BACKGROUND: The death of aging cells is virtually apoptosis. To a certain extent, it can be interpreted as a series of results of gere activities.Therefore, the inhibition of oncogene's expression can lengthen the life span of cells and delay aging of brain tissues.OBJECTIVE: To study the effect of abstragulus mongholicus bung (AMB) on apoptosis of nerve cells and the expression of relevant gene in aging mice brain.DESIGN: Completely randomized design and controlled experiment.SETTING: Department of Biochemistry, School of Basic Medical Sciences of Jiamusi University.MATERIALS: The experiment was conducted at the Experimental Animal Center and Biochemical Laboratory of Jiamusi University from December 2003 to May 2004. Totally 24 healthy Kunming mice were recruited in this study. There were 8 two-month-old mice (young group) and 16 twelve-month-old mice. All the 16 mice were randomized into abstragulus mongholicus bung group and old control group with 8 in each group.METHODS:① AMB group:Mice in AMB group received gastric gavage was provided by the Department of Traditional Chinese Medicine of the First Hospital Affiliated to Jiamusi University, and evaluated by Jiamusi Drug Inspection Bureau. Water decoction was prepared with 2 kg/L raw materials. Mice in old control group and young group were filled with lukewarm boiled water.② All the animals were treated as above for 30 consecutive days before put to death. Their brains were taken out immediately and the middle parts of the brains were removed to fix with neutral formaldehyde. The remaining brain tissues were made into mitochondria suspension. Content of manganese superoxide dismutase (Mn-SOD) and malondialdehyde (MDA) was determined with xanthosine oxidase method and TBA chemical colorimetry. Apoptotic cells (cells with yellow nuclei were positive ones) were assayed with in situ end-labeling (ISEL) and expression of bcl-2 gene was assayed with immunohistochemical method. The cells stained brown were positive ones. A total of 400 cells were counted under the 400× microscope. We graded the samples according to the percentage of the positive cells: the number of positive cells ＜ 5% -; 5%-10％ +; 11％-50％ ++; ＞ 51％ ().③ Grade and quantitative data were compared with rank sum test and t-test.MAIN OUTCOME MEASURES: Effects of AMB on the rate of neu-ronal apoptosis, the activity of Mn-SOD, the concentration of MDA in mitochondria, and the intensity of the expression of bcl-2 gene.RESULTS: Totally 24 mice entered the final analysis.① Content of MnSOD was higher in young group and AMB group than in old control group (P＜0.05).② Concentration of MDA and apoptotic rate in young group and AMB group were lower than those in old control group (P ＜ 0.01).③Expression of bcl-2 gene was significantly different in young group and AMB group from that in old control group (P ＜ 0.01).CONCLUSION: AMB is found to be able to obviously inhibit neuronal apoptosis in aging mice brain by affecting the activity of Mn-SOD, the concentration of MDA and the expression of bcl-2.%背景:衰老的细胞死亡的实质就是细胞凋亡,凋亡从某种程度上可理解为一系列基因活动的结果.因此抑制癌基因的表达即可延长细胞的寿命,延缓脑组织衰老.目的:观察老年小鼠脑神经细胞凋亡和相关基因表达的变化及黄芪的干预效应.设计:完全随机分组设计,对照实验.单位:佳木斯大学基础医学院生物化学教研室.材料:实验于2003-12/2004-05佳木斯大学实验动物中心及生物化学实验室完成.选用健康昆明种小鼠24只.其中2月龄小鼠8只(青年组),12月龄小鼠16只.将16只12月龄小鼠随机分为2组:黄芪治疗组和老年对照组,各8只.方法:①黄芪治疗组:灌胃10.8 g/(kg·d)黄芪水煎剂(黄芪购于佳木斯大学第一附属医院中药局,由佳木斯市药品检验所鉴定,制成含生药浓度为2 kg/L的水煎剂);老年对照组和青年组:灌服与黄芪水煎剂体积相等的温开水.均连续干预30 d.②各组小鼠连续干预30 d后断头处死,立即取出脑组织,取大脑中部中性甲醛固定.余脑组织制成制成线粒体悬液,采用黄嘌呤氧化酶法及TBA化学比色法测定脑线粒体锰超氧化物歧化酶活性及丙二醛含量.用原位末端标记法观察老年小鼠神经细胞凋亡率(凋亡的细胞核呈黄色,为阳性细胞),并用免疫组织化学法检测相关基因bcl-2的表达.细胞液染成棕色者为基因表达阳性细胞,400倍显微镜下计数400个细胞,-:阳性细胞所占百分比＜5%;+:阳性细胞所占百分比5%～10%;H:阳性细胞所占百分比11%～50%;卅:阳性细胞所占百分比＞51%.③等级和计量资料差异比较要用秩和检验和t检验.主要观察指标:黄芪对老龄小鼠脑线粒体锰超氧化物歧化酶活性、丙二醛含量、脑细胞凋亡率、脑组织bcl-2基因表达强度的影响.结果:小鼠24只均进入结果分析.①脑线粒体锰超氧化物歧化酶含量:青年组和黄芪治疗组明显高于老年对照组(P＜0.05).②脑线粒体丙二醛含量和脑细胞凋亡率:青年组和黄芪治疗组明显低于老年对照组(P＜0.01).③脑组织bcl-2基因表达:青年组和黄芪治疗组与老年对照组比较,差异明显(P＜0.01).结论:黄芪通过影响锰超氧化物歧化酶活性和丙二醛含量及bcl-2基因表达对老年小鼠脑神经细胞凋亡有明显的抑制作用.
展开▼
