补肾法、疏肝法对促性腺激素预处理小鼠排卵机制影响的比较研究
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摘要
一、补肾法、疏肝法对促性腺激素预处理小鼠排卵数目的影响
目的:研究补肾调经Ⅱ号方、Ⅲ号方、逍遥丸对促性腺激素预处理小鼠排卵数目的影响,以探讨并比较补肾法、疏肝法诱发排卵的机制。
方法:雌性未成年昆明小鼠102只,随机分为17组,每组6只。其中1-4组为补肾法组,5-8组为疏肝法组,9-12组为补肾调经Ⅱ号方+逍遥丸组,13-16组为逍遥丸+补肾调经Ⅲ号方组,17组为对照组。实验第1、2、3天,除对照组外,补肾法组和补肾调经Ⅱ号方+逍遥丸组、疏肝法组和逍遥丸+补肾调经Ⅲ号方组,每只小鼠分别灌胃Ⅱ号方(高剂量或低剂量)、逍遥丸(高剂量或低剂量)0.4 ml,共3天。实验第3天13:00所有小鼠均腹腔注射PMSG(5 IU/只);实验第4、5天补肾法组和补肾调经Ⅱ号方+逍遥丸组、疏肝法组和逍遥丸+补肾调经Ⅲ号方组,每只小鼠分别灌胃Ⅲ号方(高剂量或低剂量)、逍遥丸(高剂量或低剂量)0.4 ml共2天;第5天13:00各组小鼠均腹腔注射hCG(5 IU/只)。第6天上午8:00处死小鼠,取输卵管、卵巢,体式显微镜下刺破输卵管壶腹部,计数卵子数。
结果:单因素方差分析:与对照组比较,补肾法以补肾调经Ⅱ号方高剂量与Ⅲ号方高剂量序贯使用排卵数目多(P=0.003);疏肝法以逍遥丸高剂量促进卵泡发育、逍遥丸低剂量诱发排卵序贯使用排卵数目多(P=0.007)。析因设计分析:补肾法促排卵使用补肾调经Ⅱ号方高剂量与低剂量有显著差异(P=0.004),Ⅱ号方、Ⅲ号方高剂量序贯使用排卵数目多,二者有交互作用(P=0.047)。疏肝法促排卵使用逍遥丸高、低剂量有显著差异(P=0.039),序贯应用高、低剂量排卵数目最多,二者有交互作用(P=0.046)。单因素方差分析:与对照组比较,逍遥丸+补肾调经Ⅲ号方组中逍遥丸低剂量与Ⅲ号方高剂量序贯使用排卵数增多(P=0.013),析因设计分析二者无交互作用(P=0.838);单因素方差分析:与对照组比较,Ⅱ号方高剂量与逍遥丸低剂量序贯使用排卵数多(P=0.039),析因设计分析二者无交互作用(P=0.604)。
结论:对促性腺激素预处理小鼠序贯使用补肾调经Ⅱ号方、Ⅲ号方高剂量卵巢排卵数目多,与阴精盛助卵泡发育、阳气足使阴阳转化顺利有关;逍遥丸高、低剂量序贯使用排卵数目多,与血足精充促卵泡发育、疏肝理气助阴阳转化顺利有关。两法均有交互作用,即序贯使用可提高药物疗效。
二、补肾法、疏肝法对促性腺激素预处理小鼠卵巢COX-2、PTX3、TSG-6影响的比较
目的:研究补肾法、疏肝法对促性腺激素预处理小鼠卵巢COX-2以及透明质酸结合蛋白PTX3、TSG-6表达的影响,以探讨两法对卵丘膨胀的影响而诱发排卵的机制及其异同。
方法:将雌性昆明种未成年小鼠96只随机分为正常组、对照组、补肾组、疏肝组,每组24只。实验第1、2、3天,补肾组、疏肝组小鼠分别灌服补肾调经Ⅱ号方高剂量、逍遥丸高剂量,对照组、正常组灌服蒸馏水,共3天;实验第3天8:00,补肾组、疏肝组、对照组小鼠腹腔注射PMSG(5 IU/只),48 h后腹腔注射hCG(5 IU/只),正常组在相同时间注射等体积生理盐水;实验第4、5天,补肾组、疏肝组小鼠分别灌服补肾调经Ⅲ号方高剂量、逍遥丸低剂量,对照组、正常组灌服蒸馏水,共2天。灌胃剂量均为0.4 ml/只。各组小鼠均在注射hCG后0、4、8、12 h处死,取双侧卵巢,液氮保存备检。RT-PCR检测COX-2、PTX-3 mRNA在卵巢的表达,免疫组化法检测COX-2、TSG-6蛋白在卵巢的表达。
结果:
1各组促性腺激素预处理小鼠在注射hCG后0、4、8、12 h卵巢COX-2 mRNA及其蛋白的表达注射hCG后0、4、8 h,各组小鼠卵巢COX-2表达呈升高趋势,12 h均下降。注射hCG后0 h:与对照组比较,疏肝组、补肾组小鼠卵巢COX-2表达增强,有统计学意义(P<0.05)。与疏肝组比较,补肾组小鼠卵巢COX-2表达下降但无统计学差异(P>0.05)。注射hCG后4 h:与对照组比较,疏肝组和补肾组小鼠卵巢COX-2表达均下降,有统计学意义(均P<0.05)。与疏肝组比较,补肾组小鼠卵巢COX-2表达下降但无统计学差异(P>0.05)。注射hCG后8 h:与对照组比较,疏肝组小鼠卵巢COX-2表达增强,有统计学意义(P<0.01);补肾组小鼠卵巢COX-2表达降低,有统计学意义(P<0.05)。与疏肝组比较,补肾组小鼠卵巢COX-2表达降低,有统计学意义(P<0.01)。注射hCG后12 h:与对照组比较,疏肝组小鼠卵巢COX-2表达增强,有统计学意义(P<0.05);补肾组小鼠卵巢COX-2无统计学差异(P>0.05)。与疏肝组比较,补肾组小鼠卵巢COX-2表达降低,有统计学意义(P<0.05)。2各组促性腺激素预处理小鼠在注射hCG后0、4、8、12 h卵巢TSG-6蛋白的表达各时间点对照组小鼠卵巢TSG-6蛋白表达逐渐增强,各时间点间比较,除4h、8h之间无统计学差异(P>0.05)外其余时间点间比较均有统计学差异(P<0.01)。疏肝组各时间点小鼠卵巢TSG-6蛋白表达逐渐增强,各时间点之间比较,除8h、12h之间比较无统计学差异(P>0.05)外其余时间点间比较均有统计学差异(P<0.01)。补肾组各时间点小鼠卵巢TSG-6蛋白表达逐渐增强,各时间点之间比较有统计学意义(P<0.01)。注射hCG后0 h:与对照组比较,疏肝组、补肾组小鼠卵巢TSG-6蛋白表达无统计学差异(P>0.05)。注射hCG后4 h:与对照组比较,疏肝组、补肾组小鼠卵巢TSG-6蛋白表达无统计学差异(P>0.05)。与疏肝组比较,补肾组小鼠卵巢TSG-6蛋白表达增强,有统计学意义(P<0.05)。注射hCG后8 h:与对照组比较,疏肝组、补肾组小鼠卵巢TSG-6蛋白表达均增强,有统计学意义(P<0.01)。与疏肝组比较,补肾组小鼠卵巢TSG-6蛋白表达降低,无统计学差异(P>0.05)。注射hCG后12h:与对照组比较,疏肝组、补肾组小鼠卵巢TSG-6蛋白表达均增强,有统计学意义(P<0.05,P<0.01)。与疏肝组比较,补肾组小鼠卵巢TSG-6蛋白表达增强,有统计学意义(P<0.01)。
