龟甲胶对肾阴虚大鼠抗氧化活性和Bax、Bcl-2蛋白表达的影响＊

目的 探讨龟甲胶对肾阴虚大鼠抗氧化活性及Bax（Bcl-2同源二聚体X,Bcl2-Associated X）、Bcl-2（B淋巴细胞瘤-2,B-cell lymphoma-2）蛋白表达的作用机制。方法 60只SPF级雌性SD大鼠适应性喂养3天后,随机分为正常组,模型组、给药组（龟甲胶低剂量组、龟甲胶中剂量组、龟甲胶高剂量组）和阳性（六味地黄丸）组,给药21天。除正常组外,其余大鼠通过灌胃甲状腺素建立肾阴虚大鼠病理模型;于造模同时正常组及模型组每天灌胃等体积生理盐水。给药期间,观测大鼠脏器（肝脏、肾脏）指数、体质量、肛温、饮水量和进食量的变化;运用酶联免疫（ELISA）法检测血清超氧化物歧化酶（SOD）活性、丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-Px）、雌二醇（E2）、睾酮（T）水平;免疫组化法检测Bax、Bcl-2蛋白表达变化;RT-qPCR（荧光定量聚合酶链式反应,Real-time quantitative PCR）法检测Bax、Bcl-2蛋白mRNA相对表达量,并进行组间比较。结果 龟甲胶各给药组均能明显缓解大鼠的阴虚状态,龟甲胶中、高剂量组能显著增加肾阴虚大鼠体质量及肝脏指数（P < 0.05,P < 0.01）,龟甲胶高剂量组能升高大鼠肾脏指数（P < 0.01）;龟甲胶各剂量组均能降低肾阴虚大鼠肛温（P < 0.05,P < 0.01）。龟甲胶中、高剂量组大鼠血清中SOD、GSH-Px、E2水平升高（P < 0.05,P < 0.01）,MDA含量降低（P < 0.05）;龟甲胶高剂量组大鼠血清中T含量显著升高（P < 0.05,P < 0.01）。龟甲胶低、高剂量组大鼠肾组织中Bax表达水平降低（P < 0.05）;龟甲胶高剂量组大鼠肾组织中Bcl-2表达水平显著升高（P < 0.01）。结论 龟甲胶具有传统滋阴功效,能增强阴虚状态下机体抗氧化能力,提高SOD活力、降低MDA含量;同时下调Bax以及上调Bcl-2等衰老相关因子的蛋白表达,从而延缓机体的衰老。

Effects of Tortoise Shell Gum on Antioxidant Activity and Protein Expression of Bax and Bcl-2 in Rats with Kidney Yin Deficiency

Objective To investigate the effect of tortoise shell gum on antioxidant activity and expression of Bax and Bcl-2 in rats with kidney-yin deficiency.Methods After 3 days of adaptive feeding, 60 SPF female SD rats were randomly divided into normal group, model group, drug administration group (low dose group, medium dose group, high dose group) and positive group (Liuwei Dihuang Pill) for 21 days. Except the normal group, the pathological model of kidney-yin deficiency was established by gavage of thyroxine. The normal group and the model group were given the same volume of normal saline every day. During administration, the changes of viscera (liver and kidney) index, body mass, anal temperature, water consumption and food intake were observed. Serum levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), estradiol (E2) and testosterone (T) were detected by enzyme-linked immunosorbent assay (ELISA). The expression of Bax and Bcl-2 was detected by immunohistochemistry. RT-qPCR was used to detect the relative mRNA expression of Bax and Bcl-2 proteins, and the results were compared between groups.Results The condition of yin deficiency of rats was significantly alleviated in all the treatment groups, and the body mass and liver index of rats with kidney yin deficiency were significantly increased in the medium and high dose groups (P < 0.05, P < 0.01), and the kidney index of rats with kidney yin deficiency was significantly increased in the high dose group (P < 0.01). The anal temperature of rats with kidney-yin deficiency was decreased in all dose groups (P < 0.05, P < 0.01). The levels of SOD, GSH-Px and E2 in the serum of rats in the medium and high dose groups increased (P < 0.05, P < 0.01), while the levels of MDA decreased (P <0.05). The serum T content of rats in the high dose group was significantly increased (P < 0.05, P < 0.01). Bax expression level in the kidney of rats in the low and high dose groups was decreased (P < 0.05). The expression level of Bcl-2 in the kidney of rats in the high dose group was significantly increased (P < 0.01).Conclusion Tortoise shell glue has the traditional yin nourishing effect, which can enhance the body"s antioxidant capacity under the state of yin deficiency, increase the activity of SOD and reduce the content of MDA; at the same time, it can down-regulate the protein expression of Bax and up-regulate the protein expression of aging-related factors such as Bcl-2, thereby delaying the body"s aging.