Abstract: 【Objective】Follicle development is a complex regulatory process, in which the proliferation and differentiation of ovine follicular granulosa cells (Granulosa Cells, GCs) will directly affect the development of follicles.TGF-β signaling pathway plays an important role in sheep ovarian development and follicle growth, and Smad7 also plays an important role as a key inhibitor of TGF-β signaling pathway. In this study, the effect of Smad7-mediated TGF-β signaling pathway on the proliferation and apoptosis of ovine follicular GCs was explored providing a basis for further research on the regulatory role of Smad7 in the process of follicular GCs proliferation and apoptosis. 【Method】20 non-pregnant Jinzhong Duhu hybrid ewes aged 4-6 months were selected. After slaughtering, bilateral ovaries were collected, and GCs were isolated and cultured in vitro; FSHR cell immunofluorescence identification; Expression localization of Smad7 cells by immunofluorescence, the cell proliferation was measured by CCK8 method, and the growth curve was drawn; After the cells were passaged, different concentrations of Smad7 agonist Asiaticoside AS (0, 100, 200, 400, 600 ng/mL) were added exogenously, and the optimal concentration of AS was selected to treat the second generation GCs, using qRT-PCR and Western blotting to detect the expression levels of key genes in the TGF-β signaling pathway, as well as apoptosis and cycle-related genes; Three siSmad7s were synthesized and transfected into follicular GCs, and the one with the best interference effect was selected. The expression levels of key genes in the TGF-β signaling pathway, apoptosis and cycle-related genes were detected by qRT-PCR and Western blotting. 【Result】The results showed that Smad7 was expressed in ovine follicular GCs; 200 ng/mL AS could significantly up-regulate the expression of Smad7 in GCs (P<0.01), siSmad7-1 could effectively tranfected ovine follicle GCs (P<0.01); Up-regulation of Smad7 could significantly increase the expressions of Smad4, TFDP-1 and EP300 in the TGF-β signaling pathway (P<0.05), extremely significantly increase the expression of SP1 in the TGF-β signaling pathway (P<0.01), and significantly increase the expression of apoptosis-related gene Caspase8 （P<0.05）, extremely significantly increased the expressions of apoptosis-related genes Caspase3 and Bim (P<0.01), significantly increased the expressions of cell cycle-related genes P21 and P27 (P<0.05), extremely significantly increased the expression of cell cycle-related gene CCND2 (P<0.01), while the expression of CCND1 was significantly decreased (P<0.05); Smad7 siRNA significantly decreased the expressions of Smad4, TFDP-1 and EP300 in the TGF-β signaling pathway (P<0.05), extremely significantly decreased the expression of SP1 in the TGF-β signaling pathway (P<0.01), and significantly decreased the apoptosis-related gene Caspase8 （P<0.05）, extremely significantly decreased the expressions of apoptosis-related genes Caspase3 and Bim (P<0.01), extremely significantly decreased the expressions of cell cycle-related genes P21, P27 and CCND2 (P<0.01), extremely significantly increased the expression of CCND1 (P<0.01); CCK8 analysis demonstrated into that when Smad7 was up-regulated, the proliferation rate of GCs was significantly down-regulated at 24 h and 48 h (P<0.01). Smad7 siRNA significantly increased the proliferation rate of GCs at 24 h and 72 h (P<0.05). extremely significantly increased the proliferation rate of GCs (P<0.01).【Conclusion】In summary, we suggest that Smad7 mediates TGF-β signaling pathway to inhibit the proliferation of ovine follicular GCs and promote cell apoptosis.

Key words: AS, siRNA, Smad7, TGF-β signaling pathway, sheep, granulosa cells