Effect Of L-3-n-butylphthalide On Hippocampus And The Expression Of Bcl-2and Bax In Mice With Vascular Dementia

Objective:To observe the improving effects of l-3-n-Butylphthalide（l-NBP） on the expression of Bcl2and Bax on the C57Bl/6mice that wereestablished the VaD model.Methods:The C57Bl/6mice were subjected to20min ischemia and10min reperfusion by ligating the bilateral common carotid arteries.Theischemia-reperfusion was repeated three times to establish the VaD model.Themice were intragastred and given l-NBP once a day and randomly assigned to5groups：sham group，model group，pretreatment group：preoperativeprophylaxis by oral gavage for7d (l-NBP30mg/kg)，L-NBP(low-dosel-3-n-Butylphthalide treatment group)：postoperative administration by oralgavage for28d (dosage:30mg/kg) and H-NBP(high-dose l-3-n-Butylphthalidetreatment group)：postoperative administration by oral gavage for28d(dosage:60mg/kg).The changes of behavior were observed through the Morriswater in the29th days after model preparation.All rats were decapitated afterbehavioral.Each mouse in sham group and normal group were used toobserver and analyse the ultrastructure of the corpus callosum by transmissionelectron microscope.To evaluate the neuropathological of hippocampusthrough thionin staining.Western-blot and immunohistochemical technologywere applied to detect the expression level of Bcl-2and Bax in the rathippocampus The mRNA levels of Bcl-2and Bax were identified throughPCR method.Results:1The Morris maze test1.1The study results of Morris water maze test in5days1.1.1There is no statistical significance in the first day among the five group.1.1.2In the second day，time is longer that model group spent than the sham group(P<0.05) L-NBP group had less time compared with modelgroup(P<0.05).1.1.3In the third day，L-NBP group had less time compared with modelgroup(P<0.05).1.1.4In the fouth day，the learning ability had a stabilized trend.The shamgroup， L-NBP group，pretreatment group had less time on appear on thestage compared with model group.(P<0.05)1.1.5In the fifth day， The sham group，L-NBP group，H-NBP group，pretreatment group had less time on appear on the stage compared with modelgroup.(P<0.05)1.2The memory results of Morris water maze test in the sixth day1.2.1time in the fourth quadrant（%）The time that stay on the fourth quadrant，which was longer that shamgroup，and pretreatment group compared with the model group.(P<0.05)2Results of electron microscopy2.1Sham group:Smooth vessel wall in the region of the corpus callosum weresmooth.The vascular endothelial cells and myelin were arrangedclosely.There were no demyelination.2.2Model group:The vessel wall were rough，thickened.The elastic membranehad been disrupted.Elastic fibers and collagen fibers was hyperplastic.Thebase film and the substrate were incomplete.Degenerative of the nerve fibersin varying degrees were visible，texture and structrue of myelin are unclear.3results of Thionine staining3.1Sham group：neurons of hippocampal CA1were dense and arranged inneat rows, Morphology was integrity and clear. The nuclus in this area werelarge and round，and the nucleolus were clear. The number of normal neuronwas203.53±11.043.2Model group:parts of neurons were dead.Some fault zones could beobserved.The remnants of the neuron were shrink and irregular. The nucluswere pyknotic and hyperchromatic.The nucleolus were unclear or disappeared.The number of normal neuron was69.23±2.14.(P<0.05compared with sham group)3.3L-NBP group：The number of the neuron was less than the sham group,butslightly more than the model group. Morphology of the cells was integrity andclear.Only a few nuclus were pyknotic and hyperchromatic. The number ofnormal neuron was114.92±2.08.(P<0.05compared with sham group)3.4H-NBP group：The cells were in neat and close order in the hippocampusCA1area.The nucleus were plump,and the nucleolus were clear.Thephenomenon of Cell rupture could hardly be see.The number of normalneuron was189.92±3.18.(P<0.05compared with sham group)3.5Pretreatment group:General observation was same as the H-NBP group. The number of normalneuron was199.92±2.96.(P<0.05compared with sham group)4Espression of Bcl-2/Bax4.1The results of immunohistochemistry and Western-blot showed that，compared with the sham group，there was a significant reduction of Bcl-2/Baxwith the model group.Athough the L-NBP intervention group showed anotable trend compared with the model group，there was no statisticalsignificance.4.2The PCR results showed that it was statistically insignificant with themRNA decreasing and the Bcl-2/Bax ascending in the five groups.Conclusions:1The study provided a perfect VaD mice model that had obviouslydisfuntion in learning and memory which had been identified through theresults of the the transmission electron microscope and the Morris water maze.Preventive application of l-3-n-Butylphthalide could significantly reduced theoccurrence and development of vascular dementia.2The number of the neurons in the hippocampus in the L-Butylphthalidetreatment group and pretreatment group were more than model group.TheL-Butylphthalide could reverse the histopathological of the VaD mouse andimprove their learning and memory abilities.3The amount of Bcl-2/Bax in model group was decreased，while the L-Butylphthalide intervention group，although the data of L-Butylphthalidetreating group and the L-Butylphthalide pretreatment group showed amakeable trend， there was no statistical significance.Repeated cerebralischemia and reperfusion activate apoptosis protein Bcl-2and Bax，andprevented nerves apoptosis，but it was not the primary mechanism for theL-Butylphthalide in the treatment of vascular dementia.