Effects Of Homocysteine On Activation Of Microglia And Mitochondrial Damage In Rats With Cerebral Ischemia-reperfusion By Regulating P-STAT3 Level

ObjectiveTo observe the effects of homocysteine(Hcy)on microglia activation and mitochondrial damage in the ischemic brain by establishing rat model of middle cerebral artery occlusion(MCAO),and investigate the possible mechanism of signal tranducer and activators of transcriptions 3(STAT3)in cerebral ischemia caused by Hcy and AG490 intervention,and provide new ideas and experimental basis for prevention and treatment of cerebral ischemia-reperfusion injury.Methods120 adult male SD rats weighing 180~200 g were randomly divided into sham operation group(SHAM),middle cerebral artery occlusion group(MCAO),MCAO+Hcy group,MCAO+Hcy+AG490 group,MCAO+AG490 group.There were twenty-four rats in each group.The rats in SHAM and MCAO group were injected with saline at 4 mL/kg·d via tail veins.The rats in MCAO+Hcy and MCAO+Hcy+AG490 group were injected with 0.8 mL/mg Hcy solution at 4 mL/kg·d via tail veins.The rats in MCAO+Hcy+AG490 and MCAO+AG490 group were intraperitoneally injected with 0.75 m L/mg AG490 solution before 1 h of surgery.The brain infact volume was measured by triphenyltetrazolium chloride(TTC)staining.Morphological changes were detected by hematoxylin-eosin(HE)staining.The mitochondrial damage of brain was observed by transmission electron microscopy.Immunofluorescence staining was used to detect the numbers of Iba-1,OX-42,IL-6,TNF-α,8-OHdG and p-STAT3 positive cells.Western blot was used to detect the protein level of Iba-1,OX-42,IL-6,TNF-α,Cytochrome c(Cyt c)and p-STAT3 in brain.Reactive oxygen species(ROS)in N2 a cells were detected by H2 DCFDA probe,and the expression of p-STAT3 in mitochondria of N2 a cells was detected by western blot.ResultsCompared with MCAO group,the TTC staining after Hcy treatment showed that the volume of cerebral infraction increased,HE staining showed that the injury of neurons was aggravated,the degree of mitochondrial damage was increased.Immunofluorescence staining showed that the numbers of OX-42(Iba-1)and p-STAT3 double-positive cells,p-STAT3 and COX IV double-positive cells,OX-42,Iba-1,IL-6,TNF-α and 8-OHdG positive cells were significantly increased(P<0.05).Western blot showed that the protein level of Iba-1,TNF-α,IL-6,TNF-α and p-STAT3 were significantly increased(P<0.05).Compared with MCAO+Hcy group,the numbers of OX-42(Iba-1)and p-STAT3 double-positive cells,p-STAT3 and COX IV double-positive cells,OX-42,Iba-1,IL-6,TNF-α and 8-OHdG positive cells were all significantly reduced,and the expression of protein level of Iba-1,TNF-α,IL-6,TNF-α and p-STAT3 were all significantly decreased in MCAO+Hcy+AG490 group(P<0.05).Measured the cellular ROS levels with the fluorescent probe H2 DCFDA,compared with the control group,Hcy also induced ROS accumulation in N2 a cells,and the level of intracellular ROS began to increase by 5 min,mitochondrial STAT3 was also activated in Hcy-treated N2 a cells,and a significant increase in p-STAT3 levels was observed as early as 30 min after Hcy treatment.Compared with the Hcy group,both AG490 and GEE could significantly suppress STAT3 activation and ROS formation after Hcy treatment.ConclusionsIt was found that Hcy could exacerbate the damage of brain tissue and mitochondria in rats,promote the activation of microglia and the expression of proinflammatory cytokines and raise the level of oxidative stress and the expression of p-STAT3 in microglia and mitochondria,and these effects above were all inhibited by AG490,therefore,Hcy promoted microglia activation and mitochondrial damage through the p-STAT3 expression.In addition,early ROS generation induced by Hcy may induce mitochondria-localized STAT3 activation,in turn,the p-STAT3 expression in mitochondria may further induce the generation of ROS.There may be a crosstalk between oxidative stress and mitochondrial p-STAT3.