GnRH激动剂在子宫腺肌病治疗中自噬现象与作用机制研究
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摘要
目的子宫腺肌病是生育期女性的常见疾病,GnRH-a是治疗腺肌病的主要药物,但其作用机制尚不十分清楚,本文通过研究GnRH-a与小鼠子宫内膜细胞自噬作用的关系,探索GnRH-a的作用机制。方法 ICR健康孕鼠(上海斯莱克实验动物有限责任公司)5只,整个孕期孕鼠均独笼饲养,出生的雌性小鼠用于后续研究。随机选取16只雌性新生小鼠造模,8只作为正常对照组。建模小鼠于出生后第2～5日滴喂2.7μmol/kg他莫昔芬,正常对照使用生理盐水。鼠龄75 d,将模型鼠分为腺肌病模型组、腺肌病GnRH-a治疗组。根据不同干预措施将小鼠分为正常对照组(生理盐水+生理盐水)、腺肌病模型组(他莫昔芬+生理盐水)、腺肌病GnRH-a治疗组(他莫昔芬+GnRH-a)3组。腺肌病GnRH-a治疗组采用单次长效GnRH-a 8 mg腹腔注射,腺肌病模型组使用等量生理盐水。4周后,雌雄鼠交配,取见栓第4天子宫内膜,通过透射电镜测试自噬小体数量、Western-blot检测自噬相关蛋白Beclin-1及LC-3的表达。结果腺肌病模型组较对照组,自噬小体数量显著减少,而腺肌病GnRH-a给药组较模型组自噬小体数量明显增多(P<0.05);Beclin-1、LC-3模型组表达水平低于正常对照组,而GnRH-a治疗组Beclin-1、LC-3蛋白表达水平显著高于模型组(P<0.05)。结论腺肌病小鼠经GnRH-a治疗后,在位内膜的自噬水平显著提高,揭示GnRH-a可通过病灶部位的自噬降解,改善子宫内膜容受性,从而可提高腺肌病患者妊娠结局。
Objective Gonadotropin-releasing hormone(GnRH) agonists are effective in the treatment of adenomyosis. Whether GnRH agonists act on autophagic mechanisms to exert their therapeutic effect remains unclear. Methods We investigated the effect of a GnRH agonist on autophagy in a mouse model of adenomyosis. The mice were randomly divided into model group(16 female newborn mice) and normal control(8 female newborn mice):model group received 2.7 μmol/kg tamoxifen; normal control treated saline solution during 2-5 days after birth. At 75 days, we further divided the mice of model group into adenomyosis model group and GnRH agonist treatment group. According to different interventions, the mice were divided into 3 groups:normal control group received saline solution; adenomyosis model group treated with tamoxifen plus saline solution; adenomyosis GnRH-a treatment group received tamoxifen plus GnRH-a. After 4 weeks, mice were mated and the eutopic endometrium was obtained at the fourth day of vaginal plug appeared. The number of autophagosomes was detected by Transmission Electron Microscopy and the expression of Beclin 1 and LC-3 were detected by Western-blot. Results Compared with the control group, mice in the adenomyosis model group showed significantly fewer autophagosomes and less Beclin 1 and LC-3 expression in the eutopic endometrium(P<0.05). However, there were more autophagosomes and Beclin 1 and LC-3 expression in the GnRH agonist treatment group than those in the adenomyosis model group(P<0.05). Conclusion After treatment with GnRH-a in mice with adenomyosis, the level of autophagy in eutopic endometrium is significantly increased. It revealed that GnRH agonists could improve endometrial receptivity by autophagy in the lesion site, thereby reducing pregnancy outcomes.
