Wang Jun-Li, Liu Bing, Zhang Chao, Wang Xin-Mei, Zhen Di, Huang Xiao-Meng, Chen Wu, Gao Jian-Ming
Animal Science and Technology College, Beijing University of Agriculture, Beijing, China.
College of Animal Science and Technology, China Agricultural University, Beijing, China.
Theriogenology. 2019 Feb;125:157-167. doi: 10.1016/j.theriogenology.2018.10.028. Epub 2018 Nov 1.
In this study, effects of icariin (Ica) on were examined in a mouse model of d-galactose (D-gal)-induced ovarian aging. Kunming white mice were divided into three groups: aging group induced with D-gal, experiment group treated with Ica at low (50 mg/kg), middle (100 mg/kg) and high (200 mg/kg) concentrations, and control group with no treatment. Ovarian histomorphology, serum FSH, LH and E levels, and reproductive function were compared among the groups. Ovarian expression of Amh, Bax and Bcl-2 was examined by qPCR and western blotting. Our results showed that diameters of secondary and tertiary follicles were significantly reduced in the aging group when compared with control group (P < 0.01), and were restored to normal in Ica 100 and Ica 200 treatment groups. The diameter of atretic follicles was significantly smaller in the aging group compared with control group and Ica 200 treatment group (P < 0.05). The proportion of secondary and atretic follicles was higher in the aging group compared with control group, Ica 100 and 200 treatment groups, whereas the proportion of tertiary and mature follicles was reduced in the aging group versus control, Ica 100 and 200 groups. The aging group lacked mature follicles, whereas Ica treatment induced mature follicle development. Primary and secondary follicles exhibited similar theca cell numbers and theca interna and externa cell layers in all groups examined, whereas theca interna and externa cell layers were decreased and increased, respectively, in tertiary follicles of aging group compared with control and I 200 groups. In the aging group, FSH and LH levels were significantly higher than those in control and Ica 200 groups (P < 0.05), and the E level was significantly reduced compared with control (P < 0.01), Ica 200 (P < 0.01), and Ica 100 (P < 0.05) groups. Serum hormone levels were equivalent in the control, Ica 100 and Ica 200 groups. The pregnancy rate was reduced in the aging group compared with other groups. The average litter size per birth, birth litter weight, and weaning weight of litters were all significantly lower in the aging group compared with control, Ica 100 and 200 groups (P < 0.05). The ovarian expression of AMH and Bcl-2 mRNA was significantly reduced in the aging group compared with those in control and Ica-treated groups (P < 0.01). In contrast, Bax expression was significantly higher in the aging group compared with all other groups (P < 0.01), and the Bcl-2/Bax ratio was markedly reduced in aging group compared with control, Ica 100 and 200 groups (P < 0.01), and Ica 50 group (P < 0.05). Ovarian expression of AMH protein was elevated in the Ica 100 group compared with the aging, control and Ica 50 groups (P < 0.01) and Ica 200 group (P < 0.05). Ovarian Bcl-2 protein levels and the Bcl-2/Bax ratio were significantly higher in the Ica 100 group than those in the Ica 50, 200 and aging groups (P < 0.05), and were similar or reduced (P < 0.05), respectively, compared to those in control group. Ovarian Bax expression was similar in each group. These findings suggest that Ica can improve ovarian follicular development, inhibit follicular atresia, decrease FSH and LH levels and increase E, upregulate ovarian AMH expression and increase the Bcl-2/Bax ratio in aging mice. Therefore, Ica can partially restore ovarian function of aging mice and enhance their fertility. Optimal reproductive effects were obtained with the Ica 100 group.
在本研究中，在D-半乳糖（D-gal）诱导的小鼠卵巢衰老模型中检测了淫羊藿苷（Ica）的作用。昆明小白鼠分为三组：D-gal诱导的衰老组、低（50mg/kg）、中（100mg/kg）、高（200mg/kg）浓度Ica处理的实验组和未处理的对照组。比较各组的卵巢组织形态学、血清促卵泡生成素（FSH）、促黄体生成素（LH）和雌二醇（E）水平以及生殖功能。通过qPCR和蛋白质印迹法检测卵巢中抗缪勒管激素（Amh）、促凋亡蛋白（Bax）和抗凋亡蛋白（Bcl-2）的表达。我们的结果显示，与对照组相比，衰老组次级和三级卵泡直径显著减小（P<0.01），而在Ica 100和Ica 200处理组中恢复正常。与对照组和Ica 200处理组相比，衰老组闭锁卵泡直径显著更小（P<0.05）。与对照组、Ica 100和200处理组相比，衰老组次级和闭锁卵泡比例更高，而衰老组三级和成熟卵泡比例低于对照组、Ica 100和200组。衰老组缺乏成熟卵泡，而Ica处理可诱导成熟卵泡发育。在所有检测组中，初级和次级卵泡的卵泡膜细胞数量以及卵泡膜内层和外层细胞层数相似，而与对照组和Ica 200组相比，衰老组三级卵泡的卵泡膜内层和外层细胞层数分别减少和增加。在衰老组中，FSH和LH水平显著高于对照组和Ica 200组（P<0.05）；与对照组（P<0.01）、Ica （P<0.01）和Ica 100（P<0.05）组相比，E水平显著降低。对照组、Ica 100和Ica 200组血清激素水平相当。与其他组相比，衰老组妊娠率降低。与对照组、Ica 100和200组相比，衰老组每窝平均产仔数、出生窝重和断奶窝重均显著降低（P<0.05）。与对照组和Ica处理组相比，衰老组卵巢中AMH和Bcl-2 mRNA表达显著降低（P<0.01）。相反，与所有其他组相比，衰老组Bax表达显著更高（P<0.01），与对照组、Ica 100和200组相比，衰老组Bcl-2/Bax比值显著降低（P<0.01），与Ica 50组相比显著降低（P<0.05）。与衰老组、对照组和Ica 50组相比，Ica 100组卵巢中AMH蛋白表达升高（P<0.01），与Ica 200组相比显著升高（P<0.05）。与Ica 50、200和衰老组相比，Ica 100组卵巢Bcl-2蛋白水平和Bcl-2/Bax比值显著更高（P<0.05），与对照组相比分别相似或降低（P<0.05）。各组卵巢Bax表达相似。这些发现表明，Ica可改善衰老小鼠的卵巢卵泡发育，抑制卵泡闭锁，降低FSH和LH水平并增加E，上调卵巢AMH表达并增加Bcl-2/Bax比值。因此，Ica可部分恢复衰老小鼠的卵巢功能并提高其生育能力。Ica 100组获得了最佳生殖效果。
