卵巢储备功能下降(Diminished ovarian reserve, DOR)是指卵巢产生卵子的能力减弱,表现为卵母细胞数量和质量的双重下降,是导致女性生育力下降的重要原因,约占卵巢疾病的20%[1-3]。美国疾病控制与预防中心的全国辅助生殖技术数据显示DOR患者约占助孕人群的30% [4]。近年来,研究发现氧化应激(Oxidative stress,OS)过程中产生过量的活性氧(Reactive oxygen species,ROS)蓄积在卵巢,影响卵泡发育和颗粒细胞功能,导致卵母细胞质量下降,可能是其导致DOR的重要途径[5-6]。课题组前期研究表明DOR患者卵泡液微环境中存在氧化/抗氧化失衡和脂质过氧化差异代谢产物,氧化/抗氧化指标和COX2途径产生的AA差异代谢物与卵母细胞质量参数具有相关性,提示卵泡内可能同时存在OS和脂质过氧化,与DOR卵母细胞质量下降密切相关[7-8]。

沉默调控蛋白3(Sirtuin3,SIRT3)是一种重要的烟酰胺腺嘌呤二核苷酸依赖性去乙酰化酶,涉及多种生物过程,通过调控氧化应激(OS)和脂质代谢在卵泡发育过程中发挥关键作用[9-10]。研究表明,SIRT3不仅可直接激活超氧化物歧化酶(superoxide dismutase, SOD)的活性,还能通过去乙酰化叉头蛋白O3α(forkhead box protein, FOXO3a)促进其核转位,进而上调过氧化氢酶(Catalase, CAT)和SOD等抗氧化酶的表达,显著增强细胞清除ROS的能力[11-12]。与此同时,SIRT3还参与调控花生四烯酸(arachidonic acid, AA)代谢通路,该通路主要由磷脂酶A2(phosphorlipase A2, PLA2)和环氧合酶2(cyclooxygenase2, COX2)介导:PLA2作为限速酶催化AA从膜磷脂中释放,而COX2则负责将AA转化为前列腺素等活性代谢物[12]。相关研究证实SIRT3通过对FOXO3a抗氧化通路与PLA2/COX2代谢通路的同步调控,改善线粒体功能,共同促进卵泡发育和成熟[13-14]。

课题组自2008年开始对行IVF-ET治疗的DOR患者予以中医辅助治疗,认为DOR的主要病机在于脾肾两虚,以健脾补肾为治疗大法,经临床反复筛选验证优化为养卵方,前期临床研究显示养卵方可双重改善DOR患者的卵母细胞质量和数量,提高胚胎发育潜能,改善卵巢功能[15]。实验研究表明养卵方能改善DOR模型小鼠卵巢功能及胚胎发育潜能,作用机制与促进SIRT3表达以提高抗氧化能力有关[16]。因此,基于以上论述,推测养卵方可能通过激活SIRT3,去乙酰化修饰FOXO3a和PLA2/COX2通路调控OS和AA代谢,改善线粒体功能,促进卵泡发育,故本研究基于SIRT3/FOXO3a和PLA2/COX2通路探究养卵方改善DOR卵巢功能的作用机制。

资料与方法

一、实验动物及伦理审查

SPF级SD雌鼠60只,7～8周龄,体重(207.49±13.56)g,购自并饲养于北京维通利华实验动物有限公司上海分公司,许可证号:SYXK(沪)2022-0018,自由饮水进食,室温22～25 ℃,湿度60%～70%。本实验方案对实验动物的处置均符合实验动物福利与伦理相关规范,通过上海中医药大学实验动物福利与伦理委员会审查,批准编号:PZSHUTCM190912004。

