The Protective Effect And Mechanism Of NR(Nicotinamide Riboside) On ICH(Intracerebral Hemorrhage)

Aim:Hemorrhagic stroke has become a global health problem due to its high morbidity and mortality.This study explored the protective effect of nicotinamide riboside(NR)on collagenase-induced hemorrhagic stroke and its possible mechanisms.Methods:The intracerebral hemorrhage(ICH)model was established by stereotactic injection of collagenase into the right striatum of adult male ICR mice.Thirty minutes after the model was successfully established,NR was injected via tail vein,the mice were sacrificed at different time points as indicated,and the corresponding indicators were measured.The cerebral water content,neurobehavioral,HE staining and other indicators were used to evaluate the protective effect of NR on hemorrhagic brain injury and to determine the optimal dose of NR for hemorrhagic stroke.SOD activity,MDA content,H2O2 content,GSH/GSSG ratio and ATP content were detected with corresponding detection kits,Western blot detection of oxidative stress-related protein Nuclear respiration factor 2(Nrf2)levels in the nucleus and cytoplasm and the levels of antioxidant protein HO-1 to evaluate the effects of NR on oxidative stress induced by collagenase.Activation of microglia and astrocyte in peri-hematoma tissues by immunofluorescence assay,and protein and mRNA levels of inflammatory factors including COX-2,NLRP3,ASC,Caspase-1,tnf-α and il-1βwere detected by Western blot and RT-QPCR to evaluate the effects of NR on inflammatory response induced by collagenase.Results:This study successfully established a hemorrhagic stroke model.Cerebral water content,neurobehavioral,HE staining results showed that the cerebral water content increased,the neurological deficit was aggravated and the number of normal neurons significantly decreased in ICH group.Different doses of NR were given 30 min after injection of collagenase,it reduced cerebral water content,restored the neurobehavioral function of mice and increased the number of normal morphological neurons to varying degrees.These indicators suggest that NR has a protective effect on hemorrhagic stroke,and the optimal dose is 300 mg/kg.The results of SOD,MDA,H2O2,GSH/GSSG and ATP showed that SOD activity,GSH/GSSG ratio and ATP content in ICH group were significantly decreased,MDA content and H2O2 content were significantly increased.NR administration inhibited collagenase-induced increase in MDA content and H2O2 content,and inhibited collagenase-induced decrease in SOD activity,GSH/GSSG ratio and ATP content.The results of immunofluorescence showed that the microglia and astrocyte in perihematoma tissues were significantly activated in ICH group,while NR administration inhibited this activation.Western blot results showed that the protein levels of COX-2,HO1,NLRP3,ASC and Caspase-1 increased in ICH group,Nrf2 entered into the nucleus,NR administration inhibited the protein levels of COX-2,NLRP3,ASC and Caspase-1 induced by collagenase,and further increased the protein levels of HO-1 and promoted Nrf2 localization to the nucleus.RT-QPCR results showed that the mRNA levels of nlrp3,caspase-1,tnf-α and il-1β increased in ICH group,and NR administration inhibited collagenase-induced increase in nlrp3,caspase-1,tnf-α and il-1β mRNA levels.Conclusions:(1)NR reduced cerebral water content,promoted neurological function recovery and reduced histopathological damage after intracerebral hemorrhage,suggesting that NR has a protective effect on hemorrhagic stroke.(2)NR reduced the levels of MDA,H2O2;increased the levels of HO-1,SOD,GSH/GSSG and ATP;promoted Nrf2 entry into the nucleus;suggesting that improving the ability of anti-oxidative stress and maintaining energy metabolism may be the protective mechanisms of NR against hemorrhagic stroke.(3)NR inhibited the activation of microglia and astrocyte;reduced the levels of inflammatory factors COX-2,tnf-α,il-1β;inhibited the activation of inflammasomes(NLRP3,ASC,Caspase-1);suggesting that reducing inflammatory response may be also the protective mechanisms of NR against hemorrhagic stroke.