二甲双胍对种鸽卵巢衰老的缓解作用研究

doi:10.16431/j.cnki.1671-7236.2025.11.044

摘要/Abstract

摘要： 【目的】本研究旨在探讨二甲双胍对种鸽卵巢衰老的缓解作用。【方法】①选用40对6年龄种鸽，随机分为2组，分别饲喂含0(对照组)和62.5 mg/kg二甲双胍的饮用水，试验期为40 d。试验结束后采集各组种鸽血液进行血清抗氧化能力指标测定，采集卵巢组织检测糖酵解相关基因表达水平，同时通过 Western blotting检测葡萄糖转运蛋白 1(glucose transporter 1，GLUT1)、己糖激酶2(hexokinase 2，HK2)、乳酸脱氢酶(lactate dehydrogenase，LDH)表达量及AMPK和AKT磷酸化水平。②选用3年龄种鸽，屠宰后采集小白卵泡并洗净。试验分为4组：对照组、H₂O₂组、二甲双胍+H₂O₂组和二甲双胍组，培养72 h后收集小白卵泡免疫荧光染色检测LDHA、HK2和GLUT1蛋白定位；Western blotting试验，检测磷酸化AMPK、磷酸化AKT、B细胞淋巴瘤/白血病-2(B-cell lymphoma/leukemia-2，Bcl2)、Bcl-2 相关X蛋白(Bcl-2 associated X protein，BAX)和增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)蛋白含量；实时荧光定量PCR检测PFKP、ENO1、LDH、ALDOB和GLUT1等基因表达量。③建立卵泡颗粒细胞培养模型，试验共分为4组：对照组、H₂O₂组、二甲双胍+H₂O₂组和二甲双胍组，培养72 h后收集颗粒细胞进行EdU染色。【结果】①体内试验结果显示，与对照组相比，饲喂二甲双胍种鸽产蛋量和卵巢重显著提高(P＜0.05)。实时荧光定量PCR结果显示，饲喂二甲双胍显著提高了种鸽卵巢糖酵解相关基因PFKP、PFKL、ENO1、LDHA、LDHC、LDHB、ALDOB和GLUT1 mRNA表达量和抗氧化能力(P＜0.05)。②H₂O₂诱导体外卵泡衰老模型免疫荧光染色结果显示，LDHA、HK2和GLUT1蛋白主要定位于卵泡颗粒层。Western blotting结果显示，与对照组相比，H₂O₂组颗粒层中LDHA、HK2、GLUT1、p-AKT/AKT和PCNA表达量显著下调(P＜0.05)，Bax∶Bcl2值显著上调(P＜0.05)。与H₂O₂组相比，二甲双胍+H₂O₂组颗粒层中LDHA、HK2、GLUT1、p-AKT/AKT和PCNA的表达量显著上调(P＜0.05)，Bax∶Bcl2值显著下调(P＜0.05)。实时荧光定量PCR结果显示，与对照组相比，H₂O₂组PFKP、ENO1、LDH、ALDOB和GLUT1等基因mRNA表达量显著下调(P＜0.05)；与H₂O₂组相比，二甲双胍+H₂O₂组PFKP、ENO1、LDH、ALDOB和GLUT1等基因mRNA表达量显著上调(P＜0.05)。③在卵泡颗粒细胞培养模型中，EdU结果显示，与对照组相比，H₂O₂组颗粒细胞增殖减少。与H₂O₂组相比，二甲双胍+H₂O₂组颗粒细胞增殖增加。【结论】二甲双胍能通过激活AMPK/Akt信号通路，提高卵泡颗粒细胞糖酵解水平，缓解种鸽卵巢衰老，提高产蛋性能，进而提高种鸽的经济效益。

关键词: 二甲双胍; 种鸽; 糖酵解; AMPK/Akt信号通路; 卵巢

Abstract: 【Objective】 This study aimed to explore the alleviating effect of metformin on ovarian aging in breeding pigeons. 【Method】 ①Forty pairs of 6-year-old breeding pigeons were randomly divided into two groups.The breeding pigeons were provided drinking water containing either 0 (control group) or 62.5 mg/kg metformin for a 40-day feeding trial.After the experiment was completed,blood was collected to measure serum antioxidant capacity indicators,and ovarian tissues were harvested to assess glycolysis-related gene expression levels.Western blotting analysis was performed to detect the expression of glucose transporter 1 (GLUT1),hexokinase 2 (HK2) and lactate dehydrogenase (LDH),and phosphorylation levels of AMP-activated protein kinase (AMPK) and protein kinase B (AKT).② Small white follicles (SWFs) were collected from 3-year-old breeding pigeons post-slaughter and rinsed.The experiment was divided into four groups:Control group,H₂O₂ group,metformin＋H₂O₂ group and metformin group.After 72 h of culture,SWFs were collected for Western blotting to evaluate the expression levels of LDHA,HK2,GLUT1,p-AKT/AKT,pAMPK/AMPK,Bcl2,BAX and PCNA.The localization of LDHA,HK2 and GLUT1 were evaluated by immunofluorescence staining.Real-time quantitative PCR revealed elevated mRNA expression of glycolysis-related genes PFKP,ENO1,LDH,ALDOB, GLUT1, and so on.③A granulosa cell culture model was established,with the experiment divided into four groups:Control group,H₂O₂ group,metformin＋H₂O₂ group and metformin group.After 72 h of culture, granulosa cells were collected for EdU staining. 【Result】 ①In vivo test results showed that compared with control group,metformin supplementation significantly improved egg production quantity and ovarian weight (P＜0.05).Real-time quantitative PCR revealed mRNA expression of glycolysis-related genes PFKP,PFKL,ENO1,LDHA,LDHC,LDHB,ALDOB and GLUT1 and enhanced antioxidant capacity (P＜0.05).②Immunofluorescence of in vitro follicular aging model showed that LDHA,HK2 and GLUT1 were predominantly localized in the granulosa layer.Western blotting results showed that,compared with control group,H₂O₂ treatment downregulated LDHA,HK2,GLUT1,p-AKT/AKT and PCNA levels (P＜0.05),while increased Bax∶Bcl2 ratio (P＜0.05).Metformin＋H₂O₂ reversed these effects (P＜0.05).Real-time quantitative PCR results showed that compared with control group,the mRNA expression levels of genes PFKP,ENO1, LDH,ALDOB, GLUT1, and so on were significantly downregulated in H₂O₂ group (P＜0.05),Metformin＋H₂O₂ reversed these effects (P＜0.05).③In the granulosa cell model,EdU results showed that H₂O₂ treatment reduced cell proliferation compared with control group,while metformin＋H₂O₂ treatment increased proliferation. 【Conclusion】 Metformin could alleviate ovarian aging in breeding pigeons by activating the AMPK/Akt signaling pathway,enhancing glycolysis in granulosa cells,and improving egg-laying performance,thereby increasing economic benefits in poultry farming.

Key words: metformin; breeding pigeon; glycolysis; AMPK/Akt signaling pathway; ovary

中图分类号:

S836
Q41

赵星晨, 高求炜, 梁雅妍, 高铭, 王玮, 周烁. 二甲双胍对种鸽卵巢衰老的缓解作用研究[J]. 中国畜牧兽医, 2025, 52(11): 5499-5509.

ZHAO Xingchen, GAO Qiuwei, LIANG Yayan, GAO Ming, WANG Wei, ZHOU Shuo. Study on the Alleviating Effect of Metformin on Ovarian Aging in Breeding Pigeons[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(11): 5499-5509.

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