3各组促性腺激素预处理小鼠在注射hCG后0、4、8、12 h卵巢PTX-3 mRNA的表达注射hCG后0、4、8 h,对照组小鼠卵巢PTX3 mRNA表达逐渐增强,各组之间均有统计学意义(P<0.01);注射hCG后12h,小鼠卵巢PTX3 mRNA表达降低,与注射hCG后8 h比较有统计学意义(P<0.01)。注射hCG后0、4 h:与对照组比较,疏肝组、补肾组小鼠卵巢PTX3 mRNA表达增强,有统计学意义(P<0.01)。与疏肝组比较,补肾组小鼠卵巢PTX3 mRNA表达降低,有统计学意义(P<0.01)。注射hCG后8 h:与对照组比较,疏肝组、补肾组小鼠卵巢PTX3 mRNA的表达降低,有统计学意义(P<0.01)。与疏肝组比较,补肾组小鼠卵巢PTX3 mRNA表达降低,有统计学意义(P<0.01)。注射hCG后12 h:与对照组比较,疏肝组、补肾组小鼠卵巢PTX3 mRNA的表达增强,有统计学意义(P<0.01)。与疏肝组比较,补肾组小鼠卵巢PTX3 mRNA表达降低,无统计学差异(P>0.05)。
结论:补肾法、疏肝法对促性腺激素预处理小鼠排卵前卵巢COX-2 mRNA及其蛋白的作用机制不同,疏肝法主要上调卵巢COX-2 mRNA及其蛋白的表达,补肾法主要是下调卵巢COX-2 mRNA及其蛋白的表达。补肾法、疏肝法对卵巢TSG-6、PTX3作用机制相似,均可上调排卵前卵巢TSG-6蛋白、PTX3 mRNA的表达,从而促进了卵丘细胞外基质形成,使卵丘充分膨胀有利于排卵。疏肝法、补肾法上调TSG-6蛋白的表达可能是使排卵数目增多的原因之一。
三、补肾法、疏肝法对促性腺激素预处理小鼠卵巢ADAMTS-1、Cat L影响的比较
目的:研究补肾法、疏肝法对促性腺激素预处理小鼠卵巢ADAMTS-1、Cat L mRNA及其蛋白表达的影响,以探讨两种治法对排卵过程中促使卵泡壁破裂机制的排卵机制的异同。方法:动物分组及取材同第二部分。RT-PCR检测卵巢ADAMTS-1、Cat L mRNA的表达,Western blot检测卵巢ADAMTS-1、Cat L蛋白的表达,免疫组化检测卵巢PR、ADAMTS-1、Cat L蛋白的表达。
结果:
1各组促性腺激素预处理小鼠在注射hCG后0、4、8、12h卵巢PR蛋白的表达注射hCG后0h、8h、12h,与对照组比较,疏肝组、补肾组小鼠卵巢PR蛋白表达均增强,有统计学意义(P<0.01)。注射hCG 4 h,与对照组比较,疏肝组、补肾组小鼠卵巢PR蛋白表达均无统计学差异(P>0.05)。疏肝组与补肾组之间在4个时间点比较,小鼠卵巢PR蛋白表达均无统计学差异(P>0.05)。
2各组促性腺激素预处理小鼠在注射hCG后0、4、8、12h卵巢ADAMTS-1 mRNA及其蛋白的表达4个时间点各组小鼠卵巢ADAMTS-1表达呈上升趋势,12h达高峰。注射hCG后0 h:各组小鼠卵巢ADAMTS-1表达不明显。注射hCG后4 h:与对照组比较,疏肝组、补肾组小鼠卵巢ADAMTS-1表达增强,有统计学意义(P<0.01,P<0.05)。与疏肝组比较,补肾组小鼠卵巢ADAMTS-1表达降低但无统计学差异(P>0.05)。注射hCG后8 h:与对照组比较,疏肝组小鼠卵巢ADAMTS-1表达增强,有统计学意义(P<0.01);补肾组小鼠卵巢ADAMTS-1表达降低但无统计学差异(P>0.05)。与疏肝组比较,补肾组小鼠卵巢ADAMTS-1表达降低,有统计学意义(P<0.01)。注射hCG后12 h:与对照组比较,疏肝组小鼠卵巢ADAMTS-1表达无统计学差异(P>0.05);补肾组卵巢ADAMTS-1表达增强,有统计学意义(P<0.01)。与疏肝组比较,补肾组小鼠卵巢ADAMTS-1表达增强,有统计学意义(P<0.01)。3各组促性腺激素预处理小鼠在注射hCG后0、4、8、12 h卵巢Cat L mRNA及其蛋白的表达4个时间点各组小鼠卵巢Cat L表达呈上升趋势,12h达高峰。注射hCG后0 h:各组小鼠卵巢Cat L表达均不明显。注射hCG后4 h:与对照组比较,疏肝组、补肾组小鼠卵巢Cat L表达均增强,有统计学意义(P<0.01)。与疏肝组比较,补肾组小鼠卵巢Cat L表达较低但无统计学差异(P>0.05)。注射hCG后8 h:与对照组比较,疏肝组小鼠卵巢Cat L表达增强,有统计学意义(P<0.01);补肾组无统计学差异(P>0.05)。与疏肝组比较,补肾组小鼠卵巢Cat L表达降低,有统计学意义(P<0.01)。注射hCG后12 h:与对照组比较,疏肝组小鼠卵巢Cat L表达增强但无统计学差异(P>0.05);补肾组小鼠卵巢Cat L表达增强,有统计学意义(P<0.01)。与疏肝组比较,补肾组小鼠卵巢Cat L表达增强,有统计学意义(P<0.015)。
结论:补肾法、疏肝法均可上调促性腺激素预处理小鼠排卵前卵巢PR蛋白的表达,在注射hCG后12h,补肾法上调PR蛋白表达的作用较疏肝法强。补肾法、疏肝法均可上调促性腺预处理小鼠卵巢排卵关键蛋白水解酶ADAMTS-1、Cat L mRNA及其蛋白的表达,促进了排卵斑的形成、卵泡破裂而有利于排卵。补肾法两种蛋白酶表达高峰在排卵时(注射hCG后12h),疏肝法两种蛋白酶表达在排卵前(注射hCG后8h)迅速达到高水平并缓慢增高至排卵时。补肾法卵巢ADAMTS-1、Cat LmRNA及其蛋白的表达高峰高于疏肝法。据此可指导补肾法、疏肝法的临床用药时点。
PartⅠEffects of Bushen Treatment and Shugan Treatment on Ovulation Number in Gonadotropin Pretreatment Mice
Objective: To study the effects of BushenTiaojingⅡRecipe,ⅢRecipe and Xiaoyao Pill on ovulation number of Gonadotropin pretreatment mice.
