基于“阴阳平衡”理论探讨补肾调冲方对SIRT1介导的卵母细胞线粒体“融合-分裂”稳态的影响

The homeostasis of "fusion-division" of mitochondria plays an important role in the function of mitochondria. Previous studies have found that bu shen tiao chong recipe can promote oocytes’ maturation, improve oocytes’ quality, and upregulate the expression of SIRT1 in oocytes. However, it is not known that if bu shen tiao chong recipe promote oocytes’ maturation by upregulating the expression of SIRT1, regulating the homeostasis of "fusion-division" in oocytes and improving the function of mitochondria. In this study, to obtain the direct evidence of bu shen tiao chong recipe regulating the homeostasis of "fusion-division" of oocytes’ mitochondria and verifie whether bu shen tiao chong recipe regulate the the homeostasis of "fusion-division" of mitochondria through SIRT1 pathway, the effect of bu shen tiao chong recipe on ovarian function will be observed in animal experiments and in vitro culture of oocytes, and the direct evidence of regulating the homeostasis of "fusion and division" of mitochondria by bu shen tiao chong recipe will be obtained. The aim of this study is to find out whether bu shen tiao chong recipe regulates the homeostasis of "fusion-division" of mitochondria through SIRT1 pathway. This study sought to find out direct evidence for bu shen tiao chong recipe mediating the expression of Mfn1/2, OPA1, FISl and DRPl through SIRT1, regulating the homeostasis of "fusion-division" of mitochondria and improving the function of mitochondria, and then provide scientific basis for the combination of bu shen tiao chong recipe and assisted reproductive technology to improve the oocytes’ quality and pregnancy outcome of elderly infertile women.

线粒体 “融合-分裂”稳态对线粒体功能具有重要意义。前期研究发现，补肾调冲方可促进卵母细胞成熟、提高卵母细胞质量，且可上调卵母细胞SIRT1的表达。然而，补肾调冲方是否通过调节SIRT1的表达，调控卵母细胞“融合-分裂”稳态，改善线粒体功能，进而促进卵母细胞成熟，目前尚不可知。本研究拟从整体动物及卵母细胞体外培养两方面观察补肾调冲方对卵巢功能的影响，获取补肾调冲方调控卵母细胞线粒体“融合-分裂”稳态的直接证据，验证补肾调冲方是否通过SIRT1途径调控线粒体“融合-分裂”稳态。本研究力图寻找补肾调冲方通过SIRT1介导Mfn1/2和OPA1，FISl和DRPl的表达，调控线粒体“融合-分裂”稳态，提高线粒体功能的直接证据，以期为补肾调冲方改善高龄不孕女性卵母细胞质量及妊娠结局提供科学依据。

阴阳学说是中医学基本理论之一，其调节重视调节事物相反两方面，使其达到平衡的理念贯穿于整个妇科诊疗过程中。线粒体融合-分裂稳态失常是导致卵母细胞质量降低的重要因素。本课题立足于中医基本理论，结合辅助生殖技术及生殖生物学，提出假说：增龄可导致卵巢及卵母细胞氧化损伤，进而影响线粒体形态变化及能量供应，降低卵母细胞质量。补肾调冲方可发挥中药双向调节优势，调控线粒体“融合-分裂”稳态，进而提高卵母细胞质量。实验选取氧化应激小鼠，随机分为空白组、模型组和治疗组，治疗组大鼠每日灌服调补冲任方水溶液6.26g／kg，空白组及模型组小鼠每日灌服等体积生理盐水，共4周。于灌胃第26 天腹腔注射PMSG 0.2ml，48h后腹腔注射HCG 0.2ml。14~16h后收集卵母细胞和卵巢。实验结束后，计算小鼠增重情况及卵巢指数；计算获卵数及第一极体排出率；检测卵巢组织SOD、GSH-Px、CAT活性；利用DCFH-DA法检测卵母细胞 ROS 水平；应用Mito-Tracker Green法观察卵母细胞线粒体分布情况；利用FITC-PNA法观察卵母细胞皮质颗粒分布情况；应用RT-PCR法检测卵母细胞内Sirt1、FOXO3a及Mn-SOD表达；应用微量蛋白质组学技术检测卵母细胞内Mfn1/2和OPA1、FISl和DRP表达情况。研究结果显示：①小鼠卵巢氧化应激可导致促排卵后获卵数减少、卵母细胞成熟率降低及卵母细胞质量下降。②预先给与补肾调冲方可保护卵巢免受氧化应激损伤。③小鼠卵母细胞内Sirt1可能通过FOXO3a的去乙酰化上调Mn-SOD表达。④补肾调冲方可能通过调控Mfn1/2和OPA1、FISl和DRP的表达，调控线粒体“融合-分裂”稳态，进而发挥中药双向调节优势，提高线粒体功能，改善卵母细胞质量。本研究结果表明：氧化应激可导致卵巢储备功能降低，卵母细胞线粒体“融合-分裂”稳态失常。补肾调冲方可调控SIRT1及Mfn1/2、OPA1、FISl、DRP表达，维持线粒体“融合-分裂”动态平衡，进而提高线粒体功能，改善卵母细胞质量。本研究运用现代生殖生物学技术，为补肾调冲方提高卵母细胞质量提供了现代科学实证基础，为临床治疗DOR提供了新的治疗思路，为中医学“阴阳学说”提供了新的理论依据。

内容获取失败，请点击重试