二、药物与制备

养卵方(党参15 g、黄芪15 g、白术9 g、茯苓9 g、菟丝子15 g、巴戟天15 g、女贞子12 g、山茱萸9 g、陈皮9 g、当归9 g、白芍15 g、山药12 g、炙甘草6 g),按照“人和动物体表面积折算的等效剂量比率表”进行换算得出大鼠每日等效剂量为14.98 g/kg,设定为养卵方中剂量(YLF-M)组,养卵方低剂量(YLF-L)组为7.49 g/kg,养卵方高剂量(YLF-H)组为29.95 g/kg,养卵方所有饮片购于上海中医药大学附属曙光医院中药房,对药材来源进行鉴定,筛选优质上品,加10倍蒸馏水浸泡0.5 h,煎煮2次,第1次1.5 h,第2次1.0 h,合并2次滤液,过滤,加入旋转蒸发仪中分别浓缩成7.49 g/mL、14.98 g/mL和29.95 g/mL的生药浓度,相当于成人临床用量0.5、1、2 倍,置于4℃冰箱备用;补佳乐(批号J20171038)购自拜耳医药保健有限公司,配置成0.02 mg/mL的混悬液备用;去氧乙烯基环己烯(4-vinylcyclonhexenediepoxide,VCD,货号:V820469)购自MACKLIN,根据课题组前期造模及预实验结果确定VCD的浓度为160 mg/kg,质量浓度:1.094 g/mL,50只大鼠每天实验用量:160 mg/kg×1 d×0.2 kg×50=1 600 mg,体积:1.0 mL×50 =50 mL,需要的浓度:1 600/50=32 mg/mL,设原液为a,计算如下:1 094 a/(a+50)=32,a=1.5066 mL(1.51 mL原液配在48.49 mL大豆油中),4 ℃冰箱备用。

三、试剂与仪器

大鼠卵泡刺激素(follicle stimulating hormone, FSH)试剂盒(QZ-10015)、黄体生成素(luteinizing hormone, LH)试剂盒(QZ-12009)、抗苗勒管激素(anti miillerian hormone, AMH)试剂盒(QZ-14689)均购自泉州市九邦生物科技有限公司,SIRT3抗体(10099-1-AP,Proteintech),FOXO3a抗体(66428-1-Ig,Proteintech),PLA2抗体(ABP55601,Abbkine),COX2(66351-1-Ig,proteintech),ß-Actin(ABL1010,Abcam);酶标仪(BIO-TEK,Synergy LX)、石蜡切片机(德国Leica,RM2235)、倒置显微镜(OLMYPUS,BX51)、电泳仪(北京六一仪器厂,DYCZ-24FN)、电泳槽(北京六一仪器厂, DYCP-36)、紫外可见分光光度计(赛默飞,Evolution 350)、高压灭菌锅(SANYO, MLS-3020U)、核酸蛋白测定仪(Eppendorf,BioPhotometer D30)、实时荧光定量PCR仪器(Applied Biosystems,ABI7500)。

四、研究方法

1.造模。采用阴道细胞学涂片判断动情周期,将连续2个动情周期正常的大鼠纳入实验,随机选取10只大鼠作为对照组,其余50只进行造模,根据预实验及本课题组前期实验方法进行造模[17],每日上午9点腹腔注射VCD(160 mg/kg),每天1次,连续15 d,空白对照组腹腔注射等体积的大豆油,每天1次,连续15 d。采用光镜显微镜观察大鼠阴道细胞形态学判断动情周期的变化,若动情周期紊乱则证明造模成功。

2.分组与给药。造模期间由于腹腔注射操作不当引起腹腔感染或大量腹水导致5只大鼠死亡,将造模成功的45只大鼠,随机分为模型组(Model组)、YLF低剂量组(YLF-L组)、YLF中剂量组(YLF-M组)、YLF高剂量组(YLF-H组)组和补佳乐组(BJL组),各9只。另有原未造模的对照组(Control组)10只。每日上午9点,YLF-L组、YLF-M组和YLF-H组分别予浓度为7.49 g/mL、14.98 g/mL和29.95 g/mL的养卵方水溶液灌胃;BJL组予0.02 mg/mL浓度的补佳乐混悬液灌胃;对照组和模型组予等量生理盐水灌胃,连续15 d。灌胃期间,模型组、YLF-H组和 BJL组分别有1只大鼠死亡。

3.取材。末次给药结束后,采用阴道涂片筛查各组大鼠动情周期,均于动情前期采集标本;予腹腔注射3%戊巴比妥钠进行麻醉,分别取①子宫与卵巢称重并记录,随机选一侧卵巢置于4%多聚甲醛固定后行HE染色;另一侧卵巢组织于冻存管置-80 ℃冰箱保存;②腹主动脉取血,室温静置2 h,3 000(×g)离心15 min,取上清液,-80 ℃冰箱保存。

