葛根素对顺铂诱导小鼠卵巢早衰的作用研究

Effect of puerarin on cisplatin induced premature ovarian failure in mouse

摘要:

目的 探讨葛根素对顺铂诱导小鼠卵巢早衰的作用。方法 选取雌性C57BL/6J小鼠，随机分为3组，每组10只。给药途径均为腹腔注射。模型组给予生理盐水（4.0 ml/kg×9 d），顺铂（4.5 mg/kg×5 d）及生理盐水（4.0 ml/kg×5 d）；实验组给予葛根素（200.0 mg/kg×9 d），葛根素（200.0 mg/kg×5 d）及顺铂（4.5 mg/kg×5 d）；对照组给予同体积生理盐水14 d。 实验第9天，称重小鼠并记录，观察小鼠一般活动。酶联免疫吸附试验（ELISA）检测血浆中激素水平；HE染色观察卵巢组织形态结构；免疫荧光染色检测小鼠卵巢组织中Cleaved caspase-3、Bcl-2、Bax的表达；Western blotting法检测小鼠卵巢组织中Cleaved caspase-3、Bcl-2、Bax蛋白的相对表达量。结果 模型组小鼠一般情况差；实验组小鼠相对于模型组，一般情况有所改善；对照组、模型组及实验组小鼠的体重有差异，其中模型组与对照组相比体重呈现下降趋势，实验组相比模型组体重下降更加平缓（P <0.05）。模型组小鼠动情间期占动情周期的比例高于对照组（P <0.05）；实验组小鼠动情间期占动情周期的比例低于模型组（P <0.05）。模型组小鼠血清FSH水平升高（P <0.05），E2、AMH水平降低（P <0.05），实验组小鼠血清E2、AMH水平较模型组升高（P <0.05），FSH水平降低（P <0.05）。对照组小鼠卵巢组织可见不同发育阶段的卵泡，成熟卵泡位于卵巢的边缘，颗粒细胞规则排列；模型组小鼠卵泡闭锁，颗粒细胞减少、排列紊乱；实验组小鼠闭锁卵泡减少，颗粒细胞有序排列且数目多。Cleaved caspase-3、Bcl-2、Bax在3组小鼠卵巢组织中均有表达，且以颗粒细胞为主。模型组与对照癌比较，卵巢组织细胞中Bax、Cleaved caspase-3蛋白相对表达量增加（P <0.05），Bcl-2蛋白相对表达量减少（P <0.05）；实验组与模型组相比，卵巢组织细胞中Bax、Cleaved caspase-3蛋白相对表达量减少（P <0.05），Bcl-2蛋白相对表达量增加（P <0.05）。结论 葛根素可以抑制卵巢组织细胞凋亡，恢复顺铂损伤小鼠卵巢的功能，改善小鼠一般活动。

Abstract:

Objective To reveal the anti-apoptotic effect of puerarin on ovarian tissue cells, and to explain its protection of premature ovarian failure induced by cisplatin in mice.Methods Female C57BL / 6J mice were randomly divided into three groups, 10 mices in each group receiving medicine by intraperitoneal injection. Model group was given normal saline (9 days) and cisplatin with normal saline (4.5 mg/kg ×5 days). The experimental group was given puerarin (200.0 mg/kg× 9 d) and puerarin with cisplatin (200.0 mg/kg and 4.5 mg/kg× 5 d). The Reference group was given the same volume of normal saline for 14 days. On the 9th day of the experiment, weight of mice were recorded to observe general activities. Concentration of hormone in plasma through the ELISA method and expression of apoptosis related proteins (cleaved Caspase-3, Bcl-2, Bax) in mouse ovary through the Western blotting were detected.Results The general condition of mice in the experimental group was better than that in the model group；the body weight of the control group, model group, and experimental group were different. Compared with the control group, the weight of the model group showed a downward trend, and the weight loss of the experimental group was more gentle than that of the model group, and the difference was statistically significant (P < 0.05). The proportion of estrous intervals in the estrous cycle was significantly higher than that in the control group (P < 0.05), while the proportion of estrous intervals in the experimental group was lower in the model group (P < 0.05); in the model group, the levels of serum FSH increased (P < 0.05), while the levels of E2 and AMH decreased (P < 0.05). The levels of serum E2 and AMH in the experimental group were higher than those in the model group (P < 0.05), and FSH level decreased (P < 0.05). HE staining showed that follicles at different developmental stages could be observed in the ovarian tissue of mice in the control group. Besides, the mature follicles were located at the edge of the ovary, and the granulosa cells arranged regularly. In the model group, the follicles were atresia, and granulosa cells decreased and arranged disorderly. However, in the experimental group, atresia follicles decreased, granulosa cells arranged orderly, and the number of granulosa cells was larger. Apoptosis-related proteins were expressed in all the three groups, mainly granulosa cells. In the model group, the expression of Bax and Cleaved caspase-3 increased significantly (P < 0.05), while the expression of Bcl-2 decreased (P < 0.05). Compared with the model group, the expression of Bax and Cleaved caspase-3 decreased (P < 0.05), and the expression of Bcl-2 increased (P < 0.05) in the experimental group.Conclusion Puerarin can restrain apoptosis of ovarian tissue, restore the function of ovary injured by cisplatin, and improve the general activities of mice.