| Dyslipidemia is a common metabolic disease and related to the occurrence and development of atherosclerotic cardiovascular disease(ASCVD).Although comprehensive management of traditional risk factors such as LDL-C can achieve considerable results,some patients are still at risk of macrovascular or microvascular events,which is known as residual cardiovascular risk.The residual cardiovascular risk is mainly related to hypertriglyceridemia and can be reduced by decreasing triglyceride(TG)level.Apolipoproteins(Apos)play an important role in the metabolism of TG,among which ApoCⅢ can lead to dyslipidemia through multiple pathways.A number of clinical trials have confirmed that individuals with high ApoCⅢ level are prone to atherosclerosis,and it is an effective method to prevent hyperlipidemia and cardiovascular disease by reducing TG level.However,there is still no specific drug that can reduce ApoCⅢ level.ApoCⅢ is mainly produced in the liver,and the synthesis and secretion of ApoCⅢ are obviously affected by dietary factors.Therefore,changing dietary structure is an important way to regulate the level of ApoCⅢ.Resveratrol(Res),as a natural polyphenol compound,widely exists in many plants such as grape,and has many functions including anti-inflammatory,anti-cancer,anti-oxidation,anti-atherosclerosis and regulation of lipid metabolism.It is still unclear whether resveratrol can regulate lipid metabolism by affecting the expression of ApoCⅢ.In this study,we conducted a high-fat meal test to evaluate the postprandial TG metabolism of paticipants,and compared the differences of ApoCⅢ levels among people with different lipid metabolism.Then we investigated the effect of high fat feeding on the expression of ApoCⅢ in the liver in both animal models and cell models.By observing whether resveratrol can regulate lipid metabolism by affecting the expression of ApoCⅢ,we hope to provide new ideas for the prevention and treatment of dyslipidemia and cardiovascular diseases.Part one The level of ApoCⅢ and postprandial hypertriglyceridemiaObjective:To investigate possible mechanisms of postprandial hypertriglyceridemia(PPT),we analyzed serum lipid,ApoA I,ApoB,ApoCⅡ and ApoCⅢ levels before and after a high-fat meal.Methods:We recruited 143 volunteers with normal fasting triglyceride(TG)levels.All subjects consumed a high-fat test meal.Venous blood samples were obtained during fasting and at 2,4,and 6 hours after the high-fat meal.PPT was defined as TG≥2.5 mmol/L at any time after the meal.Subjects were divided into two groups according to the high-fat meal test results:postprandial normal triglyceride(PNT)and PPT.We compared the fasting and postprandial lipid and ApoA I,ApoB,ApoCⅡ and ApoCⅢ levels between the two groups.Results:1.Comparison of fasting indicatorsFasting insulin,HOMA-IR,TG,TC,LDL-C,non-HDL-C,TG-rich lipoproteins remnants(TRLR),ApoB,ApoCⅢ levels and ApoCⅡ/ApoCⅢ in the PPT group were higher than those in the PNT group,while ApoA I/ApoCⅢ in the PPT group was lower than that in the PNT group,and the difference was statistically significant.2.Comparison of lipid and apolipoprotein levels after the high-fat mealThe levels of TG,TC,LDL-C,non-HDL-C,TRLR,ApoB and ApoCⅢ at each time point after the high-fat meal in the PPT group were higher than those in the PNT group,and the difference was statistically significant.The postprandial TG level peaked in the PNT group 2 hours after the meal but was significantly higher in the PPT group and peaked at 4 hours.TRLRs gradually increased within 6 hours after the high-fat meal in both groups.The area under the curve(AUC)of TG and TRLRs and the AUC increment were higher in the PPT group(P<0.001).ApoCⅢ peaked in the PNT group 2 hours after the meal and gradually decreased.ApoCⅢ gradually increased in the PPT group within 6 hours after the meal,exhibiting a greater AUC increment(P<0.001).3.Correlation and regression analysis of ApoCⅢ and other indicatorsFasting ApoCⅢ was positively correlated with age,systolic and diastolic blood pressure,body mass index(BMI),waist circumference,TC,TG,LDL-C,non-HDL-C,TRLRs,and ApoB(P<0.05).ApoCⅢ was an independent risk factor of PPT after adjustment for BMI,waist circumference,TC,LDL-C,and ApoB(P<0.001,OR=1.188).Summary:Increased expression of ApoCⅢ is an important cause of postprandial hypertriglyceridemia.Part two Effects of resveratrol on the expression of ApoCⅢ in the liver of high-fat fed miceObjective:The aim of this study was to establish animal models with a high-fat diet and to