Preliminary Study On The Cognitive Protective Effect And Mechanism Of Angelica Polysaccharide On Alzheimer’s Disease Model Rats Through Inhibiting Neuronal Apoptosis And Pyroptosis

Background:Alzheimer’s disease（AD）is a common central nervous degenerative disease in the elderly and the most common dementia.Currently,the pathogenesis and etiology of AD are not still unknown.In recent years,numerous evidences have pointed out that neuroinflammation plays a crucial role in the pathogenesis of AD.Pyroptosis is a type programmed cell death which has attracted extensive attention in recent years.Pyroptosis is a necrotic cell death different from apoptosis,which could release various inflammatory cytokines and induce systemic inflammatory responses.It has been well-established that pyroptosis play a doubles role in immune response.On the one hand,it could eliminate intracellular risk factors through inflammatory reaction.On the other hand,exaggerated pyroptosis also lead to excessive inflammatory reaction.Pyroptosis mainly was triggered by intracellular pattern recognition receptor among them,the nucleotide-binding oligomerization domain-like receptor protein 3（NLRP3）inflammasome is a key position in the neuroinflammatory pathway of AD.Accumulating evidence reveal that pyroptosis is closely related to the occurrence and development of AD,and inhibition of NLRP3 mediated pyroptosis could effectively alleviate the development of AD.To explore the role of pyroptosis in Aβ_1-42-induced cognitive dysfunction in AD rats,and elucidate whether the protective effect of angelica polysaccharides on neuronal cells is related to the inhibition of neuronal pyroptosis and apoptosis injury induced by high expression of lncRNA H19.Methods:1.Six-week-old healthy Sprague Dawley male rats were selected for rat AD animal model building via injecting Aβ_1-42 into the bilateral hippocampus.The spatial cognition function of SD rats was evaluated using water maze behavioral experiment.Hippocampus neuron apoptosis was examined using TUNEL assay.The expression levels of NLRP3 m RNA and lncRNA H19 in rat hippocampus tissue were measured using q RT-PCR.Protein expression levels of apoptosis and pyroptosis related genes in SD rat hippocampus tissue were analyzed using western blotting.2.Primary neural cells were isolated from newborn Sprague Dawley hippocampus and identified using immunofluorescence for MAP-2 expression.AD cell model was established using Aβ_1-42 and neuronal apoptosis（Annexin V-FITC+/PI+）and pyroptosis（caspase-1⁺/PI⁺）were detected by flow cytometry.Ed U staining and flow cytometry were used to evaluate the proliferation and apoptosis of neuronal cells.The transfection efficiency was detected by q RT-PCR after transfection NLRP3 si-RNA and lncRNA H19overexpressing adenovirus in neuronal cells.The expression and distribution of NLRP3in cells were detected by immunofluorescence,while the expression levels of pyroptosis and apoptosis-related proteins were detected by western blotting.The contents of IL-1βand IL-18 in cell supernatant were detected by ELISA kit.Results:1.The results of water maze test showed that compared with the control group,the escape latency of AD model group was significantly increased,while the residence time in platform quadrant and the number of crossing platform were significantly reduced（P<0.05）.Compared with AD model group,the escape latency of rats in Angelica polysaccharide administration group was significantly shortened（P<0.05）,while the residence time in platform quadrant and the number of crossing platform were significantly increased（P<0.05）.The results of TUNEL experiment showed that compared with the control group,the proportion of TUNEL positive cells in AD model group increased significantly（P<0.01）,the expression level of anti apoptotic gene Bcl-2decreased significantly,while the expression levels of Pro apoptotic gene Bax and apoptosis executive protein cleaved caspase-3 increased significantly（P<0.05）.Compared with AD group,the positive rate of TUNEL decreased significantly（P<0.01）,the expression level of Bcl-2 increased,and the levels of Bax and cleaved caspase-3decreased significantly（P<0.05）.q RT-PCR showed that NLRP3 and ASC m RNA levels in AD model group were significantly higher than those in control group（P<0.001）,while NLRP3 and ASC m RNA levels in Angelica polysaccharide group were significantly lower than those in AD group（P<0.01）;The results of Western blotting showed that compared with the control group,the ad model group had NLRP3,ASC,GSDMD-N,cleaved caspase-1 and IL-1β、The expression level of IL-18 protein increased significantly（P<0.01）.Compared with AD model group,angelica polysaccharide administration group had NLRP3,ASC,GSDMD-N,cleaved caspase-1and IL-1β.The expression level of IL-18 decreased significantly（P<0.01）,while the expression of pro-caspase-1 and gsdmd did not change significantly（P>0.05）.In addition,compared with the control group,the level of lncRNA H19 in the AD model group was significantly up-regulated（P<0.001）,while Angelica polysaccharides treatment could significantly reverse the increased expression of lncRNA H19 in the AD group（P<0.001）.2.Immunofluorescence results showed that the cells isolated from the hippocampus of neonatal rats highly expressed MAP2,suggesting that the successful isolation of primary neurons.Compared with control group,after treatment the viability of neuronal cells was significantly decreased,while the rate of apoptosis was significantly increased after incubation with 2μM Aβ_1-42 for 24 h（P<0.01）.In addition,Aβ_1-42 treatment can obviously increase the protein expression levels of NLRP3,ASC,GSDMD-N,cleaved caspase-1,IL-1β,and IL-18 in neuronal cells（P<0.01）,while transfection of NLRP3si-RNA could effectively reverse Aβ_1-42-induced inhibition of cell viability,apoptosis,and up-regulation of NLRP3,ASC,GSDMD-N,cleaved caspase-1,IL-1β,and IL-18levels（P<0.01）.Compared with the Aβ_1-42 group,the apoptosis,pyroptosis ratio,NLRP3,ASC,GSDMD-N,cleaved caspase-1,IL-1β,and IL-18 levels in the angelica polysaccharide treatment group were significantly decreased（P<0.01）.3.The expression of lncRNA H19 in Aβ_1-42-induced AD cell model was significantly upregulated when compared with control group,while Angelica polysaccharides could effectively reduce the up-regulation of lncRNA H19.In addition,lncRNA H19overexpression could obviously inhibit the neurprotection role of angelica polysaccharide,as evidenced by the decreased neuronal cell viability,and increased apoptotic and pyroptotic cell death,increased levels of NLRP3,ASC,GSDMD-N,cleaved caspase-1,IL-1β,and IL-18（P<0.01）.Conclusions:NLRP3-mediated pyroptosis is one of the important pathogenesis mechanisms for Aβ_1-42-induced neuronal cell injury,and the increased level of lncRNA H19 induced by Aβ_1-42 is involved in the process of NLRP3-mediated pyroptosis.Angelica polysaccharides can inhibit Aβ_1-42-induced neuronal apoptosis and pyroptosis at least partially throughing down-regulation lncRNA H19 expression.