CAJ | 학술논문

目的：探讨丙泊酚和醒脑静注射液对大鼠离体脑片不同性质损伤的保护作用及其机制。方法取出生7 d的SD乳鼠脑皮质制备离体脑片，2，3，5-氯化三苯基四氮唑（TTC）染色判断脑片活性后按随机数字表法分为正常对照组（A组）、谷氨酸（Glu）损伤组（B组）、过氧化氢（H2O2）损伤组（C组）、丙泊酚预处理Glu损伤组（D组）、醒脑静注射液预处理Glu损伤组（E组）、丙泊酚预处理H2O2损伤组（F组）、醒脑静预处理H2O2损伤组（G组）7组，每组12片。D、E、F、G组于培养3 d时进行药物预处理24 h（丙泊酚浓度为20 mg/L，醒脑静注射液浓度为10μg/mL）；脑片成功培养至4 d时用1 mmol/L Glu或0.1 mmol/L H2O2复制损伤模型，损伤后30 min所有组转入正常培养液中孵育至7 d。A组不予任何处理。观察脑组织及细胞形态学变化，尼氏体染色计数尼氏小体，碘化丙啶-吖啶橙（PI-AO）双染观察各组脑片细胞的红绿染色比例，流式细胞仪测定细胞凋亡率。结果①脑片形态学观察：3d肉眼可见脑片轻度水肿，3～5d水肿逐渐消失，6d镜下可见大量典型的神经细胞及少量胶质细胞；7 d细胞生长达到顶点，之后凋亡逐渐占据主导。②苏木素-伊红（HE）染色观察细胞形态：A组神经元呈多边或梭形；B组及C组细胞数量较A组明显减少；D、E、F、G组神经元数量介于A组与B、C组之间，且组间存在差异。③尼氏体染色：A组尼氏小体清晰可见；B、C组染色后尼氏小体阳性细胞数较A组明显减少（个/HP：8.8±2.5、10.3±2.5比28.9±5.1，均P＜0.05）；D组、E组尼氏小体阳性细胞数较B组明显增多，且以D组增多更显著（21.5±4.7比13.4±3.1，P＜0.05）；F组、G组较C组明显增多，且以F组增加更显著（23.9±1.9比19.2±4.1，P＜0.05）。④PI-AO双染：A组细胞几乎全部呈现胞核绿色荧光；B组、C组大量神经元出现两种荧光，轮廊不清；D组、E组细胞胞质红染比例较B组有所减少，D组下降更为明显；F组、G组细胞胞质红染比例较C组有所减少，以F组更为明显。⑤细胞凋亡率：B组、C组凋亡率均较A组升高〔（22.00±0.64）%、（21.28±1.44）%比（8.57±0.67）%，P＜0.05〕；D组、E组细胞凋亡率较B组明显降低，且以D组降低更显著〔（11.94±0.57）%比（18.17±0.65）%，P＜0.05〕；F组、G组较C组明显降低，且以F组降低更显著〔（10.54±1.24）%比（13.12±0.13）%，P＜0.05〕。结论丙泊酚和醒脑静注射液对大鼠离体脑片的Glu和H2O2损伤均有保护作用，且对于同一种损伤，丙泊酚的脑保护作用较醒脑静注射液强。目的：探討丙泊酚和醒腦靜註射液對大鼠離體腦片不同性質損傷的保護作用及其機製。方法取齣生7 d的SD乳鼠腦皮質製備離體腦片，2，3，5-氯化三苯基四氮唑（TTC）染色判斷腦片活性後按隨機數字錶法分為正常對照組（A組）、穀氨痠（Glu）損傷組（B組）、過氧化氫（H2O2）損傷組（C組）、丙泊酚預處理Glu損傷組（D組）、醒腦靜註射液預處理Glu損傷組（E組）、丙泊酚預處理H2O2損傷組（F組）、醒腦靜預處理H2O2損傷組（G組）7組，每組12片。D、E、F、G組于培養3 d時進行藥物預處理24 h（丙泊酚濃度為20 mg/L，醒腦靜註射液濃度為10μg/mL）；腦片成功培養至4 d時用1 mmol/L Glu或0.1 mmol/L H2O2複製損傷模型，損傷後30 min所有組轉入正常培養液中孵育至7 d。A組不予任何處理。觀察腦組織及細胞形態學變化，尼氏體染色計數尼氏小體，碘化丙啶-吖啶橙（PI-AO）雙染觀察各組腦片細胞的紅綠染色比例，流式細胞儀測定細胞凋亡率。結果①腦片形態學觀察：3d肉眼可見腦片輕度水腫，3～5d水腫逐漸消失，6d鏡下可見大量典型的神經細胞及少量膠質細胞；7 d細胞生長達到頂點，之後凋亡逐漸佔據主導。②囌木素-伊紅（HE）染色觀察細胞形態：A組神經元呈多邊或梭形；B組及C組細胞數量較A組明顯減少；D、E、F、G組神經元數量介于A組與B、C組之間，且組間存在差異。③尼氏體染色：A組尼氏小體清晰可見；B、C組染色後尼氏小體暘性細胞數較A組明顯減少（箇/HP：8.8±2.5、10.3±2.5比28.9±5.1，均P＜0.05）；D組、E組尼氏小體暘性細胞數較B組明顯增多，且以D組增多更顯著（21.5±4.7比13.4±3.1，P＜0.05）；F組、G組較C組明顯增多，且以F組增加更顯著（23.9±1.9比19.2±4.1，P＜0.05）。④PI-AO雙染：A組細胞幾乎全部呈現胞覈綠色熒光；B組、C組大量神經元齣現兩種熒光，輪廊不清；D組、E組細胞胞質紅染比例較B組有所減少，D組下降更為明顯；F組、G組細胞胞質紅染比例較C組有所減少，以F組更為明顯。⑤細胞凋亡率：B組、C組凋亡率均較A組升高〔（22.00±0.64）%、（21.28±1.44）%比（8.57±0.67）%，P＜0.05〕；D組、E組細胞凋亡率較B組明顯降低，且以D組降低更顯著〔（11.94±0.57）%比（18.17±0.65）%，P＜0.05〕；F組、G組較C組明顯降低，且以F組降低更顯著〔（10.54±1.24）%比（13.12±0.13）%，P＜0.05〕。結論丙泊酚和醒腦靜註射液對大鼠離體腦片的Glu和H2O2損傷均有保護作用，且對于同一種損傷，丙泊酚的腦保護作用較醒腦靜註射液彊。
