The Effects Of Multiple Trace Elements Supplementation On Antioxidant,Anti-inflammatory And Immune Functions In Rats Fed A High-fat Diet

ObjectiveA high-fat diet is a dietary pattern characterized by high fat energy supply ratio,which causes various complications due to obesity,accompanied by impaired lipid metabolism,oxidative stress,inflammatory response and immune imbalance,which endangers the health of the population and increases the incidence and prevalence of chronic diseases.Trace elements play an important role in maintaining human health as essential nutrients by effectively promoting metabolism and improving antioxidant capacity,anti-inflammatory capacity and immunity in the organism.The aim of this study was to investigate the effects of trace element supplementation on antioxidant,anti-inflammatory and immune functions in rats fed a high-fat diet,since trace elements intake in the population is unbalanced and there are few studies on balanced trace elements supplementation in people on a long-term high-fat diet.Methods1.Grouping and feeding of experimental animalsThe 160 male Wistar rats were randomly divided into 4 groups(40 rats/group)after 1 week of adaptation feeding:control group;trace element supplementation group(TE);high-fat diet group(HFD);and a high-fat diet supplemented with trace elements group(HFD+TE).The control group and the trace element supplementation group were fed a special maintenance diet without trace elements,and the high-fat group and the high-fat diet supplementation group were fed a special high-fat diet without trace elements;the control group and the high-fat diet group were gavaged with saline,and the trace element supplementation group and the high-fat diet supplementation group were gavaged with a mixture solution of trace elements containing boric acid(1.76 mg/mL),vanadium oxide sulfate(13.5μg/mL),chromium chloride(4.1 μg/mL),and chromium acetate(4.1 μg/mL),chromium chloride(4.1 μg/mL),manganese chloride(0.68 mg/mL),iron chloride(1.62 mg/mL),cobalt chloride(8.1 μg/mL),strontium chloride(0.54 mg/mL),copper chloride(108μg/mL),zinc chloride(1.69 mg/mL),sodium selenite(8.1μg/mL),and sodium molybdate(13.5 μg/mL).The rats were weighed weekly,and their diet and growth activity were observed and recorded daily.Ten rats were randomly selected from each batch at the end of week 4,week 8,week 12,and week 20,and whole blood was collected after anesthesia.The spleens of the rats were taken on ice after blood collection,partially ground and analyzed by flow cytometry,and the rest were frozen in liquid nitrogen and stored at-80℃ for testing.2.Determination of serum indexes of ratsThe levels of triglycerides(TG),total cholesterol(TC),glutathione(GSH),malondialdehyde(MDA),total antioxidant capacity(T-AOC),glutathione peroxidase(GSHPx),glutathione reductase(GR),superoxide dismutase(SOD),catalase(CAT),myeloperoxidase(MPO),xanthine oxidase(XOD),interleukin 2(IL-2),interleukin 4(IL-4),interleukin 6(IL-6),interleukin 10(IL-10),and tumor necrosis factor-α(TNF-α)were determined in rat serum according to the kit instructions.3.Determination of various indicators in rat spleen tissue fluidThe levels of glutathione(GSH)and malondialdehyde(MDA),glutathione peroxidase(GSH-Px),superoxide dismutase(SOD),and catalase(CAT)were measured in the spleen of rats according to the instructions of the kit.4.Evaluation of immune function in ratsThe CD3+、CD4+、CD8+T lymphocyte ratios and CD4+/CD8+T lymphocyte ratios in whole blood and spleen of rats were determined using flow cytometry after pretreatment.5.Determination of trace elements in rat serumThe selenium,chromium,copper,iron,manganese,zinc,boron,vanadium,molybdenum,strontium,and cobalt in rat serum were determined by ICP-MS after pretreatment.Results1.Rat growth statusThe rats in the TE and HFD+TE groups showed better growth and development compared with the control and HFD rats.With increasing feeding time,the body weight of HFD rats continued to increase,and