摘要
目的:研究硒在糖尿病代谢紊乱中的治疗效果及其可能机理,选择可行的疗效评价指标,初步探讨补硒形态的优劣。
方法:选用昆明种雄性小鼠120只,体重19.09±1.3 g,适应性饲养3天,按体重随机抽取24只分为两组备用,剩余部分为造模组。禁食24h后,造模组严格按体重一次性腹腔注射180 mg/kg,2%的四氧嘧啶,造糖尿病(DM)模型,备用的两组小鼠给予相应容积的生理盐水腹腔注射。注射后恢复食、水,5d、10d、14d监测三次尿糖,以尿糖持续3个“+”以上者为成模小鼠。然后将成模小鼠随机分为4组,每组17只。实验共分六组:Ⅰ组为正常对照组,Ⅱ组正常+硒蛋白组(100μg Se /kg 体重),Ⅲ组为糖尿病对照组(DM组),Ⅳ组为DM+硒蛋白低剂量组(100μg Se /kg 体重),Ⅴ组为DM+硒蛋白高剂量组(300μg Se /kg 体重),Ⅵ为DM+亚硒酸钠组(100μg Se /kg 体重),补硒量均在常备饲料(经本实验室测定硒含量为0.159 mg/kg)硒水平之上另加补予,饮水方式给予。每周称体重一次,测定日进食进水量3次,以平均进水量和体重调整一次补硒液浓度。各组自由进食、进水,饲料为常备饲料,正常组饮用自来水,Ⅱ~Ⅵ组饮用自来水配制的相应剂量补硒液,硒蛋白每日配制,亚硒酸钠一周配制一次。补硒8周,小鼠禁食12h,摘眼球取血,肝素抗凝,并及时离心,分离血浆,低温保存备用,小鼠脱颈处死,取部分肝脏及时用10%甲醛固定以用于今后HE、PAS染色的病理检
验,剩余的肝脏、肾脏用冷生理盐水漂洗后滤纸吸干,低温保存,在短期内及时测定肝硒、肝糖原、肌糖原、肾脏Na+-K+-ATPase、Ca2+-ATPase、全血GPX、血浆葡萄糖、MDA、NOS、NO、TG、TC等指标。
结果:1. 正常补硒组(5.93±1.64 mmol/L)血糖较正常对照组(8.33±1.35 mmol/L)明显降低(P<0.01),DM补高剂量硒蛋白组(20.40±6.32 mmol/L)血糖与糖尿病对照组(45.29±3.26 mmol/L)相比明显降低(P<0.01),但未能降至正常组水平(P<0.05),DM补亚硒酸钠和硒蛋白组的降糖效果没有差异(P>0.05);正常鼠补硒后肝脏硒含量高于正常对照组(P<0.05),糖尿病补硒组中,硒蛋白高剂量组(1.40±0.26μg/g)和亚硒酸钠组(1.52±0.35μg/g)的硒含量要明显高于DM对照组(0.99±0.31μg/g)(P<0.05)。
2. DM对照组(6.78±2.22 mg/g肝湿重)与正常对照组(2.14±0.95 mg/g肝湿重)比较肝糖原含量明显升高(P<0.01),各DM补硒组与DM对照组比较均明显降低(P<0.01),且降至正常对照组水平(P>0.05);DM对照组与正常对照组比较肌糖原含量有升高趋势,但还未达到统计学检验差异(P>0.05),各DM补硒组与DM对照组比较均无差异(P>0.05)。
3. DM对照组(3.04±0.99 mmol/L)血浆TC含量明显高于正常对照组(2.00±0.26 mmol/L)(P<0.05),DM+亚硒酸钠组(1.89±0.56 mmol/L)TC含量与DM对照组比较明显降低(P<0.05),且降至正常对照组水平(P>.05);各实验组之间血浆TG含量均无明显差异(P>0.05)。
4. DM对照组(30.16±5.79 U/ml)全血中GPX活性明显高于正常对照组(11.19±1.37 U/ml)(P<0.01),各DM补硒组GPX
活性与DM对照组相比均无明显差异(P>.05);DM对照组(6.64±1.61 nmol/ml)血浆MDA含量明显高于正常对照组(4.27±1.64 nmol/ml)(P<0.05),DM补硒蛋白组与DM对照组比较MDA含量无明显差异(P>.05)。
5. DM对照组(7.26±0.87μMpi.mg-1蛋白.小时-1)与正常对照组(4.85±0.76μMpi.mg-1蛋白.小时-1)比较酶活性明显升高(P<0.01),各DM补硒组与DM对照组比较酶活性均无差异(P>0.05);DM补高剂量硒蛋白组(0.90±0.51μMpi.mg-1蛋白.小时-1)肾脏Ca2+-ATPase活性明显高于DM对照组(0.35±0.11μMpi.mg-1蛋白.小时-1)(P<0.05)。
6. 正常补硒蛋白组(35.57±2.97 U/ml)NOS活性明显高于正常对照组(P<0.05),DM对照组(35.20±4.38 U/ml)与正常对照组(29.84±3.85 U/ml)比较NOS活性明显升高(P<0.05),DM补硒组中,高硒蛋白组(24.98±4.28 U/ml)和亚硒酸钠组(29.27±4.52 U/ml)与DM对照组相比均明显降低(P<0.05);正常补硒蛋白组(10.10±4.94μmol/L)NO含量明显低于正常对照组(P<0.05),DM对照组(60.62±20.90μmol/L)与正常对照组(24.43±9.60μmol/L)比较NO含量明显升高(P<0.05),DM补硒组中,亚硒酸钠组(32.76±14.32μmol/L)与DM对照组相比明显降低(P<0.05),并降至正常水平(P>.05)。
结论:1. 糖尿病发生后血糖明显升高,补硒具有降血糖的功效,不但可以降低糖尿病小鼠血糖浓度,而且可以降低正常小鼠血糖浓度。
2. 长期糖尿病状态下肝糖原会升高,补硒后肝糖原会明显的降低,肝糖原含量与血糖浓度有一定的消长趋势。
3. 糖尿病发生后有血脂升高现象,补硒有降脂功效,血中TC
比TG更敏感。
4. 四氧嘧啶所造的糖尿病小鼠模型具有明显的肝肿大现象,镜下肝脏病理改变较轻,多以灶性细胞肿大、水样变性为主,PAS染色细胞内有大量糖原蓄积,补硒后能明显减轻以上病变。
5. 补硒具有提高糖尿病鼠血中GPX活性的作用,但喂饲糖尿病小鼠饲料硒达到0.159mg /kg 时,糖尿病小鼠全血中GPX活性会?
