The Study Of Effects Of Lycopene On Nonalcoholic Fatty Liver Disease In Mice And Its Mechanism Based On Lipidomics

Objective:By establishing the model of Non-alcoholic fatty liver disease(NAFLD)in C57BL/6J mice,and using different doses of Lycopene(LYC)to intragastric intervention,the effect of lycopene on serum and liver biochemical indices in NAFLD mice was explored.Lipidomics was employed to investigate the regulatory influence of lycopene on hepatic lipid metabolism in NAFLD mice,and molecular biotechnology was used to investigate probable mechanisms.It will give fresh proof regarding lycopene’s health impacts as well as new approaches for treating NAFLD effectively.Methods:1.C57BL/6J mice were continuously fed with high-fat diet(accounted for 60% energy)for 12 weeks to establish NAFLD model,meanwhile the control group(CON group)was administered a regular maintenance diet.Then all mice were randomly divided into model control group(MON group),low-dose lycopene group(50 mg/kg·d,LYC-L group),lycopene medium-dose group(100mg/kg·d,LYC-M group)and lycopene high-dose group(200 mg/kg·d,LYC-H group).During the intervention,the body weight and food intake were measured weekly.The animals were anesthetized and euthanized at the end of the 24-week period to obtain blood and liver tissues,and the final body weight and liver weight were recorded,as well as the liver index.Biochemical analysis was used to determine the levels of fasting blood glucose(FBG),alanine aminotransferase(ALT),aspartate aminotransferase(AST),total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),free fatty acids(FFAs),bile acid,and adiponectin in serum.In addition,the levels of TC,TG and adiponectin in liver tissue were determined.2.High performance liquid chromatography-mass spectrometry(HPLC-MS)was used to determine the lipid profiles in the liver of each group.To examine the changes in lipid metabolism between two groups,principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLA-DA)were used.The difference in lipids between the MON and other groups was shown using hierarchical clustering technique.KEGG database was used for pathway enrichment analysis to explore the potential metabolic pathways of lycopene regulating lipid metabolism in the liver.3.The gene expression levels of farnesoid X receptor(FXR),fibroblast growth factor 21(FGF21),sterol regulatory element binding protein 1C(SREBP-1C),fatty acid synthetase(FAS),peroxisome proliferator-activated receptor(PPAR-),and AMP-activated protein kinase(AMPK)in the liver of mice were detected using RT-PCR,and the relevant protein expression was detected using the western blotting(WB).Results:1.The NAFLD model was successfully established with more than 30% hepatic cell steatosis and huge fat vacuoles in the MON group’s liver.In the model group,the area of fat vacuoles in the microscopic field accounted for 31.11%.With an increase in lycopene dose,the number and area of fat vacuoles were reduced,and the degree of steatosis was dramatically reduced.Between the MON and lycopene intervention groups,there was no significant difference in the final body weight,food intake,liver index,and FBG levels.The levels of serum bile acid and adiponectin did not significantly differ among groups.When compared to the MON group,the LYC-H group had a significantly lower AST/ALT ratio(P<0.05).The serum levels of TC,HDL-C and FFAs in the LYC-H group were significantly lower than in the MON group,while serum TG and LDL-C levels in the LYC-L and LYC-M groups were statistically significant lower(P <0.05).The TC level in the liver was significantly lower in all intervention groups compared with MON group,while the TG level was significantly lower in the LYC-L and LYC-M groups,and the adiponectin level was significantly higher in the LYC-H group(P<0.05).2.Lipidomics research revealed a total of 551 metabolites in liver samples from five groups,with glycerolipids(GL),glycerophospholipids(GP),and sphingolipids(SP)being the most common.In the scatter plot of PCA analysis,the liver samples of the CON and MON groups were dispersed in different quadrants,showing a clear difference in lipid composition between the two groups,and the NAFLD model was successfully established.The OPLS-DA models compared MON group with other groups were robust and showed significant partitions.Differential lipids were mostly concentrated in glycerolipids,glycerophospholipids,and sphingolipids,according to the screen and hierarchical clustering analysis.The most obvious lipid difference between the CON and MON groups was the up-regulation of 37 TG levels,the down-regulation of 48 PC levels,and the downregulation of 18 PE levels in the MON group.The number of distinct lipid species between groups gradually increased as the lycopene intervention dose was increased.Sphingolipids mainly include ceramide non-hydroxy fatty acid – sphingosine(Cers),cers hexosyl ceramide non-hydroxy fatty acid--sphingosine(Her Cer)and sphingomyelin(SM).The levels of SM(d14:0/24:2)and Hex Cer/NS(d16:1/35:0)in the LYC-M and LYC-H groups were significantly lower than the MON group,and the levels of Cer/NS(d18:2/20:0)in the LYC-H group were significantly lower,and Hex Cer/NS(d18:1/31:0)in each intervention group were significantly lower.Among glycerophospholipids,there was an upward trend for PE(13:0/22:3),PC(17:1/17:1),PC(6:0/27:0)and PC(16:2e/24:4)after the lycopene intervention.The levels of diacylglycerol(DG)in glycerol were compared in glycerolipid.When compared to the MON group,DG(18:2/20:3)and DG(20:3/20:4)levels were considerably lower in the LYC-H group,while DG(20:3/22:6)and DG(22:4/22:6)levels were significantly lower in the LYC-M group and LYC-H group(P<0.05).KEGG metabolic pathway enrichment analysis showed that high fat altered glycerophospholipid metabolism,choline metabolism,and the retrograde enannabinoid signaling pathway in C57BL/6J mice.Lycopene supplementation regulated cholesterol metabolism,fat digestion and absorption,the regulation of lipolysis in adipocytes,lipid and atherosclerosis signaling pathway in NAFLD mice.3.The PCR revealed that the FXR gene was significantly up-regulated in the LYC-L group,the gene expressions of PPAR-α and AMPK in the LYC-H group were significantly up-regulated compared to the MON group,and the gene expression of SREBP-1C gene was significantly downregulated in the LYC-L and LYC-H groups(P<0.05).The WB results showed that protein expressions of FGF21 and PPAR-α in the LYC-H group were considerably higher than in the MON group,whereas the protein expressions of SREBP-1c in the LYC-H and LYC-L groups were significantly lower(P<0.05).The expression of FAS protein in LYC-M group was significantly decreased(P<0.05).Conclusion1.Lycopene could reduce the lipid levels in serum and liver of NAFLD mice,and improve the lipid accumulation and the liver damage.2.The regulatory effect of lycopene on the liver lipid metabolism profile is mainly concentrated in glycerolipids,glycerophospholipids and sphingolipids,suggesting that lycopene intervention may alleviate the liver lipid toxicity by reducing sphingolipid levels,and reduce hepatic lipid accumulation by reducing glycerolipid levels in the liver.Lycopene may regulate hepatic lipid metabolism through cholesterol metabolism,fat digestion and absorption,lipolysis pathway regulation,and lipid and atherosclerosis signaling pathway in NAFLD mice.3.Lycopene may limit hepatic fatty acid and TG synthesis via the FXR-SREBP-1C-FAS pathway,while also promoting fatty acid oxidation and controlling hepatic lipid and energy metabolism via the FXR-PPAR-FGF21-AMPK pathway.The greatest significant regulating impact on hepatic lipid metabolism was found in LYC-H group.