| Objective: Alzheimer’s disease(AD)is a progressive degenerative disease of the central nervous system.Beta-amyloid protein(Aβ)deposition in the brain is an important part of the pathogenesis of AD.Lipoprotein lipase(LPL)is closely related to the pathogenesis of AD.LPL localized on brain microvascular endothelial cells is thought to be derived from peripheral such as adipose.Previous studies have found that LPL expression in brain microvascular endothelial cells in AD models is reduced,which may be caused by the reductionof LPL expression in adipose tissue,and time-limited feeding can inhibit the decreaseof LPL expression in brain microvascular endothelial cells of AD mouse.However,whether time-limited feeding can antagonize the decrease of LPL expression in the brain microvascular endothelial cells of AD model mice by regulating the expression of LPL in adipose tissue remains to be confirmed.Whether the upstream transcription factor peroxisome proliferator-activated receptor γ(PPARγ)of LPL is involved in the regulation of LPL expression in AD adipose by time-limited feeding has not been reported.Earlier studies also found that histone deacetylase 2(HDAC2)and HDAC3 are important in the regulation of LPL expression of βOHB in SH-SY5 Y,which treated by Aβ,during time-limited feeding.The expressions and regulations of HDACs and LPL are tissue-specific.Therefore,whether HDAC2/3 is involved in time-limited feeding to regulate LPL expression in AD adipocytes via βOHB which still remain and whether PPARγ is involved in the above effects,remains to be studied.The main reason for the deposition of Aβ in the brain is due to the obstacle to its clearance.LPL can promote Astrocyte uptake and clear Aβ through the autophagolysosome pathway.The main way of Aβ clearance in the brain is to cross the blood-brain barrier(BBB).Brain microvascular endothelial cells are an important part of BBB.Our previous studies have shown that time-limited feeding can reduce Aβ deposition in the brain of AD mice.Whether brain microvascular endothelial cells LPL participate in intermitten fasting to reduce Aβ deposition in AD brain by regulating Aβ cross-BBB transport in the brain has not been reported.Low-density lipoprotein receptor-related protein 1(LRP1),which is located on the basement membrane side of brain microvascular endothelial cells,plays an important role in the translocation of Aβ across BBB in the brain.LPL can promote cell uptake of low-density lipoprotein(LDL)and increase the level of intracellular cholesterol.There is evidence that LDL can regulate the expression of LRP1 in vascular smooth muscle cells,and cholesterol can also affect the function of LRP1 by regulating the expression of depolymerin-like metalloproteinase 10(ADAM10)and ADAM17 in HEK cells.However,does LPL promote the entry of Aβ in the brain into the cell through the basement membrane side of the cell via LRP1,and whether LPL affects the primary endosomes(Rab5),sorting endosomes(Rab11)and the Aβ exflow pump ATP-binding cassette transporter B1(ABCB1),which participates in intermitten fasting to reduce Aβdeposition in the brain of AD model mice,has yet to be confirmed.In summary,this study combined with in vivo and in vitro experiments explored the role and possible mechanism of time-limited feeding to change the expression of LPL in adipose tissue of AD model mice to promote the transfer of Aβ across the blood-brain barrier in the brain.Methods: 1.Animals and Treatment: At 5-month-old,APPswe / PS1 d E9 double transgenic mice and littermate-born wild-type mice were divided into ADgroup and WT group(fed ad libitum),ADF groupand WT+ADF group(fed ad libitum everyother day and fasted the following day).5 months later,mice were excutedafter etheranesthesia.Abdominal adipose and brain of mice were taken.2.Cell culture: 2.1 First,3T3-L1 was selected and divided into control,βOHB,Aβ(0.5,2 μM)and βOHB + Aβ(0.5,2 μM)group,βOHB and βOHB + Aβ(0.5,2 μM)group were taken pretreatment with 1 m MβOHB for 3 h,then added 0.5 or 2 μM Aβ to Aβ(0.5,2 μM)and βOHB + Aβ(0.5,2 μM)group to continue culturing,andcollected cells after 12 h.Second,The si RNA interference experiment was divided into si RNA-HDAC2 or si RNA-HDAC3 group and Scrambled si RNA group.Add 50 n M HDAC2 or HDAC3 to cells in s si RNA-HDAC2 or si RNA-HDAC3 group,respectively.1% fetal bovine serum-free monoclonal antibody-containing cell culture medium of si RNA,1% fetal bovine serum-free monoclonal