关键词: 益经汤, 生殖干细胞, 卵巢储备功能, 骨形态发生蛋白, 抗DPP同源物, 果蝇

Abstract:

Objective To investigate the ameliorative effect of Yijing Decoction （YJD） on ovarian reserve function in the naturally aged female Drosophila， and to elucidate its potential mechanism. Methods A total of 1 920 female Drosophila melanogaster were randomly divided into young group， older group， low dose of YJD （0.25 g·L^-1） group， medium dose of YJD （0.50 g·L^-1） group， high dose of YJD （1.00 g·L^-1） group， and positive drug 20-hydroxyecdysone （20E） group， with 320 Drosophila in each group. A hundred female Drosophila were selected from each group and male Drosophila were matched to each group at a ratio of 1∶2 （male∶female） and co-cultured for 7 d. Then the female Drosophila were collected and allowed to lay eggs alone for 48 h. The numbers of eggs laid and pupae formed were counted， and the pupation rates were calculated. Ovaries of the Drosophila in various groups were dissected under a stereomicroscope， and the lengths and diameters were measured to evaluate the morphology of the ovaries. Enzyme-linked immunosorbent assay （ELISA） method was used to detect the levels of 20E and juvenile hormone （JH） in ovarian tissues of the Drosophila in various groups. Immunofluorescence staining method was used to observe the numbers of germline stem cells （GSCs） in ovarian tissues of the Drosophila in various groups. Western blotting method was used to detect the expression levels of proteins related to the bone morphogenetic protein （BMP）/mothers against decapentaplegic homolog （Smad） signaling pathway， including Bam， Nos， Dpp， Smad4， Smad1/5/9， and phosphorylated Smad1/5/9 （p-Smad1/5/9）， in the ovarian tissue of the Drosophila in various groups. Results Compared with young group， the numbers of eggs laid and pupae formed as well as the pupation rate in older group were significantly decreased （P<0.01）， the ovarian morphology was atrophic and the ovarian length and diameter were significantly decreased （P<0.01）， the levels of 20E and JH in ovarian tissue of the Drosophila were significantly decreased （P<0.01）， and the number of GSCs in ovarian tissue of the Drosophila was significantly decreased （P<0.01）. Compared with older group， the numbers of eggs laid and pupae formed as well as the pupation rates in low dose of YJD group， medium dose of YJD group， high dose of YJD group， and 20E group were significantly increased （P<0.05 or P<0.01）， the ovarian recovered to full and normal morphology， the ovarian lengths and diameters of the Drosophila were significantly increased （P<0.05 or P<0.01）， the levels of 20E and JH in the ovarian tissue of the Drosophila were significantly increased （P<0.05 or P<0.01）； the numbers of GSCs in the ovarian tissue of the Drosophila in medium dose of YJD group， high dose of YJD group， and 20E group were significantly increased （P<0.01）. Compared with young group， the expression levels of Bam， Nos， Dpp， Smad4， and p-Smad1/5/9 proteins in the ovarian tissue of the Drosophila were significantly decreased in older group （P<0.01）. Compared with older group， the expression levels of Bam， Nos， Dpp， Smad4， and p-Smad1/5/9 proteins in the ovarian tissue of the Drosophila in low dose of YJD group， medium dose of YJD group， high dose of YJD group， and 20E groupwere significantly increased （P<0.05 or P<0.01）. Conclusion YJD ameliorates ovarian reserve function in older female Drosophila， enhances their reproductive capacity， restores ovarian morphology， up-regulates ovarian hormone levels， and increases the number of GSCs. Its mechanism is associated with regulating the BMP/Smad signaling pathway to promote GSC regeneration.

Key words: Yijing Decoction, Germline stem cells, Ovarian reserve function, Bone morphogenetic protein, Mothers against decapentaplegic homolog, Drosophila melanogaster