瓜石汤对初老雌性大鼠卵巢储备功能影响的实验研究
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摘要
目的：探索中药复方瓜石汤对初老雌性大鼠卵巢储备功能的影响及其作用机理,为改善卵巢储备功能下降、延缓卵巢衰老、增进绝经前期妇女身体健康、防治相关病症提供新的思路。
方法：选4～6月龄雌性SD大鼠为正常对照组。10～12月龄雌性SD大鼠、阴道细胞学涂片显示动情周期延长者为初老大鼠模型来模拟人女性卵巢储备功能下降阶段；将初老模型大鼠随机分为5组：模型组、瓜石汤高剂量组、瓜石汤中剂量组、六味地黄丸组和金匮肾气丸组。全部实验大鼠灌药4周选择动情间期称重,断头取血,离心取上清液,采用放射免疫分析法测定血清中E2、P和IGF-1水平,采用酶联免疫吸附法检测血清FSH和VEGF;迅速剖腹取出卵巢、子宫、脾脏、肾上腺,剔除周围组织,滤纸稍拭后称重,计算脏器指数；子宫、卵巢标本均用4%多聚甲醛溶液固定,常规脱水,浸蜡包埋,切片4μm,HE染色,普通光学显微镜观察子宫、卵巢组织形态学变化,用免疫组织化学法检测卵巢中INHB、ER、FSHR、Bcl-2和Caspase-3的表达情况。
结果：在血清检测方面：与正常对照组比较,模型组大鼠的血清E2、P水平明显降低(P<0.05)；经瓜石汤治疗后,初老模型大鼠的血清E2、P水平显著提高,与模型组比较差异有统计学意义(P<0.05)；六味地黄丸组和金匮肾气丸组有升高初老大鼠血清E2、P水平的趋势,但与模型组比较差异无统计学意义(P>0.05)；各组大鼠血清FSH比较差异均无统计学意义(P>0.05)。瓜石汤、六味地黄丸和金匮肾气丸均可不同程度增加初老模型大鼠的子宫、卵巢、脾脏、肾上腺的指数,能显著升高初老模型大鼠血清VEGF和IGF-1的水平,与模型组比较均有显著性差异(P<0.05)。
大鼠卵巢组织形态学的改变：正常对照组大鼠卵巢形态结构正常,卵泡生长活跃,可见各期卵泡及黄体组织；模型组大鼠卵巢可见各级卵泡但数量明显减少,成熟卵泡及黄体组织偏少,血管管径变细,与正常对照组比较有显著性差异(P<0.05)；瓜石汤中剂量组、瓜石汤高剂量组、六味地黄丸组和金匮肾气丸组可见各级卵泡和黄体组织,血管基本正常,与模型组比较其差异均有统计学意义(P<0.05)。与模型组比较,各用药组大鼠的卵巢血管腔面积增加,血管壁厚度面积比降低,提示各用药组初老大鼠卵巢血供均有所增加。
在Bcl-2、Caspase-3蛋白表达方面：模型组大鼠卵巢Bcl-2蛋白表达较正常对照组明显下降,其Caspase-3蛋白的表达较正常组明显升高,两组间比较均有极显著性差异(P<0.01)；六味地黄丸组、金匮肾气丸组和瓜石汤高剂量组均可以升高初老模型大鼠卵巢Bcl-2、降低Caspase-3蛋白的表达,与模型组比较均有显著性差异(P<0.05)。在INHB蛋白表达方面：模型组大鼠卵巢INHB的表达较正常对照组明显降低,两组间比较有极显著性差异(P<0.01)；六味地黄丸组和瓜石汤高剂量组均可以明显升高初老大鼠卵巢INHB蛋白的表达,与模型组比较均有显著性差异(P<0.05)。瓜石汤和补肾中药还可以上调初老模型大鼠卵巢ERp和FSHR的表达,与模型组比较均有显著性差异(P<0.05),但均不能达到正常对照组水平。
结论：①瓜石汤和补肾中药都能升高初老模型大鼠血清E2、P水平,可能存在改善初老大鼠卵巢生殖内分泌功能的作用。②细胞凋亡蛋白Bcl-2、Caspase-3及卵巢局部调节因子INHB与卵巢储备功能密切相关,瓜石汤和补肾中药可以上调初老大鼠Bcl-2、INHB,下调Caspase-3蛋白的表达,从而抑制卵巢颗粒细胞凋亡、减少卵泡闭锁。③瓜石汤和补肾中药还具有增加卵巢局部血供,改善卵巢局部微循环,调节初老卵巢功能的作用。以上这些都可能是瓜石汤和补肾中药改善卵巢储备功能下降、延缓初老模型大鼠卵巢生殖机能衰老的作用机制。
Objective:To discuss the effect and mechanism of Gua Shi Tang to improve ovary reserve of the primary senile rat ovary.The study may offer a new thought to improve ovary store founction, delay the age of ovary, improve premenopausal women health, prevent and treat relative diseases.
