摘要
目的观察雷公藤甲素(TP)不同浓度和作用时间对大鼠卵巢氧化应激及颗粒细胞线粒体自噬的影响。方法取3个月龄雌性Sprague-Dawley大鼠50只,连续观察2个动情周期,筛选出25只动情周期正常的大鼠,按随机数字表法分为空白对照组、实验1组、实验2组、实验3组和实验4组,每组5只。实验1组大鼠每日给予400μg·kg^(-1)的TP灌胃1次,连续灌胃30 d;实验2组大鼠每日给予400μg·kg^(-1)的TP灌胃1次,连续灌胃40 d;实验3组大鼠每日给予500μg·kg^(-1)的TP灌胃1次,连续灌胃30 d;实验4组大鼠每日给予500μg·kg^(-1)的TP灌胃1次,连续灌胃40 d;空白对照组大鼠每日给予10 mL·kg^(-1)蒸馏水灌胃1次,连续灌胃40 d。应用酶联免疫吸附测定法检测各组大鼠血清抗苗勒管激素(AMH)、雌二醇(E_(2))、促卵泡生成素(FSH)水平,苏木精-伊红染色观察各组大鼠卵巢组织病理变化,使用酶标仪检测各组大鼠卵巢组织中丙二醛(MDA)水平及超氧化物歧化酶(SOD)活性,透射电子显微镜观察各组大鼠卵巢颗粒细胞线粒体形态结构变化及线粒体自噬情况,流式细胞仪检测各组大鼠卵巢颗粒细胞凋亡率。结果与空白对照组比较,实验1组、实验2组、实验3组和实验4组大鼠血清中AMH、E_(2)水平及卵巢组织中SOD活性显著降低,血清中FSH及卵巢组织中MDA水平显著增高(P<0.05)。与实验1组比较,实验2组、实验3组、实验4组大鼠血清中AMH、E_(2)水平及卵巢组织SOD活性显著降低,血清中FSH及卵巢组织中MDA水平显著增高(P<0.05)。实验2组、实验3组、实验4组大鼠血清中AMH、E_(2)、FSH水平及卵巢组织中SOD活性、MDA水平比较差异无统计学意义(P>0.05)。实验1组、实验2组、实验3组和实验4组大鼠卵巢颗粒细胞凋亡率均显著高于空白对照组(P<0.05);实验2组、实验3组和实验4组大鼠卵巢颗粒细胞凋亡率显著高于实验1组(P<0.05);实验3组、实验4组大鼠卵巢颗粒细胞凋亡率显著高于实验2组(P<0.05);实验4组大鼠卵巢颗粒细胞凋亡率显著高于实验3组(P<0.05)。空白对照组大鼠卵巢组织被膜完整,皮质区原始卵泡、初级卵泡及次级卵泡等数量较为正常,各级卵泡发育正常,闭锁卵泡少见,黄体数量较少;髓质区纤维结缔组织排列紧密,未见明显水肿或坏死。实验1组、实验2组、实验3组和实验4组大鼠卵巢组织可见卵泡数量减少,闭锁卵泡增多,颗粒层卵泡细胞坏死、脱落和卵泡囊性扩张等病理改变;实验1组大鼠卵巢组织病理改变程度相对较轻,闭锁卵泡少,颗粒层细胞坏死不多;实验4组大鼠卵巢组织病变程度相对最重,可见较多闭锁卵泡和颗粒细胞层坏死、脱落;实验2组和实验3组大鼠卵巢组织病变程度相对轻于实验4组,闭锁卵泡较少,颗粒细胞层不同程度坏死和卵泡囊性扩张。空白对照组大鼠的卵巢颗粒细胞形态结构正常,其细胞核呈不规则多边形,染色质分布均匀,以常染色质为主,核膜清晰完整,细胞质中可见线粒体等细胞器且结构完整清晰。实验1组、实验2组、实验3组和实验4组大鼠卵巢颗粒细胞的细胞质中可见不同程度的线粒体自噬,实验2组、实验3组、实验4组大鼠线粒体自噬程度重于实验1组,实验3组、实验4组大鼠线粒体自噬程度重于实验2组,实验3组与实验4组自噬程度相当;实验3组和实验4组电镜下见卵巢颗粒细胞的细胞质中含较多线粒体自噬小体,多数线粒体发生轻度肿胀,部分线粒体嵴结构消失,粗面内质网扩张呈囊状。结论采用TP灌胃给药可成功构建大鼠卵巢功能减退模型,大鼠卵巢损伤与TP剂量和干预时间有关;TP诱导的卵巢氧化应激损伤可能是引发卵巢功能低下的重要因素,其作用机制可能是氧化应激影响卵巢内分泌功能和诱导颗粒细胞凋亡等病理生理过程。随着氧化应激损伤加重,线粒体自噬增加,线粒体自噬到达一定程度后不再随着TP的干预时间继续增加而持续增强。
Objective To observe the effects of different concentration and intervention time of triptolide(TP)on ovarian oxidative stress and mitophagy in rats.Methods Fifty 3-month-old female Sprague-Dawley rats were observed for 2 consecutive estrus cycles,and 25 rats with normal estrus cycles were selected and divided into the blank control group,experiment group 1,experiment group 2,experiment group 3 and experiment group 4 according to random number table method,with 5 rats in each group.Rats in experiment group 1 were administered intragastrically 400μg·kg^(-1) TP once a day for 30 d.Rats in experiment group 2 were given 400μg·kg^(-1) TP once a day for 40 d.Rats in experiment group 3 were given 500μg·kg^(-1) TP once a day for 30 d.Rats in the experiment group 4 were given 500μg·kg^(-1) TP once a day for 40d.Rats in the blank control group were given 10 mL·kg^(-1) distilled water once a day for 40 d.Serum anti-mullerian hormone(AMH),estradiol(E_(2))and follicle-stimulating hormone(FSH)levels were detected by enzyme-linked immunosorbent assay;hematoxylin-eosin staining was used to observe the pathological changes of ovarian tissue;malondialdehyde(MDA)level and superoxide dismutase(SOD)activity in ovarian tissue were detected by microplate reader.The morphological and structural changes of mitochondria in ovarian granulosa cells of rats in each group were observed by transmission electron microscope,and the apoptosis rate of ovarian granulosa cells was detected by flow cytometer.Results Compared with the blank control group,the levels of AMH and E_(2) in serum and SOD activity in ovarian tissue of rats in the experiment groups 1,2,3 and 4 significantly decreased,while the levels of FSH in serum and MDA in ovarian tissue significantly increased(P<0.05).Compared with the experiment group 1,the levels of AMH and E_(2) in serum and SOD activity in ovarian tissue of rats in experiment groups 2,3 and 4 significantly decreased,while the levels of FSH in serum and MDA in ovarian tissue significantly increased(P<0.05).There was no significant difference in the levels of AMH,E_(2) and FSH in serum,SOD activity