摘要
目的 探究复幼合剂通过磷脂酰肌醇3-激酶-蛋白激酶B (PI3K/Akt)信号通路对性早熟(PP)雌鼠下丘脑促性腺激素释放激素(GnRH)分泌的影响。方法 取75只SD雌鼠,用随机数字表法将其分为正常组、模型组、复幼合剂组、亮丙瑞林组、槲皮素组各15只。母乳喂养至23日龄。在第5日龄时,除正常组外,其余4组均一次性颈背部皮下注射25μL达那唑溶液建立PP模型。大鼠阴道开口时间、首次发情时间及正常发情周期显著提前为造模成功。造模10 d,亮丙瑞林组于左侧下腹部皮下注射亮丙瑞林100μg/kg;复幼合剂组每天给予6 mL/kg复幼合剂灌胃;槲皮素组每天给予4 mg/kg槲皮素灌胃;正常组、模型组给予2 mL/kg生理盐水灌胃。20日龄开始检查阴道开口情况,通过苏木精-伊红(HE)染色观察性腺发育情况;酶联免疫吸附检测血清中促黄体生成素(LH)、促卵泡刺激素(FSH)、雌二醇(E2);免疫组织化学法检测下丘脑GnRH蛋白表达;实时荧光定量聚合酶链反应(RT-qPCR)检测下丘脑GnRH、吻素(Kiss-1)、G蛋白偶联受体54(GPR54)、胰岛素生长因子(IGF-1)、雌激素受体α (ESR1) mRNA表达,Western blotting法检测GnRH、磷脂酰肌醇3-激酶(PI3K)、磷酸化磷脂酰肌醇3-激酶(p-PI3K)、蛋白激酶B (Akt)、磷酸化蛋白激酶B (p-Akt)蛋白表达。结果 与正常组比较,模型组阴道开口提前。模型组子宫系数、卵巢系数较正常组大(P<0.05)。复幼合剂组子宫系数较模型组小(P<0.01),亮丙瑞林组子宫系数、卵巢系数较模型组小(P<0.05)。模型组、复幼合剂组、亮丙瑞林组、槲皮素组子宫、卵巢细胞形态结构无明显病理变化,表明达那唑不会造成性器官病理性增长。各组卵巢发育情况大致相同,均可见初级、次级、成熟卵泡。模型组子宫壁厚度与正常组比较差异无统计学意义(P>0.05),复幼合剂组子宫壁厚度较模型组小,但差异无统计学意义(P>0.05),亮丙瑞林组子宫壁厚度较模型组小(P<0.05),槲皮素子宫壁厚度与模型组比较差异无统计学意义(P>0.05)。模型组血清E2、LH、FSH水平较正常组高(P<0.05);亮丙瑞林组E2及FSH水平较模型组低(P<0.05),复幼合剂组、槲皮素组血清E2水平较模型组低(P<0.01)。模型组下丘脑GnRH细胞平均光密度值较正常组高(P<0.05);复幼合剂组、亮丙瑞林组光密度值较模型组低(P<0.01),槲皮素组光密度值与模型组比较差异无统计学意义(P>0.05)。显微镜下可见各组深浅不等的棕黄色GnRH阳性细胞,细胞结构清晰,核仁明显,且以Broca斜角带分布最为集中。模型组下丘脑GnRH、Kiss-1、GPR54、ESR1、IGF-1 mRNA表达较正常组高(P<0.01)。复幼合剂组、亮丙瑞林组下丘脑GnRH、Kiss-1、GPR54、ESR1、IGF-1 mRNA表达较模型组低(P<0.05),槲皮素组下丘脑GnRH、Kiss-1、ESR1 mRNA表达较模型组低(P<0.05)。模型组下丘脑GnRH蛋白相对表达量及p-Akt/Akt、p-PI3K/PI3K均较正常组高(P<0.01),复幼合剂组、亮丙瑞林组、槲皮素组GnRH蛋白相对表达量及p-Akt/Akt、p-PI3K/PI3K均较模型组低(P<0.05)。结论 复幼合剂通过下调IGF-1mRNA以及p-Akt/Akt、p-PI3K/PI3K蛋白表达,抑制IGF-1/PI3K/Akt信号通路激活,进而降低Kiss-1、GPR54 mRNA表达,减少GnRH以及血清E2的分泌,从而抑制下丘脑-垂体-性腺轴(HPGA)的启动,发挥治疗大鼠PP的作用。
Objective To investigate the effects of Fuyou Mixture on the secretion of gonadotropin-releasing hormone(GnRH)in the hypothalamus of female rats with precocious puberty through the phosphoinositide 3-kinase-protein kinase B(PI3K/Akt)signaling pathway.Methods A total of 75 female SD rats were randomly divided into a normal group,a model group,a Fuyou Mixture group,a leuprolide group,and a quercetin group according to a random number table,with 15 rats in each group.The rats were fed breast milk until 23 days of age.On day 5,except for the normal group,the other four groups received a single subcutaneous injection of 25μL of danazol solution in the neck to establish the precocious puberty model.Successful modeling was confirmed by an earlier vaginal opening time,earlier first estrus,and abnormal estrous cycles.After 10 days,the leuprolide group received subcutaneous injections of leuprolide(100μg/kg)on the left lower abdomen,the Fuyou Mixure group received Fuyou Mixture at 6 mL/kg by gavage daily,and the quercetin group received quercetin at 4 mg/kg by gavage daily.The normal and model groups received normal saline at 2 mL/kg by gavage.Vaginal opening was checked starting from day 20,and ovarian development was observed using hematoxylin-eosin(HE)staining.Serum levels of luteinizing hormone(LH),follicle-stimulating hormone(FSH),and estradiol(E2)were measured by enzyme-linked immunosorbent assay(ELISA).Immunohistochemistry was used to detect GnRH protein expression in the hypothalamus.