| Background and Purpose:Schizophrenia is a group of severe mental disorders.Icariin is one of the main active ingredient of herba epimedii,and previous studies have shown that it has therapeutic effects on a variety of neurodegenerative diseases.This study investigated whether icariin can effectively relieve schizophrenia-like symptoms and explore its potential molecular mechanism.In our previous study,the expression of microRNA-144-3p(miR-144-3p)was up-regulated in the schizophrenic rat brains.In addition,many other neurological and psychiatric diseases have also been reported to be associated with miR-144-3p dysfunction,including Parkinson’s disease,Alzheimer’s disease,Huntington’s disease,amyotrophic lateral sclerosis,bipolar affective disorder,etc.Therefore,due to the extensive involvement of miR-144-3p in neurological and psychiatric diseases,the signaling pathway mediated by miR-144-3p seems to be a promising target for the treatment of schizophrenia,which is worthy of further study.TargetScanHuman database predicted that ZBTB20(zinc finger and BTB domain containing 20)is the target gene of miR-144-3p.ZBTB20 plays an important role in brain development and cortical genesis.Its dysfunction induces a variety of neurological disorders,suggesting that ZBTB20 might be a potential mechanism of the pathogenesis of schizophrenia.Therefore,this study investigated the therapeutic effects of icariin on the schizophrenic-like abnormal behavior induced by MK-801 and its inhibition of neural cell damage;this study also explored the connection between the miR-144-3p/ZBTB20 signaling and the pathogenesis of schizophrenia and the regulatory effects of ZBTB20 on this signaling pathway.Method:Young Sprague Dawley rats were injected intraperitoneally with MK-801 for two weeks to build a neurodevelopmental schizophrenic rat model.Using behavioral experiments:open field experiment,new object recognition experiment,and rod rotation experiment to explore the improvement effect of icariin on MK-801-induced schizophrenic like abnormal behavior;the safe concentration of icariin at the cell level was screened through the MTT test,then,the control group(normal complete culture medium,without drugs),MK-801 injury group,positive drug(risperidone)group,and different icariin concentration groups were set up,respectively,and the inhibitory effects of icariin on MK-801-induced neural cell injury was detected by the Calcein/PI experiment.Detection of miR-144-3p expression in hippocampus and caudate nucleus of schizophrenic model rats by RT-PCR.In the SK-N-SH cells overexpressing miR-144-3p,the differentially expressed genes(DEGs)were screened by second-generation transcriptome sequencing(RNA-seq).In addition,the DEGs in the hippocampus of schizophrenic model rats were also screened by RNA-seq.Next,TargetScanHuman(Version 8.0)database was used to predict the target genes of miR-144-3p.Analyze the above three gene sets to find key downstream genes of miR-144-3p;The regulatory relationship between miR-144-3p and key genes was detected through dual luciferase reporting experiments.Measure the mRNA and protein expression levels or phosphorylation levels of key genes in constructed cell and animal models;Western blot was used to detect key genes and related signal pathways;Immunohistochemical techniques were used to detect the expression level of key genes in the hippocampus and caudate nucleus of rats.Results:1.The open-field experiment has been proved that icariin treatment can alleviate anxiety induced by MK-801 in rats with schizophrenia;The new object recognition experiment has been proved that icariin treatment can partially recover the decline in the ability to explore new things induced by MK-801 in schizophrenic rats;The rotating rod experiment showed that icariin treatment can partially recover the sports ability damage caused by MK-801 in schizophrenic rats.2.The MTT test results showed that the safe concentration of icariin at the cell level was 0-40 μM.The Calcein/PI staining results showed that icariin could inhibit the damages in neural cells induced by MK-801.3.The RT-PCR data showed that the expression levels of miR-144-3p was significantly increased in the hippocampus and caudate nucleus of the schizophrenic model rats.4.A total of 426 DEGs were screened from the hippocampus of the schizophrenic model rats;the DEG analysis on the SK-N-SH cells overexpressing miR-144-3p revealed 906 DEGs;the TargetScanHuman(Version 8.0)database predicted 1048 genes that might be the targets of miR-144-3p.Finally,ZBTB20 was found by intersecting the above three gene sets.5.The targeted regulatory relationship between ZBTB20 and miR-144-3p was confirmed using the dual luciferase report probe technology.6.In the hippocampus and caudate nucleus of schizophrenic model rats,the mRNA and protein expressions of ZBTB20 were significantly reduced,and the phosphorylation levels of ERK and CREB in MK-801 group were significantly reduced.Icariin can restore these abnormal changes to near normal levels.7.In the 293T cells overexpressing miR-144-3p,the RT-PCR results showed that the levels of ZBTB20 mRNA was significantly decreased.The Western blot results showed that the expression of ZBTB20 protein was significantly decreased by MK-801,and the phosphorylation levels of ERK and CREB were also significantly decreased by MK-801.Icariin can restore these abnormal changes to near normal levels.8.The immunohistochemical results showed that the expression levels of ZBTB20 were significantly decreased in the hippocampus and caudate nucleus of the schizophrenic model rats,but restored to near the normal levels by icariin.Conclusion:Icariin can improve the anxiety,exploratory ability to new things,and sports ability of schizophrenia model rats;The safe concentration of icariin at the cellular level is 0-40 μM and it has an inhibitory effect on SK-N-SH cell damage induced by MK-801;The pathogenesis of schizophrenia may be related to dysfunction of miR-144-3p/ZBTB20/ERK/CREB signaling pathway;In addition,icariin may treat schizophrenia by regulating this signaling pathway. |
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