Effects Of Melatonin On The Expressions Of Nox2and Nox4in The Brain In A Rat Experimental Cerebral Ischemia And Reperfusion Model

Objective：The therapeutic time window for ischemic stroke is very short and earlyrecanalization by intravenous administration of thrombolytics is currently the onlyapproved treatment for stroke. Unfortunately, even if blood flow is restored,ischemia-reperfusion (I/R) injury induced by reactive oxygen species (ROS), whichespecially increases in the reperfusion phase and generates from the newly arriving oxygen,is a complex phenomenon and leads to unfavorable clinical outcome of the therapy. Studieshave suggested that Nox family proteins, especially Nox2and Nox4, act as importantgenerators of ROS during I/R injury. As a radical scavenger, melatonin (N-acetyl5-methoxytryptamine) has been demonstrated to play a neuroprotective role in a rattransient middle cerebral artery occlusion (MCAO) stroke model. However, littleinformation has been reported on the mechanism for the protective role of melatoninduring brain I/R injury. The aim of this study is to evaluate whether melatonin plays itsanti-oxidant and anti-apoptotic effects via Nox2and Nox4in a rat brain I/R injury modelinduced by MCAO.Methods:30male Sprague-Dawley (SD) rats were randomly divided into threegroups: sham operation group, ischemia-reperfusion group, melatonin pretreatment+ischemia-reperfusion group, Middle cerebral artery occlusion (MCAO) model in rats wasprepared by Suture-occluded method(Corresponding nerve dysfunction after rat middlecerebral artery embolism to verify model was successful).Then use Fluorescencespectrophotometer to detect ROS levels in brain tissue,TUNEL staining to detect braintissue cell apoptosis, and Nox2, Nox4protein levels were detected by Western blot. WhileThe number of cells of TUNEL and Nox2Nox4immunofluorescence staining. thecorresponding experimental data and results were analyzed by SPSS18.0statisticalanalysis software, the data for statistical analysis. Results：1)Compared with the sham operation group, the ROS levels of the ischemia-reperfusion group increased significantly, especially in the ischemic hemisphere;Melatonin pretreatment can inhibite the rising of the ROS levels in ischemic hemisphereand the contralateral hemisphere significantly2)Compared with the sham operation group, the apoptotic cells increased significantlyin the ischemia-reperfusion group, especially in the ischemic hemisphere; The melatoninpretreatment can significantly inhibit the cell apoptosis which caused by ischemia-reperfusion that in ischemic hemisphere and the contralateral hemisphere.3)Compared with the sham group, Nox2and Nox4protein levels increasedsignificantly in ischemia-reperfusion group; And melatonin pretreatment can significantlyinhibit the rising of Nox2and Nox4protein levels that caused by ischemia-reperfusion.This result suggests that melatonin may inhibit the production of ROS, and inhibitingapoptosis by lowering the expression of Nox2and Nox4.4) Immunofluorescence staining showed that: TUNEL positive cells accompanied byhigh expression of Nox2or Nox4; Melatonin pretreatment significantly reduced thenumber of Nox2/TUNEL double positive cells and Nox4/TUNEL double positive cells.Conclusion：Our findings suggested that the inhibition of Nox2and Nox4expressions bymelatonin may essentially contribute to its anti-oxidant and anti-apoptotic effects duringbrain I/R.