五、检测指标

1.性腺湿重及性腺指数。取各组大鼠性腺(卵巢和子宫),去除其周围的脂肪及筋膜,立即分别称量双侧卵巢和子宫湿重,计算卵巢和子宫指数。卵巢指数(%)=双侧卵巢湿重量(g)/体质量(g)×100%;子宫指数(%)=子宫湿重量(g)/体质量(g)×100%。

2.HE染色观察卵巢组织形态学。取出固定好的卵巢组织,依次脱水、透明、浸蜡与包埋、切片、脱蜡、染色、脱水透明、封片,显微镜下观察各组卵巢组织形态。

3.ELISA检测血清FSH、LH、AMH 水平。采用ELISA法检测各组大鼠血清FSH、LH、AMH的水平,具体步骤严格按照说明书进行。

4.Western blot检测卵巢组织SIRT3、FOXO3a、PLA2、COX2蛋白表达量。将卵巢组织按试剂说明书进行蛋白提取,并测定蛋白浓度,配胶、上样与电泳、转膜,然后进行染色、洗膜、封闭,加入稀释的一抗SIRT3、FOXO3a、PLA2、COX2后4 ℃孵育过夜,次日洗膜,加入稀释的二抗,室温孵育1h,洗膜后采用ECL化学发光法检测,以β-action为内参,用Image J软件分析目的蛋白条带的光密度值,并统计分析。

5.PCR检测卵巢组织SIRT3、FOXO3a、PLA2、COX2、SOD、GSH-Px、CAT的mRNA表达量。使用Trizol裂解卵巢组织并测定总RNA含量,按试剂盒说明书配置成20 μL体系反转录成cDNA,然后按照TB Green Premix Ex Taq Ⅱ (2×,10 μL)、ROX Reference Dye(50×,0.4 μL)、RNase-free Water(6 μL)、样本cDNA(2 μL)配置成反应体系,反应条件为:95 ℃预变性30 s,95 ℃变性30 s,60 ℃退火30 s,60 ℃延伸15 s,扩增40个循环。反应结束后导出数据,以GAPDH作为内参基因,用2-ΔΔCt法进行目的基因的相对表达量分析。引物序列见表1,由生工生物工程(上海)有限公司合成。

六、统计分析

应用SPSS 26.0和 GraphPad Prism 8.0 软件进行数据分析和制图,计量资料符合正态分布用均数±标准差进行统计描述,符合正态分布且方差齐性采用单因素方差分析(One-Way ANOVA)进行组间比较,若组间差异有统计学意义进一步采用Bonferroni法进行两两比较;若不符合正态分布用中位数及四分位数间距(IQR)进行统计描述,选择非参数检验(Kruskal-Wallis H检验)进行组间比较,当组间总的差异有统计学意义,进一步采用Dunn法进行多重比较,以P<0.05为差异有统计学意义。

结 果

一、卵巢和子宫指数比较

与对照组比较,模型组大鼠卵巢和子宫指数均下降(P<0.05);与模型组相比,YLF-M组、YLF-H组及BJL组大鼠卵巢和子宫指数增高(P<0.05);而YLF-L组大鼠卵巢和子宫指数与模型组相比无统计学差异(P>0.05);与YLF-L组相比,YLF-M组、YLF-H组及BJL组大鼠子宫指数增高(P<0.05),见图1。

二、卵巢组织病理形态学比较

对照组大鼠卵巢可见各级卵泡,卵泡形态、数量正常,卵泡颗粒细胞层次多,排列整齐,间质未见炎性细胞浸润(图2A);模型组大鼠卵巢各级卵泡数均明显减少,趋于闭锁卵泡较多,颗粒细胞层数少,排列紊乱,间质可见不同程度的炎性细胞浸润(图2B);与模型组相比,YLF-L组、YLF-M组、YLF-H组及BJL组大鼠卵巢各级卵泡增多,闭锁卵泡减少,颗粒细胞增加(图2C、2D、2E、2F)。

