Studies On Effects And Mechanism Of High-Fat Diet On Oxidative Stress And Lipid Metabolism In Mice

Diet is established among the most important influences on health in modern society.The incidence of chronic metabolic diseases such as obesity is causally related to the globally upward trends of westernized dietary pattern,which is characterized by excessive intake of fat,saturated fatty acids and processed meat products with insufficient consumption of fresh fruits and vegetable.High-fat diet not only increases mitochondrion-derived reactive oxygen species,but also increases reactive oxygen species generated by NADPH oxidase,leading to the oxidative stress,which in turn induced inflammation and immune response.Gut microbiota is considered as an critical bridge between diet and health.Both dietary fat level and dietary protein sources（soybean protein and dry-cured pork protein）affect gut microflora composition and health.High-fat-diet-induced obesity is usually associated with oxidative stress,gut microbiota disorders and dysregulated lipid metabolism in the liver.Meanwhile,gender is also considered to be one of the important factors in human studies.Glrx1 is an critical member of the antioxidant enzyme system which essentially regulates the redox reactions and participate in oxidative stress.Recently,a great deal of attention has been focused on high-fat-diet-induced oxidative stress and obesity,but it is rarely reported and need to explore the mechanisms which Glrx1 impacts on oxidative stress,gut flora,inflammation level and glycolipid metabolism combining high-fat diet with different protein sources.In this study,after C57BL6/J wild type and Glrx1^-/-mice in both genders were fed with high-fat diet（45%calories from fat）and normal control diet（20%calories from fat）with dry-cured pork protein or soybean protein respectively for 12 weeks,the blood,fecal,liver tissue,colonic tissue and colonic content samples were harvested.The effects of Glrx1on oxidative stress,intestinal flora,inflammatory level,immune response and lipid metabolism in mice were studied from the perspectives of diet,genotype and gender,to provide scientific evidence in the role of dietary pattren and gender regulating body health.The main research contents and results are as follows:1.Effect of high-fat diet on physiological responses in miceIn order to explore the influence of high-fat diet on the physiological response of mice,four diets with different fat content and protein sources（soybean or dry-cured pork protein）were prepared and administrated to WT and Glrx1^-/-mice in different gender for 12 weeks.During feeding period,body weight and daily food intake were measured and the diets and drinking water were renewed every two days.Glucose tolerance tests were performed one week before excuted.The liver,spleen,brown adipose tissue,epididymal visceral adipose tissue,perirenal adipose tissue and cecum were weighed during sampling,and the length of the small intestine and colon was measured.The results showed that the obese phenotype occured in high-fat soybean protein treated Glrx1^-/-male mice with glucose tolerance.High-fat diet with dry-cured pork protein did not lead to the significant body weight gain in WT mice,but the effect was amplified 30.73 times in Glrx1 defeciency mice（female especially）.Gender differences exsited in response of body weight gain to high-fat diet with different dietary proteins（soybean protein and dry-cured pork protein,P<0.05）.High-fat diet with soybean protein induced body weight gain in WT mice with gender differences.Glrx1defiencey further promoted high-fat diet induced fat accumulation with distinct gender differences（P<0.05）.In brief,The above results suggested that Glrx1 knockout amplifies the body changes induced by high-fat diets with gender differences.As a bridge between diet and host health,the changes of intestinal flora and its metabolites in male and female Glrx1^-/-mice fed with high-fat diet need to be further studied.2.Effect of high-fat diet on gut microbiota and its metabolites of miceTo explore the effects of dietary fat levels,dietary protein species,genotype and gender on the gut flora and its metabolites in mice,genomic DNA from fresh feces of mice was extracted and subjected to bioinformatics analysis of OTUs by 16S r RNA gene amplicon sequencing.Blood samples were collected to measure the concentration of LBP,and colonic contents