Methods: One hundred and two female immature mice were randomly divided into 17 groups with 6 mice each group. 1 to 4 were Bushen Treatment groups, 5 to 8 were Shugan Treatment groups, 9 to 12 were BushenTiaojingⅡRecipe add Xiaoyao Pill groups, 13 to 16 were Xiaoyao Pill add BushenTiaojingⅢRecipe groups, 17 was control group. All groups except control group were treated by BushenTiaojingⅡRecipe (high or low does) or Xiaoyao Pill (high or low does) 0.4 ml or during the 1-3 day to promot follicular development. All groups were injected PMSG (5IU/each) by intraperitoneal at 1pm 3rd day, and then with HCG (5 IU/each) after 48 h. All groups except control group were treated with 0.4 ml BushenTiaojingⅢRecipe (high or low does) or Xiaoyao Pill (high or low dose) on the 4-5 day. Mice were executed at 8am of the 6th day, and ampulla portion of uterine tubes were punctured with somatotype microscope to count the number of ovulated oocytes.
Results: Compared with control group by One way ANOVA, ovulation number was more in BushenTiaojingⅡRecipe high-dose add BushenTiaojingⅢRecipe high-dose of Bushen Treatment(P=0.003) and Xiaoyao Pill high-dose group add Xiaoyao Pill low-dose group of Shugan Treatment(P=0.007). Factorial experiment design analysis in each group, treatment BushenTiaojingⅡRecipe high-dose and low-dose group had significant difference(P=0.004), and treatment Xiaoyao Pill high-dose group and low-dose group in induced ovulation had significant differenc(eP=0.039), and they have interaction(P=0.047, P=0.046). Compared with control group by one way ANOVA, ovulation number in Xiaoyao Pill low-does addⅢRecipe high-does group andⅡRecipe high-does add Xiaoyao Pill low-does group were significantly more(P=0.013, P=0.039), but they had no interaction by factorial design analysis(P=0.838, P=0.604).
Conclusions: Ovulation number in BushenTiaojingⅡaddⅢRecipe high-dose group was more, related to YinJing was magnificent to promote follicle’s development and YangQi was easy to transformation between Yin and Yang. Xiaoyao Pill high-dose promoted follicular development, while low-dose induced ovulation, and ovulation number in this group was the most. Jing and Xue was magnificent to ovarian follicle’s development; adjusted liver was useful to transformation between Yin and Yang. Two methods had interaction, so can improve curative effect.
PartⅡEffects of Bushen Treatment and Shugan Treatment on COX-2、PTX3、TSG-6 in Gonadotropin Pretreatment Mice Ovary
Objective: To study and compare the effects of Bushen Treatment and Shugan Treatment on COX-2、PTX3、TSG-6 of gonadotropin pretreatment mice ovary in ovulatory processes.
Methods: Ninety-six female immature KM mice were randomly divided into normal group, control group, Bushen group and Shugan group of 4 mice. Bushen group and Shugan group were treated with BushenTiaojingⅡRecipe high-dose and Xiaoyao Pill high-dose respectively for 3 days from experiment started, while normal group and control group gave distilled water. All groups except normal group were injected intraperitoneally with PMSG (5 IU/each) at 8am on the 3rd day, and 48h later injected with hCG (5 IU), and the normal group injected with distilled water at the same time. On the 4-5 day, Bushen group and Shugan group were treated with BushenTiaojingⅢRecipe and Xiaoyao Pill low-does 0.4 ml respectively, and normal group and control group were treated with distilled water 0.4 ml. Mice were executed respectively at 0h、4h、8h、12h after injecting hCG, and ovaries were preserved in liquid nitrogen. Examined expression of COX-2、PTX-3 mRNA with RT-PCR and COX-2、TSG-6 protein with Immunohistochemical method.
