Study On Mechanisms Of Improving Alzheimer's Disease By Vitamin D Combined With Resveratrol

Alzheimer's disease(AD),a progressive neurodegenerative disease,is one of the main reasons resulting in increased global incidence of dementia.Currently,there is no effective measure for the prevention and treatment of AD,so it is urgent to explore cost-effective measures for prevention and treatment of AD etiology.Vitamin D(VD)and resveratrol(RSV)are two substances concerned by nutrition academic circles,studies have revealed that they have protective role in the pathogenesis of AD,and they play similar effects in some biological processes.This suggests that VD and RSV may have synergistic effects in terms of controlling AD.Therefore it should be further investigated whether VD combined with RSV has cooperative or synergistic effects in regards to control AD,as well as specific mechanisms involved in.Clarifying possible mechanisms of preventing and curing AD by VD combined with RSV could provide theoretical basis for the implementation of effective measures based on food active ingredients in the prevention and treatment of AD.Objective:1.To determine the effects of vitamin D and RSV on endoplasmic reticulum stress(ER)stress and A?25-35 mediated neurotoxicity using human neuroblastoma SH-SY5Ycells.2.To determine the effects of vitamin D and RSV on ER stress,beta amyloid protein(A?),Tau pathology and cognitive function using the senescence accelerated prone mouse 8(SAMP8).Method:1.SH-SY5Y cells experimentSH-SY5Y cell were cultured,Tunicamycin(TM)induced cell toxicity,then were administered with the following intervention:VD(10-9,10-8,10-7M)or RSV(25 ?M)or VD(10-9,10-8,10-7 M)plus RSV(25 ?M);A beta 25-35 induced cell toxicity,then were administered with the following intervention:VD(10-9M)or RSV(25 ?M)or VD(10-9,M)plus RSV(25 ?M)The following markers were detected:1.1 MTT and LDH test measured cell viability.1.2 Western bloting was used to measure endoplasmic reticulum stress marker protein,including glucose regulation 78(GRP78),phosphorylated eukaryotic initiation 2alpha(p-eIF2a)and CHOP;key protein involved in insulin signaling pathway including phosphorylated IRS-1 at serine 307 and Akt at serine 407;phosphorylated tau at serine 396 and 404 and the expression of tau related kinases.1.3 RT-PCR measured endoplasmic reticulum stress marker,including GRP78,CHOP and XBP-1mRNA.2.Animal experimentTen-week male the senescence accelerated prone mouse 8(SAMP8)mouse(n=40)and senescence-accelerated mouse resistant 1 SAMR1(n=10)were randomized into 5 groups:SAMR1(R1)group?SAMP8(P8)group?VD SAMP8 group(the mice were administered a intramuscular injection of 1000 IU vitamin D3 once every 1 week),RSV SAMP8 group(mice fed with a AIN-93G diet supplemented with 0.3%RSV),VDRSV SAMP8 group(VD combined with RSV).At the end of the 20 weeks' intervention,we examined:2.1 Basic index and blood biochemical index:glucose tolerance test(GTT)and insulin tolerance tests(ITT)were measured,and the area under the curve(AUC)was calculated for each.We also determined serum markers,including fasting serum glucose,insulin,adiponectin,glycerol,free fatty acid and 25hydroxyvitamin D3.2.2 Behavior functional test:Morris water maze experiment were conducted to measure cognitive function in mice.2.3 Abeta pathology:ELISA method was used for the determination of A?42 in hippocampus;Western blot measured A? related protein of hippocampus and cortex including APP,BACE1,and cathepsin B.2.4 Tau pathology:Western blot measured phosphorylated tau protein and tau related protein kinase in the cortex,immunohistochemistry was used to measure the relative distribution of p-Tauser 404 in the cortex.2.5 Neuroinflammation and synaptic function related proteins:Western blot measured key proteins involved in neuroinflammation including GFAP and p-NF-?B p65,and synaptic function related proteins in hippocampus and cortex.2.6 Endoplasmic reticulum stress and apoptosis index:Western blot measured endoplasmic reticulum stress markers,including p-eIF2??GRP78 and p-PERK protein in hippocampus and cortex,and apoptosis protein p-p53 level in cortex;TUNEL experiment was performed to detect cell apoptosis in cortex.Results:1.The cell experiment1.1 Effects of VD?RSV and joint intervention on TM and A?25-35 induced cytotoxicity in SH-SY5Y cells.MTT suggest that,compared with normal control(Veh)group,the viability of SH-SY5Y cells in TM group and A?25-35 group dropped significantly,but VD?RSV or joint intervention significantly increased cellular activity.LDH suggest that compared with normal Veh group,the viability of SH-SY5Y cells in TM group dropped significantly,but VD?RSV or joint intervention significantly increased cellular activity1.2 Effects of VD,RSV and joint intervention on TM and A?25-35 induced ER stress.Western bloting showed that he key markers of ER stress(GRP78,p-eIF2a and CHOP)in TM group and A?25-35 group was significantly higher than veh group,VD combined with RSV significantly reduced the above protein expression.RT-PCR demonstrated that the mRNA expression of GRP78,CHOP and XBP-1)in TM group were significantly increased compared with veh group,VDcombined with RSV significantly reduced GRP78,CHOP and XBP-1 mRNA expression.1.3 Effects of VD,RSV and joint