| Background：Thioredoxin (TRX) is a small cellular redox enzyme with multiplefunctions ubiquitously expressed in all living cells. However, in TRXfamily, Trx1is the member which has been studied mostly. Studies haveshown that Trx1has multiple actions such as anti-apoptotic, anti-oxidantanti-tumor and anti-inflammatory effects. In recent years, studies havefound that Trx1could obviously reduce infarct volume and improve theneurological function in rats, which induced by cerebral ischemia andreperfusion injury.Trx1was been found that it has potential prospect incerebral ischemia and reperfusion injury diseases.Objecitve：To explore neuroprotective effects of Trx1on focal cerebral ischemiaand reperfusion injury in rats and explore its possible mechanisms andrelated signal transduction pathways．Methods：Transient cerebral ischemia and reperfusion was induced by middle cerebral artery occlusion (MCAO) using modified suture occlusiontechniquein in rats. Rats were randomly divided into6groups includingsham group, MCAO group, MCAO treated with non-specific controlsiRNA and three specific Trx1siRNA groups. siRNAs were injected intorat brains24h prior to MCAO by intracerebroventricular injection. Theneuroprotective effects of Trx1and its possible mechanisms were studied.(1) Proteins were extracted from the ischemic cortex24h after reperfusion.Western blot and Quantitative PCR were applied to detect Trx1protein andmRNA expression in rat brain tissue to screen for an optimized siRNAsequence targeting Trx1.(2) Mortality and neurological scores wereevaluated; TTC staining was used to measure infarct volume; HE-stainingand Nissl-staining were applied to observe the cell morphologic changes.TUNEL staining was used to count apoptotic cells.(3) Co-IP assay wasused to detect whether Trx1siRNA could dissociate the ASK1/Trx1binding complex. Meanwhile, the expression of ASK1、P-ASK1、JNK、P-JNK p38、P-p38、cytochrome c and cleaved caspase-3proteins in theischemic cortex were detected by Western blot.Results:(1) Compared with the MCAO+Control group, three pairs of siRNAstargeting Trx1significantly suppressed the Trx1expression; theinhibitory effect of siRNA2was the strongest one, with an interferenceefficiency over50%. (2) Trx1siRNA obviously increased mortality, behavioral deficits,and cerebral infarction volume and exacerbated neuronal cell apoptoticdeath after MCAO injury.(3) Co-immunoprecipitation assay suggested an interaction betweenTrx1and ASK1in normal rat brains and Trx1siRNA dissociatedASK1-Trx1binding complex. Western blot revealed increased expressionof apoptotic proteins such as P-ASK1, P-JNK, P-p38, Cytochrome c andCleaved Caspase-3in the Trx1siRNA-treated group.Conclusion:(1) Direct delivery of Trx1siRNA into the lateral ventricle of the brainduring middle cerebral artery occlusion-induced focal ischemia in ratmodel is effective for exacerbating cerebral ischemia and reperfusioninjury.(2) The neuroprotective effects of Trx1against focal cerebral I/R injuryare likely related to the attenuation of apoptosis. It was one of importantneuroprotective mechanisms of Trx1that inhibiting apoptosissignal-regulated kinase1(ASK1) and ASK1-mediated downstreamJNK/P38signaling pathway. |
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