Study On Lipid-lowering Effect Of ?-oryzanol And Its Mechanism

Oryzanol is one of the most important active substances in rice bran.It is a natural mixture of ferulic acid ester of ferulicillin and ferulic ester of sterol,which has the functions of scavenging free radicals,oxidation,hypoglycemia,anti-tumor and other physiological activity.The lipid-lowering effect of the study is still in its infancy;its molecular mechanism of suppressing hyperlipidemia and relieving obesity is still unclear.In this thesis,y-oryzanol was used as the research object to induce the differentiation of 3T3-L1 preadipocytes by using a high fat diet-induced mouse model of hyperlipidemia and isobutyl-methyl-xanthine(IBMX),insulin and dexamethasone In vitro model to evaluate the hypolipidemic effect of oryzanol,and on this basis to explore the molecular mechanism of oryzanol lipid-lowering,for the development of functional foods to provide a theoretical basis,but also for the scientific diet guidance.Hyperlipidemia mice model was established by long-term feeding of high-fat diet to evaluate the lipid-lowering effect of oryzanol on hyperlipidemia.Thirty ICR experimental male mice were randomly divided into control group,high fat group and protective group,with 10 mice in each group.High fat diet induces hyperlipidemia in mice induced by high fat diet.After 11 weeks of oryrosin administration,the mice are sacrificed and the effect of oryzanol on body weight,liver and epididymal fat pad of hyperlipemia mice and the effect on serum lipoprotein are analyzed.Liver and fat pathological observation,the liver grinding,RNA and protein extraction,RT-qPCR technology and western blot were used to study the molecular mechanism of oryzanol lipid-lowering.The results showed that oryzanol can alleviate the weight gain of mice during high-fat diets;from the point of view of the appearance phenotype of liver size and color,the protective group can.Oryzanol on mice with high fat and mouse organ has some protective effect.Effects of oryzanol on the level of blood lipid in high fat diet mice.In order to test the effect of oryzanol on the blood lipid level of mice,we obtained the serum for biochemical detection.The results showed that oryzanol can effectively reduce the levels of total cholesterol(TG),total triglyceride(TC),low density lipoprotein(LDL-c)and increase high density lipoprotein(HDL-c).HE staining of the liver and adipose tissue sections showed that the pericyte of the liver cells of the hyperlipidemic group increased with vacuolar vacuoles;the volume of the adipose tissue became larger,and the oryzanol significantly inhibited the effects of the high-fat diet on it.The liver RNA and protein were extracted for real-time PCR and Western blot respectively.The results showed that lipid metabolism related factors such as PPARa,PPAR8 and SIRT1 in the protective group were significantly lower than those in the hyperlipidemia group,PPAR?,SREBP-1c,CD36,ACC and FASN were up-regulated,and the expression level of protective group was significantly lower than that of high-fat group.The expression of TNF-?,IL-1?,IL-6 and iNOS were significantly up-regulated while the expression of inflammatory cytokines in the protection group was lower than that in the hyperlipidemia group.The RNAs extracted from liver tissues of mice were analyzed by gene chip,and the genes of Cyp51,Fads1,Srfp5,Sult3a1,Ywhang and Cyp4a12a,Eif4ebp2,K1f10,Mt2,Scd3,Slc27a2 and Tsku were selected for qPCR analysis.The results are basically consistent with gene chip data,and we speculated that these genes involved in lipid metabolism-related reactions.The data of the chip was analyzed by IP A pathway network:two protein molecular networks were constructed,and important network node proteins such as retinoic acid-related orphan receptor alpha and Ikaros zinc finger protein were obtained.It discloses a variety of biological functions such as cancer,glucose metabolism,lipid metabolism and mineral metabolism.Based on in vivo experiments,we continued to explore the hypolipidemic physiological functions of oryzanol by in vitro cell model.Hoechst staining was used to determine the effect of different concentrations of oryzanol on cytotoxicity.The results indicate that oryzanol has no toxic effect on 3T3-L1 cells at a concentration range of 0-200?g/mL.The final concentrations of oryzanol test were 50,100,200 ?g/mL.In the control group,a large number of lipid droplets stained with oil red O were observed.When the dose of oryzanol was increased,the amount of lipid droplets dyed orange gradually decreased,and the quantitative results of extraction confirmed the accumulation of fat in the protective group gradually decreases.Real-time PCR and Western blotting were used to extract RNA and protein respectively.The results showed that the concentration of oryzanol from 0 to 200 ?g/mL PPAR-?,PPAR-? gradually increased;PPAR-?,SREBP-1c,ACC and FASN expression decreased gradually.The activation of ERK,p38,JNK in MAPK pathway in each group of cells was tested.The phosphorylation of ERK and JNK increased,while the phosphorylation of p38 decreased.Oryzalin may inhibit adipocyte differentiation through regulating MAPK signaling pathway.To sum up,in the aspect of animals,oryzanol can reduce macroscopical phenotype,reduce body weight,liver weight and fat pad weight of obese mice induced by high-fat diet,decrease blood lipid index,regulate lipid metabolism related factors and reduce inflammatory cytokines.In the cell,oryzanol can inhibit the differentiation of preadipocytes and regulate the gene of lipid metabolism,confirmed that oryzanol in the 3T3-L1 preadipocytes ERK,JNK,p38 pathway regulation,the pathways involved in transcription,lipid metabolism also need to do further research and enhance to understand the lowering-lipid molecular mechanism of oryzanol.