Study On The Mechanism Of MiRNA-592 Down-Regulation To Inhibit Oxidative Stress Injury In Astrocytes Of Alzheimer’s Disease Rats

Background and objective The etiology and pathogenesis of Alzheimer’s disease are multifactorial.Abnormal accumulation of Aβand tau protein is the core factor in the pathogenesis of AD.Oxidative stress and neuroinflammation promote the pathogenesis and development of AD.Current studies have shown that micro RNAs are involved in the regulation of these pathogenic mechanisms.Micro RNA-592（miRNA-592）plays an important role in mediating the activity of neural cells,but its relationship with AD is unknown.In this study,we investigated the mechanism of miRNA-592 in astrocyte oxidative stress injury by establishing a rat model of AD and primary astrocyte experiments to provide a theoretical basis for the treatment of AD.Methods Animal experiment:（1）54 SD rats,2 months old with a body weight of 264.80±26.80g were divided into the control group（n=26）and AD group（n=28）.（2）The AD rat model was prepared by subcutaneous injection of D-galactose and bilateral hippocampus injection of Aβ_25-35to SD rats,and the Morris water maze test was used to evaluate the reliability of the established AD model rats.（3）The pathological changes in the CA1 area of hippocampus of control group and AD model rats were observed by staining with Congo red,hematoxylin and eosin（HE）.（4）After the AD model was successfully induced,the ELISA method was used to determine the rat serum oxidative stress indicators.（5）RT-q PCR to detect the changes of miRNAs and KIAA0319 m RNA in rat brain tissue.（6）Western blot was used to analyze the expression of Aβand tau protein in rat brain tissue.Cell experiments:（1）Astrocyte（AST）cells were extracted,isolated and cultured from the brain tissue of AD model rats,and were identified by immunocytochemistry,GFAP and DAPI immunofluorescence staining,and their purity was determined.The morphological and structural changes of the ASTs were observed under inverted fluorescence microscope.（2）Dual-luciferase reporter assay combined with bioinformatics analysis was used to investigate the relationship between miRNA-592 and KIAA0319 gene.（3）Construct p GL3 KIAA0319 Wt（wild-type luciferase reporter gene plasmid）,miRNA-592 NC（mir NA-592 negative control）,p GL3 KIAA0319 Wt,miRNA-592 mimic（mir NA-592 simulation group）,PGL3 KIAA0319 Mut（mutant luciferase reporter gene plasmid）,miRNA-592 NC,p GL3 KIAA0319 Mut,miRNA-592 mimic and recombinant plasmid were transfected with different combinations to verify whether KIAA0319 was the target gene of miRNA-592.（4）Use reporter gene vector to construct miRNA-592 and si RNA-Ki AA0319 plasmids,and transfect miRNA-592inhibitor+sir NA-Ki AA0319 plasmids.Mimic NC,inhibitor NC,and blank control were used.（5）The viability of ASTs cells in each group was measured by MTT assay and the OS activity level in each group was measured with a full-wavelength fluorescence microplate reader（Spectra Max Gemini EM,Molecular Devices）.（6）The m RNA levels of miRNA-592,KIAA0319,Keap1,Nrf2 and NQO1 in each group were measured by RT-q PCR and the expression levels of KIAA0319,C-Keap1,N-Nrf2,T-Nrf2 and NQO1 in each group were analyzed by Western blot.Results Animal experimental results:（1）Morris water maze test:AD rats induced by subcutaneous injection of D-galactose combined with hippocampal injection of Aβ_25-35were evaluated for behavior and learning and memory ability.Morris water maze results revealed that the escape latency and swimming distance of rats were the longest on the second day（P>0.05）,and the escape latency and swimming distance gradually shortened with the increase of training days.From day 3,the escape latency and swimming distance of AD group rats were significantly longer than those of the control group（P<0.05）.In the spatial exploration test on day 7,rats in the AD group had prolonged initial reach to the target area,shorter residence time in the target area,and reduced number of crossings of the target area compared with the control group（P<0.05）.（2）The results of serum ACh E,MAO,MDA,TNF-α,IL-6,and SOD in the control and AD rats showed that the serum ACh E,MAO,MDA,and IL-6 levels in the AD group were significantly higher than those in the control group（P<0.05）,and the serum SOD level was significantly decreased（P<0.05）,while there was no significant difference in TNF-αlevels between the two groups（P>0.05）.（3）HE staining of brain tissue:The cells in the CA1 region of the hippocampus in the AD group were unclear and reduced,the cytoplasm was light red,and the nuclei were dark and pyknotic.However,the cells in the CA1 region of the hippocampus in the control group were arranged orderly and dense,with clear cell boundaries and clear nuclei and cytoplasm.Congo red staining:In AD,most of the stained deposits were distributed in the hippocampus,with different sizes and staining,the background color was brownish-red,while the staining color was close to red.Most cells were round,irregular,and disorganized.In the control group,the hippocampus cells had clear boundaries,obvious nucleoli,and no obvious deposits.