| Objective: Alcoholic fatty liver is a kind of disease caused by long-term heavy drinking.In recent years,the global prevalence of alcoholic fatty liver has been increasing,which has greatly affected people’s physical and mental health.Tartary buckwheat is a kind of precious medicinal and edible cereals,rich in flavonoids,and has many health effects such as anti-oxidation,anti-inflammatory,lowering blood pressure,lowering blood sugar,and lowering lipids.Our study intends to construct a mouse model of alcoholic fatty liver,from the perspective of lipid metabolism,to study the regulatory effect of tartary buckwheat extract on the lipid metabolism of alcoholic fatty liver in mice.Methods: 1.C57BL/6J mice were divided into 5 groups: the normal control group(Normal),the alcoholic fatty liver model group(Ethanol),and the low-dose(100 mg/kg)tartary buckwheat extract administration group(LTBE),the medium-dose(200 mg/kg)tartary buckwheat extract administration group(MTBE),and the high-dose(400 mg/kg)tartary buckwheat extract administration group(HTBE).The tartary buckwheat extract group was pre-administered(1 time/day)for 2 weeks.Then the mice in the model group and the tartary buckwheat extract administration group were fed with Lieber-De Carli alcohol liquid to make them suffer from alcoholic fatty liver,while the normal control group was fed with Lieber-De Carli control liquid.Two weeks later,blood was collected from the saphenous vein,and the levels of AST,ALT and TG in plasma were measured to evaluate the effect of the therapeutic effect of tartary buckwheat extract.Then,the frequency of administration of tartary buckwheat extract was changed to 2 times/day,and the modeling process was continued.After another two week administration,the animals were sacrificed,and the blood and liver were collected.H&E staining and Oil Red O staining are used for liver slices,and levels of AST,ALT,TG,TC,LDL-C,and HDL-C in plasma were measured,the levels of TG and TC in liver were detected.Western blot and q PCR were performed on several lipid metabolism-related pathways of interest.2.The liver samples of mice in the Normal,Ethanol and LTBE group were used for non-targeted lipidomics detection by UHPLC-Q-Exactive MS/MS technology.Multivariate statistical analysis methods such as PCA,PLS-DA,and OPLS-DA were used to screen the differential lipid metabolites between the Normal and the Ethanol group,and between the Ethanol and the LTBE group.The pathway analysis of the differential lipid metabolites between the Ethanol group and the LTBE group was carried out to explore the possible mechanism pathways of lipid-regulating effects for tartary buckwheat.Results: 1.The results of the lipid regulation effect of tartary buckwheat extract in mice:(1)Alcohol induced an increase in liver index in mice,while tartary buckwheat extract decreased the elevated linver index level;H&E staining and Oil Red O staining results showed that tartary buckwheat extract could improve liver lipid accumulation to a certain extent;(2)Alcohol induced an increase of AST,ALT,TG,TC,and LDL-C in plasma,a decrease of HDL-C in plasma,as well as an increase in TG and TC in liver.Tartary buckwheat extract could reverse these effects induced by alcohol.(3)Alcohol induced the expression of liver lipid synthesis-related regulatory factor SREBP1 and its downstream target molecules such as ACC and HMGR,while inhibiting the expression of lipid oxidation-related regulatory factor PPARα and its downstream target molecules such as CPT1 and CPT2.In addition,alcohol inhibited the LKB1-AMPK pathway by inhibiting the expression of SIRT1,reducing the phosphorylation of ACC,and in turn increasing its expression.Tartary buckwheat extract could reverse these effects.2.The results of lipidomics research of tartary buckwheat extract:(1)QC sample verification results showed that our lipidome detection method was stable,and the data was reliable,which could be used for subsequent analysis.(2)Sample correlation analysis and PCA analysis showed that the Normal group,the Ethanol group and the LTBE group had large differences between each other.There was minor intra-group difference for Normal and LTBE groups,while large intra-group difference was observed for Ethanol group.(3)Using the OPLS-DA model VIP>1,p<0.05 as the screening criteria,691 differential lipid metabolites between the Normal group and Ethanol group were obtained,and 556 differential lipid metabolites between the Ethanol group and the LTBE group were obtained.(4)Using OPLS-DA model VIP>3,p<0.05 as the screening criteria for further screening,7 different lipid metabolites between the Normal group and the Ethanol group were obtained(PG(20:5/22:6),TG(18:1/22:6/23:0),TG(20:3/22:3/22:3),WE(3:0/20:3),CL(17:1/22:6/22:6/22:6),PEt(18:0/18:1),and PEt(18:1/20:4)),and 3 different lipids between the Ethanol group and the LTBE group were obtained(PEt(18:0/18:1),PEt(18:1/20:4),and PE(15:0/18:2)).(5)The heat map showed that the LTBE group could effectively reverse the abnormalities of liver lipid profile in mice induced by alcohol.(6)KEGG pathway enrichment analysis and KEGG topology analysis results showed that tartary buckwheat extract was most likely to exert its lipid-regulating effect by affecting the glycerophospholipid metabolism pathway.Conclusions: 1.Tartary buckwheat extract could regulate liver lipid metabolism in mice by affecting SIRT1 and its downstream genes AMPK,SREBP1,PPARα,etc.2.Tartary buckwheat extract could effectively reverse alcohol-induced abnormal changes in mouse liver lipid profile,which was most related to the glycerophospholipid metabolism pathway. |
