The Effects And Mechanisms Of CTRP 3 On Oxidative Stress Injury In Rats After Intracerebral Hemorrhage Via PKA Pathway

Background:Intracerebral hemorrhage(ICH)is a common disease in nervous system.it is characteristic of speedy development,high morbidity,high mortality and high disability rate.Increasingly research is showing that oxidative stress may play an important role in the development of intracerebral hemorrhage.Recent studies have demonstrated that CTRP 3 plays an important role in inflammation,metabolism,apoptosis,angiogenesis,and cardiac protection.Study shows that CTRP 3 can decrease the infarct size,attenuated cardiomyocyte apoptosis and increase angiogenesis,which has protective effects on I/R myocardium in myocardial ischemia in mice.studies have shown that NADPH enzymes plays an important role in oxidative stress injury,NOX 2 is one of the most important subunits mediated oxidative stress injury.CTRP 3 may function as a mediator of oxidative stress injury after intracerebral hemorrhage has never been previously investigated.Objective:(1)To study whether CTRP 3 possess protective effects against oxidative stress in ICH injury.(2)To study whether PKA is involved in CTRP3-mediated NOX 2 and activation of oxidative stress and illustrate its possible mechanism.Methods:(1)The experiment time was chosed to research after oxidative stress-mediated injury.The adult SD rats were divided into sham group,6 h,12 h,24 h,48 h,72 h intracerebral hemorrhage model groups at random,the expression of NOX 2 was observed by immunoblot,the time with highest level of NOX 2 expression was chosed to our research.(2)The protective effects of CTRP 3 on brain after intracerebral hemorrhage.Rats were randomly divided into sham group,ICH group,ICH+vehicle group and ICH+CTRP 3(recombinant or lentivirus CTRP 3).Recombinant or lentivirus CTRP3 were injected into brain by intracerebroventricular(ICV)injection.The neuroprotective effects of CTRP 3 and its possible mechanisms were researched.The neurological score,brain water content and blood-brain barrier were measured after ICH.The expression of CTRP 3,PKA and NOX 2 proteins around the hematoma in experimental ICH were detected by Western blot.The dynamic changes of CTRP 3 mRNA and NOX 2 mRNA were measured by real-time Polymerase chain reaction(RT-PCR).Brain striatal tissues were collected and superoxide dismutase(SOD),malondialdehyde(MDA),reduced glutathione(GSH)and the ratio of oxidized to reduced glutathione(GSSG/GSH)were measured 24 h after ICH.The expression of NOX 2 was assayed by immunohistochemical staining.(3)The activator or inhibitor of PKA was injected into brain after ICH,The neurological score,brain water content and blood-brain barrier were measured after ICH.The expression of PKA and NOX 2 proteins around the hematoma in experimental ICH were detected by Western blot.Brain striatal tissues were collected and superoxide dismutase(SOD)and malondialdehyde(MDA)were measured 24 h after ICH.Results:(1)The expression of NOX 2 protein was significantly increased after ICH,and 24 h after ICH was most significant.(2)Treatment with recombinant or lentivirus CTRP3 significantly reduced neurological impairment,brain water content and blood-brain barrier injury,increased the expression of CTRP 3 and PKA proteins.The expression of NOX 2 protein,mRNA,positive expression in brain tissue and MDA were reduced after treatment with CTRP 3.The expression level of SOD and GSH were increased in CTRP 3 treatment group relative to ICH group.(3)PKA agonist treatment increased PKA protein expression,decreased NOX 2 protein expression,reduced brain water content,blood-brain barrier injury and oxidative stress injury.PKA inhibitor treatment increased NOX 2 protein expression and increased brain injury.Conclusion:(1)Treatment with CTRP 3 is beneficial to protect brain against oxidative stress injury after ICH.(2)CTRP 3 treatment significant reduced the expression of NOX 2 and oxidative stress injury.It may contribute to the upregulation of PKA expression.