| Background:Late-onest Alzheimer’s disease(LOAD)is Alzheimer’s disease(AD)that develops after the age of 65 years and is associated with neuroinflammation caused by an abnormal accumulation of beta-amyloidprotein(Aβ).Klothohoid The Klotho(KL)gene is an antiageing gene and has been shown in several studies to be associated with AD,to reduce neuroinflammation in AD and to block fibroblast growth factor23(FGF23)expression,as well as to have a correlation with VD.KL,FGF23 and VD form an endocrine axis(KL-FGF23-VD axis)that may play an anti-inflammatory role in the inflammatory environment.Activation of the transcriptional nuclear factor kappa-B(NF-κB)signalling pathway amplifies the inflammatory response and plays a key role in the inflammatory mechanism of LOAD.However,there are no studies on the role of the KL-FGF23-VD axis and NF-κB signalling pathway in delaying neuroinflammation in LOAD.Objective:(1)Silencing and enhancement of the KL gene were used to confirm the protective and ameliorative neuroinflammatory effects of the KL gene in LOAD animal models(LOAD rats)and cellular models(Aβ microglia)through animal and cellular experiments.(2)Using KL gene silencing and enhancement,animal and cellular experiments were conducted to test the hypothesis that KL gene acts on the rate-limiting enzyme IKK through the KL-FGF23-VD axis to inhibit the activation of NF-κB signaling pathway,thereby reducing the activation of microglia,apoptosis of rat hippocampal neurons and the degree of inflammatory response in LOAD model rats,thereby exploring the mechanism of action of KL gene to alleviate LOAD The mechanism of action of KL gene to alleviate neuroinflammation in LOAD.Materials and Methods:(1)LOAD rat model and LOAD cell model were constructed:The LOAD rat model was constructed by injecting Aβ and IBO into the CA1 region of the hippocampus,and simultaneously injecting empty vector,KL-interfering adenovirus and KL-overexpressing adenovirus,thus constructing Control group,empty vector/NS group(NC/NS group),empty vector/Aβ group(NC/Aβ group),KLsilenced/NS group(KL-si/NS group),KL-silenced/Aβ group(KL-si/Aβ group),KLenhanced/NS group(KL-OE/NS group)and KL-enhanced/Aβ group(KL-OE/Aβ group).Microglia were isolated by primary isolation,infected and intervened with microglia using empty vector,KL-interfering adenovirus,KL overexpressing adenovirus and IKK inhibitor(IKK-16),respectively,then microglia were co-induced with Aβ and IBO to construct LOAD cell model(Aβ microglia),and to construct Control group,empty vector group,KL silent group(KL-si group),KL-enhanced group(KL-OE group),IKK-16 group and KL-enhanced + IKK-16 group(KL-OE/IKK-16 group).(2)Zoological studies: KL gene transduction was detected by quantitative real-time PCR(q PCR)in each group;morphological and structural changes of hippocampal neurons were observed under transmission electron microscope in each group;the deposition of Aβ in the brain of each group was detected by immunohistochemical staining;and the histopathological changes in the brain of each group were observed by HE staining.Histopathological changes and surrounding inflammation were observed by HE staining;apoptosis of hippocampal neurons was detected by flow cytometry;the protein expression levels of IL-1,IL-2,IL-6 and TNF-α were measured by western blot(WB).Cytological studies: flow cytometry to identify the degree of microglia purification;fluorescence microscopy and q PCR to detect KL gene transduction in each group of Aβmicroglia;transmission electron microscopy to observe the morphological and structural changes of each group of Aβ microglia;ELISA to detect IL-1,IL-2,IL-6 and TNF-αsecreted by each group of Aβ microglia.levels.(3)Zoological studies: The expression of KL,FGF23 and β-amyloid precursor protein(APP)in each group of rats was measured by WB;the expression of nuclear transcription factor NF-κB in each group of rats was measured by immunohistochemistry.Cytological studies: expression of FGF23,VD and APP in Aβ microglia by ELISA;expression of KL,NF-κB and IKK in Aβ microglia by WB;expression of NF-κB(p65)by immunofluorescence staining.Results:(1)Zoological studiesA LOAD rat model was constructed by injecting Aβ and IBO in the CA1 region of the hippocampus and infecting LOAD rats with KL overexpression adenovirus,KL interference adenovirus,and empty vector.q PCR was used to detect KL gene expression after the intervention,and the results showed that compared with the Control group,the KL gene expression level was significantly higher in the KL-OE group,while the KL gene expression in the KL-si group levels were significantly reduced,suggesting successful KL gene transduction.The results of the experiment were as follows:(1)Aβ depositionImmunohistochemical staining showed that no significant Aβ deposition and brown positive cells were seen in the brains of rats in the Control,NC/NS,KL-si/NS and KLOE/NS groups;compared to the Control group,the NC/Aβ,KL-si/Aβ and KL-OE/Aβgroups showed significantly increased Aβ deposition and an increased number of brown positive cells in the brains of rats;The deposition of Aβ in the brains of rats in the KLOE/Aβ group was reduced compared to the NC/Aβ and KL-si/Aβ groups.(2)Brain histopathologyHE staining showed that neurons in the Control group were tightly arranged,with no obvious neuronal cell vacuolation degeneration and injury deformation;neuronal cells in the NC/Aβ group were loosely arranged and glial cells were increased;inflammatory cell infiltration,neuronal cell vacuolation degeneration and injury deformation were observed in the KL-si/Aβ group;histopathological manifestations in the KL-OE/Aβgroup were similar to those in the Control group.(3)Cell morphology and structureUnder transmission electron microscopy,rat hippocampal microglia in the NC/Aβ,KL-si/Aβ and KL-OE/Aβ groups were activated and intracellular lysosomes were increased.