Effects of Naringenin on Proliferation, Apoptosis and Estradiol Synthesis of Granulosa cells in Tibetan Sheep (Ovis aries)
LIN Qian1, WANG Li-Bin1,2,*, ZHAO Bo1, WANG Meng1,2, PAN Yang-Yang1,2, JIAO Zheng-Xing1, XU Rui-Hua1, ZHANG Hui1, YU Si-Jiu1,2
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 2 Technology and Research Center of Gansu Province for Embryonic Engineering of Livestock, Lanzhou 730070, China
Abstract：As the main economic animal in the Qinghai-Tibet plateau of China, the development of Tibetan sheep (Ovis aries) population is limited due to its weak reproductive ability, low conception rate and low pregnancy rate. Therefore, it is of great significance to study the problems related to Tibetan sheep reproduction. This study was designed to investigate the effects of naringenin (NAR) on proliferation, apoptosis and estradiol (E2) synthesis of granulosa cells (GCs) in Tibetan sheep. The primary GCs of Tibetan sheep were collected and cultured in vitro to logarithmic growth phase, different concentrations of NAR (0, 5, 10, 15 and 20 μmol/L) were added to culture GCs for 12 h. qPCR and Western blot were used to detect the expressions of genes and proteins related to proliferation (PCNA, CCND1), apoptosis (Bcl-2, BAX, Caspase-3) and E2 synthesis (CYP19A1, CYP17A1, CYP11A1, STAR). The proliferation of GCs was detected by CCK-8, and the apoptosis of GCs was detected by immunofluorescence. The concentration of E2 in the culture medium was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that adding 10 μmol/L of NAR significantly up-regulated the expression of CCND1 mRNA (P<0.05). And the expressions of proliferation-related proteins (PCNA, CCND1) in NAR treatment groups were significantly higher than those of the control group (P<0.05). The expressions of genes and proteins of BAX and Caspase3 in the treatment groups decreased significantly (P<0.05). The expression levels of E2 synthesis-related genes and proteins (CYP11A1, CYP17A1 and CYP19A1) in the 10 μmol/L NAR treatment group rose significantly (P<0.05), while those of STAR mRNA and protein decreased significantly (P<0.05). In summary, NAR of 10 μmol/L promoted the proliferation of GCs and inhibited their apoptosis by up-regulating PCNA, CCND1, Bcl-2 mRNA and down-regulating BAX and Caspase3 protein expression; E2 was synthesized and secreted by up-regulating CYP11A1, CYP17A1, CYP19A1 and down-regulating STAR mRNA and proteins. This study provides a foundation for further study on the effects of NAR on functions of mammalian ovaries and improvement the animal reproductive potential.
[1] 段香茹, 康佳, 杨若晨, 等. 2024. L-半胱氨酸对绵羊卵巢颗粒细胞增殖、凋亡和类固醇激素分泌的影响[J]. 畜牧兽医学报, 55(01): 179-191. (Duan X R, Kang J, Yang R C, et al.2024. Effects of L-cysteine on proliferation, apoptosis and steroid hormone secretion of ovine ovarian granulosa cells[J]. Journal of Veterinary Science and Animal Industry, 55(01): 179-191.) [2] 皇甫奕雯, 宋莉娜, 侯丽娥, 等. 2023. 维生素E通过抑制氧化应激缓解鹅卵泡颗粒细胞凋亡[J]. 农业生物技术学报, 31(08): 1659-1670. (HuangFu Y W, Song L N, Hou L E, et al.2023. Vitamin E relieves the apoptosis of goose (Anser cygnoides) granulosa cells by inhibiting oxidative stress[J]. Journal of Agricultural Biotechnology, 31(08): 1659-1670.) [3] 张配颖, 宋鹏琰, 周荣艳, 等. 2023. 转录因子ID1对绵羊卵泡颗粒细胞增殖和凋亡的影响[J].农业生物技术学报, 31(08): 1641-1650. (Zhang P Y, Song P Y, Zhou R Y, et al.2023. Effects of transcription factor ID1 on proliferation and apoptosis of sheep (Ovis aries) follicular granulosa cells[J]. Journal of Agricultural Biotechnology, 31(08): 1641-1650.) [4] Arafah A, Rehman M U, Mir T M, et al.2020. Multi-therapeutic potential of naringenin (4',5,7-trihydroxyflavonone): Experimental evidence and mechanisms[J]. Plants (Basel, Switzerland), 9(12): 1784. [5] Gudipaty S A, Conner C M, Rosenblatt J, et al.2018. Unconventional ways to live and die: Cell death and survival in development, homeostasis, and disease[J]. Annual Review of