antibody-containing cell culture medium containing 50 n M negative control si RNA was added to the Scrambled si RNA group cells,and the cells were further cultured for 24 h,and all cells were collected.2.2 First,in the experiment of treating HBMEC with MβCD(which can significantly reduce the free cholesterol level of brainmicrovascular endothelial cells),the cells were divided into control and MβCD group(5 m M MβCD treatment for 30 min).Second,in the experiment of LPL intervention on HBMEC,the cells were divided into control,LPL,LDL and LPL + LDL group.LPL and LPL + LDL group were pretreated with 1 μg/m L LPL for 3 h,and the cells of these two groups were treated with 62.5 μg/m L LDL,and collect cells after 24 h.Last,HBMEC in vitro of BBB were divided into control,LPL,LDL and LPL + LDL group.After the cells in the upper chamber were treated with LPL or LDL(the operation method is the same as above),1 n M Aβ1-40 was added to the lower chamber,and collect cells and medium 1 hour later.3.Detection indicators and methods: 3.1 RT-PCR and Western Blotting were used to detect LPL,PPARγ,HDAC2/3 m RNA and protein expression and Ace-H3K9/H4K12 levels in adipose.3.2 The oil red "O" staining method was used to detect the lipid droplet level and the RT-PCR method was used to detect the PPARγ expression to determine the 3T3-L1 differentiation.RT-PCR and Western Blotting were used to detect LPL,PPARγ,HDAC2/3 m RNA and protein expression and Ace-H3K9 H4K12 levels in 3T3-L1 cells.3.3 RT-PCR and Western Blotting were used to detect the HDAC2/3 m RNA and protein expression(to determine the effect of silencing),and the LPL,PPARγ m RNA and protein expression.3.4 Using immunofluorescence to detect the expression levels of Aβ,LRP1,ADAM10,ADAM17,Rab5,Rab11 and ABCB1 in mouse brain microvascular endothelial cells(CD31 as a marker of brain microvascular endothelial cells).The Aβ level in serum were tested by ELISA.The cholesterol level of HBMEC cells were tested by ELISA.3.5 The expression of LRP1,ADAM10,ADAM17,Rab5,Rab11 and ABCB1 m RNA and protein were detected by RT-PCR and Western Blotting.The level of s LRP1 in the culture medium was detected by ELISA.3.6 The expression of tight junction protein ZO-1 in vitro of BBB detected by immunofluorescence.The Aβ levels in HBMEC and culture medium were detected by ELISA.4.SPSS 12.0 was used for statistical analysis of the data,and the difference was considered statistically significant when P <0.05.Results: 1.Time-limited feeding can inhibit the expression of LPL,PPARγ,HDAC2/3,and the expressionlevel of Ace-H3K9/H4K12 in the APPswe/PS1 d E9 double transgenicmice: Compared with the WT model group,the expressions of LPL,PPARγand Ace-H3K9/H4K12 in theadipose tissueof the AD model group were decreased,and the expressions of HDAC2/3 were increased(P <0.01).Compared with the AD mice,the expressions of LPL,PPARγ and Ace-H3K9/H4K12 were increased,while the expressionsof HDAC2/3 were decreased(P <0.01)in the ADF.2.βOHB can inhibit 0.5μMAβ-treated 3T3-L1 cells,LPL,PPARγ expression levels increase and inhibit 2μMAβ-treated 3T3-L1 cells,LPL,PPARγ expression levels or decrease.βOHB can inhibit Ace-H3K9/H4K12 levels decrease and inhibit the HDAC2/3 expressions increase.After 3T3-L1 differentiation,the cells showed a large number of dark red lipid droplets,and the expression of PPARγ was increased(P <0.01).Compared with the control group,the expressions of LPL,PPARγ and Ace-H3K9/H4K12 in the cellsoftheβOHB and Aβ(0.5 μM)group were increased,while the expressions of HDAC2/3 were decreased.In the Aβ(2 μM)group,the expressionsof LPL,PPARγ and Ace-H3K9/H4K12 were decreased,and the expressions level of HDAC2/3 were increased(P <0.01).Compared with the Aβ-treated(0.5 μM)group,the expressions of LPL,PPARγ and Ace-H3K9/H4K12 cells were decreased,theexpressions of HDAC2/3 were increased(P<0.01)of the Aβ(2 μM)and βOHB + Aβ(0.5 μM)groups.Compared with the Aβ-treated(2 μM)group,the expressions of LPL,PPARγ and Ace-H3K9/H4K12 were increased,while the expressions of HDAC2/3 were decreased(P <0.01)in cells of βOHB+ Aβ(2 μM)group.3.Silent cells HDAC2 or HDAC3 can reduce or increase the expression of LPL and PPARγ: Compared with the Scrambled si RNA group,LPL and PPARγ m RNA and protein expression levels of the cells in the si RNA-HDAC2 group are reduced.And compared with the Scrambled si RNA group,the si RNA-HDAC3 group The expression levels of LPL and PPARγ m RNA and protein in cells were increased.4.Intermitten