连翘酯苷A通过HIF-1α调控牦牛卵丘细胞雌激素合成与分泌

doi:10.16431/j.cnki.1671-7236.2025.09.019

摘要/Abstract

摘要： 【目的】以健康成年(5～8岁)雌牦牛卵丘细胞为研究对象，探究连翘酯苷A(forsythiaside A，FA)对卵丘细胞雌激素合成酶的影响及作用机制，为FA应用于牦牛繁殖管理提供科学依据。【方法】取对数生长期牦牛卵丘细胞，通过设置浓度梯度(0、5、10、20、30 和40 μmol/L)和时间梯度(12、24、36和48 h)进行细胞毒性试验，筛选FA最佳作用条件。采用实时荧光定量PCR和Western blotting技术检测FA对卵丘细胞低氧诱导因子1α(hypoxia-inducible factor 1α，HIF-1α)、雌激素合成酶(CYP11A1、CYP17A1和CYP19A1)基因及蛋白表达的影响。通过HIF-1α特异性抑制剂LW6干扰HIF-1α的合成，将卵母细胞分为对照组、FA组及FA+LW6组，体外培养12 h后检测卵丘细胞中HIF-1α、CYP11A1、CYP17A1和CYP19A1基因及蛋白的表达量，并检测细胞上清液中雌二醇浓度。【结果】细胞毒性试验结果显示，与0 μmol/L FA处理组相比，5 μmol/L FA孵育牦牛卵丘细胞12 h后细胞活性提升最为显著(P＜0.05)，且对HIF-1α、CYP11A1、CYP17A1和CYP19A1基因和蛋白表达量具有显著促进作用(P＜0.05)。LW6抑制试验结果显示，与对照组相比，FA组牦牛卵丘细胞中HIF-1α、CYP11A1、CYP17A1和CYP19A1基因和蛋白(除CYP19A1外)相对表达量均显著升高(P＜0.05)，HIF-1α荧光强度明显增强，雌二醇浓度显著升高(P＜0.05)；FA+LW6组牦牛卵丘细胞中HIF-1α、CYP11A1、CYP17A1和CYP19A1基因和蛋白相对表达量均显著降低(P＜0.05)，雌二醇浓度显著降低(P＜0.05)。【结论】 FA可通过HIF-1α的表达来调控雌激素合成酶的合成，从而促进牦牛卵丘细胞雌激素分泌。本研究结果为FA作为天然生殖调控剂改善牦牛繁殖性能提供了试验依据。

关键词: 牦牛; 卵丘细胞; 连翘酯苷A(FA); 低氧诱导因子-1α(HIF-1α); 雌激素

Abstract: 【Objective】 This study was aimed to investigate the effects of forsythiaside A (FA) on estrogen synthase in cumulus cells of healthy adult female yak (5-8 years old) and explore its mechanism,so as to provide a scientific basis for FA application in yak reproductive management. 【Method】 Yak cumulus cells during the logarithmic growth phase were used for cytotoxicity assays with concentration gradients (0,5,10,20,30,and 40 μmol/L) and time gradients (12,24,36,and 48 h) to determine optimal FA treatment conditions.Real-time quantitative PCR and Western blotting were performed to analyze the expression of hypoxia-inducible factor 1α (HIF-1α),estrogen synthetase (CYP11A1,CYP17A1,and CYP19A1) genes and proteins.HIF-1α inhibitor LW6 was used to block HIF-1α synthesis,with cells divided into control group,FA group,and FA+LW6 group,after 12 h of in vitro culture，the expression of HIF-1α,CYP11A1,CYP17A1,and CYP19A1 genes and proteins in cumulus cells and estradiol concentration in supernatants of yaks were detected. 【Result】 Cytotoxicity assays showed that compared with 0 μmol/L FA group,5 μmol/L FA incubation for 12 h significantly increased cell viability and upregulated the expression of HIF-1α,CYP11A1,CYP17A1,and CYP19A1 genes and proteins in yak cumulus cells (P＜0.05).LW6 inhibition experiment revealed that compared with control group,FA treatment significantly increased the relative expression of HIF-1α,CYP11A1,CYP17A1,and CYP19A1 genes and proteins (except for CYP19A1) in yak cumulus cells (P＜0.05),the fluorescence intensity of HIF-1α was significantly enhanced,and the estradiol concentration was significantly increased (P＜0.05).FA+LW6 treatment significantly decreased the expression of all four genes and proteins in yak cumulus cells (P＜0.05),with significantly decreased estradiol concentration (P＜0.05). 【Conclusion】 FA could regulate the synthesis of estrogen synthase through the expression of HIF-1α gene,thereby promote estrogen secretion in yak cumulus cells.The results provided experimental evidence for FA as a natural reproductive regulator to improve yak breeding performance.

Key words: yak; cumuluscells; forsythiaside A (FA); hypoxia-induciblefactor-1α (HIF-1α); estrogen

中图分类号:

S814.1

杨娟, 潘阳阳, 王萌, 杨晓晴, 李天昊, 马鑫, 张小坤, 常剑英, 王立斌, 樊江峰. 连翘酯苷A通过HIF-1α调控牦牛卵丘细胞雌激素合成与分泌[J]. 中国畜牧兽医, 2025, 52(9): 4205-4215.

YANG Juan, PAN Yangyang, WANG Meng, YANG Xiaoqing, LI Tianhao, MA Xin, ZHANG Xiaokun, CHANG Jianying, WANG Libin, FAN Jiangfeng. Effect of Forsythiaside A on Estrogen Synthase Secretion of Yak Cumulus Cells via HIF-1α[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(9): 4205-4215.

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