大黄-桃仁药对调控子宫腺肌病在位内膜线粒体稳态的机制研究

Mechanism of Rhei Radix et Rhizoma-Persicae Semen drug pair on regulating endometrial mitochondrial homeostasis in adenomyosis

目的 探索大黄-桃仁药对对子宫腺肌病(adenomyosisi,AM)在位内膜线粒体稳态的调控机制。方法 同种异体垂体移植法构建AM小鼠模型,光镜及透射电镜(TEM)观察大黄-桃仁药对治疗前后子宫组织病理及超微结构改变;免疫荧光检测线粒体自噬通路PTEN诱导的推定激酶1(PTEN-induced putative kinase 1,PINK1)/帕金森蛋白(Parkinson protein,Parkin)蛋白表达。培养人永生化子宫内膜异位症在位内膜间质细胞hEM15A,观察大黄-桃仁药对处理前后细胞增殖能力、活性氧(reactive oxygen species,ROS)、抗氧化酶系、线粒体膜电位、线粒体自噬小体、线粒体自噬蛋白的变化。结果 病理切片显示,经大黄-桃仁药对治疗后,中、重度的浸润比例较模型组减少。TEM观察发现模型组子宫内膜上皮细胞及间质细胞线粒体肿胀、线粒体脊消失,部分形成线粒体自噬体;经大黄-桃仁药对治疗后子宫内膜上皮细胞及间质细胞线粒体数量增加,无明显肿胀、线粒体脊清晰可见。与对照组比较,造模后子宫内膜层PINK1、Parkin表达显著上调(P<0.001);经大黄-桃仁药对治疗后,PINK1、Parkin表达显著下降(P<0.05、0.001)。体外实验结果表明,hEM15A细胞线粒体膜电位下降,ROS呈较高水平;经大黄-桃仁药对处理后,hEM15A细胞的增殖及侵袭能力下降(P<0.05、0.001),线粒体膜电位升高,ROS水平下降(P<0.001),抗氧化酶系统Cu-Zn超氧化物歧化酶(superoxide dismutase,SOD)、Mn-SOD和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性上调(P<0.01、0.001),氧化损伤指标丙二醛(malondialdehyde,MDA)水平下调(P<0.01)。hEM15A细胞线粒体与溶酶体之间存在较强的共定位关系,经大黄-桃仁药对处理后,线粒体与溶酶体共定位的数量显著减少(P<0.05),PINK1、Parkin及其下游视神经蛋白(Optineurin,OPTIN)、核点蛋白52(nuclear dot protein 52,NDP52)经大黄-桃仁药对处理后显著下调(P<0.01、0.001)。结论 大黄-桃仁药对可通过改善线粒体抗氧化酶系、调控线粒体自噬水平、恢复线粒体膜电位等多途径维护AM在位内膜细胞乏氧线粒体稳态,从而减轻氧化应激损伤。

Objective To explore the regulation mechanism of Dahuang(Rhei Radix et Rhizoma)-Taoren(Persicae Semen) drug pair(RP)on endometrial mitochondrial homeostasis in adenomyosis(AM). Methods AM mice model was established by allogeneic pituitary transplantation. Histopathological and ultrastructural changes of uterus before and after treatment with RP were observed by light microscope and transmission electron microscope(TEM). Immunofluorescence was used to detect PTEN-induced putative kinase 1(PINK1)/Parkinson protein(Parkin) expressions. Human immortalized endometriosis eutopic endometrial stromal cells hEM15A were cultured, and the effects of RP on cell proliferation, reactive oxygen species(ROS), antioxidant enzymes, mitochondrial membrane potential, mitophagosomes and mitophagy proteins changes. Results Pathological sections showed that compared with model group,proportion of moderate and severe infiltration was reduced after treatment with RP. TEM observation showed that mitochondria of endometrial epithelial cells and interstitial cells in model group were swollen, mitochondrial ridges were disappeared and some mitophagosomes were formed;The number of mitochondria in endometrial epithelial cells and interstitial cells were increased after treatment with RP, there was no obvious swelling, and mitochondrial ridges were clearly visible. Compared with control group, PINK1and Parkin expressions in endometrial layer after modeling were significantly up-regulated(P < 0.001);After treatment with RP, PINK1and Parkin expressions were significantly decreased(P < 0.05, 0.001). The results of in vitro experiments showed that mitochondrial membrane potential of hEM15A cells was decreased, and ROS level was higher;After treatment with RP, proliferation and invasion abilities of hEM15A cells were decreased(P < 0.05, 0.001), mitochondrial membrane potential was increased, ROS level was decreased(P < 0.001), antioxidant enzyme systems Cu-Zn superoxide dismutase(SOD), Mn-SOD and glutathione peroxidase(GSH-Px) activities were up-regulated(P < 0.01, 0.001), and oxidative damage index malondialdehyde(MDA) level was down-regulated(P < 0.01). There was a strong co-localization relationship between mitochondria and lysosomes in hEM15A cells. After treatment with RP, number of mitochondria and lysosomes co-localized was significantly reduced(P < 0.05), PINK1, Parkin and their downstream proteins Optineurin(OPTIN), nuclear dot protein 52(NDP52) were significantly down-regulated after treatment with RP(P < 0.01, 0.001). Conclusion RP can maintain hypoxic mitochondrial homeostasis of AM resident endomembrane cells by improving mitochondrial antioxidant enzymes,regulating level of mitophagy and restoring mitochondrial membrane potential, thereby reducing oxidative stress injury.