목적：탐토병박분화성뇌정주사액대대서리체뇌편불동성질손상적보호작용급기궤제。방법취출생7 d적SD유서뇌피질제비리체뇌편，2，3，5-록화삼분기사담서（TTC）염색판단뇌편활성후안수궤수자표법분위정상대조조（A조）、곡안산（Glu）손상조（B조）、과양화경（H2O2）손상조（C조）、병박분예처리Glu손상조（D조）、성뇌정주사액예처리Glu손상조（E조）、병박분예처리H2O2손상조（F조）、성뇌정예처리H2O2손상조（G조）7조，매조12편。D、E、F、G조우배양3 d시진행약물예처리24 h（병박분농도위20 mg/L，성뇌정주사액농도위10μg/mL）；뇌편성공배양지4 d시용1 mmol/L Glu혹0.1 mmol/L H2O2복제손상모형，손상후30 min소유조전입정상배양액중부육지7 d。A조불여임하처리。관찰뇌조직급세포형태학변화，니씨체염색계수니씨소체，전화병정-아정등（PI-AO）쌍염관찰각조뇌편세포적홍록염색비례，류식세포의측정세포조망솔。결과①뇌편형태학관찰：3d육안가견뇌편경도수종，3～5d수종축점소실，6d경하가견대량전형적신경세포급소량효질세포；7 d세포생장체도정점，지후조망축점점거주도。②소목소-이홍（HE）염색관찰세포형태：A조신경원정다변혹사형；B조급C조세포수량교A조명현감소；D、E、F、G조신경원수량개우A조여B、C조지간，차조간존재차이。③니씨체염색：A조니씨소체청석가견；B、C조염색후니씨소체양성세포수교A조명현감소（개/HP：8.8±2.5、10.3±2.5비28.9±5.1，균P＜0.05）；D조、E조니씨소체양성세포수교B조명현증다，차이D조증다경현저（21.5±4.7비13.4±3.1，P＜0.05）；F조、G조교C조명현증다，차이F조증가경현저（23.9±1.9비19.2±4.1，P＜0.05）。④PI-AO쌍염：A조세포궤호전부정현포핵록색형광；B조、C조대량신경원출현량충형광，륜랑불청；D조、E조세포포질홍염비례교B조유소감소，D조하강경위명현；F조、G조세포포질홍염비례교C조유소감소，이F조경위명현。⑤세포조망솔：B조、C조조망솔균교A조승고〔（22.00±0.64）%、（21.28±1.44）%비（8.57±0.67）%，P＜0.05〕；D조、E조세포조망솔교B조명현강저，차이D조강저경현저〔（11.94±0.57）%비（18.17±0.65）%，P＜0.05〕；F조、G조교C조명현강저，차이F조강저경현저〔（10.54±1.24）%비（13.12±0.13）%，P＜0.05〕。결론병박분화성뇌정주사액대대서리체뇌편적Glu화H2O2손상균유보호작용，차대우동일충손상，병박분적뇌보호작용교성뇌정주사액강。
Objective To investigate the protective effects and mechanisms of propofol and Xingnaojing injection on different characteristic injuries in cerebrocortical slices of newborn rats in vitro. Methods The cerebral cortex of the 7 days-old Sprague-Dawley(SD)neonatal rat was cut into slices. 2,3,5-Triphenyl Tetrazolium(TTC) was used to stain the slices to judge their activities,afterwards they were randomly divided into seven different groups (each,n=12):normal control group(group A),glutamate(Glu)injury group(group B),hydrogen dioxide(H2O2) injury group(group C),propofol preconditioning before Glu injury group(group D),Xingnaojing preconditioning before Glu injury group(group E),propofol preconditioning before H2O2 injury group(group F), Xingnaojing preconditioning before H2O2 injury group(group G). On the 3rd day,the pre-medical treatments or pre-conditionings for D,E,F and G groups were carried out for 24 hours(the concentration of propofol:20 mg/L,the concentration of Xingnaojing:10 μg/mL);the slices were successfully incubated for 4 days,afterwards they were immersed in 1 mmol/L Glu and 0.1 mmol/L H2O2 for 30 minutes respectively to establish the injury models which had no pre-treatment,finally all the groups were transferred into normal cultural medium to incubate till the 7th day. In the above processes,the group A had no specific medical treatment. After all the operations,the changes in brain tissue and cell morphology,the quantity of Nissl body(NISSL)stained by its stain,and the proportion of cells stained with red and green dye after the slices in various groups stained with propidium iodide-acridine orange(PI-AO)were observed,and the apoptosis rate was tested by flow cytometry. Results ①Morphological observation of brain slices:on the 3rd day,the slices appeared mild edematous,3-5 days later,the edema gradually disappeared. Until the 6th day, a large number of typical nerve cells and a few glial cells were seen;on the 7th day,the growth of cells reached the peak,and afterwards,gradually apoptosis played the leading role.