Study On The Effect Of Loganin On Astrocytes In ApoE4 Transgenic AD Model

Background: Alzheimer’s disease(AD)is a degenerative disease of the central nervous system.It is a devastating age-related neurodegenerative disease.According to the time of onset,AD can be divided into early-onset Alzheimer’s Disease(EOAD)and late-onset Alzheimer’s Disease(LOAD),the E4 allele of apolipoprotein E4(ApoE4)is one of the strongest genetic risk factors for LOAD,Apolipoprotein E(ApoE)is mainly produced and expressed in astrocytes.In pathological conditions astrocytes are overactivated,and it leads to the accumulation of amyloid plaques and the aggravation of AD.Loganin is one of the main components of cornus officinalis.Previous studies have found that Loganin can improve the cognitive ability and spatial memory ability of ApoE4 mice to some extent.However,the effect and mechanism of Loganin on reactive astrocytes and LOAD in ApoE4 model are still unclear.Objective: Based on the ApoE4 transgenic AD model in vitro and in vivo,we investigated the effect and the related molecular mechanisms of loganin on ApoE4 overexpressed astrocytes and the potential of loganin for the treatment of AD was explored.To reveal the mechanisms by which ApoE4 regulates astrocyte function and thus promotes AD progression.Methods: 1.In vivo experiments the genotypes of ApoE3 and ApoE4 mice at 8 months of age were first identified.Select mice with the correct genotype for the experiment.To construct ApoE3 and ApoE4 cell models in vitro experiments,first synthesize ApoE4 gene fragments according to the ApoE4 gene sequence,the ApoE4 gene fragment was integrated into the PLenti CRISPR-V2 vector to construct the complete ApoE4 plasmid by homologous recombination experiment.Using the PLenti CRISPR-V2-ApoE4 plasmid as a vector,ApoE4 was point-mutated into ApoE3 through restriction endonuclease cutting,point mutation,and homologous recombination.Afterwards,PCR agarose gel experiments and sequencing experiments were performed to verify the sequences.The lentiviral packaging plasmid was transfected into HEK-293 T cells to prepare the virus,and the supernatant medium containing the virus was collected 24 h after transfection.ApoE was transfected into U87 cells,and the results were verified by Protein immunoblotting(Western blot)assay..2.In vivo experiments,according to the identification results,the mice were divided into three groups:ApoE3 control group,ApoE4 model group and ApoE4+Loganin group(40 mg/kg,i.p.)for 28 days.The expressions of astrocyte markers Glial fibrillary acidic protein(GFAP),and A1/A2 type astrocyte markers complement 3(C3)and S100 calcium binding protein A10(S100A10)in mouse brain tissue were detected by immunohistochemistry,Western blot and Quantitative reverse transcription polymerase chain reaction(qRT-PCR)experiments.In vitro assays using MTT and EdU assays to determine the concentration of drug used on cells,the U87 cells were grouped into ApoE3,ApoE3+Loganin(60 μmol/L),ApoE4 and ApoE4+Loganin(60 μmol/L),The expression of astrocyte markers and A1/A2 phenotypic markers was detected by Western blot assay,the expression of A1/A2 specific transcription factors in reactive astrocytes was detected by qRT-PCR,this was used to evaluate the effect of Loganin on the activation and phenotypic changes of ApoE4 astrocytes.3.Enzyme-Linked Immuno Sorbent Assay(ELISA)was used to detect the expression of inflammatory factors Tumour Necrosis Factor-α(TNF-α),Interleukin-6(IL-6)and Interleukin-1β(IL-1β),and Western blot was used to detect the expression of Inducible nitric oxide synthase(iNOS)to evaluate the effect of marganyin on inflammatory factors.The expressions of Postsynaptic density(PSD95),Synaptophysin(SYP),Amyloid precursor protein(APP)and Beta-site amyloid precursor protein cleaving enzyme 1(BACE1)in vivo models were detected by Western blot,and use Nissl staining and Thioflavin-S staining experiments to verify the effect of Loganin on Nissl bodies and Aβ aggregation in nerve cells Then validated in vitro models,SH-SY5 Y cells were incubated with ApoE3/ApoE4 astrocyte conditioned medium(ACM),and the effect of Loganin on cell viability was detected by MTT assay.And The expressions of PSD95,SYP,APP,BACE1 and β-Amyloid(Aβ)in SH-SY5 Y cells were detected by Western blot.4.Western blot was used to detect the expression levels of LDLR-related protein 1(LRP1),Brain-derived neurotrophic factor(BDNF)and Tropomyosin related kinase(TrkB)in vivo and in vitro experiments,the expressions of GFAP and LRP1 were detected by immunofluorescence double staining,this study aimed to explore the molecular mechanism of Loganin regulating the function of reactive astrocytes.LRP1 siRNA and TrkB inhibitor(ANA-12)were introduced in vitro to verify the LRP1/BDNF/TrkB signaling pathway,and the expressions of A1 marker C3 and A2 marker S100A10 were detected by Western blot,ELISA was used to detect the inflammatory factors in the supernatant(TNF-α,IL-1β and IL-6)expression.In order to verify the role of LRP1/BDNF/TrkB signaling pathway in regulating astrocytes by Loganin.Results: 1.Identification of in vivo models of ApoE4 transgenes and construction of in vitro models1)After the animal model was identified by PCR,dosing and follow-up experiments based on results.2)Constructing ApoE overexpression cell models.The PLenti CRISPR-V2 recombinant expression plasmid of ApoE4,ApoE3 and GFP-negative control was successfully constructed by enzyme digestion identification,point mutation amplification,colony PCR identification and gene sequencing results analysis.3)Lentiviral supernatant solutions were obtained by infecting HEK-293 T cells with pMD2G