菟丝子总黄酮对大鼠卵巢储备功能减退的改善作用

Improving Effect of Total Flavones from Semen Cuscutae on Hypofunction of Ovarian Reserve in Rats

【目的】探讨菟丝子总黄酮通过卵泡刺激素(FSH)-环磷酸腺苷(c AMP)信号轴对大鼠卵巢储备功能减退的改善作用。【方法】从68只雌性SD大鼠中抽取10只为正常组,其余构建卵巢储备功能减退模型。将成功建模的48只大鼠随机分为抑制剂组、模型组、抑制剂+菟丝子总黄酮组、菟丝子总黄酮组,每组12只。抑制剂组灌胃H-89 0.1 mg/kg,抑制剂+菟丝子总黄酮组灌胃H-89 0.1 mg/kg+菟丝子总黄酮530.1 mg/kg,菟丝子总黄酮组灌胃菟丝子总黄酮530.1 mg/kg,模型组、正常组灌胃等体积生理盐水,均连续4周,每周休息1 d。给药4周后,称取各组大鼠卵巢湿质量,计算卵巢指数,酶联免疫吸附分析(ELISA)检测大鼠血清性激素水平,苏木素-伊红(HE)染色观察卵巢组织形态学变化,ELISA法检测卵巢组织中cAMP含量,蛋白免疫印迹(Western Blot)法检测卵巢组织卵泡刺激素受体(FSHR)、蛋白激酶A(PKA)及磷酸化PKA(p-PKA)表达。【结果】模型组大鼠进食、二便、毛色、动情周期出现异常,卵巢萎缩明显,卵泡数量、黄体减少;与模型组比较,抑制剂组大鼠进食、二便、毛色、动情周期异常情况更严重,卵巢萎缩更明显,卵泡与黄体数量更少,而抑制剂组+菟丝子总黄酮组、菟丝子总黄酮组大鼠进食、二便、毛色、动情周期,卵巢、卵泡与黄体数量明显改善。与正常组比较,抑制剂组、模型组、抑制剂+菟丝子总黄酮组、菟丝子总黄酮组的左侧和右侧卵巢湿质量及卵巢指数降低,FSH、FSH/LH、E;水平升高,卵巢组织中cAMP含量降低,FSHR、p-PKA蛋白相对表达量降低(P<0.05);与抑制剂组比较,模型组、抑制剂+菟丝子总黄酮组、菟丝子总黄酮组的左侧和右侧卵巢湿质量及卵巢指数升高,FSH、FSH/LH、E;水平降低,卵巢组织中cAMP含量升高,FSHR、p-PKA蛋白相对表达量升高(P<0.05);与模型组比较,抑制剂+菟丝子总黄酮组、菟丝子总黄酮组的左侧和右侧卵巢湿质量及卵巢指数升高,FSH、FSH/LH、E;水平降低,卵巢组织中c AMP含量升高,FSHR、p-PKA蛋白相对表达量升高(P<0.05);与抑制剂+菟丝子总黄酮组比较,菟丝子总黄酮组的左侧和右侧卵巢湿质量及卵巢指数升高,FSH、FSH/LH、E;水平降低,卵巢组织中cAMP含量升高,FSHR、p-PKA蛋白相对表达量升高(P<0.05)。【结论】菟丝子总黄酮可能通过FSH-cAMP信号轴改善大鼠卵巢储备功能减退。

Objective To explore the improvement effect of total flavones from Semen Cuscuta on hypofunction of ovarian reserve in rats through follicle stimulating hormone(FSH)-cyclic adenosine monophosphate(cAMP)signa axis.Methods Ten from sixty-eight female SD rats were taken out as the normal group,the rest were used to construct ovarian reserve dysfunction model.Forth-eight successfully modeled rats were randomly divided into inhibitor group,model group,inhibitor+total flavones from Semen Cuscuta group and total flavones from Semen Cuscuta group,with 12 rats in each group.The inhibitor group was given gastric gavage of H-89 0.1 mg/kg,the inhibitor+total flavones from Semen Cuscuta group was given gastric gavage of H-89 0.1 mg/kg plus total flavones from Semen Cuscuta 530.1 mg/kg,the total flavones from Semen Cuscuta group was given gastric gavage of 530.1 mg/kg,the model group and the control group were given gastric gavage of equal volume of normal saline.All the treatments lasted for 4 consecutive weeks and one day rest per week.After 4 weeks of administration,the wet mass of ovaries of rats in each group was weighed and the ovarian index was calculated,the serum sex hormone levels of rats were detected by enzyme-linked immunosorbent assay(ELISA),the morphological changes of ovarian tissue were observed by hematoxylin-eosin(HE)staining,Western Blot was used to detect the expression of follicle stimulating hormone receptor(FSHR),protein kinase A(PKA)and phosphorylated PKA(p-PKA)in ovarian tissue.Results Rats in the model group showed anomalies of feeding,defecation,hair color and estrous cycle,obvious atrophy of ovary,decreased number of follicles and luteum;compared with the model group,the anomalies of feeding,defecation,hair color and estrous cycle was much severer,atrophy of ovary was more obvious,number of follicles and luteum was more decreased in the inhibitor group,while the condition of feeding,defecation,hair color and estrous cycle,atrophy of ovary,number of follicles and luteum in the inhibitor plus total flavones from Semen Cuscuta group and total flavones from Semen Cuscuta group were significantly improved.Compared with the normal group,the wet mass and ovarian index of left and right ovaries in the inhibitor group,model group,inhibitor plus total flavones from Semen Cuscuta group and total flavones from Semen Cuscuta group were decreased,the levels of FSH,FSH/LH and E;were increased,the content of cAMP in ovarian tissue was decreased,and protein relative expression contents of FSHR and p-PKA were decreased(P<0.05);compared with the inhibitor group,the wet mass and ovarian index of left and right ovaries in the model group,inhibitor+total flavones from Semen Cuscuta group and total flavones from Semen Cuscuta group were increased,the levels of FSH,FSH/LH and E;were decreased,the content of cAMP in ovarian tissue was increased,and the protein relative expression contents of FSHR and p-PKA were increased(P<0.05);compared with the model group,the wet mass and ovarian index of left and right ovaries in the inhibitor plus total flavones from Semen Cuscuta group and total flavones from Semen Cuscuta group were increased,the levels of FSH,FSH/LH and E;were decreased,the content of cAMP in ovary was increased,and the protein relative expression contents of FSHR and p-PKA were increased(P<0.05);compared with the inhibitor plus total flavones from Semen Cuscuta group,the wet mass and ovarian index of left and right ovaries in the total flavones from Semen Cuscuta group were increased,the levels of FSH,FSH/LH and E;were decreased,the content of cAMP in ovary was increased,and the protein relative expression contents of FSHR and p-PKA were increased(P<0.05).Conclusion Total flavones from Semen Cuscuta may improve the hypofuntion of ovarian reserve function in rats through FSH-cAMP signal axis.