②Morphological observation of brain slices stained by hematoxylin-eosin(HE)stain:the neurons of group A presented multilateral-or shuttle-shaped. The cell numbers of groups B and C were significantly lower than the number of group A. In the neuron number sequence,the positions of the numbers of groups D,E,F,G were located in the interval between the number of group A and those of groups B and C,and there were differences in number among groups.③NISSL:Nissl-body of group A could be clearly seen. The numbers of Nissl-body in groups B and C were significantly reduced compared with the number in group A(cell/HP:8.8±2.5,10.3±2.5 vs. 28.9±5.1,both P<0.05). The numbers of Nissl-body in groups D and E were obviously increased compared with the number in group B,and the greater increase being in group D(21.5±4.7 vs. 13.4±3.1, P<0.05). The numbers of Nissl-body in groups F and G were markedly increased compared with the number in group C ,and the greater increase being in group F(23.9±1.9 vs. 19.2±4.1,P<0.05). ④ Double staining with PI-AO:in group A,nearly the total number of nuclei presented fluorescent green in color. In groups B and C,a large number of neurons appeared two types of fluorescence and their edges blurred. The proportions of red staining of neuron cytoplasm in groups D and E were lower than the proportion in group B,and the greater decrease being in group D. The proportions of red staining of neuron cytoplasm in groups F and G were lower than the proportion in group C ,and the greater decrease being in group F.⑤Apoptosis rate:the apoptosis rates of groups B and C were higher than the rate of group A〔(22.00±0.64)%,(21.28±1.44)%vs.(8.57±0.67)%,P<0.05〕;the apoptosis rates of groups D and E were lower than group B,the decrease in group D being greater〔(11.94±0.57)%vs.(18.17±0.65)%,P<0.05〕;the apoptosis rates of groups F and G were much lower than the rate of group C,the decrease in group F being greater〔(10.54±1.24)% vs.(13.12±0.13)%,P<0.05〕. Conclusion The pre-treatment of propoful or Xingnaojing injection has protective effect on Glu or H2O2 injury of in vitro rat cerebrocortical slices,and upon the same injury,the brain protective effect of propoful is more powerful than that of Xingnaojing injection.