Objective:To investigate the effect and possible mechanism of selenium cure metabolize turbulence on diabetes mice, choose feasible estimate index of curative effect, and discuss the excellent or inferior of supply selenium forms primarily.
Methods:Use 19.09±1.3 g ,male Kuming mice were feed three days to adapting circumstance ,and were randomly taken out twenty four mice into two groups,after 24 hours fasting ,the odd mice were injected 180mg/kg weight ,2% alloxan in abdomen to make diabetes model(DM),and the two groups mice were injected correspond cubage 0.9% brine in abdomen. After injection ,resume to serve food and water,and measure three urine glucose at 5、10、14 day.According to urine glucose level, It’s continue to present three “ + ” that can be regarded as become diabetes model (DM) ,then divided randomly diabetes mice into four groups,each group have seventeen mice.Experiment have six groups: normal control group (Ⅰ),normal+ selenoprotein group(100μg Se /kg weight) (Ⅱ), DM control group (Ⅲ),DM+ lower dose selenoprotein group(100μg Se /kg weight) (Ⅳ), DM+ higher dose selenoprotein group(300μg Se /kg weight) (Ⅴ),DM+ Na2SeO3 group(100μg Se /kg weight) (Ⅵ). Selenium was supplied except selenium in ordinary feedstuff,and the
selenium level of ordinary feedstuff is 0.159 mg /kg by our lab mensurating. The way of feeding selenium is by drinking liquid.We measure once weight and thrice dose of take food and drink water of mice every week,and adjust once thickness of supply selenium liquid by measure average drink water dose and weight of every group every week. Mice free eating and drinking, the supplied feedstuff is ordinary feedstuff. The normal group drink tap water , Ⅱ~Ⅵ groups drink supply selenium liquid by tap water dissolving. Selenoprotein liquid were confect everyday and Na2SeO3 liquid were confect every week. After supplied selenium eight weeks,mice were forbided eating to 12 hours,were taken blood by extirpating eyeball, after resisted cruor , were separating blood plasma from blood in time to be keep in low temperature.Then ,break neck to kill mice,and extirpate part of liver ,put into 10% formaldehyde liquid for fixation and used for pathology examination by HE and PAS dyeing. The odd liver and kidney wash by cool 0.9% brine and sip up by filter paper,then to keep in low temperature,used to measure these index : Selenium content of liver, Hepatin content of liver and muscle, Na+-K+-ATPase and Ca2+-ATPase activity of kidney, GPX activity in blood ,and Glucose、MDA、NOS、NO、TG、TC of plasma, et al.
Results:1. Compare with Ⅰ group(8.33±1.35 mmol/L), the blood glucose of Ⅱ group(5.93±1.64 mmol/L ) could be significantly decreased , p<0.01 ; compare with Ⅲ group(45.29±3.26 mmol/L), the blood glucose of Ⅴ group(20.40
±6.32 mmol/L ) could be significantly decreased , p<0.01 ,but can’t be dropped to the level of Ⅰ group; blood glucose among Ⅳ、Ⅴ and Ⅵ groups have not significant difference, P>0.05. The selenium content of liver of Ⅱ group are higher thanⅠ group, P<0.05; the selenium content of liver of Ⅴ(1.40±0.26μg/g) and Ⅵ groups(1.52±0.35μg/g) are higher than Ⅲ group(0.99±0.31μg/g), P<0.05.
2. Hepatin content of liver of Ⅲ group(6.78±2.22 mg/g liver) are higher than Ⅰ group(2.14±0.95 mg/g liver), P<0.01; Ⅳ、Ⅴ and Ⅵ groups compare with Ⅲ group , hepatin content of liver could be significantly decreased , P<0.01, and that can be dropped to the level of Ⅰ group, P>0.05. Ⅲ group compare with Ⅰ group ,hepatin content of muscle have increase trend, but have not statistic significance, P>0.05;Ⅳ、Ⅴ and Ⅵ groups compare with Ⅲ group, hepatin content of muscle have not statistic significance, P>0.05.
3. TC content in plasma of Ⅲ group(3.04±0.99 mmol/L) compare with Ⅰ group(2.00±0.26 mmol/L) have significantly increase, P<0.05;Ⅵ group(1.89±0.56 mmol/L) compare with Ⅲ group , TC content in plasma have significantly decrease, P<0.05,and that can be dropped to the level of Ⅰ group, P>.05. TG content
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