三、血清FSH、LH和AMH水平的比较

与对照组比较,模型组大鼠血清FSH、LH 水平升高,AMH水平降低,FSH/LH增大(P<0.05);与模型组相比,YLF-L组、YLF-M组、YLF-H组及BJL组大鼠血清FSH、LH 水平降低,AMH水平升高,FSH/LH减小(P<0.05);与YLF-L组相比,YLF-H组及BJL组大鼠血清AMH水平升高(P<0.05),见表2。

四、养卵方对大鼠卵巢组织SIRT3、FOXO3a、PLA2、COX2蛋白表达的影响

Western blot结果显示,与对照组比较,模型组大鼠卵巢组织SIRT3、FOXO3a、PLA2、COX2蛋白表达均降低(P<0.05);与模型组相比,YLF-L组、YLF-M组、YLF-H组及BJL组大鼠卵巢组织SIRT3、FOXO3a、PLA2、COX2蛋白表达量升高(P<0.05),与YLF-L组相比,YLF-H组、BJL组大鼠卵巢组织SIRT3、FOXO3a、PLA2、COX2蛋白相对表达量上调(P<0.05),见图3、表3。

五、养卵方对大鼠卵巢组织SIRT3、FOXO3a、PLA2、COX2、SOD、GSH-Px、CAT mRNA表达的影响

与对照组比较,模型组大鼠卵巢组织SIRT3、FOXO3a、PLA2、COX2、SOD、GSH-Px、CAT mRNA表达降低(P<0.05);与模型组相比,YLF-L组大鼠卵巢组织PLA2 mRNA表达量增加(P<0.05),SIRT3、FOXO3a、COX2、SOD、GSH-Px、CAT mRNA表达无统计学差异(P>0.05);YLF-M组、YLF-H组及BJL组大鼠卵巢组织SIRT3、FOXO3a、PLA2、COX2、SOD、GSH-Px、CAT mRNA表达量均较模型组升高(P<0.05);与YLF-L组相比,YLF-M组、YLF-H组和BJL组大鼠卵巢组织FOXO3a、COX2、SOD、GSH-Px、CAT mRNA相对表达量上调(P<0.05),YLF-H组大鼠卵巢组织SIRT3 mRNA相对表达量上调(P<0.05),YLF-H组和BJL组大鼠卵巢组织PLA2 mRNA相对表达量上调(P<0.05),见表4、图4。

讨 论

在祖国医学中无“DOR”的病名,根据临床表现可归属于“月经后期、月经过少、闭经、绝经前后诸证、不孕”等范畴。中医药治疗DOR具有独特优势和疗效,在辅助生殖中应用中医药可改善卵巢功能,并在此基础上改善卵巢反应性,提高卵子质量,提高优质胚胎着床率和临床妊娠率,同时避免卵巢过度刺激综合征的发生[18]。临床中,本课题组以健脾补肾为基本治法,用上海市名中医齐聪教授经验方——养卵方,方中党参、白术、茯苓、炙甘草源自四君子汤,具有健脾益气的功效,黄芪加强补气之功,当归补血活血,陈皮健脾燥湿,以资气血生化之源;菟丝子性温,归肝肾脾经,功能补肾益精、健脾止泻,具有温而不燥、补而不滞的特点,巴戟天温补肾阳,山茱萸和女贞子滋补肾阴,四药水火既济,补肾益精;白芍酸甘敛阴而柔肝,得当归相助,养血调冲,山药补肾固精,健脾益气,全方共奏健脾补肾、调冲养卵之效。课题组前期研究结果显示养卵方改善卵巢功能及卵母细胞质量的作用机制与抗氧化有关[19-20]。

