棉酚对2型糖尿病大鼠大脑皮层及海马神经元凋亡相关基因表达的影响

The effect of gossypol on expression of apotosis-related gene in the cerebral cortex and hippocampus neuron of type Ⅱ diabetic rats

目的:观察棉酚对2型糖尿病大鼠脑内神经元的保护作用,并探讨其机制。方法:30只SD雄性大鼠,随机均分成3组:正常组、2型糖尿病组、棉酚干预组。后2组给予高脂饮食加小剂量(30 mg/kg)链脲佐菌素(STZ)诱导2型糖尿病模型,棉酚干预组(第1～第4周棉酚按15 mg·kg-1·d-1剂量灌胃,第5～第12周棉酚按15 mg·kg-1·周-1剂量灌胃)。用Morris水迷宫法评价大鼠学习记忆能力;生化法检测血糖;放射免疫法检测血胰岛素水平;Western blot法检测大脑皮层及海马组织Bax、Bcl-2、caspase-3、凋亡诱导因子(AIF)蛋白表达水平;电镜、光镜下观察大脑皮层及海马组织的形态学改变。结果:与正常组比,2型糖尿病组大脑皮层及海马神经元可见较明显的核固缩、核膜皱缩、胞浆空泡化,血糖、血胰岛素水平明显升高(P<0.01),海马中,caspase-3、AIF蛋白表达升高(P<0.05),Bax蛋白表达明显升高(P<0.01),Bcl-2蛋白表达无明显变化,皮层中,Bax、AIF蛋白表达升高(P<0.05),caspase-3呈升高趋势,Bcl-2蛋白表达无明显变化。行为测试潜伏期明显延长(P<0.01),搜索策略明显变差(P<0.01);经棉酚干预后,大脑皮层及海马组织形态学病变减轻,血糖、血胰岛素水平明显下降(P<0.01),海马中,Bax蛋白表达明显降低(P<0.01),caspase-3、AIF蛋白表达降低(P<0.05),皮层中,Bax、AIF蛋白表达降低(P<0.05),caspase-3呈下降趋势,行为测试潜伏期明显缩短(P<0.01),搜索策略明显好转(P<0.01)。结论:棉酚能保护2型糖尿病大鼠脑内神经元,提高大鼠空间记忆能力,这可能与其下调神经元凋亡基因caspase-3、Bax、AIF的表达有关。

Objective： To study the protective effect of gossypol on the neuron of type Ⅱ diabetic rats, andexplore its mechanism. Methods- Thirty male Sprague-Dawley rats were divided into three groups randomly： normal group, type Ⅱ diabetic group and gossypol treated group. After fed with high-fat diet for 4 weeks, the later two groups were injected with strepozotocin intraperitoneally to induce type Ⅱ diabetic rats model. The gossypol treated group was given gossypol at the dosage of 15 nag .kg-1 once per day for 4 weeks by gavage. And since 5th week, the times of gavages had been changed into once per week at the same dosage and lasted to 12th week. Learning and memory abilities of rats were assayed with Morris water maze test. The concentration of blood glucose was measured with biochemical method, the levels of serum insulin were detected by ELISA （enzyme- linked immunosorbent assay） and RIA （radio immunoassay） respectively. The protein expression of Bax, Bcl-2, caspase-3, AIF of cerebral cortex and hippocampus were determined by Western blot. The morphological changes of cerebral cortex and hippocampus were studied under light microscopy （LM） and transmission electron micros- copy （TEM） respectively. Results： Compared to normal groups, the karyopyknosis, shrinkage of nuclear membrane,vacuolization of cytoplasm from cerebral cortex and hippocampus were prominent in diabetic group. The concen- trations of the blood glucose, serum insulin rose significantly （P〈0.01）. In hippocampus, caspase-3, AIF protein expression increased obviously （P〈0.05）. Bax protein expression increased significantly （P〈0.01）. Bcl-2 protein expression did not change. In cortex, Bax and AIF increased obviously （P〈0.05）, caspase-3 protein tended to increase and Bcl-2 protein did not change. Platform searching score was lower （P〈0.01） and escape latency was longer （P〈0.01） in diabetic group. After treated with gossypol, the concentrations of blood glucose and serum insulin declined （P〈0.01）. In hippocampus, the protein expression of Bax, caspase-3 and AIF was decreased obviously （P〈0.05）. In cortex, Bax and AIF protein was decreased obviously （P〈0.05） while caspase-3 protein tended to decrease. Escape latency was shorter （P〈0.01） and platform searching score was higher （P〈0.01）. Conclusion： Gossypol may protect the neuron in the brain of type II diabetic rats, improve the space-memorizing ability, it may relate with its function of down-regulating the expression of caspase-3, Bax and AIF gene.