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect mRNA expression of GnRH,Kiss-1,G protein-coupled receptor 54(GPR54),insulin-like growth factor(IGF-1),and estrogen receptorα(ESR1)in the hypothalamus.Western blotting was used to assess the protein expression of GnRH,PI3K,phosphorylated PI3K(p-PI3K),Akt,and phosphorylated Akt(p-Akt).Results Compared with the normal group,the model group showed an earlier vaginal opening.The model group had higher uterine and ovarian coefficients than the normal group(P<0.05).The Fuyou Mixture group had a smaller uterine coefficient than the model group(P<0.01),and the leuprolide group showed smaller uterine and ovarian coefficients than the model group(P<0.05).No significant pathological changes were observed in the morphology of ovarian and uterine cells among the model,Fuyou Mixture,leuprolide,and quercetin groups,indicating that danazol did not cause pathological growth of the sexual organs.Ovarian development in all groups showed primary,secondary,and mature follicles.There was no significant difference in the uterine wall thickness between the model group and the normal group(P>0.05).The Fuyou Mixture group showed a smaller uterine wall thickness than the model group,but the difference was not statistically significant(P>0.05),while the leuprolide group had a significantly thinner uterine wall than the model group(P<0.05).No significant differences in uterine wall thickness were observed between the quercetin group and the model group(P>0.05).The serum levels of E2,LH,and FSH were significantly higher in the model group than in the normal group(P<0.05).E2 and FSH levels were lower in the leuprolide group than in the model group(P<0.05),and both the Fuyou Mixture and quercetin groups showed significantly lower serum E2 levels than the model group(P<0.01).The average optical density of GnRH cells in the hypothalamus was higher in the model group than in the normal group(P<0.05).The Fuyou Mixture and leuprolide groups showed lower optical density values than the model group(P<0.01),while the quercetin group showed no significant difference from the model group(P>0.05).Under the microscope,GnRH-positive cells with varying shades of brown-yellow were observed in all groups,with clear cell structure and prominent nucleoli,most concentrated in the Broca's diagonal band.The mRNA expression levels of GnRH,Kiss-1,GPR54,ESR1,and IGF-1 in the hypothalamus were significantly higher in the model group than in the normal group(P<0.01).The Fuyou Mixture and leuprolide groups showed lower mRNA expression of GnRH,Kiss-1,GPR54,ESR1,and IGF-1 than the model group(P<0.05),while the quercetin group showed lower mRNA expression of GnRH,Kiss-1,and ESR1 than the model group(P<0.05).The relative protein expression of GnRH,p-Akt/Akt,and p-PI3K/PI3K in the hypothalamus was significantly higher in the model group than in the normal group(P<0.01).The Fuyou Mixture,leuprolide,and quercetin groups showed lower protein expression of GnRH,p-Akt/Akt,and p-PI3K/PI3K than the model group(P<0.05).Conclusion Fuyou Mixture exerts therapeutic effects on precocious puberty in rats by downregulating IGF-1 mRNA expression and the protein expression of p-Akt/Akt and p-PI3K/PI3K and inhibiting the IGF-1/PI3K/Akt signaling pathway to reduce the expression of Kiss-1 and GPR54 mRNA,thereby decreasing GnRH secretion and serum E2 levels and inhibiting the activation of the hypothalamic-pituitary-gonadal axis.
作者
郭春彦
柳静
张怡
丁倩
王谦
张萌
王晓玲
GUO Chunyan;LIU Jing;ZHANG Yi;DING Qian;WANG Qian;ZHANG Meng;WANG Xiaoling(Pharmacy Department of Beijing Children's Hospital Affiliated to Capital Medical University,National Children's Medical Center,Beijing 100045,China;Department of Traditional Chinese Medicine,Beijing Children's Hospital Affiliated to Capital Medical University,National Children's Medical Center,Beijing 100045)
出处
《北京中医药》
2024年第10期1131-1137,共7页
Beijing Journal of Traditional Chinese Medicine
基金
重大新药创制科技重大专项(2018ZX09721003-002-001)
首都医科大学附属北京儿童医院“萌芽杯”儿童用药专项(MYQY202201)。
