补肾调经方对体外受精–胚胎移植技术中重复控制性卵巢刺激模型小鼠囊胚质量及凋亡的影响

Effects of Bushen Tiaojing Formula on quality and apoptosis of blastocyst in mice induced by repeated controlled ovarian stimulation

目的观察补肾调经方对重复控制性卵巢刺激(COS)模型小鼠囊胚质量及凋亡的影响。方法将160只雌性ICR小鼠按照随机数字表法分为正常组、模型组、补肾低剂量组(25.6 g/kg)和补肾高剂量组(51.2 g/kg)。除正常组外,其余3组建立重复COS模型(COS 3次)。每日9时正常组及模型组雌鼠灌胃蒸馏水,补肾低、高剂量组雌鼠灌胃低、高剂量补肾调经方,连续10 d,各组均于第11天16时进行腹腔注射,除正常组注射生理盐水0.1 mL外,其余3组腹腔注射孕马血清促性腺激素(PMSG)10 IU/只,并于48 h后腹腔注射人绒毛膜促性腺激素(hCG)10 IU/只。灌胃及腹腔注射连续3个周期,每次间隔4 d。各组小鼠第3次腹腔注射后立即与同品系雄鼠合笼,次日清晨检查阴栓,见栓或阴道涂片见精子的雌鼠记为妊娠。妊娠小鼠于末次腹腔注射96 h后处死,获取囊胚,倒置显微镜下观察囊胚质量并计数,计算优质囊胚率;Tunel法检测囊胚细胞凋亡率;实时荧光定量PCR检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)mRNA表达;Simple Western检测凋亡相关因子Bcl-2、Bax及活化的半胱氨酸蛋白酶-3(cleaved caspase-3)的蛋白表达;免疫荧光法检测细胞色素C表达情况。结果与正常组比较,模型组获取囊胚数增多,优质囊胚率下降(P<0.05);囊胚内细胞总数减少,凋亡率升高(P<0.05);Bcl-2、Bcl-2/Bax mRNA及蛋白比值降低,Bax mRNA及蛋白表达升高,cleaved caspase-3蛋白表达升高(P<0.05);细胞色素C从线粒体释放,共定位系数M1、M2降低(P<0.05),差异有统计学意义。与模型组比较,补肾低、高剂量组获取囊胚数差异无统计学意义(P>0.05),优质囊胚率升高(P<0.05);囊胚内细胞总数增多,凋亡率降低(P<0.05);Bcl-2、Bcl-2/Bax mRNA及蛋白比值升高,Bax mRNA及蛋白表达降低,cleaved caspase-3蛋白表达降低(P<0.05);细胞色素C无明显释放,共定位系数M1、M2升高(P<0.05)。结论补肾调经方可抑制重复COS引起的囊胚细胞凋亡,提高早期胚胎质量,其作用机制可能与线粒体凋亡途径有关。

Objective To observe the effect of Bushen Tiaojing(Kidney-tonifying Menstruation-regulating)Formula(BSTJF)on quality and apoptosis of blastocyst in mice induced by repeated controlled ovarian stimulation(COS).Methods 160 female ICR mice were randomly divided into normal group,model group,low-dose BSTJF group and high-dose BSTJF group(25.6,51.2 g/mL).Except for the normal group,in the other three groups repeated COS model was established(COS three times).The mice in the normal group and model group were gavaged with distilled water,and the mice in the low-dose and high-dose BSTJF groups were gavaged with low-and high-dose BSTJF at 9 a.m.every day for 10 consecutive days.The model group,low-and high-dose BSTJF groups were intraperitoneally injected with pregnant mare serum gonadotropin(PMSG)10 IU at 4 p.m.on the 11day,and human chorionic gonadotropin(hCG)10 IU 48 hours later.The normal group was intraperitoneally injected with saline 0.1 mL at 4 p.m.on the 11day.There were 3 consecutive cycles with an interval of 4 days between every two cycles.Mice in all groups were caged with male mice of the same strain immediately after the third intraperitoneal injection of hCG(for the model group,low-and high-dose BSTJF groups)or normal saline(for the normal group).It was recorded as pregnancy if a vaginal plug was identified or sperm was observed on the vaginal smear in the following morning.Pregnant mice were killed 96 hours after the last intraperitoneal injection to obtain blastocysts.The quality of blastocysts was observed and number of blastocysts were counted under inverted microscope,and the rate of high quality blastocysts was calculated.The apoptosis rate of blastocysts was detected by TUNEL,and the levels of mRNA expression of Bcl-2 and Bax were detected by real-time fluorescence quantitative PCR.The levels of expression of apoptosis-related factors(Bcl-2,Bax and cleaved caspase-3)were detected with Simple Western,and the expression of cytochrome c was detected with immunofluorescence.Results Compared with the normal group,in the model group the number of blastocysts obtained was increased(P<0.05),while the rate of high-quality blastocysts was decreased(P<0.05);the total number of cells in blastocysts decreased,and the apoptosis rate increased(P<0.05);the RNA and protein ratio of Bcl-2 and Bcl-2/Baxm decreased(P<0.05),while the mRNA and protein expression levels of Bax increased(P<0.05),and the protein expression level of cleaved caspase-3 increased(P<0.05);cytochrome C was released from mitochondria and co-localization coefficients M1 and M2 decreased(P<0.05).Compared with the model group,there was no significant difference in the number of blastocysts obtained in the low-and high-dose BSTJF groups(P>0.05),but the rate of high-quality blastocysts increased significantly(P<0.05);the total number of cells in blastocysts increased and the apoptosis rate decreased(P<0.05);the mRNA and protein ratio of Bcl2 and Bcl-2/Bax increased(P<0.05),while the mRNA protein expression level of Bax decreased(P<0.05),and protein expression level of cleaved caspase-3 decreased(P<0.05);there was no significant release of cytochrome C,but the co-localization coefficients M1 and M2 increased(P<0.05).Conclusion Bushen Tiaojing Formula has the potential to inhibit blastocyst cell apoptosis induced by repeated COS and improve the quality of early embryos,and its mechanism may be related to mitochondrial apoptosis pathway.