Neuroprotective Effects And Its Mechanism Of 3-n-butylphthalide Pretreatment On Cerebral Ischemia Reperfusion Injury In Rats

Objective:To observe the effects of 3-n-butylphthalide(NBP) pretreatment on the score of neurological deficit, infarction volume of brain, pathomorphology change observed under light microscope, oxidative stress, brain edema, the expression of MMP-9, permeability and ultrastructural changes of brain blood barrier(BBB) in rats. To explore protective effects and its mechanism of NBP pretreatment on oxidative stress and BBB in cerebral ischemia reperfusion injury.Methods:210 male SD rats were randomly divided into sham operation group(Sham group), ischemia-reperfusion group(IR group), NBP pretreatment low dose group(NBPⅠgroup), NBP pretreatment medium dose group(NBPⅡ group) and NBP pretreatment high dose group(NBPⅢgroup), 42 rats per group. Pretreatment was given once a day within 1 week before establishing the model of cerebral ischemia reperfusion injury. The model of middle cerebral artery occlusion(MCAO) was subjected by suture method. The score of neurological deficit was executed after ischemia for 2h and reperfusion for 24 h in all the rats; the cerebral infarction was observed by TTC staining; the pathologic change of brain was observed by HE staining under the microscope; Hydroxylamine method was used to detect activity of SOD, chemical colorimetry method was used to measure activity of GSH-PX, and TBA method was used to detect content of MDA; the water content was analyzed through dry/wet weight measurement; the permeability of BBB was detected by collecting extravascular Evans blue(EB) in the brain cortex; the protein level of MMP-9 was measured by immunohistochemical techniques, the mRNA expression of MMP-9 was determined by real-time fluorescence quantitative PCR; pathological changes on the ultrastructure of blood brain barrier(BBB) were observed by transmission electron microscopy.Result:1 The score of neurological deficit: rats in Sham group emerged no any abnormal activity, the score was 0; IR group animals displayed independently automatical activity tend to the left side, even dumped, the score significantly increased(P<0.01); Compared with IR group, the score of neurological deficit were obviously reduced in NBP pretreatment groups(all P<0.01), there was no significantly different(P>0.05) between NBPⅡgroup and NBP Ⅲ group.2 The measurement of infarcted volume of brain: the infarction size was measured by 2,3,5-Triphenyltetrazolium chloride(TTC) staining technique. The right side of the brain tissue of the Sham group manifest uniform red without local infarction; IR group showed a large volume of pale infarcts(P<0.01); Compared with IR group, the infarction volume obviously reduced in NBP pretreatment groups(all P<0.01), there is no significantly different(P>0.05) between NBP Ⅱ group and NBP Ⅲ group.3 The pathologic changes of neurons observed by TTC staining: the neurons displayed normal shape, the nuclear membrane and nucleolus were clearly visible in the Sham group; There were a wide range of necrotic foci like holes of a sieve and edema in IR group; The number of degeneration and necrosis neurons obviously decreased, and edema in intercellular substance was alleviated in NBP pretreatment groups.4 Effect of NBP pretreatment on oxidative stress: activity of SOD, GSH-PX and content of MDA of brain tissue were normal in Sham group; Compared Sham group, activity of SOD, GSH-PX was largely decreased, and content of MDA was greatly increased in IR group(all P<0.01); Compared IR group, activity of SOD, GSH-PX went up progressively in turn, and contents of MDA were cut down progressively in turn in NBPⅠ,Ⅱ,Ⅲ group(all P<0.01), there is no significantly different(P>0.05) between NBP Ⅱ group and NBP Ⅲ group.5 The content of water in brain: water content in Sham group was normal; Compared with Sham group, IR group animals displayed significant edema, the water content sharply increased(P<0.01); Compared with IR group, the water content were obviously reduced in NBP pretreatment groups(all P<0.01), there was no significantly different(P>0.05) between NBP Ⅱ group and NBP Ⅲ group.6 The content of EB in brain: EB content in Sham group was normal; Compared with Sham group, the permeability of BBB in IR group added significantly, the EB content sharply increased(P<0.01); Compared with IR group, the EB content were obviously reduced in NBP pretreatment groups(all P<0.01), there was no significantly different(P>0.05) between NBP Ⅱ group and NBP Ⅲ group.7 The expression of MMP-9: the expression of MMP-9 in Sham group was little; Compared with Sham group, the expression of MMP-9 in IR group added significantly(P<0.01); Compared with IR group, the expression of MMP-9 obviously reduced in NBP pretreatment groups(all P<0.01), there was no significantly different(P>0.05) between NBPⅡgroup and NBP Ⅲ group.8 The pathological changes on the ultrastructure of BBB: the shape of BBB was regular in Sham group, endothelial cells and tight junction were intact condition; The lumen of BBB was stenosed and deformed significantly, ight junction was fused or expansive in IR group; Compared with IR group, NBP pretreatment markedly alleviated BBB histopathological changes, and different doses of NBP pretreatment with different improvement.Conclusions:1 NBP can reduce the score of neurological deficit and infarcted volume of brain, alleviate neuronal injury when it pretreats cerebral ischemia reperfusion injury in rats, NBP has preventive protective effect by improving morphology injury.2 NBP pretreatment can relieve harmful effects from oxidative stress in cerebral ischemia reperfusion injury.3 The pretreatment of 3-n-butylphthalide has preventive protective effect against cerebral ischemia reperfusion injury in rats, which may be related to down-regulate the expression of MMP-9 and ease BBB injury, then reduce the permeability of BBB and the degree of brain edema.4 The medium dose from NBP pretreatment is the best dosage for prophylactic usage to the rats at(250±20)g, which provides fundamental basis for clinical application.