Results:
1 Expression of COX-2 mRNA and protein in each group at 0h, 4h, 8h, 12 h after injecting hCG Expression of COX-2 was increased at 0h、4h、8h after injecting hCG,and decreased at 12h. Compared with control group, expression of COX-2 in Bushen group and Shugan group were significantly increased(P<0.05). Expression of COX-2 in Bushen group was decreased but had no significant difference(P>0.05)at 0h after injecting hCG. At 4 h, expression of COX-2 in Bushen group and Shugan group were significantly decreased(P<0.05)when compared with control group. Expression of COX-2 in Bushen group was decreased but had no significant difference(P>0.05)when compared with Shugan group. At 8h, compared with control group, expression of COX-2 in Shugan group were significantly increased(P<0.01), and Bushen group was decreased(P<0.05). Compared with Shugan group, expression of COX-2 in Bushen group was significantly decreased(P<0.01). At 12h, compared with control group, expression of COX-2 in Shugan group were significantly increased(P<0.05), Bushen group was decreased but had no significant difference(P>0.05). Compared with Shugan group, expression of COX-2 in Bushen group was significantly decreased(P<0.05). 2 Expression of TSG-6 protein expression in each group at 0h, 4h, 8h, 12h after injecting HCG Expression of TSG-6 protein in control group was gradually increased, but had no significant difference between 4h and 8h(P>0.05). Expression TSG-6 protein in Shugan group was gradually increased, but had no significant difference between 8h and 12h(P>0.05). At 0h, expression TSG-6 protein in Bushen group was gradually increased, and all group had significant difference(P<0.01). Expression TSG-6 protein in Bushen group and Shugan group had no significant difference(P>0.05)when compared with control group. At 4h, expression TSG-6 protein in Bushen group and Shugan group had no significant difference(P>0.05)when compared with control group, and in Bushen group was significantly increased(P<0.05)when compared with Shugan group. At 8h, expression TSG-6 protein in Bushen group and Shugan group were significantly increased(P<0.01)when compared with control group, and in Bushen group was decreased but had no significant differenc(eP>0.05)when compared with Shugan group. At 12h, expression TSG-6 protein in Bushen group and Shugan group were significantly increased(P<0.05, P<0.01)when compared with control group, and expression TSG-6 protein in Bushen group was significantly increased(P<0.01)when compared with Shugan group.
3 Expression of PTX-3 mRNA in each group at 0h, 4h, 8h, 12h after injecting hCG Expression of PTX-3 mRNA in control group was gradually increased, and had significant difference among 0h、4h、8h after injecting hCG(P<0.01). Expression of PTX-3 mRNA at 12 h was significantly decreased(P<0.01)when compared with that at 8h. At 0h、4h, expression of PTX-3 mRNA in Bushen group and Shugan group were significantly increased(P<0.01 ) when compared with control group, and in Bushen group was significantly decreased(P<0.01)when compared with Shugan group. At 8h, expression of PTX-3 mRNA in Bushen group and Shugan group were significantly decreased(P<0.01)when compared with control group, and in Bushen group was significantly decreased(P<0.01)when compared with Shugan group. At 12h, expression of PTX-3 mRNA in Bushen group and Shugan group were significantly increased(P<0.01)when compared with control group, and in Bushen group had no significant difference(P>0.05)when compared with Shugan group.
Conclusions: Bushen Treatment can up-regulate COX-2 expression, while Shugan Treatment reduce it. Both of Bushen Treatment and Shugan Treatment could up-regulate expression of TSG-6 protein and PTX3 mRNA. Both of Bushen Treatment and Shugan Treatment could up-regulate expression of TSG-6 protein, and this maybe one reason to influence ovulation number.
PartⅢEffects of Bushen Treatment and Shugan Treatment on ADAMTS-1、Cat L in Gonadotropin pretreatment mice Ovary
Objective: To study the effects of Bushen Treatment and Shugan Treatment on the expression of ADAMTS-1、Cat L mRNA and protein of Gonadotropin pretreatment mice. To analyze the mechanisms of those two treatments in prompting follicle break.
Methods: Group and materials are the same as that in partⅡ. Reverse transcription-polymerase chain reaction (RT-PCR) was used to semiquantitatively detect the expression of ADAMTS-1 mRNA and Cat L mRNA during ovulation, and Western blot was used to detect the expression of the protein. Immunohistochemical method was used to detect the expression of PR, ADAMTS-1 and Cat L protein.
Results:
1 Expression of PR protein in each group ovary at 0h, 4h, 8h, 12h after injecting hCG At 0h, 8h and 12h after injecting hCG, expression of PR in Bushen group and Shugan group ovary were significantly increased(P<0.01)when compared with control group. At 4h, expression PR protein in Bushen group and Shugan group had no significant difference(P>0.05)when compared with control group. At 0h, 4h, 8h, 12h after injecting hCG, expression of PR protein in Bushen group had no significant difference(P>0.05)when compared with Shugan group. 2 Expression of ADAMTS-1 mRNA and its protein in each group ovary at 0h, 4h, 8h, 12h after injecting hCG Expression of ovary ADAMTS-1 at these four timings was gradually increased, and was the highest at 12h. Expression of ADAMTS-1 in each group ovary was low at 0h. At 4h, expression of ADAMTS-1 in Bushen group and Shugan group was significantly increased(P<0.05 or P<0.01)when compared with control group. Expression of ADAMTS-1 in Bushen group was decreased, but had no significant difference(P>0.05)when compared with Shugan group. At 8h, compared with control group, expression of ADAMTS-1 in Shugan group was significantly increased(P<0.01), and in Bushen group had no significant difference ( P > 0.05 ) . Compared with Shugan group, expression of ADAMTS-1 in Bushen group was significantly decreased(P<0.01). At 12h, compared with control group, expression of ADAMTS-1 in Shugan group had no significant difference(P>0.05), and in Bushen group was significantly increased(P<0.01). Expression of ADAMTS-1 in Bushen group was significantly increased(P<0.01)when compared with Shugan group. 3 Expression of Cat L mRNA and protein in each group ovary a at 0h, 4h, 8h, 12h after injecting hCG Expression of Cat L in each group ovary at these four timings was gradually increased, and was the highest at 12h. Expression of Cat L in each group ovary was low. At 4h, expression of Cat L in Bushen group and Shugan group was significantly increased(P<0.01)when compared with control group. Expression of Cat L in Bushen group was decreased, had no significant difference(P>0.05)when compared with Shugan group,. At 8h, expression of Cat L in Shugan group was significantly increased(P<0.01)when compared with control group, and in Bushen group had no significant difference(P>0.05). Compared with Shugan group, expression of Cat L in Bushen group was significantly decreased(P<0.01). At 12h, compared with control group, expression of Cat L in Shugan group was no significant difference(P>0.05), and in Bushen group was significantly increased(P<0.01). Expression of Cat L in Bushen group was significantly increased(P<0.01) when compared with Shugan group.
Conclusions: Ovary PR protein expression at preovulation of Gonadotropin pretreatment mice was increased with Bushen Treatment and Shugan Treatment. But expression of PR protein at 12h was higher in Bushen group than in Shugan group. Expression of ADAMTS-1 and Cat L of Gonadotropin pretreatment mice ovary was increased with Bushen Treatment and Shugan Treatment. Expression of ovary ADAMTS-1、Cat L was highest at ovulation (hCG injecting 12h )with Bushen Treatment, while expression was quickly increased at preovulation (hCG injecting 8h) then slow-moving to the highest at ovulation with Shugan Treatment. Peak of ADAMTS-1、Cat L expression in Bushen group was higher than in Shugan group.