(4)ApoptosisCompared with the Control group,there was no significant difference in the apoptosis rate of rat hippocampal neurons in the NC/NS and KL-si/NS groups;the apoptosis rate in the KL-si/NS,NC/Aβ,KL-si/Aβ and KL-OE/Aβ groups was significantly increased,and the difference was statistically significant,while the apoptosis rate in the KL-si/Aβ group > NC/Aβ group apoptosis rate > KL-OE/Aβ group.(5)Inflammatory indexesCompared with the Control group,there was no significant difference in the expression of inflammatory indexes IL-1,IL-2,IL-6 and TNF-α in the brains of rats in the NC/NS and KL-OE/Aβ groups;the expression of inflammatory indexes IL-1,IL-2,IL-6 and TNF-α in the brains of rats in the KL-si/NS,NC/Aβ and KL-si/Aβ groups was enhanced,and the difference was statistically significant;the expression of inflammatory indicators IL-1,IL-2,IL-6 and TNF-α decreased in the KL-OE/NS group,and the differences were statistically significant.Cytological studiesMicroglia were isolated by primary isolation and flow cytometry showed that microglia were more than 95% pure and could be used for experiments.A LOAD cell model was constructed using Aβ and IBO induction,and cells were intervened with KL overexpressing adenovirus,KL interfering adenovirus,and IKK inhibitor(IKK-16).48 h after infection,a strong green fluorescent signal was observed in the cells under inverted fluorescence microscopy,and compared with the Control group,KL gene in the KL-OE and KL-OE/IKK-16 groups The KL gene expression level was significantly higher in the KL-OE and KL-OE/IKK-16 groups compared to the Control group,while the KL gene expression level was significantly lower in the KL-si group,suggesting that the KL gene transfection was successful,with the following experimental results:(1)Cell morphology and structureUnder transmission electron microscopy,the nuclei of Aβ microglia in the Control group were deformed and the mitochondria were swollen and flocculent,with multiple lysosomes visible in them;the nuclei of cells in the KL-OE and KL-OE/IKK-16 groups were round and full,with uniform chromatin distribution and normal subcellular structures.(2)Inflammatory factorsCompared with the Control group,the levels of IL-1,IL-2,IL-6 and TNF-α secreted by Aβ microglia in the NC group were not significantly different;the levels of IL-1,IL-2,IL-6 and TNF-α secreted by Aβ microglia in the KL-si group were significantly higher and the differences were statistically significant;the KL-OE group and the KL-OE/IKK-16 group secreted The levels of IL-1,IL-2,IL-6 and TNF-α were significantly lower in the KL-OE and KL-OE/IKK-16 groups,and the differences were statistically significant;the levels of TNF-α secreted by Aβ microglia were not significantly different in the IKK-16 group,but the levels of IL-1,IL-2 and IL-6 secreted were significantly lower,and the differences were statistically significant.(2)Zoological studies(1)Expression of KL,FGF23,APPCompared with the Control group,there was no significant difference in KL protein expression in the brain tissue of rats in the NC/NS group;KL protein expression was enhanced in the KL-OE/NS and KL-OE/Aβ groups,and the difference was statistically significant;KL protein expression was weakened in the KL-si/NS,NC/Aβ and KL-si/Aβgroups,and the difference was statistically significant.Compared with Control,there were no significant differences in FGF23 and APP protein expression in the NC/NS and KL-OE/Aβ groups;enhanced FGF23 and APP protein expression in the KL-si/NS,NC/Aβ and KL-si/Aβ groups,and the differences were statistically significant;weakened FGF23 and APP protein expression in the KL-OE/NS group,and the differences were The differences were statistically significant.(2)NF-κB expressionCompared with the Control group,NF-κB expression was increased in the brains of rats in the NC/Aβ,KL-si/Aβ and KL-OE/Aβ groups,especially in the KL-si/Aβ group,where NF-κB-positive cells were significantly aggregated.NF-κB expression was reduced in the KL-OE/Aβ group compared to the NC/Aβ and KL-si/Aβ groups.Cytological studies(1)Expression of FGF23,VD and APPFGF23 and APP levels were significantly increased and VD levels were decreased in Aβ microglia in the KL-si group compared to the Control group,and the differences were statistically significant;FGF23 and APP levels were significantly decreased and VD levels were increased in the KL-OE,IKK-16 and KL-OE/IKK-16 groups,and the differences were statistically significant.(2)Expression of KL,NF-κB and IKKCompared with the Control group,KL protein expression was enhanced and NF-κB and IKK protein expression was diminished in Aβ microglia in the KL-OE and KLOE/IKK-16 groups,and the differences were all statistically significant;KL protein expression was diminished and NF-κB and IKK protein expression was enhanced in the KL-si group,and the differences were statistically significant.In contrast,NF-κB and IKK protein expression was further decreased in the KL-enhanced/IKK-16 group compared to the KL-OE and IKK-16 groups.(3)NF-κB expression in cellsImmunofluorescence staining showed that NF-κB expression was significantly enhanced in the nuclei of Aβ microglia in the KL-si group,diminished in the KL-OE and IKK-16 groups,and almost absent in the cytoplasm and nuclei of the KL-OE/IKK-16 group compared to the Control group.Conclusions:(1)KL gene can reduce Aβ accumulation in LOAD rat brain,alleviate Aβ-mediated neuroinflammation,reduce brain tissue damage and neuronal apoptosis.KL gene can alleviate Aβ-mediated inflammation and damage to cell structure in Aβ microglia.(2)The anti-inflammatory effect of KL genes on LOAD may be mediated through the KL-FGF23-VD axis that regulates the IKK and NF-κB signaling pathways. |
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