Methods:4～6 months old SD female rats were set to be normal control group and 10～12 months old SD female rats whose oestrus elongation were set as primary senile female rat model. Primary senile female rats were devided into 5 groups randomly as following:Model group; High dose GuaShiTang group; Middle dose GuaShiTang group; Liuwei dihuang Wan group; Ginkui shenqi Wan group. After 4 weeks, weighed and sacrificed the rats by decapitation during anestrum underlying the result of vagina cell smear.Get blood by decapitation, centrifugalization, separating serum, placed them respectively, determined the level E2, P, IGF-1, FSH and VEGF by means of radio immunoassay and enzyme-linked immunosorbent assay, dissected and got rats ovary, uterus, spleen and paranephros quikly,rejected the surrounding tissues,slightly wiped filter paper,weighed and calculated the ponderal index. Fixed ovary and uterus in 4% polyoxymethylene, dehydrated normally, soaked with wax,embedding,slice in 4μm,HE dyeing,observed the constructure of uterus and ovarian histomorphology under light microscope. The ovaries were treated by immune histochemistry method to observe the expression of INHB, ER, FSHR, Bcl-2 and Caspase-3
Results:The contents of serum:Compared with normal control group,the contents of serum E2 and P in model group are obviously decreased (P<0.05).Treated with GuaShiTang, the contents of serum E2 and P raised obviously in middle dose and high dose group,compared with model group,there are statistical significance (P<0.05); Liuwei dihuang Wan group and Ginkuishenqi Wan group could increase the contents of serum E2 and P, while compared with model group, there are no obviously statistical significance (P>0.05); GuaShiTang, Liu wei dihuang Wan and Ginkuishenqi Wan had no obvious influence to FSH. GuaShiTang, Liuweidihuang Wan and Ginkuishenqi Wan could increase the ponderal index of ovary, uterus, spleen and paranephros, raise the contents of serum VEGF and IGF-1 obviously, as compared with model group, there are significant difference (P<0.05).
Histomorphology changes in ovary of rats:the appearance of ovary is normal,grades of follicles and corepus luteums can be seen on it in normal control group.While in model group,there can be seen very less grades of follicles, mature follicles and corepus luteums are less,the caliber of vessels become thinner, compared with normal control group,there are statistical significance (P<0.05).There can be seen grades of follicles on it in the middle dose and high dose groups of GuaShiTang, Liuweidihuang Wan group and Ginkuishenqi Wan group, the blood vessel in the ovary is normal basically,grades of follicles can be seen on these groups, compared with model group,there are statistical significance (P<0.05). Compared with model group, the hilus vessel wall in other groups'rats became thinner and its cavity expanded to increase blood supply to the ovary.
The expression of Bcl-2, Caspase-3:Compared with normal control group? the model group has a very significant difference (P<0.01); As compared with the model group, Liuweidihuang Wan group, Ginkui shenqi Wan group and the high dose GuaShiTang group have significant difference (P<0.05). The expression of INHB:compared with normal control group, the model group has a very significant difference (P<0.01); as compared with the model group, Liuweidihuang Wan group and the high dose GuaShiTang group have significant difference (P<0.05). Compared with the model group, GuaShiTang could increase the expression of ERβ和FSHR and there are statistical significance (P<0.05), but not to the degree of the normal control group.
