The Effect And Mechanism Of Notoginsenoside R1 In Alleviating Aβ_25-35-Induced PC12 Cell Damage Through The MiRNA-216a-5p/SRR Axis

Background:Alzheimer’s disease（AD）is the most common form of dementia in old age and is currently the fourth leading cause of death among the elderly.The pathogenesis of AD is complex and the main drugs currently available in China are cholinesterase inhibitors,meperidine and mannitol,which are limited in their variety and effects and cannot prevent progressive neuronal loss,ventricular atrophy or cognitive deterioration.ventricular atrophy or cognitive deterioration.Traditional Chinese medicine is a treasure of China,and the search for effective Chinese medicinal preparations to treat AD has become a hot topic in current research on AD treatment.Notoginsenoside R1（NR1）is an active ingredient derived from P.notoginseng saponins,which has anti-inflammatory,antioxidant and neuroprotective effects and can reverse ischemia/reperfusion-induced injury.NR1 reduces apoptosis of neuronal cells in AD,promotes the degradation of Aβ,and improves learning and memory in APP/PS1double transgenic AD model mice.ability.Therefore,NR1 may be a new drug candidate for the treatment of AD,and the mechanism of NR1 action in AD needs to be further investigated.Serine Racemase（SRR）is a Pyridoxal-5-phosphate（PLP）-dependent enzyme whose product,D-serine,is one of the N-methyl-D-aspartic acid receptor（NMDAR）activators.D-serine is one of the activators of NMDAR.Our group showed that SRR expression was increased in Aβ-treated PC12 cells;down-regulation of SRR could reduce NMDAR2A protein expression in PC12 cells,alleviate the over-activation of NMDAR,reduce apoptosis,improve cell survival and protect Aβ-injured PC12 cells,and also increase PSD95 protein expression to alleviate synaptic damage.We analysed miRNAs that may pair with SRRs through the OncomiR database and further identified miR-216a-5p as a target binding site in the 3’UTR region of SRR m RNAs through Target Scan database analysis.miR-216a-5p is a non-coding micro RNA.It has been demonstrated that in spinal cord injury,miR-216a-5p promotes functional behavioural recovery from spinal cord injury by facilitating the conversion of microglia from an M1 pro-inflammatory phenotype to an M2 anti-inflammatory phenotype.It has also been demonstrated that miR-216a-5p expression is elevated in the male brain in cerebral ischemia-reperfusion injury thereby achieving neuroprotective effects.Whether NR1 can reduce AD neuronal damage through the miR-216a-5p/SRR axis and its mechanism of action remain unclear.Therefore,this study used Aβ_25-35 to intervene in PC12 cells to construct an in vitro cell model of AD.NR1 was applied to intervene and the changes of miR-216a-5p and SRR expression in PC12 cells were investigated to explore the role of NR1 on Aβ-induced damage in PC12 cells and related mechanisms,providing a new strategy for the future treatment of Alzheimer’s disease.Methods:PC12 cells were treated withβ-amyloid fragment 25-35(Aβ_25-35)to establish an AD cell model.Cell viability was detected using the Cell Counting Kit-8（CCK-8）method.Cell morphology was observed using an inverted microscope and nuclear morphology was observed using Hoechst 33258 nuclear staining.Apoptosis and apoptotic protein markers（Cleaved PARP,Caspase 3 and Cleaved caspase 3）were detected by flow cytometry and protein immunoblotting.Protein immunoblotting was used to detect the expression of serine racemase（SRR）protein.Real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of miR-216a-5p,SRR.The interaction sites of miR-216a-5p and SRR were predicted using the online database Target Scan and confirmed by dual luciferase reporter gene analysis and Western Blot analysis.Results:（1）Aβ_25-35 reduced PC12 cell viability and miR-216a-5p expression and upregulated SRR expression.（2）NR1 attenuated cell damage in an in vitro cell model of AD.（3）miR-216a-5p attenuated PC12 cell injury in an in vitro cell model of AD.（4）NR1 attenuated PC12 cell injury by promoting miR-216a-5p expression in an in vitro cell model of AD.（5）SRR promotes PC12 cell injury in an in vitro cell model of AD.（6）In an in vitro cell model of AD,NR1 attenuated PC12 cell injury by downregulating SRR expression.（7）miR-216a-5p targets SRR m RNA,and in AD cell models,NR1regulates SRR expression through miR-216a-5p targeting.Conclusion:In an in vitro cell model of AD,Aβ_25-35PC12 cell damage was promoted by the downregulation of miR-216a-5p expression and the upregulation of SRR expression.Targeted binding of miR-216a-5p to the 3′UTR region of SRR m RNA,and downregulation of SRR protein expression.NR 1 could also downregulate SRR expression by up-regulating miR-216a-5p and promoting its m RNA targeting binding to SRR and thereby alleviating PC12 cell damage.In conclusion,we suggest that Panax notoginseng saponin R1 alleviated Aβvia the miR-216a-5p/SRR axis_25-35This finding of inducing damage in PC12 cells may be a novel therapeutic strategy to improve the progression of Alzheimer’s disease.