The Expression And Role Of NOX1and NOX4in Ischemia Reperfusion In Mice

Objective To investigate the expression and role of NOX1and NOX4after ischemia strokeand reperfusion.Methods36KM male mice were divided into the Sham group, the middle cerebral arteryocclusion(MCAO) group and the inhibitor (Betulinic) group. The experiments were carriedout in1day and3day time points after establishment of MCAO model，6mice for each timepoint. The Betulinic group were treated with betulinic acid dissolved in dimethyl sulfoxide(DMSO) and treated the mice via gavage for7days, while the Shame group and the MCAOgroup were treated with DMSO of the same dose for7days as well. Then the middlecerebral artery of the mice in the MCAO group and the Betulinic group were embolized for2h and reperfused in1st day and3rd day, while the Sham group were only exposed thecarotid artery. The brain slices were strained with2%2,3,5-triphenyltetrazoliumchloridemonohydrate (TTC) to detect the infarction volume rate. Expressions and locationsof NOX1and NOX4in the infarcted sides and the contralateral non-infarctionwere detectedbyreal-time PCR and in situ hybridization, respectively. The apoptosis of neuron in theinfracted district were evaluated by TUNEL, while the production of ROS were measured byDCFH2-DA.In analysis, the MCAO group were compired with the Sham group and theBetulinic group were compired with the MCAO group, respectively. Results TTC staining showed that infarctions were significant in the MCAO group in1stand3rdday (13.6_i1.23%and8.460.82%，respectively, P<0.05), while in the Betulinic group,they decreased to1.6_i0.28%and1.7_i0.35%, respectively (P<0.05).The expression ofNOX1in the MCAO group were up-regulatedinto3.20.24folds in1stday (P<0.05) andappeared in the cortex, then returned to normal levels in3rdday and disappeared in thecortex. In the Betulinic group, the expression were decreased into2.50.18folds and1.7_i0.13folds, respectively(P<0.05).Meanwhile, the expression of NOX4graduallyincreased from2.1_i0.21folds in1stday to3.70.34folds in3rdday(P<0.05). But the regionsof the expression had no change. When treated by Betulinic,NOX4were downregulated(2.7_i0.26folds and2.3_i0.22folds,respectively,P<0.05). ROS production significantlyincreased to492.1_i15.27%in1stday and478.914.23%in the3rdday relative to22.3_i1.35and21.42.46in the Sham group, respectively(P<0.05), while decreased to78.6_i4.31and92.3_i8.34in the Betulinic group(P<0.05).Apoptosis could be ignored in the Sham group.However, it significantly increased to74.36.58%and47.94.35%in the MCAO groupversus0.20.05%and0.30.07%in the Sham group (P<0.05)， while decreased to21.22.16%and5.70.49%in the Betulinic group (P<0.05).Conclusion The time-dependent expression of NOX1and NOX4suggested that NOX1isinvolved in the first(reactive) phase while NOX4involved in the second(regeneration) phaseof ischemic response. Reduction of NOX1and NOX4and its oxidative stress production bybetulinic decreased neuron apoptosis rate which may protect the brain from ischemiareperfusion injury.