泻脏方法促进再生障碍性贫血小鼠骨髓造血功能恢复

Effect of reducing zang method in the promotion of hematopoietic function of bone marrow in mice with aplastic anemia

目的:观察泻脏方法(六味地黄丸之三泻,即泽泻、茯苓、丹皮)治疗再生障碍性贫血效果,并分析其作用途径。方法:实验于2004-08/2005-01在成都医学院第一附属医院康复医学科完成。①选用昆明种雄性小鼠60只,鼠龄90d,体质量(20.0±0.3)g。按随机抽签法将小鼠分为6组:空白对照组,造模组,阳性药物对照组,高、中、低剂量中药治疗组,每组10只。②小鼠驯养1周后,造模组及4个治疗组采用理化因素复合造模:一次全身2.5Gy60Co-γ射线照射(剂量率1.1053Gy/min,距离240cm,时间135s),照射后4,5,6d腹腔注射环磷酰胺40mg/kg,氯霉素50mg/kg。空白对照组小鼠假照射(铅砖屏蔽)照射后4,5,6d腹腔注射等容量生理盐水。③造模完成后第1天开始用药,阳性药物对照组:灌胃4mg/穴kg·d雪司坦唑醇(广西佳兆药业有限责任公司,批号020305,2.0mg/片)混悬液;高、中、低剂量中药治疗组:灌胃0.5mL/(20g·d)相应高、中、低浓度泻脏方法治疗组方(组方:六味地黄丸之三泻泽泻、丹皮、茯苓各28g;制备:两煎,每煎加水1050mL,各煎得525mL,混匀,分为150,300,600mL3组,水浴浓缩600和300mL各得150mL,分别为高、中浓度,未浓缩的150mL为低浓度。

AIM： To observe the curative effect （The reducing part of Liuwei Dihuang Wan, rhizoma alismatis, poria, cortex moutan） of the reducing zang method on aplastic anemia and discuss its mechanism. METHODS： The experiment was carried out in the Department of Rehabilitation Medicine of First Affiliated Hospital of Chengdu Medical College between August 2004 and January 2005. ①Sixty male Kunming male mice, aged 90 days weighting （20.0±0.3） g were selected and randomly divided into six groups： blank control group, model group, drug control group, and high, middle and low dose traditional Chinese medicine （TCM） group with 10 mice in each group. ② At one week after domestication, mice in the model group and four treatment groups received modeling with complex factors： whole body exposed to 2.5 Gy^ 60Co -γ once （the dose rate was 1.105 3 Gy/minute, with the distance of 240 cm and timeof 135 seconds）, at 4, 5 and 6 days after radiation, mice were intraperitoneally injected with cyclophosphamide （40 mg/kg） and chloramphenicol （50 mg/kg）. Mice in the blank control group received intraperitoneal injection of normal saline at 4, 5 and 6 days after false radiation （screened with lead brick）. ③Mice received medication on the first day after modeling. Positive drug control group： mice were given gastric perfusion of stanozolol suspension 4 mg/kg per day （manufactured by Guangxi Jiayao Pharmaceutical Co., Ltd with the lot number of 020305, 2.0 mg/pill）. High, middle and low dose TCM group： mice received gastric perfusion of corresponding decoction of reducing zang （Liuwei Dihuang Wan： rhizoma alismatis, poria and cortex moutan, 28 g for each） at the dose of 0.5 mL/20 g per day. Preparation： two-decoction with each time added into 1 050 mL water. 525 mL that obtained each time were mixed and then divided into 3 groups： 150, 300 and 600 mL group, being condensed to 600 and 300 mL by water bath, and 150 mL was obtained respectively, which were at high and middle concentration, while 150 mL of uncondensed were at low concentration. There were 0.32, 0.16 and 0.08 g of crude drug in each milliliter of drug respectively at high, middle and low concentration）. Mice in the blank control group and model group were given gastric perfusion of normal saline at the same volume with the total intervention time of 13 days. ④The animals were put to death right on the day of drug withdrawal, and the left thighbone was obtained to fix with formalin. Mias-2000 image analytical system was adopted. The nucleus of bone marrow cells was detected with Olympus BX50 optics microscope, and number of bone marrow cells was inspected with hematine-eosin stain. The proliferation status was inspected with immunohistochemical stain Ki-67, and the cell apoptosis was determined with eysteine and aspartic proteinase 3. CD34 was used to reflect the microcirculation of bone marrow. ⑤The differences of measurement data were compared by one-way analysis of variance （ANOVA）,and comparison between two groups was conducted with LSD and SNK analysis. RESULTS： When the experiment ended,there were 4, 4, 3 and one mice died respectively in the high, middle and low dose TCM treatment group, and there were no mice died in the other two groups. Six mice were randomly selected from the blank control group to detect all the index. There were 6,10,6,7 and 9 mice respectively in the model group, positive drug control group, and high, middle and low dose group were stained by hematoxylin-eosin and detected. 6 mice were randomly selected from each group to detect the remaining 3 items.①The accumulated point A value and area of cell nucleus in the positive drug control group and low dose TCM treatment group were significantly higher than modeling group （P 〈 0.01）, whereas the average A value and blackness were remarkably lower than modeling group （P 〈 0.01）. The A value in high and middle dose TCM treatment were obviously higher than modeling group （P 〈 0.05）, and the blackness was similar to the modeling group （P 〈 0.05）, and the average A value was similar to the modeling group （P 〉 0.05）, which indicated that the curative effect of low dose TCM treatment group was the most significant.②The A value, area and average A value of relative indexes in bone marrow sections after immunohistochemical stain Ki-67 in the modeling group were significantly lower than blank control group and low dose TCM treatment group （P 〈 0.01） .③The A value of relative indexes of cysteine and aspartic proteinase 3 after immunohistochemical stain was obviously lower in the modeling group than blank control group and low dose TCM treatment group （P 〈 0.01 ）, whereas the area and average A value were remarkably higher than blank control group and low dose TCM treatment group （P 〈 0.01）. ④The perimeter, maximum diameter and minimum diameter of relative indexes after immunohistochemical stain CD34 were markedly lower in the modeling group than blank control group and low dose TCM treatment group （P 〈 0.01）, and the area was obviously lower than low dose TCM treatment group （P 〈 0.01）. CONCLUSION： The method of reducing zang, which is represented by the reducing part of Liuwei Dihuang Wan, rhizoma alismatis, poria, cortex moutan can promote the proliferation of cell in marrow, inhibit the apoptosis of medulla cell, ameliorate the microcireulation in the marrow, and thus promote the recovery of hematopoietic function of marrow in mice with aplastic anemia caused by physical and chemical factors.