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The Role and Regulation of SMADs in Rat Model of Cerebral Ischemia and Reperfusion

Author: WangFengZhang
Tutor: LiuQun
School: Jilin University
Course: Neurology
Keywords: TGF-β1 SMAD2 cerebral infarction SMAD3 inflammation apoptosis
CLC: R743.31
Type: PhD thesis
Year: 2013
Downloads: 103
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Abstract


This experiment selected PC12cells and rats of ischemia reperfusion model asobject to investigate SMAD2and SMAD3expression and regulation in the ischemicarea and their effects on inflammation and apoptosis after cerebral infarction. Thisexperiment was divided into3parts:1. Dynamic expression of SMAD2and SMAD3in the ischemic area andrelevance to inflammation and apoptosis.Rats of ischemia reperfusion model were sacrificied at6h,24h,72h and7dafter surgery, respectively. Rt-PCR was used to detect SMAD2/3mRNA expression,western blot was used to detect SMAD2/3protein and phosphorylated protein, Tunelstaining was used to detect apoptotic cells, and ELISA was used to detect theexpression of IL-1β. The results showed that in area surrounding infartion of modelgroup, SMAD2/3mRNA were higher than that of control group and sham group at6h, gradually increases to the peak at72h thereafter, and decreased at7d. The trend ofexpression of phosphorylated SMAD2/3(p-SMAD2/3) was consistent with SMAD2/3mRNA. At6h,24h and72h after ischemia/reperfusion, there was no obviousdifference of expression of SMAD2/3protein around ischemia area between themodel group and control group and sham group, but at7d is higher than the controlgroup and sham group. Expression of SMAD3protein was negatively correlated withapoptosis, inflammation, whereas there is no clear correlation between SMAD2protein expression levels and apoptosis, inflammation. The results showed that thecerebral ischemia reperfusion can cause SMAD2/3expression changes and SMAD3may have nerve protective effect such as anti-inflammatory and anti apoptosis.2. MiR-145downregulated SMAD2and SMAD3protein expression at cellularlevels.microRNAs are a kind of18-25nt long non-coding small RNA molecules thatcan regulate gene expression at the transcription level. After cerebral ischemiareperfusion, in the area around infarction a series of microRNAs expression levelschanged. We hypothesized that after cerebral ischemia reperfusion, SMAD2/3protein and mRNA expression is not parallel was due to the regulation of microRNAs. Wefound that the human SMAD2/3gene contained a theoretical miR-145binding site intheir3’ UTR. To confirm that these sites contributed to the regulation of SMAD2/3mRNA, we constructed a luciferase reporter vector with the SMAD2/33’UTR andperformed the luciferase reporter activity assay. We also constructed plasmids inwhich the conserved miR-145binding sequence, the poorly conserved miR-145binding sequence, or both were mutated. When a wild type SMAD2/33’-UTRluciferase reporter was co-transfected with miR-145mimics into HEK-293cells, therelative luciferase activity was significantly reduced by almost60%compared withthat of the negative control, whereas mutation of one sequence or the other attenuatedthe repression mediated by the miR-145mimics. In contrast, the luciferase activity incells transfected with empty vector or the reporter carrying both mutant sequenceswas not affected. Furthermore, we found that transfection of PC12cells with miR-145mimics efficiently reduced SMAD2/3protein levels, whereas transfection withmiR-145inhibitors notably increased the expression of SMAD2/3.3. Overexpression of SMAD3in the brain attenuated apoptosis and inflammationafter cerebral ischemia/reperfusion.We injected adenovirus plasmid contained SMAD3cDNA to lateral ventricle ofrats to study the effects of SMAD3expression on apoptosis and inflammatoryresponse after cerebral ischemia/reperfusion using gene transferring technology. Theresults showed that overexpression of SMAD3could significantly decrease tunelstaining positive cells and caspase3levels, increase the bcl2levels and downregulatethe expression of TNF-α and IL-1β mRNA significantly. The results showed that theSMAD3has nerve protective effects such as antiapoptosis and anti-inflammatoryeffects and could be used as a potential drug target for the clinical treatment ofcerebral infarction.

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CLC: > Medicine, health > Neurology and psychiatry > Neurology > Cerebrovascular disease > Acute cerebrovascular disease ( stroke) > Transient ischemic
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