Objective Gonadotropin-releasing hormone(GnRH) agonists are effective in the treatment of adenomyosis. Whether GnRH agonists act on autophagic mechanisms to exert their therapeutic effect remains unclear. Methods We investigated the effect of a GnRH agonist on autophagy in a mouse model of adenomyosis. The mice were randomly divided into model group(16 female newborn mice) and normal control(8 female newborn mice):model group received 2.7 μmol/kg tamoxifen; normal control treated saline solution during 2-5 days after birth. At 75 days, we further divided the mice of model group into adenomyosis model group and GnRH agonist treatment group. According to different interventions, the mice were divided into 3 groups:normal control group received saline solution; adenomyosis model group treated with tamoxifen plus saline solution; adenomyosis GnRH-a treatment group received tamoxifen plus GnRH-a. After 4 weeks, mice were mated and the eutopic endometrium was obtained at the fourth day of vaginal plug appeared. The number of autophagosomes was detected by Transmission Electron Microscopy and the expression of Beclin 1 and LC-3 were detected by Western-blot. Results Compared with the control group, mice in the adenomyosis model group showed significantly fewer autophagosomes and less Beclin 1 and LC-3 expression in the eutopic endometrium(P<0.05). However, there were more autophagosomes and Beclin 1 and LC-3 expression in the GnRH agonist treatment group than those in the adenomyosis model group(P<0.05). Conclusion After treatment with GnRH-a in mice with adenomyosis, the level of autophagy in eutopic endometrium is significantly increased. It revealed that GnRH agonists could improve endometrial receptivity by autophagy in the lesion site, thereby reducing pregnancy outcomes.
引文
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13 Paludan C,Schmid D,Landthaler M,et al.Endogenous MHC class II processing of a viral nuclear antigen after autophagy.Science,2005,307:593-596.
14 Wang F,Wen Z,Li H,et al.Human leukocyte antigen—G is expressed by the eutopic and ectopic endometrium of adenomyosis.Fertil Steril,2008,90:1599-1604.
15 Choi J,Jo M,Lee E,et al.Dienogest enhances autophagy induction inendometriotic cells by impairing activation of Akt,Erk1/2,and mtor.Fertil Steril,2015,104:655-664,e1.
2 He C,Bassik MC,Moresi V,et al.Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis.Nature.2012,481:511-515.
3 Li X,Zhu F,Jiang J,et al.Simultaneous inhibition of the Ubiquitin-proteasome system and autophagy enhances apoptosis induced by Er stress aggravators in human pancreatic cancer cells.Autophagy,2016,12:1521-1537.
4 Velazquez AP,Graef M.Autophagy Regulation Depends on Er Homeostasis Controlled by Lipid Droplets.Autophagy,2016,12:1409-1410.
5 Ciura S,Sellier C,Campanari ML,et al.The Most Prevalent Genetic Cause of Als-Ftd,C9orf72 Synergizes the Toxicity of Atxn2 Intermediate Polyglutamine Repeats through the Autophagy Pathway.Autophagy,2016,12:1406-1408.
6 Koike N,Tsunemi T,Uekuri C,et al.Pathogenesis and malignant transformation of adenomyosis (review).Oncol Rep,2013,29:861-867.
7 李雁,张绍芬,夏贤,等.口服他莫昔芬法建立ICR小鼠AM模型.中国实验动物学报,2009,17:345-350.
8 Green AR,Edwards RE,Greaves P,et al.Comparison of the effect of oestradiol,tamoxifen and raloxifene on nerve growth factor-alpha expression in specific neonatal mouse uterine cell types using laser capture microdissection.J Mol Endocrinol,2003,30:1-11.
9 Eskelinen EL,Reggiori F,Baba M,et al.Seeing is believing:The impact of electronmicroscopy on autophagy research.Autophagy,2011,7:935-956.
10 Qu X,Yu J,Bhagat G,et al.Promotion oftumorigenesis by heterozygous disruption of the beclin l autophagy gene.J ClinInvest,2003,112:1809-1820.
11 Takahashi K,Nagata H,Kitao M.Clinical usefulness of determination of estradiol level in the menstrual blood of patients with endometriosis.Acta Obstet Gynecol Jpn,1989,41:1849-1850.
12 Ota H,Igarashi S,Hatazawa J,et al.Is adenomyosis an immune disease?Hum Reprod Update,1998,4:360-367.
13 Paludan C,Schmid D,Landthaler M,et al.Endogenous MHC class II processing of a viral nuclear antigen after autophagy.Science,2005,307:593-596.
14 Wang F,Wen Z,Li H,et al.Human leukocyte antigen—G is expressed by the eutopic and ectopic endometrium of adenomyosis.Fertil Steril,2008,90:1599-1604.
15 Choi J,Jo M,Lee E,et al.Dienogest enhances autophagy induction inendometriotic cells by impairing activation of Akt,Erk1/2,and mtor.Fertil Steril,2015,104:655-664,e1.