激活血管紧张素1-7/Mas受体轴下调HT22细胞α-突触核蛋白表达的机制研究OA

Objective To investigate the role and potential mechanism of the angiotensin 1-7(Ang 1-7)/Mas receptor(MasR)axis in regulating the expression of hippocampal neuronal α-synuclein(α-syn)in the context of perioperative neurocognitive disorders(PND).Methods The conditioned medium(CM)from lipopolysaccharide-induced macrophages was used to stimulate the mouse hippocampal HT22 cell line,mimicking the central neuroinflammatory state induced by anesthesia and surgery.Cells were divided into six groups:control(Con),CM model,CM+Ang 1-7 analog AVE 0991 intervention,CM+AVE 0991+MasR antagonist A779 intervention,CM+AVE 0991+PP2A knockdown negative control(small interfering negative control,siNC),and CM+AVE 0991+protein phosphatase 2A gene knockdown(via small interfering RNA,siPP2A)groups.Except for the Con group,HT22 neurons were subjected to CM stimulation.The CM+AVE group received co-intervention with an optimal dose of AVE 0991.The CM+AVE+A779 group was pretreated with 10-5 mol/L A779 for 30 minutes before CM+AVE 0991 intervention.In the CM+AVE+siNC group,HT22 cells were transfected with negative control siRNA and then subjected to intervention with CM+AVE 0991.The CM+AVE+siPP2A group underwent PP2A knockdown in HT22 neurons prior to CM+AVE 0991 intervention.Cell viability was assessed by the Cell Counting Kit-8 assay.Ang 1-7 levels were measured by enzyme-linked immunosorbent assay.α-Syn protein expression was detected by dot blot.The protein expression levels of MasR,PP2A catalytic subunit(PP2A-C),methylated PP2A(Me-PP2A),and serine 129-phosphorylated α-syn(pS129-α-syn)were determined by Western blot.Results CM stimulation significantly may increase the protein expression levels of total α-syn and pS129-α-syn in HT22 neurons(P<0.05).CM stimulation for 6 h significantly suppressed the activity of the Ang 1-7/MasR axis,as evidenced by downregulated MasR protein expression,accompanied by decreasing protein levels of PP2A-C and Me-PP2A(P<0.05).A dose-gradient experiment indicated that 10-6 mol/L was the optimal concentration of AVE 0991 for upregulating MasR,PP2A-C,and Me-PP2A protein levels.At this concentration,AVE 0991 significantly reduced the expression levels of α-syn and pS129-α-syn(P<0.05).These protective effects were blocked by the MasR antagonist A779 or by specific PP2A knockdown.Conclusions CM stimulation of HT22 hippocampal neurons may inhibit the activity of the Ang 1-7/MasR axis and increase α-syn expression.The exogenous Ang 1-7 mimetic AVE 0991 downregulates α-syn expression by upregulating PP2A expression and its methylation,suggesting its potential involvement in the pathological process of PND.This study provides new experimental clues and a potential therapeutic target for elucidating the pathogenesis of PND.