目的:研究补肾调经Ⅱ号方、Ⅲ号方、逍遥丸对促性腺激素预处理小鼠排卵数目的影响,以探讨并比较补肾法、疏肝法诱发排卵的机制。
方法:雌性未成年昆明小鼠102只,随机分为17组,每组6只。其中1-4组为补肾法组,5-8组为疏肝法组,9-12组为补肾调经Ⅱ号方+逍遥丸组,13-16组为逍遥丸+补肾调经Ⅲ号方组,17组为对照组。实验第1、2、3天,除对照组外,补肾法组和补肾调经Ⅱ号方+逍遥丸组、疏肝法组和逍遥丸+补肾调经Ⅲ号方组,每只小鼠分别灌胃Ⅱ号方(高剂量或低剂量)、逍遥丸(高剂量或低剂量)0.4 ml,共3天。实验第3天13:00所有小鼠均腹腔注射PMSG(5 IU/只);实验第4、5天补肾法组和补肾调经Ⅱ号方+逍遥丸组、疏肝法组和逍遥丸+补肾调经Ⅲ号方组,每只小鼠分别灌胃Ⅲ号方(高剂量或低剂量)、逍遥丸(高剂量或低剂量)0.4 ml共2天;第5天13:00各组小鼠均腹腔注射hCG(5 IU/只)。第6天上午8:00处死小鼠,取输卵管、卵巢,体式显微镜下刺破输卵管壶腹部,计数卵子数。
结果:单因素方差分析:与对照组比较,补肾法以补肾调经Ⅱ号方高剂量与Ⅲ号方高剂量序贯使用排卵数目多(P=0.003);疏肝法以逍遥丸高剂量促进卵泡发育、逍遥丸低剂量诱发排卵序贯使用排卵数目多(P=0.007)。析因设计分析:补肾法促排卵使用补肾调经Ⅱ号方高剂量与低剂量有显著差异(P=0.004),Ⅱ号方、Ⅲ号方高剂量序贯使用排卵数目多,二者有交互作用(P=0.047)。疏肝法促排卵使用逍遥丸高、低剂量有显著差异(P=0.039),序贯应用高、低剂量排卵数目最多,二者有交互作用(P=0.046)。单因素方差分析:与对照组比较,逍遥丸+补肾调经Ⅲ号方组中逍遥丸低剂量与Ⅲ号方高剂量序贯使用排卵数增多(P=0.013),析因设计分析二者无交互作用(P=0.838);单因素方差分析:与对照组比较,Ⅱ号方高剂量与逍遥丸低剂量序贯使用排卵数多(P=0.039),析因设计分析二者无交互作用(P=0.604)。
结论:对促性腺激素预处理小鼠序贯使用补肾调经Ⅱ号方、Ⅲ号方高剂量卵巢排卵数目多,与阴精盛助卵泡发育、阳气足使阴阳转化顺利有关;逍遥丸高、低剂量序贯使用排卵数目多,与血足精充促卵泡发育、疏肝理气助阴阳转化顺利有关。两法均有交互作用,即序贯使用可提高药物疗效。
二、补肾法、疏肝法对促性腺激素预处理小鼠卵巢COX-2、PTX3、TSG-6影响的比较
目的:研究补肾法、疏肝法对促性腺激素预处理小鼠卵巢COX-2以及透明质酸结合蛋白PTX3、TSG-6表达的影响,以探讨两法对卵丘膨胀的影响而诱发排卵的机制及其异同。
方法:将雌性昆明种未成年小鼠96只随机分为正常组、对照组、补肾组、疏肝组,每组24只。实验第1、2、3天,补肾组、疏肝组小鼠分别灌服补肾调经Ⅱ号方高剂量、逍遥丸高剂量,对照组、正常组灌服蒸馏水,共3天;实验第3天8:00,补肾组、疏肝组、对照组小鼠腹腔注射PMSG(5 IU/只),48 h后腹腔注射hCG(5 IU/只),正常组在相同时间注射等体积生理盐水;实验第4、5天,补肾组、疏肝组小鼠分别灌服补肾调经Ⅲ号方高剂量、逍遥丸低剂量,对照组、正常组灌服蒸馏水,共2天。灌胃剂量均为0.4 ml/只。各组小鼠均在注射hCG后0、4、8、12 h处死,取双侧卵巢,液氮保存备检。RT-PCR检测COX-2、PTX-3 mRNA在卵巢的表达,免疫组化法检测COX-2、TSG-6蛋白在卵巢的表达。
结果:
1各组促性腺激素预处理小鼠在注射hCG后0、4、8、12 h卵巢COX-2 mRNA及其蛋白的表达注射hCG后0、4、8 h,各组小鼠卵巢COX-2表达呈升高趋势,12 h均下降。注射hCG后0 h:与对照组比较,疏肝组、补肾组小鼠卵巢COX-2表达增强,有统计学意义(P<0.05)。与疏肝组比较,补肾组小鼠卵巢COX-2表达下降但无统计学差异(P>0.05)。注射hCG后4 h:与对照组比较,疏肝组和补肾组小鼠卵巢COX-2表达均下降,有统计学意义(均P<0.05)。与疏肝组比较,补肾组小鼠卵巢COX-2表达下降但无统计学差异(P>0.05)。注射hCG后8 h:与对照组比较,疏肝组小鼠卵巢COX-2表达增强,有统计学意义(P<0.01);补肾组小鼠卵巢COX-2表达降低,有统计学意义(P<0.05)。与疏肝组比较,补肾组小鼠卵巢COX-2表达降低,有统计学意义(P<0.01)。注射hCG后12 h:与对照组比较,疏肝组小鼠卵巢COX-2表达增强,有统计学意义(P<0.05);补肾组小鼠卵巢COX-2无统计学差异(P>0.05)。与疏肝组比较,补肾组小鼠卵巢COX-2表达降低,有统计学意义(P<0.05)。2各组促性腺激素预处理小鼠在注射hCG后0、4、8、12 h卵巢TSG-6蛋白的表达各时间点对照组小鼠卵巢TSG-6蛋白表达逐渐增强,各时间点间比较,除4h、8h之间无统计学差异(P>0.05)外其余时间点间比较均有统计学差异(P<0.01)。疏肝组各时间点小鼠卵巢TSG-6蛋白表达逐渐增强,各时间点之间比较,除8h、12h之间比较无统计学差异(P>0.05)外其余时间点间比较均有统计学差异(P<0.01)。补肾组各时间点小鼠卵巢TSG-6蛋白表达逐渐增强,各时间点之间比较有统计学意义(P<0.01)。注射hCG后0 h:与对照组比较,疏肝组、补肾组小鼠卵巢TSG-6蛋白表达无统计学差异(P>0.05)。注射hCG后4 h:与对照组比较,疏肝组、补肾组小鼠卵巢TSG-6蛋白表达无统计学差异(P>0.05)。与疏肝组比较,补肾组小鼠卵巢TSG-6蛋白表达增强,有统计学意义(P<0.05)。注射hCG后8 h:与对照组比较,疏肝组、补肾组小鼠卵巢TSG-6蛋白表达均增强,有统计学意义(P<0.01)。与疏肝组比较,补肾组小鼠卵巢TSG-6蛋白表达降低,无统计学差异(P>0.05)。注射hCG后12h:与对照组比较,疏肝组、补肾组小鼠卵巢TSG-6蛋白表达均增强,有统计学意义(P<0.05,P<0.01)。与疏肝组比较,补肾组小鼠卵巢TSG-6蛋白表达增强,有统计学意义(P<0.01)。
