脾虚证小鼠脾肾组织核酸和蛋白质含量变化及强肌健力口服液的影响

背景:强肌健力口服液对重症肌无力有较好的防治作用,核酸和蛋白质水平的变化是否与药物作用机制有关。目的:观察强肌健力口服液对实验性脾虚证小鼠核酸及蛋白质合成的影响,探讨该方防治脾胃虚损型重症肌无力的作用途径及中医脾肾相关的分子生物学基础。设计:随机对照动物实验。单位:广州中医药大学测试中心和基础医学院。材料:选用4周龄健康雄性NIH小鼠,清洁级,体质量17～21g,由广东省医学实验动物中心提供。强肌健力口服液(广东东方神草药业有限公司);利血平注射液(广东邦民制药厂有限公司);[5-甲基-3H]TdR、[5-3H]尿苷、L-[4,5-3H]亮氨酸(原子高科核技术应用股份有限公司)。方法:实验于2005-06/11在广州中医药大学核医学实验室完成。选择健康雄性NIH小鼠180只,抽签法随机分为正常对照组、脾虚模型组、强肌健力口服液26g/kg组、强肌健力口服液13g/kg组、四君子汤组,每组36只。除正常对照组外,各组小鼠均采用利血平复制脾虚证动物模型,腹腔注射利血平0.2mg/(kg·d),①正常对照组小鼠腹腔注射生理盐水0.1mL/(只·d)。②强肌健力口服液26g/kg组及13g/kg组灌胃强肌健力口服液(由北芪、党参、升麻、白术、甘草等组成,每毫升含生药1.204g),给药剂量分别为26,13g/kg,1次/d。③四君子汤组灌服四君子汤(由党参、白术、茯苓、灸甘草组成)26g/kg,1次/d。各组连续用药16d,给药体积均为0.5mL/只,给药结束后,测定各组小鼠脾、肾组织DNA、RNA和蛋白质含量;并测定各组小鼠造模前及用药结束后体质量和脾、肾质量/体质量比值的变化。主要观察指标:①各组动物造模前及用药16d后体质量的变化。②各组动物脾、肾组织质量的变化。③用药16d后各组动物脾、肾组织DNA、RNA和蛋白质含量的比较。结果:180只小鼠全部进入结果分析,无脱失。①实验前各组小鼠体质量17.4-21g差异不显著(P=0.993;实验结束后脾虚模型组小鼠体质量显著低于正常对照组(Q=22.84,P<0.01);强肌健力口服液(26,13g/kg)和四君子汤灌服16d后,小鼠体质量显著高于脾虚模型组(Q=8.66～10.24,P<0.01)。②脾虚模型组小鼠脾、肾组织质量/体质量比值均比正常对照组显著下降(Q=4.02～12.93,P<0.01),强肌健力口服液26、13g/kg组和四君子汤组脾、肾组织质量较脾虚模型组增加(Q=3.21～9.69,P<0.05～0.01),强肌健力口服液13g/kg组脾组织比值和肾组织比值均大于脾虚模型组(Q=4.07,5.92,P<0.05,0.01)。③脾虚模型组小鼠脾组织DNA、RNA、蛋白质含量和肾组织RNA、蛋白质含量低于正常对照组(Q=7.15～19.2,P<0.01),肾组织DNA高于正常对照组(Q=4.19,P<0.05)。强肌健力口服液26g/kg组和四君子汤组脾组织DNA、RNA、蛋白质含量和肾组织RNA、蛋白质含量高于脾虚模型组(Q=2.91～14.12,P<0.05～0.01)。结论:强肌健力口服液能促进核酸和蛋白质合成;脾虚证的发生与核酸及蛋白质合成减少可能有关。

Effect of qiangji jianli liquid on changes of nucleic acid and protein of spleen and kidney tissue in mice with spleen deficiency syndrome