Conclusion:①GuashiTang and nourishing kidney herbs might have the effect of improving ovaries function and raising the contents of serum E2 and P.②poptosis regulator Bcl-2、Caspase-3 protein and INHB are closely related with the ovarian reserve. The inhibition of ovary apoptosis and the reduction of follicular atresia are potential mechanism of improvement of ovary reservation.③Gua Shi Tang can delay ovary aging possibly by regulating vasomotion and vascularization to improve blood supply to the ovary in order to improve the function of the ovary. These might be the mechanisms of GuaShiTang delaying the aging of ovary in primary senile rat
方法：选4～6月龄雌性SD大鼠为正常对照组。10～12月龄雌性SD大鼠、阴道细胞学涂片显示动情周期延长者为初老大鼠模型来模拟人女性卵巢储备功能下降阶段；将初老模型大鼠随机分为5组：模型组、瓜石汤高剂量组、瓜石汤中剂量组、六味地黄丸组和金匮肾气丸组。全部实验大鼠灌药4周选择动情间期称重,断头取血,离心取上清液,采用放射免疫分析法测定血清中E2、P和IGF-1水平,采用酶联免疫吸附法检测血清FSH和VEGF;迅速剖腹取出卵巢、子宫、脾脏、肾上腺,剔除周围组织,滤纸稍拭后称重,计算脏器指数；子宫、卵巢标本均用4%多聚甲醛溶液固定,常规脱水,浸蜡包埋,切片4μm,HE染色,普通光学显微镜观察子宫、卵巢组织形态学变化,用免疫组织化学法检测卵巢中INHB、ER、FSHR、Bcl-2和Caspase-3的表达情况。
结果：在血清检测方面：与正常对照组比较,模型组大鼠的血清E2、P水平明显降低(P<0.05)；经瓜石汤治疗后,初老模型大鼠的血清E2、P水平显著提高,与模型组比较差异有统计学意义(P<0.05)；六味地黄丸组和金匮肾气丸组有升高初老大鼠血清E2、P水平的趋势,但与模型组比较差异无统计学意义(P>0.05)；各组大鼠血清FSH比较差异均无统计学意义(P>0.05)。瓜石汤、六味地黄丸和金匮肾气丸均可不同程度增加初老模型大鼠的子宫、卵巢、脾脏、肾上腺的指数,能显著升高初老模型大鼠血清VEGF和IGF-1的水平,与模型组比较均有显著性差异(P<0.05)。
大鼠卵巢组织形态学的改变：正常对照组大鼠卵巢形态结构正常,卵泡生长活跃,可见各期卵泡及黄体组织；模型组大鼠卵巢可见各级卵泡但数量明显减少,成熟卵泡及黄体组织偏少,血管管径变细,与正常对照组比较有显著性差异(P<0.05)；瓜石汤中剂量组、瓜石汤高剂量组、六味地黄丸组和金匮肾气丸组可见各级卵泡和黄体组织,血管基本正常,与模型组比较其差异均有统计学意义(P<0.05)。与模型组比较,各用药组大鼠的卵巢血管腔面积增加,血管壁厚度面积比降低,提示各用药组初老大鼠卵巢血供均有所增加。
在Bcl-2、Caspase-3蛋白表达方面：模型组大鼠卵巢Bcl-2蛋白表达较正常对照组明显下降,其Caspase-3蛋白的表达较正常组明显升高,两组间比较均有极显著性差异(P<0.01)；六味地黄丸组、金匮肾气丸组和瓜石汤高剂量组均可以升高初老模型大鼠卵巢Bcl-2、降低Caspase-3蛋白的表达,与模型组比较均有显著性差异(P<0.05)。在INHB蛋白表达方面：模型组大鼠卵巢INHB的表达较正常对照组明显降低,两组间比较有极显著性差异(P<0.01)；六味地黄丸组和瓜石汤高剂量组均可以明显升高初老大鼠卵巢INHB蛋白的表达,与模型组比较均有显著性差异(P<0.05)。瓜石汤和补肾中药还可以上调初老模型大鼠卵巢ERp和FSHR的表达,与模型组比较均有显著性差异(P<0.05),但均不能达到正常对照组水平。
结论：①瓜石汤和补肾中药都能升高初老模型大鼠血清E2、P水平,可能存在改善初老大鼠卵巢生殖内分泌功能的作用。②细胞凋亡蛋白Bcl-2、Caspase-3及卵巢局部调节因子INHB与卵巢储备功能密切相关,瓜石汤和补肾中药可以上调初老大鼠Bcl-2、INHB,下调Caspase-3蛋白的表达,从而抑制卵巢颗粒细胞凋亡、减少卵泡闭锁。③瓜石汤和补肾中药还具有增加卵巢局部血供,改善卵巢局部微循环,调节初老卵巢功能的作用。以上这些都可能是瓜石汤和补肾中药改善卵巢储备功能下降、延缓初老模型大鼠卵巢生殖机能衰老的作用机制。
Objective:To discuss the effect and mechanism of Gua Shi Tang to improve ovary reserve of the primary senile rat ovary.The study may offer a new thought to improve ovary store founction, delay the age of ovary, improve premenopausal women health, prevent and treat relative diseases.