intervention on TM and A?25-35 induced impaired insulin signaling.Compared with veh group,the p-IRS1 serine307 level increased significantly,and p-Akt serine 473 levels significantly decreased in both TM and A?25-35 group,VD plus RSV reduced p-IRS1 serine307 and increased p-Akt serine 473.1.4 Effects of VD,RSV and joint intervention on TM and A?25-35 induced tau pathology.Compared with veh group,phosphorylated GSK-3? at serine 9 protein were significantly reduced,p-Tau at serine396 and p-Tau at serine404 were significantly increased in TM and A?25-35 group,VD plus RSV induced p-GSK 3 p at serine 9 expression and reduced p-Tau at serine396 and p-Tau at serine404.In TM treated experiment,VD or RSV intervention independentlyalso significantly increased p-GSK 3? at serine 9 and decreased p-Tau at serine396.In A?25-35 treated experiment,VD or RSV intervention independently also increased p-GSK 3? at serine 9 and decreased p-Tau at serine396 and p-Tau at serine404.2.Animal experiment2.1 Basic index and serum biochemical indexAll groups' body weight grew steadily during the experiment,the body weight of mice in RSV group or VD plus RSV were significantly heavier than R1 group since the eight week.Fasting serum insulin and HOMA-IR were lower in VD group than P8 group.In GTT test,P8 group,VD group,RSV group or VD plus RSV group have significantly increased AUC compared with R1 group;in ITT experiment,RSV group's AUC decreased notably compared with P8 group.Serum adiponectin level was significantly lower in all other groups than R1 group,and compared with group P8,VD plus RSV group has significantly decreased serum adiponectin levels.The serum concentration of 25(OH)D3 in VD or VD plus RSV group was obviously higher than P8 group.2.2 Water maze testDuring place navigation test,compared with R1 groups,the time of arrival in platform from P8 group was extended.During space exploration test,the time spent in the target quadrant and the number of crossing of P8 group were significantly lower than R1 groups,but VD plus RSV group has increased time spent in the target quadrant and the number of crossing.2.3 A? pathologycompared with R1 groups,APP and BACE1 expression of hippocampus and APP?BACE1and cathepsin B of cortex expression from P8 group have significantly increasd,VD plus RSV reduced the expression of BACE1 in hippocampus and cortex.The hippocampal A?42 level in P8 group was significantly higher than R1 groups,VD plus RSV reduced the level of A?42 compared with P8 group.2.4 Tau pathologyThe phosphorylation of Tau(p-Tau)at serine396 and at serine404 of P8 group were significantly higher than R1 group,VD plus RSV reduced the above protein expression.Besides,VD or RSV single intervention reduced p-Tau at serine396.2.5 Neuro-inflammation and synaptic function related proteinCompared with R1 group,the protein expression of GFAP and p-NF?B p65 in hippocampus and cortex of P8 group were significantly increased VD plus RSV significantly reduced the above protein expression.Compared with R1 groups P8 group have increased phosphorylated STAT3 level,VD plus RSV decreased p-STAT3.There is no difference for synaptophysin,PSD95 and BDNF protein expressionamong groups.2.6 Endoplasmic reticulum stress and apoptosis related proteinIn the hippocampus,there is no difference for endoplasmic reticulum stress protein including p-eIF2a,GRP78 and p-PERK among groups.In the cortex,P8 group have increased expression of p-eIF2a and p-PERK protein than R1 groups,there was no significant difference between other groups.Compared with R1 groups,P8 group has significantly increased phosphorylation level of p-53 in the cortex,but VD group?RSV or VD plus RSV group has significantly decreased p-p53 level than P8 group.Conclusion:1.In SH-SY5Y cells,vitamin D plus resveratrol intervention could improve tunicamycin and A?25-35 induced neurotoxicity.2.In SH-SY5Y cells,vitamin D plus resveratrol intervention could improve tunicamycin and A?25-35 induced endoplasmic reticulum,insulin signaling pathway disorders and highly phosphorylated tau protein.3.In SAMP8 mice,vitamin D plus resveratrol intervention had no effect on body weight,liver weight and epididymal fat fat weight,fatty acid,glucose and glycerol;while significantly reduced the serum adiponectin levels.4.In SAMP8 mice,vitamin D or resveratrol intervention could partially improve cognitive function,while vitamin D plus resveratrol exertedgreater effects in improving cognitive function than intervention independently.5.In SAMP8 mice,vitamin D plus resveratrol intervention could reduce A?production.Vitamin D,resveratrol and joint intervention improve the p-Tau ser396 level,only vitamin D plus resveratrol could improve the p-Tau ser404.Vitamin D,resveratrol and concerted intervention could improve the neuroinflammatory factors including GFAP and p-NF-?B p65 protein,and p-p53 expression.Vitamin D plus resveratrol could significantly increase p-STAT3 of hippocampus and p-CREB expression of cortex.Vitamin D,resveratrol and joint intervention had no significant effect on key protein of endoplasmic reticulum stress.6.In SAMP8 mice,vitamin D plus resveratrol could improve the cognitive dysfunction,it may be related to improvements of A? production,p-tau ser404,nerve inflammation and apoptosis.