Cell and Developmental Biology, 34: 311-332. [6] He H, Su X, Yang H, et al.2024. Effects of prolactin on the proliferation and hormone secretion of ovine granulosa cells in vitro[J]. Animal Bioscience, 37(10): 1712-1725. [7] Li H, Liu, L Cao Z, et al.2021a. Naringenin ameliorates homocysteine induced endothelial damage via the AMPKα/Sirt1 pathway[J]. Journal of Advanced Research, 34: 137-147. [8] Li L, Zhang J, Lu C, et al.2022a. MicroRNA-7a2 contributes to estrogen synthesis and is modulated by FSH via the JNK signaling pathway in ovarian granulosa cells[J]. International Journal of Molecular Sciences, 23(15): 8565. [9] Li W, Zeng W, Jin X, et al.2022b. High-altitude stress orchestrates mRNA expression and alternative splicing of ovarian follicle development genes in tibetan sheep[J]. Animals (Basel), 12(20): 2812. [10] Li X, Chen H, Zhang Z, et al.2021b. Isorhamnetin promotes estrogen biosynthesis and proliferation in porcine granulosa cells via the PI3K/Akt signaling pathway[J]. Journal of Agricultural and Food Chemistry, 69(23): 6535-6542. [11] Lossi L, 2022. The concept of intrinsic versus extrinsic apoptosis[J]. The Biochemical Journal, 479(3): 357-384. [12] Man Y, Zhao R, Gao X, et al.2020. TOX3 Promotes ovarian estrogen synthesis: An RNA-sequencing and network study[J]. Frontiers in Endocrinology, 11: 615846. [13] Manabe H, 2004. Evaluations of the ring shaped EMG measurement system[C]. The 26th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, San Francisco, CA, USA, pp. 2188-2191. [14] Nikhil Kumar Tej J, Johnson P, Krishna K, et al.2021. Copper and selenium stimulates CYP19A1 expression in caprine ovarian granulosa cells: Possible involvement of AKT and WNT signalling pathways[J]. Molecular Biology Reports, 48(4): 3515-3527. [15] Rytelewska E, Kisielewska K, Gudelsk M, et al.2020. The effect of orexin a on the StAR, CYP11A1 and HSD3B1 gene expression, as well as progesterone and androstenedione secretion in the porcine uterus during early pregnancy and the oestrous cycle[J]. Theriogenology, 143: 179-190. [16] Solak K A, Wijnolts F M J, Nijmeijer S M, et al.2014. Excessive levels of diverse phytoestrogens can modulate steroidogenesis and cell migration of KGN human granulosa-derived tumor cells[J]. Toxicology Reports, 1: 360-372. [17] Stringer J M, Alesi L R, Winship A L, et al.2023. Beyond apoptosis: Evidence of other regulated cell death pathways in the ovary throughout development and life[J]. Human Reproduction Update, 29(4): 434-456. [18] Wang J J, Ge W, Liu J Cet al.2017. Complete in vitro oogenesis: Retrospects and prospects[J]. Cell Death and Differentiation, 24(11): 184-1852. [19] Wang Q, Ou Y, Hu G, et al.2020. Naringenin attenuates non-alcoholic fatty liver disease by down-regulating the NLRP3/NF-κB pathway in mice[J]. British Journal of Pharmacology, 177(8): 1806-1821. [20] Wang T, Lei H, Zhou L, et al.2022. Effect of fumonisin B1 on proliferation and apoptosis of intestinal porcine epithelial cells[J]. Toxins, 14(7): 471. [21] Xiang C, Zhang Y, Zhang Y, et al.2020. lncRNA LEF1-AS1 promotes proliferation and induces apoptosis of Non-Small-cell lung cancer cells by regulating miR-221/PTEN signaling[J]. Cancer Management and Research, 12: 3845-3850. [22] Zhang B, Wan S, Liu H, et al.2022. Naringenin alleviates renal ischemia reperfusion injury by suppressing ER stress-induced pyroptosis and apoptosis through activating Nrf2/HO-1 signaling pathway[J]. Oxidative Medicine and Cellular Longevity, 2022: 5992436. [23] Zhang C H, Liu X Y, Wang J, 2023. Essential role of granulosa cell glucose and lipid metabolism on oocytes and the potential metabolic imbalance in polycystic ovary syndrome[J]. International Journal of Molecular Sciences, 24(22): 16247. [24] Zhao J, Xu Y, Yu H, et al.2025. Effects of PPARG on the proliferation, apoptosis, and estrogen secretion in goat granulosa cells[J]. Theriogenology, 231: 62-72.