基础性激素水平是评估DOR的常用指标,其中FSH、LH与卵巢功能密切相关,当卵巢储备功能下降时,下丘脑-垂体-卵巢轴的激素反馈作用减弱,导致FSH、FSH/LH水平升高[21]。AMH主要由卵泡颗粒细胞合成,其分泌不受下丘脑-垂体-卵巢轴的影响,且不随月经周期变化,血清AMH水平随着年龄增长逐渐降低,是卵巢储备功能的重要标志物[22]。有研究显示育龄期及超育龄期女性随年龄增加,AMH水平呈下降趋势,而FSH、E2及LH水平变化相对不明显,提示AMH评判卵巢储备功能较性激素具有更高的敏感性及特异性[23]。本实验结果发现模型组大鼠血清FSH、LH水平升高,FSH/LH比值增大,AMH水平下降,提示模型组大鼠卵巢储备功能减退。予以养卵方干预后能降低血清FSH、LH水平及FSH/LH比值,升高AMH水平,提示养卵方能有效改善DOR大鼠血清性激素和AMH水平。

生理状态下,机体的酶类,如SOD、CAT和谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)等与非酶类抗氧化系统协同工作,维持ROS的动态平衡,然而当机体产生过量的ROS,或机内抗氧化酶相对缺乏无法清除过量的ROS,导致OS损伤状态[24]。多项研究表明天然化合物(如姜黄素、辣椒素、木黄酮)及中药(如补肾疏肝方、二仙汤)能通过提升抗氧化酶(SOD、GSH-Px、CAT)的活性或表达,有效缓解由药物、辐射或化学物质诱导的卵巢OS损伤[25-29]。本实验亦发现,模型组大鼠卵巢SOD、GSH-Px、CAT抗氧化酶基因的相对表达量降低,养卵方干预后其mRNA表达水平升高,与上述研究结果一致,提养卵方能上调这些抗氧化酶基因的表达,从而减轻VCD诱导的OS损伤,改善卵巢储备功能。

如前所述,SIRT3在调控OS和脂质代谢过程中发挥重要作用,其不仅可直接调节SOD的活性提高清除ROS的能力,还通过去乙酰化FOXO3a,进入细胞核促进SOD、CAT等抗氧化酶的表达,减少ROS累积[30-31]。SIRT3对卵泡发育、成熟、卵母细胞老化、颗粒细胞功能及胚胎发育等过程具有重要调控作用[32],如Pacella-Ince等[33]发现高龄妇女和DOR患者壁层颗粒细胞和卵丘细胞中SIRT3表达均下降,其去乙酰化功能受限,导致谷氨酸脱氢酶活性下降,影响线粒体能量代谢过程,导致卵泡微环境发生改变,进而影响卵母细胞发育。FOXO3a转录因子参与调控原始卵泡的激活,其下调后可使原始卵泡大量且不受控制地激活[34-35],FOXO3a过表达可提高小鼠的卵巢生殖能力[36]。本研究结果显示模型组大鼠卵巢组织SIRT3、FOXO3a蛋白和mRNA相对表达量明显降低,予以养卵方干预后恢复其表达,提示养卵方可能通过调控SIRT3、FOXO3a的表达,抑制VCD诱导的卵巢功能下降。

PLA2作为AA的限速酶,激活后水解膜磷脂释放AA,启动AA代谢[37]。在小鼠卵母细胞中,PLA2在生发泡破裂时从细胞膜动态转移至核膜[38]。Ajmat等[39]研究表明PLA2激活剂及AA能触发蟾蜍卵母细胞激活。PLA2基因缺陷雌性小鼠表现为严重的生殖缺陷,如排卵数减少、延迟种植、妊娠发育障碍甚至妊娠失败、产仔数减少等[40]。COX2则是AA合成前列腺素的关键酶,对卵泡排卵、受精、胚胎发育和着床至关重要[41]。COX2基因缺陷雌鼠呈现排卵、受精、着床障碍等[42-43]。McKenzie等[44]指出卵巢颗粒细胞中COX2的表达与卵母细胞受精及胚胎发育紧密相关。以上研究均表明PLA2、COX2与卵母细胞发育密切相关,但其与DOR的相关性研究有限。本研究发现DOR模型组大鼠卵巢组织PLA2、COX2蛋白和基因表达显著下调,而养卵方干预则促进两者表达上调,提示养卵方可能通过促进PLA2、COX2的表达,缓解VCD诱导的卵巢损伤。

综上所述,推测VCD诱导大鼠卵巢发生OS和脂质过氧化,影响AA代谢,制约卵泡发育,进而诱导卵泡异常闭锁,最终导致DOR,而养卵方干预可能通过调控SIRT3/FOXO3a和PLA2/COX2信号通路,增强抗氧化防御,调节AA代谢,改善生殖内分泌功能,缓解VCD诱导的卵泡闭锁,改善卵巢储备功能。

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Effects of Yangluanfang on SIRT3/FOXO3a and PLA2/COX2 pathways in ovary tissue of rats with diminished ovarian reserve

HU Hui, ZHANG Xiaole, ZHANG Qinhua, LIANG Chengcheng, ZHANG Jie, YANG Hong, QI Cong, HU Panwei, LI Miao, LU Ziman.

