PARP9通过抑制PI3K/AKT/mTOR信号通路促进脑缺血再灌注大鼠神经元凋亡及炎症反应而加重认知障碍OA

Objective Cerebral ischemia-reperfusion(CIR)refers to a pathological process in which restoration of blood flow exacerbates neuronal injury in the ischemic area,with the underlying mechanisms involving cascade reactions such as oxidative stress,inflammation,and apoptosis.The biological function of poly(ADP-ribose)polymerase 9(PARP9)in CIR remains unclarified.This study aims to investigate the effects of PARP9 on neuronal apoptosis,neuroinflammation,cognitive function,and the PI3K/AKT/mTOR signaling pathway in rats with CIR.Methods ① Experiments of injury effects:Ten male SD rats(8 to 10 weeks old,weighing 250 to 260 g)were randomly divided into a sham operation group and a CIR group,with 5 rats in each group.Rats in the CIR group underwent middle cerebral artery occlusion using intraluminal suture technique,followed by reperfusion 2 h later by removing the suture to establish the CIR model;rats in the sham operation group only had the blood vessels exposed without incision or insertion of a paraffin-coated suture.At 7 d after surgery,Western blotting was applied to detect the protein levels PARP9 in the brain tissues of both groups.2 PARP9 intervention experiment:60 male SD rats(8 to 10 weeks old,weighing 250 to 260 g)were randomly divided into an AAV-siScramble group,an AAV-siPARP9 group,an AAV-Scramble group,and an AAV-PARP9 group,with 15 rats in each group.Rats in each group received an intracerebroventricular injection of the corresponding adenovirus(3 µL),and the CIR model was established 2 weeks after adenovirus injection.At 24 h postoperatively,neurological function was assessed using Zea-Longa method,and cognitive function was evaluated using Morris water maze test.At 7 d after surgery,TTC staining and Nissl staining were used to measure cerebral infarct volume and evaluate pathological damage in the cerebral cortex,respectively.TUNEL staining was used to detect the neuronal apoptosis rate;immunohistochemical staining was employed to detect the positive expression of TNF-α and IL-1β;and Western blotting was utilized to detect the protein expression of PARP9,p-PI3K,p-AKT,and p-mTOR in brain tissue.Results ① Experiments of injury effects:The expression level of PARP9 in the ischemic cortical area of brain tissue was significantly increased in the CIR group than the sham operation group(P<0.001).2 PARP9 intervention experiment:Compared with the AAV-siScramble group,the AAV-siPARP9 group exhibited decreased neurological function scores,shortened escape latency,and increased number of platform quadrant crossings(P<0.001).The percentage of cerebral infarct area was significantly reduced(P<0.05).The expression level of PARP9,neuronal apoptosis rate,and positive expression levels of TNF-α and IL-1β in the ischemic cortical area were all decreased(P<0.05).The protein levels of p-PI3K,p-AKT,and p-mTOR in the ischemic cortical area were increased(P<0.05).Compared with the AAV-Scramble group,the AAV-PARP9 group showed increased neurological function scores,prolonged escape latency,and reduced number of platform quadrant crossings(P<0.001).The percentage of cerebral infarct area was significantly increased(P<0.05).The expression level of PARP9,neuronal apoptosis rate,and positive expression of TNF-α and IL-1β in the ischemic cortical area were all increased(P<0.05).The protein levels of p-PI3K,p-AKT,and p-mTOR in the ischemic cortical area were decreased(P<0.05).In the AAV-siPARP9 group,cortical neurons were regularly arranged,with abundant Nissl bodies,blue-stained cytoplasm,and uniformly stained nuclei.In the AAV-PARP9 group,cortical neurons were scattered with locally reduced neuronal numbers,lighter blue-stained cytoplasm,and significantly reduced Nissl bodies;while in the AAV-siScramble group and AAV-Scramble group,the dissolution of Nissl bodies was alleviated and the number of surviving neurons was increased.Conclusion PARP9 functions as a key pathogenic facilitator in CIR injury,which may be mediated through inhibition of the PI3K/AKT/mTOR signaling cascade.Knockdown of PARP9 alleviates neuronal apoptosis and neuroinflammatory responses,and improve cognitive function,suggesting that PARP9 may serve as a new molecular target for CIR intervention.