Neuroprotective Effets And Mechanisms Of Sulfiredoxin-1on Cerebral Ischemia/Reperfusion Oxidative Stress

Backgroud: Oxidative stress injury could aggravate irreversibledamage induced by cerebral ischemia/reperfusion. Recently endogenousantioxidant defense system was discovered to play a crucial role inmaintaining cell survival oxidant/antioxidant balance, resisting to oxidativestress in brain ischemia-reperfusion injury. As an endogenous antioxidantprotein, Srxn1could protect cells against oxidative stress. However, the roleof Srxn1in the nervous system and the underlying signaling mechanisismhas not been fully elucidated.Objective：We explored the effects of Srxn1knockdown in vitro and invivo ischemia reperfusion modles in oxidative stress, and investigated therelated neuroprotective mechanisms.Methods:(1) Primary cortical neuronal cultures were transfected withthe interfere lentivirus. The transfection efficiency was estimated bycalculating fluorescence expression rate under inverted fluorescencemicroscope, and the knockdown efficiency was determinated by real-timeqPCR and Western Blot assays. (2) Oxygen-glucose deprivation (OGD) was conducted after Srxn1knockdown. MTS and LDH assay was used to measure cell viability and celldamage, and superoxide dismutase (SOD) kit and reduced glutathione (GSH)kit was used to assay intracellular oxidative stress state. The relationshipbetween Srxn1and Prdx1-4, Prdx-SO3activity was further measured byimmunoblot analysis.(3) Three siRNA of Srxn1and one negative siRNA was build, andsiRNA was injected intracerebroventricularly24h before MCAO. Theknockdown efficiency was determined by real-time qPCR and Western Blotassays.(4) After Srxn1knockdown, neurological deficits, infarct volume andmorphological detection were performed. Oxidative stress was evaluated bydetecting SOD activity and MDA level. The relationship between Srxn1andPrdx1-4, Prdx-SO3activity was further measured by immunoblot analysis.(5) Three siRNA of Nrf2and one negative siRNA was build, andsiRNA was injected intracerebroventricularly24h before MCAO, theknockdown efficiency was determinated by real-time qPCR and WesternBlot assays. After Nrf2knockdown, the mRNA and protein level of Srxn1further detected by real-time qPCR and immunoblot analysis.Results:(1) After neurons were transfected with the lentivirus, themost effective interference fragement was GR545. Cell death was increased,cell viability was reduced and oxidative stress injury was attenuated after knockdown Srxn1. Furthermore, the decreased Srxn1was accompaniedwith a reduction of Prdx1-4and an increase of Prdx-SO3.(2) In MCAO modles, the most effective interference siRNA wasSrxn1-631. Srxn1interference resulted in a significant increase of cerebralinfarction, neurological deficits, histological injury and oxidative stressinjury at24h after ischemic stroke. After knockdown of Srxn1, theexpression of Prdx1-4was reduced, while Prdx-SO3was increased.(3) In MCAO modles, the most effective interference siRNA wasNrf2-1008. Interference of Nrf2could ruduce the mRNA and protein level ofSrxn1.Conclusion:(1) Srxn1was activated during ischemia-reperfusion injury, however,interfering Srxn1could aggravate oxidative stress injury of nerve tissue.(2) Srxn1might be as a downstream of Nrf2, could resist cerebralischemia reperfusion injury by reversing the peroxidation of Prdxs andrecovering the antioxidant activity of Prdxs.