investigate the effect of the high-fat diet on the expression of ApoCⅢ in the liver and the intervention effect of resveratrol.Methods:C57BL/6J mice were randomly divided into control group(Con)and high-fat diet group(HFD).After 8 weeks,eight mice were chosen randomly from HFD as high-fat diet+resveratrol group(HFD+Res).HFD+Res group was given resveratrol 60 mg/kg/d;while Con and HFD group were given sodium chloride solution by daily gavage.After 8 weeks of resveratrol treatment,fasting blood glucose was measured.Serum and liver tissue samples were taken.The blood lipids and insulin level was measured.Morphological changes of liver were observed by H&E staining and Oil red O staining.The expression of ApoCⅢ,Fox O1,HNF-4α,PGC-1β and PPARα were tested by real-time polymerase chain reaction(RT-PCR)and western blot.Results:1.General indicators after resveratrol treatment for 8 weeksBody weights of mice in the HFD group were significantly higher than in the Con group,and were not altered by resveratrol treatment.Fasting blood glucose and insulin levels of the HFD group were significantly higher compared with the Con group.After resveratrol treatment,fasting blood glucose levels were significantly reduced in the HFD+Rev group,while with no changes on insulin levels.The QUICKI result for the HFD group was significantly lower than that of the Con group,and was significantly increased in the resveratrol group,indicating resveratrol improved the insulin resistance of HFD mice.2.Lipid profilesTC,TG and LDL-C levels were significantly higher in the HFD group compared with the Con group,and 8 weeks of resveratrol reduced TC,TG and LDL-C level.There were no differences of HDL-C among the three groups.This result indicated that resveratrol reduced blood lipid levels to some extent.3.Morphological comparisons between the three groupsH&E staining:no lipid droplets were observed in the Con group.Hepatocytes of the HFD group had a disordered and swollen structure,with different sizes of cytoplasmic lipid droplets.After resveratrol treatment,liver cell morphology was improved and lipid droplets were reduced.Oil red O staining:the Con group showed less red lipid droplets and a large number of red lipid droplets were observed in the HFD group.The number of lipid droplets in the HFD+Rev group was between the Con and HFD group.4.Hepatic expression of ApoCⅢ and transcription factors in miceThe levels of ApoCⅢ,Fox O1,HNF-4α,PPARα and PGC-1β in the HFD group mice were significantly higher than those in the Con group.After resveratrol intervention,the levels of ApoCⅢ,Fox O1 and HNF-4αwere decreased.5.Relative mRNA levels of ApoCⅢ and transcription factors in miceThe mRNA levels of ApoCⅢ,Fox O1,HNF-4α,PPARα and PGC-1β in the HFD group mice were significantly higher than those in the Con group.After the treatment of resveratrol,the mRNA levels of Fox O1,HNF-4α and ApoCⅢ significantly decreased.Summary:High fat diet increased the expression of ApoCⅢ,Fox O1,HNF-4α,PPARα and PGC-1β in liver of mice.Resveratrol reduced the expression of ApoCⅢ,Fox O1 and HNF-4α,and improved the hyperlipidemia of mice.Part three Resveratrol reduces the expression of ApoCⅢ in hepatocytes via HNF-4α and Fox O1Objective:The aim of this part study was to verify whether resveratrol can inhibit ApoCⅢ expression via HNF-4α and Fox O1 by up-regulating and down-regulating the expression of HNF-4α and Fox O1 in HepG2 cells.Methods:The cells were treated with 0.25 mmol/L palmitic acid,and were detected by Oil red O staining after 24 hours.Different resveratrol concentrations(100 μM,50 μM,40 μM,30 μM,20 μM and 10 μM)were added to investigate cell viability.HepG2 cells were divided into three groups:control group(Con),PA intervention group(PA),PA+Res 20 μM group(PA+Res),RT-PCR and western blot were performed to detect the levels of ApoCⅢ,Fox O1,HNF-4α,PGC-1β and PPARαafter intervention treatment for 24 hours.Cells were transfected with siRNA of Fox O1,and the HepG2 cells were divided into the following groups:control group(Con),PA group(PA),PA+Fox O1-negative control group(siRNA-NC),PA+Fox O1-knockdown group(siRNA-Fox O1).After transfection for 24 h,the protein and RNA was extracted.Western blot and RT-PCR was the same as before.Then we used the same method to knock down HNF-4α and repeat the above steps.HepG2 cells were transfected with plasmid carrying the Fox O1 gene to overexpress Fox O1,and the HepG2 cells were divided into:control group(Con),Fox O1-negative control group(Pl-NC),Fox O1overexpress group(Pl-Fox