二陈汤调控谷胱甘肽过氧化物...多囊卵巢综合征的作用及机制_丛培玮.pdf

中华中医药杂志(原中国医药学报)2023年2月第38卷第2期 CJTCMP,February 2023,Vol.38,No.2 615 二陈汤调控谷胱甘肽过氧化物酶4/环氧化酶介导的氧化损伤及铁死亡改善多囊卵巢综合征的 作用及机制丛培玮1，张丽娜2，王丹3，尚冰4，张林5（1辽宁中医药大学中医脏象理论及应用国家教育部重点实验室，沈阳 110847；2辽宁中医药大学教学实验中心，沈阳 110847；3辽宁中医药大学医学检验学院，沈阳 110847；4辽宁中医药大学 中医文献研究院，沈阳 110847；5辽宁中医药大学中医学院，沈阳 110847）摘要：目的：基于多囊卵巢综合征（PCOS）“痰湿不孕”理论探讨二陈汤对PCOS的防治作用机制。方法：选取动情周期规律SD大鼠32只随机分为对照组（n=10），模型组（n=22），模型组给予高脂饲料+来曲唑溶液灌胃。造模结束后随机选取周期延长的大鼠（n=10）为治疗组，1 mL/100 g剂量连续4周灌胃给予二陈汤煎剂。HE染色法观察大鼠卵巢形态学变化，比色法检测血清及卵巢超氧化物歧化酶（SOD）、丙二醛（MDA）和谷胱甘肽过氧化物酶（GSH-Px）水平变化，Western Blot法检测卵巢谷胱甘肽过氧化物酶4（GPX4）、环氧化酶-2（COX-2）和铁蛋白重链（FTH1）蛋白水平变化。结果：与对照组比较，模型组大鼠卵巢可见大量囊性扩张卵泡，闭锁卵泡及早期发育的小卵泡；血清、卵巢中SOD、GSH-Px水平显著降低（P0.01），MDA水平显著升高（P0.01）；COX-2蛋白表达显著升高（P0.05），GPX4、FTH1蛋白表达显著降低（P0.05）；与模型组比较，二陈汤治疗后大鼠卵巢囊性卵泡明显减少，黄体数量有所增加，出现少量正常发育的早期小卵泡；血清、卵巢中SOD、MDA、GSH-Px水平及卵巢中GPX4、COX-2和FTH1蛋白水平均有显著改善（P0.01，P0.05）。结论：二陈汤能显著改善PCOS卵巢情况，其机制可能与调控GPX4/COX-2介导的氧化损伤及抑制卵巢细胞铁死亡相关。关键词：二陈汤；多囊卵巢综合征；氧化损伤；铁死亡；痰湿不孕基金资助：国家自然科学基金青年科学基金项目（No.82104735），中国博士后科学基金第71批面上资助“地区专项支持计划”项目（No.2022MD713764），辽宁中医药大学中医脏象理论及应用教育部重点实验室开放基金资助（No.zyzx2202），辽宁省自然科学基金项目（No.2019-ZD-0953），辽宁省教育厅科学技术研究项目（No.L202038）Effects and mechanism of Erchen Decoction on the regulation of phospholipid hydroperoxide glutathione peroxidase/cyclooxygenase-2 mediated oxidative damage and ferroptosis on polycystic ovary syndromeCONG Pei-wei1,ZHANG Li-na2,WANG Dan3,SHANG Bing4,ZHANG Lin5(1Key Laboratory of Ministry of Education for TCM Viscera-State Theory and Applications,Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China;2Teaching and Experimental Center,Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China;3School of Medical Laboratory,Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China;4Academy of Traditional Chinese Medicine Literature,Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China;5College of Traditional Chinese Medicine,Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China)论著通信作者：张林，辽宁省沈阳市皇姑区崇山东路79号辽宁中医药大学中医学院，邮编：110847，电话：024-31207185E-mail：内文1.indd 6152023/2/10 16:57:32中华中医药杂志(原中国医药学报)2023年2月第38卷第2期 CJTCMP,February 2023,Vol.38,No.2 616 Abstract:Objective:To explore the effects of Erchen Decoction on PCOS and its possible mechanism based on the theory of phlegm-dampness infertility of polycystic ovary syndrome(PCOS).Methods:Thirty-two SD rats with regular estrus cycle were randomly divided into control group(n=10)and model group(n=22).The model group was given high fat diet and letrozole solution by gavage.After modeling,rats in the extended model group(n=10)were randomly selected as the treatment group and given 1 mL/100 g intragastric administration for 4 consecutive weeks.The morphologic changes of ovaries were observed by HE staining,the levels of SOD,MDA and GSH-Px in serum and ovaries were detected by colorimetry,and the protein levels of GPX4,COX-2 and FTH1 in ovaries were detected by Western Blot.Results:Compared with the control group,there were a lot of cystic dilated follicles,atretic follicles and early developing small follicles in the ovary of the model group.The levels of SOD and GSH-Px in serum and ovary were significantly decreased(P0.01),while the levels of MDA were significantly increased(P0.01),the protein expression of COX-2 was significantly increased(P0.05),and the protein expression of GPX4 and FTH1 was significantly decreased(P0.05).Compared with model group,after treatment with Erchen Decoction,cystic ovarian follicles were significantly reduced,the number of corpus luteum was increased,and there were a few small follicles in the early stage of normal development,the levels of SOD,MDA and GSH-Px in serum and ovary and the protein levels of GPX4,COX-2 and FTH1 in ovary were significantly improved(P0.01,P0.05).Conclusion:Erchen Decoction can significantly improve the ovarian condition of PCOS,the mechanism may be related to the regulation of GPX4/COX-2 mediated oxidative damage and inhibition of iron death of ovarian cells.Key words:Erchen Decoction;Polycystic ovary syndrome(PCOS);Oxidative damage;Ferroptosis;Phlegm-dampness infertilityFunding:Young Scientists of the National Natural Science Foundation of China(No.82104735),The 71st General Program of Regional Special Support Program by China Postdoctoral Science Foundation(No.2022MD713764),Open Project of Key Laboratory of Ministry of Education for TCM Viscera-State Theory and Applications,Liaoning University of Traditional Chinese Medicine(No.zyzx2202),Natural Science Foundation of Liaoning Province(No.2019-ZD-0953),Science and Technology Research Project of Education Department of Liaoning Province(No.L202038)多囊卵巢综合征（polycystic ovary syndrome，PCOS）是一种常见的女性生殖和内分泌功能紊乱综合征，居无排卵性不孕病因的首位1。PCOS属于中医学“月经不调”“闭经”“不孕”等范畴。现代医家多认为“痰湿壅塞冲任”是PCOS的主要临床证候，“化痰健脾”为其治疗根本原则2-3。现代医学研究证实，氧化损伤是PCOS卵巢病变的重要病理因素之一，已有研究4表明“痰湿壅塞冲任”与氧化应激反应关系密切，而铁死亡是一种以脂质过氧化为特征的非凋亡调控的细胞死亡，表现为活性氧（reactive oxygen species，ROS）累积引发细胞毒性，进而破坏细胞膜结构，诱导细胞死亡发生5-6。本课题组前期研究7发现，二陈汤能够显著改善氧化修饰的低密度脂蛋白（oxidized low density lipoprotein，ox-