and pSPAX2 packaging target plasmids.The recombinant lentivirus infected U87 cells were screened with medium containing puromycin and the transfected cells showed clear fluorescence and normal cell morphology under fluorescence microscopy.4)The results of Western blot experiments showed that,compared with the control group,the expression of ApoE in the ApoE3+GFP group and the ApoE4+GFP group was significantly increased,Compared with the ApoE3+GFP group,the ApoE4 protein expression level in the ApoE4+GFP group was up-regulated,the results showed that ApoE3 and ApoE4 cell models were constructed successfully.2.Loganin inhibits ApoE4 mediated overactivation of astrocytes and regulates phenotypic transition of reactive astrocytes in vivo and in vitro.1)In vivo and in vitro models,overexpression of ApoE4 led to up-regulated expression of GFAP,a marker of reactive astrocytes,and the expression of GFAP was significantly down-regulated after administration of Loganin.2)In vitro and in vivo models,the A1 neurotoxic phenotype C3 protein expression was up-regulated in the ApoE4 overexpression model,and the A2 neuroprotective phenotype S100A10 protein expression was down-regulated,the results of qRT-PCR experiment showed that the expression of A1 specific transcription factors was significantly up-regulated,and the expression of A2 specific transcription factors was significantly down-regulated,the administration of Loganin can exert neuroprotective effect and promote the conversion of A1 neurotoxic phenotype to A2 neuroprotective phenotype.3.Administration of Loganin can inhibit neuroinflammation and affect neuronal synaptic function and Aβ production.1)The results of ELISA experiments showed that the overexpression of ApoE4 in vivo and in vitro experiments would lead to an increase in the content of pro-inflammatory factors TNF-α,IL-1β and IL-6.Western blot experiments also showed that the expression of i NOS was significantly increased in the ApoE4 model,and administration of Loganin could reverse the inflammatory response.2)In the hippocampus and cortex of ApoE4 model mice,the expression of synapse-related proteins was significantly reduced,and the expression of Aβ generation-related proteins was significantly increased,the loss of Nissl bodies was obvious,the cell vacuolization was severe,and there were more β-amyloid plaques.After the intervention of Loganin,the synaptic damage was alleviated,the vacuolization of Nissl bodies was reduced,and Aβ plaque aggregation was reduced.3)In vitro experiments,ApoE astrocyte conditioned medium(ACM)was constructed SH-SY5 Y cells,resulted in reduced SH-SY5 Y cell viability,reduced expression of synapse-associated proteins and increased Aβ production and aggregation,and Loganin administration reduced synaptic damage and Aβ production in SH-SY5 Y cells.4.Loganin regulates the functional of astrocytes through the LRP1/BDNF/TrkB signaling pathway.1)The expressions of LRP1,BDNF and p-TrkB are decreased in the hippocampus and cortex of ApoE4 mice.Loganin administration can up-regulate the expression of LRP1 and activate the BDNF/TrkB signaling pathway.The results of in vitro experiments were consistent with those of in vivo experiments.2)In order to further verify the molecular mechanism of Loganin acting on the ApoE4 model,we introduced TrkB inhibitor(ANA-12)and LRP1 siRNA for verification.First,we used LRP1 siRNA to silence the expression of LRP1 to explore the effect of Loganin on the BDNF/TrkB pathway.Western blot results showed that,compared with ApoE4 group,the expressions of BDNF and p-TrkB in ApoE4+LRP1 siRNA group were down-regulated,when Loganin and LRP1 siRNA acted together,LRP1 siRNA inhibited the effect of loganin,it suggested that the BDNF/TrkB signaling pathway was located downstream of ApoE4-mediated LRP1 and that Loganin played a subsequent role through LRP1.3)Treatment of U87-ApoE4 cells with ANA-12 showed that the expression of BDNF and p-TrkB was significantly inhibited in the ANA-12 group compared to the ApoE4 group,but did not affect the expression of LRP1,confirming that LRP1 is located upstream of the BDNF/TrkB signalling pathway.And when ANA-12 and Loganin acted at the same time,the neuroprotective effect of loganin was inhibited by ANA-12,suggesting that Loganin may exert its neuroprotective effect through mediating LRP1/BDNF/TrkB.4)Next,LRP1 siRNA and ANA-12 were used to verify the effect of loganin on U87-ApoE4 cells.The results showed that LRP1 siRNA and ANA-12 treatment resulted in upregulation of C3 expression and downregulation of S100A10 expression compared to the ApoE4 group,and that LRP1 siRNA and ANA-12 treatment inhibited strychnidine-induced downregulation of C3 expression of the A1 neurotoxic phenotype and upregulation of S100A10 expression of the A2 neuroprotective phenotype compared to the strychnidin-administered group.In addition the expression of inflammatory factors IL-1β,IL-6 and TNF-α was significantly increased after LRP1 siRNA and ANA-12 induction,while when Loganin was combined with LRP1 siRNA and ANA-12,LRP1 siRNA and ANA-12 reversed the inhibitory effect of Loganin on IL-1β,IL-6 and TNF-α.Conclusions: In this study,we found that Loganin could modulate the transformation of astrocyte phenotype,inhibit neuroinflammation,reduce Aβ pathology and synaptic damage,and thus slow down the pathological process of AD in vivo and in vitro model of ApoE4.It was revealed that Loganin may slow down the pathological process of AD by regulating the LRP1/BDNF/TrkB signaling pathway,providing a new perspective for the in-depth analysis of the pathogenesis of AD and proposing new strategies for AD prevention and treatment.