基于SIRT5-FOXO3a信号通路研究不同浓度环磷酰胺诱导早发性卵巢功能不全小鼠模型及作用机制

doi:10.12300/j.issn.1674-5817.2024.194

摘要/Abstract

摘要：

目的观察比较不同浓度环磷酰胺（cyclophosphamide CTX）诱导小鼠早发性卵巢功能不全（premature ovarian insufficiency，POI）模型的效果并探讨损伤机制。方法根据不同文献记载，分别用75 mg/kg CTX、120 mg/kg CTX以及120 mg/kg CTX+12 mg/kg 白消安（Busulfan）处理C57BL/6J雌性小鼠来建立POI模型，测定卵巢系数、血清中雌二醇（estradiol，E₂）和卵泡刺激素（follicle stimulating hormone，FSH）水平，并通过Western blot检测观察不同造模时间内卵巢组织内SIRT5和FOXO3a蛋白表达水平的变化规律。结果与对照组比较，不同浓度的CTX（75 mg/kg和120 mg/kg）和120 mg/kg CTX+12 mg/kg Busulfan均可以诱导POI损伤，但120 mg/kg CTX造模后，卵巢系数和E₂水平变化较小，而120 mg/kg CTX+12 mg/kg Busulfan毛色粗糙，光泽度下降，反应迟钝，状态最差。Western blotting结果显示，与对照组相比，75mg/kg CTX造模后，FOXO3a表达显著下调，SIRT5表达没有显著变化；而120 mg/kg CTX造模后，SIRT5和FOXO3a表达均显著下调；120 mg/kg CTX造模后后第2天和第7天，卵巢组织SIRT5和FOXO3a表达均显著下调，且第7天表达水平变化最大。结论 120mg/kg CTX诱导小鼠POI模型的卵巢损伤小于75mg/kg CTX诱导的POI模型，且其作用机制可能与SIRT5-FOXO3a通路活性受抑相关。

关键词: 环磷酰胺, 早发性卵巢功能不全, 小鼠模型, SIRT5-FOXO3a通路, 机制

Abstract:

Objective To observe the effects of different concentrations of cyclophosphamide (CTX) induced premature ovarian insufficiency (POI) model in mice and investigate the mechanism of injury. Method 75 mg/kg CTX, 120 mg/kg CTX and 120 mg/kg CTX +12 mg/kg Busulfan were used to induce POI model in C57BL/6J female mice according to the study. Ovarian index, estradiol (E₂) and follicle stimulating hormone (FSH) were determined. The expression level and pattern of SIRT5 and FOXO3a protein were investigated by western blots. Result Compared with control group, all concentration of CTX (75 mg/kg, 120 mg/kg, 120 mg/kg CTX+12 mg/kg Busulfan) could induced POI injury. However, the mild change in ovarian index and E₂ compared to other two group after 120 mg/kg CTX induced, while rough coat, reduced gloss, unresponsive and in worst condition was showed in the treatment of 120 mg/kg CTX+12 mg/kg Busulfan. Western blot results showed that FOXO3a expression was significantly down-regulated and SIRT5 expression did not significantly change after 75 mg/kg CTX modeling, whereas both SIRT5 and FOXO3a expression were significantly down-regulated after 120 mg/kg CTX modeling; on days 2 and 7 after 120 mg/kg CTX modeling, SIRT5 and FOXO3a expression were significantly down-regulated in the ovarian tissue on day 2 and day 7 after 120 mg/kg CTX modeling, and the greatest change in expression level was observed on day 7. Conclusion 120 mg/kg CTX is the optimal concentration for inducing POI in mice, and the SIRT5-FOXO3a pathway may be the mechanism of its injury action.

Key words: Cyclophosphamide, Premature ovarian insufficiency, Mice model, SIRT5-FOXO3a pathway, Mechanism

中图分类号:

Q95-33

贡磊磊,王晓霞,封学伟,等.基于SIRT5-FOXO3a信号通路研究不同浓度环磷酰胺诱导早发性卵巢功能不全小鼠模型及作用机制[J]. 实验动物与比较医学.. DOI: 10.12300/j.issn.1674-5817.2024.194.

GONG Leilei,WANG Xiaoxia,FENG Xuewei,et al. Model and Mechanism of Action of Premature Ovarian Insufficiency Induced by Cyclophosphamide at Different Concentrations Based on SIRT5-FOXO3a Signaling Pathway[J]. Laboratory Animal and Comparative Medicine. DOI: 10.12300/j.issn.1674-5817.2024.194.

图/表 3

图1 CTX或CTX+Busulfan造模后卵巢系数和血清激素变化水平注：Ctl示对照组；CTX示环磷酰胺；Busulfan示白消安；E2示雌二醇；FSH示卵泡刺激素；与对照组比较（n=8），*P<0.05；**P<0.01；***P<0.001。

Figure 1 ovarian index and serum hormone change levels after CTX or CTX+Busulfan modelling.Note：Ctl represents control group； CTX represents Cyclophosphamide； Busulfan represents Busulfan； E2 represents Estradiol； FSH represents follicle-stimulating hormone； Compared with Ctl （n=8）， *P<0.05； **P<0.01； ***P<0.001.

图2 不同浓度的CTX和CTX+Busulfan造模后卵巢组织SIRT5和FOXO3a表达情况注：Ctl示对照组；CTX示环磷酰胺；Busulfan示白消安；与对照组比较（n=8），*P<0.05；**P<0.01。

Figure 2 The expression of SIRT5 and FOXO3a in ovarian tissue after modelling with different concentrations of CTX and CTX+BusulfanNote：Ctl represents control group； CTX represents Cyclophosphamide； Busulfan represents Busulfan； Compared with Ctl （n=8）， *P<0.05； **P<0.01.

图3 120 mg/kg CTX造模不同时间后卵巢组织SIRT5和FOXO3a表达变化情况注：Ctl示对照组；120 CTX示120 mg/kg CTX。A和E：造模后第1天FOXO3a和SIRT5表达变化；B和F：造模后第2天FOXO3a和SIRT5表达变化；C和G：造模后第7天FOXO3a和SIRT5表达变化；D和I：造模后第14天FOXO3a和SIRT5表达变化。

Figure 3 The expression of SIRT5 and FOXO3a in ovarian tissue at different times of 120 mg/kg CTX modellingNote：Ctl represents control group； 120 CTX represents 120 mg/kg CTX. A and E： The expression of FOXO3a and SIRT5 on day 1 post-modeling； B and F： The expression of FOXO3a and SIRT5 on day 2 post-modeling； C and G： The expression of FOXO3a and SIRT5 on day 7 post-modeling； D and I： The expression of FOXO3a and SIRT5 on day 14 post-modeling.

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