| Non-alcoholic fatty liver disease is the most common chronic disease,accompanied by complications such as hyperlipidemia,hypertension,and insulin resistance,which seriously threaten human health.At present,clinical drug treatment is the main treatment,but the drug effect is unstable and has strong side effects.The treatment of non-alcoholic fatty liver disease should focus on prevention,so it may be the right idea to develop functional foods or beverages to reduce the incidence.Sinapine is the most important rapeseed polyphenol.It is known to have antioxidant,anti-inflammatory and anti-tumor properties.Current studies have shown that sinapine can improve cardiovascular diseases and myocardial infarction.The protective effect of sinapine on liver injury has also been reported,but the specific impact mechanism is still unclear.This work takes sinapine as the research target,based on the theory of "multiple hits" in non-alcoholic fatty liver disease,systematically observes the mechanism of sinapine on nonalcoholic fatty liver disease from multiple angles such as in vitro and in vivo experiments,as well as intrahepatic and intestinal pathways,and is expected to provide a theoretical basis for the development of functional foods and clinical trials.The main research content and results of the study include:(1)The effect of sinapine on the lipid metabolism of THLE-2 hepatocytes was studied.The lipid accumulation model induced by oleic acid in vitro was established by THLE-2 cell line,the nutritional function of sinapine was evaluated,and the biochemical indexes,lipid metabolism indexes and oxidative stress indexes of liver cells were measured.The influence and function of these indicators are finally verified at the molecular level to determine the relevant pathways of sinapine on lipid accumulation.The working concentrations of sinapine were chosen for 5,20,and 80 μM through cytotoxicity experiments,and the oleic acid modeling concentration was 0.4 m M.The results showed that sinapine showed a concentration gradient to increase the survival rate of THLE-2 cells treated with oleic acid.The results of oil red O staining and lipid level detection showed that sinapine pretreatment reduced oleic acid-induced lipid metabolism disorders,and the TG and TC contents of cells treated with 80 μM sinapine were reduced by 52.18% and 23.21%,respectively,compared with the model group.Sinapine can reduce the production of lipid peroxides and maintain the mitochondrial membrane potential of liver cells,protect mitochondria from performing normal functions,and can also inhibit DNA damage caused by oxidative stress and protect the integrity of liver cells.The intervention of sinapine can up-regulate the expression of lipid oxidation-related gene PPARαand fatty acid oxidation gene ACOX1,thereby reducing the damage of oleic acid to β oxidation and reducing lipid accumulation.(2)The effect of sinapine on the oxidative stress of THLE-2 hepatocytes was studied.The oxidative stress and inflammation model induced by lipopolysaccharide was established by THLE-2 hepatocyte in vitro experiment,the nutritional function of sinapine was evaluated,and the related pathways of sinapine on oxidative stress were observed.The results showed that within a certain concentration range,sinapine increased the survival rate of THLE-2 cells treated with lipopolysaccharide in a concentration-dependent manner.The AST levels of sinapine at 20 and 80 μM concentrations were down-regulated by 24.77% and 44.43%respectively compared with the model group.Furthermore,sinapine can increase the activity of oxidase,and the levels of GSH-Px and SOD in the sinapine(80 μM)group were significantly increased(p<0.01),increasing by 49.60% and 40.61% to 2.06±0.14 and 3.57±0.22 U/mg protein,respectively,and the MDA level was reduced by 27.67%(p<0.01).Sinapine prevents the increase in ROS levels induced by LPS,improves the mitochondrial membrane potential of liver cells,inhibits DNA damage caused by oxidative stress,and protects the integrity of liver cells.In addition,sinapine can be used as an inhibitor of the mitochondrial membrane protein MCJ protein to reduce the damage of oxidative stress to liver cells.The molecular docking results show that sinapine has a good docking with MCJ protein and can be used as a protein ligand.(3)The dynamic distribution of sinapine in mice was studied.After intragastric administration of sinapine to C57BL/6 J mice,an HPLC method was established for the determination of sinapine and its hydrolysate erucic acid in the three organs and tissues of the liver,stomach,and small intestine.The precision and accuracy were investigated,and the results showed that the HPLC determination methods established for the two active components in the three organs of mice met the requirements of in vivo component analysis.Subsequently,a tissue distribution study was carried out.C57BL/6 J mice were gavaged with sinapine and sacrificed at 15 min,30 min,120 min,and 300 min after the administration.The liver,stomach,and small intestine tissues were taken out immediately.HPLC analyzes the content of the target component.The results showed that the concentration of sinapine in the organs from high to low after entering the body was: small intestine>stomach>colon>liver,and reached the peak value in the liver 120 min after intragastric administration,which was 4389.1±747.49 ng/g,and the concentration decreased after 300 min.Sinapine is far greater than the concentration of sinapine in the liver,and sinapine can enter the liver to directly exert its nutritional functions.(4)The mechanism of the effect of sinapine on liver injury in mice was investigated in vivo.The potential effect of sinapine in the C57BL/6 J mouse model induced by high-fat diet was studied.The mice were given diet intervention in the form of free intake,including low-fat diet,high-fat diet,high-fat diet containing common rapeseed oil and high-fat diet containing fortified sinapine oil(500 mg/kg oil),and feeding for 12 weeks.Then observe the protective effect of sinapine on the liver from the pathways of liver and intestines.The results showed that the NAS scores of the high-fat diet group and the sinapine-fortified group were 4 and 1,respectively.Compared with mice fed a high-fat diet,the addition of sinapine can significantly reduce the body weight and the formation of non-alcoholic fatty liver disease in mice.Sinapine supplementation effectively reduced blood lipid levels and inhibited insulin resistance,and liver AST/ALT levels were down-regulated by 26.08% compared with the high-fat group,and liver antioxidant enzyme levels were increased by 44.00%.The addition of sinapine improved the degeneration of mouse adipocytes and the accumulation of lipids in the liver.The results of gene expression levels showed that this change was achieved by down-regulating the SREBP-1c/FAS pathway and up-regulating the PPAR-α/ACOX1 pathway.In addition,the results of the intestinal flora analysis showed that the enhancement of sinapine increased the abundance of Akkermansia and Blautia in the gut,thereby increasing the production of SCFAs and downregulating the expression of downstream inflammatory factors by activating the SCFAs/GPR43 pathway.The level of intestinal inflammation is reduced to protect the normal function of the insulin receptor in adipose tissue,prevent the reduction of insulin resistance and even reverse NAFLD. |
PDF Full Text Request