and MDA level in ovarian tissue of rats among experiment groups 2,3 and 4(P>0.05).The apoptosis rate of ovarian granulosa cells in experiment groups 1,2,3 and 4 was significantly higher than that in the blank control group(P<0.05);the apoptosis rate of ovarian granulosa cells in experiment groups 2,3 and 4 was significantly higher than that in experiment group 1(P<0.05);the apoptosis rate of ovarian granulosa cells in experiment groups 3 and 4 was significantly higher than that in experiment group 2(P<0.05);the apoptosis rate of ovarian granulosa cells in experiment group 4 was significantly higher than that in experiment group 3(P<0.05).In the blank control group,the ovarian tissue capsule was intact,and the number and development of primordial,primary and secondary follicles in the cortex were normal,with rare atretic follicles and less corpus luteum.The fibrous connective tissue in the medullary area was closely arranged,and no obvious edema or necrosis was observed.In experiment groups 1,2,3 and 4,the number of follicles in ovarian tissue decreased,the number of atretic follicles increased,and necrosis and shedding of follicles in the granular layer and cystic dilatation of follicles were observed.The pathological changes of ovarian tissue in experiment group 1 were relatively light,with fewer atretic follicles and less cell necrosis in the granulosa cell layer.The degree of ovarian tissue lesions in experiment group 4 was the most severe,with more atretic follicles and cell necrosis and shedding in the granulosa cell layer.The degree of ovarian tissue lesions in experiment groups 2 and 3 was less than that in experiment group 4,with fewer atretic follicles,necrosis of granulosa cell layer and follicle cystic dilatation.In the blank control group,the ovarian granulosa cells were normal in morphology and structure,with irregular polygon nuclei,uniform distribution of chromatin,mainly euchromatin,clear and complete nuclear membrane,mitochondria and other organelles with complete and clear structure visible in the cytoplasm.Different degrees of mitophagy were observed in the cytoplasm of granulosa cells of rats in experiment groups 1,2,3 and 4;the mitophagy of rats in experiment groups 2,3 and 4 was more severe than that in experiment group 1;that in experiment groups 3 and 4 was more severe than experiment group 2;the mitophagy of rats in experiment group 3 was similar to that in experiment group 4.Under electron microscopy,more mitochondrial autophagosomes were found in the cytoplasm of ovarian granulosa cells in experiment groups 3 and 4.Most mitochondria were mildly swollen,some mitochondrial crista structures disappeared,and the rough endoplasmic reticulum showed cystic dilatation.Conclusion The ovarian dysfunction model in rats can be successfully established by TP intragastric administration.The ovarian injury of rats is related to TP dose and intervention time.TP-induced ovarian oxidative stress may be an important factor triggering ovarian hypofunction,and it may work through oxidative stress affecting ovarian endocrine function and inducing granulosa cell apoptosis and other pathophysiological processes.With the aggravation of oxidative stress damage,the mitophagy increases.After reaching a certain degree,mitophagy will not continue to increase with the increase of TP intervention time.
作者
严谨
邓蒂斯
吴克明
YAN Jin;DENG Disi;WU Keming(School of Basic Medical Science,Shaanxi University of Chinese Medicine,Xianyang 712046,Shaanxi Province,China;Department of Gynecology,Affiliated Hospital of Chengdu University of Traditional Chinese Medicine,Chengdu 610000,Sichuan Province,China)
出处
《新乡医学院学报》
CAS
2024年第2期115-121,共7页
Journal of Xinxiang Medical University
基金
陕西省教育厅科学研究项目(编号:21JS017)
2021年度陕西中医药大学校级科研课题。
