Smac, NAIP Expression In Cognitive Dysfunction Rat Brain Tissue Induced By Chronic Cerebral Ischemia, And The Efefct Caused By Butylphthalide

Chronic cerebral ischemia as long-term chronic cerebral hypoperfusion,is a common pathological process in the development of many cognitive impairmentdiseases, such as Alzheimer’s disease （AD）, vascular dementia (VD) andBinswanger’s disease, etc. The exact mechanism of cognitive dysfunction injurycaused by chronic cerebral ischemia still requires further exploration and theprevention and treatment are invalid. Therefore, the study of injury and protectionmechanisms is of great significance.Recent studies have indicated that Smac and NAIP play important roles in theregulatory mechanisms of apoptosis. Inhibitor of apoptosis protein (IAPs) family is agroup of highly conserved apoptotic protein discovered these years. They participatein the regulation of apoptosis mainly by inhibiting caspases andare closely relatedwith the nerve system diseases and malignant tumors. Among them the Neuronalapoptosis inhibitory protein (NAIP) has important effect in many neurologicaldiseases. Smac, a kind of mitochondrial protein, mainly regulates the progress ofapoptosis by reversing the role of apoptosisproteins (IAPs) inhibition. This importanteffect of Smac and NAIP can participate in the regulating process of acute cerebralischemic neurons apoptosis but is rarely reported in chronic cerebral ischemia.Butylphthalide (dl-n-butylphthalide, dl-NBP) is a natural chemicalsynthetic drug.Previous studies have already shown that Butylphthalide can improve the ischemicarea of brain perfusion in patients with acute hypoxic-ischemic encephalopathy. Thischemical can improve the neurological deficit, save the penumbra brain cells andprotect the neurons in the brain after ischemia mitochondriadue to improve the brainperfusion in ischemic area.When chronic cerebral ischemia occurs, it’s not clear whether the expression of Smac and NAIP are involved in the regulation of neuronal apoptosis, so as to thefunction that Butylphthalide may be involved in chronic cerebral ischemia inducedcognitive dysfunction by affecting the expression of Smac, NAIP.Objective: To observe the nerve cells apoptosis phenomenon and the expressionchange of Smac, NAIP, the apoptosis regulatory factor in the chronic cerebralischemia of rat brain and the impact of Butylphthalide of both. Explore the neuronprotective mechanism by Butylphthalide and further clarify the mechanism ofcognitive dysfunction impacted by chronic cerebral ischemia.Provide a basis for the prevention and treatment of cognitive dysfunction after chroniccerebral ischemia in theory and provide new ideas for clinical prevention andtreatment.Methods: By Morris water maze navigation test, healthy male Wistar rats arerandomly divided into sham-operated group,2VO+saline control group and2VO+Butylphthalide intervention group. Each group according to ischemic time arerandomly divided into1,2,3months, eventually assign each group of10rats.This experiment uses a permanent bilateral common carotid artery ligation (2VO) toprepare a chronic cerebral ischemia rat model. In48h after2VO, Butylphthalide9mg,per kg of body weight/day, intraperitoneal injection, administered continuously for20days; Saline control group at the same time give equal dose intraperitonealinjection of0.9%sodium chloride injection. At3time point: the postoperative1month,2months,3months, take a four-day test about the ability of learning andmemory in each group. Record the time of find and climb the platform within2minutes as the escape latency, record the walking path as the swimming distance.Take the grade point average as a rat’s learning and memory evaluation score.Withinthe specified time, randomly select six rats in each group, remove the brain and make4μm thick sections to stain. Use HE staining to observe the pathological changes inthe brain of rats, by TUNEL method to detect apoptosis in rat brain cells, useRT-PCR to detect the expression of Smac NAIP gene and Western blot to detect thegene expression of NAIP. Results: The result of Morris water maze navigation test shows that there is nosignificant difference in the search duration (escape latency) and the search for thewalking path (swimming distance) between1month,2month,3month insham-operated group. The search duration (escape latency) and the search walkingpath (swimming distance) extend when ischemia in a month’s time, it has a significantdifference compared with the sham-operated group. The extension and difference aremore obvious in2month’s time and3month’s time. In the ischemic3month’s time,it changes the most significant and great difference compared with ischemia1month’s group. This suggests that the ability of learning and memory in rats aresignificantly decreased with the extension of ischemic time. In the butylphthalideintervention group, at each time point the rats escape latency and swimming distance aresignificantly shorter, but still longer than the sham-operated group. The number,morphology and distribution of the nerve cells in sham-operated group are normal.The prefrontal cortex nerve cells arrange tightly and orderly. There is round and largenuclei, light staining and clear nucleolus. Ischemia one month, no nerve cellsdepigmentation, but can see many scattered ischemic changes, cell deformation,nuclear condensation, deeply stained nerve cells show triangle shape, Nucleolus andnuclear membrane are unclear, normal and ischemic nerve cells can be seenalternately. Ischemia2month’s time can see depigmentation of the nerve cells clearly.Changes in nerve cells ischemia increase, scattered eosinophilic red blood cells appear,but still visible some complete form nerve cells.Ischemia3month’s time, the demyelination of nerve cells is more obviously, cellarrangement is sparse, normal morphology. The number of pyramidal cells issignificantly reduced and it can be seen the deformation of nerve cells, pyknoticnuclear, cytoplasmic disappear, dark staining, unclear structure, a few of them lostcomplete structure. In the butylphthalide intervention group, the pathological changes inrat frontal cortical at three time points compared with the saline control group of the samepoint in time have lessened in different degrees. The neurons arrange orderly, the amountgrows and the number of eosinophilic red blood cells reduces. The number ofTUNEL-positive cell in chronic cerebral ischemic group is significantly higher thanthe corresponding sham-operated group, and will gradually increase with theextension of ischemic time. When it comes3month’s ischemic, the number is thelargest. After the intervention of butylphthalide, the number of positive cell in ischemic brain in all the3time point has reduced obviously. The RT-PCR results show that theSmac mRNA expression of frontal lobe of the ischemic group at the three time pointsis significantly higher than the sham-operated group, and there is no differencebetween this three time points. The Smac mRNA expression is less than thebutylphthalide intervention group at3time points, and the difference is obvious. Theexpression of NAIP mRNA are much lower in the frontal lobe of the ischemic groupthan the sham-operated group. With the extension of ischemic time,the number willgetting lower, and at the3month’s time point it will be the lowest. In thebutylphthalide intervention group, the expression of NAIP mRNA and protein are muchhigher than the ischemic team at each time point, the difference between groups issignificant. There’s rich NIAP positive expression in the frontal lobe of the ischemicgroup by Western blot analysis of sham-operated group, and the level of ischemicgroup is much lower than sham-operated group at each time point. With the extensionof ischemic time, the NAIP protein content decreased, the difference between groupsis significant.3months of ischemia reduce the most obvious, although thebutylphthalide intervention group rats’ NAIP protein content is still lower than thesham-operated group, but significantly increased compared with the ischemia rats.Conclusion: Chronic cerebral ischemia may cause cognitive impairment,Butylphthalide can improve the cognitive dysfunction in chronic cerebral ischemiarats. Cell Apoptosis is involved in the onset of cognitive dysfunction due to chroniccerebral ischemia. Proapoptotic factor Smac is activated, apoptosis inhibitory factorNAIP expression is suppressed, are both involved in the regulation of neuronalapoptosis during chronic cerebral ischemia. Butylphthalide may play aneuroprotective effect on neuronal apoptosis by inhibiting Smac and enhance the roleof the NAIP.