（4）RT-q PCR:the expression of miRNA-132-3p,miRNA-129-5p and miRNA-136-5p did not change in the brain of AD rats,while the expression of miRNA-592 was significantly up-regulated.The expression level of m RNA-KIAA0319 was relatively low in AD model rats.（5）Western blot showed that the expression levels of Aβ,T-tau,and p-tau protein were significantly increased in the AD group.Cell experimental results:（1）the morphology observation and identification of experimental rats observed under inverted microscope cell immune cell chemical dyeing,according to the AD group most of the cytoplasm protuberant brown,color shades,clear cell borders,cell morphological diversity,the number of ridges and length were significantly different,at high magnification,the cell body and raised a fiber sample brown staining;In the control group,only the nuclei were stained,and the cytoplasm was not brown.The cultured cells were identified by immunofluorescence staining and observed under inverted fluorescence microscope.GFAP staining（+）and DAPI staining（+）showed green cytoplasm and processes and blue nuclear ASTs respectively,and the purity reached more than 95%after counting.The morphology of the ASTs from day 6 to day 14 was similar,with flat and polygonal shapes,abundant cytoplasm and round or oval nuclei.On day 16,most of the ASTs were mature cells,and some showed hypertrophy and hyperplasia.（2）Construct p GL3KIAA0319 Wt（wild-type luciferase reporter gene plasmid）,miRNA-592 NC（miRNA-592 negative control）,p GL3 KIAA0319 Wt,miRNA-592 mimic（miRNA-592simulation group）,Plasmid p GL3 KIAA0319 Mut（mutant luciferase reporter plasmid）,miRNA-592 NC,p GL3 KIAA0319 Mut and miRNA-592 mimic were transfected and double luciferase reporter gene analysis was performed.The results showed that,miRNA-592 mimic had no significant effect on luciferase activity of p GL3 KIAA0319Mut（P>0.05）,while mir NA-592mimic down-regulated luciferase activity of p GL3KIAA0319 Wt by about 53%（P<0.05）.This indicates that miRNA-592 is bound to KIAA0319,and KIAA0319 is the target gene of miRNA-592.（3）MTT assay showed that cell viability was significantly lower in the miRNA-592 simulated si KIAA0319group compared with the control group（P<0.05）,while the miRNA-592 inhibitor group showed the opposite trend（P<0.05）.There was no significant difference in cell viability between the simulated NC group and the inhibitor NC miRNA-592inhibitor+si KIAA0319 group（P>0.05）.Gemini EM showed that compared with the control group,the SOD,CAT,and GSH levels were significantly decreased,while the MDA and ROS levels were increased（P<0.05）.The miRNA-592 inhibitor group showed the opposite trend（P<0.05）.There was no significant difference between the simulated NC group and the inhibitor NC miRNA-592 inhibitor+si KIAA0319 group（P>0.05）.（4）RT-q PCR results showed that miRNA-592 simulation+si KIAA0319,the expression of miRNA-592 and Keap1 was enhanced,and the expression of KIAA0319,Nrf2,and NQO1 was significantly decreased compared with the blank control group,while the miRNA-592 inhibitor group showed the opposite trend（all P<0.05）.There were no significant differences in the expression of miRNA-592,KIAA0319,Keap1,Nrf2,and NQO1 among the simulated NC group,inhibitor NC group,miRNA-592 inhibitor+si KIAA0319,and blank control group（P>0.05）.（5）Western blot analysis showed that the expression of miRNA-592 mimic+si KIAA0319C-Keap1 was increased and the expression of KIAA0319,N-Nrf2,and NQO1 were decreased compared with the blank control group.In addition,the expression of C-Keap1 was decreased and the expression of KIAA0319,N-Nrf2,and NQO1 was enhanced in the miRNA-592 inhibitor group relative to the blank group（p<0.05）.There were no significant differences in KIAA0319,C-Keap1,N-Nrf2,T-Nrf2,and NQO1 between the simulated NC group,the inhibitor NC group,and the inhibitor+si KIAA0319 blank control group（P>0.05）.Conclusion（1）The AD model induced by subcutaneous injection of D-galactose combined with Aβ_25-35in hippocampus is reliable;The expression of Aβ,T-Tau and p-Tau protein increased in brain tissue,and AD was pathologically altered in CA1region.（2）The results of animal experiments showed that there was oxidative stress injury in AD rats,and miRNAs expression was abnormal in brain tissue.Mi RNA-592expression was high,while KIAA0319 expression was low.（3）Cell experiment results showed that down-regulation of miRNA-592 could reduce OS injury of AD rats through activation of Keap1/Nrf2/ARE signaling pathway,and the mechanism of action might be up-regulation of KIAA0319 expression.That is,down-regulation of miRNA-592 can inhibit oxidative stress injury of AD rats through up-regulation of KIAA0319 expression and activation of Keap1/Nrf2/ARE signaling pathway.