| objective: In the present study we investigated the effect and the preliminary mechanism of Naoshu Capsule on the VD rats . Methods:The normal learning and memory male Sprague-Dawley(SD) rats were selected with Morris water maze experiment, which were randomly divided into sham operation group,model group,naofukang group(0.2g/kg) , naoshu capsule group(2g/kg), naoshu capsule group (4g/kg), naoshu capsule group(8g/kg);Except for the rats in the sham operation group,the other rats were repeatedly induced cerebral ischemia and reperfused for twice,combined with intraperitoneal injection of sodium nitroprusside to establish the VD model ;Naoshu capsule was intragastricly administrated into the VD rats at one time a day in one week before operation and in three weeks after operation respectivly;Morris water maze examination was used to detect learning and memory ability of the VD rats;The content of IL-6,sIL-2R in blood serum were detected with radioimmunoassay and enzyme-linked immunos- orbent assay;Hippocampus homogenate or cerebral cortex were prepared in ice bath and were centrifuged at 3000rpm,10miniuts,then superoxide dismutase activity,malondialdbelryde content,nitrogen monoxide content,inducible nitric oxide synthase activity or glutathione peroxidase activity were determined with chemical-colorimetric method;VD rats were administered conventional perfusion fixing by 4% paraformaldehyde and then the brain tissues were obtained .After that ,the slices of brain tissues were made with paraffin imbedding and successive coronal sectioning. Hematoxylin-eosin staining and the immunohistochemical staining (SABC) were employed to observe the degree of injuried neuron and the positive expression of TNF-αon CA1 in hippocampus,and Image pro plus 6.0 software analysis ID of positive expression of TNF-α. Results:①Place navigation:the escape latency of rats in each group have no difference from the first day to the second day;Compared with sham operation group,the escape latency of model group rats increased from(45.92±8.77)seconds to(62.74±9.84)seconds(P<0.01)from the third day ; Compared with model group, the escape latency of naoshu capsule group rats(4g/kg) and naoshu capsule group rats(8g/kg) decreased from (62.74±9.84)seconds to(52.56±11.11)seconds,(50.17±10.97)seconds(P<0.05,0.01)from the third day. Compared with model group, the escape latency of naoshu capsule group rats(2g/kg) and naoshu capsule group rats(4g/kg),naoshu capsule group rats(8g/kg) decreased from (41.54±8.13)seconds to(32.67±7.25)seconds,(30.72±10.53)seconds,(27.38±11.01)seconds(P<0.05,0.01)from the forth day. Spatial probe: Compared with sham operation group, numbers of rats crossing platform ,the time spent in target quadrant and the percentage of the distance spent in target quadrant of model group decreased from (7.30±2.00)/120seconds,(47.51±13.60)seconds and(48.39±8.51)% to(4.60±1.26)/120seconds, (34.59±9.67)seconds and(35.81±5.56)%(P<0.01). Compared with model group, the time spent in target quadrant of naofukang group(0.2g/kg) and naoshu capsule group(2g/kg) increased from ( 34.59±9.67 ) seconds to (44.49±6.23)seconds,(44.87±7.30)秒(P<0.05). Compared with model group,numbers of rats crossing platform ,the time spent in target quadrant and the percentage of the distance spent in target quadrant of naoshu capsule group(4g/kg) increased from (4.60±1.26)/120seconds,(34.59±9.67)seconds,(35.81±5.56)% to (6.20±1.87)/120seconds(45.52±6.17)seconds,(42.87±7.12)%(P<0.05);numbers of rats crossing platform ,the time spent in target quadrant and the percentage of the distance spent in target quadrant of naoshu capsule group increased from (4.60±1.26)/120seconds,(34.59±9.67)seconds,(40.87±8.82)% to (6.90±1.45)/120seconds,(46.93±10.30)seconds,(44.58±8.13)%(P<0.01,0.05)②Compared with sham operation group, interleukin-6 content increased from(76.20±10.34)pg/ml to (128.73±18.51)pg/ml(P<0.01)in blood serum of model group; Compared with model group, interleukin-6 content of naofukang group(0.2g/kg),naoshu group(2g/kg),naoshu group(4g/kg),naoshu group(8g/kg) decreased from( 128.73±18.51 ) pg/ml to (111.78±17.30 ) pg/ml ( P<0.05 ),(113.38±15.12)pg/ml,(102.65±13.71)pg/ml,(89.32±12.48)pg/ml(P<0.05,0.01)in blood serum of rats.③Compared with sham operation group, soluble interleukin-2 receptor content increased from(345.88±42.43)pg/ml to (686.92±88.95)pg/m(lP<0.01)in blood serum of model group; Compared with model group, soluble interleukin-2 receptor content of naofukang group(0.2g/kg),naoshu group(2g/kg),naoshu group(4g/kg),naoshu group(8g/kg) decreased from(686.92±88.95)pg/ml to (539.36±65.33)pg/ml,(547.20±54.17)pg/ml,(515.20±44.75)pg/ml,(464.37±49.08)pg/ml(P<0.01)in blood serum of rats.