摘要
目的 观察益气血法经后增殖方含药血清基于p38 MAPK/核转录因子-κB(NF-κB)信号通路调节生长分化因子9(GDF9)、细胞凋亡抑制蛋白1/2(c-IAP1/2)、c-Jun氨基末端激酶1/2(JNK1/2)、肿瘤坏死因子受体1/2(TNFR1/2)表达对控制性超排卵大鼠卵巢颗粒细胞凋亡的影响.方法 取12 只雌性大鼠,随机分为控制性超排卵+中药组(6 只大鼠给予经后增殖方4.5 g/kg灌胃)和控制性超排卵组(6 只大鼠给予等体积生理盐水灌胃),后续经腹腔注射醋酸曲普瑞林、孕马血清促性腺激素、HCG后,取 2 组大鼠腹主动脉血制备含药血清和空白血清.另取 15只雌性大鼠,经腹腔注射醋酸曲普瑞林、孕马血清促性腺激素、HCG后,收集卵巢颗粒细胞,分别与空白血清(空白血清组)、含药血清(含药血清组)、含药血清+GDF9 抗体(含药血清+GDF9 抗体组)在体外共同培养.TUNEL检测卵巢颗粒细胞凋亡率,qRT-PCR法检测卵巢颗粒细胞中p38 MAPK、NF-κB、GDF9、c-IAP1、c-IAP2、JNK1、JNK2、TNFR1、TNFR2 mRNA 表达情况,Western blot法检测卵巢颗粒细胞中GDF9 蛋白表达情况.结果 含药血清组和含药血清+GDF9 抗体组细胞凋亡率均明显低于空白血清组(P均<0.05),且含药血清组明显低于含药血清+GDF9 抗体组(P<0.05).含药血清组p38 MAPK、NF-κB mRNA相对表达量均明显低于空白血清组(P均<0.05).含药血清组和含药血清+GDF9 抗体组 c-IAP1、c-IAP2、GDF9 mRNA 相对表达量和GDF9 蛋白相对表达量均明显高于空白血清组(P均<0.05),且含药血清组均明显高于含药血清+GDF9 抗体组(P均<0.05);含药血清组和含药血清+GDF9 抗体组JNK1、JNK2、TNFR1、TNFR2 mRNA相对表达量均明显低于空白血清组(P均<0.05),且含药血清组均明显低于含药血清+GDF9 抗体组(P均<0.05).结论 益气血法经后增殖方可通过调控p38 MAPK/NF-κB信号通路,上调控制性超排卵大鼠卵巢颗粒细胞中GDF9、c-IAP1、c-IAP2 的表达,下调JNK1、JNK2、TN-FR1、TNFR2 的表达,抑制卵巢颗粒细胞凋亡.
Abstract
Objective It is to observe the effects of post-menstruation proliferation decoction for tonifying and replenis-hing Qi and blood on ovarian granulosa cells apoptosis of controlled ovarian hyperstimulation rats via regulating the expres-sions of growth/differentiation factor 9(GDF9)and cellular inhibitors of apoptosis proteins 1/2(c-IAP1/2),c-Jun N-ter-minal kinase 1/2(JNK1/2),TNF receptor 1/2(TNFR1/2)based on p38MAPK/NF-κB signaling pathway.Methods Twelve female rats were taken and randomly divided into controlled ovarian hyperstimulation+herbal medicine group(6 rats were given post-menstruation proliferation decoction 4.5 g/kg by gavage)and controlled ovarian hyperstimulation group(6 rats were given equal volume of saline by gavage),and after subsequent intraperitoneal injections of triptorelin acetate,serum gonadotropin,and HCG of pregnant horse,the blood from the abdominal aorta of the two groups were taken to pre-pare medicated serum and blank serum.Another 15 female rats were taken and their ovarian granulosa cells were collected after intraperitoneal injections of triptorelin acetate,serum gonadotropin,and HCG of pregnant horse,and were co-cultured in vitro with blank serum(blank serum group),medicated serum(medicated serum group),medicated serum+GDF9 an-tibody(medicated serum+GDF9 antibody group).The apoptosis rate of ovarian granulosa cells was detected by TUNEL,the mRNA expressions of p38 MAPK,NF-κB,GDF9,c-IAP1,c-IAP2,JNK1,JNK2,TNFR1,and TNFR2 in ovarian granulosa cells were detected by qRT-PCR,and the protein expression of GDF9 in ovarian granulosa cells were detected by Western blot.Results The apoptosis rates of both medicated serum group and medicated serum+GDF9 antibody group were significantly lower than that of blank serum group(both P<0.05),and the medicated serum group was significantly lower than the medicated serum+GDF9 antibody group(P<0.05).The relative expressions of p38 MAPK and NF-κB mRNA in the medicated serum group were significantly lower than those in the blank serum group(both P<0.05).The relative expression of c-IAP1,c-IAP2,GDF9 mRNA and GDF9 protein in the medicated serum group and drug-containing serum+GDF9 antibody group were all significantly higher than those in the blank serum group(all P<0.05),and the medicated serum group was significantly higher than the medicated serum+GDF9 antibody group(all P<0.05);the relative expres-sions of JNK1,JNK2,TNFR1 and TNFR2 mRNA in the medicated serum group and medicated serum+GDF9 antibody group were all significantly lower than those in the blank serum group(all P<0.05),and the medicated serum group was significantly lower than the medicated serum+GDF9 antibody group(all P<0.05).Conclusion Post-menstruation prolif-eration decoction for tonifying and replenishing Qi and blood can up-regulate the expressions of GDF9,c-IAP1,c-IAP2,and down-regulate the expressions of JNK1,JNK2,TNFR1,and TNFR2 in ovarian granulosa cells of controlled ovarian hy-perstimulation rats via regulating p38MAPK/NF-κB signaling pathway.
基金项目
广东省自然科学基金资助项目(2020A1515011175)
广东省中医药局科研基金项目(20201412)
维普
万方数据