Study On The Effects And Mechanism Of Peripheral Aβ Mediated Lipid Metabolism Disorders On Cognitive Function

Mild cognitive impairment is an intermediate stage between normal cognition and Alzheimer’s disease(AD),which is a critical period for the prevention of AD.Epidemiological evidence suggests that there is a significant positive association between lipid metabolism disorders and cognitive impairment.It is important to explore the potential mechanism of lipid metabolism disorders-induced cognitive impairment for the early prevention of AD.β-amyloid(Aβ)is one of the characteristic markers of AD,and the accumulation of Aβ in the brain is an important cause of cognitive impairment.More than40% of intracerebral Aβ can be transported to the periphery for clearance,and impaired peripheral Aβ clearance is closely related to the intracerebral Aβ accumulation and cognitive impairment.Studies have shown that plasma Aβ is a reliable marker of intracerebral Aβaccumulation and cognitive impairment.Several epidemiological studies have investigated the association between blood lipid levels and plasma Aβ levels,but the results are inconsistent.Animal studies have found that lipid metabolism disorders can promote Aβdeposition in the brain.However,the effect of lipid metabolism disorders on peripheral Aβclearance and the mechanism involved remain unclear.Therefore,this study intends to investigate the association of blood lipids levels with plasma Aβ levels in a large sample cross-sectional study.Using in vivo and in vitro experiments to investigate the role and mechanisms of peripheral Aβ clearance on cognitive impairment induced by lipid metabolism disorders.Part I The associations of blood lipid levels with plasma Aβ40 and Aβ42 levels:a population-based cross-sectional studyObjective: To explore cross-sectional associations of blood lipid levels with plasma Aβ40 and Aβ42 levels.Methods: All participants came from Tongji-Ezhou cohort study.A total of 3250 subjects who met the inclusion and exclusion criteria were chosen.Plasma Aβ40 and Aβ42levels were measured by electrochemiluminescence immunoassay.Plasma triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C)were determined by enzymatic method.Multiple linear regression analysis was used to analyze the association of blood lipid levels with plasma Aβ40 and Aβ42 concentrations.The generalized additive model was built to assess the curvilinear relationship of lipid levels with plasma Aβ40 and Aβ42 levels.The confounding factors including sex,age,body mass index,smoking,drinking,physical activity,and APOE genotype were adjusted.Result: In the hyperlipidemic population and normal population,the median(interquartile ranges)plasma Aβ40 levels were 136.74(122.27,158.50)ng/L and 131.41(116.60,150.97)ng/L,respectively,and median plasma Aβ42 levels were 13.85(11.24,17.03)ng/L and 12.85(10.49,16.00)ng/L,respectively.Compared to people with normal blood lipids,the plasma Aβ40 and Aβ42 levels were higher in the hyperlipidemia group(P< 0.001).After adjusting for confounding factors,the results of multiple linear regression analysis showed that the plasma Aβ40 and Aβ42 levels were increased by 7.91%(95% CI:6.06%,9.79%;P-trend < 0.001)and 10.46%(95% CI: 7.22%,13.81%;P-trend < 0.001)respectively in the highest tertiles of TG levels compared to the lowest.Comparing the highest tertiles of TC levels to the lowest,plasma Aβ40 levels increased by 2.30%(95% CI:0.57%,4.05%;P-trend < 0.01),plasma Aβ42 levels increased by 4.68%(95% CI: 1.66%,7.77%;P-trend < 0.01).The plasma Aβ42 levels were increased by 6.14%(95% CI: 2.97%,9.42%;P-trend < 0.001)in the highest tertiles of LDL-C levels compared to the lowest.In multivariable generalized additive model,TG levels showed a non-linear association with plasma Aβ levels(Aβ40: EDF = 3.60,P < 0.001;Aβ42: EDF = 2.41,P < 0.001).TC levels also showed a non-linear association with plasma Aβ levels(Aβ40: EDF = 2.07,P < 0.01;Aβ42: EDF = 2.80,P < 0.001).LDL-C levels showed a linear association with plasma Aβ42levels(EDF = 1.00,P < 0.001).Conclusions: This cross-sectional study showed a nonlinear positive correlation of TG and TC levels with both plasma Aβ40 and Aβ42 levels,and a linear positive correlation of LDL-C levels and plasma Aβ42 levels.Part II The effect of lipid metabolism disorders on cognitive function and Aβ levelsin ratsObjective: To investigate the effect of lipid metabolism disorders on intracerebral Aβand cognitive function as well as to investigate the role of peripheral Aβ in it.Methods: Twenty-four 4-month-old Sprague-Dawley(SD)female rats were randomly allocated into control group(n = 12)and high fat diet(HFD)group(n = 12).The Control and HFD group of rats were given normal diet or HFD(45 kcal% fat)for 12 months.The body weight and food intake of rats were recorded every week.After 12 months,the openfield test and elevated plus maze were performed in order to examine dietary effects on spontaneous rodent behaviors and anxiety-like behaviors.The novel object recognition and Morris water maze tests were performed in order to examine learning memory ability.Finally,all of the rats were sacrificed,the plasma and tissues were collected.Enzymatic method was used to detect plasma lipid levels.ELISA was used to detect the levels of Aβ40and Aβ42 in the cortex,hippocampus and liver.ECLIA was used to detect plasma Aβ40 and Aβ42 levels.WB and RT-q PCR were used to detect the expressions of proteins and m RNAs associated with Aβ production(APP)and Aβ degradation(IDE,NEP)in the cortex,hippocampus and Aβ clearance(LRP-1,IDE)in the liver.Result:(1)General performance of rats: compared to the control group,HFD-fed rats had more weight gain(P < 0.001),but there was no difference in food intake.Furthermore,HFD caused an increase in plasma TC(P < 0.05),TG and LDL-C(P < 0.01)levels but had no effect on HDL-C levels.