Abstract：Oxidative stress mediates apoptosis of goose (Anser cygnoides) follicular granulosa cells (GCs), thus affecting laying performance. To investigate the effects of vitamin E (VE) on goose follicular GCs, an oxidative stress model was established by adding H2O2 (100 μmol/L), and the effects of oxidative stress on apoptosis in goose follicular granulosa cells were examined in vitro. Four different concentrations of proanthocyanidins, resveratrol, gallic acid and VE were added to the model, and the best reliever and optimum concentration were screened out. Furthermore, ELISA, qPCR and immunofluorescence detection techniques were used to explore the effect of the best alleviator VE on the apoptosis of goose follicle GCs mediated by oxidative stress. The results showed that 100 μmol/L H2O2 treatment for 12 h extremely significantly increased the level of apoptosis in follicular GCs (P<0.01). Among the 4 different antioxidants, 40 μmol/L VE had the best alleviation effect on the oxidative stress model (P<0.01). The addition of VE significantly inhibited the release of lactate dehydrogenase (LDH) (P<0.05), and reduced the expression of oxidative stress-related genes superoxide dismutase 1 (SOD-1), SOD-2, catalase (CAT), cyclooxygenase 2 (COX-2) and reactive oxygen species (ROS) content (P<0.05). Besides, GCs proliferation viability increased significantly (P<0.05) and there was a phenomenon of dose dependence. The expression levels of anti-apoptotic genes B-cell lymphoma/leukemia-2 (Bcl2) were significantly increased (P<0.05), but pro-apoptotic genes cysteinyl aspartate specific proteinase 3 (Caspase3), Caspase9 and tumor protein p53 (p53) were significantly decreased (P<0.05), as well as the number of apoptotic cells were significantly reduced after VE treatment (P<0.05). In conclusion, the goose follicle GCs had obvious apoptosis under oxidative stress. Adding VE could inhibit oxidative stress and relieve goose follicle GCs apoptosis. The study can provide reference for improving goose laying performance.
[1] 曹剑锋.2008. 几种抗氧化剂体外抗氧化活性的比较研究[D]. 硕士学位论文, 西北师范大学, 导师: 刘国安, pp.24-29. (Cao J F.2008. A comparative study on the anti-oxidant activities of several antioxidants in vitro[D]. Thesis for M.S., Northwest Normal University, Supervi-sor: Liu G A, pp. 24-29.) [2] 葛颖华, 钟晓明. 2007. 维生素 C 和维生素 E 抗氧化机制及其应用的研究进展[J]. 吉林医学, 28(5): 707-708. (Ge Y H, Zhong X M.2007. Research progress on antioxi-dant mechanism and application of vitamin C and vita-min E[J]. Jilin Medical Journal, 28(5): 707-708.) [3] 弓浩杰, 丁雪梅, 白世平, 等. 2022. 小鼠卵巢颗粒细胞氧化应激损伤及对沉默信息调节因子 2 相关酶 1 信号通路的影响[J]. 动物营养学报, 34(3): 1942-1954. (Gong H J, Ding X M, Bai S P, et al. 2022. Oxidative stress injury of mouse ovarian granulosa cells and its effects on si-lencing information regulator 2-related enzyme 1 signal-ing pathway[J]. Chinese Journal of Animal Nutrition, 34(3): 1942-1954.) [4] 郭小虎, 张宏馨, 陈辉, 等. 2013. 维生素 E 对蛋种鸡卵巢等级前颗粒细胞增殖的影响[J]. 中国家禽, 35(4): 10-13. (Guo X H, Zhang H X, Chen H, et al. 2013. Effect of vi-tamin E on proliferation of pre-ovarian pellet cells in egg breeders[J]. China Poultry, 35(4): 10-13.) [5] 韩飞, 周孟良. 2011. 过氧化氢诱导HepG2 细胞产生氧化应激细胞模型的建立[J]. 食品科学, 32(5): 55-57. (Han F, Zhou M L.2011. Establishment of a cell model for hy-drogen peroxide inducing oxidative stress in HepG2 cells[J]. Food Science, 32(5): 55-57.) [6] 李杨, 谢莉, 刘建, 等. 2019. 二仙汤对过氧化氢诱导的人卵巢颗粒细胞氧化损伤的保护作用[J]. 中国中西医结合杂志, 39(3): 317-322. (Li Y, Xie L, Liu J, et al. 2019. Protective effects of erxian decoction on oxidative inju-ry of human ovarian granulosa cells induced by H2O2[J]. Chinese Journal of Integrative Medicine, 39(3): 317-322.) [7] 零小妹, 张绪慧, 杨晶晶, 等. 2017. 哈蟆油通过激活 PI3K/ Akt/NF-κB 途径保护氧化应激损伤的大鼠卵巢颗粒细胞[J]. 天然产物研究与开发, 29(6): 941-946. (Ling X M, Zhang X H, Yang J J, et al. 2017. Protective effect of oviductus ranae on oxidative stress-induced injury in rat ovarian granulosa cells by activation of PI3K /Akt /NF- κB signal transduction pathway[J]. Research and Devel-opment of Natural Products, 29(6): 941-946.) [8] 刘红林, 孟繁星. 2019. 氧化应激对动物有腔卵泡闭锁的影响及机制[J]. 南京农业大学学报, 42(1): 6-13. (Liu H L, Meng F X.2019. Effect of oxidative stress on an-tral follicular atresia in animals and its mechanism[J]. Journal of Nanjing Agricultural University, 42(1): 6-13.) [9] 娄亚萍.2015. 