular defense against ROS-mediated oxidative damage (Nazima et al., 2014). GSH is one of the significant intracellular non-enzymatic antioxidants and the most abundant component of an endogenous cellular "redox buffer" (Kurutas, 2015). The significant decrease could be owing to increased utilization of GSH by cells either as a scavenger of free radicals (Deng et al., 2019). Vitamin D increases GSH levels through the upstream regulation of glutathione reductase (GR) gene expression, a crucial enzyme involved in the synthesis of GSH (Kanikarla-Marie & Jain, 2016). Also, This upward trend of GSH levels after SIM administration was confirmed by (Feng et al., 2015).
Reactive oxygen species also activate NF-κB, a redox-sensitive transcription factor that acts as a master regulator of the inflammatory response (Uciechowski & Dempke, 2020). It is required to induce many inflammatory genes, including those encoding IL-6  which could explain the increased levels of IL-6 during obesity. IL-6 is a member of the proinflammatory cytokine family, which induces various proteins expression responsible for acute inflammation (Uciechowski & Dempke, 2020). Decrease IL-6 levels in vitamin D groups was matched with Kim et al., which confirmed vitamin D impact in the downregulation of IL-6 secretion (Kim et al., 2020). Also, Nicholls et al. illustrated that statins could suppress the modification of proteins by myeloperoxidase-catalyzed reactive nitrogen species. Thus, these species have been demonstrated to promote multiple inflammatory pathways (Nicholls et al., 2006).

Conclusion and Suggestion
The previous biochemical investigations confirmed the therapeutic effect of vitamin D and Simvastatin against the deleterious effect of oxidative stress and inflammation induced by HFD, which could reduce the risk of obesity-related disease.