Methods:4～6 months old SD female rats were set to be normal control group and 10～12 months old SD female rats whose oestrus elongation were set as primary senile female rat model. Primary senile female rats were devided into 5 groups randomly as following:Model group; High dose GuaShiTang group; Middle dose GuaShiTang group; Liuwei dihuang Wan group; Ginkui shenqi Wan group. After 4 weeks, weighed and sacrificed the rats by decapitation during anestrum underlying the result of vagina cell smear.Get blood by decapitation, centrifugalization, separating serum, placed them respectively, determined the level E2, P, IGF-1, FSH and VEGF by means of radio immunoassay and enzyme-linked immunosorbent assay, dissected and got rats ovary, uterus, spleen and paranephros quikly,rejected the surrounding tissues,slightly wiped filter paper,weighed and calculated the ponderal index. Fixed ovary and uterus in 4% polyoxymethylene, dehydrated normally, soaked with wax,embedding,slice in 4μm,HE dyeing,observed the constructure of uterus and ovarian histomorphology under light microscope. The ovaries were treated by immune histochemistry method to observe the expression of INHB, ER, FSHR, Bcl-2 and Caspase-3
Results:The contents of serum:Compared with normal control group,the contents of serum E2 and P in model group are obviously decreased (P<0.05).Treated with GuaShiTang, the contents of serum E2 and P raised obviously in middle dose and high dose group,compared with model group,there are statistical significance (P<0.05); Liuwei dihuang Wan group and Ginkuishenqi Wan group could increase the contents of serum E2 and P, while compared with model group, there are no obviously statistical significance (P>0.05); GuaShiTang, Liu wei dihuang Wan and Ginkuishenqi Wan had no obvious influence to FSH. GuaShiTang, Liuweidihuang Wan and Ginkuishenqi Wan could increase the ponderal index of ovary, uterus, spleen and paranephros, raise the contents of serum VEGF and IGF-1 obviously, as compared with model group, there are significant difference (P<0.05).
Histomorphology changes in ovary of rats:the appearance of ovary is normal,grades of follicles and corepus luteums can be seen on it in normal control group.While in model group,there can be seen very less grades of follicles, mature follicles and corepus luteums are less,the caliber of vessels become thinner, compared with normal control group,there are statistical significance (P<0.05).There can be seen grades of follicles on it in the middle dose and high dose groups of GuaShiTang, Liuweidihuang Wan group and Ginkuishenqi Wan group, the blood vessel in the ovary is normal basically,grades of follicles can be seen on these groups, compared with model group,there are statistical significance (P<0.05). Compared with model group, the hilus vessel wall in other groups'rats became thinner and its cavity expanded to increase blood supply to the ovary.
The expression of Bcl-2, Caspase-3:Compared with normal control group? the model group has a very significant difference (P<0.01); As compared with the model group, Liuweidihuang Wan group, Ginkui shenqi Wan group and the high dose GuaShiTang group have significant difference (P<0.05). The expression of INHB:compared with normal control group, the model group has a very significant difference (P<0.01); as compared with the model group, Liuweidihuang Wan group and the high dose GuaShiTang group have significant difference (P<0.05). Compared with the model group, GuaShiTang could increase the expression of ERβ和FSHR and there are statistical significance (P<0.05), but not to the degree of the normal control group.
Conclusion:①GuashiTang and nourishing kidney herbs might have the effect of improving ovaries function and raising the contents of serum E2 and P.②poptosis regulator Bcl-2、Caspase-3 protein and INHB are closely related with the ovarian reserve. The inhibition of ovary apoptosis and the reduction of follicular atresia are potential mechanism of improvement of ovary reservation.③Gua Shi Tang can delay ovary aging possibly by regulating vasomotion and vascularization to improve blood supply to the ovary in order to improve the function of the ovary. These might be the mechanisms of GuaShiTang delaying the aging of ovary in primary senile rat
引文
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