鹅卵泡闭锁过程中氧化应激诱导颗粒细胞自噬的研究[D]. 硕士学位论文, 浙江农林大学, 导师: 赵阿勇, pp. 27-37. (Lou Y P.2015. Study on follicular granulosa cell autophagy induced by oxidative stress in goose[D]. Thesis for M. S., Zhejiang A&F University, Supervisor: Zhao A Y, pp. 27-37.) [10] 卢长柱, 焦润生, 孔亚坤, 等. 2008. 维生素 E 对老年雌性大鼠卵巢颗粒细胞形态及凋亡的影响[J]. 哈尔滨医科大学学报, 42(3): 231-234. (Lu C Z, Jiao R S, Kong Y K, et al. 2008. Effect of vitamin E on apoptosis and mor-phous of ovarialn granular cells in the senile female rats[J]. Journal of Harbin Medical University, 42(3): 231-234.) [11] 施振旦, 孙爱东, 郭彬彬, 等. 2022. 后疫情时期我国鹅产业高质量发展策略建议[J]. 中国禽业导刊, 39(9): 13-16. (Shi Z D, Sun A D, Guo B B, et al. 2022. Suggestions on high quality development strategy of Chinese goose industry in post-epidemic period[J]. Guide to Chinese poultry Industry, 39(9): 13-16.) [12] 孙岳丞, 张婧, 宋文涛, 等. 2018. 维生素 E 对动物机体损伤的保护作用研究进展[J]. 动物营养学报, 30(1): 44-49. (Sun Y S, Zhang J, Song W T, et al. 2018. Research progress on the protective effect of vitamin E on animal body damage[J]. Journal of Animal Nutrition, 30(1): 64-69.) [13] 王建萍, 张克英. 2021. 氧化应激对家禽卵泡闭锁的影响及机制[J]. 动物营养学报, 33(6): 3001-3009. (Wang J P, Zhang K Y.2021. Effect and mechanism of oxidative stress on follicular atresia in poultry[J]. Journal of Ani-mal Nutrition, 33(6): 3001-3009.) [14] 张宏馨.2013. 维生素 E 对种母鸡繁殖性能的影响及其机理研究[D]. 博士学位论文, 河北农业大学, 导师: 黄仁录, pp. 4-5. (Zhang H X.2013. Study on the Influence of vi-tamin E on the reproductive performance and it's mecha-nism in breeding hens[D]. Thesis for M. S., Hebei Agri-cultural University, Supervisor: Huang R L, pp. 4-5.) [15] 张家庆.2013. 小鼠卵巢氧化应激模型建立以及卵巢保护性抗氧化剂的筛选[D]. 博士学位论文, 南京农业大学, 导师: 李奎, 刘红林, pp. 23-30. (Zhang J Q.2013. Es-tablishment of ovarian oxidative stress model and screening of ovarian protective antioxidants in mice[D]. Thesis for M. S., Nanjing Agricultural University, Super-visor: Li K, Liu H L, pp. 23-30.) [16] 邹坤, 路丽丽, Amponsah C. A., 等. 2020. 家禽卵泡闭锁机制的研究进展[J].生物技术通报 36(4): 185-191. (Zou K, Lu L L, C. A. Amponsah, et al. 2020. Research prog-ress on mechanism of poultry follicular atresia[J]. Bio-technology Bulletin, 36(4): 185-191.) [17] Chen Z G, Luo L L, Xu J J, et al. 2010. Effects of plant poly-phenols on ovarian follicular reserve in aging rats[J]. Biochemistry and Cell Biology-Biochimie et Biologie Cellulaire, 88(4): 737-745. [18] Freitas C, Neto A C, Matos L, et al. 2017. Follicular fluid re-dox involvement for ovarian follicle growth[J]. Journal of Ovarian Research, 10(1): 44-54. [19] Garrido C, Galluzzi L, Brunet M, et al. 2006. Mechanisms of cytochrome c release from mitochondria[J]. Cell Death and Differentiation, 13(9): 1423-1433. [20] Hu Y, Rosen D G, Zhou Y, et al. 2005. Mitochondrial manga-nese-superoxide dismutase expression in ovarian can-cer: Role in cell proliferation and response to oxidative stress[J]. The Journal of Biological Chemistry, 280(47):39485-39492. [21] Matos L, Stevenson D, Gomes F, et al. 2009. Superoxide dis-mutase expression in human cumulus oophorus cells[J]. Molecular Human Reproduction, 15(7): 411-419. [22] Özcan P, Fıçıcıoğlu C, Yıldırım ÖK, et al. 2015. Protective ef-fect of resveratrol against oxidative damage to ovarian reserve in female sprague-dawley rats[J]. Reproductive Biomedicine Online, 31(3): 404-410. [23] Schieber M, Chandel N S.2014. ROS function in redox sig-naling and oxidative stress[J]. Current Biology, 24(10): R453-462. [24] Shen M, Cao Y, Jiang Y, et al. 2018. Melatonin protects mouse granulosa cells against oxidative damage by in-hibiting Foxo1-mediated autophagy: Implication of an antioxidation-independent mechanism[J]. Redox Biolo-gy, 18(1): 138-157. [25] Tao Y, Zhou B, Xia G, et al. 2004. Exposure to L-ascorbic ac-id or alpha-tocopherol facilitates the development of porcine denuded oocytes from metaphase I to meta-phase Ⅱ and prevents cumulus cells from fragmentation[J]. Reproduction in Domestic Animals-Zuchthygiene,39(1): 52-57. [26] Zhang J, Ren Q, Chen J, et al. 2021. Autophagy contributes to oxidative stress-Induced apoptosis in porcine granulosa cells[J]. Reproduction Science, 28(8): 2147-2160.