3各组促性腺激素预处理小鼠在注射hCG后0、4、8、12 h卵巢PTX-3 mRNA的表达注射hCG后0、4、8 h,对照组小鼠卵巢PTX3 mRNA表达逐渐增强,各组之间均有统计学意义(P<0.01);注射hCG后12h,小鼠卵巢PTX3 mRNA表达降低,与注射hCG后8 h比较有统计学意义(P<0.01)。注射hCG后0、4 h:与对照组比较,疏肝组、补肾组小鼠卵巢PTX3 mRNA表达增强,有统计学意义(P<0.01)。与疏肝组比较,补肾组小鼠卵巢PTX3 mRNA表达降低,有统计学意义(P<0.01)。注射hCG后8 h:与对照组比较,疏肝组、补肾组小鼠卵巢PTX3 mRNA的表达降低,有统计学意义(P<0.01)。与疏肝组比较,补肾组小鼠卵巢PTX3 mRNA表达降低,有统计学意义(P<0.01)。注射hCG后12 h:与对照组比较,疏肝组、补肾组小鼠卵巢PTX3 mRNA的表达增强,有统计学意义(P<0.01)。与疏肝组比较,补肾组小鼠卵巢PTX3 mRNA表达降低,无统计学差异(P>0.05)。
结论:补肾法、疏肝法对促性腺激素预处理小鼠排卵前卵巢COX-2 mRNA及其蛋白的作用机制不同,疏肝法主要上调卵巢COX-2 mRNA及其蛋白的表达,补肾法主要是下调卵巢COX-2 mRNA及其蛋白的表达。补肾法、疏肝法对卵巢TSG-6、PTX3作用机制相似,均可上调排卵前卵巢TSG-6蛋白、PTX3 mRNA的表达,从而促进了卵丘细胞外基质形成,使卵丘充分膨胀有利于排卵。疏肝法、补肾法上调TSG-6蛋白的表达可能是使排卵数目增多的原因之一。
三、补肾法、疏肝法对促性腺激素预处理小鼠卵巢ADAMTS-1、Cat L影响的比较
目的:研究补肾法、疏肝法对促性腺激素预处理小鼠卵巢ADAMTS-1、Cat L mRNA及其蛋白表达的影响,以探讨两种治法对排卵过程中促使卵泡壁破裂机制的排卵机制的异同。方法:动物分组及取材同第二部分。RT-PCR检测卵巢ADAMTS-1、Cat L mRNA的表达,Western blot检测卵巢ADAMTS-1、Cat L蛋白的表达,免疫组化检测卵巢PR、ADAMTS-1、Cat L蛋白的表达。
结果:
1各组促性腺激素预处理小鼠在注射hCG后0、4、8、12h卵巢PR蛋白的表达注射hCG后0h、8h、12h,与对照组比较,疏肝组、补肾组小鼠卵巢PR蛋白表达均增强,有统计学意义(P<0.01)。注射hCG 4 h,与对照组比较,疏肝组、补肾组小鼠卵巢PR蛋白表达均无统计学差异(P>0.05)。疏肝组与补肾组之间在4个时间点比较,小鼠卵巢PR蛋白表达均无统计学差异(P>0.05)。
2各组促性腺激素预处理小鼠在注射hCG后0、4、8、12h卵巢ADAMTS-1 mRNA及其蛋白的表达4个时间点各组小鼠卵巢ADAMTS-1表达呈上升趋势,12h达高峰。注射hCG后0 h:各组小鼠卵巢ADAMTS-1表达不明显。注射hCG后4 h:与对照组比较,疏肝组、补肾组小鼠卵巢ADAMTS-1表达增强,有统计学意义(P<0.01,P<0.05)。与疏肝组比较,补肾组小鼠卵巢ADAMTS-1表达降低但无统计学差异(P>0.05)。注射hCG后8 h:与对照组比较,疏肝组小鼠卵巢ADAMTS-1表达增强,有统计学意义(P<0.01);补肾组小鼠卵巢ADAMTS-1表达降低但无统计学差异(P>0.05)。与疏肝组比较,补肾组小鼠卵巢ADAMTS-1表达降低,有统计学意义(P<0.01)。注射hCG后12 h:与对照组比较,疏肝组小鼠卵巢ADAMTS-1表达无统计学差异(P>0.05);补肾组卵巢ADAMTS-1表达增强,有统计学意义(P<0.01)。与疏肝组比较,补肾组小鼠卵巢ADAMTS-1表达增强,有统计学意义(P<0.01)。3各组促性腺激素预处理小鼠在注射hCG后0、4、8、12 h卵巢Cat L mRNA及其蛋白的表达4个时间点各组小鼠卵巢Cat L表达呈上升趋势,12h达高峰。注射hCG后0 h:各组小鼠卵巢Cat L表达均不明显。注射hCG后4 h:与对照组比较,疏肝组、补肾组小鼠卵巢Cat L表达均增强,有统计学意义(P<0.01)。与疏肝组比较,补肾组小鼠卵巢Cat L表达较低但无统计学差异(P>0.05)。注射hCG后8 h:与对照组比较,疏肝组小鼠卵巢Cat L表达增强,有统计学意义(P<0.01);补肾组无统计学差异(P>0.05)。与疏肝组比较,补肾组小鼠卵巢Cat L表达降低,有统计学意义(P<0.01)。注射hCG后12 h:与对照组比较,疏肝组小鼠卵巢Cat L表达增强但无统计学差异(P>0.05);补肾组小鼠卵巢Cat L表达增强,有统计学意义(P<0.01)。与疏肝组比较,补肾组小鼠卵巢Cat L表达增强,有统计学意义(P<0.015)。
结论:补肾法、疏肝法均可上调促性腺激素预处理小鼠排卵前卵巢PR蛋白的表达,在注射hCG后12h,补肾法上调PR蛋白表达的作用较疏肝法强。补肾法、疏肝法均可上调促性腺预处理小鼠卵巢排卵关键蛋白水解酶ADAMTS-1、Cat L mRNA及其蛋白的表达,促进了排卵斑的形成、卵泡破裂而有利于排卵。补肾法两种蛋白酶表达高峰在排卵时(注射hCG后12h),疏肝法两种蛋白酶表达在排卵前(注射hCG后8h)迅速达到高水平并缓慢增高至排卵时。补肾法卵巢ADAMTS-1、Cat LmRNA及其蛋白的表达高峰高于疏肝法。据此可指导补肾法、疏肝法的临床用药时点。
PartⅠEffects of Bushen Treatment and Shugan Treatment on Ovulation Number in Gonadotropin Pretreatment Mice
Objective: To study the effects of BushenTiaojingⅡRecipe,ⅢRecipe and Xiaoyao Pill on ovulation number of Gonadotropin pretreatment mice.
