基于AMPK/PGC-1α信号通路探讨乌萸汤对初老小鼠卵母细胞的作用机制

Discussion on the mechanism of Wuyu decoction on oocytes of primary senile mice based on AMPK/PGC-1αsignaling pathway

目的基于磷酸腺苷活化蛋白激酶(AMPK)/过氧化物酶体增殖活化受体γ共激活因子1α(PGC-1α)信号通路探讨乌萸汤对初老小鼠卵母细胞的作用机制。方法采用雄性6周龄SD大鼠制备乌萸汤含药血清;采用未成熟卵体外培养技术,原代细胞培养6周龄、24周龄小鼠卵母细胞后提取卵丘卵母细胞复合体(COC)。实验分为8组:对照组(6周龄小鼠COC培养于基础培养基)、初老组(24周龄小鼠COC培养于基础培养基)、初老+AICAR组(AMPK激动剂AICAR预处理24周龄小鼠COC 2 h)、初老+Compound C组(AMPK抑制剂Compound C预处理24周龄小鼠COC 2 h)、初老+乌萸汤含药血清组(24周龄小鼠COC培养于乌萸汤含药血清培养基)、初老+乌萸汤含药血清组+AICAR组(AMPK激动剂AICAR预处理24周龄小鼠COC 2 h后培养于乌萸汤含药血清培养基)、初老+乌萸汤含药血清组+Compound C组(AMPK抑制剂Compound C预处理24周龄小鼠COC 2 h后培养于乌萸汤含药血清培养基)、初老+山茱萸环烯醚萜苷类提取物组(24周龄小鼠COC培养于山茱萸环烯醚萜苷类提取物培养基)。采用PCR法及Western blot法检测各组卵母细胞中AMPK/PCG-1α通路相关因子基因及蛋白表达情况。结果初老+AICAR组、初老+乌萸汤含药血清组卵母细胞中AMPKα、PGC-1α、Nrf1基因相对表达量及AMPKα、p-AMPKα(Thr172)、PGC-1α、Nrf1蛋白相对表达量和初老+乌萸汤含药血清+AICAR组卵母细胞中AMPKα、PGC-1α、Nrf1基因相对表达量及AMPKα、p-AMPKα(Thr172)、PGC-1α蛋白相对表达量均明显高于初老组(P均<0.05);初老+AICAR组、初老+乌萸汤含药血清组、初老+乌萸汤含药血清+AICAR组卵母细胞中Nrf2基因相对表达量及初老+Compound C组卵母细胞中AMPKα、PGC-1α、Nrf1、Nrf2基因相对表达量均明显低于初老组(P均<0.05);初老+乌萸汤含药血清+Compound C组卵母细胞中AMPKα、PGC-1α、Nrf1、Nrf2基因相对表达量及p-AMPKα(Thr172)、PGC-1α、Nrf1蛋白相对表达量均明显低于初老+乌萸汤含药血清组(P均<0.05)。结论初老小鼠未成熟卵体外培养过程中,AMPK/PGC-1α通路可通过线粒体能量代谢从而影响卵母细胞质量;乌萸汤可通过AMPK/PGC-1α通路增强线粒体能量代谢及氧化应激状态,促进卵母细胞成熟,从而改善卵母细胞功能。

Objective It is to explore the mechanism of Wuyu decoction on oocytes of primary senile mice based on adenosine phosphate activated protein kinases(AMPK)/peroxisome proliferator-activated receptor gamma coactivator 1 alpha(PGC-1α)signaling pathway.Methods Male 6-week-old SD rats were used to prepare Wuyu Decoction-containing serum;the cumulus-oocyte complexes(COC)were extracted after the primary cells were cultured from 6-week-old and 24-week-old mice by in vitro immaturation.The experiment was divided into 8 groups:control group(6-week-old mice COC cultured in basal medium),early-aged group(24-week-old mice COC cultured in basal medium),early-aged+AICAR group(24-week-old mice COC pretreated with AMPK agonist AICAR for 2 h),early-aged+Compound C group(24-week-old mice COC pretreated with AMPK inhibitor Compound C for 2h),early-aged+Wuyu decoction-containing serum group(24-week-old mice COC cultured in Wuyu decoction-containing serum medium),early-aged+Wuyu decoction-containing serum group+AICAR group(24-week-old mice COC pretreated with AMPK agonist AICAR for 2 h and culture in Wuyu decoction-containing serum medium),early-aged+Wuyu decoction-containing serum group+Compound C group(24-week-old mice COC pretreated with AMPK inhibitor Compound C for 2 h and cultured in Wuyu decoction-containing serum medium),early-old+Cornus officinalis iridoid glycoside extract group(24-week-old mice COC cultured in Cornus officinalis iridoid glycoside extract medium).The gene and protein expressions of AMPK/PCG-1αpathway-related factors in oocytes of each group were detected by PCR and Western blot.Results The relative expression levels of AMPKα,PGC-1αand Nrf1 genes and the relative expression levels of AMPKα,p-AMPKα(Thr172),PGC-1α,Nrf1 proteins in oocytes of early-aged+AICAR group and early-aged+Wuyu decoction-containing serum group,and the relative expression levels of AMPKα,PGC-1αand Nrf1 genes and the relative protein expressions of AMPKα,p-AMPKα(Thr172)and PGC-1αin oocytes of the early-aged+Wuyu decoction-containing serum+AICAR group were significantly higher than those of the early-aged group(all P<0.05).The relative expression of Nrf2 gene in oocytes of early-aged+AICAR group,early-aged+Wuyu decoction-containing serum group,early-aged+Wuyu decoction-containing serum+AICAR group and the relative expression levels of AMPKα,PGC-1α,Nrf1,and Nrf2 genes in oocytes of early-aged+Compound C group were significantly lower than those of the early-old group(all P<0.05).The relative expressions of AMPKα,PGC-1α,Nrf1 and Nrf2 genes and the relative expressions of p-AMPKα(Thr172),PGC-1αand Nrf1 proteins in oocytes of early-aged+Wuyu decoction containing serum+Compound C group were significantly lower than those of early-aged+Wuyu decoction-containing serum group(all P<0.05).Conclusion During in-vitro maturation of from the primary senile mice,the AMPK/PGC-1αpathway can affect oocyte quality through mitochondrial energy metabolism,Wuyu decoction can enhance mitochondrial energy metabolism and oxidative stress state through AMPK/PCG-1αpathway,promote oocyte maturation,and improve oocyte function.During the in vitro culture of immature eggs of early-aged mice,the AMPK/PGC-1αpathway can affect the quality of oocytes through mitochondrial energy metabolism;Wuyu decoction can enhance mitochondrial energy metabolism and oxidative stress,promote oocyte maturation through the AMPK/PGC-1αpathway,thereby improving oocyte function.