BACKGROUND: Qiangji jianli liquid plays a key role in prevention and cure of myasthenia gravis; however, whether changes of nucleic acid and protein are related to its mechanism or not should be studied further.OBJECTIVE: To observe the effect of qiangji jianli liquid on synthesis of nucleic acid and protein in mice with experimental spleen deficiency syndrome, and investigate the effect on myasthenia gravis and the molecular biological basis.DESIGN: Randomized controlled animal study.SETTING: Testing Center and Basic Medical College of Guangzhou University of Traditional Chinese Medicine.MATERIALS: Healthy male NIH mice, aged 4 weeks, of clean grade, weighing 17-21 g, were provided by Medical Experimental Animal Center of Guangdong Province. Qiangji jianli liquid (Guangdong Dongfang Shencao Pharmaceutical Factory); serpate solution (Guangdong Bangmin Pharmaceutical Factory); [5-methyl-3H] TdR, [5-3H] uridine and L-[4,5-3H]leucine (Atom High-nuclear Technology Application Limited Company).METHODS: The experiment was carried out in the Laboratory of Nuclear Medicine of Guangzhou University of Traditional Chinese Medicine from June to November 2005. A total of 180 healthy male NIH mice were randomly divided into normal control group, model group, 26 g/kg qiangji jianli liquid group, 13 g/kg qiangjijianli liquid group and sijunzi decoction group with 36 in each group. Except normal control group, serpate was used to establish animal models of spleen deficiency syndrome. 0.2 mg/(kg ·d) serpate was intraperitoneally injected into mice. ① 0.1 mL/(mouse·d) saline was intraperitoneally injected into mice in normal control group. ② 26 g/kg and 13 g/kg were perfused into mice in 26 g/kg and 13 g/kg qiangji jianli liquid groups, respectively, once a day. Qiangji jianli liquid consisted of beiqi, dangshen, shengma,baizhu, gaicao, etc., with 1.204 g raw drugs a milliliter. ③ 26 g/kg sijunzi decoction, which consisted of dangshen,baizhu, fuling, jiugancao, etc., was perfused into mice with the dosage of 0.5 mL/mouse in si junzi decoction group once a day. After 16 successive days, contents of DNA, RNA and protein were measured in spleen and kidney tissue; meanwhile, body mass, ratio between spleen mass and body mass, and ratio between kidney mass and body mass were also measured before modeling and after administration.MAIN OUTCOME MEASURES: ① Changes of body mass before modeling and at 16 days after administration; ② changes of mass of spleen and kidney tissues; ③ Comparisons of DNA, RNA and protein in spleen and kidney tissues at 16 days after administration.RESULTS: All 180 mice were involved in the final analysis without any loss. ① There was no significant difference of body mass (17.4-21 g) among groups before experiment (P =0.993); however, after experiment, body mass in model group was lower than that in normal control group (Q =22.84, P ＜ 0.01); 16 days after administration, body mass was higher in qiangji jianli liquid groups (26 g/kg, 13 g/kg) and sijunzi decoction group than that in model group (Q =8.66-10.24, P ＜ 0.01). ② Values of spleen mass/body mass and kidney mass/body mass were decreased in model group as compared with those in normal control group (Q =4.02-12.93, P ＜ 0.01); masses of spleen and kidney tissues were increased in qiangji jianli liquid groups (26 g/kg, 13 g/kg) and sijunzi decoction group as compared with those in model group (Q =3.21-9.69, P ＜ 0.05-0.01); ratios in 13 g/kg qiangji jianli liquid group were higher than those in model group (Q =4.07, 5.92; P ＜ 0.05, 0.01). ③ Contents of DNA, RNA and protein in spleen tissue and contents of RNA and protein in kidney tissue were lower in model group than those in normal control group (Q =7.15-19.2, P＜ 0.01); however, content of DNA in kidney tissue was higher in model group than that in normal control group (Q =4.19, P ＜ 0.05). Contents of DNA, RNA and protein in spleen tissue and contents of RNA and protein in kidney tissue were higher in 26 g/kg qiangji jianli liquid group and sijunzi decoction group than those in model group (Q =2.91-14.12, P ＜ 0.05-0.01 ).CONCLUSION: Qiangji jianli liquid can accelerate synthesis of nucleic acid and protein; additionally, onset of spleen deficiency syndrome may be related to decreasing synthesis of nucleic acid and protein.