Methods: One hundred and two female immature mice were randomly divided into 17 groups with 6 mice each group. 1 to 4 were Bushen Treatment groups, 5 to 8 were Shugan Treatment groups, 9 to 12 were BushenTiaojingⅡRecipe add Xiaoyao Pill groups, 13 to 16 were Xiaoyao Pill add BushenTiaojingⅢRecipe groups, 17 was control group. All groups except control group were treated by BushenTiaojingⅡRecipe (high or low does) or Xiaoyao Pill (high or low does) 0.4 ml or during the 1-3 day to promot follicular development. All groups were injected PMSG (5IU/each) by intraperitoneal at 1pm 3rd day, and then with HCG (5 IU/each) after 48 h. All groups except control group were treated with 0.4 ml BushenTiaojingⅢRecipe (high or low does) or Xiaoyao Pill (high or low dose) on the 4-5 day. Mice were executed at 8am of the 6th day, and ampulla portion of uterine tubes were punctured with somatotype microscope to count the number of ovulated oocytes.
Results: Compared with control group by One way ANOVA, ovulation number was more in BushenTiaojingⅡRecipe high-dose add BushenTiaojingⅢRecipe high-dose of Bushen Treatment(P=0.003) and Xiaoyao Pill high-dose group add Xiaoyao Pill low-dose group of Shugan Treatment(P=0.007). Factorial experiment design analysis in each group, treatment BushenTiaojingⅡRecipe high-dose and low-dose group had significant difference(P=0.004), and treatment Xiaoyao Pill high-dose group and low-dose group in induced ovulation had significant differenc(eP=0.039), and they have interaction(P=0.047, P=0.046). Compared with control group by one way ANOVA, ovulation number in Xiaoyao Pill low-does addⅢRecipe high-does group andⅡRecipe high-does add Xiaoyao Pill low-does group were significantly more(P=0.013, P=0.039), but they had no interaction by factorial design analysis(P=0.838, P=0.604).
Conclusions: Ovulation number in BushenTiaojingⅡaddⅢRecipe high-dose group was more, related to YinJing was magnificent to promote follicle’s development and YangQi was easy to transformation between Yin and Yang. Xiaoyao Pill high-dose promoted follicular development, while low-dose induced ovulation, and ovulation number in this group was the most. Jing and Xue was magnificent to ovarian follicle’s development; adjusted liver was useful to transformation between Yin and Yang. Two methods had interaction, so can improve curative effect.
PartⅡEffects of Bushen Treatment and Shugan Treatment on COX-2、PTX3、TSG-6 in Gonadotropin Pretreatment Mice Ovary
Objective: To study and compare the effects of Bushen Treatment and Shugan Treatment on COX-2、PTX3、TSG-6 of gonadotropin pretreatment mice ovary in ovulatory processes.
Methods: Ninety-six female immature KM mice were randomly divided into normal group, control group, Bushen group and Shugan group of 4 mice. Bushen group and Shugan group were treated with BushenTiaojingⅡRecipe high-dose and Xiaoyao Pill high-dose respectively for 3 days from experiment started, while normal group and control group gave distilled water. All groups except normal group were injected intraperitoneally with PMSG (5 IU/each) at 8am on the 3rd day, and 48h later injected with hCG (5 IU), and the normal group injected with distilled water at the same time. On the 4-5 day, Bushen group and Shugan group were treated with BushenTiaojingⅢRecipe and Xiaoyao Pill low-does 0.4 ml respectively, and normal group and control group were treated with distilled water 0.4 ml. Mice were executed respectively at 0h、4h、8h、12h after injecting hCG, and ovaries were preserved in liquid nitrogen. Examined expression of COX-2、PTX-3 mRNA with RT-PCR and COX-2、TSG-6 protein with Immunohistochemical method.
Results:
1 Expression of COX-2 mRNA and protein in each group at 0h, 4h, 8h, 12 h after injecting hCG Expression of COX-2 was increased at 0h、4h、8h after injecting hCG,and decreased at 12h. Compared with control group, expression of COX-2 in Bushen group and Shugan group were significantly increased(P<0.05). Expression of COX-2 in Bushen group was decreased but had no significant difference(P>0.05)at 0h after injecting hCG. At 4 h, expression of COX-2 in Bushen group and Shugan group were significantly decreased(P<0.05)when compared with control group. Expression of COX-2 in Bushen group was decreased but had no significant difference(P>0.05)when compared with Shugan group. At 8h, compared with control group, expression of COX-2 in Shugan group were significantly increased(P<0.01), and Bushen group was decreased(P<0.05). Compared with Shugan group, expression of COX-2 in Bushen group was significantly decreased(P<0.01). At 12h, compared with control group, expression of COX-2 in Shugan group were significantly increased(P<0.05), Bushen group was decreased but had no significant difference(P>0.05). Compared with Shugan group, expression of COX-2 in Bushen group was significantly decreased(P<0.05). 2 Expression of TSG-6 protein expression in each group at 0h, 4h, 8h, 12h after injecting HCG Expression of TSG-6 protein in control group was gradually increased, but had no significant difference between 4h and 8h(P>0.05). Expression TSG-6 protein in Shugan group was gradually increased, but had no significant difference between 8h and 12h(P>0.05). At 0h, expression TSG-6 protein in Bushen group was gradually increased, and all group had significant difference(P<0.01). Expression TSG-6 protein in Bushen group and Shugan group had no significant difference(P>0.05)when compared with control group. At 4h, expression TSG-6 protein in Bushen group and Shugan group had no significant difference(P>0.05)when compared with control group, and in Bushen group was significantly increased(P<0.05)when compared with Shugan group. At 8h, expression TSG-6 protein in Bushen group and Shugan group were significantly increased(P<0.01)when compared with control group, and in Bushen group was decreased but had no significant differenc(eP>0.05)when compared with Shugan group. At 12h, expression TSG-6 protein in Bushen group and Shugan group were significantly increased(P<0.05, P<0.01)when compared with control group, and expression TSG-6 protein in Bushen group was significantly increased(P<0.01)when compared with Shugan group.
