The Effects And Mechanism Of Tanshinone ⅡA On Endoplasmic Reticulum Stress Mediated By SIRT1 In Brain Microvascular Endothelial Cells To Improve Alzheimer’s Disease

ObjectiveAlzheimer’s disease(AD)is the most common cause of cognitive impairment,its incidence is increasing year by year,but there is no effective treatment.In recent years,more and more evidences have shown that the damage of blood-brain barrier(BBB)is an early pathological feature of AD,which is earlier than the deposition of Aβ and tau proteins,and earlier than the presence of cognitive impairment.BBB is not a single structure,but composed of a variety of cell components,such as brain microvascular endothelial cells(BMEC),pericytes,and astrocytes.BMEC is the most important part of BBB.The Aβclearance is also achieved mainly through the LRP1/RAGE receptor system on BMEC.Tanshinone Ⅱ A(Tan ⅡA)is the main active component of Salvia miltiorrhiza,which has been proved to improve cognitive impairment,but its mechanism remains unclear.This study aims to explore the role and mechanism of Tan ⅡA in improving cognitive impairment from the perspective of Tan ⅡA regulating BMEC function.MethodsIn this study,bEnd.3 cells induced by Aβ1-42 was employed for in vitro experiments.The cell viability was detected by MTT assay to screen the concentration of Tan ⅡA and Ap1-42.Molecular docking and quantitative polymerase chain reaction(qPCR)were used to determine the targeted molecules of Tan ⅡA in the Sirtuins family.Western blotting(WB)was used to detect SIRT1,ER stress pathway related proteins(BIP,P-PERK,P-eIF-2α,P-IRE-1α,XBP1,ATF6,PDI,CHOP),Aβ transport related proteins(LRP1,RAGE).Immunofluorescence was used to detect SIRT1,P-PERK,P-IRE-1α,LRP1,RAGE.The APP/PS1 double transgenic mice were used for in vivo experiments.The open field experiment,new object recognition experiment,and Morris water maze experiment were used to evaluate the cognitive dysfunction.The oxidative stress levels(CAT,MDA,SOD,GSH-PX)were detected by the kit.The neurotrophic function(PSD95,BDNF,NGF)and neuronal apoptosis(Caspase-3,Bax,Bcl-2)were detected by WB.The Nissl’s staining was used to detect neurodegeneration.The Thioflavin T staining and immunofluorescence were used to detect Aβ deposition.WB was used to detect SIRT1,ER stress pathway(BIP,P-PERK,P-eIF-2α,P-IRE-1α,XBP1,ATF6,PDI,CHOP)and Aβ transport related proteins(LRP1,RAGE).Immunofluorescence was used to detect the co-localization of CD31 and SIRT1,P-PERK,P-IRE-1α,Aβ,LRP1,RAGE,in which CD31 was the endothelial marker protein.ResultsMTT assay was used to screen the concentration of Aβ1-42 at 10μM and Tan ⅡA at 20μM.Molecular docking and qPCR results showed that Tan ⅡA mainly acted on SIRT1 of Sirtuins family.SIRT1 inhibitor EX527 was added,to further observe the effect of SIRT1 on endoplasmic reticulum stress pathway.Compared.with Aβ group,in Aβ+Tan ⅡA group,the expressions of SIRT1,PDI,LRP1 were increased,while the expressions of BIP,P-PERK,P-eIF-2α,P-IRE-1α,XBP1,CHOP protein expression were decreased,ATF6 protein expression remained unchanged-After adding SIRT1 inhibitor EX527,the above protein expression showed an opposite trend.Immunofluorescence results were consistent with WB results.In APP/PS1 double transgenic mice,the open field experiment,new object recognition experiment,and Morris water maze experiment showed that Tan ⅡA could increase the central region distance,improve the recognitive index,shorten the escape latency,increase the crossing times of the platform and time spent in target quadrant.In APP/PS1 double transgenic mice,Tan ⅡA could improve the activity of CAT,T-SOD,and GSH-PX,and reduce the MDA level.The results of Nissl’s staining showed that Tan ⅡA could increase the number of Nissl bodies in the hippocampus and cortex of APP/PS1 transgenic mice.Tan ⅡA could increase the expression of PSD95,BDNF and NGF,and reduce the expression Caspase-3,Bax and Bcl-2.Tan ⅡA could increase the expression of SIRT1,PDI,LRP1 and reduce the expression of BIP,P-PERK,P-eIF-2α,P-IRE-1α,XBP1,CHOP,and RAGE,while Tan ⅡA had no effect on the expression of ATF6.Furthermore,Tan ⅡA could reduce the Aβ deposition in vascular endothelium,increase the expression of SIRT1 and LRP1,and reduce the expression of P-PERK,P-IRE-1α and RAGE in vascular endothelium.ConclusionIn APP/PS1 double transgenic mice,Tan ⅡA can alleviate oxidative stress,inhibit neuronal degeneration,and reduce neuronal apoptosis.In addition,Tan ⅡA can also alleviate SIRT1-mediated ER stress in brain microvascular endothelial cells,promote Aβtransendothelial transport,and improve cognitive impairment in APP/PS1 mice,providing a new direction for the treatment of AD patients.