| Objectives:Cytoxan was given to mice by intraperitoneal injection to establish Decreasing Ovarian Reserve(DOR) model through which to observe the level of hormone AMH, FSH, E2 and the expression of GDF-9 m RNA, BPM-15 m RNA, SCFm RNA factors in follicular signaling pathway, and to preliminarily study Guishen Pill’s effects on strengthening ovarian reserve, improving follicle qualilty and signal transduction of GDF-9, BPM-15, SCF factors. Further more, the prescription’s clinical curative effect on infertility caused by decreasing ovarian reserve and follicle development dysfunction was verified, aiming to provide scientific evidences to some degrees for the research in developing and clinical studying patterned Chinese herbal products. Method:Totally 60 BALB/c female mice aged 8 to 10 weeks with normal estrus cycle were selected. They were randomized into six groups: the normal group, the model control group(the model group), the estradiol valerate group(containing estradiol valerate tablets 0.01mg/ml) referred to as the western medicine group, the Chinese medicine of high dose group(including Guishen pill 1.8g/m L) referred to as the high dose group,the Chinese medicine of medium dose group(including Guishen pill 0.9g/m L) referred to as the medium dose group, and the Chinese medicine of low dose group(including Guishen pill 0.45g/m L) referred to as the low dose group. Except for the normal group, mice in other groups were given cytoxan to 90mg/kg dose by single-intraperitoneal injection to copy the model of follicle development dysfunction caused by decreasing ovarian reserve. From the seconed day of inducing model, viginal exfoliate cells test was conducted daily to observe the change in estrus cycle of mice. Every BALB/c mouse was perfused by dose in 0.3ml/day for continuous 30 days, and mice in the normal group and model group were perfused with physiological saline. The mice were killed at the second day after the completion of intragastric administration. Adopting enzyme linked immunosorbent assay(ELISA) and chemiluminescence immuno assay(CLIA) to detect the serum levels of E2, anti- mullerian hormone(AMH), and follicle stimulating hormone(FSH) respectively. After drawing blood, the bilateral ovarian tissues of the killed mice were extracted, and ovarian wet weight was obtained. What’s more, the expression levels of GDF-9, BPM-15, and SCF m RNA were detected by real-time PCR. Results:1. The general condition of BALB/c mice: The mice in normal group had normal diet, dry and smooth skin, good mental state, like to move without lying, and their stool was black grain shape, while the other groups of mice showed appetite loss, fur sweat wet erected sparse, dispirited spirit, camponotus atrophy, like lying crowded without moving, action dull,breathing deep and late, lethargy, weight loss, loose stools, light gray fur color. Compared with the normal group, the ovarian wet weight of the model group, the western medicine group, and the Chinese medicine of high, medium and low dose groups had no difference(P>0.05).2. Change of estrus cycle of mice: Compared with the normal group, days of reproductive cycle of female BALB/c mice in the model group significantly increased with a significant difference in statistics(P<0.05), while days of estrus cycle in the model group apparently declined, and the difference was statistically significant(P≤0.01). Results above illustrated that the model was established successfully.3. Effects on the level of serum AMH: Compared with the normal group, the level of serum AMH in the model group obviously decreased with statistical significance(P<0.05), while in the western medicine group, and the Chinese herb groups of high, middle and low dose there was no significant difference(P>0.05). The level of serum AMH in the western medicine group, and the Chinese medicine of high, medium and low dose groups were higher than that in the model group(P<0.05). There was no significant difference in the western medicine and Chinese medicine groups(P>0.05). The level of serum AMH in the Chinese medicine of low dose group was lower than high and medium dose groups with significant differences(P<0.05).4. Effects on the level of serum FSH and E2: Compared with the normal group, in the model group, the western medicine group, and the Chinese medicine of high, medium and low dose groups the level of serum FSH increased(P<0.05), while E2 decreased with statistical significance in the modern group and in the Chinese medicine of medium dose group(P<0.05). FSH in the western medicine group, the Chinese medicine of high, medium and low dose groups was lower than that in the model group(P<0.05), while E2 was higher than that in the model group(P<0.05). Compared with the western medicine group, FSH and E2 in the Chinese medicine groups had no significant difference(P>0.05). E2 in the low dose group was lower than that in the high and medium dose groups compared between the Chinese medicine groups(P<0.05).5. Effects on the relative expression of GDF-9 m RNA in ovarian: Compared with the normal group, the relative protein expressions of GDF-9 m RNA in the model group, the western medicine group, and the Chinese medicine of high, medium and low dose groups were lower than that in the normal group, and there was significant difference in the model group(P< 0.01). The relative protein expressions of GDF-9 m RNA in the western medicine group, the Chinese medicine of high and medium dose groups were higher than that in the model group(P<0.05). Compared with the western medicine group, the relative protein expressions of GDF-9 m RNA in the Chinese medicine groups had no significant difference(P>0.05). There was no difference with statistical significance compared between the Chinese medicine groups(P>0.05).6. Effects on the relative expression of BPM-15 m RNA in ovarian: Compared with the normal group, the relative expressions of BPM-15 m RNA in the model group, the western medicine group, and the Chinese medicine of high, medium and low dose groups were lower than that in the normal group, and there were significant differences in the model group and in the Chinese medicine of low dose group(P<0.01). The relative expressions of BPM-15 m RNA in the western medicine group, the Chinese medicine of high, medium, and low dose groups were higher than that in the model group(P< 0.01). Compared with the western medicine group, the relative expressions of BPM-15 m RNA in the Chinese medicine groups had no significant difference(P>0.05). The relative expressions of BPM-15 m RNA in the Chinese medicine of high and medium dose groups were higher than that in the low dose group(P<0.01).7. The effects on the relative expression of SCF m RNA in ovarian: Compared with the normal group, the relative expressions of SCF m RNA in the model group, the western medicine group, and the Chinese medicine of high, medium and low dose groups were lower than that in the normal group, and there were significant differences in the model group and in the Chinese medicine of low dose group(P<0.01). The relative expressions of SCF m RNA in the western medicine group, the Chinese medicine of high, medium and low dose groups were higher than that in the model group, and there were significant differences in the western medicine group, the Chinese medicine of high and medium dose groups(P<0.05). The relative expressions of SCF m RNA in the Chinese medicine groups were lower than that in the western medicine group, and there was significant difference in the Chinese medicine of low dose group(P<0.05). There was no difference with statistical significance compared between the Chinese medicine groups(P>0.05). Conclusion:1. The method of single-intraperitoneal injection with cytoxan can successfully copy the model of decreasing ovarian reserve(DOR).2. Guishen Pill can effectively raise the level of serum AMH and E2, and reduce the level of serum FSH.3. Guishen pill has potential to improve the expression level of GDF-9, BPM-15, and SCF m RNA in the ovarian, adjust “dialogues” between the oocyte and granulosa cells, promote the development of primordial follicles, and improve the function of ovarian reserve. |
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