3 Expression of PTX-3 mRNA in each group at 0h, 4h, 8h, 12h after injecting hCG Expression of PTX-3 mRNA in control group was gradually increased, and had significant difference among 0h、4h、8h after injecting hCG(P<0.01). Expression of PTX-3 mRNA at 12 h was significantly decreased(P<0.01)when compared with that at 8h. At 0h、4h, expression of PTX-3 mRNA in Bushen group and Shugan group were significantly increased(P<0.01 ) when compared with control group, and in Bushen group was significantly decreased(P<0.01)when compared with Shugan group. At 8h, expression of PTX-3 mRNA in Bushen group and Shugan group were significantly decreased(P<0.01)when compared with control group, and in Bushen group was significantly decreased(P<0.01)when compared with Shugan group. At 12h, expression of PTX-3 mRNA in Bushen group and Shugan group were significantly increased(P<0.01)when compared with control group, and in Bushen group had no significant difference(P>0.05)when compared with Shugan group.
Conclusions: Bushen Treatment can up-regulate COX-2 expression, while Shugan Treatment reduce it. Both of Bushen Treatment and Shugan Treatment could up-regulate expression of TSG-6 protein and PTX3 mRNA. Both of Bushen Treatment and Shugan Treatment could up-regulate expression of TSG-6 protein, and this maybe one reason to influence ovulation number.
PartⅢEffects of Bushen Treatment and Shugan Treatment on ADAMTS-1、Cat L in Gonadotropin pretreatment mice Ovary
Objective: To study the effects of Bushen Treatment and Shugan Treatment on the expression of ADAMTS-1、Cat L mRNA and protein of Gonadotropin pretreatment mice. To analyze the mechanisms of those two treatments in prompting follicle break.
Methods: Group and materials are the same as that in partⅡ. Reverse transcription-polymerase chain reaction (RT-PCR) was used to semiquantitatively detect the expression of ADAMTS-1 mRNA and Cat L mRNA during ovulation, and Western blot was used to detect the expression of the protein. Immunohistochemical method was used to detect the expression of PR, ADAMTS-1 and Cat L protein.
Results:
1 Expression of PR protein in each group ovary at 0h, 4h, 8h, 12h after injecting hCG At 0h, 8h and 12h after injecting hCG, expression of PR in Bushen group and Shugan group ovary were significantly increased(P<0.01)when compared with control group. At 4h, expression PR protein in Bushen group and Shugan group had no significant difference(P>0.05)when compared with control group. At 0h, 4h, 8h, 12h after injecting hCG, expression of PR protein in Bushen group had no significant difference(P>0.05)when compared with Shugan group. 2 Expression of ADAMTS-1 mRNA and its protein in each group ovary at 0h, 4h, 8h, 12h after injecting hCG Expression of ovary ADAMTS-1 at these four timings was gradually increased, and was the highest at 12h. Expression of ADAMTS-1 in each group ovary was low at 0h. At 4h, expression of ADAMTS-1 in Bushen group and Shugan group was significantly increased(P<0.05 or P<0.01)when compared with control group. Expression of ADAMTS-1 in Bushen group was decreased, but had no significant difference(P>0.05)when compared with Shugan group. At 8h, compared with control group, expression of ADAMTS-1 in Shugan group was significantly increased(P<0.01), and in Bushen group had no significant difference ( P > 0.05 ) . Compared with Shugan group, expression of ADAMTS-1 in Bushen group was significantly decreased(P<0.01). At 12h, compared with control group, expression of ADAMTS-1 in Shugan group had no significant difference(P>0.05), and in Bushen group was significantly increased(P<0.01). Expression of ADAMTS-1 in Bushen group was significantly increased(P<0.01)when compared with Shugan group. 3 Expression of Cat L mRNA and protein in each group ovary a at 0h, 4h, 8h, 12h after injecting hCG Expression of Cat L in each group ovary at these four timings was gradually increased, and was the highest at 12h. Expression of Cat L in each group ovary was low. At 4h, expression of Cat L in Bushen group and Shugan group was significantly increased(P<0.01)when compared with control group. Expression of Cat L in Bushen group was decreased, had no significant difference(P>0.05)when compared with Shugan group,. At 8h, expression of Cat L in Shugan group was significantly increased(P<0.01)when compared with control group, and in Bushen group had no significant difference(P>0.05). Compared with Shugan group, expression of Cat L in Bushen group was significantly decreased(P<0.01). At 12h, compared with control group, expression of Cat L in Shugan group was no significant difference(P>0.05), and in Bushen group was significantly increased(P<0.01). Expression of Cat L in Bushen group was significantly increased(P<0.01) when compared with Shugan group.
Conclusions: Ovary PR protein expression at preovulation of Gonadotropin pretreatment mice was increased with Bushen Treatment and Shugan Treatment. But expression of PR protein at 12h was higher in Bushen group than in Shugan group. Expression of ADAMTS-1 and Cat L of Gonadotropin pretreatment mice ovary was increased with Bushen Treatment and Shugan Treatment. Expression of ovary ADAMTS-1、Cat L was highest at ovulation (hCG injecting 12h )with Bushen Treatment, while expression was quickly increased at preovulation (hCG injecting 8h) then slow-moving to the highest at ovulation with Shugan Treatment. Peak of ADAMTS-1、Cat L expression in Bushen group was higher than in Shugan group.
引文
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