CAJ | 학술논문

目的：观察中药脑康Ⅱ号对脑缺血大鼠空间学习记忆功能及海马神经发生的影响。方法将54只SD大鼠按随机数字表法分为对照组，假手术组，模型组，脑康Ⅱ号小、中、大剂量组（小、中、大剂量组用药量分别是成人用量的5、10、20倍），每组各9只。采用永久结扎大鼠双侧颈总动脉法造模。对假手术组大鼠只分离双侧颈总动脉，不予结扎；对照组大鼠不予任何处理；造模后给予治疗组脑康Ⅱ号治疗，给予对照组、假手术组和模型组等量（2 ml）等渗盐水。造模后9周采用Morris水迷宫定向航行实验和空间搜索实验评价大鼠学习记忆能力。采用免疫荧光染色法、免疫组化法进行神经干细胞（NSCs）增殖、分化的观察。采用Western Blot检测自噬相关蛋白Beclin-1和LC-3Ⅱ的表达。结果（1）造模后9周模型组逃避潜伏期第1～5天分别为（100±13）、（98±13）、（82±14）、（72±15）、（46±7）s，较假手术组和对照组明显延长，差异有统计学意义（均P＜0.05）；搜索距离分别为（1239±127）、（1011±122）、（959±123）、（553±66）、（407±29）cm，较假手术组和对照组明显增加（均P＜0.01）；第5天脑康Ⅱ号小、中、大剂量组大鼠逃避潜伏期和搜索距离均较模型组明显缩短（P＜0.05），逃避潜伏期分别为（20±6）、（19±8）、（15±6）s，搜索距离分别为（234±38）、（297±23）、（199±22）cm。造模后9周模型组大鼠在原平台所在象限搜索时间为（30±9）s，较假手术组明显缩短（P＜0.01）；脑康Ⅱ号小、中、大剂量组在原平台所在象限搜索时间分别为（34±6）、（38±8）、（40±10）s，均较模型组明显延长（P＜0.01）。（2）造模后9周免疫荧光与免疫组化结果显示，模型组海马齿状回BrdU阳性细胞数、Nestin阳性细胞数多于对照组和假手术组（P＜0.05）；脑康Ⅱ号中、大剂量组BrdU阳性细胞数和Nestin阳性细胞数较模型组明显增多（P＜0.05）。模型组中LC-3Ⅱ和Beclin-1表达较对照组和假手术组明显升高（P＜0.01）；脑康Ⅱ号中、大剂量组LC-3Ⅱ的表达分别为143±9、129±9，Beclin-1的表达分别为139±10、124±7，明显低于模型组（P＜0.01）。结论中、大剂量的脑康Ⅱ号能改善脑缺血大鼠的认知功能，促进海马齿状回神经发生，抑制自噬性细胞死亡。目的：觀察中藥腦康Ⅱ號對腦缺血大鼠空間學習記憶功能及海馬神經髮生的影響。方法將54隻SD大鼠按隨機數字錶法分為對照組，假手術組，模型組，腦康Ⅱ號小、中、大劑量組（小、中、大劑量組用藥量分彆是成人用量的5、10、20倍），每組各9隻。採用永久結扎大鼠雙側頸總動脈法造模。對假手術組大鼠隻分離雙側頸總動脈，不予結扎；對照組大鼠不予任何處理；造模後給予治療組腦康Ⅱ號治療，給予對照組、假手術組和模型組等量（2 ml）等滲鹽水。造模後9週採用Morris水迷宮定嚮航行實驗和空間搜索實驗評價大鼠學習記憶能力。採用免疫熒光染色法、免疫組化法進行神經榦細胞（NSCs）增殖、分化的觀察。採用Western Blot檢測自噬相關蛋白Beclin-1和LC-3Ⅱ的錶達。結果（1）造模後9週模型組逃避潛伏期第1～5天分彆為（100±13）、（98±13）、（82±14）、（72±15）、（46±7）s，較假手術組和對照組明顯延長，差異有統計學意義（均P＜0.05）；搜索距離分彆為（1239±127）、（1011±122）、（959±123）、（553±66）、（407±29）cm，較假手術組和對照組明顯增加（均P＜0.01）；第5天腦康Ⅱ號小、中、大劑量組大鼠逃避潛伏期和搜索距離均較模型組明顯縮短（P＜0.05），逃避潛伏期分彆為（20±6）、（19±8）、（15±6）s，搜索距離分彆為（234±38）、（297±23）、（199±22）cm。造模後9週模型組大鼠在原平檯所在象限搜索時間為（30±9）s，較假手術組明顯縮短（P＜0.01）；腦康Ⅱ號小、中、大劑量組在原平檯所在象限搜索時間分彆為（34±6）、（38±8）、（40±10）s，均較模型組明顯延長（P＜0.01）。（2）造模後9週免疫熒光與免疫組化結果顯示，模型組海馬齒狀迴BrdU暘性細胞數、Nestin暘性細胞數多于對照組和假手術組（P＜0.05）；腦康Ⅱ號中、大劑量組BrdU暘性細胞數和Nestin暘性細胞數較模型組明顯增多（P＜0.05）。模型組中LC-3Ⅱ和Beclin-1錶達較對照組和假手術組明顯升高（P＜0.01）；腦康Ⅱ號中、大劑量組LC-3Ⅱ的錶達分彆為143±9、129±9，Beclin-1的錶達分彆為139±10、124±7，明顯低于模型組（P＜0.01）。結論中、大劑量的腦康Ⅱ號能改善腦缺血大鼠的認知功能，促進海馬齒狀迴神經髮生，抑製自噬性細胞死亡。
목적：관찰중약뇌강Ⅱ호대뇌결혈대서공간학습기억공능급해마신경발생적영향。방법장54지SD대서안수궤수자표법분위대조조，가수술조，모형조，뇌강Ⅱ호소、중、대제량조（소、중、대제량조용약량분별시성인용량적5、10、20배），매조각9지。채용영구결찰대서쌍측경총동맥법조모。대가수술조대서지분리쌍측경총동맥，불여결찰；대조조대서불여임하처리；조모후급여치료조뇌강Ⅱ호치료，급여대조조、가수술조화모형조등량（2 ml）등삼염수。조모후9주채용Morris수미궁정향항행실험화공간수색실험평개대서학습기억능력。채용면역형광염색법、면역조화법진행신경간세포（NSCs）증식、분화적관찰。채용Western Blot검측자서상관단백Beclin-1화LC-3Ⅱ적표체。결과（1）조모후9주모형조도피잠복기제1～5천분별위（100±13）、（98±13）、（82±14）、（72±15）、（46±7）s，교가수술조화대조조명현연장，차이유통계학의의（균P＜0.05）；수색거리분별위（1239±127）、（1011±122）、（959±123）、（553±66）、（407±29）cm，교가수술조화대조조명현증가（균P＜0.01）；제5천뇌강Ⅱ호소、중、대제량조대서도피잠복기화수색거리균교모형조명현축단（P＜0.05），도피잠복기분별위（20±6）、（19±8）、（15±6）s，수색거리분별위（234±38）、（297±23）、（199±22）cm。조모후9주모형조대서재원평태소재상한수색시간위（30±9）s，교가수술조명현축단（P＜0.01）；뇌강Ⅱ호소、중、대제량조재원평태소재상한수색시간분별위（34±6）、（38±8）、（40±10）s，균교모형조명현연장（P＜0.01）。（2）조모후9주면역형광여면역조화결과현시，모형조해마치상회BrdU양성세포수、Nestin양성세포수다우대조조화가수술조（P＜0.05）；뇌강Ⅱ호중、대제량조BrdU양성세포수화Nestin양성세포수교모형조명현증다（P＜0.05）。모형조중LC-3Ⅱ화Beclin-1표체교대조조화가수술조명현승고（P＜0.01）；뇌강Ⅱ호중、대제량조LC-3Ⅱ적표체분별위143±9、129±9，Beclin-1적표체분별위139±10、124±7，명현저우모형조（P＜0.01）。결론중、대제량적뇌강Ⅱ호능개선뇌결혈대서적인지공능，촉진해마치상회신경발생，억제자서성세포사망。