fasting can inhibit the increase of Aβ levels,the decrease of LRP1 expression and the increase of ADAM10/17 expression in brain microvascular endothelial cells of AD model mice: Compared with WT mice,the Aβ levels in the brain microvascular endothelial cells were increased in AD and ADF mice(P <0.01),and the Aβ levels in the brain microvascular endothelial cells were decreased in ADF mice compared with that in AD(P <0.05).There was no significant difference inserum Aβ levels between ADF and AD group(P> 0.05).Compared with the WT mice,the expression of LRP1 in the brain microvascular endothelial cells of the AD mice weredecreased(P <0.01),and compared with AD mice,the expression of LRP1 in brain microvascular endothelial cells of ADF were increased(P <0.01).Compared with WT mice,the expression of ADAM10/17 in the brain microvascular endothelial cells of AD mice were increased(P <0.01),and the expression of ADAM10/17 in brain microvascular endothelial cells were decreased in ADF mice compared with AD mice(P <0.01).There was no significant difference in the expression of Rab5 and Rab11 of cerebral microvascular endothelial cells among each group(P> 0.05).The expression of ABCB1 in the brain microvascular endothelial cells were descreased in AD and ADF mice compared with WT mice(P <0.01),and there was no significant difference in the expression of ABCB1 between the brain microvascular endothelial cell of ADF and AD mice(P> 0.05).5.Decreased cholesterol levels of HBMEC can down-regulate LRP1 expression and up-regulate ADAM10/17 expression:In MβCD group,the intracellular cholesterol levelswere decreased,the LRP1 expression were decreased,s LRP1 levels in the culture medium were decreased,and the expression of ADAM10/17 were increasedcompared with those in control group(P <0.01).There were no significant differences in the expression of Rab5,Rab11 and ABCB1 m RNA and protein between control and MβCD group(P> 0.05).6.LPL can increase HBMEC cholesterol level through modulating LDL,and then up-regulate LRP1 and down-regulate ADAM10/17 expression: There were no significant differences in the levels of intracellular cholesterol,LRP1 expression,culture media s LRP1,and ADAM10/17 expression between control and LPL group(P> 0.05).In LDL and LPL+LDL groups,the levels of intracellular cholesterol,LRP1 expression and medium s LRP1 were higher than control group,and ADAM10/17 expression were lower than control group(P <0.01).Compared with LPL group,the intracellular cholesterol,LRP1 expression and culture media s LRP1 were increased in the LDL and LPL+LDL groups.Compared with LDL group,the levels of intracellular cholesterol and LRP1 were increased,and the expression of ADAM10/17 were decreased in LPL+LDL groups(P<0.01).However,there was no difference in s LRP1 levels between LPL and LPL+LDL groups in culture medium(P> 0.05)(P <0.01).There were no significant differences in the expression of Rab5,Rab11 and ABCB1 among each group of cells(P> 0.05).7.LPL can promote Aβ transport into HBMEC through LDL,but has no effect on Aβ transport out of cells: Tight junction protein ZO-1 appeared between adjacent cells of HBMEC in vitro BBB model.In the lower chamber,Aβ levels in LDL and LPL + LDL groups were lower than those in control group(P <0.01),but there was no significant difference in Aβlevels between LPL and control groups(P> 0.05).Compared with LPL group,Aβ levels were decreased in LDL and LPL+LDL groups(P <0.01),and compared with LDL groups,Aβ levels in LPL+LDL groups were decreased(P <0.01).In HBMEC cells,there was no significant difference in Aβ levels between the LPL group and control group(P> 0.05),and Aβ levels in the LDL and LPL+LDL were increased(P <0.01).Compared with LPL group,the levels of Aβ were increased in LDL and LPL+LDL(P <0.01).In LPL+LDL group,Aβ levels were increased compared with that in LDL group(P <0.01).There was no significant difference in Aβ levels between the groups in the above chamber(P> 0.05).Conclusion: Intermitten fasting can regulate the expression of LPL in adipose of AD mice.The mechanism may be that βOHB is involved in the regulation of LPL expression in adipose through inhibiting HDAC2/3,and PPARγ can participate in the above regulation.LPL upregulates the expression of LRP1 in microvascular endothelial cells and then promotes the transport of brain Aβ via BBB,which mediates the anti-AD effect of intermitten fasting. |
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