| 引用本文: |
李涵,刘慧萍,肖贾丽珏,杨开锋,辛雨霏.补肾活血方对卵巢功能早衰大鼠卵巢组织外泌体相关膜蛋白的影响[J].湖南中医药大学学报,2022,42(6):885-892[点击复制] |
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| 补肾活血方对卵巢功能早衰大鼠卵巢组织外泌体相关膜蛋白的影响 |
| 李涵,刘慧萍,肖贾丽珏,杨开锋,辛雨霏 |
| (湖南中医药大学, 湖南 长沙 410208) |
| 摘要: |
| 目的 观察补肾活血方对卵巢功能早衰模型大鼠卵巢组织中外泌体相关膜蛋白表达情况及卵巢组织形态和功能的影响。方法 选取30只大鼠，腹腔注射顺铂建立卵巢功能早衰模型，造模成功后随机分为模型组、阳性对照组和中药低、中、高剂量组，每组6只。另取6只大鼠为空白组。空白组和模型组大鼠每天灌胃蒸馏水；中药低、中、高剂量组分别按每天9.72、19.44、38.88 g/kg剂量补肾活血方灌胃；阳性对照组大鼠按每天0.09 mg/kg剂量灌胃给药戊酸雌二醇片，连续21 d。每周称体质量，计算体质量增量，每天观察大鼠动情周期。末次给药后于第2天取血和卵巢、胸腺、脾脏，称脏器质量，计算脏器指数；ELISA检测大鼠血清中雌二醇（estradiol 2,E2）和抗米勒管激素（anti-Müllerian hormone,AMH）水平变化含量；HE染色观察卵巢形态变化；免疫组织化学法检测卵巢组织中外泌体膜蛋白CD63、CD81、CD82的表达水平；透射电镜观察大鼠外周血外泌体形态；Western blot检测大鼠外周血外泌体相关蛋白。结果 与空白组相比，模型组动情周期紊乱、AMH和E2减少（P<0.05，P<0.01），体质量和体质量增量明显减少（P<0.01），卵巢指数、胸腺指数明显减少而脾脏指数明显升高（P<0.01），卵泡数和成熟卵泡明显减少，闭锁卵泡增多，卵巢组织CD63、CD81、CD82蛋白表达明显下降（P<0.01）。与模型组相比，中药中、高剂量组和阳性对照组AMH和E2显著升高（P<0.01），中药高剂量组和阳性对照组动情周期恢复正常，体质量及体质量增量明显升高（P<0.01），卵巢指数、胸腺指数明显升高而脾脏指数降低（P<0.05，P<0.01），给药组卵泡数依次增多，中药高剂量组和阳性对照组蛋白表达明显升高（P<0.01）。透射电镜和Western blot证明大鼠外周血外泌体提取成功。结论 补肾活血方可以明显改善卵巢功能早衰的情况，其改善卵巢形态和功能的机制可能与提高外周血和卵巢中外泌体活性有关。 |
| 关键词: 卵巢功能早衰 补肾活血方 外泌体 抗米勒管激素 雌二醇 动情周期 |
| DOI：10.3969/j.issn.1674－070X.２０22.06.003 |
| 投稿时间：2021-12-25 |
| 基金项目:湖南省教育厅科学研究项目(21A0233,18C0388);湖南省中医药科研计划重点项目(2021025);国家级大学生创新项目(202110541042);湖南省大学生研究性学习与创新性实验项目(2021-2394);湖南中医药大学中西结合一流学科开放基金项目(2020ZXYJH66,2020ZXYJH71);湖南中医药大学科研基金项目重点项目(2020XJJJ003);中医方证研究转化医学湖南省重点实验室资助项目(2018TP1021);湖南省性与生殖健康中医临床医学研究中心项目(湘科计2020SK4014)。 |
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| Effect of Bushen Huoxue Recipe on exosome-related membrane proteins in ovarian tissue of rats with premature ovarian failure |
| LI Han,LIU Huiping,XIAO Jialijue,YANG Kaifeng,XIN Yufei |
| (Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To observe the effect of Bushen Huoxue Recipe on the expression of exosome-related membrane proteins in ovarian tissue and the morphology and function of ovarian tissue in rats with premature ovarian failure. Methods A total of 30 rats were selected and injected with cisplatin intraperitoneally to establish the mode of premature ovarian failure. After successful modeling, they were randomly divided into model group, positive control group, low, middle and high dose groups of traditional Chinese medicine, with six rats in each group. Another six rats were selected as blank group. Rats in the blank group and model group were gavaged with distilled water every day, the rats in the low, middle and high dose groups of traditional Chinese medicine were given intragastric administration of Bushen Huoxue Recipe at the dose of 9.72, 19.44 and 38.88 g/kg, respectively, and the rats in the positive control group were given estradiol valerate tablets at the dose of 0.09 mg/kg every day for 21 d. The body weight was weighed every week, the body weight increment was calculated, and the estrous cycle of rats was observed every day. After the last administration, on the second day, the blood, ovary, thymus and spleen were taken, the organ weight was weighed and the organ index was calculated. The levels of estradiol (estradiol 2, E2) and anti-Müllerian hormone (AMH) in serum were detected by ELISA; the morphological changes of ovary were observed by HE staining; the expression levels of CD63, CD81 and CD82 in ovarian tissue were detected by immunohistochemical method. The morphology of exocrine body in rat peripheral blood was observed by transmission electron microscope, the exocrine related protein in rat peripheral blood was detected by Western blot. Results Compared with the blank group, the estrous cycle disorder, AMH and E2 were decreased (P<0.01, P<0.05), the body weight and body weight increment were significantly decreased (P<0.01), the ovarian index and thymus index were significantly decreased, and the spleen index was significantly decreased, the number of follicles and mature follicles decreased significantly, the atretic follicles increased, and the protein expression levels of CD63, CD81, and CD82 in ovarian tissue decreased significantly (P<0.01) in the model group. Compared with the model group, AMH and E2 in the medium and high dose groups of traditional Chinese medicine and the positive control group were significantly increased (P<0.01). The estrous cycle in the traditional Chinese medicine high-dose group and the positive control group returned to normal, and the body weight and body weight increment increased significantly (P<0.01), the ovarian index and thymus index were significantly increased while the spleen index was decreased (P<0.01, P<0.05), the number of follicles in the administration group increased in turn, and the protein expression in the traditional Chinese medicine high-dose group and the positive control group was significantly increased (P<0.01). Transmission electron microscopy and Western blot proved that the extraction of exosomes from peripheral blood of rats was successful. Conclusion Bushen Huoxue Recipe can significantly improve the situation of premature ovarian failure, and the mechanism of improving ovarian morphology and function may be related to the increase of peripheral blood and ovarian exocrine activity. |
| Key words: premature ovarian failure Bushen Huoxue Recipe exosome anti-Müllerian hormone estradiol 2 estrous cycle |
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