摘要
背景:卵巢组织玻璃化冻存是保存生育力的重要方法之一。丹参酮ⅡA具有多种药理活性,包括抗氧化、抑制炎症反应、减少凋亡等,但其作为卵巢组织玻璃化冷冻保护添加剂的作用尚不清楚。目的:探讨丹参酮ⅡA对小鼠卵巢组织冷冻保存的保护作用。方法:获取25只6周龄雌性KM小鼠的卵巢组织,随机分为5组,每组10个卵巢:新鲜组不进行冷冻保存,冷冻对照组使用玻璃化冷冻保护剂,0.5,2.5,5μmol/L丹参酮ⅡA组分别使用含0.5,2.5,5μmol/L丹参酮ⅡA的玻璃化冷冻保护剂,置于液氮中冷冻保存。保存3 d后取出冻存管解冻,采用苏木精-伊红染色观察各组卵巢组织及卵泡形态,分析卵泡形态正常率及存活率;通过酶联免疫法检测卵巢内超氧化物歧化酶、过氧化氢酶、丙二醛、肿瘤坏死因子α、白细胞介素1β及白细胞介素17水平;采用RT-qPCR和Western blot检测小鼠卵巢内核因子E2相关因子2(nuclear factor erythroid derived 2-like 2,Nrf2)和血红素加氧酶1(heme oxygenase-1,HO-1)的mRNA及蛋白表达。结果与结论:①与新鲜组比较,冷冻对照组卵巢内各级卵泡形态异常、卵泡存活率下降(P<0.05),超氧化物歧化酶、过氧化氢酶活性下降(P<0.05),丙二醛、肿瘤坏死因子α、白细胞介素1β及白细胞介素17水平均升高(P<0.05),Nrf2、HO-1的mRNA及蛋白表达均下降(P<0.05);②与冷冻对照组比较,不同浓度丹参酮ⅡA均可改善卵巢内各级卵泡形态、提升卵泡存活率,提升超氧化物歧化酶、过氧化氢酶活性,降低丙二醛、肿瘤坏死因子α、白细胞介素1β及白细胞介素17水平,提升Nrf2、HO-1的mRNA及蛋白表达,并且呈现浓度依赖性,以5μmol/L丹参酮ⅡA的作用最显著;③结果表明,丹参酮ⅡA可能通过介导Nrf2/HO-1信号通路降低小鼠卵巢组织的氧化应激水平和炎症反应,减轻小鼠卵巢玻璃化冷冻保存而出现的生殖损伤。
BACKGROUND:Ovarian tissue vitrification cryopreservation is one of the important methods for preserving fertility.Tanshinone IIA has various pharmacological activities,including anti-oxidation,inhibition of inflammatory response,and reduction of apoptosis,but its role as an additive for vitrification cryoprotection of ovarian tissue is still unclear.OBJECTIVE:To explore the protective effect of tanshinone IIA on vitrification cryopreservation of mouse ovarian tissue.METHODS:Twenty-five 6-week-old female KM mice were randomly selected and their ovarian tissues were randomly divided into five groups,with 10 ovaries per group.The fresh group was not cryopreserved.The frozen control group used vitrification cryoprotectant.The 0.5,2.5,and 5μmol/L tanshinone IIA groups used vitrification cryoprotectant containing 0.5,2.5,and 5μmol/L tanshinone IIA,respectively,and were cryopreserved in liquid nitrogen.After 3 days of storage,the cryopreserved tubes were taken out and thawed.The ovarian tissue and follicle morphology of each group were observed by hematoxylin-eosin staining,and the normal follicle morphology and survival rate were analyzed.The levels of superoxide dismutase,catalase,malondialdehyde,tumor necrosis factor-α,interleukin-1β,and interleukin-17 in the ovary were detected by enzyme-linked immunosorbent assay.RT-qPCR and western blot assay were used to detect the mRNA and protein expressions of nuclear factor E2-related factor 2(Nrf2)and heme oxygenase 1(HO-1)in the mouse ovary.RESULTS AND CONCLUSION:(1)Compared with the fresh group,the frozen control group had abnormal morphology of follicles at all levels in the ovary,decreased follicle survival rate(P<0.05),decreased superoxide dismutase and catalase activities(P<0.05);the levels of malondialdehyde,and tumor necrosis factorα,interleukin 1β,and interleukin 17 were all increased(P<0.05),and the mRNA and protein expressions of Nrf2 and HO-1 were decreased(P<0.05).(2)Compared with the frozen control group,different concentrations of tanshinone IIA could improve the morphology of follicles at all levels in the ovary,increase the survival rate of follicles,enhance the activities of superoxide dismutase and catalase,and reduce the levels of malondialdehyde,tumor necrosis factorα,interleukin 1β,and interleukin 17,increased the mRNA and protein expression of Nrf2 and HO-1 in a concentration-dependent manner,with 5μmol/L tanshinone IIA having the most significant effect.(3)The results show that tanshinone IIA may reduce the oxidative stress level and inflammatory response of mouse ovarian tissue by mediating the Nrf2/HO-1 signaling pathway,thereby alleviating the reproductive damage caused by vitrification cryopreservation of mouse ovaries.
作者
吴小绸
王慧颖
王婕
张彩凤
侯雁云
金波
Wu Xiaochou;Wang Huiying;Wang Jie;Zhang Caifeng;Hou Yanyun;Jin Bo(Department of Gynecology,Shenzhen Hospital of Beijing University of Chinese Medicine(Longgang),Shenzhen 518000,Guangdong Province,China;Department of Pathology,Shenzhen Hospital of Beijing University of Chinese Medicine(Longgang),Shenzhen 518000,Guangdong Province,China)
出处
《中国组织工程研究》
北大核心
2025年第29期6198-6204,共7页
Chinese Journal of Tissue Engineering Research
基金
深圳市“医疗卫生三名工程”项目资助(SZZYSM202311019),项目负责人:王慧颖
深圳市龙岗区医疗卫生技术攻关项目(LGKCYLWS2023002),项目负责人:王慧颖
深圳市龙岗区科技创新局医疗卫生科技计划项目(LGKCYLWS2021000019),项目负责人:王慧颖
育龙计划-三龙人才项目(2023-BUCMSZYLRC13),项目负责人:吴小绸
山东银丰生命科学研究项目资助(KJKHX2023001),项目负责人:王慧颖。