(2)Cognitive-behavioral assessment: there were no difference in the time and distance spent in the center zone of the open-field test between two groups.There was also no difference in the time spent on the open arms of the elevated plus maze test between two groups.The HFD-fed rats had lower recognition index and discrimination index in the novel object recognition test(P < 0.001).In the Morris water maze tests,compared with the control group,the HFD-fed rats had increased latency time and total swimming distance on the fifth day of place navigation test(P < 0.01),as well as decreased time and distance travelled in the target quadrant and reduced platform crosses in the spatial probe test(P < 0.01).(3)The levels of Aβ40 and Aβ42 were increased both in the cortex(P< 0.05)and hippocampus(P < 0.01).Plasma Aβ40 levels were significantly higher in the HFD-fed rats(P < 0.05).And the levels of Aβ40 and Aβ42 were decreased in the liver(P <0.01).(4)Key genes and protein changes: HFD had no significant effect on the m RNA and protein expression levels of APP,IDE and NEP in the brain and IDE in the liver.But the LRP-1 protein and m RNA expression levels were decreased in the liver of HFD-fed rats(P< 0.01).(5)Pearson correlation analysis: plasma Aβ40 was positively correlated with Aβlevels in the cortex and hippocampus(cortex: r = 0.66,P < 0.01;hippocampus: r = 0.68,P< 0.01).LRP-1 in liver was positively correlated with Aβ40 and Aβ42 levels in liver(Aβ40:r = 0.77,P < 0.001;Aβ 42: r = 0.68,P < 0.01);LRP-1 in liver was negatively correlated with plasma Aβ40 levels(r =-0.55,P < 0.05)and with Aβ levels in the cortex and hippocampus(cortex: r =-0.53,P < 0.05;hippocampus: r =-0.56,P < 0.05).Conclusions: Lipid metabolism disorders can lead to the accumulation of Aβ in the brain and cognitive impairment.Aβ accumulation in the brain is closely associated with elevated plasma Aβ levels and reduced hepatic LRP-1 expression.It is suggested that LRP-1-mediated hepatic Aβ uptake impairment may play an important role in lipid metabolism disorders-induced cognitive impairment.Part III The effect and mechanism of lipid metabolism disorders on Aβ uptake capacity in liverObjective: To investigate the effect and mechanism of lipid metabolism disorders on LRP-1-mediated hepatic Aβ clearance both in vivo and in vitro.Methods: In vivo studies,the rats were grouped in the same way as in Part II.After treatment,enzymatic assay was used to detect the lipid levels and function of liver.Using HE staining to observe the pathological state of rat liver and oil red O staining to observe the lipid deposition in liver.WB and RT-q PCR were used to detect the LRP-1-regulated(SREBPs and PPARs)proteins and m RNAs levels in the liver.(2)In vitro studies,Palmitic acid(PA)was used to treat Hep G2 cells to establish an in vitro hepatocyte model of lipid metabolism disorder.The dose of PA treatment was determined to be 400 μM for 24 h based on the CCK-8 cell activity assay.The uptake capacity of Aβ40 and Aβ42 by Hep G2 cells was assayed by flow cytometry.WB and RT-q PCR were used to detect LRP-1,SREBPs and PPARs proteins and m RNA levels in Hep G2 cells.SREBP-1c antagonist,PPARγ antagonist and PPARα agonist were respectively added subsequently on the basis of PA treatment to observe their effects on the Aβ uptake capacity of Hep G2 cells and the effects on the modulation of LRP-1.Result:(1)In vivo studies: HFD-fed rats showed disordered hepatic cords and significant lipid deposition in the liver.Compared to the control group,the TG(P < 0.001),TC(P < 0.05)levels in liver and ALT(P < 0.05),AST(P < 0.01)levels in plasma were elevated in the HFD-fed rats.The SREBP-1c(P < 0.01)and PPARγ(P < 0.05)protein expression levels were elevated and PPARα protein and m RNA expression levels(P < 0.05)were reduced in the liver of HFD-fed rats.(2)In vitro studies: Hep G2 cells treated with PA showed significant lipid deposition and decreased uptake of both Aβ40 and Aβ42(P < 0.001).Furthermore,PA treatment significantly reduced the expression levels of LRP-1 protein(P< 0.001)and m RNA(P < 0.05),increased the SREBP-1c(P < 0.05)and PPARγ(P < 0.001)proteins and decreased the PPARα proteins and m RNAs(P < 0.05)expression levels.Treatment with PPARα agonists reversed the PA-induced decrease in LRP-1 protein expression levels(P < 0.05),but neither SREBP-1c nor PPARγ antagonists had effect on LRP-1 expression levels.Meanwhile,PPARα agonist significantly improved Aβ40(P < 0.05)and Aβ42(P < 0.01)uptake capacity in the PA-treated group.Conclusions: Lipid metabolism disorders inhibit hepatic LRP-1 levels through downregulation of PPARα and subsequently reduced hepatic Aβ uptake capacity.