O1),Pl-Fox O1+Res 30 μM,Pl-Fox O1+Res 20 μM,Pl-Fox O1+Res 10 μM.Resveratrol intervention was performed 24 hours after transfection.After intervention for 24 h,we extracted the protein and RNA for western blot and RT-PCR detection.Then we repeated the above steps to overexpress HNF-4α.Results:1.Oil red O stainingThe cytoplasm of HepG2 cells was light blue in the Con group.A large amount of red lipid droplets was observed in the PA group,suggesting lipid deposition in HepG2 cells.The number of lipid droplets in the PA+Res group was between the two groups.2.Resveratrol cytotoxicity testThe survival rate of the Con group of cells was 96.6%.After treatment with 100 μM,50 μM,40 μM,30 μM,20 μM and 10 μM resveratrol,the cell viability was 52.6%、83.7%、83.1%、92.4%、93.0%and 95.1%,respectively.These results suggested that high concentrations of resveratrol had a cytotoxic effect on cells.3.ApoCⅢ and transcription factors protein expression in HepG2 cellsThe levels of Fox O1,HNF-4α,PPARα and PGC-1β in the PA group were significantly higher than those in the Con group,and the level of ApoCⅢ was also higher.Under the effect of resveratrol,the levels of Fox O1 and HNF-4α in the PA+Res group were lower than those in the PA group,and the level of ApoCⅢ also decreased.4.Relative mRNA levels of ApoCⅢ and transcription factors in HepG2 cellsThe mRNA levels of ApoCⅢ,Fox O1,HNF-4α,PPARα and PGC-1β in the PA group were significantly higher than those of the Con group.After resveratrol intervention,the mRNA levels of Fox O1,HNF-4α and ApoCⅢ in PA+Res group were decreased compared with PA group.5.Changes of ApoCⅢ mRNA and protein levels after knockdown the Fox O1 geneCompared with the Con group,the mRNA and protein levels of Fox O1 and ApoCⅢ in the PA group were significantly increased,while the mRNA and protein levels of Fox O1 and ApoCⅢ were decreased in siRNA-Fox O1 group,but there was no difference between the siRNA-NC group and Con group.After PA intervention,the mRNA and protein levels of Fox O1 and ApoCⅢ in the siRNA-Fox O1 group were lower than those in the PA group.These results indicated that Fox O1 could up-regulate both mRNA and protein expression of ApoCⅢ,and the knockdown of Fox O1 gene could reduce the expression of ApoCⅢ.6.Changes of ApoCⅢ mRNA and protein levels after knockdown of HNF-4α geneThe mRNA and protein levels of HNF-4α and ApoCⅢ in the PA group were higher than those in the Con group,while the mRNA and protein levels of HNF-4α and ApoCⅢ after siRNA-HNF-4α intervention were significantly lower than those in the Con group.The mRNA and protein levels of HNF-4α and ApoCⅢ in the siRNA-HNF-4α group after PA intervention were also lower than those in the PA group.This also confirms that HNF-4α can upregulate both mRNA and protein expression of ApoCⅢ,and knockdown of HNF-4α gene can reduce the level of ApoCⅢ.7.Effects of resveratrol on protein and mRNA levels in HepG2 cells overexpressing Fox O1 geneThe level of Fox O1 in HepG2 cells after Pl-Fox O1 intervention was significantly increased,and the level of ApoCⅢ was also higher than that in the Con group.After the treatment of resveratrol,the levels of Fox O1 and ApoCⅢ protein and mRNA in the Pl-Fox O1+Res groups were lower than those in the Pl-Fox O1 group,and the effect of 30 μM was stronger than that of 10 μM.It suggested that resveratrol can down-regulate the expression of ApoCⅢ by affecting Fox O1.8.Effects of resveratrol on protein and mRNA levels in HepG2 cells overexpressing the HNF-4α geneHNF-4α and ApoCⅢ protein and mRNA levels in the PL-HNF-4αgroup were higher than those in the Con group,while the effect of PL-HNF-4α was weakened with the intervention of resveratrol.The levels of HNF-4α and ApoCⅢ in the PL-HNF-4α+Res group were lower than those in the PL-HNF-4α group,and the effect of resveratrol at 30μM was stronger than that at 10 μM.These results indicated that resveratrol could down-regulate ApoCⅢ expression by affecting HNF-4α.Summary:Resveratrol reduced the expression of ApoCⅢ in HepG2 cells by inhibiting the expression of HNF-4α and Fox O1.Higher concentrations of resveratrol had a stronger inhibitory effect.Conclusion:1.Elevated ApoCⅢ level is an important cause of postprandial hypertriglyceridemia.2.Resveratrol can improve hypertriglyceridemia and dyslipidemia by inhibiting the expression of ApoCⅢ in liver.3.The mechanism of resveratrol inhibiting the expression of liver ApoCⅢ is to reduce the expression of HNF-4α and Fox O1. |
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