丹参多酚酸对免疫性不孕大鼠的治疗作用及对下丘脑-垂体-卵巢轴的影响

Therapeutic effect of salvia miltiorrhiza polyphenolic acid on immune infertility rats and its effect on hypothalamic-pituitary-ovarian axis

目的探讨丹参多酚酸对免疫性不孕大鼠的治疗作用及对下丘脑-垂体-卵巢轴(HPOA)的影响。方法取50只未孕雌性SD大鼠,采用人精液注射免疫的方法建立抗精子抗体阳性的免疫性不孕大鼠模型,将建模成功大鼠采用随机数字表法分为模型组(9只)、强的松组(10只)和丹参多酚酸低剂量组(9只)、中剂量组(10只)、高剂量组(10只),另取10只未孕雌性大鼠为正常对照组。强的松组经腹腔注射给予5 mg/kg强的松,丹参多酚酸低、中、高剂量组分别经腹腔注射给予10、20、40 mg/kg丹参多酚酸,正常对照组和模型组均给予等量生理盐水,每日1次,共14 d。各组雌性大鼠与正常雄性大鼠按3∶1数量比合笼2周,统计各组大鼠受孕率;放射免疫法测定血清中促性腺激素释放激素(GnRH)、卵泡刺激素(FSH)、黄体生成素(LH)以及雌二醇(E2)水平;苏木素-伊红(HE)染色观察大鼠卵巢组织病理改变情况;实时定量逆转录聚合酶链反应(qPCR)和蛋白质免疫印迹法检测大鼠卵巢卵泡刺激素受体(FSHR)、黄体生成素受体(LHR)、雌激素受体α(ERα)mRNA和蛋白表达。结果与正常对照组相比,模型组大鼠受孕率,血清GnRH、FSH、LH、E2含量降低(P<0.05),卵巢内黄体和成熟卵泡明显减少,颗粒细胞层数减少,囊状卵泡较多,FSHR、LHR、ERαmRNA及蛋白表达降低(P<0.05)。与模型组相比,强的松组和丹参多酚酸中、高剂量组大鼠受孕率,血清GnRH、FSH、LH、E2含量升高(P<0.05),囊状卵泡数目减少,颗粒细胞层数增多,有成熟卵泡和黄体生成,FSHR、LHR、ERαmRNA及蛋白表达升高(P<0.05)。结论丹参多酚酸可通过调控HPOA促进卵泡成熟治疗免疫性不孕。

Objective To investigate the therapeutic effect of salvia miltiorrhiza polyphenolic acid on immune infertility rats and its effect on hypothalamus-pituitary-ovarian axis(HPOA).Methods Fifty non-pregnant female SD rats were selected.A rat model of immune infertility with anti-sperm antibody(+)was established by human semen injection immunization.The model rats were divided into the model group(n=9),the prednisone group(n=10),the alvia miltiorrhiza polyphenolic acid low dose group(n=9),the salvia miltiorrhiza polyphenolic acid medium dose group(n=10)and the salvia miltiorrhiza polyphenolic acid high dose group(n=10)by random array table method.Another 10 non-pregnant female rats were selected as the normal control group.The prednisone group was given 5 mg/kg prednisone by intraperitoneal injection,and the salvia miltiorrhiza polyphenolic acid low,medium and high dose groups were given 10,20 and 40 mg/kg salvia miltiorrhiza polyphenolic acid by intraperitoneal injection.The normal control group and model group were given the same amount of normal saline,once a day for 14 days.The female rats and normal male rats in each group were caged in a ratio of 3:1 for 2 weeks,and the pregnancy rate of rats in each group was observed.The serum levels of gonadotropin-releasing hormone(GnRH),follicle-stimulating hormone(FSH),luteinizing hormone(LH)and estradiol(E2)were determined by radioimmunoassay.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of rat ovarian tissue.Real-time quantitative reverse transcription polymerase chain reaction(qPCR)was used to detect rat ovarian folliclestimulating hormone receptor(FSHR),luteinizing hormone receptor(LHR)and estrogen receptor alpha(ERα)messenger ribonucleic acid(mRNA)expression.Western blotting(WB)was used to detect rat ovarian FSHR,LHR and ERαprotein expressions.Results Compared with the normal control group,the conception rate and the serum levels of GnRH,FSH,LH and E2 were decreased in the model group(P<0.05),the corpus luteum and mature follicles in the ovary were significantly reduced,the number of granulosa cell layers was reduced,and there were more cystic follicles,and the mRNA and protein expressions of FSHR,LHR and ERαwere decreased(P<0.05).Compared with the model group,the conception rate and the serum levels of GnRH,FSH,LH,and E2 were increased in the prednisone group and the salvia miltiorrhiza polyphenolic acid medium and high dose groups(P<0.05),the number of cystic follicles was significantly decreased,the number of granulosa cell layers was increased,and mature follicles and corpus luteum were formed.The mRNA and protein expressions of FSHR,LHR and ERαwere increased(P<0.05).Conclusion Salvia miltiorrhiza polyphenolic acid can promote follicle maturation by regulating HPOA in the treatment of immune infertility.