Objective ToobservetheeffectsofNaokangⅡ,atraditionalChinesemedicineon spatial learning and memory abilities and hippocampal neurogenesis in rats with cerebral ischemia. Methods Atotalof54SDratsweredividedinto6groupsaccordingtothedigitalrandommethod:a control,a sham operation,a model,a low-dose,a medium-dose,and a high-dose NaokangⅡ group (the low-,medium-,and high-dose groups were 5,10,and 20 times of adults (n=9 in each group). A rat model was induced by ligate the bilateral common carotid arteries permanently. The bilateral common carotid arteries were only isolated but not ligated in rats of the sham operation group. The rats in the control group were not given any treatment. After modeling,the rats in the treatment groups were treated with NaokangⅡ. The rats in the control,sham operation,and model groups were given equal volume of isotonic saline (2 ml). Morris water maze test and space search experiment were used to evaluate the learning and memory abilities in rats 9 weeks after modeling. Immunofluorescence staining and immunohistochemical methods were used to observe proliferation and differentiation of neural stem cells (NSCs). Western blot was used to detect the expression of autophagy related protein Beclin-1 and LC-3Ⅱ. Results (1 )The escape latencies of the model group from 1 to 5 days were 100 ± 13,98 ± 13,82 ± 14,72 ± 15,and 46 ± 7 s,respectively. They were significantly longer than those of the sham operation group and the control group (all P<0. 05). The search distances were 1239 ± 127,1011 ± 122,959 ± 123,553 ± 66,and 407 ± 29 cm,respectively. They were significantly increased as compared with those of the sham operation group and control group (all P<0. 01). Both the escape latencies and search distances of the rats in the Naokang Ⅱ low-,medium-,and high-dose groups were significantly shorter than those of the model group (P<0. 05). The escape latencies on the fifth day were 20 ± 6,19 ± 8,and 15 ± 6 s,respectively,and the search distances were 234 ± 38, 297 ± 23,and 99 ± 22 cm,respectively. Nine weeks after modeling,the quadrant search time on the original platform in the rats of the model group was 30 ± 9 s,it was significantly shorter than that of the sham operation group (P<0. 01). The quadrant search time on the original platform of the Naokang Ⅱ low-, medium-,and high-dose groups were 34 ± 6,38 ± 8,and 40 ± 10 s,respectively. They were significantly longer than those of the model group (P <0. 01). (2)Nine weeks after modeling,the results of immunofluorescence and immunohistochemistry showed that the number of BrdU-positive cells and the Nestin positive cells in hippocampal dentate gyrus of the model group were higher than those of the control group and the sham operation group (P<0. 05). The number of BrdU-positive cells and Nestin positive cells of Naokang Ⅱ each dose group were significantly higher than those of the model group (P<0. 05). The expression of LC-3Ⅱand Beclin-1 in the model group was significantly higher than those in the control and sham operation groups (P<0. 01). The expression of LC-3Ⅱ in the medium- and high-dose groups was 143 ± 9 and 129 ± 9 respectively,and the expression of Beclin-1 was 139 ± 10 and 124 ± 7 respectively, andtheyweresignificantlylowerthanthemodelgroup(P<0.01).Conclusion NaokangⅡcanimprove the cognitive function in rats with cerebral ischemia,promote neurogenesis in the dentate gyrus,and inhibit autophagic cell death.