the difference with control and HFD+TE rats gradually increased(P<0.05,P<0.01),and trace elements supplementation reduced body weight for either control or HFD rats in all weeks of feeding(P<0.05,P<0.01).Trace elements supplementation in all batches reduced serum TG levels in control and HFD rats(P<0.05,P<0.01).With increasing feeding time,trace elements supplementation reduced serum TC levels in HFD rats(P<0.01).2.Effect of multiple trace elements supplementation on antioxidant levels in rats on highfat dietWith the increase of feeding time,high-fat diet could decrease GSH level(P<0.01)and increase MDA content(P<0.05,P<0.01)in serum and spleen of control rats,respectively,while supplementation with trace elements could increase GSH level(P<0.05,P<0.01)and decrease MDA content(P<0.01).Trace elements supplementation in all batches increased the serum TAOC levels in the control and high-fat diet groups(P<0.01).With increasing feeding time,serum and spleen SOD,CAT,and GSH-Px enzyme activities decreased in HFD rats compared with control rats in all batches with statistically significant differences(P<0.05,P<0.01),while serum MPO and XOD enzyme activities increased in all batches(P<0.01),while trace elements supplementation increased the serum and spleen SOD,CAT,and GSH-Px enzyme activities in all batches(P<0.05,P<0.01),respectively.SOD,CAT,and GSH-Px enzyme activities in serum and spleen of control and HFD rats(P<0.05,P<0.01),increased GR enzyme activities in serum(P<0.01),and decreased MPO and XOD enzyme activities in serum(P<0.01),respectively.3.Effect of multiple trace elements supplementation on serum levels of inflammatory factors in rats on high-fat dietWith increasing feeding time,the high-fat diet increased the serum levels of IL-2,IL-6,and TNF-α(P<0.05,P<0.01)and decreased the levels of IL-4 and IL-10(P<0.05,P<0.01)in the control rats,respectively.In all batches,TE and HFD+TE rats showed different decreases in serum levels of IL-2,IL-6,and TNF-α(P<0.05,P<0.01)and increases in levels of IL-4 and IL-10(P<0.05,P<0.01)compared with controls and HFD.4.Effect of multiple trace elements supplementation on immune function in rats on highfat dietCompared with control rats,high-fat diet decreased the proportion of CD3+T lymphocytes in whole blood(P<0.01),decreased the proportion of CD4+T lymphocytes in spleen(P<0.01),increased the proportion of CD8+T lymphocytes in whole blood(P<0.01),and decreased the proportion of CD4+/CD8+ T lymphocytes in whole blood and spleen(P<0.01),and supplementation with trace elements all reversed the changes in these indicators in HFD(P<0.05,P<0.01).In contrast,trace elements supplementation reduced the proportion of CD8+T lymphocytes in whole blood and spleen(P<0.05,P<0.01),increased the proportion of CD4+/CD8+T lymphocytes in whole blood(P<0.05,P<0.01),and CD3+T lymphocytes in spleen(P<0.01)compared with the control group.5.Effect of multiple trace elements supplementation on serum elemental content in rats on high-fat dietWith increasing feeding time,high-fat diet could reduce the serum levels of boron,vanadium,chromium,cobalt,copper,and zinc compared with control rats,respectively(P<0.05,P<0.01).Trace elements supplementation increased the serum levels of boron,vanadium,chromium,iron,cobalt,copper,zinc,selenium,and strontium in all batches of rats compared with control and HFD rats,respectively(P<0.05,P<0.01).ConclusionSupplementation of multiple trace elements increased the lipid metabolism ability of rats on high-fat diet,improved dyslipidemia,reduced body weight and decreased the occurrence of obesity.Trace elements may be the active center of antioxidant enzymes,and supplementation with multiple trace elements can effectively improve the activity of antioxidant enzymes and enhance the resistance to oxidative stress.Supplementation of multiple trace elements can improve the anti-inflammatory ability of the body,reduce inflammatory response,enhance immunity,and improve immune disorders.