were collected to measure the level of SCFAs.The results showed that the composition of gut microbiota were mainly affected by diet,followed by gender and genotypes in both soybean protein and dry-cured pork protein groups.Glrx1 knockout and high-fat soybean protein diet treatment significantly increased the relative abundance of Firmicutes and decreased the relative abundance of Bacteroidetes（P<0.05）.High-fat diet with dry-cured pork protein significantly increased the relative abundance of Firmicutes and decreased the relative abundance of Deferribacteres（P<0.05）.Responses of gut microbiota to the same diet varied with gender and enlarged by Glrx1 deficiency.High-fat diets induced gut microbiota disorder,which in turn led to significate increase of LBP in blood and significate decrease of SCFAs in colon contents（P<0.05）.Consequently,the influence of gut microbiota metabolites on oxidative stress and inflammation in colonic tissue were needed to be investigated.3.Effects of high-fat diet on colonic oxidative stress and inflammation in miceIn order to explore the influence of oxidative stress and inflammation in colonic tissue and systemic immune response,blood samples,spleen tissue,colonic tissue and content were harvested and the relative indicators were determined.The percentage of immune cells were determinated by flow cytometry in spleen tissue.The results showed that high-fat diet（soybean protein and dry-cured pork protein）triggered the imbalance of redox reaction and subsequently induced oxidative stress in colonic tissue.In female WT colonic tissue,high-fat diets up-regulated the of My D88 in Toll-like receptor signaling pathway,thereby activated NFκB signaling pathway by up-regulating Ikkβ.On the other hand,high-fat diets activated MAPK signaling pathway through promoting Jun gene expression,thus up-regulated pro-inflammatory factor IL1βand TNFα,chemokine Ccl2 and Cxcl10 gene expression.The proportion of immune cells in spleen decreased in high-fat diet groups and gender response difference exsited.Glrx deficiency would further enhanced the gender response difference.Collectively,high-fat diets induced oxidative stress in colonic tissue,and oxidative stress induced by Glrx1 deficiency further activated inflammation-related pathways,leading to inflammatory responses in colonic tissue,and chemokine CXCL10recruited immune cells such as NK,which in turn caused systemic immune responses.4.Effects of high-fat diet to oxidative stress and lipid metabolism in liver tissueIn order to explore the mechanism which Glrx1 had an influence on oxidative stress,lipid metabolism in live tissue induced by high-fat diet.Serum lipids,oxidative stress and genes related to lipid metabolism in liver tissue were determined.The results showed that high-fat diets（soybean protein and dry-cured pork protein）increased the levels of serum triglyceride,total cholesterol,low density lipoprotein cholesterol,reduced the level of high density lipoprotein cholesterol（P<0.05）.Glrx1 knockout exacerbated high-fat diet induced hyperlipoidemia.In the case of feeding the same diet,the levels of triglycerides total cholesterol,high density lipoprotein cholesterol and low density lipoprotein in the same genetic background male mice were significantly higher than female ones.High-fat diet induced liver oxidative stress and liver injury in mice（P<0.05）.Glrx1 defiency would further enhanced oxidative stress and liver injury,and gender response difference exsited.Gender difference in high-fat diet activated Keap1-Nrf2/ARE signaling pathway（P<0.05）.Glrx1 defiency would further stimulated high-fat diet to activate Keap1-Nrf2/ARE signaling pathway,especially male mice.High-fat diets（soybean protein and dry-cured pork protein）would up-regulated genes expression（Hmgcr,Srebf2,Srebf1c,Cd36,Fas and Scd1）related to cholesterol metabolism and lipid metabolism in liver（P<0.05）.Glrx1knockout further up-regulated above genes expression,but decreased Scd1 gene expression in soybean protein group（P<0.05）.The m RNA levels of above six genes in female mice fed with soybean protein were significantly lower than male mice（P<0.05）.In summary,high-fat diet induced liver oxidative stress and liver damage.Glrx1 knockout would activated Keap1-Nrf2/ARE signaling pathway and up-regulated Srebps gene expression,which in turn promotes de novo lipogenesis and cholesterol synthesis,and ultimately induced hyperlipoidemia.