Rehmapicroside Ameliorates Cerebral Ischemia-reperfusion Injury Via Attenuating Peroxynitrite-mediated Mitophagy Activation

ObjectiveIschemic stroke is characterized with insufficient blood supply to brain with oxygen and glucose deprivation of brain tissues,subsequently leading devastative and irreversible brain damage.Peroxynitrite(ONOO-)-mediated mitophagy activation represents a vital pathogenic mechanism in ischemic stroke.Our previous study suggests that ONOO-mediates Drpl recruitment to the damaged mitochondria for excessive mitophagy,aggravating cerebral ischemia/reperfusion injury and the ONOO--mediated mitophagy activation could be a crucial therapeutic target for improving outcome of ischemic stroke.Radix Rehmanniae,a medicinal plant,has been used in Traditional Chinese Medicine(TCM)for centuries as an important component of TCM formulae for stroke.Rehmapicroside is a main active principle of the root of Radix Rehmanniae.However,the bioactivity of Rehmapicroside has been seldom investigated and whether rehmapicroside has neuroprotective effects on ischemic stroke is unknown yet.In the present study,we hypothesize that rehmapicroside could be a promising active compound to ameliorate cerebral ischemia-reperfusion injury and its underlying mechanisms could be related to inhibit the ONOO--mediated excessive mitophagy activation.Methods1.PC12 cells were exposed to oxygen-glucose deprivation and reoxygenation(OGD/RO)condition to mimic cerebral ischemia-reperfusion injury in vitro.The experimental groups were as follows:control group(Ctrl),ODG/RO,ODG/RO+Reh 25 ?M(Reh 25),ODG/RO+Reh 50 ?M(Reh 50),and ODG/RO+Reh 100 ?M(Reh 100).At the onset of reperfusion,the culture medium was replaced with high-glucose DMEM containing rehmapicroside(100?M)and cultured for 22 h.In vehicle control group,the cell medium was replaced with high-glucose DMEM and the cells were transferred to the in cubator under normoxic condition for 22 h to induce reoxygenation.2.We investigated the direct reaction between ONOO-and rehmapicroside by using UPLC for evaluating direct ONOO scavenging property of rehmapicroside.The groups were divided into the following groups:Reh 500 ?M,Reh 500 ?M+ONOO-1mM,Reh 500 ?M+ONOO-4mm.3.Adult male Sprague-Dawley(SD)rats were subjected to middle cerebral artery occlusion(MCAO)to induce cerebral ischemia.Rats were randomly divided into following groups including sham control,sham control plus rehmapicroside(10 mg/kg),MCAO,MCAO plus rehmapicroside(2.5,5,10 mg/kg wt)and MCAO plus FeTMPyP(peroxynitrite decomposition catalyst,PDC)(3mg/kg).Both rehmapicroside and PDC dissolved in saline at designed dosages were intraperitoneally administrated into the rats at the onset of reperfusion.The volume for the injection treatment was 5 mL/kg.In a parallel experiment,the same volume of saline was used as a control.4.Modified neurological severity score(mNSS)scale was used to comprehensively assess the neurological dysfunction.Then,the slices of the brains were placed in a 2%2,3,5-triphenyltetrazolium chloride(TTC).The infarct size was measured and analyzed by the white infarcted area and reddish-purple non-infarcted area.Western blot analysis,mitochondrial isolation analysis,immunoprecipitation and immunofluorescence were used to detect the biological conditions of the rats.ResultsThe major discoveries include following aspects:(1)Rehmapicroside reacted with ONOO-directly to scavenge ONOO-;(2)Rehmapicroside decreased O2-and ONOO-,up-regulated Bcl-2 but down-regulated Bax,Caspase-3 and cleaved Caspase-3,and down-regulated PINK1,Parkin,p62 and the ratio of LC3-? to LC3-? in the OGD/RO-treated PC cells;(3)Rehmapicroside suppressed 3-nitrotyrine formation,Drp1 nitration as well as NADPH oxidases and iNOS expression in the ischemia-reperfused rat brains;(4)Rehmapicroside prevented the translocations of PINK1,Parkin and Drp1 into the mitochondria for mitophagy activation in the ischemia-reperfused rat brains;(5)Rehmapicroside ameliorated infarction sizes and neurological deficit scores in the rats with transient MCAO cerebral ischemia.ConclusionTaken together,rehmapicroside could be a potential drug candidate against cerebral ischemia reperfusion injury and its underlying neuroprotective mechanisms could be attributed to inhibiting the ONOO--mediated mitophagy activation.