Department of Gynecology,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (Hu Hui, Zhang Xiaole, Zhang Qinhua, Liang Chengcheng, Qi Cong, Hu Panwei); Graduate Student, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (Lu Ziman), 201203; Laboratory of Science and Technology Center, Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (Zhang Jie), 200032; Department of Gynecology, Municipal Hospital of Traditional Chinese Medicine Affiliated to Shanghai University of Traditional Chinese Medicine (Yang Hong), 200071; Graduate Student, Yueyang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (Li Miao), 200437.

[Abstract] Objective To observe the effects of YangLuanfang (YLF) on SIRT3/ FOXO3-a and PLA2/COX2 signaling pathways in ovarian tissue of rats with diminished ovarian reserve (DOR), and explore the effect and mechanism of YLF on improving ovarian reserve function in rats with DOR.MethodsSD rats were randomly divided into control group and model group. DOR model rats were made by intraperitoneal injection of vinylcyclohexene dioxide (VCD). The successful DOR rats were randomly divided into model group, YLF low-dose (YLF-L) group, YLF middle-dose (YLF-M) group, YLF high-dose (YLF-H) group and progynova (BJL) group. Each group was given corresponding drugs by gavage for 15 days. The indexes of gonads (ovaries and uterus) were compared in each group, and observe the pathological morphological changes of ovarian tissues by HE staining. Serum FSH, LH and AMH levels were detected. Western blot was used to detect the expression levels of SIRT3,FOXO3a,PLA2 and COX2 proteins in ovarian tissues. PCR was used to detect the mRNA expression levels of SIRT3, FOXO3a, PLA2, COX2, SOD,GSH-Px and CAT in ovarian tissue.ResultsCompared with the control group, the index of ovary and uterus in the model group decreased (P<0.05), the levels of serum FSH,LH and the FSH/LH ratio increased (P<0.05), AMH level decreased (P<0.05), and the expression levels of SIRT3, FOXO3a, PLA2, COX2 protein and mRNA, SOD, GSH-Px, and CAT mRNA in ovarian tissue decreased (P<0.05). Compared with the model group, the moderate to high doses of YLF improved the ovary pathological morphology of DOR rats, increased the ovarian and uterine index (P<0.05), decreased the levels of serum FSH, LH and the FSH/LH ratio (P<0.05), increased the AMH level (P<0.05), and increased the expressions of SIRT3, FOXO3a, PLA2, COX2 protein and mRNA, and the mRNA of SOD, GSH-Px and CAT in ovarian tissue (P<0.05).ConclusionYLF may improve the activity of antioxidant enzymes, regulate endocrine function, and alleviate ovarian damage induced by VCD by regulating SIRT3/FOXO3a and PLA2/COX2 signaling pathways.

[Key words] diminished ovarian reserve; YangLuanfang; oxidative stress; lipid peroxidation; arachidonic acid

【中图分类号】 R71

基金项目：国家重点研发计划“中医药现代化研究”重点专项(SQ2018YFC170061)

作者单位：201203,上海中医药大学附属曙光医院妇科(胡慧,章晓乐,张勤华,梁程程,齐聪,胡攀伟),上海中医药大学附属曙光医院肝病科研究生(陆紫蔓);200032,上海中医药大学附属龙华医院科技中心实验室(张婕);200071,上海中医药大学附属市中医医院妇科(杨红);200437,上海中医药大学附属岳阳医院皮肤科研究生(李淼)

通信作者：齐聪(qicongshuguang@126.com)

▲共同第一作者

(收稿日期：2025-08-08)