④Compared with sham operation group, superoxide dismutase activity decreased from(77.14±11.93)U/mgprot to (44.36±7.67)U/mgprot(P<0.01) ,and malondialdbelryde content increased from (5.12±1.03)nmol/mgprot to(7.25±0.84)nmol/mgprot(P<0.01) in hippocampus of model group;nitrogen monoxide activity and inducible nitric oxide synthase content increased from (21.24±5.06)umol/gprot,(2.48±0.25)U/mgprot to(35.70±7.12)umol/gprot,(3.91±0.24)U/mgpro(tP<0.01) in hippocampus of model group;glutathione peroxidase activity decreased from(779.60±75.52) to (432.32±58.44)(P<0.01)in cerebral cortex of model group. Compared with model group, superoxide dismutase activity of naoshu group(2g/kg),naoshu group(4g/kg),naoshu group(8g/kg) increased from(42.36±7.67)U/mgprot to (52.89±9.41)U/mgprot,(60.28±8.49)U/mgprot,(70.63±11.82)U/mgprot(P<0.05, 0.01 ) in hippocampus of rats ; Compared with model group, malondialdbelryde content of naofukang group(0.2g/kg),naoshu group(4g/kg),naoshu group(8g/kg) decreased from ( 7.25±0.84 )nmol/mgprot to (6.00±0.75)nmol/mgprot,(6.35±0.70)nmol/mgprot,(5.71±0.66)nmol/mgprot(P<0.01,0.05)in hippocampus of rats;Compared with model group, nitrogen monoxide activity of naofukang group(0.2g/kg),naoshu group(4g/kg),naoshu group(8g/kg) decreased from(35.70±7.12)umol/gprot to (29.66±3.62)umol/gprot,(29.18±4.31)umol/gprot,(25.63±4.99)umol/gprot(P<0.05,0.01)in hippocampus of rats;Compared with model group, inducible nitric oxide synthase content of naofukang group,naoshu group(2g/kg),naoshu group (4g/kg),naoshu group(8g/kg) decreased from(3.91±0.24)U/mgprot to (3.46±0.72)U/mgprot,(3.43±0.48)U/mgprot,(3.12±0.33)U/mgprot,(2.76±0.34)U/mgpro(tP<0.05,0.01)in hippocampus of rats;Compared with model group, glutathione peroxidase activity of naofukang group(0.2g/kg),naoshu group(4g/kg),naoshu group(8g/kg) increased from ( 432.32±58.44 ) to (538.85±67.94 ),( 518.98±58.38 ),(602.22±62.19),(743.33±75.82)(P<0.05,0.01)in cerebral cortex of rats.⑤Histopathological detection:Under the low power lens(×100),arrangement of neurons in hippocampus of sham operation group rats was ordered,while arrangement of neurons in hippocampus of model group rats was disordered, cell boundaries were fuzzy, especially CA1 area; Compared with model group rats, as dose of naoshu capsule increases,the numbers of pyramidal cells of naoshu group(2g/kg),naoshu group(4g/kg),naoshu group(8g/kg) raise gradually. Under high power lens(×400),pyramidal cells of hippocampal of sham operation group in CA1 area were normal,nucleus was large and clear, cytoplasm was rich;pyramidal cells of hippocampal of model group in CA1 area were narrowed and degenerated, cytopla- sm appear obvious degenetation,nuclear was like a triangle or cord ,nucleolus was not clear; as dose of naoshu capsule increases, pyramidal cells of hippocampal of naoshu group (2g/kg),naoshu group(4g/kg),naoshu group(8g/kg)in CA1 area increased gradullly, neurons of degenetation were decreased, and nucleolus were clear; pyramidal cells of hippocampal of naofukang group were narrowed and dark-blue stained, nucleoli were fuzzy.⑥Immu- nohistochemistry detection:Under low power lens(×100), except for the rats in the sham operation group,the others groups appeared positive expression of TNF-α. Under high power lens(×400),pyramidal cells of hippocampal of model group in CA1 area were shuttle,nuclei were oval or irregular,dark-blue stained, and vacuolation of cytoplasm were increased and brown stained . ID of TNF-αin hippocampus of model group increased from(0.09±0.02)to(0.39±0.08)(P<0.01)compared with control group;ID of TNF-αin hippocampus of naoshu group(0.2g/kg),naoshu capsule group(2g/kg),naoshu capsule group(4g/kg),naoshu capsule group(8g/kg) decreased from (0.39±0.08),to(0.24±0.03),(0.26±0.06),(0.21±0.06),(0.15±0.05)(P<0.01)compared with model group. Conclusion:①The VD rats models were successfully established by two-vessel occlusion,and they were related with increasion of free radicals and inflammatory factors induced by Ischemia-reperfusion.②All kinds of naoshu capsule groups can improve the learning and memory of VD rats and are dose-dependent .③The mechanism on improving the learning and memory of naoshu capsule related with several factors below:counteracting free radicals damage to hippocampal neurons induced by Cerebral ischemia-reperfusion;inhibitt- ing hippocampal neurons injury of immune inflammmatory cascade induced by Cerebral ischemia-reperfusion and restraining hippocampal neurons injury of inflammmatory reaction caused by the increase of free radicals induced by Cerebral ischemia-reperfusion .
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